cyclin-d1 and Prostatic-Neoplasms

Cyclin D1 (CCND1) is critical in the transition of the cell cycle from the G1 to S phases, and unbalanced cell cycle regulation is a hallmark of carcinogenesis. Numerous epidemiological studies have evaluated the association between the CCND1 A870G polymorphism and the risk of prostate cancer (PCa). However, these studies have yielded conflicting results.. In the present study, the possible association above was assessed by a meta-analysis. Eligible articles were identified for the period up to July 2014. Pooled odds ratios (ORs) with 95% confidence intervals (95% CI) were appropriately derived from fixed effects or random effects models.. A total of ten case-control studies, which included 3,820 cases and 3,825 controls, were identified. Overall, the allelic/genotypic association between the G870A polymorphism and prostate cancer was nonsignificant (OR = 1.045, 95% CI = 0.947 to 1.153 for A versus G, P = 0.380; OR = 1.088, 95% CI = 0.896 to 1.321 for AA versus GG, P = 0.393; OR = 1.044, 95% CI = 0.941 to 1.158 for GA versus GG, P = 0.414; OR = 1.053, 95% CI = 0.955 to 1.161 for the dominant model AA + GA versus GG, P = 0.303; OR = 1.072, 95% CI = 0.881 to 1.306 for the recessive model AA versus AA + GA, P = 0.486). Moreover, subgroup analyses according to ethnicity failed to demonstrate a significant association between this polymorphism and prostate cancer. In addition, we also performed a stratified analysis of cases with PCa metastasis, and the results supported the findings of no significant association between CCND1 A870G polymorphism and metastasis risk of PCa.. Our results suggest that the CCND1 A870G polymorphism might not be a potential candidate for predicting prostate cancer risk, including metastasis risk.

Topics: Case-Control Studies; Cyclin D1; Genetic Predisposition to Disease; Humans; Male; Polymorphism, Genetic; Prognosis; Prostatic Neoplasms; Risk Factors

The NF-kappaB family of transcription factors has been shown to be constitutively activated in various human malignancies, including leukemias, lymphomas, and a number of solid tumors. NF-kappaB is hypothesized to contribute to development and/or progression of malignancy by regulating the expression of genes involved in cell growth and proliferation, anti-apoptosis, angiogenesis, and metastasis. Prostate cancer cells have been reported to have constitutive NF-kappaB activity due to increased activity of the IkappaB kinase complex. Furthermore, an inverse correlation between androgen receptor (AR) status and NF-kappaB activity was observed in prostate cancer cell lines. NF-kappaB may promote cell growth and proliferation in prostate cancer cells by regulating expression of genes such as c-myc, cyclin D1, and IL-6. NF-kappaB may also inhibit apoptosis in prostate cancer cells through activation of expression of anti-apoptotic genes, such as Bcl-2, although pro-apoptotic activity of NF-kappaB has also been reported. NF-kappaB-mediated expression of genes involved in angiogenesis (IL-8, VEGF), and invasion and metastasis (MMP9, uPA, uPA receptor) may further contribute to the progression of prostate cancer. Constitutive NF-kappaB activity has also been demonstrated in primary prostate cancer tissue samples and suggested to have prognostic importance for a subset of primary tumors. The limited number of samples analyzed in those studies and the relative lack of NF-kappaB target genes identified in RNA expression microarray analyses of prostate cancer cells suggest that further studies will be required in order to determine if NF-kappaB actually plays a role in human prostate cancer development, and/or progression, and to characterize its potential as a therapeutic target.

Topics: Cell Division; Cyclin D1; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; Male; Metalloendopeptidases; NF-kappa B; Prostatic Neoplasms; Receptors, Androgen

The development and progression of a prostate carcinoma from prediagnosis to death can be characterized as a series of clinical states. The states are milestones that can be used to assess prognosis, define therapeutic objectives, and assess outcomes. The antitumor effects of hormone therapies and cytotoxic agents in patients with prostate carcinoma are placed in context along with the bidrectional tumor-host interactions that contribute to the growth and resistance of osseous lesions. Identifying the factors that contribute to the growth of the disease at different points in the illness has lead to novel, systemic approaches. Proving the benefit of these approaches requires a series of unique trials with unique endpoints relevant to the clinical state of the patients and the specific therapy under evaluation.

Topics: Androgen Antagonists; Bone Neoplasms; Clinical Trials as Topic; Cyclin D1; Disease Progression; Hormone Antagonists; Humans; Hypothalamo-Hypophyseal System; Ki-67 Antigen; Male; Neoplasm Recurrence, Local; Orchiectomy; Pituitary-Adrenal System; Prostate-Specific Antigen; Prostatic Neoplasms; Radionuclide Imaging; Survival Rate

Prostate cancer (PCa) is prevalent among older men and difficult to survive after metastasis. It is urgent to find new drugs and treatments. Several studies show that taraxasterol (TAX) has important anti-inflammatory, anti-oxidative and anti-tumor effects. However, the function and mechanisms of TAX in PCa remain unclear. Here, we found that TAX could significantly suppress the viability and growth of androgen-independent PCa cells and down-regulate the expression of c-Myc and cyclin D1 in vitro. Mechanistically, PI3K/AKT signaling pathway was weakened and the expression of FGFR2 was reduced after TAX treatment in androgen-independent PCa cells. Moreover, TAX evidently inhibited the tumor growth in nude mice and the expression of c-Myc, cyclin D1, p-AKT and FGFR2 were down-regulated in xenograft tumor. These results indicate that TAX suppresses the proliferation of androgen-independent PCa cells via inhibiting the activation of PI3K/AKT signaling pathway and the expression of FGFR2, which means TAX may be a novel anti-tumor agent for later PCa treatment.

Topics: Androgens; Animals; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Humans; Male; Mice; Mice, Nude; Phosphatidylinositol 3-Kinases; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Receptor, Fibroblast Growth Factor, Type 2; Signal Transduction

Dysregulation of the cell cycle contributes to tumor progression. Cell division cycle‑associated 3 (CDCA3) is a known trigger of mitotic entry and has been demonstrated to be constitutively upregulated in tumors. It is therefore associated with carcinogenic properties reported in various cancers. However, the role of CDCA3 in prostate cancer is unclear. In the present study, western blotting and analysis of gene expression profiling datasets determined that CDCA3 expression was upregulated in prostate cancer and was associated with a poor prognosis. CDCA3 knockdown in DU145 and PC‑3 cells led to decreased cell proliferation and increased apoptosis, with increased protein expression levels of cleaved‑caspase3. Further experiments demonstrated that downregulated CDCA3 expression levels induced G0/G1 phase arrest, which was attributed to increased p21 protein expression levels and decreased cyclin D1 expression levels via the regulation of NF‑κB signaling proteins (NFκB‑p105/p50, IKKα/β, and pho‑NFκB‑p65). In conclusion, these results indicated that CDCA3 may serve a crucial role in prostate cancer and consequently, CDCA3 knockdown may be used as a potential therapeutic target.

Topics: Apoptosis; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Disease Progression; Down-Regulation; G1 Phase; Humans; Male; NF-kappa B; Prostatic Neoplasms; Resting Phase, Cell Cycle; Signal Transduction; Up-Regulation

Prostate cancer continues to be one of the main global health issues in men. Neuropeptide substance P (SP) acting via neurokinin-1receptor (NK1R) promotes tumorigenicity in many human malignant tumors. However, its pro-tumorigenic functions and the therapeutic effects of its inhibition in prostate cancer remain unclear.. MTT assay was employed for measuring cellular proliferation and cytotoxicity. mRNAs and proteins expression levels were evaluated by qRT-PCR and western blot assay, respectively. Gelatinase activity was assessed by zymography. The migration ability was defined using wound-healing assay. Flow cytometry was employed to evaluate the cell cycle distribution. We also performed an in vivo experiment in a mouse model of prostate cancer to confirm the in vitro therapeutic effect of targeting the SP/NK1R system.. We found a noticeable increase in the expression of the truncated isoform of NK1R as an oncogenic NK1R splice variant in tumor cells. We also demonstrated that SP promotes both proliferative and migrative phenotypes of prostate cancer through modifying cell cycle-related proteins (c-Myc, cyclin D1, cyclin B1, p21), and apoptosis-related genes (Bcl-2 and Bax), promoting cell migration and increasing MMP-2 and MMP-9 expression and activity, while aprepitant administration could remarkably reverse these effects. SP also stimulated tumor growth in vivo, which was correlated with shorter survival times, while aprepitant reversed this effect and led to significantly longer survival time.. Our findings suggest that SP/NK1R system may serve as a novel therapeutic target in prostate cancer and support the possible candidacy of aprepitant in future prostate cancer therapy.

Topics: Animals; Antineoplastic Agents; Aprepitant; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; G2 Phase Cell Cycle Checkpoints; Humans; Male; Matrix Metalloproteinases; Mice; Mice, Nude; Prostatic Neoplasms; Protein Isoforms; Proto-Oncogene Proteins c-bcl-2; Receptors, Neurokinin-1; Substance P; Survival Rate; Transplantation, Heterologous

The cold-shock protein Y-box-binding protein (YB)-1 regulates the expression of various chemokines and their receptors at the transcriptional level. Expression of the orphan chemokine CXCL14 is repressed by EGF induced signaling. The possible links between EGF-mediated YB-1 and CXCL14 as well as the functions of critical kinase pathways in the progression of prostate cancer have remained unexplored. Here we examined the correlation between YB-1 and CXCL14, and the ERK/AKT/mTOR pathways in prostate cancer. Knockdown of YB-1 decreased cyclinD1 expression with an upregulation of cleaved-PARP in human prostate cancer cells. EGF treatment upregulated phospho-YB-1 expression in a time-dependent manner, while treatment with an ERK inhibitor completely silenced its expression in prostate cancer cells. EGF treatment stimulates CyclinD1 and YB-1 phosphorylation in an ERK-dependent pathway. Positive and negative regulation of YB-1 and CXCL14 was observed after EGF treatment in prostate cancer cells, respectively. EGF rescues cell cycle and apoptosis via the AKT and ERK pathways. Furthermore, YB-1 silencing induces G1 arrest and apoptosis, while knockdown of CXCL14 facilitates cell growth and inhibits apoptosis in prostate cancer cells. YB-1 and CXCL14 were inversely correlated in prostate cancer cells and tissues. A significant association between poor overall survival and High YB-1 expression was observed in human prostate cancer patients. In conclusion, our data reveal the functional relationship between YB-1 and CXCL14 in EGF mediated ERK signaling, and YB-1 expression is a significant prognostic marker to predict prostate cancer.

Topics: Apoptosis; Cell Cycle; Cell Line, Tumor; Chemokines, CXC; Cyclin D1; Disease Progression; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Gene Silencing; Humans; Male; MAP Kinase Signaling System; Middle Aged; Phosphorylation; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Signal Transduction; Up-Regulation; Y-Box-Binding Protein 1

Lysine (K)-specific demethylase 6B (KDM6B), a stress-inducible H3K27me3 demethylase, plays oncogenic or antitumoral roles in malignant tumors depending on the type of tumor cell. However, how this histone modifier affects the progression of prostate cancer (PCa) is still unknown. Here we analyzed sequenced gene expression data and tissue microarray to explore the expression features and prognostic value of KDM6B in PCa. Further, we performed in vitro cell biological experiments and in vivo nude mouse models to reveal the biological function, upstream and downstream regulation mechanism of KDM6B. In addition, we investigated the effects of a KDM6B inhibitor, GSK-J4, on PCa cells. We showed that KDM6B overexpression was observed in PCa, and elevated KDM6B expression was associated with high Gleason Score, low serum prostate-specific antigen level and shorted recurrence-free survival. Moreover, KDM6B prompted proliferation, migration, invasion and cell cycle progression and suppressed apoptosis in PCa cells. GSK-J4 administration could significantly suppress the biological function of KDM6B in PCa cells. KDM6B is involved in the development of castration-resistant prostate cancer (CRPC), and combination of MDV3100 plus GSK-J4 is effective for CRPC and MDV3100-resistant CRPC. Mechanism exploration revealed that androgen receptor can decrease the transcription of KDM6B and that KDM6B demethylates H3K27me3 at the cyclin D1 promoter and cooperates with smad2/3 to prompt the expression of cyclin D1. In conclusion, our study demonstrates that KDM6B is an androgen receptor regulated gene and plays oncogenic roles by promoting cyclin D1 transcription in PCa and GSK-J4 has the potential to be a promising agent for the treatment of PCa.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Cyclin D1; DNA Methylation; Gene Expression Regulation, Neoplastic; Heterografts; Humans; Jumonji Domain-Containing Histone Demethylases; Male; Mice; Mice, Nude; PC-3 Cells; Promoter Regions, Genetic; Prostatic Neoplasms; Signal Transduction; Tissue Array Analysis; Transfection

Emerging evidence has implied the importance of long non-coding RNAs in cancer development, including prostate cancer (PCa). Bioinformatic analyses have identified that ADAMTS9-AS1 may play a role in cancer progression.. A quantitative real-time polymerase chain reaction was conducted to measure ADAMTS9-AS1 expression level at messenger RNA level. In vitro functional analyses were performed to investigate cell behaviors status under different conditions. Moreover, rescue assays were conducted to explore the potential mechanisms of ADAMTS9-AS1 in PCa progression.. ADAMTS9-AS1 expression is down-regulated in PCa. Forcing ADAMTS9-AS1 expression impedes PCa cell proliferation via initiating cell apoptosis. Importantly, microRNA-142-5p (miR-142-5p) mimic and small-interfering RNA targeting cyclin D1 (CCND1, si-CCND1) could attenuate the inhibitory effects of ADAMTS9-AS1 overexpression on PCa cell growth.. Collectively, our results indicate that ADAMTS9-AS1 suppresses PCa progression by regulating the miR-142-5p/CCND1 axis, which provides a new mechanism for the progression of PCa.

Topics: Apoptosis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Disease Progression; Gene Expression Regulation, Neoplastic; Humans; Male; MicroRNAs; Prostatic Neoplasms; RNA, Long Noncoding; Signal Transduction

Eight novel ERβ selective daidzein analogues (NCE1-8) were synthesized and their anti-cancer activity was evaluated by

Topics: Animals; Caspases; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Dose-Response Relationship, Drug; Estrogen Receptor beta; Female; Genistein; Humans; Isoflavones; Male; Mice; Mice, Inbred C57BL; Prostatic Neoplasms; Rats; Rats, Sprague-Dawley; Receptors, Estrogen; Tumor Burden; Xenograft Model Antitumor Assays

Histone deacetylases (HDACs) play a vital role in the epigenetic regulation of gene expression due to their overexpression in several cancer forms. Therefore, these enzymes are considered as a potential anticancer drug target. Different synthetic and natural structures have been studied as HDACs inhibitors; based on available structural design information, the capping group is important for the biological activity due to the different interactions in the active site entrance. The present study aimed to analyze high substituted pyridine as a capping group, which included carrying out the synthesis, antiproliferative activity analysis, and docking studies of these novel compounds.. To achieve the synthesis of these derivatives, four reaction steps were performed, generating desired products 15a-k. Their effects on cell proliferation and gene expression of p21, cyclin D1, and p53 were determined using the sulphorhodamine B (SRB) method and quantitative real-time polymerase chain reaction. The HDAC1, HDAC6, and HDAC8 isoforms were used for performing docking experiments with our 15a-k products.. The products 15a-k were obtained in overall yields of 40-71%. Compounds 15j and 15k showed the highest antiproliferative activity in the breast (BT-474 and MDA-MB-231) and prostate (PC3) cancer cell lines at a concentration of 10 µM. These compounds increased p21 mRNA levels and decreased cyclin D1 and p53 gene expression. The docking study showed an increment in the strength, and in the number of interactions performed by the capping moiety of the tested molecules compared with SAHA; interactions displayed are mainly van der Waals, π-stacking, and hydrogen bond.. The synthesized compounds 2-thiophene (15j) and 2-furan (15k) pyridine displayed cell growth inhibition, regulation of genes related to cell cycle progression in highly metastatic cancer cell lines. The molecular coupling analysis performed with HDAC1, HDAC6 and HDAC8 showed an increment in the number of interactions performed by the capping moiety and consequently in the strength of the capping group interaction.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Epigenesis, Genetic; Female; Furans; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Male; Molecular Docking Simulation; PC-3 Cells; Pregnancy; Prostatic Neoplasms; Pyridines; Thiophenes; Tumor Suppressor Protein p53

BACKGROUND This study aimed to investigate the effects of the 6-nitroindazole compound and amino analog of ludartin, (11R)-13-(6-nitroindazole)-11,13-dihydroludartin (NDHL), on human prostate carcinoma cells in vitro and in mouse tumor xenografts in vivo. MATERIAL AND METHODS DU-145 and LNCaP human prostate carcinoma cells were cultured with increasing concentrations of NDHL. Cell viability was measured using the MTT assay, and cell apoptosis was measured by fluorescence flow cytometry. Mouse tumor xenografts were created by implanting 2×10⁶ of DU-145 cells subcutaneously in the left flank. On the second day following DU-145 cell implantation, the mice in the treatment groups were injected intraperitoneally with 2, 5, and 10 mg/kg of NDHL. RESULTS Treatment of DU-145 and LNCaP cells with NDHL (range, 2.5-20.0 μM) significantly reduced cell proliferation in vitro (P<0.05). The proliferation rate of DU-145 and LNCaP cells was reduced to 27% and 24%, respectively, following treatment with 20.0 μM of NDHL. Treatment with NDHL significantly increased cell apoptosis and the formation of reactive oxygen species (ROS) formation in DU-145 cells at 48 h (P<0.05). NDHL significantly increased the proportion of DU-145 cells in the G1 phase of the cell cycle and significantly increased the expression of cyclin D1 and p21 (P<0.05). Treatment of the mice in the xenograft tumor model with NDHL significantly increased survival and suppressed tumor growth (P<0.02). CONCLUSIONS NDHL inhibited cell proliferation, increased apoptosis, and caused cell cycle arrest in human prostate carcinoma cells in vitro and inhibited mouse tumor xenograft growth in vivo.

Topics: Animals; Apoptosis; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Humans; Indazoles; Male; Mice; Prostatic Neoplasms; Reactive Oxygen Species; Santonin; Xenograft Model Antitumor Assays

This research aimed to examine the antitumor mechanisms of selenium nanoparticles (SeNPs) specifically against prostate cancers.. The antitumor activities of SeNPs against cancer cells were determined via MTT assay. The cell cycle was determined by detecting the DNA content, and apoptosis was determined via annexin V-Fluos staining kit. The microRNA expressions in cancer cells were analyzed via microarray and qRT-PCR. The potential targets of miR-16 were identified via luciferase analysis and mRNA expression determination. miR-16 functions in cancer cells were explored via the transient transfection of miR-16 mimic or inhibitor.. SeNPs were most potent in prostate cancer cells, regardless of whether or not they were androgen-dependent. Furthermore, SeNP stimulation can induce cell cycle arrest and the apoptosis enhancement of prostate cancer cells. Microarray and molecular mechanism studies demonstrated that miR-16 could directly target cyclin D1 and BCL-2 to mediate SeNP apoptosis enhancement. Results show that the serum selenium levels positively correlate with miR-16 expressions, and they correlate with the overall and disease-free survival rates.. These results signify the cytotoxic potential of SeNPs in prostate cancer treatment.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cyclin D1; Disease-Free Survival; Drug Screening Assays, Antitumor; Gene Expression Regulation, Neoplastic; Humans; Male; MicroRNAs; Nanoparticles; Prognosis; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Selenium; Up-Regulation

Prostate cancer (PC) is common cancer worldwide. Several markers have been developed to differentiate between benign prostatic hyperplasia (BPH) from PC. A descriptive retrospective hospital-based study aimed at determining the expression of Cyclin D1 in BPH and PC. The study took place at different histopathology laboratories in Khartoum state, Sudan, from December 2016 to January 2019. Formalin-fixed paraffin-embedded blocks were sectioned and fixed in 3-aminopropyltriethoxysilane coated slides incubated into primary antibody for Cyclin D1. The assessment of immunoreactivity of Cyclin D1 of each section was done using the Gleason scoring system.. A total of 153 males' prostate sections included in this study, of them, 120 (78.4%) were PC, and 33 (21.6%) were BPH. Their age ranged from 45 to 88 years, mean age was 66.19 ± 8.599. 142 (92.8%) did not have a family history of PC, while 11 (7.2%) patients reported having a family history. The Gleason scoring showed a total of 81 (52.9%) patients with high-grade and 39 (25.5%) with low-grade. 118 (97.5%) patients had PC showed positive results for Cyclin D1, while BPH was 3 (2.5%). P value < 0.001. Cyclin D1 staining was associated with high-grade Gleason score and perineural invasion, P value 0.001.

Topics: Aged; Aged, 80 and over; Cyclin D1; Humans; Immunohistochemistry; Male; Middle Aged; Neoplasm Grading; Prostatic Hyperplasia; Prostatic Neoplasms; Retrospective Studies; Sudan

Cyclin D1 (CCND1) has been revealed as a key regulating protein in cell cycle (G1 phase) and plays a critical role in promoting tumor development. The purpose of our study was to investigate the associations between CCND1 and biochemical recurrence of prostate cancer (PCa). We performed immunostaining of CCND1 on a tissue microarray and evaluated the CCND1 expression levels based on the intensity and extent of staining. The clinical data was collected, and the follow-up data was received by searching our follow-up database called "PC-follow". We revealed that CCND1 expression patterns were different between cytoplasm and nucleus in this study, and the expression of CCND1 in adjacent normal tissues was higher than that in PCa tissues (

Topics: Aged; Aged, 80 and over; Biomarkers, Tumor; Cyclin D1; Humans; Male; Middle Aged; Prognosis; Prostate; Prostatic Neoplasms

Prostate cancer is the most common malignancy in men in developed countries. In previous study, we identified HNF1B (Hepatocyte Nuclear Factor 1β) as a downstream effector of Enhancer of zeste homolog 2 (EZH2). HNF1B suppresses EZH2-mediated migration of two prostate cancer cell lines via represses the EMT process by inhibiting SLUG expression. Besides, HNF1B expression inhibits cell proliferation through unknown mechanisms. Here, we demonstrated that HNF1B inhibited the proliferation rate of prostate cancer cells. Overexpression of HNF1B in prostate cancer cells led to the arrest of G1 cell cycle and decreased Cyclin D1 expression. In addition, we re-explored data from ChIP-sequencing (ChIP-seq) and RNA-sequencing (RNA-seq), and demonstrated that HNF1B repressed Cyclin D1 via direct suppression of SMAD6 expression. We also identified CDKN2A as a HNF1B-interacting protein that would contribute to HNF1B-mediated repression of SMAD6 expression. In summary, we provide the novel mechanisms and evidence in support HNF1B as a tumour suppressor gene for prostate cancer.

Topics: Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Gene Expression Regulation, Neoplastic; Hepatocyte Nuclear Factor 1-beta; Humans; Immunohistochemistry; Male; Prostatic Neoplasms; Protein Binding; Smad6 Protein

Prostate cancer (PCa) is one of the major health problems of the aging male. The roles of dysregulated microRNAs in PCa remain unclear. In this study, we mined the public published data and found that miR-487a-3p was significantly downregulated in 38 pairs of clinical prostate tumor tissues compared with the normal tissues. We further verified this result by in situ hybridization on tissue chip and quantitative real-time polymerase chain reaction (qRT-PCR) in PCa/normal cells. miR-487a-3p targeting of cyclin D1 (CCND1) was identified using bioinformatics, qRT-PCR and western blot analyses. The cellular proliferation, cell cycle, migration, and invasion were assessed by cell counting kit-8, flow cytometry analysis and transwell assay. We discovered that overexpression of miR-487a-3p suppressed PCa cell growth, migration, invasion by directly targeting CCND1. Knockdown of CCND1 in PCa cells showed similar results. Meanwhile, the expression level of CCND1 was significantly upregulated in the PCa tissues and cell lines, which presented negative correlation with the expression of miR-487a-3p. More important, we demonstrated significantly reduced growth of xenograft tumors of stable miR-487a-3p-overexpressed human PCa cells in nude mice. Taken together, for the first time, our results revealed that miR-487a-3p as a tumor suppressor of PCa could target CCND1. Our finding might reveal miR-487a-3p could be potentially contributed to the pathogenesis and a clinical biomarker or the new potential therapeutic target of PCa.

Topics: Animals; Cell Movement; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Heterografts; Humans; Male; Mice; Mice, Nude; MicroRNAs; Neoplasm Invasiveness; Prostatic Neoplasms

Transgenic adenocarcinoma mouse prostate (TRAMP) model is established to mimic human prostate cancer progression, where seminal vesicle lesions often occur and has been described as phyllodes-like epithelial-stromal tumors. However, the molecular mechanism regulating tumorigenesis and progression in seminal vesicles of TRAMP mice remains largely unknown. In this study, C57BL/6 TRAMP mice were found to have a significantly shorter lifespan than wild-type (WT) mice and all of the seminal vesicles were markedly increased in size and weight with age from 24 weeks exhibiting a clearly papillary-phyllode pattern, though no obvious difference was observed in multiple organs including heart, liver, spleen, lungs, kidneys, testicles and bone between TRAMP and WT mice, and less than 10% of TRAMP mice developed prostate tumors. Western blotting showed Cyclin (CCN) B1 and CCND1 were remarkably overexpressed in seminal vesicle tumors of TRAMP mice at 24 weeks of age and increased with age till the end of trial, which was confirmed by Immunohistochemistry (IHC). P21 and P27 were also significantly augmented, whereas P53 and phosphorylated P53 (p-P53) were constantly expressed in normal controls and P53 did not appear to be mutated. Not only cyclin-dependent kinase (CDK) 1 and phosphorylated forkhead box protein (FOX) O1 but also CDK4, CDK6 and phosphorylated retinoblastoma-associated protein (RB) had similar increase trends, so did epidermal growth factor receptor (EGFR), AKT serine/threonine kinase (AKT), and their respective phosphorylation levels. Signal transducer and activator of transcription (STAT) 3, p-STAT3, enhancer of zeste homolog 2 (EZH2) and EZH2 mediated trimethylation of histone H3 lysine 27 (H3K27me3) were considerably elevated, too. Taken together, this finding suggests P21 and P27 promote carcinogenesis and development in seminal vesicles of TRAMP mice via accelerating cell cycle progression, in which oncogenic transformation of P21 and P27 might be through regulation of EGFR-AKT signaling.

Topics: Animals; Carcinogenesis; Cell Cycle; Cell Transformation, Neoplastic; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Disease Models, Animal; Disease Progression; ErbB Receptors; Immunohistochemistry; Male; Mice; Prostate; Prostatic Neoplasms; Receptors, Tumor Necrosis Factor, Member 25; Seminal Vesicles; Signal Transduction

Topics: Cell Cycle Checkpoints; Cell Line, Tumor; Cyclin D1; Cyclin E; E2F1 Transcription Factor; Gene Expression Regulation, Neoplastic; Humans; Male; MicroRNAs; Oncogene Proteins; Prostatic Neoplasms; Radiation Tolerance; Retinoblastoma Protein; Signal Transduction

The in vitro antiprostate cancer activity of lipophilic grape seed proanthocyanidin (LGSP) against the PC3 cell line was evaluated by MTT assay, flow cytometry, and immunoblot analysis, and the in vivo antiprostate cancer effect was evaluated by a PC3-derived mouse xenograft model via oral gavage LGSP. Ki67 and cleaved caspase 3 immunostaining experiments were performed in tumor tissues. LGSP exhibited a strong inhibitory effect on PC3 cell proliferation by inducing apoptosis. Treatment with LGSP resulted in a G1 phase cell cycle arrest in PC3 cells, which was further confirmed by decreasing the expression of cyclin D1 and CDK 4 and increasing the expression of the tumor suppressors p21 and p27. Furthermore, activation of cleaved fragments of caspases 3, caspases 9, and PARP indicated that LGSP-induced apoptosis is caspase-dependent. Upstream of caspase cascade, LGSP increased the cytochrome c release in cytoplasm. After treatment with LGSP, the Bcl-2/Bax ratio also decreased in PC3 cells. In tumor studies, LGSP inhibited the growth of PC3-derived mouse xenografts by inhibiting tumor cell proliferation and inducing apoptosis. Our findings suggest that LGSP is an effective antiprostate cancer component and deserves further study.

Topics: Animals; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Grape Seed Extract; Humans; Male; Mice; Mice, Nude; Proanthocyanidins; Prostatic Neoplasms; Xenograft Model Antitumor Assays

MicroRNA-501-3p (miR-501-3p) has been reported as a novel cancer-related miRNA in many types of cancer. However, the precise biological function of miR-501-3p in prostate cancer remains unknown. In this study, we aimed to investigate the regulatory effect and mechanism of miR-501-3p on cell growth of prostate cancer cells. We found that miR-501-3p expression was significantly downregulated in prostate cancer tissues and cell lines. Gain-of-function experiments showed that upregulation of miR-501-3p expression significantly decreased cell proliferation and colony formation, and induced cell cycle arrest in the G0/G1 phase. Bioinformatics analysis predicted that cell cycle-related and expression-elevated protein in tumor (CREPT) was a potential target gene of miR-501-3p., and the results of our luciferase reporter assay confirmed that miR-501-3p bound to the 3'-untranslated region of CREPT at the predicted binding site. Moreover, miR-501-3p was shown to negatively regulate CREPT expression in prostate cancer cells. Correlation analysis showed that miR-501-3p was inversely correlated with CREPT expression in prostate cancer tissues. Knockdown studies revealed that miR-501-3p regulated the expression of cyclin D1 by targeting CREPT. Additionally, the inhibitory effect of miR-501-3p on prostate cancer cell growth was partially reversed by CREPT overexpression. Overall, these results suggest that miR-501-3p restricts prostate cancer cell growth by targeting CREPT to inhibit the expression of cyclin D1. These findings indicate that the miR-501-3p/CREPT/cyclin D1 axis plays a crucial role in the progression of prostate cancer and may serve as potential therapeutic target.

Topics: Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Male; MicroRNAs; Neoplasm Proteins; Prostatic Neoplasms

Mammary serine protease inhibitor or Maspin has been characterized as a class II tumor suppressor gene in several cancer types, among them prostate cancer (CaP). Androgen ablation is an effective therapy for CaP, but with short-term effectiveness, thus new therapeutic strategies are actively sought. The present study is aimed to explore the effects of a glucosamine derivative, 2-(N-Carbobenzyloxy)l-phenylalanylamido-2-deoxy-β-d-glucose (NCPA), on two CaP cell lines, PC3 and LNCaP. In particular we analyzed the impact of NCPA on Maspin production, cell viability and cell cycle progression and apoptosis/necrosis pathway activation in PC3 and LNCaP cell lines. NCPA is able to stimulate Maspin production in PC3 and not in LNCaP cell lines. NCPA blocks the PC3 cell cycle in G1 phase, by inhibiting Cyclin D1 production and induces the apoptosis, therefore interfering with aggressiveness of this androgen-insensitive cell line. Moreover, NCPA is able to induce the expression of Maspin in LNCaP cell line treated with androgen receptor inhibitor, Bicalutamide, and in turn to stimulate the apoptosis of these cells. These findings suggest that NCPA, stimulating the endogenous production of a tumor suppressor protein, could be useful in the design of new therapeutic strategies for treatment of CaP.

Topics: Anilides; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Glucosamine; Humans; Male; Nitriles; Prostatic Neoplasms; Receptors, Androgen; Serpins; Tosyl Compounds

von Willebrand factor C and EGF domain‑containing protein (URG11), a cell growth regulator, is involved in the progression of a variety of types of cancer, including prostate cancer (Pca). However, the functions of the URG11 gene in Pca cells require in‑depth investigation. The mRNA and protein levels of URG11 were measured by reverse transcription quantitative polymerase chain reaction (RT‑qPCR) and western blot analysis. Cell Counting kit‑8 (CCK‑8), wound‑healing and Transwell assays were used to detect cell viability, migration and invasion, respectively. Apoptosis and cell cycle analyses were performed using flow cytometry. The mRNA and protein expression levels of epithelial (E)‑cadherin, vimentin, α‑smooth muscle actin (α‑SMA), cyclin D1 and MYC proto‑oncogene protein (c‑Myc) were analyzed by RT‑qPCR and western blot analysis. In the present study, the mRNA and protein levels of URG11 were markedly upregulated in Pca cell lines compared with those in the normal prostate epithelial cell line. With functional experiments, the cell viability, migration and invasion of Pca cells were markedly promoted by URG11 overexpression. The cell cycle was effectively induced by URG11 and apoptosis was inhibited by the overexpression of URG11. Concomitantly, the epithelial marker E‑cadherin was downregulated, and the mesenchymal markers vimentin and α‑SMA were upregulated following URG11 overexpression. By contrast, genetic knockout of URG11 elicited the opposite effects. The present study also identified that the downstream effector genes of the Wnt/β‑catenin signal pathway, cyclin D1 and c‑Myc, were increased following the overexpression of endogenous URG11, which are known to regulate cell proliferation. In addition, the Wnt/β‑catenin inhibitor FH535 ameliorated the promotive effects of URG11 on LNCaP cells viability, migration and invasion, and the Wnt/β‑catenin agonist LiCl reversed the inhibitory effects of siURG11 in LNCaP cells on cell viability, migration and invasion. The present study demonstrated that URG11 served an oncogenic role in the development of Pca cells and provided evidence that URG11 has potential as a novel therapeutic target in Pca.

Topics: Actins; Apoptosis; Cadherins; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Cyclin D1; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Lithium Chloride; Male; Neoplasm Invasiveness; Prostatic Neoplasms; Proto-Oncogene Proteins c-myc; RNA, Small Interfering; Sulfonamides; Trans-Activators; Vimentin

Accumulating evidence suggests that miRNAs play a crucial role in the development of prostate cancer (PC); however, the role of miR-500 in PC remains poorly understood. The data presented here reveal abnormal increases in miR-500 expression in PC tissues and cell lines. Suppression of miR-500 expression significantly inhibited the proliferation of PC-3 and LnCap cells and was negatively regulative with low-density lipoprotein receptor-related protein 1B (LRP1B). Increased cell cycle arrest at the G1 stage and decreased protein expression of cyclinD1 and CDK2 was observed in response to miR-500 knockdown in PC-3 and LnCap cells, in combination with LRP1B overexpression. LRP1B was identified as a target of miR-500 and was significantly decreased in PC tissues. Taken together, these findings demonstrate that miR-500 plays an important role in the proliferation of PC cells via the inhibition of LRP1B expression.

Topics: Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 2; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Male; MicroRNAs; PC-3 Cells; Prostatic Neoplasms; Receptors, LDL

Prostate cancer (PCa) remains a challenge worldwide. Due to the development of castration-resistance, traditional first-line androgen deprivation therapy (ADT) became powerlessness. Epidermal growth factor receptor (EGFR) is a well characterized therapeutic target to treat colorectal carcinoma and non-small cell lung cancer. Increasing studies have unraveled the significance of EGFR and its downstream signaling in the progression of castration-resistant PCa.. MTS, colony formation and Edu staining assays were used to analyze the cell proliferation of PCa cells. Flow cytometry was used to analyze PCa cell cycle distribution and cell apoptosis. Western blot was used to measure the expression of key proteins associated with cell cycle progression, apoptosis and EGFR signaling pathways. Transfection of exogenous small interfering RNA (siRNA) or plasmid was used to intervene specific gene expression. Nude mouse model was employed to test the in vivo effect of Spautin-1.. The current study reveals that Spautin-1, a known inhibitor of ubiquitin-specific peptidase 10 (USP10) and USP13, inhibits EGFR phosphorylation and the activation of its downstream signaling. Inhibition of EGFR signaling induced by Spautin-1 leads to cell cycle arrest and apoptosis of PCa in a USP10/USP13 independent manner. The application of Spautin-1 reduces the expression of glucose transporter 1 (Glut1) and dramatically induces cell death under glucose deprivation condition. In vivo experiments show a potent anti-tumor effect of Spautin-1 alone and in combination with Enzalutamide.. This study demonstrates the therapeutic potential of EGFR signaling inhibition by the use of Spautin-1 for PCa treatment.

Topics: Animals; Apoptosis; Autophagy; Benzamides; Benzylamines; Biomarkers, Tumor; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Disease Models, Animal; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Glucose; Glucose Transporter Type 1; Humans; JNK Mitogen-Activated Protein Kinases; Male; Mice; Models, Biological; Molecular Targeted Therapy; Nitriles; Phenylthiohydantoin; Prostatic Neoplasms; Quinazolines; Signal Transduction; Xenograft Model Antitumor Assays

Estrogens play a role in the patho-physiology of the prostate. In the present work we studied the effects of nonylphenol (NP), a xenoestrogen, on human adenocarcinoma prostate cells (LNCaP). In order to understand molecular and cellular involvement, we observed the effects on cell cycle and we investigated the expression and the cellular localization of estrogen receptors and gene expression of cyclin D1, ki-67, c-myc, IL-8, IL-1β. We performed the same experiments with 17β-estradiol (E2), the most abundant estrogen circulating in nonpregnant humans in order to compare these two different substances. We demonstrated the ability of 1 × 10

Topics: Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Environmental Pollutants; Estradiol; Estrogen Receptor alpha; Gene Expression; Humans; Interleukin-1beta; Male; Phenols; Prostatic Neoplasms

Estrogens act as key factors in prostate biology, cellular proliferation and differentiation as well as cancer development and progression. The expression of estrogen receptor (ER)-β appears to be lost during prostate cancer progression through hypermethylation mechanism. Epigenetic drugs such as 5-aza-2'-deoxycytidine (5-AZAC) and Trichostatin A (TSA) showed efficacy in restoring ERβ expression in prostate cancer cells. This study was designed to explore the potential anti-carcinogenic effects resulting from re-expressing ERβ1 using 5-AZAC and/or TSA, followed by its stimulation with Diarylpropionitrile (DPN), a selective ERβ1 agonist, in prostate cancer cell line PC-3.. Cells were treated with 5-AZAC, TSA, DPN and their combination. Subsequently, they were subjected to proliferation assays, determinations of ERβ1 expression, protein levels of active caspase-3, cyclin D1, β-catenin and VEGF.. Treatment with these drugs exhibited an increase in ERβ1 expression to different extents as well as active caspase-3 levels. Meanwhile, a significant reduction in cyclin D1, VEGF and β-catenin levels was achieved as compared to the vehicle control group (p < 0.05). Interestingly, the triple combination regimen led to the most prominent anti-tumor responses in terms of increased apoptosis, reduced proliferation as well as angiogenesis.. The results support the notion that ERβ1 acts as a tumor suppressor protein and suggest that sequential ERβ1 expression and activation can offer significant anti-tumor responses. The study highlights that the strategy of merging epigenetic and hormonal therapies may be beneficial in treating advanced prostate cancer.

Topics: Anticarcinogenic Agents; Azacitidine; beta Catenin; Caspase 3; Cell Proliferation; Cyclin D1; Decitabine; Disease Progression; Epigenesis, Genetic; Estrogen Receptor beta; Hormones; Humans; Hydroxamic Acids; Male; Methylation; Nitriles; Propionates; Prostate; Prostatic Neoplasms; Vascular Endothelial Growth Factor A

Increasing evidence has indicated the important roles of long non-coding RNAs (lncRNAs) in tumorigenesis and cellular progression, including prostate cancer. In this study, we aim to investigate the expression level of SNHG7 and its biological functions on prostate cancer cells. Results indicated that SNHG7 expression was significantly up-regulated in prostate cancer tissue and cell lines. Besides, the overexpression of SNHG7 was closely correlated with the poor prognosis. In vitro and in vivo, experiments demonstrated that SNHG7 knockdown markedly inhibited prostate cancer proliferation and cycle-related protein (CDK4, CDK6, Cyclin D1), induced cell cycle arrest at G0/G1 phase and suppressed tumor growth. Moreover, miR-503 was predicted by bioinformatics tools and validated using luciferase reporter assay to both directly inhibited SNHG7 and Cyclin D1 expression by targeting their RNA 3'-UTR. In conclusion, results present that SNHG7 regulates the cycle progression and acts as an oncogenic gene in the prostate cancer tumorigenesis via miR-503/Cyclin D1 pathway, revealing the vital role of lncRNA/miRNA/mRNA axis in prostate cancer carcinogenesis.

Topics: Aged; Animals; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Gene Knockdown Techniques; Humans; Male; Mice, Nude; MicroRNAs; Middle Aged; Prostatic Neoplasms; RNA, Long Noncoding; Up-Regulation; Xenograft Model Antitumor Assays

Traditional Chinese medicine (TCM) has the synergistic effect of the combination of a single ingredient and a monomer, and systemic and local therapeutic effects in cancer treatment, through which TCM is able to enhance the curative effect and reduce the side effects. The present study analyzed the effect of TCM‑1 (an anti‑cancer TCM) on prostate cancer (PCa) cell lines, and studied in detail the mechanism of cell death induced by TCM‑1 in vitro and in vivo. From the present results, it was identified for the first time, to the best of our knowledge, that TCM‑1 arrested the cell cycle at the G1 phase, decreased cell viability and increased nuclear rupture in a dose‑dependent manner; these effects finally resulted in apoptosis in PCa cells. At the molecular level, the data demonstrated that TCM‑1 competitively acted on epidermal growth factor receptor (EGFR) with EGF, and suppressed the auto‑phosphorylation and activity of EGFR. Inhibition of EGFR further suppressed the downstream phosphatidylinositol 3‑kinase (PI3K)/RAC‑α serine/threonine‑protein kinase (AKT) and RAF proto‑oncogene serine/threonine‑protein kinase/extracellular signal regulated kinase signaling pathways and resulted in a decrease in the phosphorylated‑forkhead box protein O1 (at Ser256, Thr24 and Ser319) expression level, and induced cell growth inhibition and apoptosis by regulating the expression of apoptosis‑and cell cycle‑associated genes. In addition, TCM‑1 markedly inhibited the PI3K/AKT/serine/threonine‑protein kinase mTOR signaling pathway and induced cell autophagy by downregulating the phosphorylation of p70S6K and upregulating the levels of Beclin‑1 and microtubule‑associated protein light chain‑3II. In vivo, the TCM‑1‑treated group exhibited a significant decrease in tumor volume compared with the negative control group in subcutaneous xenograft nude mice by inhibiting EGFR‑associated signaling pathways. Therefore, the bio‑functions of Chinese medicine TCM‑1 in inducing PCa cell growth inhibition, autophagy and apoptosis suggested that TCM‑1 may have clinical potential for the treatment of patients with PCa.

Topics: Animals; Apoptosis; Autophagy; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; ErbB Receptors; Humans; Immunohistochemistry; Male; Medicine, Chinese Traditional; Mice; Phosphatidylinositol 3-Kinase; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Signal Transduction; Tumor Suppressor Protein p53

Prostate carcinoma contain foci of neuroendocrine transdifferentiation, resulting in an increase of androgen-independent neuroendocrine-like (NE) tumor cells, whose number significantly correlates with tumor aggressiveness and thus lower survival rate. Neuroendocrine transdifferentiation of prostate cancer cells and a potential role of miRNAs within this process are poorly understood. MicroRNAs are small non-coding RNAs which post-transcriptionally regulate gene expression. The aim of this project was to identify new genes and miRNAs involved in neuroendocrine transdifferentiation. LNCaP prostate cancer cells were differentiated to NE-like cancer cells and microarray analyses were performed. Microarray results have been validated for the eight most deregulated mRNAs and microRNAs via qRT-PCR and analyzed with different algorithms to predict new targets for deregulated microRNAs. The induced CyclinD1 gene could be validated as new target gene for the repressed miR-17 family containing miR-17, miR-20a, miR-20b, miR-106a and miR-106b via reporter gene assays and Western Blot. Functional analysis of miR-17 family shows a high influence on cell proliferation, colony forming ability and apoptosis in LNCaP cells. Our data demonstrate wide changes in mRNA and microRNA expression during neuroendocrine transdifferentiation of LNCaP cells and confirm new mRNA-miRNA interactions with potential roles in NE-transdifferentiation of prostate carcinoma.

Topics: Cell Line, Tumor; Cell Transdifferentiation; Cyclin D1; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Male; MicroRNAs; Neuroendocrine Cells; Prostatic Neoplasms; RNA, Neoplasm

Radiation therapy (XRT) is a standard treatment for prostate cancer (PCa). Although dose escalation increases local control, toxicity hampers further escalation. Broader improvement will be possible by the addition of adjuvant therapies, which can synergize with radiation and thus improve efficacy. We have identified a natural compound (Nexrutine, Nx) that inhibits the survival and growth of PCa cells in combination with radiation. Combination studies demonstrated strong interaction between Nx and radiation both in vitro in multiple PCa cell lines and in the Transgenic adenocarcinoma of mouse prostate (TRAMP) model. Nx potentiated growth inhibitory effects of IR by down regulating ribosomal protein S6K (RPS6KB1), CyclinD1, Chk1 and HIF-1 α and prolonging G2/M checkpoint block. RPS6KB1 is upregulated in prostate cancers and its expression is correlated with tumor grade. Knockdown of RPS6KB1 in PCa cells increased their sensitivity toward radiation-induced survival inhibition. Overall, we provide scientific evidence (i) in support of Nx as an adjuvant in PCa patients receiving XRT (ii) suggesting that RPS6KB1 is an important player in Nx-mediated combinatorial benefits and emphasizes that RPS6KB1 is a novel target for PCa treatment. These data underscore the need to test the agent in additional preclinical models to validate these observations.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Cell Survival; Checkpoint Kinase 1; Cyclin D1; G2 Phase Cell Cycle Checkpoints; Gene Knockdown Techniques; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Mice; PC-3 Cells; Plant Extracts; Prostatic Neoplasms; Ribosomal Protein S6 Kinases, 70-kDa

Prostate cancer is one of the most common types of cancer in men and the leading cause of death in developed countries. With the aid of molecular and genetic profiling of cancers, cancer molecular subtypes are paving the way for tailored cancer therapy. FOXA1 has been identified as one of the seven molecular subtypes of prostate cancer. FOXA1 is involved in a variety of metabolic process such as glucose homeostasis and deregulation of its expression is crucial in prostate cancer progression. In this study, we investigated the effects of FOXA1 gene knock-out on the expression levels of various cancer cell metabolism and cell cycle-related protein expressions. FOXA1 gene was knocked-out by using CRISPR/Cas9 technique. While FOXA1 gene knock-out significantly altered Casp-9, Bax, CCND1, CDK4, and fibronectin protein expressions (P < 0.05, fold change: ∼40, 4.5, 2.5, 4.5, and 4, respectively), it did not affect the protein expression levels of Casp-3, Bcl-2, survivin, β-catenin, c-Myc, and GSK-3B. Knocking-out FOXA1 gene in androgen-dependent LNCaP prostate cancer cells inhibited CCND1 protein expression. Our pre-clinical results demonstrate the importance of FOXA1 as a drug target in the treatment of prostate cancer. Impact statement Knock-out studies offer a unique way of studying the function of genes especially for developmentally lethal genes. FOXA1 has prominent roles both in breast and prostate cancer pathogenesis due to its role in ER receptor signaling pathway. FOXA1 has also been identified as one of the seven molecular subtypes of primary prostate cancer. In the present study, we used an efficient gene knock-out method, CRISPR/Cas9, in order to investigate FOXA1 function on LNCaP prostate cancer cells in vitro. FOXA1 knock-out altered cell-cycle regulator CCND1 protein expression levels. Therefore, our results suggest that FOXA1 might be a plausible drug target for prostate cancer treatment.

Topics: Apoptosis; bcl-2-Associated X Protein; Caspase 9; Cell Cycle; Cell Line, Tumor; Cell Proliferation; CRISPR-Cas Systems; Cyclin D1; Cyclin-Dependent Kinase 4; Epithelial-Mesenchymal Transition; Fibronectins; Gene Expression Regulation, Neoplastic; Gene Knockout Techniques; Hepatocyte Nuclear Factor 3-alpha; Humans; Male; Mutation; Prostatic Neoplasms; Protein Binding; Protein Interaction Mapping; Signal Transduction

Prostate cancer is one of the most commonly diagnosed cancers in man. Studies have shown that phthalates may act as promoters in various types of cancer; however, the role of phthalates in prostate cancer has been rarely reported. The MAPK/AP-1 pathway is a vital regulator of cell proliferation in cancer. In this report we found that three typical phthalates, diethylhexyl phthalate (DEHP), Butyl benzyl phthalate (BBP) and Dibutyl phthalate (DBP), up-regulated cyclinD1 and PCNA, down-regulated P21, inducing proliferation of prostate cancer cells. Furthermore, we found that phthalates increased the expression of p-ERK5 and p-p38, along with upregulation of AP-1 (p-c-fos and p-c-jun). In studies with ERK5 and a p38 inhibitor, our data showed that downregulation of p-ERK5 or p38 inhibited phthalate-triggered cell proliferation. Taken together, findings from this study suggest that phthalates activate MAPK/AP-1 pathway and may potentially promote cell proliferation in prostate cancer, thus providing new insight into the effects and the underlying mechanism of phthalates on prostate cancer.

Topics: Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Dibutyl Phthalate; Diethylhexyl Phthalate; Gene Expression Regulation, Neoplastic; Humans; Male; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 7; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Phthalic Acids; Proliferating Cell Nuclear Antigen; Prostatic Neoplasms

Prostate cancer is one of the most frequent malignancies affecting men. Long non-coding RNAs (lncRNAs) are involved in the pathogenesis of prostate cancer. LncRNA LOXL1-AS1 participates in the pathogenesis of the exfoliation syndrome. However, the role of LOXL1-AS1 in cancer remains largely unknown. Here, we found that LOXL1-AS1 down-regulation inhibited prostate cancer cell proliferation and cell cycle progression. RNA sequencing analysis revealed that it regulates the expression of cell cycle-related genes. LOXL1-AS1 is predominantly distributed in the cytoplasm, where it interacts with miR-541-3p. In addition, miR-541-3p targets the cell cycle regulator CCND1 in prostate cancer cells. LOXL1-AS1 down-regulation inhibits the expression of CCND1 and cell cycle progression, whereas these effects are abolished upon miR-541-3p suppression. In summary, our study revealed that LOXL1-AS1 regulates prostate cancer cell proliferation and cell cycle progression through miR-541-3p and CCND1. Modulation of their levels may be used to treat prostate cancer.

Topics: 3' Untranslated Regions; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Humans; Male; MicroRNAs; Prostatic Neoplasms; RNA, Long Noncoding

Bromodomain-containing protein 4 (BRD4) overexpression participates in prostate cancer progression by enhancing the transcriptional activity and expression of several key oncogenes. AZD5153 is a novel BRD4 inhibitor.. Prostate cancer cells were treated with AZD5153. Cell survival was tested by MTT assay and clonogenicity assay. Cell proliferation was tested by [H3] DNA incorporation assay. Cell apoptosis was tested by caspase-3/-9 activity assay, Histone DNA ELISA assay, Annexin V FACS assay and TUNEL staining assay. Cell cycle progression was tested by propidium iodide (PI) FACS assay. Signaling was tested by Western blotting assay. The nude mice PC-3 xenograft model was applied to test AZD5153's activity in vivo.. AZD5153 inhibited proliferation and survival of established and primary prostate cancer cells. AZD5153 induced apoptosis activation and cell cycle arrest in prostate cancer cells. AZD5153 was non-cytotoxic to the prostate epithelial cells. AZD5153 downregulated BRD4 targets (cyclin D1, Myc, Bcl-2, FOSL1 and CDK4) in PC-3 and primary prostate cancer cells. Further studies show that AKT could be the primary resistance factor of AZD5153. Pharmacological inhibition or genetic depletion of AKT induced BRD4 downregulation, sensitizing AZD5153-induced cytotoxicity in PC-3 cells. In vivo, AZD5153 oral administration inhibited PC-3 xenograft tumor growth in nude mice. Its anti-tumor activity was further enhanced with co-treatment of the AKT specific inhibitor MK-2206.. Together, our results indicate a promising therapeutic value of the novel BRD4 inhibitor AZD5153 against prostate cancer cells.

Topics: Administration, Oral; Animals; Apoptosis; B-Cell Lymphoma 3 Protein; Cell Cycle Checkpoints; Cell Cycle Proteins; Cell Proliferation; Cyclin D1; Down-Regulation; Heterocyclic Compounds, 2-Ring; Heterocyclic Compounds, 3-Ring; Humans; Male; Mice; Mice, Nude; Nuclear Proteins; Piperazines; Prostatic Neoplasms; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Pyrazoles; Pyridazines; RNA Interference; RNA, Small Interfering; Transcription Factors; Tumor Cells, Cultured

Rosmarinic acid (RA), a main phenolic compound contained in rosemary which is used as tea, oil, medicine and so on, has been known to present anti-inflammatory, anti-oxidant and anti-cancer effects. Histone deacetylases (HDACs) are enzymes that play important roles in gene expression by removing the acetyl group from histone. The aberrant expression of HDAC in human tumors is related with the onset of human cancer. Especially, HDAC2, which belongs to HDAC class I composed of HDAC 1, 2, 3 and 8, has been reported to be highly expressed in prostate cancer (PCa) where it downregulates the expression of p53, resulting in an inhibition of apoptosis. The purpose of this study is to investigate the effect of RA in comparison with suberoylanilide hydroxamic acid (SAHA), an HDAC inhibitor used as an anti-cancer agent, on survival and apoptosis of PCa cell lines, PC-3 and DU145, and the expression of HDAC. RA decreased the cell proliferation in cell viability assay, and inhibited the colony formation and tumor spheroid formation. Additionally, RA induced early- and late-stage apoptosis of PC-3 and DU145 cells in Annexin V assay and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, respectively. In western blot analysis, RA inhibited the expression of HDAC2, as SAHA did. Proliferating cell nuclear antigen (PCNA), cyclin D1 and cyclin E1 were downregulated by RA, whereas p21 was upregulated. In addition, RA modulated the protein expression of intrinsic mitochondrial apoptotic pathway-related genes, such as Bax, Bcl-2, caspase-3 and poly (ADP-ribose) polymerase 1 (

Topics: Annexin A5; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cinnamates; Cyclin D1; Cyclin E; Depsides; DNA Fragmentation; Gene Expression Regulation; Histone Deacetylase 2; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; In Situ Nick-End Labeling; Male; Oncogene Proteins; Poly (ADP-Ribose) Polymerase-1; Proliferating Cell Nuclear Antigen; Prostatic Neoplasms; Rosmarinic Acid; Rosmarinus; Signal Transduction; Teas, Herbal; Teas, Medicinal; Vorinostat

FAM46B is a member of the family with sequence similarity 46. Little is known about the expression and functional role(s) of FAM46B in prostate cancer (PC). In this study, the expression of FAM46B expression in The Cancer Genome Atlas, GSE55945, and an independent hospital database was measured by bioinformatics and real-time PCR analysis. After PC cells were transfected with siRNA or a recombinant vector in the absence or presence of a β-catenin signaling inhibitor (XAV-939), the expression levels of FAM46B, C-myc, Cyclin D1, and β-catenin were measured by western blot and real-time PCR. Cell cycle progression and cell proliferation were measured by flow cytometry and the CCK-8 assay. The effects of FAM46B on tumor growth and protein expression in nude mice with PC tumor xenografts were also measured. Our results showed that FAM46B was downregulated but that β-catenin was upregulated in patients with PC. FAM46B silencing promoted cell proliferation and cell cycle progression in PC, which were abrogated by XAV-939. Moreover, FAM46B overexpression inhibited PC cell cycle progression and cell proliferation in vitro and tumor growth in vivo. FAM46B silencing promoted β-catenin protein expression through the inhibition of β-catenin ubiquitination. Our data clearly show that FAM46B inhibits cell proliferation and cell cycle progression in PC through ubiquitination of β-catenin.

Topics: beta Catenin; Carcinogenesis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Computational Biology; Cyclin D1; Heterocyclic Compounds, 3-Ring; Humans; Male; Nucleotidyltransferases; Prostatic Neoplasms; Proteins; Proto-Oncogene Proteins c-myc; RNA, Small Interfering; Ubiquitination

Inositol polyphosphate 4-phosphatase type II emerges as a tumor suppressor in prostate cancer, and its loss of expression is associated with poor prognosis for prostate cancer. However, the mechanism of downregulation of inositol polyphosphate 4-phosphatase type II in prostate cancer development has not yet been fully clarified. In this study, microRNA-590-3p was found to be upregulated in both prostate cancer tissues and cell lines. Overexpression of microRNA-590-3p by microRNA-590-3p mimics promoted prostate cancer cell proliferation and invasion and accelerated the growth of xenografted tumors, while microRNA-590-3p inhibitors contributed to inhibition of cellular proliferation and invasion as well as tumor growth. A dual-luciferase reporter assay and expression analysis further confirmed that inositol polyphosphate 4-phosphatase type II was a direct target of microRNA-590-3p. Enforced expression of microRNA-590-3p led to repression of inositol polyphosphate 4-phosphatase type II messenger RNA and protein expression, as well as upregulation of p-Akt, p-FoxO3a, and cyclin D1 and downregulation of p21 expression in prostate cancer cell lines. Overexpression of inositol polyphosphate 4-phosphatase type II could reduce microRNA-590-3p-induced cell proliferation and invasion as well as tumor growth, and decrease microRNA-590-3p-mediated upregulation of cyclin D1 and downregulation of p21 expression in prostate cancer cells. Taken together, our findings reveal that microRNA-590-3p is a potential onco-microRNA that participates in carcinogenesis of human prostate cancer by suppressing inositol polyphosphate 4-phosphatase type II expression and involving the Akt/FoxO3a pathway. MicroRNA-590-3p may represent a potential therapeutic target for prostate cancer patients.

Topics: Animals; Carcinogenesis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Female; Forkhead Box Protein O3; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; HEK293 Cells; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Neoplasm Invasiveness; Neoplasm Transplantation; Phosphoric Monoester Hydrolases; Prognosis; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; RNA, Messenger; Transplantation, Heterologous

Recent studies have shown that miRNAs have potent abilities to activate gene expression by targeting promoter elements, a phenomenon known as RNA activation (RNAa). In the present study, we identified a new endogenous miR-3619-5p which was decreased in prostate cancer tissues and cells compared to corresponding normal controls. Moreover, overexpression of miR-3619-5p readily induced CDKN1A gene expression by directly targeting the putative site in the promoter. Besides, miR-3619-5p possessed considerable capacity to inhibit prostate cancer DU145 and PC3 cell growth, and downregulate several CDKN1A downstream genes, such as cyclin D1, CDK4 and CDK6. Notably, this antitumor function of miR-3619-5p was mainly achieved by stimulating CDKN1A expression.

Topics: Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p21; Gene Expression Regulation, Neoplastic; Humans; Male; MicroRNAs; Promoter Regions, Genetic; Prostatic Neoplasms

Sex determining region Y (SRY)-box 18 (SOX18) gene encodes transcription factors that have been recently confirmed to be overexpressed in various human types of cancer and maintain the malignant behavior of cancer cells. However, the role and its potential function in prostate cancer (PCa) has not been demonstrated and the mechanisms of SOX18 involved in tumor progression remain largely unclear. In the present study, the expression of SOX18 was analyzed in 98 PCa and 81 adjacent non-tumor tissues using immunohistochemistry. The data showed that SOX18 was overexpressed in 72 of 98 (73.5%) PCa tissues compared with that in 28 of 81 (34.6%) non-tumor tissues. In addition, the expression of SOX18 was related with the clinical features of patients with PCa. To explore the potential role of SOX18 in PCa cells, Cell Counting Kit-8 (CCK-8), migration, invasion and xenograft assays were performed. Our data showed that knockdown of SOX18 decreased the proliferation, migration and invasion of PCa cells in vitro, in addition to the tumor growth in vivo. Markedly, SOX18 knockdown caused the decreased expression of TCF1, c-Myc, cyclin D1 and MMP-7. In conclusion, SOX18 was overexpressed in PCa and may regulate the malignant capacity of cells via the upregulation of TCF1, c-Myc, cyclin D1 and MMP-7.

Topics: Aged; Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Hepatocyte Nuclear Factor 1-alpha; Humans; Male; Matrix Metalloproteinase 7; Mice, Nude; Prostatic Neoplasms; Proto-Oncogene Proteins c-myc; SOXF Transcription Factors; Xenograft Model Antitumor Assays

Evidence has revealed that some microRNAs play a critical role in tumor proliferation. We demonstrated that miR-141-3p appears to be a novel oncogene miRNA, which promotes prostate tumorigenesis and facilitates the stemness of prostate cancer cells via suppressing a key transcription factor kruppel-like factor-9 (KLF9). KLF9 is the core effector protein that might suppress tumor growth. MiR-141-3p is upregulated in prostate cancer cells and tissues compared to non-tumorigenic prostate epithelial cells and prostate tissues. MiR-141-3p positively regulated proliferation, spheroid formation, and expression of the stemness factors OCT-4, Nanog, SOX-9, Bmil, CCND1, and CD44 in PC-3 cells. Restoration of miR-141-3p suppresses the expression of the transcription factor KLF9 in PC-3 and accelerates prostate tumorigenesis via targeted binding with its 3'-UTR. Downregulation of KLF9 enhances spheres formation of prostate cancer cells. Our results suggest that miR-141-3p/KLF9 may play an important role in regulating the growth of prostate cancer and is a potential target of prevention and therapy.

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Epithelial Cells; Gene Expression Regulation, Neoplastic; Humans; Hyaluronan Receptors; Kruppel-Like Transcription Factors; Male; MicroRNAs; Nanog Homeobox Protein; Octamer Transcription Factor-3; Polycomb Repressive Complex 1; Prostate; Prostatic Neoplasms; SOX9 Transcription Factor; Spheroids, Cellular

Liver X receptors (LXRs) are nuclear receptors family of ligand-dependent transcription factors that play a crucial role in regulating cholesterol metabolism and inflammation. Recent studies show that LXR agonists exhibit anti-cancer activities in a variety of cancer cell lines including prostate. To further identify the potential mechanisms of LXRα activation on prostate cancer, we investigated the effect of LXR agonist T0901317 on PC3 prostate cancer cell and in which activity of beta-catenin pathway involved.. Prostate cancer PC3 cells were transfected with LXR-a siRNA and treated with LXR activator T0901317. qRT-PCR and western blot were used to detect the LXR-a expression. beta-catenin, cyclin D1 and c-MYC were analyzed by western blot. Cell apoptosis was examined by flow cytometry and Cell proliferation was assessed by Cell Counting Kit-8 assay. Cell migration was detected by Transwell chambers.. Data showed that T0901317 significantly inhibited PC3 cell proliferation as well as invasion and increased apoptosis in vitro. Furthermore, we found that LXRα activation induced the reduction of beta-catenin expression in PC3 cells, and this inhibitory effect could be totally abolished when cells were treated with LXRα. Meanwhile, the expression of beta-catenin target gene cyclin D1 and c-MYC were also decreased.. This study provided additional evidence that LXR activation inhibited PC-3 prostate cancer cells via suppressing beta-catenin pathway.

Topics: Apoptosis; beta Catenin; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Humans; Hydrocarbons, Fluorinated; Liver X Receptors; Male; Prostate; Prostatic Neoplasms; Proto-Oncogene Proteins c-myc; Signal Transduction; Sulfonamides

Therapy resistance and poor outcome in prostate cancer is associated with increased expression of cyclin D1. Androgens promote DNA double-strand break repair to reduce DNA damage, and cyclin D1 was also shown to enhance DNA damage repair (DDR). In this study, we investigated the significance of cyclin D1 in androgen-induced DDR using established prostate cancer cells and prostate tissues from cyclin D1 knockout mice. We demonstrate that endogenous cyclin D1 further diminished the dihydrotestosterone (DHT)-dependent reduction of γH2AX foci in vitro. We also show that cyclin D1 was required for the androgen-dependent DNA damage response both in vitro and in vivo. Furthermore, cyclin D1 was required for androgen-enhanced DDR and radioresistance of prostate cancer cells. Moreover, microarray analysis of primary prostate epithelial cells from cyclin D1-deficient and wild-type mice demonstrated that most of the DHT-dependent gene expression changes are also cyclin D1 dependent. Collectively, our findings suggest that the hormone-mediated recruitment of cyclin D1 to sites of DDR may facilitate the resistance of prostate cancer cells to DNA damage therapies and highlight the need to explore other therapeutic approaches in prostate cancer to prevent or overcome drug resistance.

Topics: Animals; Cell Line, Tumor; Cyclin D1; Dihydrotestosterone; DNA Damage; DNA Repair; Histones; Humans; Male; Mice; Mice, Knockout; Neoplasms, Hormone-Dependent; Prostatic Neoplasms; Transfection

The aim of the present study was to determine whether hexabromocyclododecane (HBCD) or 4-nonylphenol (NP) may induce prostatic cancer progression in LNCaP cells. Androgenic effects of HBCD and NP were examined in LNCaP prostate cancer cells expressing androgen receptors (ARs). HBCD and NP increased LNCaP cell viability similar to dihydrotestosterone (DHT) by MTT assay. This phenomenon was reversed by treatment with Casodex, an AR antagonist, suggesting that they act as xenoandrogens via AR signaling pathway. In cell migration assay, HBCD and NP also enhanced LNCaP cell migration similar to DHT. To elucidate underlying mechanisms of their actions on LNCaP, transcriptional levels of cell cycle- and apoptosis-related markers, including cyclin D1, cyclin E, p27, bcl-2, and bax, were determined by reverse transcription (RT)-PCR. An increase in expression cyclin D1 and cyclin E and reduction in p27 and bax mRNA levels were observed by their treatments. Western blot assay showed their alterations in translational levels of cyclin D1, cyclin E, p21, bax, and cathepsin D. Expressions of genes related to a G1/S transition of cell cycle and cathepsin D were elevated, while expression of p21 and bax was decreased. Taken together, these results indicate that HBCD and NP may enhance progression of prostate cancer by modulating growth and migration of LNCaP prostate cells by acting on cell cycle, apoptosis, and metastasis.

Topics: Androgens; Apoptosis; bcl-2-Associated X Protein; Cathepsin D; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase Inhibitor p21; Endocrine Disruptors; Humans; Hydrocarbons, Brominated; Male; Oncogene Proteins; Phenols; Prostatic Neoplasms

Aminomethylphosphonic acid (AMPA) has been shown to inhibit prostate cancer cell growth in vitro. The purpose of the present study was to determine if AMPA could inhibit growth and metastasis of prostate cancer in vivo. Human prostate cancer PC-3-LacZ-luciferase cells were implanted into the ventral lateral lobes of the prostate in 39 athymic Nu/Nu nude male mice. Seven days later, mice were randomized into the control group (n = 14, treated intraperitoneally with phosphate buffered saline), low dose group (n = 10, treated intraperitoneally with AMPA at 400 mg/kg body weight/day), and high dose group (n = 15, treated intraperitoneally with AMPA at 800 mg/kg body weight/day). Tumor growth and metastasis were examined every 4-7 days by bioluminescence imaging of live mice. We found that AMPA treatment significantly inhibited growth and metastasis of orthotopic xenograft prostate tumors and prolonged the survival time of the mice. AMPA treatment decreased expression of BIRC2 and activated caspase 3, leading to increased apoptosis in the prostate tumors. AMPA treatment decreased expression of cyclin D1. AMPA treatment also reduced angiogenesis in the prostate tumors. Taken together, these results demonstrate that AMPA can inhibit prostate cancer growth and metastasis, suggesting that AMPA may be developed into a therapeutic agent for the treatment of prostate cancer.

Topics: Animals; Antineoplastic Agents; Apoptosis; Caspase 3; Cell Line, Tumor; Cyclin D1; Disease Models, Animal; Enzyme Activation; Humans; Inhibitor of Apoptosis Proteins; Isoxazoles; Male; Mice; Mice, Nude; Neoplasm Metastasis; Neovascularization, Pathologic; Organophosphonates; Prostatic Neoplasms; Tetrazoles; Ubiquitin-Protein Ligases; Xenograft Model Antitumor Assays

Prostate cancer is a leading cause of cancer-related mortality in men worldwide and there is a lack of effective treatment options for advanced (metastatic) prostate cancer. Long non-coding RNAs (lncRNAs) play important roles in diverse biological processes, such as cell growth, apoptosis and migration. However, little is known about the molecular mechanism of lncRNA-HOTTIP-mediated prostate cancer cell proliferation and apoptosis. The aim of this study was to elucidate the involvement of lncRNA HOTTIP in prostate cancer tumorigenesis and further investigate the role of HOXA13 in this process. Here, we showed that HOTTIP silencing inhibited cell survival pathway in vitro and in vivo by reducing the protein expression of Bcl-2 and enhancing Bax. We further demonstrated that knockdown of HOTTIP inhibited the expression of cell cycle regulatory protein Cyclin D1 and induced cell cycle arrest in G0/G1 phase. Additionally, depletion of HOXA13 by RNA interference (si-HOXA13) revealed that HOTTIP silencing suppressed cell growth at least partly through regulating HOXA13. In conclusion, down-regulation of HOTTIP and HOXA13 was associated with cell growth and cell cycle, and exerts tumor-suppressive functions in the genesis and progression of prostate cancer, providing a potential attractive therapeutic approach for this malignancy.

Topics: Apoptosis; bcl-2-Associated X Protein; Carcinogenesis; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Male; Prostate; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; RNA, Long Noncoding

Cyclin D1 (Ccnd1) is a proto-oncogen amplified in many different cancers and nuclear accumulation of Ccnd1 is a characteristic of tumor cells. Ccnd1 activates the transcription of a large set of genes involved in cell cycle progress and proliferation. However, Ccnd1 also targets cytoplasmic proteins involved in the regulation of cell migration and invasion. In this work, we have analyzed by immunohistochemistry the localization of Ccnd1 in endometrial, breast, prostate and colon carcinomas with different types of invasion. The number of cells displaying membranous or cytoplasmic Ccnd1 was significantly higher in peripheral cells than in inner cells in both collective and pushing invasion patterns of endometrial carcinoma, and in collective invasion pattern of colon carcinoma. Also, the cytoplasmic localization of Ccnd1 was higher when tumors infiltrated as single cells, budding or small clusters of cells. To evaluate cytoplasmic function of cyclin D1, we have built a variant (Ccnd1-CAAX) that remains attached to the cell membrane therefore sequestering this cyclin in the cytoplasm. Tumor cells harboring Ccnd1-CAAX showed high levels of invasiveness and metastatic potential compared to those containing the wild type allele of Ccnd1. However, Ccnd1-CAAX expression did not alter proliferative rates of tumor cells. We hypothesize that the role of Ccnd1 in the cytoplasm is mainly associated with the invasive capability of tumor cells. Moreover, we propose that subcellular localization of Ccnd1 is an interesting guideline to measure cancer outcome.

Topics: Amino Acid Motifs; Animals; Biomarkers, Tumor; Breast Neoplasms; Cell Line, Tumor; Cell Membrane; Cells, Cultured; Colonic Neoplasms; Cyclin D1; Cytoplasm; Endometrial Neoplasms; Female; Humans; Immunohistochemistry; Male; Mice, Nude; Mice, SCID; Microscopy, Confocal; Neoplasm Invasiveness; Neoplasms; Prostatic Neoplasms

The aim of the present study was to characterize the mechanism underlying estrogen effects on the androgen-independent prostate cancer cell line PC-3. 17β-estradiol and the ERβ-selective agonist DPN, but not the ERα-selective agonist PPT, increased the incorporation of [methyl-(3)H]thymidine and the expression of Cyclin D2, suggesting that ERβ mediates the proliferative effect of estrogen on PC-3 cells. In addition, upregulation of Cyclin D2 and incorporation of [methyl-(3)H]thymidine induced by 17β-estradiol and DPN were blocked by the ERβ-selective antagonist PHTPP in PC-3 cells. Upregulation of Cyclin D2 and incorporation of [methyl-(3)H]thymidine induced by DPN were also blocked by PKF118-310, a compound that disrupts β-catenin-TCF (T-cell-specific transcription factor) complex, suggesting the involvement of β-catenin in the estradiol effects in PC-3 cells. A diffuse immunostaining for non-phosphorylated β-catenin was detected in the cytoplasm of PC-3 cells. Low levels of non-phosphorylated β-catenin immunostaining were also detected near the plasma membrane and in nuclei. Treatment of PC-3 cells with 17β-estradiol or DPN markedly increased non-phosphorylated β-catenin expression. These effects were blocked by pretreatment with the ERβ-selective antagonist PHTPP, PI3K inhibitor Wortmannin or AKT inhibitor MK-2206, indicating that ERβ-PI3K/AKT mediates non-phosphorylated β-catenin expression. Cycloheximide blocked the DPN-induced upregulation of non-phosphorylated β-catenin, suggesting de novo synthesis of this protein. In conclusion, these results suggest that estrogen may play a role in androgen-independent prostate cancer cell proliferation through a novel pathway, involving ERβ-mediated activation of β-catenin.

Topics: beta Catenin; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin D2; Cycloheximide; Estradiol; Estrogen Receptor beta; Humans; Male; Nitriles; Phenols; Phosphorylation; Prostatic Neoplasms; Protein Kinase Inhibitors; Pyrazoles; Pyrimidines; Thymidine

Long non-coding RNAs (lncRNAs) have been identified to be critical mediators in various tumors associated with cancer progression. Long non-coding RNA activated by TGF-β (lncRNA-ATB) is a stimulator of epithelial-mesenchymal transition (EMT) and serves as a novel prognostic biomarker for hepatocellular carcinoma. However, the biological role and clinical significance of lncRNA-ATB in human prostate cancer have yet to be fully elucidated. The present study was designed to explore the expression of lncRNA-ATB in human prostate cancer patients and the role of lncRNA-ATB in prostate cancer cells. We showed that lncRNA-ATB expression was significantly upregulated in tumor tissues in patients with prostate cancer in comparison with adjacent non-tumor tissues. Further analysis indicted that high lncRNA-ATB expression may be an independent prognostic factor for biochemical recurrence (BCR)-free survival in prostate cancer patients. Overexpression of lncRNA-ATB promoted, and knockdown of lncRNA-ATB inhibited the growth of prostate cancer cells via regulations of cell cycle regulatory protein expression levels. In addition, lncRNA-ATB stimulated epithelial-mesenchymal transition (EMT) associated with ZEB1 and ZNF217 expression levels via ERK and PI3K/AKT signaling pathways. These results indicated that lncRNA-ATB may be considered as a new predictor in the clinical prognosis of patients with prostate cancer. Overexpression of lncRNA-ATB exerts mitogenic and EMT effects of prostate cancer via activation of ERK and PI3K/AKT signaling pathways.

Topics: Cadherins; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin E; Epithelial-Mesenchymal Transition; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Humans; Male; Neoplasm Invasiveness; Oncogene Proteins; Phosphatidylinositol 3-Kinases; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; RNA Interference; RNA, Long Noncoding; RNA, Small Interfering; Signal Transduction; Trans-Activators; Vimentin; Zinc Finger E-box-Binding Homeobox 1; Zonula Occludens-1 Protein

TMPRSS4 is a novel type II transmembrane serine protease found at the cell surface that is highly expressed in pancreatic, colon, and other cancer tissues. Previously, we demonstrated that TMPRSS4 mediates tumor cell invasion, migration, and metastasis. We also found that TMPRSS4 activates the transcription factor activating protein-1 (AP-1) to induce cancer cell invasion. Here, we explored TMPRSS4-mediated cellular functions and the underlying mechanisms. TMPRSS4 induced Slug, an epithelial-mesenchymal transition (EMT)-inducing transcription factor, and cyclin D1 through activation of AP-1, composed of c-Jun and activating transcription factor (ATF)-2, which resulted in enhanced invasion and proliferation of PC3 prostate cancer cells. In PC3 cells, not only c-Jun but also Slug was required for TMPRSS4-mediated proliferation and invasion. Interestingly, Slug induced phosphorylation of c-Jun and ATF-2 to activate AP-1 through upregulation of Axl, establishing a positive feedback loop between Slug and AP-1, and thus induced cyclin D1, leading to enhanced proliferation. Using data from The Cancer Genome Atlas, we found that Slug expression positively correlated with that of c-Jun and cyclin D1 in human prostate cancers. Expression of Slug was positively correlated with that of cyclin D1 in various cancer cell lines, whereas expression of other EMT-inducing transcription factors was not. This study demonstrates that TMPRSS4 modulates both invasion and proliferation via Slug and cyclin D1, which is a previously unrecognized pathway that may regulate metastasis and cancer progression.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Disease Progression; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Prostatic Neoplasms; RNA, Small Interfering; Serine Endopeptidases; Skin Neoplasms; Snail Family Transcription Factors; Up-Regulation

Human cancers exhibit significant cellular heterogeneity featuring tumorigenic cancer stem cells (CSCs) in addition to more differentiated progeny with limited tumor-initiating capabilities. Recent studies suggest that microRNAs (miRNAs) regulate CSCs and tumor development. A previous library screening for differential miRNA expression in CD44+ (and other) prostate CSC vs. non-CSC populations identified miR-199a-3p to be among the most highly under-expressed miRNAs in CSCs. In this study, we characterized the biological functions of miR-199a-3p in CD44+ prostate cancer (PCa) cells and in tumor regeneration. Overexpression of miR-199a-3p in purified CD44+ or bulk PCa cells, including primary PCa, inhibited proliferation and clonal expansion without inducing apoptosis. miR-199a-3p overexpression also diminished tumor-initiating capacities of CD44+ PCa cells as well as tumor regeneration from bulk PCa cells. Importantly, inducible miR-199a-3p expression in pre-established prostate tumors in NOD/SCID mice inhibited tumor growth. Using target prediction program and luciferase assays, we show mechanistically that CD44 is a direct functional target of miR-199a-3p in PCa cells. Moreover, miR-199a-3p also directly or indirectly targeted several additional mitogenic molecules, including c-MYC, cyclin D1 (CCND1) and EGFR. Taken together, our results demonstrate how the aberrant loss of a miRNA-mediated mechanism can lead to the expansion and tumorigenic activity of prostate CSCs, further supporting the development and implementation of miRNA mimics for cancer treatment.

Topics: Animals; Apoptosis; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Cyclin D1; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; Hyaluronan Receptors; Male; Mice; Mice, Inbred NOD; Mice, SCID; MicroRNAs; Neoplasm Transplantation; Neoplastic Stem Cells; Prostatic Neoplasms; Signal Transduction

Lapatinib, a dual inhibitor of epidermal growth factor receptor (EGFR)/ErbB2, has antiproliferative effects and is used to treat patients with ErbB2-positive metastatic breast cancer. In the present study, we examined the effects of lapatinib on growth of oral and prostate cancer cells. Oral squamous cell carcinoma (OSCC) cell lines HSC3, HSC4 and Ca9-22 were sensitive to the antiproliferative effects of lapatinib in anchorage-dependent culture, but the OSCC cell lines KB and SAS and the prostate cancer cell line DU145 were resistant to lapatinib. Phosphorylation levels of EGFR in all cell lines decreased during lapatinib treatment in anchorage‑dependent culture. Furthermore, the phosphorylation levels of ErbB2, ErbB3 and Akt and the protein levels of cyclin D1 were decreased by lapatinib treatment of HSC3, HSC4 and Ca9-22 cells. ErbB3 was not expressed and cyclin D1 protein levels were not altered by lapatinib treatment in KB, DU145 and SAS cells. The phosphorylation of ErbB2 and AKT was not affected by lapatinib in SAS cells and was not detected in KB and DU145 cells. Lapatinib-resistant cell lines exhibited sphere-forming ability, and SAS cells developed sensitivity to lapatinib during sphere formation. The phosphorylation levels of ErbB2 and AKT and protein levels of cyclin D2 increased during sphere formation of SAS cells and decreased with lapatinib treatment. In addition, sphere formation of SAS cells was inhibited by the AKT inhibitor MK2206. AKT phosphorylation and cyclin D2 levels in SAS spheres were decreased by MK2206 treatment. SAS cells expressed E-cadherin, but not vimentin and KB cells expressed vimentin, but not E-cadherin. DU145 cells expressed vimentin and E-cadherin. These results suggested that phosphorylation of EGFR and ErbB2 by cell detachment from the substratum induces the AKT pathway/cyclin D2-dependent sphere growth in SAS epithelial cancer stem-like cells, thereby rendering SAS spheres sensitive to lapatinib treatment.

Topics: Cadherins; Carcinoma, Squamous Cell; Cell Line, Tumor; Cyclin D1; Cyclin D2; Drug Resistance, Neoplasm; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Lapatinib; Male; Mouth Neoplasms; Phosphorylation; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Quinazolines; Receptor, ErbB-2; Receptor, ErbB-3

Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in human cancers. Therefore, STAT3 is a therapeutic target of cancer drug discovery. We previously reported that natural products inhibited constitutively activated STAT3 in human prostate tumor cells. We used a dual-luciferase assay to screen 200 natural products isolated from herbal medicines and we identified ginkgetin obtained from the leaves of Ginkgo biloba L. as a STAT3 inhibitor. Ginkgetin inhibited both inducible and constitutively activated STAT3 and blocked the nuclear translocation of p-STAT3 in DU-145 prostate cancer cells. Furthermore, ginkgetin selectively inhibited the growth of prostate tumor cells stimulated with activated STAT3. Ginkgetin induced STAT3 dephosphorylation at Try705 and inhibited its localization to the nucleus, leading to the inhibition of expression of STAT3 target genes such as cell survival-related genes (cyclin D1 and survivin) and anti-apoptotic proteins (Bcl-2 and Bcl-xL). Therefore, ginkgetin inhibited the growth of STAT3-activated tumor cells. We also found that ginkgetin inhibited tumor growth in xenografted nude mice and downregulated p-STAT3(Tyr705) and survivin in tumor tissues. This is the first report that ginkgetin exerts antitumor activity by inhibiting STAT3. Therefore, ginkgetin is a good STAT3 inhibitor and may be a useful lead molecule for development of a therapeutic STAT3 inhibitor.

Topics: Active Transport, Cell Nucleus; Animals; Antineoplastic Agents; Apoptosis; Biflavonoids; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Drug Screening Assays, Antitumor; Female; G1 Phase Cell Cycle Checkpoints; Ginkgo biloba; HCT116 Cells; Humans; Inhibitor of Apoptosis Proteins; Interleukin-6; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Phosphorylation; Prostate; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Repressor Proteins; STAT3 Transcription Factor; Survivin; Xenograft Model Antitumor Assays

WW domain-containing oxidoreductase (WWOX) has been reported to be a tumor suppressor in multiple cancers, including prostate cancer. WWOX can induce apoptotic responses to inhibit tumor progression, and the other mechanisms of WWOX in tumor suppression have also been reported recently. In this study, we found significant down-regulation of WWOX in prostate cancer specimens and prostate cancer cell lines compared with the normal controls. In addition, an ectopically increased WWOX expression repressed tumor progression both in vitro and in vivo. Interestingly, overexpression of WWOX in 22Rv1 cells led to cell cycle arrest in the G1 phase but did not affect sub-G1 in flow cytometry. GFP-WWOX overexpressed 22Rv1 cells were shown to inhibit cell cycle progression into mitosis under nocodazole treatment in flow cytometry, immunoblotting and GFP fluorescence. Further, cyclin D1 but not apoptosis correlated genes were down-regulated by WWOX both in vitro and in vivo. Restoration of cyclin D1 in the WWOX-overexpressed 22Rv1 cells could abolish the WWOX-mediated tumor repression. In addition, WWOX impair c-Jun-mediated cyclin D1 promoter activity. These results suggest that WWOX inhibits prostate cancer progression through negatively regulating cyclin D1 in cell cycle lead to G1 arrest. In summary, our data reveal a novel mechanism of WWOX in tumor suppression.

Topics: Animals; Cell Cycle Checkpoints; Cell Line, Tumor; Cyclin D1; Disease Progression; G1 Phase; Gene Silencing; Humans; Male; Mice, Nude; Oxidoreductases; Prostatic Neoplasms; Proto-Oncogene Proteins c-jun; Tumor Suppressor Proteins; WW Domain-Containing Oxidoreductase

Micro (mi) RNAs are important regulators involved in various physical and pathological processes, including cancer. The miRNA-302 family has been documented as playing a critical role in carcinogenesis. In this study, we investigated the role of miRNA-302a in prostate cancer (PCa). MiRNA-302a expression was detected in 44 PCa tissues and 10 normal prostate tissues, and their clinicopathological significance was analyzed. Cell proliferation and cell cycle analysis were performed on PCa cells that stably expressed miRNA-302a. The target gene of miRNA-302a and the downstream pathway were further investigated. Compared with normal prostate tissues, miRNA-302a expression was downregulated in PCa tissues, and was even lower in PCa tissues with a Gleason score ≥8. Overexpression of miRNA-302a induced G1/S cell cycle arrest in PCa cells, and suppressed PCa cell proliferation both in vitro and in vivo. Furthermore, miRNA-302a inhibits AKT expression by directly binding to its 3΄ untranslated region, resulting in subsequent alterations of the AKT-GSK3β-cyclin D1 and AKT-p27Kip1 pathway. These results reveal miRNA-302a as a tumor suppressor in PCa, suggesting that miRNA-302a may be used as a potential target for therapeutic intervention in PCa.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Genetic Vectors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; HEK293 Cells; Humans; Lentivirus; Male; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Neoplasm Grading; Neoplasm Transplantation; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Signal Transduction

Recent epidemiological studies showed that metformin, a widely used anti-diabetic drug might prevent certain cancers. Metformin also has an anti-proliferative effect in preclinical studies of both hematologic malignancies as well as solid cancers and clinical studies testing metformin as an anti-cancer drug are in progress. However, all cancer types do not respond to metformin with the same effectiveness or acquire resistance. To understand the mechanism of acquired resistance and possibly its mechanism of action as an anti-proliferative agent, we developed metformin resistant LNCaP prostate cancer cells. Metformin resistant LNCaP cells had an increased proliferation rate, increased migration and invasion ability as compared to the parental cells, and expressed markers of epithelial-mesenchymal transition (EMT). A detailed gene expression microarray comparing the resistant cells to the wild type cells revealed that Edil2, Ereg, Axl, Anax2, CD44 and Anax3 were the top up-regulated genes and calbindin 2 and TPTE (transmembrane phosphatase with tensin homology) and IGF1R were down regulated. We focused on Axl, a receptor tyrosine kinase that has been shown to be up regulated in several drug resistance cancers. Here, we show that the metformin resistant cell line as well as castrate resistant cell lines that over express Axl were more resistant to metformin, as well as to taxotere compared to androgen sensitive LNCaP and CWR22 cells that do not overexpress Axl. Forced overexpression of Axl in LNCaP cells decreased metformin and taxotere sensitivity and knockdown of Axl in resistant cells increased sensitivity to these drugs. Inhibition of Axl activity by R428, a small molecule Axl kinase inhibitor, sensitized metformin resistant cells that overexpressed Axl to metformin. Inhibitors of Axl may enhance tumor responses to metformin and other chemotherapy in cancers that over express Axl.

Topics: Adenylate Kinase; Antineoplastic Agents; Axl Receptor Tyrosine Kinase; Benzocycloheptenes; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Docetaxel; Drug Resistance, Neoplasm; Enzyme Activation; Epithelial-Mesenchymal Transition; Gene Knockdown Techniques; Humans; Male; Metformin; Neoplasm Invasiveness; Prostatic Neoplasms; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Receptor Protein-Tyrosine Kinases; Taxoids; Triazoles; Up-Regulation

Micro-RNAs (miRNA) are important regulators of gene expression and often differentially expressed in cancer and other diseases. We have previously shown that miR-193b is hypermethylated in prostate cancer (PC) and suppresses cell growth. It has been suggested that miR-193b targets cyclin D1 in several malignancies. Here, our aim was to determine if miR-193b targets cyclin D1 in prostate cancer. Our data show that miR-193b is commonly methylated in PC samples compared to benign prostate hyperplasia. We found reduced miR-193b expression (P < 0.05) in stage pT3 tumors compared to pT2 tumors in a cohort of prostatectomy specimens. In 22Rv1 PC cells with low endogenous miR-193b expression, the overexpression of miR-193b reduced CCND1 mRNA levels and cyclin D1 protein levels. In addition, the exogenous expression of miR-193b decreased the phosphorylation level of RB, a target of the cyclin D1-CDK4/6 pathway. Moreover, according to a reporter assay, miR-193b targeted the 3'UTR of CCND1 in PC cells and the CCND1 activity was rescued by expressing CCND1 lacking its 3'UTR. Immunohistochemical analysis of cyclin D1 showed that castration-resistant prostate cancers have significantly (P = 0.0237) higher expression of cyclin D1 compared to hormone-naïve cases. Furthermore, the PC cell lines 22Rv1 and VCaP, which express low levels of miR-193b and high levels of CCND1, showed significant growth retardation when treated with a CDK4/6 inhibitor. In contrast, the inhibitor had no effect on the growth of PC-3 and DU145 cells with high miR-193b and low CCND1 expression. Taken together, our data demonstrate that miR-193b targets cyclin D1 in prostate cancer.

Topics: 3' Untranslated Regions; Cell Line, Tumor; Cyclin D1; DNA Methylation; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Humans; Male; MicroRNAs; Neoplasm Grading; Neoplasm Recurrence, Local; Neoplasm Staging; Prostatic Neoplasms; RNA Interference; RNA, Messenger

Small-cell neuroendocrine differentiation in prostatic carcinoma is an increasingly common resistance mechanism to potent androgen deprivation therapy (ADT), but can be difficult to identify morphologically. We investigated whether cyclin D1 and p16 expression can inform on Rb functional status and distinguish small-cell carcinoma from adenocarcinoma.. We used gene expression data and immunohistochemistry to examine cyclin D1 and p16 levels in patient-derived xenografts (PDX), and prostatic small-cell carcinoma and adenocarcinoma specimens.. Using PDX, we show proof-of-concept that a high ratio of p16 to cyclin D1 gene expression reflects underlying Rb functional loss and distinguishes morphologically identified small-cell carcinoma from prostatic adenocarcinoma in patient specimens (n = 13 and 9, respectively). At the protein level, cyclin D1, but not p16, was useful to distinguish small-cell carcinoma from adenocarcinoma. Overall, 88% (36/41) of small-cell carcinomas showed cyclin D1 loss by immunostaining compared with 2% (2/94) of Gleason score 7-10 primary adenocarcinomas at radical prostatectomy, 9% (4/44) of Gleason score 9-10 primary adenocarcinomas at needle biopsy, and 7% (8/115) of individual metastases from 39 patients at autopsy. Though rare adenocarcinomas showed cyclin D1 loss, many of these were associated with clinical features of small-cell carcinoma, and in a cohort of men treated with adjuvant ADT who developed metastasis, lower cyclin D1 gene expression was associated with more rapid onset of metastasis and death.. Cyclin D1 loss identifies prostate tumors with small-cell differentiation and may identify a small subset of adenocarcinomas with poor prognosis. Clin Cancer Res; 21(24); 5619-29. ©2015 AACR.

Topics: Adenocarcinoma; Animals; Biomarkers, Tumor; Carcinoma, Small Cell; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Disease Models, Animal; Gene Expression; Gene Expression Profiling; Heterografts; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Male; Mice; Neoplasm Grading; Neoplasm Metastasis; Prognosis; Prostatic Neoplasms; Retinoblastoma Protein

The prognosis and survival rate of prostate cancer are very poor. Previous studies have shown that miR-556-5p have emerged as important regulators in cancer cell biological processes. The role of miR-556-5p in prostate cancer remains unclear. In this study, expression of miR-556-5p in prostate cancer cell lines and tissues was upregulated. Result of MTT assays, colony formation and anchorage-independent growth assays demonstrated that overexpression of miR-556-5p promoted prostate cancer cell growth. Additionally, PPP2R2A was identified as a direct target of miR-556-5p. Ectopic expression of miR-556-5p led to downregulation of PPP2R2A protein, which resulted in the downregulation of p27, upregulation of cyclin D1. Taken together, our data provide compelling evidence that miR-556-5p functions as an onco-miRNA and participates in prostate cancer carcinogenesis by suppressing PPP2R2A expression.

Topics: Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Male; MicroRNAs; Prostatic Neoplasms; Protein Phosphatase 2; RNA Interference; Signal Transduction; Time Factors; Transfection

Curcumin is a nontoxic, chemopreventive agent possessing multifaceted functions. Our previous study showed that curcumin inhibits androgen receptor (AR) through modulation of Wnt/β-catenin signaling in LNCaP cells. Therefore, we investigated the in vivo effects of curcumin by using LNCaP xenografts.. LNCaP cells were subcutaneously inoculated in Balb/c nude mice. When the tumor volume reached greater than 100 mm(3), either curcumin (500 mg/kg body weight) or vehicle was administered through oral gavage three times weekly for 4 weeks. The expression of AR and intermediate products of Wnt/β-catenin were assessed.. Curcumin had an inhibitory effect on tumor growth during the early period, which was followed by a slow increase in growth over time. Tumor growth was delayed about 27% in the curcumin group. The mean prostate-specific antigen (PSA) doubling time in the curcumin group was approximately twice that in the untreated group. Curcumin significantly decreased AR expression at both the mRNA and protein level. The PSA levels tended to be reduced in the curcumin group. However, there were no significant changes in expression of Wnt/β-catenin pathway intermediates.. This study revealed that curcumin initially interferes with prostate cancer growth by inhibiting AR activity and possibly by reducing PSA expression. Further research is needed to investigate the plausible mechanism of the antiandrogenic action of curcumin.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; beta Catenin; Curcumin; Cyclin D1; Heterografts; Humans; Male; Mice, Inbred BALB C; Prostate-Specific Antigen; Prostatic Neoplasms; Receptors, Androgen; RNA, Messenger; Wnt Signaling Pathway

Nitroxoline is an antibiotic by chelating Zn2+ and Fe2+ from biofilm matrix. In this study, nitroxoline induced G1 arrest of cell cycle and subsequent apoptosis in prostate cancer cells through ion chelating-independent pathway. It decreased protein levels of cyclin D1, Cdc25A and phosphorylated Rb, but activated AMP-activated protein kinase (AMPK), a cellular energy sensor and signal transducer, leading to inhibition of downstream mTOR-p70S6K signaling. Knockdown of AMPKα significantly rescued nitroxoline-induced inhibition of cyclin D1-Rb-Cdc25A axis indicating AMPK-dependent mechanism. However, cytoprotective autophagy was simultaneously evoked by nitroxoline. Comet assay and Western blot analysis demonstrated DNA damaging effect and activation of Chk2 other than Chk1 to nitroxoline action. Instead of serving as a DNA repair transducer, nitroxoline-mediated Chk2 activation was identified to function as a pro-apoptotic inducer. In conclusion, the data suggest that nitroxoline induces anticancer activity through AMPK-dependent inhibition of mTOR-p70S6K signaling pathway and cyclin D1-Rb-Cdc25A axis, leading to G1 arrest of cell cycle and apoptosis. AMPK-dependent activation of Chk2, at least partly, contributes to apoptosis. The data suggest the potential role of nitroxoline for therapeutic development against prostate cancers.

Topics: AMP-Activated Protein Kinases; Antineoplastic Agents; Apoptosis; Blotting, Western; cdc25 Phosphatases; Cell Line, Tumor; Cell Proliferation; Checkpoint Kinase 2; Cyclin D1; DNA Damage; Drug Repositioning; Enzyme Activation; G1 Phase Cell Cycle Checkpoints; Humans; Male; Microscopy, Fluorescence; Nitroquinolines; Prostatic Neoplasms; Retinoblastoma Protein; RNA Interference; Signal Transduction; TOR Serine-Threonine Kinases

We previously identified a protein tumor signature of PTEN, SMAD4, SPP1, and CCND1 that, together with clinical features, was associated with lethal outcomes among prostate cancer patients. In the current study, we sought to validate the molecular model using time-dependent measures of AUC and predictive values for discriminating lethal from non-lethal prostate cancer.. Using data from the initial study, we fit survival models for men with prostate cancer who were participants in the Physicians' Health Study (PHS; n = 276). Based on these models, we generated prognostic risk scores in an independent population, the Health Professionals Follow-up Study (HPFS; n = 347) to evaluate external validity. In each cohort, men were followed prospectively from cancer diagnosis through 2011 for development of distant metastasis or cancer mortality. We measured protein tumor expression of PTEN, SMAD4, SPP1, and CCND1 on tissue microarrays.. During a median of 11.9 and 14.3 years follow-up in the PHS and HPFS cohorts, 24 and 32 men (9%) developed lethal disease. When used as a prognostic factor in a new population, addition of the four markers to clinical variables did not improve discriminatory accuracy through 15 years of follow-up.. Although the four markers have been identified as key biological mediators in metastatic progression, they do not provide independent, long-term prognostic information beyond clinical factors when measured at diagnosis. This finding may underscore the broad heterogeneity in aggressive prostate tumors and highlight the challenges that may result from overfitting in discovery-based research.

Topics: Aged; Aged, 80 and over; Area Under Curve; Biomarkers, Tumor; Cyclin D1; Disease Progression; Follow-Up Studies; Gene Expression Profiling; Humans; Male; Middle Aged; Neoplasm Invasiveness; Osteopontin; Prognosis; Prostate; Prostatic Neoplasms; PTEN Phosphohydrolase; Smad4 Protein

It has been noticed that crosstalk between androgen receptor (AR) and mammalian target of rapamycin (mTOR) signaling pathways plays a crucial role in the proliferation of prostate cancer cells. To clarify this mechanism, we focused on DEPTOR, a naturally occurring inhibitor of mTOR. The treatment of a human AR-positive prostate cancer cell line, LNCaP, with the AR-agonist dihydrotestosterone (DHT) repressed DEPTOR mRNA expression in a time-dependent manner. This repression was abrogated by treatment with the AR-antagonist bicalutamide. Knockdown of DEPTOR mRNA by siRNA resulted in the increased phosphorylation of 70 kDa ribosomal protein S6 kinase 1 (S6K), a substrate of mTORC1, accompanied by the elevated expression of cyclin D1, a positive regulator of cell proliferation. Furthermore, the ChIP assay demonstrated that AR could bind to AR-responsible element-like region within the 4th intron of the DEPTOR gene. The amount of acetylated histone H3 (Lys9, Lys14) was reduced by the DHT treatment in this region. Taken together, these results propose that AR-dependent prostate cancer cell proliferation requires decreased DEPTOR transcription directly controlled by AR.

Topics: Androgen Antagonists; Anilides; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Dihydrotestosterone; Histones; Humans; Intracellular Signaling Peptides and Proteins; Male; Nitriles; Phosphorylation; Prostatic Neoplasms; Receptor Cross-Talk; Receptors, Androgen; Ribosomal Protein S6 Kinases, 70-kDa; RNA, Messenger; Signal Transduction; Time Factors; TOR Serine-Threonine Kinases; Tosyl Compounds; Transcription, Genetic; Tumor Cells, Cultured

Long non-coding RNAs (lncRNAs) play important roles in diverse biological processes, such as cell growth, apoptosis and migration. Although downregulation of lncRNA maternally expressed gene 3 (MEG3) has been identified in several cancers, little is known about its role in prostate cancer progression. The aim of this study was to detect MEG3 expression in clinical prostate cancer tissues, investigate its biological functions in the development of prostate cancer and the underlying mechanism.. MEG3 expression levels were detected by qRT-PCR in both tumor tissues and adjacent non-tumor tissues from 21 prostate cancer patients. The effects of MEG3 on PC3 and DU145 cells were assessed by MTT assay, colony formation assay, western blot and flow cytometry. Transfected PC3 cells were transplanted into nude mice, and the tumor growth curves were determined.. MEG3 decreased significantly in prostate cancer tissues relative to adjacent normal tissues. MEG3 inhibited intrinsic cell survival pathway in vitro and in vivo by reducing the protein expression of Bcl-2, enhancing Bax and activating caspase 3. We further demonstrated that MEG3 inhibited the expression of cell cycle regulatory protein Cyclin D1 and induced cell cycle arrest in G0/G1 phase.. Our study presents an important role of MEG3 in the molecular etiology of prostate cancer and implicates the potential application of MEG3 in prostate cancer therapy.

Topics: Animals; Apoptosis; Caspase 3; Cell Proliferation; Cyclin D1; Enzyme Activation; Heterografts; Humans; Male; Mice; Mice, Nude; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; RNA, Long Noncoding

Increased expression of Chromosome Region Maintenance (CRM-1)/exportin-1 (XPO-1) has been correlated with poor prognosis in several aggressive tumors, making it an interesting therapeutic target. Selective Inhibitor of Nuclear Export (SINE) compounds bind to XPO-1 and block its ability to export cargo proteins. Here, we investigated the effects of a new class of SINE compounds in models of prostate cancer.. We evaluated the expression of XPO-1 in human prostate cancer tissues and cell lines. Next, six SINE (KPT-127, KPT-185, KPT-205, KPT-225, KPT-251 and KPT-330) compounds having different potency with broad-spectrum, tumor-selective cytotoxicity, tolerability and pharmacokinetic profiles were tested in a panel of prostate cancer cells representing distinct differentiation/progression states of disease and genotypes. Two SINE candidates for clinical trials (KPT-251 and KPT-330) were also tested in vivo in three cell models of aggressive prostate cancer engrafted in male nude mice.. XPO-1 is overexpressed in prostate cancer compared to normal or hyperplastic tissues. Increased XPO-1 expression, mainly in the nuclear compartment, was associated with increased Gleason score and bone metastatic potential supporting the use of SINEs in advanced prostate cancer. SINE compounds inhibited proliferation and promoted apoptosis of tumor cells, but did not affect immortalized non-transformed prostate epithelial cells. Nuclei from SINE treated cells showed increased protein localization of XPO-1, survivin and cyclin D1 followed by degradation of these proteins leading to cell cycle arrest and apoptosis. Oral administration of KPT-251 and KPT-330 in PC3, DU145 and 22rv1 tumor-bearing nude mice reduced tumor cell proliferation, angiogenesis and induced apoptosis. Our results provide supportive evidence for the therapeutic use of SINE compounds in advanced/castration resistant prostate cancers and warrants further clinical investigation.

Topics: Adult; Analgesics; Animals; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Hydrazines; Inhibitor of Apoptosis Proteins; Male; Mice; Mice, Nude; Neoplasm Grading; Oxadiazoles; Prostatic Neoplasms; Survivin; Triazoles; Up-Regulation; Xenograft Model Antitumor Assays

Improved clinical management of prostate cancer has been impeded by an inadequate understanding of molecular genetic elements governing tumor progression. Gene signatures have provided improved prognostic indicators of human prostate cancer. The TGF-β/BMP-SMAD4 signaling pathway, which induces epithelial-mesenchymal transition (EMT), is known to constrain prostate cancer progression induced by Pten deletion. Herein, cyclin D1 inactivation reduced cellular proliferation in the murine prostate in vivo and in isogenic oncogene-transformed prostate cancer cell lines. The in vivo cyclin D1-mediated molecular signature predicted poor outcome of recurrence-free survival for patients with prostate cancer (K-means HR, 3.75, P = 0.02) and demonstrated that endogenous cyclin D1 restrains TGF-β, Snail, Twist, and Goosecoid signaling. Endogenous cyclin D1 enhanced Wnt and ES cell gene expression and expanded a prostate stem cell population. In chromatin immunoprecipitation sequencing, cyclin D1 occupied genes governing stem cell expansion and induced their transcription. The coordination of EMT restraining and stem cell expanding gene expression by cyclin D1 in the prostate may contribute to its strong prognostic value for poor outcome in biochemical-free recurrence in human prostate cancer.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Disease Progression; Disease-Free Survival; Epithelial-Mesenchymal Transition; Gene Deletion; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Mice, Transgenic; Oligonucleotide Array Sequence Analysis; Prognosis; Prostatic Neoplasms; PTEN Phosphohydrolase; Recurrence; Signal Transduction; Treatment Outcome

HOXB13 is a homeobox protein that is expressed in normal adult prostate and colon tissues; however, its deregulated expression was evidenced in various malignancies. To characterize the putative role of HOXB13 in cell cycle progression, we performed overexpression and siRNA-mediated knockdown studies in PC-3 and LNCaP cells. Immunohistochemistry (IHC) analyses were also performed using formalin-fixed, paraffin-embedded tissues containing normal, H-PIN and PCa sections from 20 radical prostatectomy specimens. Furthermore, when the role of HOXB13 during cell cycle progression, association with cyclins, cell growth and colony formation using real-time cell proliferation were assessed, we observed that ectopic expression of HOXB13 accumulated cells at G1 through decreasing the cyclin D1 level by promoting its ubiquitination and degradation. This loss slowed S phase entry in both cell lines examined, with an associated decrease in pRb((S780) and (S795)) phosphorylations. Contrary, siRNA-mediated depletion of HOXB13 expression noticeably increased cyclin levels, stabilized E2F1 and CDC25C, subsequent to increased pRb phosphorylations. This increase in Cyclin B1 and CDC25C both together facilitated activation of cyclin B complex via dephosphorylating CDK1((T14Y15)), and resumed the G2/M transition after nocodazole synchronization. Despite an increase in the total expression level and cytoplasmic retention of HOXB13 in H-PIN and PCa samples that were observed via IHC evaluation of prostate tissues, HOXB13 depletion facilitated to an increase in PC-3 and LNCaP cell proliferation. Thus, we suggest that HOXB13 expression is required for cell cycle regulation, and increases by an unknown mechanism consequent to its functional loss in cancer.

Topics: CDC2 Protein Kinase; cdc25 Phosphatases; Cell Line, Tumor; Cell Proliferation; Cyclin B1; Cyclin D1; E2F1 Transcription Factor; G1 Phase Cell Cycle Checkpoints; G2 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Male; Phosphorylation; Prostate; Prostatectomy; Prostatic Neoplasms; Proteolysis; Retinoblastoma Protein; Signal Transduction; Ubiquitination

Aberrant activation of the Wnt/β-catenin signaling pathway is a critical event in advanced prostate cancer, but the genetic alterations that activate the Wnt signaling pathway in many other cancers are rarely observed in prostate cancer. Other molecular mechanisms that regulate the Wnt signaling pathway in prostate cancer remain to be identified. Here, it is demonstrated that KIF3a, a subunit of kinesin-II motor protein, functions as an agonist of the Wnt signaling pathway in prostate cancer. KIF3a is upregulated in the majority of human prostate cancer cell lines and primary tumor biopsies. The expression levels of KIF3a correlate with a higher Gleason score, tumor-node-metastasis stage, and metastatic status of prostate cancer. Moreover, exogenous expression of KIF3a promoted cell growth in the benign prostate cells, whereas silencing KIF3a in cancer cells decreased cell proliferation, anchorage-independent cell growth, and cell migration/invasion. Mechanistically, KIF3a increases CK1-dependent DVL2 phosphorylation and β-catenin activation in prostate cancer cells, leading to transactivation of the Wnt-signaling target genes such as cyclin D1, HEF1, and MMP9. These findings support the notion that upregulation of KIF3a is causal of aberrant activation of Wnt signaling in advanced prostate cancer through the KIF3a-DVL2-β-catenin axis.. Inactivation of KIF3a may improve survival of patients with advanced prostate cancer in which Wnt signaling is activated.

Topics: Adaptor Proteins, Signal Transducing; beta Catenin; Cell Growth Processes; Cell Line, Tumor; Cyclin D1; Disease Progression; Dishevelled Proteins; HEK293 Cells; Humans; Kinesins; Male; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Phosphoproteins; Phosphorylation; Prostatic Neoplasms; Tissue Array Analysis; Transfection; Up-Regulation; Wnt Proteins; Wnt Signaling Pathway

It has been reported that NKX3.1 an androgen-regulated homeobox gene restricted to prostate and testicular tissues, encodes a homeobox protein, which transcriptionally regulates oxidative damage responses and enhances topoisomerase I re-ligation by a direct interaction with the ATM protein in prostate cells. In this study, we aimed to investigate the role of NKX3.1 in DNA double-strand break (DSB) repair. We demonstrate that the DNA damage induced by CPT-11 (irinotecan, a topo I inhibitor), doxorubicin (a topo II inhibitor), and H2O2 (a mediator of oxidative damage), but not by etoposide (another topo II inhibitor), is negatively influenced by NKX3.1 expression. We also examined γH2AX((S139)) foci formation and observed that the overexpression of NKX3.1 resulted a remarkable decrease in the formation of γH2AX((S139)) foci. Intriguingly, we observed in NKX3.1 silencing studies that the depletion of NKX3.1 correlated with a significant decrease in the levels of p-ATM((S1981)) and γH2AX((S139)). The data imply that the DNA damage response (DDR) can be altered, perhaps via a decrease in the topoisomerase I re-ligation function; this is consistent with the physical association of NKX3.1 with DDR mediators upon treatment of both PC-3 and LNCaP cells with CPT-11. Furthermore, the depletion of NKX3.1 resulted in a G1/S progression via the facilitation of an increase in E2F stabilization concurrent with the suppressed DDR. Thus, the topoisomerase I inhibitor-mediated DNA damage enhanced the physical association of NKX3.1 with γH2AX((S139)) on the chromatin in LNCaP cells, whereas NKX3.1 in the soluble fraction was associated with p-ATM((S1981)) and RAD50 in these cells. Overall, the data suggest that androgens and NKX3.1 expression regulate the progression of the cell cycle and concurrently activate the DDR. Therefore, androgen withdrawal may augment the development of an error-prone phenotype and, subsequently, the loss of DNA damage control during prostate cancer progression.

Topics: Acid Anhydride Hydrolases; Ataxia Telangiectasia Mutated Proteins; Camptothecin; Cell Cycle; Cell Line, Tumor; Cyclin D1; DNA Damage; DNA Repair; DNA Repair Enzymes; DNA-Activated Protein Kinase; DNA-Binding Proteins; Doxorubicin; Etoposide; Histones; Homeodomain Proteins; Humans; Hydrogen Peroxide; Irinotecan; Male; Metribolone; Mutagens; Phosphorylation; Prostatic Neoplasms; Protein Processing, Post-Translational; Testosterone Congeners; Transcription Factors

Activation of Wnt/β-catenin signaling can result in up-regulation of mTORC1 signaling in cancer cells. The low density lipoprotein receptor-related protein-6 (LRP6) is an essential Wnt co-receptor for Wnt/β-catenin signaling. We found that rottlerin, a natural plant polyphenol, suppressed LRP6 expression and phosphorylation, and inhibited Wnt/β-catenin signaling in HEK293 cells. Furthermore, the inhibitory effects of rottlerin on LRP6 expression/phosphorylation and Wnt/β-catenin signaling were confirmed in human prostate cancer PC-3 and DU145 cells and breast cancer MDA-MB-231 and T-47D cells. Mechanistically, rottlerin promoted LRP6 degradation, but had no effects on LRP6 transcriptional activity. In addition, rottlerin-mediated LRP6 down-regulation was unrelated to activation of 5'-AMP-activated protein kinase (AMPK). Importantly, we also found that rottlerin inhibited mTORC1 signaling in prostate and breast cancer cells. Finally, we demonstrated that rottlerin was able to suppress the expression of cyclin D1 and survivin, two targets of both Wnt/β-catenin and mTORC1 signaling, in prostate and breast cancer cells, and displayed remarkable anticancer activity with IC(50) values between 0.7 and 1.7 μM for prostate cancer PC-3 and DU145 cells and breast cancer MDA-MB-231 and T-47D cells. The IC(50) values are comparable to those shown to suppress the activities of Wnt/β-catenin and mTORC1 signaling in prostate and breast cancer cells. Our data indicate that rottlerin is a novel LRP6 inhibitor and suppresses both Wnt/β-catenin and mTORC1 signaling in prostate and breast cancer cells, and that LRP6 represents a potential therapeutic target for cancers.

Topics: Acetophenones; Antineoplastic Agents; Benzopyrans; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Drug Screening Assays, Antitumor; Female; HEK293 Cells; Humans; Inhibitor of Apoptosis Proteins; Inhibitory Concentration 50; Low Density Lipoprotein Receptor-Related Protein-6; Male; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Promoter Regions, Genetic; Prostatic Neoplasms; Proteolysis; Survivin; TOR Serine-Threonine Kinases; Transcription, Genetic; Wnt Signaling Pathway; Wnt3A Protein

Multiple studies show that reactive oxygen species (ROS) play a major role in prostate cancer (PCa) development and progression. Previously, we reported an induction of Spermidine/Spermine N(1) -Acetyl Transferase (SSAT) by androgen-activated androgen receptor (AR)-JunD protein complex that leads to over-production of ROS in PCa cells. In our current research, we identify small molecules that specifically block AR-JunD in this ROS-generating metabolic pathway.. A high throughput assay based on Gaussia Luciferase reconstitution was used to identify inhibitors of the AR-JunD interaction. Selected hits were further screened using a fluorescence polarization competitor assay to eliminate those that bind to the AR Ligand Binding Domain (LBD), in order to identify molecules that specifically target events downstream to androgen activation of AR. Eleven molecules were selected for studies on their efficacy against ROS generation and growth of cultured human PCa cells by DCFH dye-oxidation assay and DNA fluorescence assay, respectively. In situ Proximity Ligation Assay (PLA), SSAT promoter-luciferase reporter assay, and western blotting of apoptosis and cell cycle markers were used to study mechanism of action of the lead compound.. Selected lead compound GWARJD10 with EC(50) 10 μM against ROS production was shown to block AR-JunD interaction in situ as well as block androgen-induced SSAT gene expression at IC(50) 5 μM. This compound had no effect on apoptosis markers, but reduced cyclin D1 protein level.. Inhibitor of AR-JunD interaction, GWARJD10 shows promise for prevention of progression of PCa at an early stage of the disease by blocking growth and ROS production.

Topics: Apoptosis; Cell Line, Tumor; Cyclin D1; Disease Progression; Humans; Male; Prostate; Prostatic Neoplasms; Proto-Oncogene Proteins c-jun; Reactive Oxygen Species; Receptors, Androgen

To construct a recombinant adenovirus expression vector containing the anti-oncogene PTEN and to investigate the effects of the PTEN gene on the proliferation of prostate cancer PC-3 cells and the expressions of cyclin D1 and p21 in the PC-3 cells.. The PTEN gene was amplified from the rat hippocampus by RT-PCR and cloned into the shuttle plasmid pEN-TR2A. The plasmids were constructed and amplified in 293A cells. Prostate cancer PC-3 cells were cultured in vitro and infected with the adenoviral vector carrying the PTEN gene (Ad-PTEN). The up-regulation of the PTEN protein was measured by indirect immuno-fluorescence assay; the expressions of PTEN, cyclin D1 and p21 in the cells infected with Ad-PTEN and Ad-LacZ were determined by. The Western blot; and the effect of PTEN on the cell proliferation was detected by MTT assay and plate colony formation. recombinant adenoviral vector Ad-PTEN was successfully constructed. Western blot showed a significantly increased expression of the PTEN protein in the PC-3 cells infected with Ad-PTIEN (0.215 +/-0.065) as compared with that in the control ([0.052 +/-0.009], t = 4. 30, P <0.05) and the Ad-LacZ group ( [0. 056 +/- 0.008 ] , t =4.21, P <0.05). The expression of cyclin D1 was significantly lower in the Ad-PTEN-infected PC-3 cells (0. 256 +/- 0. 072) than in the control ( [0. 502 +/- 0. 087 ], t = 3.77, P < 0.05) and the Ad-LacZ group ([0.498 +/-0.081] , t =3.87, P <0.05), while the expression of p21 remarkably higher in the Ad-PTEN-infected PC-3 cells (0.589 +/-0. 076) than in the control ([0. 146 +/-0.026] , t = 9.55, P<0. 01) and the Ad-LacZ group ([0. 163 +/-0. 024] , t = 9.26, P <0.01). Ad-PTEN significantly inhibited the growth of the PC-3 cells (21.98%) at 48 h (t = 6.80, P <0.01). The colony formation rate of the PC-3 cells was (37.4 +/-4. 18)% in the Ad-PTEN group, significantly lower than (54.9 +/-4.81)% in the control (t =4.76, P<0.01) and (56.5 +/- 5.42)% in the Ad-LacZ group (t=4.83, P<0.01).. The expression of PTEN induced by Ad-PTEN can significantly inhibit the proliferation of PC-3 cells, down-regulate the expression of cyclin D1, and up-regulate the expression of p21.

Topics: Adenoviridae; Animals; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Humans; Male; Prostatic Neoplasms; PTEN Phosphohydrolase; Rats; Rats, Sprague-Dawley

Both androgen and phosphatidylinositol 3-kinase (PI3K) signaling are critical for cell proliferation of androgen receptor (AR)-positive prostate cancer cells, but the underlying mechanisms are still not fully understood. Here we report that serum- and glucocorticoid-inducible kinase 3 (SGK3), a Ser/Thr kinase functioning downstream of PI3K, is an AR transcriptional target and promotes prostate cancer cell proliferation. SGK3 expression is up-regulated by androgen DHT via AR. We identified an AR-binding region at the sgk3 locus, which confers androgen responsiveness of sgk3 promoters. Interestingly, we found that androgen/AR-dependent SGK3 expression requires estrogen receptor (ER) (including both isoforms, ERα and ERβ). Depletion of ER blocked DHT-induced SGK3 expression. Functionally, knockdown of SGK3 expression significantly decreased LNCaP prostate cancer cell proliferation by inhibiting G1 to S phase cell cycle progression. We further provided evidence that SGK3 promotes p70 S6 kinase (p70S6K) activation and increases cyclin D1 levels. In summary, our study identifies SGK3 as an AR target and provides a novel androgen-induced cell proliferation mechanism mediated by the AR-SGK3-p70S6K-cyclin D1 pathway in prostate cancer cells.

Topics: Androgens; Base Sequence; Binding Sites; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Dihydrotestosterone; Enzyme Activation; G1 Phase Cell Cycle Checkpoints; Humans; Male; Molecular Sequence Data; Promoter Regions, Genetic; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Receptors, Androgen; Ribosomal Protein S6 Kinases, 70-kDa; Transcription, Genetic; Transcriptional Activation; Up-Regulation

Sperm-associated antigen 9 (SPAG9) was recently reported to be overexpressed in several cancers and associated with the malignant behavior of cancer cells. However, the expression pattern of SPAG9 and its clinical significance in human prostate cancer have not been reported. In the present study, we analyzed SPAG9 expression in human prostate cancer tissues by immunohistochemistry and found that SPAG9 was overexpressed in 36.5 % of prostate cancer specimens. There was a significant association between SPAG9 overexpression and tumor stage (p = 0.0020) and Gleason score (p = 0.0377). Transfection of SPAG9 plasmid was performed in PC-3 cell line and siRNA knockdown was carried out in DU145 cells. Colony formation and MTT showed that SPAG9 overexpression promoted while siRNA knockdown inhibited prostate cancer cell proliferation. In addition, we found that SPAG9 could regulate cyclin D1 and cyclin E protein expression. In conclusion, SPAG9 is overexpressed in human prostate cancers and contributes to prostate cancer cell growth, possibly through cyclin protein regulation.

Topics: Adaptor Proteins, Signal Transducing; Aged; Biomarkers, Tumor; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Disease-Free Survival; Gene Expression Regulation, Neoplastic; Humans; Male; Middle Aged; Neoplasm Staging; Prognosis; Prostatic Neoplasms; RNA, Small Interfering

The incidence of cancer grows annually worldwide and in Brazil it is the second cause of death. The search for anti-cancer drugs has then become urgent. It depends on the studies of natural and chemical synthesis products. The antitumor action of LQB-118, a pterocarpanquinone structurally related to lapachol, has been demonstrated to induce mechanisms linked to leukemia cell apoptosis. This work investigated some mechanisms of the in vitro antitumor action of LQB-118 on prostate cancer cells. LQB-118 reduced the expression of the c-Myc transcription factor, downregulated the cyclin D1 and cyclin B1 mRNA levels and upregulated the p21 cell cycle inhibitor. These effects resulted in cell cycle arrest in the S and G2/M phases and inhibition of tumor cell proliferation. LQB-118 also induced programmed cell death of the prostate cancer cells, as evidenced by internucleosomal DNA fragmentation and annexin-V positive cells. Except the cell cycle arrest in the S phase and enhanced c-Myc expression, all the mechanisms observed here for the in vitro antitumor action of LQB-118 were also found for Paclitaxel, a traditional antineoplastic drug. These findings suggest new molecular mechanisms for the LQB-118 in vitro antitumor action.

Topics: Apoptosis; Cell Cycle; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Male; Naphthoquinones; Prostatic Neoplasms; Proto-Oncogene Proteins c-myc; Pterocarpans; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured

The purpose of this study was to investigate the relationship between cyclin D1 expression and clinicopathological parameters in patients with prostate carcinoma. We assessed cyclin D1 expression by conventional immunohistochemistry in 85 patients who underwent radical prostatectomy for prostate carcinoma and 10 normal prostate tissue samples retrieved from autopsies. We measured nuclear immunostaining in the entire tumor area and based the results on the percentage of positive tumor cells. The preoperative prostate-specific antigen (PSA) level was 8.68±5.16 ng/mL (mean±SD). Cyclin D1 staining was positive (cyclin D1 expression in >5% of tumor cells) in 64 cases (75.4%) and negative (cyclin D1 expression in ≤5% of tumor cells) in 21 cases (including 15 cases with no immunostaining). Normal prostate tissues were negative for cyclin D1. Among patients with a high-grade Gleason score (≥7), 86% of patients demonstrated cyclin D1 immunostaining of >5% (P<0.05). In the crude analysis of cyclin D1 expression, the high-grade Gleason score group showed a mean expression of 39.6%, compared to 26.9% in the low-grade Gleason score group (P<0.05). Perineural invasion tended to be associated with cyclin D1 expression (P=0.07), whereas cyclin D1 expression was not associated with PSA levels or other parameters. Our results suggest that high cyclin D1 expression could be a potential marker for tumor aggressiveness.

Topics: Aged; Carcinoma; Cyclin D1; Humans; Immunohistochemistry; Male; Middle Aged; Neoplasm Grading; Prognosis; Prostate-Specific Antigen; Prostatectomy; Prostatic Neoplasms; Staining and Labeling; Statistics as Topic

Emerging evidence indicates that activation of Wnt/β-catenin signaling at the cell surface results in inhibition of glycogen synthase kinase 3β (GSK3β), leading to activation of mTORC1 signaling in cancer cells. The low density lipoprotein receptor-related protein-6 (LRP6) is an essential Wnt co-receptor for Wnt/β-catenin signaling. Salinomycin is a novel small molecule inhibitor of LRP6. In the present study, we found that LRP6 overexpression induced mTORC1 signaling activation in cancer cells, and that salinomycin was not only a potent Wnt/β-catenin signaling inhibitor, but also a strong mTORC1 signaling antagonist in breast and prostate cancer cells. Mechanistically, salinomycin activated GSK3β in cancer cells. Moreover, salinomycin was able to suppress the expression of cyclin D1 and survivin, two targets of both Wnt/β-catenin and mTORC1 signaling, in prostate and breast cancer cells, and displayed remarkable anticancer activity. Our results present novel mechanisms underlying salinomycin-mediated cancer cell death.

Topics: Anti-Bacterial Agents; Apoptosis; beta Catenin; Breast; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Enzyme Activation; Female; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; HEK293 Cells; Humans; Inhibitor of Apoptosis Proteins; Low Density Lipoprotein Receptor-Related Protein-6; Male; MCF-7 Cells; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Prostate; Prostatic Neoplasms; Pyrans; Survivin; TOR Serine-Threonine Kinases; Wnt Proteins; Wnt Signaling Pathway

ORAI family channels have emerged as important players in malignant transformation, yet the way in which they reprogram cancer cells remains elusive. Here we show that the relative expression levels of ORAI proteins in prostate cancer are different from that in noncancerous tissue. By mimicking ORAI protein remodeling observed in primary tumors, we demonstrate in in vitro models that enhanced ORAI3 expression favors heteromerization with ORAI1 to form a novel channel. These channels support store-independent Ca(2+) entry, thereby promoting cell proliferation and a smaller number of functional homomeric ORAI1-based store-operated channels, which are important in supporting susceptibility to apoptosis. Thus, our findings highlight disrupted dynamic equilibrium of channel-forming proteins as an oncogenic mechanism.

Topics: Adenocarcinoma; Aged; Animals; Apoptosis; Arachidonic Acid; Calcium Channels; Calcium Signaling; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Humans; Ion Channel Gating; Male; Membrane Proteins; Mice; Mice, Nude; Middle Aged; Neoplasm Proteins; NFATC Transcription Factors; ORAI1 Protein; Prostatic Neoplasms; Protein Transport; RNA Interference; Stromal Interaction Molecule 1; Time Factors; Transfection; Tumor Burden; Xenograft Model Antitumor Assays

3,19-(3-Chloro-4-fluorobenzylidene)andrographolide (SRJ23), a new semisynthetic derivative of andrographolide (AGP), exhibited selectivity against prostate cancer cells in the US National Cancer Institute (NCI) in vitro anti-cancer screen. Herein, we report the in vitro growth inhibition and mechanisms of cell cycle arrest and apoptosis induced by SRJ23.. 3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used in assessing in vitro growth inhibition of compounds against prostate cancer (PC-3, DU-145 and LNCaP) and mouse macrophage (RAW 264.7) cell lines. Flow cytometry was utilised to analyse cell cycle distribution, whereas fluorescence microscopy was performed to determine morphological cell death. DNA fragmentation and annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) flow cytometry were done to confirm apoptosis induced by SRJ23. Quantitation of cell cycle and apoptotic regulatory proteins were determined by immunoblotting.. AGP and SRJ23 selectively inhibited the growth of prostate cancer cells compared with RAW 264.7 cells at low micromolar concentrations; however, SRJ23 was more potent. Mechanistically, SRJ23-treated PC-3 cells displayed down-regulation of cyclin-dependent kinase (CDK) 1 without affecting levels of CDK4 and cyclin D1. However, SRJ23 induced down-regulation of CDK4 and cyclin D1 but without affecting CDK1 in DU145 and LNCaP cell lines. DNA histogram analysis revealed that the SRJ23 induced G2/M in PC-3 cells but G1 arrest in DU-145 and LNCaP cells. Morphologically, both compounds induced predominantly apoptosis, which was further confirmed by DNA fragmentation and annexin V-FITC staining. The DNA fragmentation was inhibited in the presence of caspase 8 inhibitor (Z-IETD-FMK). Apoptosis was associated with an increase in caspase 8 expression and activation. This thought to have induced cleavage of Bid into t-Bid. Additionally, increased expression and activation of caspase 9 and Bax proteins were apparent, with a concomitant down-regulation of Bcl-2 protein. Similar apoptosis cascade of events was observed in SRJ23-treated DU145 and LNCaP cell lines.. SRJ23 inhibited the growth of prostate cancer cells by inducing G2/M and G1 arrest via down-regulation of CDK1, and CDK4 and cyclin, respectively, and initiated caspase-8-mediated mitochondrial apoptosis. Taken together, these data support the potential of this compound as a new anti-prostate cancer agent.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 8; Caspase 9; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinases; Diterpenes; DNA Fragmentation; Down-Regulation; Guinea Pigs; Humans; Male; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2

Prostate cancer incidence and mortality rates have increased over the past years. The purpose of the present study was to examine the molecular mechanism underlying the chemopreventive effects of quercetin on prostate cancer in an in vivo model. Sprague-Dawley male rats were divided into four groups, Group I: vehicle control (propylene glycol), Group II: chemically induced cancer model (MNU + T); Group III: chemically induced cancer model + quercetin (200 mg per kg b.w.); Group IV: quercetin (200 mg per kg b.w.). Serum levels of quercetin were assessed by high performance liquid chromatography (HPLC). EGFR, PI3K/Akt protein levels were significantly increased in chemically induced cancer rats, which were brought back to normalcy in both DLP & VP (dorsolateral prostate & ventral prostate) by quercetin supplementation. Also, the protein expression levels of proliferating cell nuclear antigen (PCNA), N-cadherin, vimentin, and cyclin D1 exhibited a significant increase in both DLP & VP of chemically induced cancer rats. However, simultaneous quercetin supplementation significantly decreased PCNA, N-cadherin, vimentin, and cyclin D1 protein levels compared to chemically induced cancer rats. The E-cadherin expression was decreased in chemically induced cancer animals. Simultaneous quercetin supplementation prevented it. Real time PCR was used to study the mRNA expression of snail, slug and twist. Quercetin significantly decreased snail, slug, and twist mRNA levels in chemically induced cancer rats. To conclude from the present study, quercetin was effective in preventing prostate cancer progression by inhibiting the EGFR signaling pathway and by regulating cell adhesion molecules in Sprague Dawley rats.

Topics: Animals; Anticarcinogenic Agents; Cadherins; Cell Adhesion; Chemoprevention; Chromatography, High Pressure Liquid; Cyclin D1; Disease Models, Animal; ErbB Receptors; Male; Phosphatidylinositol 3-Kinases; Polyphenols; Proliferating Cell Nuclear Antigen; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Quercetin; Rats; Rats, Sprague-Dawley; RNA, Messenger; Signal Transduction; Vimentin

Diethylstilbestrol (DES) and other pharmaceutical estrogens have been used at ≥ µM concentrations to treat advanced prostate tumors, with successes primarily attributed to indirect hypothalamic-pituitary-testicular axis control mechanisms. However, estrogens also directly affect tumor cells, though the mechanisms involved are not well understood.. LAPC-4 (androgen-dependent) and PC-3 (androgen-independent) cell viability was measured after estradiol (E2) or DES treatment across wide concentration ranges. We then examined multiple rapid signaling mechanisms at 0.1 nM E2 and 1 µM DES optima including levels of: activation (phosphorylation) for mitogen-activated protein kinases, cell-cycle proteins, and caspase 3, necroptosis, and reactive oxygen species (ROS).. LAPC-4 cells were more responsive than PC-3 cells. Robust and sustained extracellular-regulated kinase activation with E2 , but not DES, correlated with ROS generation and cell death. c-Jun N-terminal kinase was only activated in E2-treated PC-3 cells and was not correlated with caspase 3-mediated apoptosis; necroptosis was not involved. The cell-cycle inhibitor protein p16(INK4A) was phosphorylated in both cell lines by both E2 and DES, but to differing extents. In both cell types, both estrogens activated p38 kinase, which subsequently phosphorylated cyclin D1, tagging it for degradation, except in DES-treated PC-3 cells.. Cyclin D1 status correlated most closely with disrupted cell cycling as a cause of reduced cell numbers, though other mechanisms also contributed. As low as 0.1 nM E2 effectively elicited these mechanisms, and its use could dramatically improve outcomes for both early- and late-stage prostate cancer patients, while avoiding the side effects of high-dose DES treatment.

Topics: Apoptosis; Caspase 3; Cell Cycle Proteins; Cell Line, Tumor; Cell Survival; Cyclin D1; Diethylstilbestrol; Dose-Response Relationship, Drug; Estradiol; Estrogens; Humans; Male; Mitogen-Activated Protein Kinase Kinases; Neoplasm Staging; Prostatic Neoplasms; Reactive Oxygen Species

Prostate cancer, one of the most common cancers in the Western world, affects many men worldwide. This study investigated the effects of magnolol, a compound found in the roots and bark of the magnolia tree Magnolia officinalis, on the behavior of 2 androgen insensitive human prostate cancer cell lines, DU145 and PC3, in vitro. Magnolol, in a 24-h exposure at 40 and 80 μM, was found to be cytotoxic to cells. Magnolol also affected cell cycle progression of DU145 and PC3 cells, resulting in alterations to the cell cycle and subsequently decreasing the proportion of cells entering the G2/M-phase of the cell cycle. Magnolol inhibited the expression of cell cycle regulatory proteins including cyclins A, B1, D1, and E, as well as CDK2 and CDK4. Protein expression levels of pRBp107 decreased and pRBp130 protein expression levels increased in response to magnolol exposure, whereas p16(INK4a), p21, and p27 protein expression levels were apparently unchanged post 24-h exposure. Magnolol exposure at 6 h did increase p27 protein expression levels. This study has demonstrated that magnolol can alter the behavior of androgen insensitive human prostate cancer cells in vitro and suggests that magnolol may have potential as a novel anti-prostate cancer agent.

Topics: Antineoplastic Agents, Phytogenic; Biphenyl Compounds; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cyclin A; Cyclin B1; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p27; Humans; Lignans; Magnolia; Male; Plant Extracts; Prostatic Neoplasms

Formononetin is an O-methylated isoflavone isolated from the root of Astragalus membranaceus. It has already been reported that formononetin could inhibit cell proliferation and induce cell apoptosis in several cancers, including prostate cancer. This study aimed to further investigate whether cell cycle arrest is involved in formononetin-mediated antitumor effect in human prostate cancer cells, along with the underlying molecular mechanism.. Human prostate cancer cells PC-3 and DU145 were respectively treated with various concentrations of formononetin. The inhibitory effect of formononetin on proliferation of prostate cancer cells was determined using MTT assays and flow cytometry. Next, formononetin-induced alterations in cyclin D1, CDK4 and Akt expression in PC-3 cells were detected by real-time PCR and western blot.. Formononetin dose-dependently inhibited prostate cancer cell proliferation via the induction of cell cycle arrest at G0/G1 phase in vitro, which was more evident in PC-3 cells. Meanwhile, concomitant with reduced phosphorylation of Akt in PC-3 cells, formononetin remarkably downregulated expression levels of cyclin D1 and CDK4 in a dose-dependent manner. More interestingly, in the in vivo studies, formononetin showed a noticeable inhibition of tumor growth in recipient mice.. Formononetin could exhibit inhibitory activity against human prostate cancer cells in vivo and in vitro, which is associated with G1 cell cycle arrest by inactivation of Akt/cyclin D1/CDK4. Therefore, formononetin may be used as a candidate agent for clinical treatment of prostate cancer in the future.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Down-Regulation; G1 Phase Cell Cycle Checkpoints; Humans; Isoflavones; Male; Mice; Mice, Nude; Phosphorylation; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Resting Phase, Cell Cycle

In the present study, the main natural estrogen-agonist/antagonist from Pigeonpea roots was studied by the estrogen receptor α-dependent signaling pathway in human prostate cancer cell. First, the natural products with estrogenic activity in Pigeonpea roots were screened by pER8-GFP transgenic Arabidopsis, and cajanol (5-hydroxy-3-(4-hydroxy-2-methoxyphenyl)-7-methoxychroman-4-one) was confirmed as the active compound. Further study showed that cajanol significantly arrested the cell cycle in the G1 and G2/M phase and induced nuclei condensation, fragmentation and the formation of apoptotic bodies. Western blotting showed that cajanol modulated the ERα-dependent PI3K pathway and induced the activation of GSK3 and CyclinD1 closely following the profile of PI3K activity. Based on above results, we proposed a mechanism through which cajanol could inhibit survival and proliferation of estrogen-responsive cells (PC-3 cells) by interfering with an ERα-associated PI3K pathway, following a process that could be dependent of the nuclear functions of the ERα. Above all, we conclude that cajanol represents a valuable natural phytoestrogen source and may potentially be applicable in health food industry.

Topics: Arabidopsis; Cajanus; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Estrogen Receptor alpha; Glycogen Synthase Kinase 3; Humans; Isoflavones; Male; Phosphatidylinositol 3-Kinases; Phytoestrogens; Plant Roots; Plants, Genetically Modified; Prostatic Neoplasms; Signal Transduction

To explore the mechanism and synergistic effect of vitamin A and vitamin D in inducing apoptosis of prostate cancer cells. The cell proliferation activity was determined by MTT assay. The proportion of apoptotic cells was analyzed by FACS and fluorescence intensity. TUNEL was used to evaluate vitamin A and vitamin D's induction of apoptosis in prostate cancer cells. The protein and mRNA expression level of Cyclin D1 and Bax were determined by real time-PCR and western blot. The results of MTT showed vitamin A and vitamin D's inhibition on proliferation ratio in prostate cancer cells is time and concentration dependent. FACS and fluorescence intensity analysis proved that the proportion of apoptotic cells increased after vitamin A and vitamin D treatment. TUNEL showed vitamin A and vitamin D induced prostate cancer cells apoptosis. The combination of vitamin A and vitamin D markedly enhanced the expression of Bax and reduced the expression of Cyclin D1 by real time-PCR and western blot assay. In conclusion, vitamin A and vitamin D could synergistically induce apoptosis in prostate cancer cells.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Cell Division; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Drug Synergism; Gene Expression Regulation, Neoplastic; Humans; Male; Membrane Potential, Mitochondrial; Prostatic Neoplasms; Vitamin A; Vitamin D

Ginkgetin is a natural biflavonoid isolated from leaves of Ginkgo biloba L. Though it was known to have anti-inflammatory, anti-influenza virus, anti-fungal activity, osteoblast differentiation stimulating activity and neuro-protective effects, the underlying antitumor mechanism of ginkgetin still remains unclear. Thus, in the present study, anti-cancer mechanism of ginkgetin was elucidated in human prostate cancer PC-3 cells. Ginkgetin suppressed the viability of PC-3 cells in a concentration-dependent manner and also significantly increased the sub-G1 DNA contents of cell cycle in PC-3 cells. Ginkgetin activated caspase-3 and attenuated the expression of survival genes such as Bcl-2, Bcl-xL, survivin and Cyclin D1 at protein and mRNA levels. Consistently, pan-caspase inhibitor Z-DEVD-fmk blocked sub G1 accumulation and cleavages of PRAP and caspase 3 induced by ginkgetin in PC-3 cells. Overall, these findings suggest that ginkgetin induces apoptosis in PC-3 cells via activation of caspase 3 and inhibition of survival genes as a potent chemotherapeutic agent for prostate cancer treatment.

Topics: Antineoplastic Agents; Apoptosis; bcl-X Protein; Biflavonoids; Caspases; Cell Line, Tumor; Cyclin D1; G1 Phase Cell Cycle Checkpoints; Ginkgo biloba; Humans; Inhibitor of Apoptosis Proteins; Male; Oligopeptides; Plant Leaves; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Survivin

SUMOylation plays an important role in regulating a wide range of cellular processes. Previously, we showed that ATF3, a stress response mediator, can be SUMOylated and lysine 42 is the major SUMO site. However, the significance of ATF3 SUMOylation in biological processes is still poorly understood. In the present study, we investigated the role of ATF3 SUMOylation on CCND activity and cellular proliferation in human prostate cancer cells. First, we showed that ATF3 can be SUMOylated endogenously in the overexpression system, and lysine 42 is the major SUMO site. Unlike normal prostate tissue and androgen-responsive LNCaP cancer cells, androgen-independent PC3 and DU145 cancer cells did not express ATF3 endogenously. Overexpression of ATF3 increased CCND1/2 expression in PC3 and DU145 cancer cells. Interestingly, we observed that SUMOylation is essential for ATF3-mediated CCND1/2 activation. Finally, we observed that SUMOylation plays a functional role in ATF3-mediated cellular proliferation in PC3 and DU145 cells. Taken together, our results demonstrate that SUMO modification of ATF3 influences CCND1/2 activity and cellular proliferation of prostate cancer PC3 and DU145 cells and explains at least in part how ATF3 functions to regulate cancer development.

Topics: Activating Transcription Factor 3; Amino Acid Substitution; Androgens; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin D2; Humans; Lysine; Male; Mutagenesis, Site-Directed; Neoplasms, Hormone-Dependent; Prostate; Prostatic Neoplasms; Recombinant Proteins; RNA, Messenger; RNA, Neoplasm; Sumoylation; Transfection

Prostate cancer (PCa) is the second leading cause of cancer-related deaths in men. Studies show that consumption of polyunsaturated fatty acids (PUFA) modulates the development and progression of prostate cancer. High amounts of omega-6 fatty acids have been linked with increased prostate cancer risk, whereas omega-3 fatty acids have been shown to inhibit PCa growth. However, because omega-3 and omega-6 are both essential fatty acids and part of a complete diet, it is more relevant to determine the ideal ratio of the two that would allow patients to benefit from the therapeutic properties of omega-3 fatty acids. LNCaP prostate cancer cells were treated with dietary-based ratios of omega-6 to omega-3 fatty acids under hormone-deprivation conditions, and effects on various cellular processes were determined. A low omega-6 to omega-3 PUFA ratio can delay the progression of cells toward castration-resistance by suppressing pathways involved in prostate cancer progression, such as the Akt/mTOR/NFκB axis. It also suppresses the expression of cyclin D1, and activation of caspase-3 and annexin V staining shows induction of proapoptotic events. Taken together, our data demonstrates that maintaining a low omega-6 to omega-3 fatty acids ratio can enhance efficacy of hormone ablation therapy.

Topics: Apoptosis; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Fatty Acids, Omega-3; Fatty Acids, Omega-6; Humans; Male; NF-kappa B; Phosphatidylinositol 3-Kinases; Prostatic Neoplasms; Prostatic Neoplasms, Castration-Resistant; Signal Transduction; TOR Serine-Threonine Kinases

Previous studies have demonstrated that fibroblast growth factor 8b (FGF8b) is up-regulated in a large proportion of prostate cancer patients and that it plays a key role in prostate carcinogenesis. In this study, we designed and synthesized a gN helix domain derived short peptide (termed 8b-13) based on the analysis of the FGF8b-FGFR structure. The synthetic peptides inhibited the proliferation of prostate cancer cell lines, including PC-3 and DU-145 cells. Further investigations indicated that 8b-13 arrested the cell cycle at the G0/G1 phase, reduced the activation of the Erk1/2, P38, and Akt cascades, and down-regulated the expression of G1/S-specific cyclinD1. The suppression of DNA synthesis and the G1 to S phase transition due to the expression of proteins related to proliferation and cell cycle progression may contribute to the inhibitory effect of 8b-13 peptides on cellular proliferation. Our results not only suggest that 8b-13 exerts an antitumor effect in prostate cancer but also confirm the essential role of the gN helix domain in mediating the activity of FGF8b.

Topics: 1-Alkyl-2-acetylglycerophosphocholine Esterase; Amino Acid Sequence; Carboxylesterase; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Fibroblast Growth Factor 8; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Male; Microtubule-Associated Proteins; Mitogen-Activated Protein Kinases; Peptides; Proliferating Cell Nuclear Antigen; Prostatic Neoplasms; Protein Interaction Domains and Motifs; Proteomics; Proto-Oncogene Proteins c-akt; Receptors, Retinoic Acid; Reproducibility of Results; Signal Transduction

Integrin α7 (ITGA7) is a tumor-suppressor gene that is critical for suppressing the growth of malignant tumors; however, the mechanisms allowing ITGA7 to suppress the growth of cancer cells remain unclear. Herein, we show that ITGA7 binds to tissue inhibitor of metalloproteinase 3 (TIMP3) in prostate cancer cells. The ITGA7-TIMP3 binding led to a decreased protein level of tumor necrosis factor α, cytoplasmic translocation of NF-κB, and down-regulation of cyclin D1. These changes led to an accumulation of cells in G0/G1 and a dramatic suppression of cell growth. Knocking down TIMP3 or ITGA7/TIMP3 binding interference largely abrogated the signaling changes induced by ITGA7, whereas a mutant ITGA7 lacking TIMP3 binding activity had no tumor-suppressor activity. Interestingly, knocking down ITGA7 ligand laminin β1 enhanced ITGA7-TIMP3 signaling and the downstream tumor-suppressor activity, suggesting the existence of a counterbalancing role between extracellular matrix and integrin signaling. As a result, this report demonstrates a novel and critical signaling mechanism of ITGA7, through the TIMP3/NF-κB/cyclin D1 pathway.

Topics: Amino Acid Motifs; Antigens, CD; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Humans; Integrin alpha Chains; Laminin; Male; Prostatic Neoplasms; Protein Binding; Tissue Inhibitor of Metalloproteinase-3; Transcription Factor RelA; Tumor Necrosis Factor-alpha

We previously identified SMIP004 (N-(4-butyl-2-methyl-phenyl) acetamide) as a novel inducer of cancer-cell selective apoptosis of human prostate cancer cells. SMIP004 decreased the levels of positive cell cycle regulators, upregulated cyclin-dependent kinase inhibitors, and resulted in G1 arrest, inhibition of colony formation in soft agar, and cell death. However, the mechanism of SMIP004-induced cancer cell selective apoptosis remained unknown. Here, we used chemical genomic and proteomic profiling to unravel a SMIP004-induced pro-apoptotic pathway, which initiates with disruption of mitochondrial respiration leading to oxidative stress. This, in turn, activates two pathways, one eliciting cell cycle arrest by rapidly targeting cyclin D1 for proteasomal degradation and driving the transcriptional downregulation of the androgen receptor, and a second pathway that activates pro-apoptotic signaling through MAPK activation downstream of the unfolded protein response (UPR). SMIP004 potently inhibits the growth of prostate and breast cancer xenografts in mice. Our data suggest that SMIP004, by inducing mitochondrial ROS formation, targets specific sensitivities of prostate cancer cells to redox and bioenergetic imbalances that can be exploited in cancer therapy.

Topics: Acetamides; Animals; Apoptosis; Cell Line, Tumor; Cyclin D1; Gene Expression; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Mitochondria; Oxidative Stress; Prostatic Neoplasms; Proteasome Endopeptidase Complex; Random Allocation; Signal Transduction; Ubiquitin; Unfolded Protein Response; Xenograft Model Antitumor Assays

Piperine, an alkaloid from black and long peppers (Piper nigrum Linn & Piper longum Linn), has been reported to exhibit antitumor activities in vitro and in vivo. To further understand the antitumor mechanism of piperine, we investigated the growth inhibitory effects of piperine on human prostate cancer DU145, PC-3 and LNCaP cells. Piperine treatment resulted in a dose-dependent inhibition of the proliferation of these cell lines. Cell cycle arrest at G₀/G₁ was induced and cyclin D1 and cyclin A were downregulated upon piperine treatment. Notably, the level of p21(Cip1) and p27(Kip1) was increased dose-dependently by piperine treatment in both LNCaP and DU145 but not in PC-3 cells, in line with more robust cell cycle arrest in the former two cell lines than the latter one. Although piperine induced low levels of apoptosis, it promoted autophagy as evidenced by the increased level of LC3B-II and the formation of LC3B puncta in LNCaP and PC-3 cells. The piperine-induced autophagic flux was further confirmed by assaying LC3-II accumulation and LC3B puncta formation in the presence of chloroquine, a well-known autophagy inhibitor. Taken together, these results indicated that piperine exhibited anti-proliferative effect in human prostate cancer cells by inducing cell cycle arrest and autophagy.

Topics: Alkaloids; Autophagy; Benzodioxoles; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin A1; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Dose-Response Relationship, Drug; Down-Regulation; G1 Phase; Humans; Male; Piperidines; Polyunsaturated Alkamides; Prostatic Neoplasms; Up-Regulation

Recent evidence suggests that oxidative stress contributes to the pathogenesis of prostate cancer. The present study focused on the effect of apocynin, an inhibitor of NADPH oxidase, on prostate carcinogenesis using the transgenic rat for adenocarcinoma of prostate (TRAP) model. There were no toxic effects with apocynin treatment. The percentages and numbers of carcinomas in both the ventral and lateral prostate were significantly reduced by apocynin treatment, with dose dependence. Reduction of reactive oxygen species by apocynin was confirmed by immunohistochemistry of 8-OHdG and dihydroethidium staining. Positivity of Ki67 was significantly reduced by apocynin treatment, and downregulation of clusterin expression, as well as inactivation of the MEK-ERK1/2 pathway, was a feature of the apocynin treated groups. In human prostate cancer cell line LNCaP, apocynin also inhibited reactive oxygen species production and blocked cell growth by inducing G0/G1 arrest with downregulation of clusterin and cyclin D1. These data suggest that apocynin possesses chemopreventive potential against prostate cancer.

Topics: Acetophenones; Adenocarcinoma; Animals; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Clusterin; Cyclin D1; Down-Regulation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; G1 Phase Cell Cycle Checkpoints; Humans; Ki-67 Antigen; Male; MAP Kinase Kinase Kinases; MAP Kinase Signaling System; NADPH Oxidases; Oxidative Stress; Prostate; Prostatic Neoplasms; Rats; Rats, Sprague-Dawley; Rats, Transgenic; Reactive Oxygen Species

Hyperthermia (HT) improves the efficacy of anti-cancer radiotherapy and chemotherapy. However, HT also inevitably evokes stress responses and increases the expression of heat-shock proteins (HSPs) in cancer cells. Among the HSPs, HSP70 is known as a pro-survival protein. In this study, we investigated the sensitizing effect of pifithrin (PFT)-μ, a small molecule inhibitor of HSP70, when three human prostate cancer cell lines (LNCaP, PC-3, and DU-145) were treated with HT (43°C for 2 h). All cell lines constitutively expressed HSP70, and HT further increased its expression in LNCaP and DU-145. Knockdown of HSP70 with RNA interference decreased the viability and colony-forming ability of cancer cells. PFT-μ decreased the viabilities of all cell lines at one-tenth the dose of Quercetin, a well-known HSP inhibitor. The combination therapy with suboptimal doses of PFT-μ and HT decreased the viability of cancer cells most effectively when PFT-μ was added immediately before HT, and this combination effect was abolished by pre-knockdown of HSP70, suggesting that the effect was mediated via HSP70 inhibition. The combination therapy induced cell death, partially caspase-dependent, and decreased proliferating cancer cells, with decreased expression of c-Myc and cyclin D1 and increased expression of p21(WAF1/Cip), indicating arrest of cell growth. Additionally, the combination therapy significantly decreased the colony-forming ability of cancer cells compared to therapy with either alone. Furthermore, in a xenograft mouse model, the combination therapy significantly inhibited PC-3 tumor growth. These findings suggest that PFT-μ can effectively enhance HT-induced antitumor effects via HSP70 inhibition by inducing cell death and arrest of cell growth, and that PFT-μ is a promising agent for use in combination with HT to treat prostate cancer.

Topics: Animals; Antineoplastic Agents; Cell Death; Cell Line, Tumor; Cell Survival; Cyclin D1; Gene Expression Regulation, Neoplastic; HSP70 Heat-Shock Proteins; Humans; Hyperthermia, Induced; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Prostatic Neoplasms; Proto-Oncogene Proteins c-myc; Xenograft Model Antitumor Assays

Scoparone, a natural compound isolated from Artemisia capillaris, has been used in Chinese herbal medicine to treat neonatal jaundice. Signal transducer and activator of transcription 3 (STAT3) contributes to the growth and survival of many human tumors. This study was undertaken to investigate the anti-tumor activity of scoparone against DU145 prostate cancer cells and to determine whether its effects are mediated by inhibition of STAT3 activity. Scoparone inhibited proliferation of DU145 cells via cell cycle arrest in G1 phase. Transient transfection assays showed that scoparone repressed both constitutive and IL-6-induced transcriptional activity of STAT3. Western blot and quantitative real-time PCR analyses demonstrated that scoparone suppressed the transcription of STAT3 target genes such as cyclin D1, c-Myc, survivin, Bcl-2, and Socs3. Consistent with this, scoparone decreased phosphorylation and nuclear accumulation of STAT3, but did not reduce phosphorylation of janus kinase 2 (JAK2) or Src, the major upstream kinases responsible for STAT3 activation. Moreover, transcriptional activity of a constitutively active mutant of STAT3 (STAT3C) was inhibited by scoparone, but not by AG490, a JAK2 inhibitor. Furthermore, scoparone treatment suppressed anchorage-independent growth in soft agar and tumor growth of DU145 xenografts in nude mice, concomitant with a reduction in STAT3 phosphorylation. Computational modeling suggested that scoparone might bind the SH2 domain of STAT3. Our findings suggest that scoparone elicits an anti-tumor effect against DU145 prostate cancer cells in part through inhibition of STAT3 activity.

Topics: Animals; Antineoplastic Agents; Artemisia; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Coumarins; Cyclin D1; G1 Phase; HCT116 Cells; HeLa Cells; Hep G2 Cells; HT29 Cells; Humans; Inhibitor of Apoptosis Proteins; Interleukin-6; Janus Kinase 2; Male; MCF-7 Cells; Mice; Mice, Inbred BALB C; Mice, Nude; Phosphorylation; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; Signal Transduction; STAT3 Transcription Factor; Suppressor of Cytokine Signaling 3 Protein; Suppressor of Cytokine Signaling Proteins; Survivin; Transcription, Genetic

To establish a system to study differentiation therapy drugs, we used the androgen-independent human prostate PC-3 tumor cell line as a target and α- and γ-tocopherol as inducers. Effects of α- and γ-tocopherol on the cell cycle, proliferation and differentiation, were examined. A more significant growth inhibition activity for γ- than for α-tocopherol was observed. Flow cytometry analysis of α- and γ-tocopherol-treated prostate carcinoma PC3 cells showed decreased progression into the S-phase. This effect, particularly evident for γ-tocopherol, was associated with an up-regulation and increased activity of transglutaminase 2 (TG2), a reduced DNA synthesis and a remarkable decreased levels of cyclin D1 and cyclin E. Activation of TG2 suggests that γ-tocopherol has an evident differentiative capacity on PC3 cells, leading to an increased expression of TG2, and reduced cyclin D1 and cyclin E levels, affecting cell cycle progression. It is feasible that up-regulation and activation of TG2, associated with a reduced proliferation, are parts of a large-scale reprogramming that can attenuate the malignant phenotype of PC3 cells in vitro. These data suggest further investigation on the potential use of this γ-form of vitamin E as a differentiative agent, in combination with the common cytotoxic treatments for prostate cancer therapy.

Topics: alpha-Tocopherol; Antineoplastic Agents; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin E; DNA Replication; Down-Regulation; Enzyme Induction; gamma-Tocopherol; GTP-Binding Proteins; Humans; Male; Prostatic Neoplasms; Protein Glutamine gamma Glutamyltransferase 2; Transglutaminases; Up-Regulation; Vitamins

Akt/mTOR/S6K1 signaling cascades play an important role both in the survival and proliferation of tumor cells.. In the present study, we investigated the effects of embelin (EB), identified primarily from the Embelia ribes plant, on the Akt/mTOR/S6K1 activation, associated gene products, cellular proliferation, and apoptosis in human prostate cancer cells.. EB exerted significant cytotoxic and suppressive effects on Akt and mTOR activation against androgen-independent PC-3 cells as compared to androgen-dependent LNCaP cells. Moreover, EB suppressed the constitutive activation of Akt/mTOR/S6K1 signaling cascade, which correlated with the induction of apoptosis as characterized by accumulation of cells in subG1 phase, positive Annexin V binding, down-regulation of anti-apoptotic (Bcl-2, Bcl-xL, survivin, IAP-1, and IAP-2) and proliferative (cyclin D1) proteins, activation of caspase-3, and cleavage of PARP. We also observed that EB can significantly enhance the apoptotic effects of a specific pharmacological Akt inhibitor when used in combination and also caused broad inhibition of all the three kinases in Akt/mTOR/S6K1 signaling axis in PC-3 cells.. EB inhibits multiple signaling cascades involved in tumorigenesis and can be used as a potential therapeutic candidate for both the prevention and treatment of prostate cancer.

Topics: Androgens; Apoptosis; Benzoquinones; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Dose-Response Relationship, Drug; Down-Regulation; Humans; In Vitro Techniques; Male; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Time Factors; TOR Serine-Threonine Kinases

The mechanisms responsible for the switch of prostate cancer from androgen-sensitive (AS) to androgen-insensitive (AI) form are not well understood. Regulation of androgen receptor (AR), through which androgens control the expression of genes involved in prostate cells proliferation, migration and death also involves its cross-talk with the other signaling pathways, transcription factors and coregulatory proteins, such as β-catenin. With the aim to determine their possible contribution in triggering the switch from AS to AI form, which occurs upon androgen deprivation therapy - AR, Akt and β-catenin expression were knocked-down with respective siRNAs. Treatment of LNCaP prostate cells with siRNA for AR significantly reduced their proliferation (45-70%), expression of nuclear β- catenin, cyclin-D1, cyclin-G1, c-Myc as well as activity of metalloproteinases (MMPs) -2,-7,-9 and cell migration. Surprisingly, after longer (over 72 hrs) silencing of AR in LNCaP cells, elevated levels of p-Akt were detected and enhanced proliferation as well as expression of nuclear β-catenin, cyclin-D1, c-Myc and activity of MMPs were observed. Such effects were not observed in either PC-3 or DU145 AI cells. However, silencing of Akt and /or β-catenin in those as well as in LNCaP cells led to their decreased proliferation and migration. Our findings suggest that in prostate cancer cells, either AR or Akt signaling prevails, depending on their initial androgen sensitivity and its availability. In AI prostate cancer cells, Akt takes over the role of AR and more effectively contributes through the same signaling molecule, β-catenin, to AI cancer progression.

Topics: Androgens; beta Catenin; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Male; Matrix Metalloproteinases, Secreted; Prostate; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; Receptors, Androgen; RNA Interference; RNA, Small Interfering; Signal Transduction

Estrogen receptor beta (ERβ) has been demonstrated to be expressed in prostate carcinoma cells and estrogen signals through ERβ to act as a tumor suppressor in prostate cancer patients. ERβ is thought to regulate the cell cycle of prostate carcinoma cells by controlling the expression of cell cycle regulators including cyclin D1 (CCND1). This interaction is of particular interest as CCND1 has been implicated in the development of prostate cancer.. We evaluated ERβ and CCND1 immunoreactivity in human prostate cancer (n = 112, surgical specimens), and correlated the findings with clinicopathological features of the patients. Subsequent in vitro experiments using PC-3 prostate carcinoma cells were also performed to examine whether estradiol (E2) could change the expression level of CCND1 mRNA.. CCND1 immunoreactivity was detected in 78/112 cases (70%), and was significantly correlated with incidence of perineural invasion and ERβ immunoreactivity (P < 0.05). Forty-eight hours incubation with E2 (10 nM) increased the expression level of CCND1 mRNA as well as c-jun (JUN) and c-fos (FOS) in PC-3 cells, and PHTPP (ERβ antagonist) suppressed E2 -induced expression of those mRNAs.. These findings suggest that CCND1 expression is possibly regulated by estrogens via ERβ and that this signaling pathway may influence prostate cancer development.

Topics: Aged; Carcinoma; Cell Line, Tumor; Cell Nucleus; Cyclin D1; Estrogen Receptor beta; Gene Expression Regulation, Neoplastic; Humans; Male; Middle Aged; Neoplasm Grading; Neoplasm Invasiveness; Neoplasm Staging; Prostatic Neoplasms; Signal Transduction

Accumulating evidence indicates that microRNAs play a pivotal role in the development and progression of prostate cancer. The present study was aimed at clarifying the biological functions of miR-153, one of the upregulated microRNAs in prostate cancers, and the signaling transduction induced by miR-153.. miR-153 was identified to be overexpressed in prostate cancers. The probable biological function of miR-153 in cellular proliferation was then examined by diverse assays, such as MTT, colony formation and BrdUrd incorporation assay. Moreover, real-time PCR and western blotting analysis were applied to investigate the underlying molecular mechanism induced by miR-153. Luciferase assays were used to determined the FOXO1 transactivity and the direct regulation of PTEN-3'-UTR by miR-153.. High-throughput method identified miR-153 to be upregulated in prostate cancers, which is further confirmed by the upregulated expression in four paired prostate tumor/adjacent non-cancerous tissues from the same patients. Further studies revealed that overexpression of miR-153 promoted cell cycle transition and cell proliferation, while inhibition of miR-153 reduced this effect. Moreover, miR-153 overexpression in prostate cancer cells increased the G1/S transitional promoter, cyclin D1 expression, and decreased cyclin-dependent kinase (CDK) inhibitor, p21(Cip1) expression. In addition, we demonstrated that miR-153 directly targeted the PTEN tumor suppressor gene, activated the AKT signaling and downregulated FOXO1 transcriptional activity.. Taken together, our results suggest that miR-153 play an important role in promoting proliferation of human prostate cancer cells and present a novel mechanism of microRNA-mediated direct suppression of PTEN expression in prostate cancer cells.

Topics: 3' Untranslated Regions; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Forkhead Box Protein O1; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Humans; Male; MicroRNAs; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Signal Transduction; Transcriptional Activation; Up-Regulation

Cyclin D1b is a splice variant of the cell cycle regulator cyclin D1 and is known to harbor divergent and highly oncogenic functions in human cancer. While cyclin D1b is induced during disease progression in many cancer types, the mechanisms underlying cyclin D1b function remain poorly understood. Herein, cell and human tumor xenograft models of prostate cancer were utilized to resolve the downstream pathways that are required for the protumorigenic functions of cyclin D1b. Specifically, cyclin D1b was found to modulate the expression of a large transcriptional network that cooperates with androgen receptor (AR) signaling to enhance tumor cell growth and invasive potential. Notably, cyclin D1b promoted AR-dependent activation of genes associated with metastatic phenotypes. Further exploration determined that transcriptional induction of SNAI2 (Slug) was essential for cyclin D1b-mediated proliferative and invasive properties, implicating Slug as a critical driver of disease progression. Importantly, cyclin D1b expression highly correlated with that of Slug in clinical samples of advanced disease. In vivo analyses provided strong evidence that Slug enhances both tumor growth and metastatic phenotypes. Collectively, these findings reveal the underpinning mechanisms behind the protumorigenic functions of cyclin D1b and demonstrate that the convergence of the cyclin D1b/AR and Slug pathways results in the activation of processes critical for the promotion of lethal tumor phenotypes.

Topics: Alternative Splicing; Animals; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Prostatic Neoplasms; Receptors, Androgen; Signal Transduction; Snail Family Transcription Factors; Transcription Factors; Transcriptional Activation; Transplantation, Heterologous

Transferrin (Tf) conjugates of monomeric artemisinin (ART) and artemisinin dimer were synthesized. The two conjugates, ART-Tf and dimer-Tf, retained the original protein structure, and formed stable aggregates in aqueous buffer. ART-Tf induced declines in proteins involved in apoptosis (survivin), cell cycling (cyclin D1), oncogenesis (c-myelocytomatosis oncogene product (c-MYC)), and dysregulated WNT signaling (beta-catenin) in both the human prostate (DU145) and breast (MCF7) cancer cell lines. Both ART-Tf and dimer-Tf induced down-regulation of survivin, c-MYC and mutated human epidermal growth factor receptor-2 (ERBB2 or HER2) in the BT474 breast cancer cell line. To our knowledge, this is the first demonstration that an ART derivative can cause a decline of ERBB2 in a human cancer cell line. Potential mechanisms for the observed effects are presented. Both transferrin conjugates strongly inhibited the growth of BT474 cells in the same concentration range that the conjugates caused declines in the levels of ERBB2, survivin, and c-MYC, while showing essentially no toxicity towards MCF10A normal breast cells.

Topics: Apoptosis; Artemisinins; beta Catenin; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Humans; Inhibitor of Apoptosis Proteins; Male; Prostatic Neoplasms; Protein Multimerization; Proto-Oncogene Proteins c-myc; Receptor, ErbB-2; Survivin; Transferrin; Wnt Signaling Pathway

Two isoforms of sphingosine kinase, SK1 and SK2, catalyze the formation of the bioactive lipid sphingosine 1-phosphate (S1P) in mammalian cells. We have previously shown that treatment of androgen-sensitive LNCaP prostate cancer cells with a non-selective SK isoform inhibitor, 2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole (SKi), induces the proteasomal degradation of SK1. This is concomitant with a significant increase in C22:0-ceramide and sphingosine levels and a reduction in S1P levels, resulting in the apoptosis of LNCaP cells. In contrast, we show here that a SK2-selective inhibitor, (R)-FTY720 methyl ether (ROME), increases sphingosine and decreases S1P levels but has no effect on ceramide levels and does not induce apoptosis in LNCaP cells. We also show that several glycolytic metabolites and (R)-S-lactoylglutathione are increased upon treatment of LNCaP cells with SKi, which induces the proteasomal degradation of c-Myc. These changes reflect an indirect antagonism of the Warburg effect. LNCaP cells also respond to SKi by diverting glucose 6-phosphate into the pentose phosphate pathway to provide NADPH, which serves as an antioxidant to counter an oxidative stress response. SKi also promotes the formation of a novel pro-apoptotic molecule called diadenosine 5',5'''-P(1),P(3)-triphosphate (Ap3A), which binds to the tumor suppressor fragile histidine triad protein (FHIT). In contrast, the SK2-selective inhibitor, ROME, induces a reduction in some glycolytic metabolites and does not affect oxidative stress. We conclude that SK1 functions to increase the stability of c-Myc and suppresses Ap3A formation, which might maintain the Warburg effect and cell survival, while SK2 exhibits a non-overlapping function.

Topics: Cell Line, Tumor; Ceramides; Cyclin D1; Dinucleoside Phosphates; Enzyme Inhibitors; Glycolysis; Humans; Lysophospholipids; Male; Metabolome; Microtubule-Associated Proteins; Oxidative Stress; Phosphotransferases (Alcohol Group Acceptor); Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Proto-Oncogene Proteins c-myc; Sphingosine

Cyclin D1 (CCND1) is an important cell-cycle regulator involved in carcinogenesis and progression of prostate cancer. We tested whether genetic variations within the CCND1 gene are related to clinical outcomes in prostate cancer patients receiving radical prostatectomy.. A total of 320 clinical localized prostate cancer patients who underwent radical prostatectomy in Taiwan were prospectively follow-up in this study. A total of 5 tagged single-nucleotide polymorphisms that captured the genetic variability across the CCND1 gene were genotyped, and the prognostic significance on prostate-specific antigen (PSA) recurrence was assessed using the Kaplan-Meier analysis and Cox regression model.. We found a polymorphism, rs9344, and 2 haplotypes, GAGG and CTGG, consisting of rs667515, rs2450254, rs9344, and rs678653, were associated with PSA recurrence (P ≤ 0.033). After adjusting for other clinicopathologic predictors, including age, PSA levels, pathologic stage, Gleason score, and surgical margin, rs9344 and the haplotype CTGG remained significant (P ≤ 0.044). The model based on clinical variables plus CCND1 rs9344 or haplotype showed improvement over the model without genetic information, as indicated by ≥ 7.2 % net reclassification improvement (P ≤ 0.040), integrated discrimination index (P ≤ 0.041), and likelihood ratio test (P ≤ 0.028).. Our data suggest that the CCND1 rs9344 and a specific haplotype CTGG may be prognostic factors for PSA recurrence after radical prostatectomy.

Topics: Aged; Biomarkers, Tumor; Cyclin D1; Follow-Up Studies; Humans; Male; Neoplasm Grading; Neoplasm Recurrence, Local; Neoplasm Staging; Polymorphism, Single Nucleotide; Prognosis; Prostate-Specific Antigen; Prostatectomy; Prostatic Neoplasms

Prostate cancer (CaP) is a heterogeneous, multifactorial, and multifocal disease. Therefore, the search for a combination of functional polymorphisms using cell cycle and apoptotic genes as tumor markers is fundamental for a more precise and reliable diagnosis. In the present study, we investigated the diagnostic value of 3 different genes associated with CaP carcinogenesis, encoding for cell cycle (MDM2, CCND1) and apoptotic (Fas) genes that are differentially expressed in CaP.. In a hospital-based case control study of northern India, blood samples were obtained from 192 CaP patients and 224 cancer-free age matched unrelated healthy controls of similar ethnicity. They were genotyped for MDM2 G309T, CCND1 G870A, Fas A670G, and G1377A polymorphisms using polymerase chain restriction fragment length polymorphism (PCR-RFLP) method.. MDM2 309GG variant was at reduced risk for developing CaP (P = 0.041; OR, 0.59). Whereas CCND1 AA genotype demonstrated increased risk (P = 0.018; OR, 1.86). The diplotype analysis of Fas G670A and G1377A (G-A) was observed to be associated with a significant increase in CaP risk (P = 0.024; OR, 1.63).. Findings based on current sample size our results suggested a positive association of CCND1AA genotype and diplotype analysis of Fas G670A and G1377A (G-A) to be associated with CaP risk that could influence the pathophysiology, thereby modulating the risk of CaP.

Topics: Aged; Alleles; Case-Control Studies; Cyclin D1; fas Receptor; Gene Frequency; Genetic Predisposition to Disease; Genotype; Haplotypes; Humans; Logistic Models; Male; Middle Aged; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Polymorphism, Single Nucleotide; Prostate-Specific Antigen; Prostatic Neoplasms; Proto-Oncogene Proteins c-mdm2; Risk Factors

The gastrin-releasing peptide receptor (GRPR) has emerged as an attractive target for both therapeutic and diagnostic appliances, but has only insufficiently been characterized in the human prostate so far. The aim of this study is to profile GRPR in a large cohort and correlate it with clinicopathologic and molecular parameters.. Benign and malignant (primary carcinoma, metastases, and castration-resistant prostate cancer) prostate samples from 530 patients were analyzed immunohistochemically for GRPR, androgen receptor and Cyclin D1 expression. Staining intensity was assessed assigning a semiquantitative score to each sample.. Normal prostate tissues were mostly GRPR negative, significantly higher expression rates were seen in primary carcinomas and metastases. Significant inverse correlations were found for GRPR and increasing Gleason score, PSA value, and tumor size. A stratified Kaplan-Meyer analysis for GRPR and high AR expression shows a significant prognostic advantage for high GRPR expression, whereas GRPR expression alone shows no independent prognostic value. Highly significant correlations for GRPR, AR, and Cyclin D1 were found.. Our data show that GRPR is overexpressed in prostate cancer, particularly of lower grade and smaller size. These findings constitute a caveat for the use of GRPR as a target for diagnostic or therapeutic approaches to high grade or progressed prostate cancer.

Topics: Aged; Aged, 80 and over; Biomarkers, Tumor; Castration; Cyclin D1; Follow-Up Studies; Gene Expression Profiling; Humans; Kaplan-Meier Estimate; Male; Middle Aged; Neoplasm Metastasis; Prostate; Prostatectomy; Prostatic Neoplasms; Receptors, Androgen; Receptors, Bombesin

Prostate cancer (PCa) is a potentially curable disease when diagnosed in early stages and subsequently treated with radical prostatectomy (RP). However, a significant proportion of patients tend to relapse early, with the emergence of biochemical failure (BF) as an established precursor of progression to metastatic disease. Several candidate molecular markers have been studied in an effort to enhance the accuracy of existing predictive tools regarding the risk of BF after RP. We studied the immunohistochemical expression of p53, cyclooxygenase-2 (COX-2) and cyclin D1 in a cohort of 70 patients that underwent RP for early stage, hormone naïve PCa, with the aim of prospectively identifying any possible interrelations as well as correlations with known prognostic parameters such as Gleason score, pathological stage and time to prostate-specific antigen (PSA) relapse. We observed a significant (p = 0.003) prognostic role of p53, with high protein expression correlating with shorter time to BF (TTBF) in univariate analysis. Both p53 and COX-2 expression were directly associated with cyclin D1 expression (p = 0.055 and p = 0.050 respectively). High p53 expression was also found to be an independent prognostic factor (p = 0.023). Based on previous data and results provided by this study, p53 expression exerts an independent negative prognostic role in localized prostate cancer and could therefore be evaluated as a useful new molecular marker to be added in the set of known prognostic indicators of the disease. With respect to COX-2 and cyclin D1, further studies are required to elucidate their role in early prediction of PCa relapse after RP.

Topics: Cyclin D1; Cyclooxygenase 2; Follow-Up Studies; Humans; Immunoenzyme Techniques; Male; Neoplasm Grading; Neoplasm Recurrence, Local; Neoplasms, Hormone-Dependent; Prognosis; Prospective Studies; Prostate-Specific Antigen; Prostatectomy; Prostatic Neoplasms; Retrospective Studies; Survival Rate; Tumor Suppressor Protein p53

Non-steroidal anti-inflammatory drug-activated gene (NAG-1), a divergent member of the transforming growth factor-beta superfamily, has been implicated in many cellular processes, including inflammation, early bone formation, apoptosis, and tumorigenesis. Recent clinical studies suggests that a C to G single nucleotide polymorphism at position 6 (histidine to aspartic acid substitution, or H6D) of the NAG-1 protein is associated with lower human prostate cancer incidence. The objective of the current study is to investigate the activity of NAG-1 H6D variant in prostate cancer tumorigenesis in vivo.. Human prostate cancer DU145 cells expressing the H6D NAG-1 or wild-type (WT) NAG-1 were injected subcutaneously into nude mice and tumor growth was monitored. Serum and tumor samples were collected for subsequent analysis.. The H6D variant was more potent than the WT NAG-1 and inhibited tumor growth significantly compared to control mice. Mice with tumors expressing the WT NAG-1 have greater reduced both body weight and abdominal fat than mice with H6D variant tumors suggesting different activities of the WT NAG-1 and the H6D NAG-1. A significant reduction in adiponectin, leptin, and IGF-1 serum levels was observed in the tumor-bearing mice with a more profound reduction observed with expression of H6D variant. Cyclin D1 expression was suppressed in the tumors with a dramatic reduction observed in the tumor expressing the H6D variant.. Our data suggest that the H6D variant of NAG-1 inhibits prostate tumorigenesis by suppressing IGF-1 and cyclin D1 expression but likely additional mechanisms are operative.

Topics: Adiponectin; Alleles; Animals; Cell Line, Tumor; Cyclin D1; Growth Differentiation Factor 15; Humans; Insulin-Like Growth Factor I; Leptin; Male; Mice; Mice, Nude; Neoplasm Transplantation; Polymorphism, Single Nucleotide; Prostate; Prostatic Neoplasms; Transplantation, Heterologous

Hypoxia-inducible factor-1 (HIF-1) is a master regulator of the transcriptional response to oxygen deprivation and controls genes involved in glycolysis, angiogenesis, migration and invasion. Overexpression of HIF-1α has been demonstrated in many common human cancers.. Luciferase reporter gene assay under hypoxia and normoxia was used to demonstrate transcriptional inhibition of HIF-1 by P276-00. Detailed studies such as western blotting, reverse-transcriptase-PCR and immunofluorescence were carried out to elucidate its mechanism of action. Cytotoxic potential of P276-00 under normoxia and hypoxia was determined on prostate cancer cells using CCK-8 assay, and cell-cycle analysis was carried out using flow cytometry. Antiangiogenic activity of P276-00 was demonstrated by migration assay and tube-formation assay. Efficacy study of P276-00 was performed in a PC-3 xenograft model.. P276-00 inhibits transcriptional activation of HIF-1 under hypoxia. It suppressed hypoxia-mediated nuclear HIF-1α expression, as well as phosphorylation of Akt and 4E-BP1 and abrogated expression of HIF-1-inducible gene viz. vascular endothelial growth factor. Under hypoxia, P276-00 did not exhibit enhanced cytotoxic activity in prostate cancer cells but arrested them in the G2/M phase of the cell cycle. The tubular formation of human umbilical vein endothelial cells and migration of prostate cancer cells were also inhibited by P276-00 in vitro. In addition, it demonstrated significant in vivo efficacy in the PC-3 xenograft model.. Given its low toxicity profile, its demonstrated antitumor activity and its potential to inhibit the HIF-1 pathway, P276-00 should be considered as antiangiogenic chemotherapy for prostate cancer.

Topics: Animals; Antineoplastic Agents; Cell Hypoxia; Cell Line, Tumor; Cell Movement; Cell Nucleus; Cell Proliferation; Cyclin D1; Drug Synergism; Flavones; G2 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Genes, Reporter; Human Umbilical Vein Endothelial Cells; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Luciferases; Male; Mice; Mice, SCID; Prostatic Neoplasms; Proteasome Endopeptidase Complex; Protein Kinase Inhibitors; Proteolysis; Random Allocation; RNA, Small Interfering; Signal Transduction; Transcriptional Activation; Xenograft Model Antitumor Assays

L-Mimosine, an iron chelator and a prolyl 4-hydroxylase inhibitor, blocks many cancer cells at the late G1 phase. B-cell translocation gene 2 (Btg2) regulates the G1/S transition phases of the cell cycle. N-myc downstream regulated gene 1 (Ndrg1) is a differentiation-inducing gene upregulated by hypoxia. We evaluated the molecular mechanisms of L-mimosine on cell cycle modulation in PC-3 and LNCaP prostate carcinoma cells. The effect of L-mimosine on cell proliferation of prostate carcinoma cells was determined by the [3H]thymidine incorporation and flow cytometry assays. L-Mimosine arrested the cell cycle at the G1 phase in PC-3 cells and at the S phase in LNCaP cells, thus attenuating cell proliferation. Immunoblot assays indicated that hypoxia and L-mimosine stabilized hypoxia-inducible factor-1α (HIF-1α) and induced Btg2 and Ndrg1 protein expression, but downregulated protein levels of cyclin A in both PC-3 and LNCaP cells. L-Mimosine treatment decreased cyclin D1 protein in PC-3 cells, but not in LNCaP cells. Dimethyloxalylglycine, a pan-prolyl hydroxylase inhibitor, also induced Btg2 and Ndrg1 protein expression in LNCaP cells. The transient gene expression assay revealed that L-mimosine treatment or cotransfection with HIF-1α expression vector enhanced the promoter activities of Btg2 and Ndrg1 genes. Knockdown of HIF-1α attenuated the increasing protein levels of both Btg2 and Ndrg1 by hypoxia or L-mimosine in LNCaP cells. Our results indicated that hypoxia and L-mimosine modulated Btg2 and Ndrg1 at the transcriptional level, which is dependent on HIF-1α. L-Mimosine enhanced expression of Btg2 and Ndrg1, which attenuated cell proliferation of the PC-3 and LNCaP prostate carcinoma cells.

Topics: B-Lymphocytes; Carcinoma; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Immediate-Early Proteins; Intracellular Signaling Peptides and Proteins; Male; Mimosine; Organ Specificity; Procollagen-Proline Dioxygenase; Prostate; Prostatic Neoplasms; Signal Transduction; Transcriptional Activation; Transfection; Tumor Suppressor Proteins

Aberrant expression of cyclin D1, frequently observed in human malignant disorders, has been linked to the control of G(1)→S cell cycle phase transition and development and progression in carcinogenesis. Cyclin D1 level changes are partially controlled by GSK-3β-dependent phosphorylation at threonine-286 (Thr286), which targets cyclin D1 for ubiquitination and proteolytic degradation. In our continuing studies on the mechanism of prostate cancer prevention by resveratrol, focusing on the role of its recently discovered target protein, quinone reductase 2 (NQO2), we generated NQO2 knockdown CWR22Rv1 using short hairpin RNA (shRNA)-mediated gene silencing approach. We found that, compared with cells expressing NQO2 (shRNA08), NQO2 knockdown cells (shRNA25) displayed slower proliferation and G(1) phase cell accumulation. Immunoblot analyses revealed a significant decrease in phosphorylation of retinoblastoma Rb and cyclin D1 in shRNA25 compared with shRNA08. Moreover, shRNA25 cells showed a 37% decrease in chymotrypsin-like proteasome activity. An increase in AKT activity was also observed in shRNA25, supported by a ∼1.5-fold elevation in phosphorylation and ∼50% reduction/deactivation of GSK-3α/β at Ser21/9, which were accompanied by a decrease in phosphorylation of cyclin D1 at T286. NQO2 knockdown cells also showed attenuation of resveratrol-induced downregulation of cyclin D1. Our results indicate a hitherto unreported role of NQO2 in the control of AKT/GSK-3β/cyclin D1 and highlight the involvement of NQO2 in degradation of cyclin D1, as part of mechanism of chemoprevention by resveratrol.

Topics: Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3; Humans; Male; Phosphorylation; Prostatic Neoplasms; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins c-akt; Quinone Reductases; Resveratrol; Retinoblastoma Protein; RNA Interference; RNA, Small Interfering; Stilbenes

Antihormonal and chemotherapy are standard treatments for nonorgan-confined prostate cancer. The effectivity of these therapies is limited and the development of alternative approaches is necessary. In the present study, we report on the use of the multikinase inhibitor sorafenib in a panel of prostate cancer cell lines and their derivatives which mimic endocrine and chemotherapy resistance. (3)H-thymidine incorporation assays revealed that sorafenib causes a dose-dependent inhibition of proliferation of all cell lines associated with downregulation of cyclin-dependent kinase 2 and cyclin D1 expression. Apoptosis was induced at 2  μM of sorafenib in androgen-sensitive cells, whereas a higher dose of the drug was needed in castration-resistant cell lines. Sorafenib stimulated apoptosis in prostate cancer cell lines through downregulation of myeloid cell leukemia-1 (MCL-1) expression and Akt phosphorylation. Although concentrations of sorafenib required for the antitumor effect in therapy-resistant sublines were higher than those needed in parental cells, the drug showed efficacy in cells which became resistant to bicalutamide and docetaxel respectively. Most interestingly, we show that sorafenib has an inhibitory effect on androgen receptor (AR) and prostate-specific antigen expression. In cells in which AR expression was downregulated by short interfering RNA, the treatment with sorafenib increased apoptosis in an additive manner. In summary, the results of the present study indicate that there is a potential to use sorafenib in prostate cancers as an adjuvant therapy option to current androgen ablation treatments, but also in progressed prostate cancers that become unresponsive to standard therapies.

Topics: Androgen Receptor Antagonists; Antineoplastic Agents; Apoptosis; Benzenesulfonates; Caspase 3; Caspase 7; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 2; Down-Regulation; Humans; Male; Myeloid Cell Leukemia Sequence 1 Protein; Niacinamide; Phenylurea Compounds; Prostate-Specific Antigen; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Pyridines; Receptors, Androgen; RNA, Small Interfering; Signal Transduction; Sorafenib

Prostate cancer is one of the leading causes of cancer death among men worldwide. In this study, using transgenic adenocarcinoma of mouse prostate (TRAMP) mice, the effect of diet enriched with 1% w/w ursolic acid (UA) was investigated to evaluate the stage specific chemopreventive activity against prostate cancer. We found that TRAMP mice fed with UA diet for 8 weeks (weeks 4 to 12) delayed formation of prostate intraepithelial neoplasia (PIN). Similarly, mice fed with UA diet for 6 weeks (weeks 12 to 18) inhibited progression of PIN to adenocarcinoma as determined by hematoxylin and eosin staining. Finally, TRAMP mice fed with UA diet for 12 weeks (weeks 24 to 36) demonstrated markedly reduced tumor growth without any significant effects on total body weight and prolonged overall survival. With respect to the molecular mechanism, we found that UA down-regulated activation of various pro-inflammatory mediators including, NF-κB, STAT3, AKT and IKKα/β phosphorylation in the dorsolateral prostate (DLP) tissues that correlated with the reduction in serum levels of TNF-α and IL-6. In addition, UA significantly down-regulated the expression levels of cyclin D1 and COX-2 but up-regulated the levels of caspase-3 as revealed by immunohistochemical analysis of tumor tissue sections. Finally, UA was detected in serum samples obtained from various mice groups fed with enriched diet in nanogram quantity indicating that it is well absorbed in the GI tract. Overall, our findings provide strong evidence that UA can be an excellent agent for both the prevention and treatment of prostate cancer.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents, Phytogenic; Blotting, Western; Caspase 3; Cyclin D1; Cyclooxygenase 2; DNA Primers; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation, Neoplastic; Immunohistochemistry; Kaplan-Meier Estimate; Male; Mice; Mice, Transgenic; Prostatic Neoplasms; Signal Transduction; Spectrophotometry; Survival Analysis; Triterpenes; Ursolic Acid

Myb, a cellular progenitor of v-Myb oncogenes, is amplified in prostate cancer and exhibits greater amplification frequency in hormone-refractory disease. Here, we have investigated the functional significance of Myb in prostate cancer. Our studies demonstrate Myb expression in all prostate cancer cell lines (LNCaP, C4-2, PC3 and DU145) examined, whereas it is negligibly expressed in normal/benign prostate epithelial cells (RWPE1 and RWPE2). Notably, Myb is significantly upregulated, both at transcript (>60-fold) and protein (>15-fold) levels, in castration-resistant (C4-2) cells as compared with androgen-dependent (LNCaP) prostate cancer cells of the same genotypic lineage. Using loss and gain of function approaches, we demonstrate that Myb promotes and sustains cell cycle progression and survival under androgen-supplemented and -deprived conditions, respectively, through induction of cyclins (A1, D1 and E1), Bcl-xL and Bcl2 and downregulation of p27 and Bax. Interestingly, Myb overexpression is also associated with enhanced prostate-specific antigen expression. Furthermore, our data show a role of Myb in enhanced motility and invasion and decreased homotypic interactions of prostate cancer cells. Myb overexpression is also associated with actin reorganization leading to the formation of filopodia-like cellular protrusions. Immunoblot analyses demonstrate gain of mesenchymal and loss of epithelial markers and vice versa, in Myb-overexpressing LNCaP and -silenced C4-2 cells, respectively, indicating a role of Myb in epithelial to mesenchymal transition. Altogether, our studies provide first experimental evidence for a functional role of Myb in growth and malignant behavior of prostate cancer cells and suggest a novel mechanism for castration resistance.

Topics: Androgens; Apoptosis; bcl-2-Associated X Protein; bcl-Associated Death Protein; bcl-X Protein; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin A1; Cyclin D1; Cyclin E; Epithelial-Mesenchymal Transition; Humans; Male; Neoplasm Invasiveness; Oncogene Proteins; Oncogene Proteins v-myb; Orchiectomy; Proliferating Cell Nuclear Antigen; Prostate; Prostate-Specific Antigen; Prostatic Neoplasms; Pseudopodia; RNA Interference; RNA, Small Interfering; Transcription, Genetic; Up-Regulation

Anterior-gradient 2 (AGR2), overexpressed in many tumors including prostate cancer (PCa), is implicated in stimulation of cell proliferation, adhesion, anti-apoptosis and cell cycle regulation. Here, a potential role of AGR2 in cellular senescence was investigated. We first observed that AGR2 was overexpressed in Chinese Han PCa tissues and had a positive correlation with cyclin D1 and p-Rb but not with p16(INK4a). AGR2 expression profiles varied among cell lines, with PC3 cells being the highest level, LNCaP and DU145 relatively less. The expression of cyclin D1 showed similar pattern to the AGR2 in cell lines. Knockdown of AGR2 caused a decrease in cell viability in PC3 cells, whereas forced expression of AGR2 led to an increased cell proliferation of LNCaP and DU145 cells. Importantly, AGR2 depletion resulted in accumulation of cells at the G(0)/G(1) phase and induction of cellular senescence in all three PCa cell lines as indicated by an increase of flat, enlarged and senescence-associated β-galactosidase (SA-β-Gal) positive cells. Senescent response to AGR2 silencing was also evidenced by elevated γH2AX and fluorescent punctuate formation of tri-methyl-histone H3 in AGR2-depleted cells. Further studies indicated that LNCaP underwent a p21(CIP1)-dependent cellular senescence in response to AGR2 depletion that requires inactivation of ERK signaling, whereas PC-3 was also p21(CIP1) dependent but involved in suppression of PI3K/Akt. Unlike LNCaP and PC-3, senescent response of DU145 was found to be mainly p27(KIP1) dependent that may require upregulation of PTEN and inhibition of PI3K/Akt signaling. Thus, these findings suggest a novel role of AGR2 in regulation of cellular senescence.

Topics: Aged; Aged, 80 and over; beta-Galactosidase; Cell Line, Tumor; Cell Proliferation; Cellular Senescence; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; G1 Phase Cell Cycle Checkpoints; Histones; Humans; Male; MAP Kinase Signaling System; Middle Aged; Mucoproteins; Oncogene Proteins; Phosphoinositide-3 Kinase Inhibitors; Prostate; Prostatic Neoplasms; Proteins; Proto-Oncogene Proteins c-akt; Retinoblastoma Protein; RNA Interference; RNA, Small Interfering

The growing incidence of prostate cancer and the traditional use of Rubus coreanus Miquel (RCM) for prostate health led us to compare RCM extracts and to test their efficacy in inhibiting the growth of prostate cancer cells differing in androgen dependency. Ethanol extracts of unripe RCM (EUR) were more effective in reducing cell viability than water extracts or ripe RCM. EUR-induced growth inhibition, as indicated by significant reductions in numbers of proliferating cells and decreases in the protein levels of proliferating cell nuclear antigen (PCNA), cyclin D1 and CDK4, was greater in the androgen-dependent LNCaP cells than in the androgen-independent DU145 cells. EUR also induced mitochondrial-mediated apoptosis in prostate cancer cells by reducing Bcl-2 and Bcl-(X)L levels, but increased Bax levels. Nevertheless, the LNCaP cells were more sensitive to EUR-induced apoptosis and displayed sub-G1 and late apoptotic cell populations, whereas the DU145 cells did not. Our findings suggest that EUR suppresses the growth of prostate cancer cells by anti-proliferative and/or pro-apoptotic effects, and that these effects are stronger in androgen-dependent cells.

Topics: Androgens; Antineoplastic Agents, Phytogenic; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Cyclin-Dependent Kinase 4; Ethanol; Fruit; Gene Expression Regulation, Neoplastic; Humans; Male; Mitochondria; Plant Extracts; Proliferating Cell Nuclear Antigen; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Rosaceae

Persistent activation of Survivin and its overexpression contribute to the formation, progression and metastasis of several different tumor types. Therefore, Survivin is an ideal target for RNA interference mediated-growth inhibition. Blockade of Survivin using specific short hairpin RNAs (shRNA) can significantly reduce prostate tumor growth. RNA interference does not fully ablate target gene expression, owing to the idiosyncrasies associated with shRNAs and their targets. To enhance the therapeutic efficacy of Survivin-specific shRNA, we employed a combinatorial expression of Survivin-specific shRNA and gene associated with retinoid-interferon-induced mortality-19 (GRIM-19). Then, the GRIM-19 coding sequences and Survivin-specific shRNAs were used to create a dual expression plasmid vector and were carried by an attenuated strain of Salmonella enteric serovar typhimurium (S. typhimurium) to treat prostate cancer in vitro and in vivo. We found that the co-expressed Survivin-specific shRNA and GRIM-19 synergistically and more effectively inhibited prostate tumor proliferation and survival, when compared with treatment with either single agent alone in vitro and in vivo. This study has provided a novel cancer gene therapeutic approach for prostate cancer.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; bcl-X Protein; Carcinoma; Caspase 3; Cell Line, Tumor; Cyclin D1; Gene Expression; Genetic Therapy; Humans; Inhibitor of Apoptosis Proteins; Ki-67 Antigen; Male; Mice; NADH, NADPH Oxidoreductases; Plasmids; Prostate; Prostatic Hyperplasia; Prostatic Neoplasms; Proto-Oncogene Proteins c-myc; RNA, Small Interfering; Salmonella typhimurium; STAT3 Transcription Factor; Survivin; Vascular Endothelial Growth Factor A

Cyclin D1 is an important cell cycle regulatory proteins, which is a functional target of Slug in the regulation of cell growth of prostate cancer cells. But the pathway of these two factors interacting with each other is unclear.. The infectde PCa Cells were treated with proteasome inhibitor MG-132. Expression level of Slug, HA-cyclin D1 and other protein was examined by Western blot.. Increasing doses of adenovirus expressing human Slug were added to DU-145 cells separately, but there were no significantly difference on expressions of Slug and cyclin D1. We found that the protein expressions of HA-Cyclin D1 (wide-type) were all reduced through high expression of Slug, which is dose-dependent. However, there is no change for HA-Cyclin D1 (mutant) expression in PC-3 with pMIGW-Cyclin D1-HA T286A. The protein expression of HA-Cyclin D1 were all reduced three days after infection by adding adenovirus expressing human Slug to PC-3 carrying pMIGW-Cyclin D1-HA vector compared to negative control, which is dose-dependent. However, there is no change for HA-Cyclin D1 expression in PC-3 with pMIGW-Cyclin D1-HA treated by MG-132.. We found that forced expression of Slug inhibited proliferation of prostate cancer cells through downregulation of cyclin D1 expression. And Slug regulates cyclin D1 expression by ubiquitin-proteasome pathway in PCa cells.

Topics: Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Dose-Response Relationship, Drug; Down-Regulation; Endopeptidases; Gene Expression Regulation, Neoplastic; Humans; Leupeptins; Male; Mutation; Prostatic Neoplasms; RNA, Messenger; Snail Family Transcription Factors; Transcription Factors; Ubiquitin; Ubiquitin-Specific Proteases

To study the effect of siRNA targeting ADAM17 (ADAM17-siRNA) on the proliferation of prostate cancer PC-3 cells.. After transfecting PC-3 cells with ADAM17-siRNA 1 and ADAM17-siRNA 2, we detected the expressions of ADAM17 mRNA and protein by RT- PCR and Western blotting, respectively. We measured the changes in the proliferation and DNA synthesis of PC-3 cells by MTT and bromodeoxyuridine (BrdU) incorporation assay, examined the cell cycle profile by flow cytometry, and determined the expressions of the genes associated with PC-3 cell proliferation by Western blotting.. Both ADAM17-siRNA 1 and 2 effectively reduced the expressions of ADAM17 mRNA and protein in the PC-3 cells. Knockdown of ADAM17 with the two siRNAs significantly inhibited cell proliferation as compared with the control group (0.43 +/- 0.57 and 0.44 +/- 0.64 vs 0.80 +/- 0.51, P < 0.05) and down-regulated DNA synthesis (0.48 +/- 0.43 and 0.54 +/- 0.59 vs 0.79 +/- 0.72, P < 0.05). The cell cycle profile showed that the cell population of the G1 phase was markedly higher in both the ADAM17-siRNA groups than in the control ([61.83 +/- 2.41]% and [59.78 +/- 1.92]% vs [41.38 +/- 1.53]%, P < 0.05), but that of the S phase remarkably lower in the former two than in the latter ([23.64 +/- 2.56]% and [25.24 +/- 1.86]% vs [33.51 +/- 1.47]%, P < 0.05), with a concomitant decrease in the expression of the cell cycle protein cyclin D1 and increase in the cyclin-dependent kinase inhibitor p21.. ADAM17-siRNA can effectively inhibit the proliferation of PC-3 cells by up-regulating cyclin D1 and down-regulating p21 protein, and ADAM17 has a potential value in the gene therapy of prostate cancer.

Topics: ADAM Proteins; ADAM17 Protein; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; Humans; Male; Prostatic Neoplasms; RNA Interference; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Transfection

Brassinosteroids (BRs) are a group of polyhydroxylated sterol derivatives with important regulatory roles in various plant physiological processes. The aim of this study was to examine the mechanism of the antiproliferative activity of natural BRs 28-homocastasterone (28-homoCS) and 24-epibrassinolide (24-epiBL) in hormone-sensitive and -insensitive (LNCaP and DU-145, respectively) human prostate cancer cell lines. The effects of BRs on prostate cancer cells were surveyed using flow cytometry, Western blotting, TUNEL, DNA ladder assays and immunofluorescence analyses. The studied BRs inhibited cell growth and induced G(1) blocks in LNCaP cells accompanied by reductions in cyclin D(1), CDK4/6 and pRb expression. Following BR treatment of DU-145 cells, increases in proportions of cells in the G(2)/M phase of cell cycle were observed, accompanied by down-regulation of cyclins A and B(1). Changes in AR localization patterns in LNCaP cells treated with BRs were shown by immunofluorescence analysis. Furthermore, apoptotic detection methods demonstrated induction of apoptosis mediated by BRs in both cell lines, although changes in the expression of apoptosis-related proteins were modulated differently by 28-homoCS and 24-piBL in each cell line. The studied BRs seem to exert potent growth inhibitory and pro-apoptotic effects and could be therefore highly valuable new candidates for prostate anticancer drugs.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Brassinosteroids; Cell Cycle; Cell Line, Tumor; Cholestanones; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Drug Screening Assays, Antitumor; Humans; Male; Prostatic Neoplasms; Receptors, Androgen; Receptors, Estrogen; Steroids, Heterocyclic

Endocannabinoids (ECs), anandamide (AEA) and 2-arachidonoylglycerol (2-AG), inhibit proliferation of carcinoma cells. Several enzymes hydrolyze ECs to reduce endogenous EC concentrations and produce eicosanoids that promote cell growth. In this study, we determined the effects of EC hydrolysis inhibitors and a putative EC, 2-arachidonylglyceryl ether (noladin ether, NE) on proliferation of prostate carcinoma (PC-3, DU-145, and LNCaP) cells. PC-3 cells had the least specific hydrolysis activity for AEA and administration of AEA effectively inhibited cell proliferation. The proliferation inhibition was blocked by SR141716A (a selective CB1R antagonist) but not SR144528 (a selective CB2R antagonist), suggesting a CB1R-mediated inhibition mechanism. On the other hand, specific hydrolysis activity for 2-AG was high and 2-AG inhibited proliferation only in the presence of EC hydrolysis inhibitors. NE inhibited proliferation in a concentration-dependent manner; however, SR141716A, SR144528 and pertussis toxin did not block the NE-inhibited proliferation, suggesting a CBR-independent mechanism of NE. A peroxisome proliferator-activated receptor gamma (PPARγ) antagonist GW9662 did not block the NE-inhibited proliferation, suggesting that PPARγ was not involved. NE also induced cell cycle arrest in G(0)/G(1) phase in PC-3 cells. NE inhibited the nuclear translocation of nuclear factor-kappa B (NF-κB p65) and down-regulated the expression of cyclin D1 and cyclin E in PC-3 cells, suggesting the NF-κB/cyclin D and cyclin E pathways are involved in the arrest of G1 cell cycle and inhibition of cell growth. These results indicate therapeutic potentials of EC hydrolysis inhibitors and the enzymatically stable NE in prostate cancer.

Topics: Anilides; Antineoplastic Agents; Cannabinoid Receptor Modulators; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin E; Endocannabinoids; Glycerides; Humans; Male; NF-kappa B; PPAR gamma; Prostate; Prostatic Neoplasms

Mitochondria are key organelles for ATP production and apoptosis. Therefore, impairment of mitochondria can modulate or accelerate cancer progression. p32, originally identified as a pre-mRNA splicing factor SF2/ASF-associated protein, is localized predominantly in the mitochondrial matrix and involved in mitochondria respiration. Recently, p32 was implicated in apoptosis and resultantly cancer progression. However, little is known about the expression and function of p32 in human tumors including prostate cancer. Here, we investigated the expression of p32 in 148 prostate carcinoma tissues by immunohistochemistry and found a positive correlation of p32 expression to clinicopathological parameters including follow-up data. p32 is highly expressed in prostate tumor samples and its expression is significantly associated with the Gleason score, pathological stage and relapse. For localized cancers, high p32 is a strong and independent predictor of clinical recurrence in multivariate analysis (P=0.01). In addition, p32 is overexpressed in the prostate cancer cell lines examined. The selective knockdown of p32 by RNA interference inhibits the growth of prostate cancer cell lines but not of a non-cancerous cell line. The p32 RNA interference decreases cyclin D1, increases p21 expression and causes a G1/S cell cycle arrest in prostate cancer cells. These data suggest that p32 is critical for prostate cancer cell proliferation and may be a novel marker of clinical progression in prostate cancer.

Topics: Aged; Biomarkers, Tumor; Carrier Proteins; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Humans; Male; Middle Aged; Mitochondria; Mitochondrial Proteins; Prostate-Specific Antigen; Prostatectomy; Prostatic Neoplasms; Recurrence; Time Factors

D-type cyclins regulate cellular outcomes in part through cyclin-dependent, kinase-independent mechanisms that modify transcription factor action, and recent in vivo studies showed that cyclin D1 associates with a large number of transcriptional regulators in cells of the retina and breast. Given the frequency of cyclin D1 alterations in cancer, it is imperative to delineate the molecular mechanisms by which cyclin D1 controls key transcription factor networks in human disease. Prostate cancer was used as a paradigm because this tumor type is reliant at all stages of the disease on androgen receptor (AR) signaling, and cyclin D1 has been shown to negatively modulate AR-dependent expression of prostate-specific antigen (KLK3/PSA). Strategies were employed to control cyclin D1 expression under conditions of hormone depletion, and the effect of cyclin D1 on subsequent androgen-dependent gene expression was determined using unbiased gene expression profiling. Modulating cyclin D1 conferred widespread effects on androgen signaling and revealed cyclin D1 to be a selective effector of hormone action. A subset of androgen-induced target genes, known to be directly regulated by AR, was strongly suppressed by cyclin D1. Analyses of AR occupancy at target gene regulatory loci of clinical relevance demonstrated that cyclin D1 limits AR residence after hormone stimulation. Together, these findings reveal a new function for cyclin D1 in controlling hormone-dependent transcriptional outcomes and demonstrate a pervasive role for cyclin D1 in regulating transcription factor dynamics.

Topics: Androgens; Cell Line, Tumor; Cyclin D1; Gene Expression Regulation, Neoplastic; Genetic Loci; Humans; Kallikreins; Male; Prostate-Specific Antigen; Prostatic Neoplasms; Receptors, Androgen; Signal Transduction

Effective clinical management of prostate cancer (PCA) has been challenged by significant intratumoural heterogeneity on the genomic and pathological levels and limited understanding of the genetic elements governing disease progression. Here, we exploited the experimental merits of the mouse to test the hypothesis that pathways constraining progression might be activated in indolent Pten-null mouse prostate tumours and that inactivation of such progression barriers in mice would engender a metastasis-prone condition. Comparative transcriptomic and canonical pathway analyses, followed by biochemical confirmation, of normal prostate epithelium versus poorly progressive Pten-null prostate cancers revealed robust activation of the TGFβ/BMP-SMAD4 signalling axis. The functional relevance of SMAD4 was further supported by emergence of invasive, metastatic and lethal prostate cancers with 100% penetrance upon genetic deletion of Smad4 in the Pten-null mouse prostate. Pathological and molecular analysis as well as transcriptomic knowledge-based pathway profiling of emerging tumours identified cell proliferation and invasion as two cardinal tumour biological features in the metastatic Smad4/Pten-null PCA model. Follow-on pathological and functional assessment confirmed cyclin D1 and SPP1 as key mediators of these biological processes, which together with PTEN and SMAD4, form a four-gene signature that is prognostic of prostate-specific antigen (PSA) biochemical recurrence and lethal metastasis in human PCA. This model-informed progression analysis, together with genetic, functional and translational studies, establishes SMAD4 as a key regulator of PCA progression in mice and humans.

Topics: Animals; Bone Morphogenetic Proteins; Cell Proliferation; Cyclin D1; Disease Progression; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Lung Neoplasms; Lymphatic Metastasis; Male; Mice; Mice, Transgenic; Models, Biological; Neoplasm Invasiveness; Neoplasm Metastasis; Osteopontin; Penetrance; Prognosis; Prostate; Prostate-Specific Antigen; Prostatic Neoplasms; PTEN Phosphohydrolase; Smad4 Protein; Transforming Growth Factor beta

Two recent reports in Nature provide evidence for increasingly complex "disruptive" molecular alterations that occur during prostate cancer progression. They shed light on the intricacy of genetic changes that modulate PTEN's control over the phosphoinositide 3-kinase pathway and prostate cancer progression, and identify new potential biomarkers and therapeutic targets.

Topics: Adaptor Proteins, Signal Transducing; Biomarkers, Tumor; Carrier Proteins; Cyclin D1; Disease Progression; Genetic Predisposition to Disease; Guanylate Kinases; Humans; Male; Mutation; Nuclear Proteins; Phosphatidylinositol 3-Kinases; Prostatic Neoplasms; PTEN Phosphohydrolase; Repressor Proteins; Signal Transduction

To test the prognostic significance of cyclin D1 in nodal-positive prostate cancer.. Nuclear and cytoplasmic cyclin D1 expression was evaluated in 119 nodal-positive prostate cancer patients undergoing radical prostatectomy and extended lymphadenectomy. Cyclin D1 was correlated with various tumour features and biochemical recurrence-free survival (bRFS), disease-specific survival (DSS) and overall survival (OS). In the metastases, high-level cytoplasmic cyclin D1 expression independently predicted poor outcome (5-year bRFS, 12.5% versus 26.4%, P = 0.006; 5-year DSS, 56.3% versus 80.7%, P = 0.007; 5-year OS, 56.3% versus 78.7%, P = 0.011). These patients had a 2.62-fold elevated risk of dying from prostate cancer as compared with patients with low-level cytoplasmic cyclin D1 expression (P = 0.024). All other subcellular compartments of cyclin D1 expression in primary tumours and metastases were prognostically non-significant.. The subcellular location of cyclin D1 expression in prostate cancer is linked to specific clinical courses. Survival stratification according to biomarker expression in metastases indicates an important role for tumour sampling from these tissues.

Topics: Cyclin D1; Cytoplasm; Disease-Free Survival; Humans; Lymph Node Excision; Lymph Nodes; Lymphatic Metastasis; Male; Prognosis; Prostatectomy; Prostatic Neoplasms

Metformin is a widely prescribed antidiabetic drug associated with a reduced risk of cancer. Many studies show that metformin inhibits cancer cell viability through the inhibition of mTOR. We recently showed that antiproliferative action of metformin in prostate cancer cell lines is not mediated by AMP-activated protein kinase (AMPK). We identified REDD1 (also known as DDIT4 and RTP801), a negative regulator of mTOR, as a new molecular target of metformin. We show that metformin increases REDD1 expression in a p53-dependent manner. REDD1 invalidation, using siRNA or REDD1(-/-) cells, abrogates metformin inhibition of mTOR. Importantly, inhibition of REDD1 reverses metformin-induced cell-cycle arrest and significantly protects from the deleterious effects of metformin on cell transformation. Finally, we show the contribution of p53 in mediating metformin action in prostate cancer cells. These results highlight the p53/REDD1 axis as a new molecular target in anticancer therapy in response to metformin treatment.

Topics: AMP-Activated Protein Kinases; Animals; Cell Cycle; Cell Line, Tumor; Cyclin D1; Humans; Male; Metformin; Mice; Mice, Transgenic; Prostatic Neoplasms; RNA, Messenger; RNA, Small Interfering; TOR Serine-Threonine Kinases; Transcription Factors; Transfection; Tumor Suppressor Protein p53

The cell-cycle G(2)-M phase gene UBE2C is overexpressed in various solid tumors including castration-resistant prostate cancer (CRPC). Our recent studies found UBE2C to be a CRPC-specific androgen receptor (AR) target gene that is necessary for CRPC growth, providing a potential novel target for therapeutic intervention. In this study, we showed that the G(1)-S cell-cycle inhibitor-779 (CCI-779), an mTOR inhibitor, inhibited UBE2C mRNA and protein expression in AR-positive CRPC cell models abl and C4-2B. Treatment with CCI-779 significantly decreased abl cell proliferation in vitro and in vivo through inhibition of cell-cycle progression of both G(2)-M and G(1)-S phases. In addition, exposure of abl and C4-2B cells to CCI-779 also decreased UBE2C-dependent cell invasion. The molecular mechanisms for CCI-779 inhibition of UBE2C gene expression involved a decreased binding of AR coactivators SRC1, SRC3, p300, and MED1 to the UBE2C enhancers, leading to a reduction in RNA polymerase II loading to the UBE2C promoter, and attenuation of UBE2C mRNA stability. Our data suggest that, in addition to its ability to block cell-cycle G(1) to S-phase transition, CCI-779 causes a cell-cycle G(2)-M accumulation and an inhibition of cell invasion through a novel UBE2C-dependent mechanism, which contributes to antitumor activities of CCI-779 in UBE2C overexpressed AR-positive CRPC.

Topics: Animals; Antineoplastic Agents; Cell Division; Cell Growth Processes; Cell Line, Tumor; Cyclin D1; Down-Regulation; Enzyme Inhibitors; G2 Phase; Humans; Male; Mice; Mice, Inbred BALB C; Orchiectomy; Prostatic Neoplasms; RNA, Messenger; Sirolimus; Transcription, Genetic; Transfection; Ubiquitin-Conjugating Enzymes; Xenograft Model Antitumor Assays

Topics: Biomarkers, Tumor; Cyclin D1; Humans; Male; Neoplasm Metastasis; Osteopontin; Prognosis; Prostatic Neoplasms; PTEN Phosphohydrolase; Smad4 Protein

Nuclear factor-kappaB (NF-kB), a transcription factor, is abundantly expressed in prostate cancer and regulates many tumor-related genes. Given the important roles of these genes in tumor control, the present study was conducted to test the hypothesis that there was different expression of NF-kB in androgen- dependent or androgen-independent prostate cancer cells. In addition NF-kB decoy oligodeoxynucleotides (ODNs) were transfected into two prostate cancer cells to determine affects on growth and apoptosis.. First, NF-kB decoy ODNs were designed according to the NF-κB elements in the promoter region of c-myc gene. Then, NF-kB and control decoy ODNs were transfected with lipofectamine. Their influence on prostate cancer cell line proliferative activity was detected by MTT assay. Cell apoptosis was determined by flow cytometric(FCM) analysis and AO/EB study. Thirdly, nuclear extracts were prepared from PC-3M cells and DNA-protein interactions were examined by electrophoretic mobility shift assay (EMSA). Lastly, to confirm mechanisms of action, a pGL3-C-MYC luciferase expression vector containing a fragment of the c-myc promoter was constructed and co-transfected with NF-kB decoy ODNs into PC-3M cells with lipofectamineTM2000. Expression levels of related endogenous genes were assessed by western blotting.. We found overexpression of NF-kB in the androgen-independent prostate cancer cell line PC-3M compared to the androgen-independent LNCaP. Treatment with NF-kB decoy ODNs resulted in strong suppression of proliferation, especially in the PC-3M case. Induction of apoptosis of PC-3M was observed in FCM and AO/EB studies. Activity of luciferase was significantly reduced in the NF-kB decoy-transfected cells, but not in cells transfected with a control decoy. Furthermore, we found that expression of some endogenous genes was reduced, while other genes transcripts were induced. EMSA demonstrated specific binding of the NF-kB decoy to NF-kB protein.. These findings indicate that NF-kB activation plays an important role in evolution of androgen-independent prostate cancer via manipulating expression of target genes. Inhibitors of NF-kB may thus offer promise as a therapeutic approach for the treatment of androgen-independent prostate cancer. NF-kB decoy ODNs may allow development of therapeutic and investigative tools for human malignancies.

Topics: Androgens; Apoptosis; Caspase 3; Caspase 8; Caspase 9; Cell Line, Tumor; Cyclin D1; Electrophoretic Mobility Shift Assay; Forkhead Transcription Factors; Genes, myc; Humans; Inhibitor of Apoptosis Proteins; Lipids; Male; Nerve Tissue Proteins; NF-kappa B; Oligodeoxyribonucleotides; Promoter Regions, Genetic; Prostatic Neoplasms

Prostate cancer is one of the most frequently diagnosed cancers in men. Progression of these tumors is facilitated by growth factors that activate critical signaling cascades thereby promote prostate cancer cell growth, survival, and migration. Among these, insulin-like growth factors (IGFs) signaling pathway contributes a major role. In this study, we examined the effect of zinc on insulin-like growth factors signaling in prostate cancer cells.. Human androgen-independent prostatic carcinoma (PC-3) cells were treated with different concentrations of zinc (20-100micromol/l) for 24 and 48h. Cell viability was performed by 3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Insulin-like growth factor binding protein-3 (IGFBP-3), insulin-like growth factor-I receptor (IGF-IR), insulin receptor substrate-1 (IRS-1) and IRS-2, phosphatidylinositol-3 kinase (PI-3 K), protein kinase B or Akt, phosphorylated Akt (p-Akt), extracellular regulated kinase 1/2 (ERK1/2), phosphorylated ERK1/2 (p-ERK1/2), and cyclin D1 protein levels were assessed by Western blot analysis. Apoptosis was confirmed by 4',6'-diaminido-2-phenylindole dihydrochloride (DAPI) staining, and mitochondrial membrane potential was performed using rhodamine-123 staining method.. Zinc significantly reduces the cell viability of PC-3 cells. It decreases the protein levels of IGF-IR, IRS-1, and IRS-2 and increases the level of IGFBP-3. Zinc reduces the levels of PI-3 K, Akt, ERK1/2, and cyclin Dl. Loss of mitochondrial membrane potential and apoptotic cell death were also observed in zinc-treated cells.. This study suggests that zinc decreases the survival of androgen-independent prostate cancer cells by modulating the expression of IGF system components and its signaling molecules. Thus, zinc may be qualified as a potential agent for the treatment of prostate cancer.

Topics: Androgens; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Enzyme Activation; Humans; Indoles; Insulin Receptor Substrate Proteins; Insulin-Like Growth Factor Binding Protein 3; Male; Membrane Potential, Mitochondrial; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphatidylinositol 3-Kinases; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Receptor, IGF Type 1; Signal Transduction; Somatomedins; Staining and Labeling; Zinc

Human cyclin D1 is expressed as two isoforms derived by alternate RNA splicing, termed D1a and D1b, which differ for the inclusion of intron 4 in the D1b mRNA. Both isoforms are frequently upregulated in human cancers, but cyclin D1b displays relatively higher oncogenic potential. The splicing factors that regulate alternative splicing of cyclin D1b remain unknown despite the likelihood that they contribute to cyclin D1 oncogenicity. In this study, we report that Sam68, an RNA-binding protein frequently overexpressed in prostate cancer cells, enhances splicing of cyclin D1b and supports its expression in prostate cancer cells. Chromatin immunoprecipitation and RNA coimmunoprecipitation experiments showed that Sam68 is recruited to the human CCND1 gene encoding cyclin D1 and that it binds to cyclin D1 mRNA. Transient overexpression and RNAi knockdown experiments indicated that Sam68 acts to enhance endogenous expression of cyclin D1b. Minigene reporter assays showed that Sam68 directly affected alternative splicing of CCND1 message, with a preference for the A870 allele that is known to favor cyclin D1b splicing. Sam68 interacted with the proximal region of intron 4, and its binding correlated inversely with recruitment of the spliceosomal component U1-70K. Sam68-mediated splicing was modulated by signal transduction pathways that elicit phosphorylation of Sam68 and regulate its affinity for CCND1 intron 4. Notably, Sam68 expression positively correlates with levels of cyclin D1b, but not D1a, in human prostate carcinomas. Our results identify Sam68 as the first splicing factor to affect CCND1 alternative splicing in prostate cancer cells, and suggest that increased levels of Sam68 may stimulate cyclin D1b expression in human prostate cancers.

Topics: Adaptor Proteins, Signal Transducing; Alternative Splicing; Blotting, Western; Cell Line, Tumor; Cyclin D1; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Immunoprecipitation; Male; Prostatic Neoplasms; Proto-Oncogene Mas; Proto-Oncogenes; Reverse Transcriptase Polymerase Chain Reaction; RNA-Binding Proteins; RNA, Messenger; Tissue Array Analysis; Transfection

Ligation of cancer cell surface GRP78 by activated alpha2-macroglobulin (alpha2M*) triggers pro-proliferative and anti-apoptotic signaling pathways. Cancer patients who develop autoantibodies to the alpha2M* binding site in GRP78 have a poor prognosis since these antibodies are receptor agonists. The NF-kappaB family of transcription factors induces expression of genes affecting cell growth and differentiation. NF-kappaB1 plays a major regulatory role in controlling innate immunity and inflammation, whereas NF-kappaB2 plays a greater role in cancer cell proliferation. Here we report that treatment of prostate cancer cells with antibody directed against the carboxyl terminal domain of GRP78 inhibits alpha2M*-induced activation of NF-kappaB2 by approximately 50% while exerting a lesser effect of approximately 20% on NF-kappaB1 activation. Treatment of these cells nearly abolished alpha2M*-induced activation of IKKalpha involved in the activation of NF-kappaB2. This antibody also suppressed alpha2M*-induced phosphorylation of IKKalpha, IKKalpha/beta, IkappaBalpha, and IkappaBbeta as well as levels of NIK. Antibody treatment of cancer cells elevated pro-apoptotic p21WAF and p27kip while reducing cyclin D1 levels. These studies demonstrate that antibody directed against the carboxyl terminal domain of GRP78 inhibits the pro-proliferative NF-kappaB signaling cascade in cancer cells.

Topics: alpha-Macroglobulins; Antibodies, Neoplasm; Autoantibodies; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Endoplasmic Reticulum Chaperone BiP; Heat-Shock Proteins; Humans; Male; NF-kappa B p50 Subunit; NF-kappa B p52 Subunit; Prostatic Neoplasms; Protein Structure, Tertiary; Tumor Suppressor Protein p53

Epigenetic silencing of Ras-association domain family 1A (RASSF1A) protein in cancer cells results in a disruption of cell cycle control, genetic instability, enhanced cell motility, and apoptotic resistance. Ectopic expression of RASSF1A reverses this tumorigenic phenotype. Thus, small molecules with the ability to restore RASSF1A expression may represent a new class of therapeutic agents. Recently, we designed and synthesized a fluorescent carbazole analogue of mahanine (alkaloid from Murraya koenigii) that restored RASSF1A mRNA expression. Our fluorescent lead compound up-regulated RASSF1A in vitro, potently inhibited human prostate cancer cell proliferation, and fluoresced at a visible wavelength, allowing for the observation of intracellular distribution. The small molecule lead was not acutely toxic up to 550 mg/kg, and dosing at 10 mg/kg reduced human xenograft tumor volume by about 40%.

Topics: Animals; Carbazoles; Cell Line, Tumor; Cell Proliferation; Cyclin D1; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; Dose-Response Relationship, Drug; Fluorescence; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Models, Chemical; Molecular Structure; Murraya; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; Tumor Suppressor Proteins; Xenograft Model Antitumor Assays

Development of chemoresistance limits the clinical efficiency of platinum-based therapy. Although many resistance mechanisms have been demonstrated, genetic/molecular alterations responsible for drug resistance in the majority of clinical cases have not been identified. We analyzed three pairs of testicular germ cell tumor cell lines using Affymetrix expression microarrays and revealed a limited number of differentially expressed genes across the cell lines when comparing the parental and resistant cells. Among them, CCND1 was the most significantly differentially expressed gene. Analysis of testicular germ cell tumor clinical samples by quantitative reverse transcription PCR analysis revealed that overall expression of CCND1 was significantly higher in resistant cases compared with sensitive samples (P < 0.0001). We also found that CCND1 was dramatically overexpressed both in induced and intrinsically resistant samples of ovarian and prostate cancer. Finally combined CCND1 knockdown using small-interfering RNA and cisplatin treatment inhibited cell growth in vitro significantly more effectively than any of these single treatments. Therefore, deregulation of CCND1 may be a major cause of cisplatin resistance in testicular germ cell tumors and may also be implicated in ovarian and prostate cancers. CCND1 could be potentially used as a marker for treatment stratification and as a molecular target to improve the treatment of platinum-resistant tumors.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cisplatin; Comparative Genomic Hybridization; Cyclin D1; Drug Resistance, Neoplasm; Female; Gene Expression Profiling; Humans; Male; Microarray Analysis; Neoplasms, Germ Cell and Embryonal; Ovarian Neoplasms; Prostatic Neoplasms; RNA, Small Interfering; Testicular Neoplasms

The cyclin D1b oncogene arises from alternative splicing of the CCND1 transcript, and harbors markedly enhanced oncogenic functions not shared by full-length cyclin D1 (cyclin D1a). Recent studies showed that cyclin D1b is selectively induced in a subset of tissues as a function of tumorigenesis; however, the underlying mechanism(s) that control tumor-specific cyclin D1b induction remain unsolved. Here, we identify the RNA-binding protein ASF/SF2 as a critical, allele-specific, disease-relevant effector of cyclin D1b production. Initially, it was observed that SF2 associates with cyclin D1b mRNA (transcript-b) in minigene analyses and with endogenous transcript in prostate cancer (PCa) cells. SF2 association was altered by the CCND1 G/A870 polymorphism, which resides in the splice donor site controlling transcript-b production. This finding was significant, as the A870 allele promotes cyclin D1b in benign prostate tissue, but in primary PCa, cyclin D1b production is independent of A870 status. Data herein provide a basis for this disparity, as tumor-associated induction of SF2 predominantly results in binding to and accumulation of G870-derived transcript-b. Finally, the relevance of SF2 function was established, as SF2 strongly correlated with cyclin D1b (but not cyclin D1a) in human PCa. Together, these studies identify a novel mechanism by which cyclin D1b is induced in cancer, and reveal significant evidence of a factor that cooperates with a risk-associated polymorphism to alter cyclin D1 isoform production. Identification of SF2 as a disease-relevant effector of cyclin D1b provides a basis for future studies designed to suppress the oncogenic alternative splicing event.

Topics: Alleles; Alternative Splicing; Biomarkers, Tumor; Blotting, Western; Cell Line, Tumor; Cyclin D1; Disease Progression; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Immunoenzyme Techniques; Immunoprecipitation; Male; Neoplasms, Hormone-Dependent; Nuclear Proteins; Oligonucleotide Array Sequence Analysis; Polymorphism, Genetic; Prostate; Prostatic Neoplasms; Protein Isoforms; Reverse Transcriptase Polymerase Chain Reaction; RNA-Binding Proteins; RNA, Messenger; Serine-Arginine Splicing Factors

Slug is a transcription factor of the Snail/Slug zinc-finger family and is implicated in metastasis of tumors, but its role in cell proliferation of prostate cancers is unclear.. Expression level of Slug and other genes was examined by Western blot, RT-PCR, and QPCR analyses. The forced expression of Slug was mediated by retroviruses and adenoviruses. Slug was downregulated by shRNA. Cell growth was measured by the MTT assay and the quick cell proliferation assay.. Here, we demonstrated that Slug expression is elevated in mouse prostate tumors, and human prostate cancer cell lines LNCaP, PC-3, and 22RV1. Forced expression of Slug-inhibited proliferation of prostate cancer cells PC-3 and DU-145. Conversely, reduced expression of Slug by shRNA promoted growth of PC-3 cancer cells. Consistent with these data, we found that forced expression of Slug in prostate cancer cells led to G1 cell-cycle arrest. Furthermore, ectopic expression of Slug decreased cyclin D1 expression in both PC-3 and DU-145 cells, and knockdown of Slug by shRNA upregulated cyclin D1 expression in these cancer cells. In addition, we demonstrated that ectopic expression of cyclin D1 relieved Slug-mediated inhibition of proliferation of prostate cancer cells.. We provide the first compelling evidence that Slug is a negative regulator of proliferation of prostate cancer cells. Our findings in this study are distinct from the previously reported role of Slug as a promoter for tumor metastasis, and suggest that Slug is a prognostic marker and potential therapeutic target.

Topics: Animals; Blotting, Western; Cell Cycle; Cell Division; Chickens; Cyclin D1; DNA Primers; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; Snail Family Transcription Factors; Transcription Factors

BZL101 is an aqueous extract from the Scutellaria barbata plant shown to have anticancer properties in a variety of human cancers. In order to determine its efficacy on human reproductive cancers, we assessed the responses of two human breast cancer cell lines, estrogen sensitive MCF7 and estrogen insensitive MDA-MB-231, and of two human prostate cancer cell lines, androgen sensitive LNCaP and androgen insensitive PC3 which are human cell lines that represent early and late stage reproductive cancers. BZL101 inhibited reproductive cancer growth in all cell lines by regulating expression levels of key cell cycle components that differ with respect to the cancer cell phenotypes. In early stage estrogen sensitive MCF7 cells, BZL101 induced a G₁ cell cycle arrest and ablated expression of key G₁ cell cycle regulators Cyclin D1, CDK2 and CDK4, as well as growth factor stimulatory pathways and estrogen receptor-α expression. Transfection of luciferase reporter plasmids revealed that the loss of CDK2, CDK4 and estrogen receptor-α transcript expression resulted from the BZL-dependent ablation of promoter activities. BZL101 growth arrests early stage androgen sensitive LNCaP cells in the G₂/M phase with corresponding decreases in Cyclin B1, CDK1 and androgen receptor expression. In late stage hormone insensitive breast (MDA-MB-231) and prostate (PC3) cancer cells, BZL101 induced an S phase arrest with corresponding ablations in Cyclin A2 and CDK2 expression. Our results demonstrate that BZL101 exerts phenotype specific anti-proliferative gene expression responses in human breast and prostate cancer cells, which will be valuable in the potential development of BZL-based therapeutic strategies for human reproductive cancers.

Topics: Antineoplastic Agents, Phytogenic; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin A2; Cyclin B1; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Female; Flow Cytometry; G1 Phase; Gene Expression; Humans; Male; Phytotherapy; Plant Extracts; Prostatic Neoplasms; Receptors, Androgen; Receptors, Estrogen; Receptors, Growth Factor; Reverse Transcriptase Polymerase Chain Reaction; Scutellaria

The carcinogenic potential of vasoactive intestinal peptide (VIP) was analyzed in non-tumor human prostate epithelial cells (RWPE-1) and in vivo xenografts. VIP induced morphological changes and a migratory phenotype consistent with stimulation of expression/activity of metalloproteinases MMP-2 and MMP-9, decreased E-cadherin-mediated cell-cell adhesion, and increased cell motility. VIP increased cyclin D1 expression and cell proliferation that was blocked after VPAC(1)-receptor siRNA transfection. Similar effects were seen in RWPE-1 tumors developed by subcutaneous injection of VIP-treated cells in athymic nude mice. VIP acts as a cytokine in RWPE-1 cell transformation conceivably through epithelial-mesenchymal transition (EMT), reinforcing VIP role in prostate tumorigenesis.

Topics: Animals; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Humans; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Prostatic Neoplasms; Receptors, Vasoactive Intestinal Polypeptide, Type I; Vasoactive Intestinal Peptide

The androgen receptor (AR) regulates growth and progression of androgen-dependent as well as androgen-independent prostate cancer cells. Peroxisome proliferator-activated receptor gamma (PPARγ) agonists have been reported to reduce AR activation in androgen-dependent LNCaP prostate cancer cells. To determine whether PPARγ ligands are equally effective at inhibiting AR activity in androgen-independent prostate cancer, we examined the effect of the PPARγ ligands ciglitazone and rosiglitazone on C4-2 cells, an androgen- independent derivative of the LNCaP cell line. Luciferase-based reporter assays and Western blot analysis demonstrated that PPARγ ligand reduced dihydrotestosterone (DHT)-induced increases in AR activity in LNCaP cells. However, in C4-2 cells, these compounds increased DHT-induced AR driven luciferase activity. In addition, ciglitazone did not significantly alter DHT-mediated increases in prostate specific antigen (PSA) protein or mRNA levels within C4-2 cells. siRNA-based experiments demonstrated that the ciglitazone-induced regulation of AR activity observed in C4-2 cells was dependent on the presence of PPARγ. Furthermore, overexpression of the AR corepressor cyclin D1 inhibited the ability of ciglitazone to induce AR luciferase activity in C4-2 cells. Thus, our data suggest that both PPARγ and cyclin D1 levels influence the ability of ciglitazone to differentially regulate AR signaling in androgen-independent C4-2 prostate cancer cells.

Topics: Androgen-Binding Protein; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Dihydrotestosterone; Gene Expression; Genes, Reporter; Humans; Hypoglycemic Agents; Male; Mutation; Neoplasms, Hormone-Dependent; PPAR gamma; Prostate-Specific Antigen; Prostatic Neoplasms; Receptors, Androgen; RNA, Small Interfering; Rosiglitazone; Thiazolidinediones; Transfection

MicroRNAs (miRNAs) are a class of small non-coding RNAs (ncRNAs) that regulate gene expression by repressing translation or triggering the degradation of complementary mRNA sequences. Certain miRNAs have been shown to function as integral components of the p53 and/or retinoblastoma (Rb) regulatory networks. As such, miRNA dysregulation can have a profound effect on cancer development. Previous studies have shown that miR-449a is down-regulated in human prostate cancer tissue and possesses potential tumor suppressor function. In the present study, we identify miR-449a-mediated growth arrest in prostate cancer cells is dependent on the Rb protein. We show that mutant Rb prostate cancer cells (DU- 145) are resistant to cell cycle arrest and cellular senescence induced by miR- 449a, while overexpression of wild-type Rb in DU-145 sublines (DU-1.1 and B5) restores miR-449a function. In silico analysis of 3'UTR regions reveal a putative miR-449a target site in the transcript of Cyclin D1 (CCND1); an oncogene involved in directly regulating Rb activity and cell cycle progression. Luciferase 3'UTR reporter constructs and inhibitory oligonucleotides confirm that Cyclin D1 is a direct downstream target of miR-449a. We also reveal that miR-449a suppresses Rb phosphorylation through the knockdown of Cyclin D1 and previously validated target HDAC1. By targeting genes involved in controlling Rb activity, miR- 449a regulates growth and senescence in an Rb-dependent manner. These data indicate that miR-449a is a miRNA component of the Rb pathway and its tumor suppressor-like effects, in part, depends on Rb status in prostate cancer cells.

Topics: 3' Untranslated Regions; Apoptosis; Binding Sites; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cellular Senescence; Cyclin D1; Gene Expression Regulation, Neoplastic; Genes, Reporter; Histone Deacetylase 1; Humans; Male; MicroRNAs; Mutation; Phosphorylation; Prostatic Neoplasms; Retinoblastoma Protein; RNA Interference; Transfection

To investigate the mechanism of influence of phosphoprotein associated with glycosphingolipid microdomains 1 (PAG1) on the biological behavior of human prostatic cancer cell line PC-3M-1E8.. The expression of GST-Raf1-RBD recombinant protein, a specific binding domain of active Ras (GTP-Ras), was induced by IPTG in JM109 bacterium. SDS-PAGE and coomassie brilliant blue staining were performed using cleaved product of the bacterium to determine the expression of the fusion protein. GST-pull down essay was designed to detect the level of active Ras in PGA1 and vector transfected, respectively and in the native PC-3M-1E8 cells. Western blotting was used to detect the expression levels of downstream proteins of Ras signal pathway which may be related to the function of PAG1. Phalloidine labeled by tetramethylrhodamine-5-(and-6) isothiocyanate (TRITC) was used for the staining of intracellular F-actin, Laser passing confocal microscopy was adopted for observing change of the cell morphology and the arrangement of F-actin.. After IPTG induction, the GST-Raf1-RBD recombinant protein, with a molecular weight of 33 000, was noticed to be highly expressed in JM109 bacterium. GST-pull down assay revealed that the expression level of active Ras markedly decreased after PAG1-transfection while the total Ras remained unchanged. The expression of p-ERK and cyclin D1 in the PAG1-transfected cells decreased in accordance with the level of active Ras. However, the expression of p21(WAF1/CIP1) and p-Akt didn't show any variation. Additionally, the structure of F-actin was turbulent and the pseudopodia of cells diminished conspicuously after PAG1-transfection. There was a high expression of PAG1 in normal human prostate tissue, however, the positive rate of PAG1 immuno-staining decreased in cases of prostatic adenocarcinoma, correspondent with increasing of the grading index of cell differentiation established in the Gleason grading system for the diagnosis of prostate adenocarcinoma.. An over-expression of PAG1 in PC-3M-1E8 cells effectively suppresses the activation of Ras and ERK, as well as the cyclin D1 expression, leading to an inhibition of the proliferation ability of tumor cells. The turbulence of F-actin and reduction of pseudopodia of cells result in an impairment of cell mobility, invasiveness and metastatic capability. In human prostate and prostatic adenocarcinoma tissues, the expression of PAG1 is related to the cellular differentiation and malignancy.

Topics: Actins; Adaptor Proteins, Signal Transducing; Adenocarcinoma; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Extracellular Signal-Regulated MAP Kinases; Humans; Male; Membrane Proteins; Neoplasm Invasiveness; Prostate; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; ras Proteins; Transfection

In androgen sensitive LNCaP prostate cancer cells, the proliferation induced by the epidermal growth factor (EGF) involves a cross-talk between the EGF receptor (EGFR) and the androgen receptor (AR). In lung cancer the role of the EGF-EGFR transduction pathway has been documented, whereas androgen activity has received less attention. Here we demonstrate that in LNCaP and A549 non-small cell lung cancer (NSCLC), AR and EGFR are required for either 5alpha-dihydrotestosterone (DHT) or EGF-stimulated cell growth. Only EGF activated ERK signaling and up-regulated early gene expression, while DHT triggered the expression of classical AR-responsive genes with the exception of the EGF-induced PSA transcript in A549 cells. DHT and EGF up-regulated cyclinD1 (CD1) at both mRNA and protein levels in A549 cells, while in LNCaP cells each mitogen increased only CD1 protein expression. In both cell contexts, CD1 up-regulation was prevented by selective inhibitors as well as by knock-down of either AR or EGFR and also inhibiting p38MAPK and the mammalian target of rapamycin (mTOR) pathways. Interestingly, p38MAPK and mTOR repression prevented the activation of the mTOR target ribosomal p70S6 kinase induced by DHT and EGF, indicating that p38MAPK acts as an upstream mTOR regulator. In addition, the proliferative effects promoted by both DHT and EGF in LNCaP and A549 cancer cells were no longer observed blocking either p38MAPK or mTOR activity. Hence, our data suggest that p38MAPK-dependent activation of the mTOR/CD1 pathway may represent a mechanism through which AR and EGFR cross-talk contributes to prostate and lung cancer progression.

Topics: Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; p38 Mitogen-Activated Protein Kinases; Prostatic Neoplasms; Protein Kinases; Receptor Cross-Talk; Receptors, Androgen; Signal Transduction; TOR Serine-Threonine Kinases

Deregulation of insulin-like growth factor (IGF)-I/IGF-IR signaling has been implicated in the development and progression of prostate cancer. Agents that can suppress the mitogenic activity of the IGF/IGF-IR growth axis may be of preventive or therapeutic value. We have previously demonstrated that apigenin, a plant flavone, modulates IGF signaling through upregulation of IGFBP-3. In this study, we investigated the mechanism(s) of apigenin action on the IGF/IGF-IR signaling pathway. Exposure of human prostate cancer DU145 cells to apigenin markedly reduced IGF-I-stimulated cell proliferation and induced apoptosis. Apigenin inhibited IGF-I-induced activation of IGF-IR and Akt in DU145 cells. Similar growth inhibitory and apoptotic responses were observed in PC-3 cells, which constitutively overexpress this pathway. This effect of apigenin appears to be due partially to reduced autophosphorylation of IGF-IR. Inhibition of p-Akt by apigenin resulted in decreased phosphorylation of GSK-3beta along with decreased expression of cyclin D1 and increased expression of p27/kip1. In vivo administration of apigenin to PC-3 tumor xenografts inhibited tumor growth, resulted in IGF-IR inactivation and dephosphorylation of Akt and its downstream signaling. These results suggest that inhibition of cell proliferation and induction of apoptosis by apigenin are mediated, at least in part, by its ability to inhibit IGF/IGF-IR signaling and the PI3K/Akt pathway.

Topics: Animals; Apigenin; Apoptosis; Cell Cycle; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Enzyme-Linked Immunosorbent Assay; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Immunoblotting; Insulin-Like Growth Factor I; Intracellular Signaling Peptides and Proteins; Male; Mice; Mice, Nude; Phosphorylation; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Receptor, IGF Type 1; Signal Transduction

Regulation of the androgen receptor (AR) is critical to prostate cancer (PCa) development; therefore, AR is the first line therapeutic target for disseminated tumors. Cell cycle-dependent accumulation of cyclin D1 negatively modulates the transcriptional regulation of AR through discrete, CDK4-independent mechanisms. The transcriptional corepressor function of cyclin D1 resides within a defined motif termed repressor domain (RD), and it was hypothesized that this motif could be utilized as a platform to develop new strategies for blocking AR function. Here, we demonstrate that expression of the RD peptide is sufficient to disrupt AR transcriptional activation of multiple, prostate-specific AR target genes. Importantly, these actions are sufficient to specifically inhibit S-phase progression in AR-positive PCa cells, but not in AR-negative cells or tested AR-positive cells of other lineages. As expected, impaired cell cycle progression resulted in a suppression of cell doubling. Additionally, cell death was observed in AR-positive cells that maintain androgen dependence and in a subset of castrate-resistant PCa cells, dependent on Akt activation status. Lastly, the ability of RD to cooperate with existing hormone therapies was examined, which revealed that RD enhanced the cellular response to an AR antagonist. Together, these data demonstrate that RD is sufficient to disrupt AR-dependent transcriptional and proliferative responses in PCa, and can enhance efficacy of AR antagonists, thus establishing the impetus for development of RD-based mimetics.

Topics: Androgen Antagonists; Cell Cycle; Cell Proliferation; Cell Survival; Cyclin D1; Cyclin-Dependent Kinase 4; Gene Expression Regulation, Neoplastic; Humans; Immunoblotting; Male; Phosphatidylinositol 3-Kinases; Promoter Regions, Genetic; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Receptors, Androgen; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transcription, Genetic; Transfection; Tumor Cells, Cultured

Because signal transducer and activator of transcription 3 (STAT3) is constitutively activated in most human solid tumors and is involved in the proliferation, angiogenesis, immune evasion, and antiapoptosis of cancer cells, researchers have focused on STAT3 as a target for cancer therapy. We screened for natural compounds that inhibit the activity of STAT3 using a dual-luciferase assay. Cryptotanshinone was identified as a potent STAT3 inhibitor. Cryptotanshinone rapidly inhibited STAT3 Tyr705 phosphorylation in DU145 prostate cancer cells and the growth of the cells through 96 hours of the treatment. Inhibition of STAT3 Tyr705 phosphorylation in DU145 cells decreased the expression of STAT3 downstream target proteins such as cyclin D1, survivin, and Bcl-xL. To investigate the cryptotanshinone inhibitory mechanism in DU145 cells, we analyzed proteins upstream of STAT3. Although phosphorylation of Janus-activated kinase (JAK) 2 was inhibited by 7 micromol/L cryptotanshinone at 24 hours, inhibition of STAT3 Tyr705 phosphorylation occurred within 30 minutes and the activity of the other proteins was not affected. These results suggest that inhibition of STAT3 phosphorylation is caused by a JAK2-independent mechanism, with suppression of JAK2 phosphorylation as a secondary effect of cryptotanshinone treatment. Continuing experiments revealed the possibility that cryptotanshinone might directly bind to STAT3 molecules. Cryptotanshinone was colocalized with STAT3 molecules in the cytoplasm and inhibited the formation of STAT3 dimers. Computational modeling showed that cryptotanshinone could bind to the SH2 domain of STAT3. These results suggest that cryptotanshinone is a potent anticancer agent targeting the activation STAT3 protein. It is the first report that cryptotanshinone has antitumor activity through the inhibition of STAT3.

Topics: bcl-X Protein; Breast Neoplasms; Cell Growth Processes; Cell Line, Tumor; Cyclin D1; Dimerization; Down-Regulation; Drugs, Chinese Herbal; HCT116 Cells; HeLa Cells; Humans; Inhibitor of Apoptosis Proteins; Luciferases; Male; Microtubule-Associated Proteins; Models, Molecular; Phenanthrenes; Phosphorylation; Prostatic Neoplasms; Protein-Tyrosine Kinases; STAT3 Transcription Factor; Stomach Neoplasms; Survivin

One of the major obstacles in the treatment of hormone-refractory prostate cancer (HRPC) is the development of chemoresistant tumors. The aim of this study is to evaluate the role of azacitidine as chemosensitizing agent in association with docetaxel (DTX) and cisplatin using two models of aggressive prostate cancer, the 22rv1, and PC3 cell lines. Azacitidine shows antiproliferative effects associated with increased proportion of cells in G0/G1 and evident apoptosis in 22rv1 cells and increased proportion of cells in G2/M phase with the absence of acute cell killing in PC3 cells. In vivo, azacitidine (0.8 mg/kg i.p.) reduced tumor proliferation and induced apoptosis in both xenografts upmodulating the expression of p16INKA, Bax, Bak, p21/WAF1, and p27/KIP1, and inhibiting the activation of Akt activity and the expression of cyclin D1, Bcl-2, and Bcl-XL. In vitro treatments with azacitidine lead to upregulation of cleaved caspase 3 and PARP. BCl2 antagonists, such as HA-14-1, enhanced the effects of azacitidine in these two prostate cancer models. In addition, azacitidine showed synergistic effects with both DTX and cisplatin. In vivo this agent caused tumor growth delay without complete regression in xenograft systems. Azacitidine sensitized PC3 and 22rv1 xenografts to DTX and cisplatin treatments. These combinations were also tolerable in mice and superior to either agent alone. As DTX is the standard first-line chemotherapy for HRPC, the development of DTX-based combination therapies is of great interest in this disease stage. Our results provide a rationale for clinical trials on combination treatments with azacitidine in patients with hormone-refractory and chemoresistant prostate tumors.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Azacitidine; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Blotting, Western; Caspase 3; Cell Proliferation; Cisplatin; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Disease Models, Animal; Docetaxel; Drug Synergism; Drug Therapy, Combination; Flow Cytometry; Humans; Male; Mice; Mice, Nude; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Taxoids; Xenograft Model Antitumor Assays

Thiazolidinediones (TZDs) are peroxisome proliferator activated receptor gamma (PPARgamma) ligands that have been reported to reduce proliferation of human prostate cancer cells. However, the mechanisms by which TZDs inhibit prostate cancer cell proliferation are not fully understood. In addition, it is not known if the anti-proliferative effects of TZDs require activation of PPARgamma or are mediated by PPARgamma-independent pathways. The goals of this study were to assess whether TZDs regulate expression of proteins that control the transition from G1 to S phase of the cell cycle and define the role of PPARgamma in these TZD-induced responses in androgen-independent human prostate cancer cell lines. Western blot analysis revealed that growth inhibitory concentrations of the TZDs rosiglitazone and ciglitazone induced expression of the cyclin dependent kinase inhibitor p21 and decreased cyclin D1 levels in the androgen independent PC-3 cell line. Phosphorylation of retinoblastoma protein at Serine 780 was also reduced in PC-3 cells exposed to ciglitazone. Furthermore, growth inhibitory concentrations of ciglitazone increased p21 and lowered cyclin D1 expression within C4-2 cells. PPARgamma-directed siRNAs inhibited the ability of rosiglitazone to regulate expression of cyclin D1 and p21. However, knockdown of PPARgamma did not significantly reduce ciglitazone-induced alterations in cyclin D1 and p21. Furthermore PPARgamma siRNA did not prevent inhibition of PC-3 cell proliferation by either TZD. Thus, activation of PPARgamma is involved in rosiglitazone-induced alterations in cell cycle protein expression. However, the alterations in protein expression and proliferation induced by ciglitazone occur primarily via PPARgamma-independent signaling pathways.

Topics: Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Humans; Male; PPAR gamma; Prostatic Neoplasms; RNA, Small Interfering; Rosiglitazone; Signal Transduction; Thiazolidinediones

Treatment of androgen-independent prostate cancer (AIPC) remains unsatisfactory. In our present experiment, natural occurring ginsenosides (NOGs) and intestinal bacterial metabolites (IBMs) were employed to investigate their anti-AIPC cell growth activity using PC-3 cells. Our results showed that the IBMs exerted more portent anti-AIPC activity than NOGs, by decreasing survival rate, inhibiting proliferation, inducing apoptosis, and leading to cell cycle arrest in AIPC PC-3 cells. The increase of LogP and decrease of C-6 steric hindrance, which were caused by deglycosylation by intestinal bacteria, may be the reason for the higher anti-AIPC activity of IBMs.

Topics: Androgen Antagonists; Antineoplastic Agents; Apoptosis; Bacteria; Biotransformation; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin A; Cyclin D1; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Ginsenosides; Glycosylation; Humans; Hydrophobic and Hydrophilic Interactions; Intestines; Male; Mitochondria; Molecular Structure; Prostatic Neoplasms; Structure-Activity Relationship

There is little known on the involvement of vasoactive intestinal peptide (VIP) in the metastatic cascade of human prostate cancer, that is, cell proliferation, cell-cell adhesion, extracellular-matrix degradation, and migration/invasion. Here we evaluated the expression of related biomarker proteins (cyclin D1, metalloproteinases MMP-2 and MMP-9, and E-cadherin) in human androgen-dependent (LNCaP) and independent (PC3) prostate cancer cells.. Reverse transcriptase (RT)-polymerase chain reaction (PCR), gelatin zymography, Western blotting, confocal immunofluorescence microscopy, and assays on cell proliferation, adhesion, wound-healing, migration and random homing were performed.. VIP increased cell proliferation and cyclin D1 expression whereas it decreased cell adhesion and E-cadherin expression in LNCaP and PC3 cells. VIP enhanced the gelatinolytic activity of MMP-2 and MMP-9. Semiquantitative RT-PCR assays showed that VIP stimulated mRNA levels of these MMPs and suppressed mRNA levels of its inhibitory protein RECK. VIP promoted cell invasion and migration, and the responses were faster according to the most aggressive status in cancer progression (androgen-independence). The involvement of nuclear factor-kappaB (NF-kappaB) was demonstrated since the anti-inflammatory agent curcumin blocked VIP effects on the above biomarkers in both cell lines.. Taken together, these results and the presence of kappaB sites on gene promoter of cyclin D1, MMPs and, possibly, E-cadherin suggest that VIP may act as a cytokine in an early metastatic stage of human prostate cancer through the NF-kappaB/MMPs-RECK/E-cadherin system. Our findings may help to define novel targets and agents with potential usefulness in prostate cancer therapy.

Topics: Anti-Inflammatory Agents; Biomarkers, Tumor; Cadherins; Cell Adhesion; Cell Growth Processes; Cell Line, Tumor; Cell Movement; Curcumin; Cyclin D1; Extracellular Matrix; GPI-Linked Proteins; Humans; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Membrane Glycoproteins; Microscopy, Confocal; Neoplasms, Hormone-Dependent; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; Vasoactive Intestinal Peptide

Lupeol, a dietary triterpene, was shown to decrease serum prostate-specific antigen levels and inhibit the tumorigenicity of prostate cancer (CaP) cells in vivo. Here, we show that Lupeol inhibits the proliferative potential of CaP cells and delineated its mechanism of action. Employing a focused microarray of human CaP-associated genes, we found that Lupeol significantly modulates the expression level of genes such as ERBB2, tissue inhibitor of metalloproteinases-3, cyclin D1 and matrix metalloproteinase (MMP)-2 that are known to be associated with proliferation and survival. A common feature of these genes is that all of them are known to either regulate or act as downstream target of beta-catenin signaling that is highly aberrant in CaP patients. Lupeol treatment significantly (1) reduced levels of beta-catenin in the cytoplasmic and nuclear fractions, (2) modulated expression levels of glycogen synthase kinase 3 beta (GSK3beta)-axin complex (regulator of beta-catenin stability), (3) decreased the expression level and enzymatic activity of MMP-2 (downstream target of beta-catenin), (4) reduced the transcriptional activation of T Cell Factor (TCF) responsive element (marker for beta-catenin signaling) in pTK-TCF-Luc-transfected cells and (5) decreased the transcriptional activation of MMP-2 gene in pGL2-MMP-2-Luc-transfected cells. Effects of Lupeol treatment on beta-catenin degradation were significantly reduced in CaP cells where axin is knocked down through small interfering RNA transfection and GSK3beta activity is blocked. Collectively, these data suggest the multitarget efficacy of Lupeol on beta-catenin-signaling network thus resulting in the inhibition CaP cell proliferation. We suggest that Lupeol could be developed as an agent for chemoprevention as well as chemotherapy of human CaP.

Topics: Anti-Inflammatory Agents; beta Catenin; Cell Division; Cyclin D1; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Humans; Male; Matrix Metalloproteinase 2; Oligonucleotide Array Sequence Analysis; Pentacyclic Triterpenes; Prostatic Neoplasms; Signal Transduction; Tissue Inhibitor of Metalloproteinase-3; Triterpenes

The interleukin-6 (IL-6) and the chemokine CCL5 are implicated in the development and progression of several forms of tumours including that of the prostate. The expression of the prostate specific membrane antigen (PSMA) is augmented in high-grade and metastatic tumors. Observations of the clinical behaviour of prostate tumors suggest that the increased secretion of IL-6 and CCL5 and the higher expression of PSMA may be correlated. We hypothesized that PSMA could be endowed with signalling properties and that its stimulation might impact on the regulation of the gene expression of IL-6 and CCL5. We herein demonstrate that the cross-linking of cell surface PSMA with specific antibodies activates the small GTPases RAS and RAC1 and the MAPKs p38 and ERK1/2 in prostate carcinoma LNCaP cells. As downstream effects of the PSMA-fostered RAS-RAC1-MAPK pathway activation we observed a strong induction of NF-kappaB activation associated with an increased expression of IL-6 and CCL5 genes. Pharmacological blockade with specific inhibitors revealed that both p38 and ERK1/2 participate in the phenomenon, although a major role exerted by p38 was evident. Finally we demonstrate that IL-6 and CCL5 enhanced the proliferative potential of LNCaP cells synergistically and in a dose-dependent manner and that CCL5 functioned by receptor-mediated activation of the STAT5-Cyclin D1 pro-proliferative pathway. The novel functions attributable to PSMA which are described in the present report may have profound influence on the survival and proliferation of prostate tumor cells, accounting for the observation that PSMA overexpression in prostate cancer patients is related to a worse prognosis.

Topics: Cell Line, Tumor; Cell Proliferation; Chemokine CCL5; Cyclin D1; Gene Expression Regulation, Neoplastic; GTP Phosphohydrolases; Humans; Interleukin-6; Male; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; NF-kappa B; Prostate-Specific Antigen; Prostatic Neoplasms; STAT5 Transcription Factor

We have recently shown that penta-1,2,3,4,6-O-galloyl-beta-D-glucose (PGG), a naturally occurring hydrolyzable gallotannin, inhibited the in vivo growth of human androgen-independent p53-mutant DU145 prostate cancer (PCa) xenograft in athymic nude mice without adverse effect on their body weight. We have also shown that PGG induced caspase-mediated apoptosis in the DU145 cells and the androgen-dependent human p53-wild-type LNCaP cells. Here, we investigated the cell cycle effects of PGG in these and other PCa cells. Our data show that treatment with subapoptotic doses of PGG induced S-arrest, whereas higher doses of PGG induced not only S-arrest but also G(1) arrest. We show, for the first time, that irrespective of the p53 functional status of the PCa cell lines, PGG exerted a rapid (within 2 h) and potent inhibition (inhibitory concentration by 50% approximately 6 microM) of 5-bromo-2'-deoxyuridine incorporation into S phase cells. In isolated nuclei, PGG inhibited DNA replicative synthesis with superior efficacy than a known DNA polymerase alpha inhibitor, aphidocolin. In addition to the S-arrest action, we have found a close association of downregulation of cyclin D1 with G(1) arrest induced by PGG. Overexpressing this G(1) cyclin abolished G(1) arrest, but hastened the S-arrest induction by PGG. Together, our data indicate that PGG induced PCa S-arrest probably through DNA replicative blockage and induced G(1) arrest via cyclin D1 downregulation to contribute to anticancer activity. Our data raise the hypothesis that PGG may be a novel inhibitor of DNA polymerases.

Topics: Amino Acid Chloromethyl Ketones; Cell Cycle; Cell Division; Cell Line, Tumor; Cell Nucleus; Cyclin D1; DNA Replication; Humans; Hydrolyzable Tannins; Male; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms

Increasing interest in the use of phytochemicals to reduce prostate cancer led us to investigate 2 potential agents, curcumin and resveratrol as preventive agents. However, there is concern about the bioavailability of these agents pertinent to the poor absorption and thereby limiting its clinical use. With the view to improve their bioavailability, we used the liposome encapsulated curcumin, and resveratrol individually and in combination in male B6C3F1/J mice. Further, we examined the chemopreventive effect of liposome encapsulated curcumin and resveratrol in combination in prostate-specific PTEN knockout mice. In vitro assays using PTEN-CaP8 cancer cells were performed to investigate the combined effects curcumin with resveratrol on (i) cell growth, apoptosis and cell cycle (ii) impact on activated p-Akt, cyclin D1, m-TOR and androgen receptor (AR) proteins involved in tumor progression. HPLC analysis of serum and prostate tissues showed a significant increase in curcumin level when liposome encapsulated curcumin coadministered with liposomal resveratrol (p < 0.001). Combination of liposomal forms of curcumin and resveratrol significantly decreased prostatic adenocarcinoma in vivo (p < 0.001). In vitro studies revealed that curcumin plus resveratrol effectively inhibit cell growth and induced apoptosis. Molecular targets activated due to the loss of phosphatase and tensin homolog (PTEN) including p-Akt, cyclin D1, mammalian target of rapamycin and AR were downregulated by these agents in combination. Findings from this study for the first time provide evidence on phytochemicals in combination to enhance chemopreventive efficacy in prostate cancer. These findings clearly suggest that phytochemicals in combination may reduce prostate cancer incidence due to the loss of the tumor suppressor gene PTEN.

Topics: Androgen Receptor Antagonists; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Western; Carrier Proteins; Cell Cycle; Cell Proliferation; Curcumin; Cyclin D1; Disease Progression; Drug Carriers; Drug Delivery Systems; Incidence; Liposomes; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Phosphotransferases (Alcohol Group Acceptor); Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Receptors, Androgen; Resveratrol; Signal Transduction; Stilbenes; TOR Serine-Threonine Kinases

Suppressor of cytokine signaling (SOCS) proteins play a pivotal role in the development and progression of various cancers. We have previously shown that SOCS-3 is expressed in prostate cancer, and its expression is inversely correlated with activation of signal transducer and activator of transcription factor 3. We hypothesized that SOCS-1, if expressed in prostate cancer cells, has a growth-regulatory role in this malignancy. The presence of both SOCS-1 mRNA and protein was detected in all tested cell lines. To assess SOCS-1 expression levels in vivo, we analyzed tissue microarrays and found a high percentage of positive cells in both prostate intraepithelial neoplasias and cancers. SOCS-1 expression levels decreased in samples taken from patients undergoing hormonal therapy but increased in specimens from patients who failed therapy. In LNCaP-interleukin-6- prostate cancer cells, SOCS-1 was up-regulated by interleukin-6 and in PC3-AR cells by androgens; such up-regulation was also found to significantly impair cell proliferation. To corroborate these findings, we used a specific small interfering RNA against SOCS-1 and blocked expression of the protein. Down-regulation of SOCS-1 expression caused a potent growth stimulation of PC3, DU-145, and LNCaP-interleukin-6- cells that was associated with the increased expression levels of cyclins D1 and E as well as cyclin-dependent kinases 2 and 4. In summary, we show that SOCS-1 is expressed in prostate cancer both in vitro and in vivo and acts as a negative growth regulator.

Topics: Aged; Androgens; Blotting, Western; Cyclin D1; Cyclin E; Cyclin-Dependent Kinases; Down-Regulation; Humans; Immunoenzyme Techniques; Interleukin-6; Male; Middle Aged; Neoplasm Recurrence, Local; Prostate; Prostatic Intraepithelial Neoplasia; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Suppressor of Cytokine Signaling 1 Protein; Suppressor of Cytokine Signaling Proteins; Tissue Array Analysis; Tumor Cells, Cultured

Gossypol (GP), a male contraceptive compound naturally present in cottonseed products, possesses anti-proliferative and anti-metastatic effects in vitro and in vivo. However, the detailed mechanisms responsible for the effects of GP on the cell cycle of prostate cancer cells remain to be elucidated. In the present study, we investigated the effects of GP on the regulation of the cell cycle of rodent prostate cancer MAT-LyLu cells and the mechanisms of GP-induced growth inhibition. Our results showed that GP inhibited the cell proliferation and colony formation in a dose-dependent manner by the up-regulation of expression and secretion of transforming growth factor beta1 (TGFbeta1) and down-regulation of expression of Akt and phospho-Akt protein. The inhibition of cell growth was also demonstrated by cell cycle arrest at G0/G1 phase. Furthermore, GP decreased the expression of cyclin D1, Cdk4 and phospho-Rb in MAT-LyLu cells. Thus, the inhibitory effects of GP on the proliferation of MAT-LyLu prostate cancer cells are associated with modulation of TGFbeta1 and Akt signaling, which influence the expression of regulatory proteins such as cyclin D1, Cdk4 and phospho-Rb which regulate cell cycle progression of prostate cancer cells.

Topics: Animals; Antineoplastic Agents; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Contraceptive Agents, Male; Cyclin D1; Gossypol; Male; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Rats; Retinoblastoma Protein; Signal Transduction; Transforming Growth Factor beta1

Alternative CCND1 splicing results in cyclin D1b, which has specialized, protumorigenic functions in prostate not shared by the cyclin D1a (full length) isoform. Here, the frequency, tumor relevance, and mechanisms controlling cyclin D1b were challenged.. First, relative expression of both cyclin D1 isoforms was determined in prostate adenocarcinomas. Second, relevance of the androgen axis was determined. Third, minigenes were created to interrogate the role of the G/A870 polymorphism (within the splice site), and findings were validated in primary tissue. Fourth, the effect of G/A870 on cancer risk was assessed in two large case-control studies.. Cyclin D1b is induced in tumors, and a significant subset expressed this isoform in the absence of detectable cyclin D1a. Accordingly, the isoforms showed noncorrelated expression patterns, and hormone status did not alter splicing. Whereas G/A870 was not independently predictive of cancer risk, A870 predisposed for transcript-b production in cells and in normal prostate. The influence of A870 on overall transcript-b levels was relieved in tumors, indicating that aberrations in tumorigenesis likely alter the influence of the polymorphism.. These studies reveal that cyclin D1b is specifically elevated in prostate tumorigenesis. Cyclin D1b expression patterns are distinct from that observed with cyclin D1a. The A870 allele predisposes for transcript-b production in a context-specific manner. Although A870 does not independently predict cancer risk, tumor cells can bypass the influence of the polymorphism. These findings have major implications for the analyses of D-cyclin function in the prostate and provide the foundation for future studies directed at identifying potential modifiers of the G/A870 polymorphism.

Topics: Alleles; Alternative Splicing; Case-Control Studies; Cyclin D1; Gene Expression Regulation, Neoplastic; Genotype; Humans; Male; Polymorphism, Genetic; Prostatic Neoplasms; Protein Isoforms; Tissue Array Analysis

Androgen receptor (AR) signaling plays a critical role in the development and progression of prostate cancer. It has been reported previously that peroxiredoxin-1 (Prx1), a member of a novel family of peroxidases, interacts physically with AR to enhance AR transactivation of target genes. In the present study, we evaluated the biological significance of Prx1 in modulating dihydrotestosterone (DHT)-stimulated growth and AR target gene expression of prostate cancer cells. We also investigated the mechanism by which Prx1 might potentiate AR signaling. The contribution of Prx1 was assessed mainly by using the approach of stable Prx1 knockdown. The major observations are as follows: (a) A low level of Prx1 desensitizes cells to growth stimulation and AR target gene induction by DHT, such that exposure to a higher level of DHT is required to reach the same magnitude of response when Prx1 is depressed; (b) Prx1 increases the affinity of AR to DHT and decreases the rate of DHT dissociation from the occupied receptor; (c) Prx1 enhances the NH2 terminus and COOH terminus interaction of AR; a stronger N-C interaction is consistent with a more robust AR activation signal by keeping DHT tight in the ligand-binding pocket; (d) the stimulatory effects of Prx1 on AR ligand binding affinity and AR N-C interaction are manifested regardless of a wild-type or mutant AR. The above findings led us to believe that Prx1 may be a therapeutic target in blocking the transition of prostate cancer from an androgen-dependent to an androgen-refractory phenotype.

Topics: Cell Growth Processes; Cell Line, Tumor; Cyclin D1; Dihydrotestosterone; Dimerization; Gene Knockdown Techniques; Humans; Male; Peroxiredoxins; Prostate-Specific Antigen; Prostatic Neoplasms; Protein Binding; Receptors, Androgen; RNA Interference; RNA, Small Interfering; Tissue Kallikreins

Constitutive activation of phosphoinositide 3-kinase (PI3K)-Akt pathway transmits growth-regulatory signals that play a central role in promoting survival, proliferation, and angiogenesis in human prostate cancer cells. Here, we assessed the efficacy of inositol hexaphosphate (IP6) against invasive human prostate cancer PC-3 and C4-2B cells and regulation of PI3K-Akt pathway. IP6 treatment of cells suppressed proliferation, induced apoptosis along with caspase-3 and poly(ADP-ribose) polymerase (PARP) cleavage, and inhibited constitutive activation of Akt and its upstream regulators PI3K, phosphoinositide-dependent kinase-1 and integrin-linked kinase-1 (ILK1). Downstream of Akt, IP6 inhibited the phosphorylation of glycogen synthase kinase-3alpha/beta at Ser(21/9) and consequently reduced cyclin D1 expression. Efficacy studies employing PC-3 tumor xenograft growth in nude mice showed that 2% (w/v) IP6 feeding in drinking water inhibits tumor growth and weight by 52% to 59% (P < 0.001). Immunohistochemical analysis of xenografts showed that IP6 significantly reduces the expression of molecules associated with cell survival/proliferation (ILK1, phosphorylated Akt, cyclin D1, and proliferating cell nuclear antigen) and angiogenesis (platelet endothelial cell adhesion molecule-1 or CD31, vascular endothelial growth factor, endothelial nitric oxide synthase, and hypoxia-inducible factor-1alpha) together with an increase in apoptotic markers (cleaved caspase-3 and PARP). These findings suggest that, by targeting the PI3K-ILK1-Akt pathway, IP6 suppresses cell survival, proliferation, and angiogenesis but induces death in prostate cancer cells, which might have translational potential in preventing and controlling the growth of advanced and aggressive prostate cancer for which conventional chemotherapy is not effective.

Topics: Animals; Apoptosis; Cell Growth Processes; Cyclin D1; DNA, Neoplasm; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Mice; Mice, Nude; Neovascularization, Pathologic; Nitric Oxide Synthase Type III; Phosphatidylinositol 3-Kinases; Phosphorylation; Phytic Acid; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

To screen the genes and possible signal transduction pathways involved in the mechanism of nucleostemin (NS) in the proliferation of prostate cancer.. Oligonucleotide DNA microarray was used to screen the genome changes after knocking-down expression of NS in PC-3 cells and quantitative real-time PCR was used to further confirm the important differentially expressed genes.. 219 differentially expressed genes were found and theses genes were involved in cell cycle, cell proliferation, signal transduction, cell apoptosis and cell differentiation, etc. INK4 family genes (p15, p16, p18) were up-regulated and cyclin D1, HDAC1 were down-regulated, the main action points were CDK4/6-cyclin D and pRb-E2F1 complexes.. NS may promote the progression of prostate cancer by inhibiting the expression of p15, p16, and p18 in PC-3 cells. NS is an important G(1)/S checkpoint regulator and its regulatory activity has been certified at gene level.

Topics: Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor Proteins; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; GTP-Binding Proteins; Histone Deacetylase 1; Humans; Male; Nuclear Proteins; Oligonucleotide Array Sequence Analysis; Prostatic Neoplasms; RNA Interference; Signal Transduction

Inhibitors of the enzyme 17alpha-hydroxylase/17,20 lyase are a new class of anti-prostate cancer agents currently undergoing preclinical and clinical development. We have previously reported the superior anticancer activity of our novel 17alpha-hydroxylase/17,20 lyase inhibitor, VN/124-1, against androgen-dependent cancer models. Here, we examined the effect of VN/124-1 on the growth of the androgen-independent cell lines PC-3 and DU-145 and found that the compound inhibits their growth in a dose-dependent manner in vitro (GI50, 7.82 micromol/L and 7.55 micromol/L, respectively). We explored the mechanism of action of VN/124-1 in PC-3 cells through microarray analysis and found that VN/124-1 up-regulated genes involved in stress response and protein metabolism, as well as down-regulated genes involved in cell cycle progression. Follow-up real-time PCR and Western blot analyses revealed that VN/124-1 induces the endoplasmic reticulum stress response resulting in down-regulation of cyclin D1 protein expression and cyclin E2 mRNA. Cell cycle analysis confirmed G1-G0 phase arrest. Measurements of intracellular calcium levels ([Ca2+]i) showed that 20 micromol/L VN/124-1 caused a release of Ca2+ from endoplasmic reticulum stores resulting in a sustained increase in [Ca2+]i. Finally, cotreatment of PC-3 cells with 5, 10, and 20 micromol/L VN/124-1 with 10 nmol/L thapsigargin revealed a synergistic relationship between the compounds in inhibiting PC-3 cell growth. Taken together, these findings show VN/124-1 is endowed with multiple anticancer properties that may contribute to its utility as a prostate cancer therapeutic.

Topics: Androgens; Androstadienes; Animals; Benzimidazoles; Calcium; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Drug Synergism; Endoplasmic Reticulum; Eukaryotic Initiation Factor-2; G1 Phase; Gene Expression Regulation, Neoplastic; Genes, Neoplasm; Male; Oligonucleotide Array Sequence Analysis; Phosphorylation; Prostatic Neoplasms; Resting Phase, Cell Cycle; Steroid 17-alpha-Hydroxylase; Thapsigargin; Up-Regulation

Epidemiologic studies inclusively indicate that "unhealthy" dietary fat intake is one of the potential risk factors for cancer. In dietary fat, there are two types of polyunsaturated fatty acids (PUFA), omega-3 (n-3) and omega-6 (n-6). Numerous studies support that the ratio of n-6/n-3 affects tumorigenesis. It was reported that adenoviral transfer of the fat-1 gene, which converts n-6 to n-3, into breast and lung cancer cells had an antitumor effect in vitro. However, the effects of the fat-1 gene expression on tumor growth in vivo have not been studied and the mechanisms remain unclear. Accordingly, prostate cancer DU145 and PC3 cells were transfected with either the fat-1 gene or a control vector. The cells that expressed the fat-1 gene had a lower n-6/n-3 PUFA ratio compared with the cells that expressed the control vector. The fat-1 gene expression significantly inhibited prostate cancer cell proliferation and invasion in vitro. The fat-1 and control vector-transfected prostate cancer cells were s.c. implanted into severe combined immunodeficient mice for 6 weeks. The fat-1 gene expression significantly diminished tumor growth in vivo, but the control vector had no effect. Finally, we evaluated signaling pathways that may be important for fat-1 gene function. Administration of n-3 PUFA induced caspase-3-mediated prostate cancer cell apoptosis in vitro. The fat-1 gene expression inhibited prostate cancer cell proliferation via reduction of GSK-3beta phosphorylation and subsequent down-regulation of both beta-catenin and cyclin D1. These results suggest that fat-1 gene transfer directly into tumor cells could be used as a novel therapeutic approach.

Topics: Animals; Apoptosis; beta Catenin; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Fatty Acid Desaturases; Fatty Acids, Omega-3; Fatty Acids, Omega-6; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Male; Mice; Neoplasm Invasiveness; Phosphorylation; Prostatic Neoplasms; Transfection

Delphinidin, a major anthocyanidin present in many pigmented fruits and vegetables, possesses antioxidant, anti-inflammatory, and antiangiogenic properties. In this study, we provide evidence that it could be developed as a novel agent against human prostate cancer (PCa). We observed that delphinidin treatment to human PCa LNCaP, C4-2, 22Rnu1, and PC3 cells resulted in a dose-dependent inhibition of cell growth without having any substantial effect on normal human prostate epithelial cells. We selected PC3 cells as a test model system because of their highly aggressive proliferative nature. Delphinidin treatment of cells resulted in a dose-dependent induction of apoptosis and arrest of cells in G(2)-M phase. This induction of apoptosis seems to be mediated via activation of caspases because N-benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluromethylketone significantly reduced apoptosis induced by delphinidin. We also observed that delphinidin treatment of cells resulted in a dose-dependent decrease in (a) phosphorylation of IkappaB kinase gamma (NEMO), (b) phosphorylation of nuclear factor-kappaB (NF-kappaB) inhibitory protein IkappaBalpha, (c) phosphorylation of NF-kappaB/p65 at Ser(536) and NF-kappaB/p50 at Ser(529), (d) NF-kappaB/p65 nuclear translocation, and (e) NF-kappaB DNA binding activity. Delphinidin administration (2 mg, i.p. thrice weekly) to athymic nude mice implanted with PC3 cells resulted in a significant inhibition of tumor growth. Analysis of tumors from delphinidin-treated mice showed significant decrease in the expression of NF-kappaB/p65, Bcl2, Ki67, and PCNA. Taken together, our data suggest that delphinidin could be developed as an agent against human PCa.

Topics: Animals; Anthocyanins; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Cell Division; Cell Line, Tumor; Cell Proliferation; Cyclin D1; DNA; Dose-Response Relationship, Drug; G2 Phase; Humans; I-kappa B Kinase; Ki-67 Antigen; Male; Mice; Mice, Nude; NF-kappa B; Phosphorylation; Poly(ADP-ribose) Polymerases; Proliferating Cell Nuclear Antigen; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Signal Transduction

MicroRNAs (miRNAs) are noncoding small RNAs that repress protein translation by targeting specific messenger RNAs. miR-15a and miR-16-1 act as putative tumor suppressors by targeting the oncogene BCL2. These miRNAs form a cluster at the chromosomal region 13q14, which is frequently deleted in cancer. Here, we report that the miR-15a and miR-16-1 cluster targets CCND1 (encoding cyclin D1) and WNT3A, which promotes several tumorigenic features such as survival, proliferation and invasion. In cancer cells of advanced prostate tumors, the miR-15a and miR-16 level is significantly decreased, whereas the expression of BCL2, CCND1 and WNT3A is inversely upregulated. Delivery of antagomirs specific for miR-15a and miR-16 to normal mouse prostate results in marked hyperplasia, and knockdown of miR-15a and miR-16 promotes survival, proliferation and invasiveness of untransformed prostate cells, which become tumorigenic in immunodeficient NOD-SCID mice. Conversely, reconstitution of miR-15a and miR-16-1 expression results in growth arrest, apoptosis and marked regression of prostate tumor xenografts. Altogether, we propose that miR-15a and miR-16 act as tumor suppressor genes in prostate cancer through the control of cell survival, proliferation and invasion. These findings have therapeutic implications and may be exploited for future treatment of prostate cancer.

Topics: Animals; Cell Line, Tumor; Cyclin D1; Humans; Male; Mice; MicroRNAs; Multigene Family; Oncogene Proteins; Prostatic Neoplasms; Wnt Proteins; Wnt3 Protein; Wnt3A Protein

Cytosolic phospholipase A2-alpha (cPLA2-alpha) provides intracellular arachidonic acid to supply both cyclooxygenase and lipoxygenase pathways. We aim to determine the expression and activation of cPLA2-alpha in prostate cancer cell lines and tissue and the effect of targeting cPLA2-alpha in vitro and in vivo.. The expression of cPLA2-alpha was determined in prostate cancer cells by reverse transcription-PCR, Western blot, and immunocytochemistry. Growth inhibition, apoptosis, and cPLA2-alpha activity were determined after inhibition with cPLA2-alpha small interfering RNA or inhibitor (Wyeth-1). Cytosolic PLA2-alpha inhibitor or vehicle was also administered to prostate cancer xenograft mouse models. Finally, the expression of phosphorylated cPLA2-alpha was determined by immunohistochemistry in human normal, androgen-sensitive and androgen-insensitive prostate cancer specimens.. cPLA2-alpha is present in all prostate cancer cells lines, but increased in androgen-insensitive cells. Inhibition with small interfering RNA or Wyeth-1 results in significant reductions in prostate cancer cell numbers, as a result of reduced proliferation as well as increased apoptosis, and this was also associated with a reduction in cPLA2-alpha activity. Expression of cyclin D1 and phosphorylation of Akt were also observed to decrease. Wyeth-1 inhibited PC3 xenograft growth by approximately 33% and again, also reduced cyclin D1. Immunohistochemistry of human prostate tissue revealed that phosphorylated cPLA2-alpha is increased when hormone refractory is reached.. Expression and activation of cPLA2-alpha are increased in the androgen-insensitive cancer cell line and tissue. Inhibition of cPLA2-alpha results in cells and xenograft tumor growth inhibition and serves as a potentially effective therapy for hormone refractory prostate cancer.

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cytosol; Enzyme Inhibitors; Group IV Phospholipases A2; Humans; Male; Phosphorylation; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt

To determine the mechanisms of glucocorticoids in inhibiting advanced prostate cancer growth.. The cell proliferation and cell cycle of prostate cancer DU145 cells following dexamethasone treatment were determined by proliferation assay and fluorescence-activated cell sorter. Western blot analysis was carried out to evaluate the effects of dexamethasone on phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and expression of cyclin D1 in DU145 cells with or without glucocorticoid receptor (GR) antagonist RU486. Reverse transcription-polymerase chain reaction verified the expression of GR mRNA in DU145 cells.. Dexamethasone significantly inhibited DU145 cell proliferation at the G(0)/G(1) phase. Western blot analysis showed a dramatic reduction of ERK1/2 activity and cyclin D1 expression in dexamethasone-treated cells. The decreased phosphorylation of ERK1/2 in dexamethasone-treated cells was attenuated by GR blockade. Additionally, the effects of dexamethasone in inhibiting cyclin D1 expression were altered by GR blockade.. Dexamethasone suppresses DU145 cell proliferation and cell cycle, and the underlying mechanisms are through the inhibition of phosphorylation of ERK1/2 and cyclin D1 expression. The inhibition of ERK1/2 phosphorylation and cyclin D1 expression is attenuated by GR blockade, suggesting that GR regulates ERK1/2 and cyclin D1 pathways. These observations suggest that dexamethasone has a potential clinical application in prostate cancer therapy.

Topics: Antineoplastic Agents, Hormonal; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Dexamethasone; Gene Expression Regulation, Neoplastic; Humans; Male; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Prostatic Neoplasms; Receptors, Glucocorticoid; RNA, Messenger; Signal Transduction

Signal transducer and activator of transcription 3 (Stat3) is constitutively active (phosphorylated) in several forms of cancer, including prostate cancer (PCa). Stat3 signaling may be an interesting target for cancer therapy since inhibition of this pathway mediates growth inhibition and apoptosis of these cells. In this study we investigated the in vitro and in vivo effects of the fungal metabolite galiellalactone, a direct inhibitor of Stat3, on PCa cells.. The human PCa cell lines DU145, PC-3, and LNCaP were used. Nude mice with subcutaneous PCa cell xenografts were subjected to daily intraperitoneal injections of galiellalactone for 3 weeks. The effect of galiellalactone on the induction of apoptosis of cultured PCa cells was investigated by Western blot analysis, immunocytochemistry, and annexin V staining. Effects of galiellalactone on Stat3 signaling were investigated by a luciferase reporter gene assay. Expression of Stat3 associated proteins and mRNA was investigated by Western blot and real-time quantitative PCR analysis.. Galiellalactone induced apoptosis of p-Stat3 positive PCa cells (androgen-insensitive DU145 and PC-3) but not in cells lacking p-Stat3 (androgen-sensitive LNCaP). Galiellalactone inhibited Stat3-mediated luciferase activity (IC(50) approximately 5 microM) and reduced the expression of Bcl-2, Bcl-x(L), c-myc, and cyclin D1. Furthermore, galiellalactone significantly suppressed DU145 xenograft growth in vivo (42% growth reduction; P<0.002) and reduced the relative mRNA expression of Bcl-x(L) and Mcl-1.. Galiellalactone induced growth inhibition and apoptosis in androgen-insensitive PCa cells expressing p-Stat3. We suggest that galiellalactone is a potential anti-tumor lead against hormone-refractory PCa with constitutively active Stat3.

Topics: Animals; Apoptosis; bcl-X Protein; Blotting, Western; Cell Line, Tumor; Cell Survival; Cyclin D1; Humans; Immunohistochemistry; Lactones; Luciferases; Male; Mice; Mice, Nude; Neoplasms, Hormone-Dependent; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; Reverse Transcriptase Polymerase Chain Reaction; STAT3 Transcription Factor

Metformin is a widely used antidiabetic agent, which regulates glucose homeostasis through inhibition of liver glucose production and an increase in muscle glucose uptake. Recent studies suggest that metformin may reduce the risk of cancer, but its mode of action in cancer remains not elucidated. We investigated the effect of metformin on human prostate cancer cell proliferation in vitro and in vivo. Metformin inhibited the proliferation of DU145, PC-3 and LNCaP cancer cells with a 50% decrease of cell viability and had a modest effect on normal prostate epithelial cell line P69. Metformin did not induce apoptosis but blocked cell cycle in G(0)/G(1). This blockade was accompanied by a strong decrease of cyclin D1 protein level, pRb phosphorylation and an increase in p27(kip) protein expression. Metformin activated the AMP kinase pathway, a fuel sensor signaling pathway. However, inhibition of the AMPK pathway using siRNA against the two catalytic subunits of AMPK did not prevent the antiproliferative effect of metformin in prostate cancer cells. Importantly, oral and intraperitoneal treatment with metformin led to a 50 and 35% reduction of tumor growth, respectively, in mice bearing xenografts of LNCaP. Similar, to the in vitro study, metformin led to a strong reduction of cyclin D1 protein level in tumors providing evidence for a mechanism that may contribute to the antineoplastic effects of metformin suggested by recent epidemiological studies.

Topics: AMP-Activated Protein Kinases; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Gene Expression Regulation, Neoplastic; Humans; Hypoglycemic Agents; Male; Metformin; Multienzyme Complexes; Prostate; Prostatic Neoplasms; Protein Serine-Threonine Kinases

To investigate the role of mitogen-activated protein kinase (MAPK) in the apoptosis and cell cycle arrest of human prostate carcinoma cells induced by raloxifene (RAL).. Human prostate carcinoma cells of the line PC3 were cultured. RAL of the concentrations of 10(-4), 10(-5), 10(-6), and 10(-7) mol/L were added into the culture fluid. MTT method was used to detect the inhibitory rate of the PC3 proliferation. RAL of the concentrations of 10(-6) mol/L or 10(-6) mol/L +10 micromol/L PD98059, a MEK1/2 inhibitor, and 10(-6) mol/L RAL +10 micromol/L SB203580, JNK inhibitor, and RAL + SB203580, a p38 inhibitor were added respectively for 48 h, and the flow cytometry (FCM) was used to detect the cell cycle. The cell apoptosis percentage was measured by TUNEL staining. The activation of extra cellular regulated protein kinases (ERK1/2), c-Jun N-terminal protein kinase (JNK), and p38 mitogen-activated protein kinase (p38), and the expressions of Bcl-2, Bax, phospho-Bcl-2 (p-Bcl-2), and caspase-3 were determined by Western blotting. The expressions of estrogen receptor (ER) alpha, ERbeta, cyclin dependent kinase inhibitor (P21(WAF1)), and cyclinD1 mRNA were determined by reverse transcription-polymerase chain reaction (RT-PCR).. A dose-dependent proliferation inhibition of RAL was demonstrated in the PC3 cells. A G(1) cell cycle arrest and apoptosis were induced in the PC3 cells exposed to 10(-6) mol/L RAL. 10(-6) mol/L RAL induced the activation of ERK1/2 and p38 with different time courses, but it did not induce the activation of JNK. Suppression ERK1/2 activation by treatment with PD98059 or p38 activation by treatment with SB203580 attenuated the cell-cycle arrest at the G(1) phase respectively. 48 h after the treatment of 10(-6) mol/L RAL the PC3 cells was arrested at G(1) stage, however, 48 h after the treatment of 10(-6) mol/L RAL +10 micromol/L PD98059 and 10(-6) mol/L RAL +10 micromol/L SB203580 the degree of PC3 cell arrest at the G(1) stage was lower. 18 h after the treatment of 10(-6) mol/L RAL, the expressions of cyclinD1 and P21(WAF1) gene were (0.50 +/- 0.02) and (4.48 +/- 0.12) times that of the control group, and the expressions of cyclinD1 and P21(WAF1) gene of the SB203580 pretreatment group and SB203580 pretreatment groups were (2.36 +/- 0.08) and (4.50 +/- 0.03) times, and (0.49 +/- 0.02) and (1.77 +/- 0.06) times respectively. 48 h after treatment with 10(-6) mol/L RAL, the apoptosis rates were 22.9% +/- 1.5%, significantly higher than those of the 10(-6) mol/L RAL +10 micromol/L PD98059 and 10(-6) mol/L RAL +10 micromol/L SB203580 groups (15.2% +/- 1.8% and 9.7% +/- 0.6% respectively, both P < 0.05). The expression of Bcl-2, Bax and caspase-3 (control, 10(-6) mol/L raloxifene, 10(-6) mol/L raloxifene +10 micromol/L PD98059 and 10(-6) mol/L raloxifene +10 micromol/L SB203580) was 1, 0.33 +/- 0.02, 0.34 +/- 0.01, 0.81 +/- 0.05; 1, 3.14 +/- 0.02, 1.67 +/- 0.11, 3.15 +/- 0.03; 1, 4.16 +/- 0.02, 2.66 +/- 0.03, 1.80 +/- 0.06, respectively. Western blotting showed that 48 h after the treatment of 10(-6) mol/L RAL the expression of Bcl-2 decreased, and the expression of caspace-3 increased. Pretreatment with PD98059 inhibited the increase of caspase-3 and Bax expression induced by RAL. 1.5 h after the treatment with RAL the p- Bcl-2 expression increased remarkable, pretreatment with SB203580 inhibited the p- Bcl-2 expression induced by RAL, and however, PD98059 did not show such effect.. RAL inhibits the proliferation and induces the apoptosis and G(1) cell cycle arrest via MAPK cascades in human prostate carcinoma cells. Up-regulation of P21(WAF1) mRNA expression by activating ERK1/2 and down-regulation of cyclinD1 by activating p38 induces G(1) cell cycle arrest in the human prostate carcinoma cells. The ERK1/2 or p38 cascade is involved in the induction of apoptosis by RAL. The activation of ERK1/2 increases the expression of Bax. The activation of p38 phosphorylates Bcl-2 then inactivates Bcl-2.

Topics: Apoptosis; Blotting, Western; Bone Density Conservation Agents; Caspase 3; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Enzyme Inhibitors; Flavonoids; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; In Situ Nick-End Labeling; JNK Mitogen-Activated Protein Kinases; Male; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Pyridines; Raloxifene Hydrochloride; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction

Insulin-like growth factor 1 receptor (IGF-1R) activation is required for prostate cell proliferation. Prostate cancer is one of the most commonly diagnosed malignant tumors in Western countries. Overexpression of IGF-1R in prostate cancer is associated with tumor growth. These suggest that IGF-1R inhibitory agents may be of preventive and/or therapeutic value. With evidence accumulating for a chemopreventive role of flavonoids, the effects of luteolin, a bioactive flavonoid, on IGF-1R signaling in prostate cancer cells were examined. Luteolin inhibited insulin-like growth factor 1 (IGF-1) induced activation of IGF-1R and AKT in prostate cancer PC-3 and DU145 cells. Inhibition of AKT by luteolin resulted in decreased phosphorylation of its downstream targets, including p70S6K1, GSK-3beta and FKHR/FKHRL1. Luteolin also inhibited the IGF-1-induced activation of EGFR and MAPK/ERK signaling. Luteolin inhibited expression of cyclin D1 and increased expression of p21. As a result, luteolin suppressed proliferation and induced apoptosis of prostate cancer cells. Knockdown of IGF-1R by siRNA led to inhibition of proliferation of prostate cancer cells. Results of in vivo tumor growth assay indicated that luteolin inhibited PC-3 tumor growth. Immunoblotting of the extracts of tumor tissues showed that luteolin inhibited IGF-1R/AKT signaling. Our results provide a new insight into the mechanisms that luteolin is against cancer cells.

Topics: Animals; Anticarcinogenic Agents; Cell Cycle; Cell Line, Tumor; Cyclin D1; Humans; Luteolin; Male; Mice; Mice, Inbred BALB C; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Receptor, IGF Type 1; RNA, Small Interfering; Signal Transduction; Transfection

To study the effect of gum mastic, a natural resin, on the proliferation of androgen-independent prostate cancer PC-3 cells, and further investigate the mechanisms involved in this regulatory system, taking nuclear factor kappaB (NF-kappaB) signal as the target.. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and a flow cytometer were used to detect the effect of gum mastic on the proliferation of PC-3 cells. Then, reporter gene assay, RT-PCR, and Western blotting were carried out to study the effects of gum mastic on the NF-kappaB protein level and the NF-kappaB signal pathway. The expression of genes involved in the NF-kappaB signal pathway, including cyclin D1, inhibitors of kappaBs (I kappaB alpha), and phosphorylated Akt (p-AKT), were measured. In addition, transient transfection assays with the 5X NF-kappaB consensus sequence promoter was also used to test the effects of gum mastic.. Gum mastic inhibited PC-3 cell growth and blocked the PC-3 cell cycle in the G1 phase. Gum mastic also suppressed NF-kappaB activity in the PC-3 cells. The expression of cyclin D1, a crucial cell cycle regulator and an NF-kappaB downstream target gene, was reduced as well. Moreover, gum mastic decreased the p-AKT protein level and increased the I kappa B alpha protein level.. Gum mastic inhibited the proliferation and blocked the cell cycle progression in PC-3 cells by suppressing NF-kappaB activity and the NF-kappaB signal pathway.

Topics: Androgens; Antineoplastic Agents, Phytogenic; Cell Nucleus; Cell Proliferation; Cyclin D1; Humans; Male; Mastic Resin; NF-kappa B; Oncogene Protein v-akt; Prostatic Neoplasms; Resins, Plant; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction

Cyclin D1 is a critical regulator of androgen-dependent transcription and cell cycle progression in prostate cancer cells. Despite the influence of D-type cyclins on prostate cancer proliferation, few studies have examined the expression of cyclin D1 in localised tumours or challenged its relevance to disease progression. Cyclin D1 status was characterised using immunohistochemistry in 38 non-neoplastic prostate samples, 138 primary human prostate carcinomas, and three lymph node metastatic specimens. Relevance of cyclin D1 to preoperative prostate-specific antigen (PSA) levels, Ki-67 index, and p21Cip1 status was also examined. Cyclin D1-positive phenotype was increased in primary carcinoma compared to non-neoplastic tissue, and was evident in all lymph node metastases cases. Interestingly, at least three distinct localisation patterns were observed in the cyclin D1-positive cohort, wherein cytoplasmic localisation was identified in a large fraction, and this pattern was predominant in lower grade tumours. Relevance of altered cyclin D1 status was observed, wherein cyclin D1-positive tumours were associated with low preoperative PSA levels, consistent with in vitro reports that cyclin D1 may alter the expression of this tumour marker. Moreover, tumours with predominantly cytoplasmic cyclin D1 showed the lowest Ki-67 index, whereas nuclear cyclin D1 was associated with higher grade, elevated Ki-67, and increased nuclear p21Cip1. These data demonstrate that differential cyclin D1 status may influence clinicopathological parameters, and reveal new insight as to the regulation and potential consequence of cyclin D1 expression in prostate cancer.

Topics: Adenocarcinoma; Biomarkers, Tumor; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Disease Progression; Humans; Ki-67 Antigen; Lymphatic Metastasis; Male; Neoplasm Metastasis; Prostate-Specific Antigen; Prostatic Neoplasms

To investigate the mechanisms of androgen-independent growth in prostate cancer (PCa), we established two PCa cell lines, LN-REC4 and LNCaP-SF, from the androgen-dependent PCa cell line, LNCaP.. LN-Pre and LN-REC4 cells were generated from LNCaP tumors grown on intact and castrated severe combined immunodeficient (SCID) mouse, respectively. After we cultured LNCaP cells under a steroid-free conditions for 6 months in vitro, LNCaP-SF cells were established. To show the character of LN-REC4 and LNCaP-SF cells, androgen sensitivity was investigated through examination of growth rate, and prostate-specific antigen (PSA), androgen receptor (AR), p21, p27, and cyclin D1 expression were examined by reverse transcription-polymerase chain reaction (RT-PCR). Angiogenesis assay in vitro was carried out using conditioned medium. To examine the expression level of vascular endothelial growth factor (VEGF), RT-PCR and enzyme-linked immunosorbent assay were also done.. LN-REC4 cells proliferated better than LNCaP cells in castrated mice and did well irrespective of castration, although responsiveness for androgen of LN-REC4 cells attenuated less than that of LNCaP cells in vitro. LNCaP-SF cells in castrated mice proliferated more rapidly than in normal mice. The PSA expression in LNCaP-SF cells was still induced by androgen. Expression of AR, p21, p27 and cyclin D1 were not changed in LN-REC4 and LNCaP-SF cells. Angiogenesis assay showed that both cells stimulated angiogenesis. LN-REC4 induced VEGF more than LNCaP and LN-Pre cells. However, expression of VEGF per cell in LNCaP-SF was lower than LNCaP cells, suggesting that other factors might be involved in angiogenesis. These cell lines might be a useful tool for researching androgen-independent growth and treatments of recurred PCa.

Topics: Androgens; Animals; Biomarkers, Tumor; Cell Line, Tumor; Cell Proliferation; Culture Media, Conditioned; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Mice, SCID; Neoplasm Transplantation; Proliferating Cell Nuclear Antigen; Prostate-Specific Antigen; Prostatic Neoplasms; Receptors, Androgen; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; Vascular Endothelial Growth Factor A

Apigenin, a dietary plant-flavonoid has shown anti-proliferative and anticancer properties, however the molecular basis of this effect remains to be elucidated. We studied the molecular events of apigenin action in human prostate cancer cells. Treatment of LNCaP and PC-3 cells with apigenin causes G0-G1 phase arrest, decrease in total Rb protein and its phosphorylation at Ser780 and Ser807/811 in dose- and time-dependent fashion. Apigenin treatment caused increased phosphorylation of ERK1/2 and JNK1/2 and this sustained activation resulted in decreased ELK-1 phosphorylation and c-FOS expression thereby inhibiting cell survival. Use of kinase inhibitors induced ERK1/2 phosphorylation, albeit at different levels, and did not contribute to cell cycle arrest in comparison to apigenin treatment. Despite activation of MAPK pathway, apigenin caused a significant decrease in cyclin D1 expression that occurred simultaneously with the loss of Rb phosphorylation and inhibition of cell cycle progression. The reduced expression of cyclin D1 protein correlated with decrease in expression and phosphorylation of p38 and PI3K-Akt, which are regulators of cyclin D1 protein. Interestingly, apigenin caused a marked reduction in cyclin D1, D2 and E and their regulatory partners CDK 2, 4 and 6, operative in G0-G1 phase of the cell cycle. This was accompanied by a loss of RNA polymerase II phosphorylation, suggesting the effectiveness of apigenin in inhibiting transcription of these proteins. This study provides an insight into the molecular mechanism of apigenin in modulating various tyrosine kinases and perturbs cell cycle progression, suggesting its future development and use as anticancer agent in humans.

Topics: Antineoplastic Agents, Phytogenic; Apigenin; Cell Cycle; Cyclin D1; Humans; Male; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Phosphorylation; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Retinoblastoma Protein; Tumor Cells, Cultured

Development of prostate cancer prevention strategies is an important priority to overcome high incidence, morbidity, and mortality. Recently, we showed that Nexrutine, an herbal extract, inhibits prostate cancer cell proliferation through modulation of Akt and cAMP-responsive element binding protein (CREB)-mediated signaling pathways. However, it is unknown if Nexrutine can be developed as a dietary supplement for the prevention of prostate cancer. In this study, we used the transgenic adenocarcinoma of mouse prostate (TRAMP) model to examine the ability of Nexrutine to protect TRAMP mice from developing prostate cancer.. Eight-week-old TRAMP mice were fed with pelleted diet containing 300 and 600 mg/kg Nexrutine for 20 weeks. Efficacy of Nexrutine was evaluated by magnetic resonance imaging at 18 and 28 weeks of progression and histologic analysis of prostate tumor or tissue at the termination of the experiment. Tumor tissue was analyzed for modulation of various signaling molecules.. We show that Nexrutine significantly suppressed palpable tumors and progression of cancer in the TRAMP model. Expression of total and phosphorylated Akt, CREB, and cyclin D1 was significantly reduced in prostate tissue from Nexrutine intervention group compared with tumors from control animals. Nexrutine also inhibited cyclin D1 transcriptional activity in androgen-independent PC-3 cells. Overexpression of kinase dead Akt mutant or phosphorylation-defective CREB inhibited cyclin D1 transcriptional activity.. The current study shows that Nexrutine-mediated targeting of Akt/CREB-induced activation of cyclin D1 prevents the progression of prostate cancer. Expression of CREB and phosphorylated CREB increased in human prostate tumors compared with normal tissue, suggesting their potential use as prognostic markers.

Topics: Adenocarcinoma; Animals; Cell Proliferation; Cyclic AMP Response Element-Binding Protein; Cyclin D1; Dietary Supplements; Disease Models, Animal; Male; Mice; Mice, Transgenic; Phosphorylation; Plant Extracts; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Receptors, Tumor Necrosis Factor, Member 25

The receptor tyrosine kinase ErbB-2 plays an important role in the regulation of growth factor-induced signal transduction cascades in the epithelium, and ErbB-2 is frequently overexpressed in epithelial tumors. Our previous studies on clinical prostate cancer specimens indicated that ErbB-2 expression was increased in patients undergoing hormone ablation therapy. We had also shown that the critical cell cycle regulatory gene cyclin D1 and its promoter were targets of proliferative signaling in prostate cancer cell lines, and that cyclin D1 was required for ErbB-2-induced mammary tumorigenesis. In the current studies, we found that increased ErbB-2 membrane expression correlated with increased nuclear cyclin D1 staining in clinical prostate cancer specimens, and that expression of ErbB-2 was capable of inducing cell cycle progression in human prostate cancer cell lines. We further showed that ErbB-2 induced the cyclin D1 promoter in DU145 cells, and that small interfering RNA knockdown of cyclin D1 protein levels blocked a significant proportion of the heregulin-induced cell cycle progression in LNCaP cells. Probasin promoter-targeted expression of an activated ErbB-2 isoform induced cyclin D1 expression in the mouse prostate, commensurate with prostate intraepithelial neoplasia. Together, these in vitro and in vivo studies identify cyclin D1 as a critical downstream target of ErbB-2 in the prostate epithelium, both of which are possible therapeutic targets for cancer intervention. Furthermore, our novel mouse model provides a useful platform for ongoing in vivo investigations of ErbB-2 signaling in the prostate epithelium.

Topics: Animals; Cell Cycle; Cell Growth Processes; Cell Line, Tumor; Cyclin D1; Epithelial Cells; Gene Expression Regulation, Neoplastic; Genes, bcl-1; Humans; Male; Mice; Promoter Regions, Genetic; Prostatic Intraepithelial Neoplasia; Prostatic Neoplasms; Receptor, ErbB-2; RNA, Small Interfering

We have shown previously that interleukin-8 (IL-8) and IL-8 receptor expression is elevated in tumor cells of human prostate biopsy tissue and correlates with increased cyclin D1 expression. Using PC3 and DU145 cell lines, we sought to determine whether IL-8 signaling regulated cyclin D1 expression in androgen-independent prostate cancer (AIPC) cells and to characterize the signaling pathways underpinning this response and that of IL-8-promoted proliferation. Administration of recombinant human IL-8 induced a rapid, time-dependent increase in cyclin D1 expression in AIPC cells, a response attenuated by the translation inhibitor cycloheximide but not by the RNA synthesis inhibitor, actinomycin D. Suppression of endogenous IL-8 signaling using neutralizing antibodies to IL-8 or its receptors also attenuated basal cyclin D1 expression in AIPC cells. Immunoblotting using phospho-specific antibodies confirmed that recombinant human IL-8 induced rapid time-dependent phosphorylation of Akt and the mammalian target of rapamycin substrate proteins, 4E-BP1 and ribosomal S6 kinase, resulting in a downstream phosphorylation of the ribosomal S6 protein (rS6). LY294002 and rapamycin each abrogated the IL-8-promoted phosphorylation of rS6 and attenuated the rate of AIPC cell proliferation. Our results indicate that IL-8 signaling (a) regulates cyclin D1 expression at the level of translation, (b) regulates the activation of proteins associated with the translation of capped and 5'-oligopyrimidine tract transcripts, and (c) activates signal transduction pathways underpinning AIPC cell proliferation. This study provides a molecular basis to support the correlation of IL-8 expression with that of cyclin D1 in human prostate cancer and suggests a mechanism by which this chemokine promotes cell proliferation.

Topics: Adaptor Proteins, Signal Transducing; Androgens; Cell Cycle Proteins; Cell Proliferation; Cyclin D1; Enzyme Activation; Gene Expression Regulation, Neoplastic; Humans; Interleukin-8; Male; Models, Biological; Phosphatidylinositol 3-Kinases; Phospholipase D; Phosphoproteins; Phosphorylation; Prostatic Neoplasms; Protein Biosynthesis; Protein Kinase C; Protein Kinases; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases; Signal Transduction; TOR Serine-Threonine Kinases

Propolis is a resinous substance collected by bees (Apis mellifera) from various tree buds which they then use to coat hive parts and to seal cracks and crevices in the hive. Propolis, a known ancient folk medicine, has been extensively used in diet to improve health and to prevent disease. In the present study, we have evaluated the effects of ethanolic extracts of Brazilian propolis group l2 and bud resins of botanical origin (B. dracunculifolia), and propolis group 3 on proliferation of metastasis (DU145 and PC-3) and primary malignant tumor (RC58T/h/SA#4)-derived human prostate cancer cells. The strongest inhibition was observed in propolis group 3 (sample #3) extracts whereas moderate growth inhibition was observed in human prostate epithelial cells. In the RC58T/h/SA#4 cells, resins of botanical origin of propolis group 12 (sample #1) and propolis group 12 (sample #2) induced growth inhibition that was associated with S phase arrest whereas propolis group 3 (sample #3) induced growth inhibition that was associated with G2 arrest. The mechanisms of cell cycle effects of propolis were investigated. The resins of botanical origin of propolis group 12 and propolis group 12 showed similar inhibition of cyclin D1, CDK4 and cyclin B1 expression. Propolis group 3 showed higher induction of p21 expression but no inhibition of cyclin D1, CDK4 and cyclin B1 expression. The results obtained here demonstrate that the Brazilian propolis extracts have significant inhibitory effect on proliferation of human prostate cancer cells. Inhibition was achieved through regulation of protein expression of cyclin D1, B1 and cyclin dependent kinase (CDK) as well as p21. Our results indicate that the Brazilian propolis extracts show promise as chemotherapeutic agents as well as preventive agents against prostate cancer.

Topics: Antineoplastic Agents; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin B; Cyclin B1; Cyclin D1; Cyclin-Dependent Kinase 4; Ethanol; Gene Expression Regulation, Neoplastic; Humans; Male; Plant Extracts; Propolis; Prostatic Neoplasms

It is becoming clear that frequent epigenetic silencing of tumor suppressor genes could be responsible for the development of cancer in various organs. Several recent reports suggest that suppression of RASSF1A is associated with the advanced grade and stage of prostate cancer and many other cancers. In this investigation, we demonstrated that, mahanine, a plant derived carbazole alkaloid, induced RASSF1A expression in both androgen-responsive (LNCaP) and androgen-negative (PC3) prostate cancer cells by inhibiting DNA methyltransferase (DNMT) activity. Mahanine-induced expression of RASSF1A in turn significantly reduced cyclin D1 but not other cyclins. To understand the inverse relationship between RASSF1A and cyclin D1, we observed that mahanine treatment down-regulates cyclin D1 and addition of RASSF1A siRNA prevented this inhibition. This study show for the first time that mahanine can reverse an epigenetically silenced gene, RASSF1A in prostate cancer cells by inhibiting DNMT activity that in turn down-regulates a key cell cycle regulator, cyclin D1. Mahanine therefore, promises an encouraging therapeutic choice for advanced prostatic cancer.

Topics: Alkaloids; Carbazoles; Cell Line, Tumor; Cyclin D1; DNA (Cytosine-5-)-Methyltransferases; Dose-Response Relationship, Drug; Down-Regulation; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Male; Prostatic Neoplasms; Tumor Suppressor Proteins

The cyclin D1 oncogene encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the Rb protein and promotes progression through G(1) to S phase of the cell cycle. Several prostate cancer cell lines and a subset of primary prostate cancer samples have increased cyclin D1 protein expression. However, the relationship between cyclin D1 expression and prostate tumor progression has yet to be clearly characterized. This study examined the effects of manipulating cyclin D1 expression in either human prostatic epithelial or stromal cells using a tissue recombination model. The data showed that overexpression of cyclin D1 in the initiated BPH-1 cell line increased cell proliferation rate but did not elicit tumorigenicity in vivo. However, overexpression of cyclin D1 in normal prostate fibroblasts (NPF) that were subsequently recombined with BPH-1 did induce malignant transformation of the epithelial cells. The present study also showed that recombination of BPH-1 + cyclin D1-overexpressing fibroblasts (NPF(cyclin D1)) resulted in permanent malignant transformation of epithelial cells (BPH-1(NPF-cyclin D1) cells) similar to that seen with carcinoma-associated fibroblasts (CAF). Microarray analysis showed that the expression profiles between CAFs and NPF(cyclin D1) cells were highly concordant including cyclin D1 up-regulation. These data indicated that the tumor-promoting activity of cyclin D1 may be tissue specific.

Topics: Animals; Cell Line; Cyclin D1; Disease Progression; Epithelial Cells; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Mice, SCID; Neoplasm Invasiveness; Organ Specificity; Prostatic Neoplasms; Rats; Stromal Cells; Transfection; Transplantation, Heterologous; Tumor Cells, Cultured

Blockade of androgen activity is a major effective therapy for advanced prostate cancer. Estrogen analogs have been used for prostate cancer therapy for years presumably by inhibiting testosterone biosyntheses, but with considerable adverse events due to their classic estrogenic activity. With the discovery of the estrogen receptor (ER) beta and its presence in prostate tumor cells, evaluation of estrogen analogs with less classic estrogenic activity in prostate cancer therapy is emerging.. The effects of 17alpha-estradiol (alphaE2), a stereo-isomer of 17beta-estradiol (betaE2), on dihydrotestosterone (DHT)-induced cell growth and gene expressions were examined in androgen-dependent LAPC-4 prostatic tumor cells and in LAPC-4 xenograft animals, and compared to those of betaE2.. Both alphaE2 and betaE2 attenuated DHT induction of PSA gene expression, cell proliferation, and cell growth in cultured LAPC-4 cells. The inhibition of cell proliferation was associated with a blockade of DHT-induced cyclin A and cyclin D1 expression by alphaE2 and betaE2. In LAPC-4 xenograft mice, alphaE2 significantly inhibited tumor growth without altering the plasma testosterone level, while betaE2 failed to inhibit tumor growth even though it significantly inhibited PSA gene expression.. alphaE2 is an effective agent for inhibition of DHT-induced PSA, cyclin A, cyclin D1 gene expression, and cell proliferation in LAPC-4 cells, and tumor growth in LAPC-4 xenograft mice.

Topics: Animals; Blotting, Western; Cell Growth Processes; Cell Line, Tumor; Cyclin A; Cyclin D1; Dihydrotestosterone; Estradiol; Gene Expression; Humans; Male; Mice; Mice, SCID; Neoplasms, Hormone-Dependent; Prostate-Specific Antigen; Prostatic Neoplasms; Random Allocation; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stereoisomerism; Xenograft Model Antitumor Assays

Nonsteroidal anti-inflammatory drugs mediate anticancer effects by modulating cyclooxygenase-2 (COX-2)-dependent and/or COX-2-independent mechanism(s); however, the toxicity issue is a concern with single agents at higher doses. In this study, we determined the combined effect of celecoxib, a COX-2 inhibitor, along with exisulind (sulindac sulfone/Aptosyn) at low doses in prostate cancer.. We used a sequential regimen of N-methyl-N-nitrosourea + testosterone to induce prostate cancer in Wistar-Unilever rats. Following carcinogen treatment, celecoxib and exisulind individually and their combination at low doses were given in NIH-07 diet for 52 weeks. We determined the incidence of prostatic intraepithelial neoplasia, adenocarcinomas, rate of tumor cell proliferation, and apoptosis. Immunohistochemical and Western blot analysis were done to determine COX-2, epidermal growth factor receptor (EGFR), Akt, androgen receptor, and cyclin D1 expression. Serum prostaglandin E2 and tumor necrosis factor-alpha levels were determined using enzyme immunoassay/ELISA assays.. The rats that received celecoxib in combination with exisulind at low doses showed a significant decrease in prostatic intraepithelial neoplasia and adenocarcinomas as well as an enhanced rate of apoptosis. An overall decrease in COX-2, EGFR, Akt, androgen receptor, and cyclin D1 expression was found associated with tumor growth inhibition. Reduced serum levels of COX-2 protein, prostaglandin E2, and tumor necrosis factor-alpha indicated anti-inflammatory effects. A strong inhibition of total and phosphorylated form of EGFR (Tyr(992) and Tyr(845)) and Akt (Ser(473)) was significant in rats given with these agents in combination.. In this study, we show for the first time that the combination of celecoxib with exisulind at low doses could prevent prostate carcinogenesis by altering key molecular events.

Topics: Animals; Anticarcinogenic Agents; Antineoplastic Combined Chemotherapy Protocols; Celecoxib; Cyclin D1; Cyclooxygenase 2; Cyclooxygenase Inhibitors; ErbB Receptors; Humans; Male; Methylnitrosourea; Prostatic Neoplasms; Pyrazoles; Rats; Rats, Wistar; Receptors, Androgen; Sulfonamides; Sulindac; Testosterone

To investigate the effects of lycopene on proliferation of human prostate cancer cell line PC-3 and explore its effecting mechanism.. The proliferation of PC-3 cells were analyzed by MTT and H3-TdR incorporation. The effects of cell cycle and apoptosis of synchronized cells were observed through flow cytometry. RT-PCR methods were used to explore the mRNA expression level of cyclin D1, bcl-2, bax.. The growth and DNA synthesis of PC-3 cell were inhibited with the lycopene concentration increased, and lycopene also could change the cell cycle distribution, i. e. increasing the proportion of G0/G1 phase and descending the proportion of S and G2/M phase, and induce the apoptosis. RT-PCR analysis showed that the mRNA expression level of cyclin D1 and bcl-2 were down regulated, while the level of bax was up regulated.. Lycopene can induce apoptosis of PC-3, change the cell cycle distribution and downregulate the expression of cyclin D1 and bcl-2 and upregulate the expression of bax and then restrain cell proliferation.

Topics: Antineoplastic Agents, Phytogenic; bcl-2-Associated X Protein; Carotenoids; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Humans; Lycopene; Male; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger

Cyclin D1 is a multifaceted regulator of both transcription and cell-cycle progression that exists in two distinct isoforms, cyclin D1a and D1b. In the prostate, cyclin D1a acts through discrete mechanisms to negatively regulate androgen receptor (AR) activity and thus limit androgen-dependent proliferation. Accordingly, cyclin D1a is rarely overexpressed in prostatic adenocarcinoma and holds little prognostic value in this tumor type. However, a common polymorphism (A870) known to facilitate production of cyclin D1b is associated with increased prostate cancer risk. Here we show that cyclin D1b is expressed at high frequency in prostate cancer and is up-regulated in neoplastic disease. Furthermore, our data demonstrate that, although cyclin D1b retains AR association, it is selectively compromised for AR regulation. The altered ability of cyclin D1b to regulate the AR was observed by using both in vitro and in vivo assays and was associated with compromised regulation of AR-dependent proliferation. Consistent with previous reports, expression of cyclin D1a inhibited cell-cycle progression in AR-dependent prostate cancer cells. Strikingly, cyclin D1b significantly stimulated proliferation in this cell type. AR-negative prostate cancer cells were nonresponsive to cyclin D1 (a or b) expression, indicating that defects in AR corepressor function yield a growth advantage specifically in AR-dependent cells. In summary, these studies indicate that the altered AR regulatory capacity of cyclin D1b contributes to its association with increased prostate cancer risk and provide evidence of cyclin D1b-mediated transcriptional regulation.

Topics: Androgens; Cell Proliferation; Cyclin D1; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Male; Polymorphism, Genetic; Promoter Regions, Genetic; Prostate-Specific Antigen; Prostatic Neoplasms; Protein Isoforms; Receptors, Androgen; Repressor Proteins; Transcription, Genetic

We demonstrate here for the first time novel positive and negative effects of the FLICE-like inhibitory protein (FLIP) on human prostate cancer cell survival. A proteaosome inhibitor, MG132, mediated cell cycle arrest at G2/M and apoptosis through p38 activation. Interestingly, FLIP was stabilized by MG132 and interacted with Raf-1, resulting in enhancement of p38 signals and cytotoxicity. In contrast, overexpression of FLIP inhibited ubiquitylation and proteasomal degradation of beta-catenin, resulting in increase of the target gene cyclin D1, colony formation and invasive activity. Immunohistochemical analysis and in vitro experiments in primary culture showed FLIP to be overexpressed, statistically associated with expression of beta-catenin/cyclin D1 in metastatic cells, the FLIP/beta-catenin/cyclin D1 signals contributing to colony formation and invasion, which were canceled by FLIP knock down. In contrast, MG132-induced cytotoxicity including apoptosis was strongly inhibited by reduction of FLIP. Taken together, the results indicate that FLIP plays an important role in development of metastatic prostate cancer by inhibiting proteasomal degradation of beta-catenin, whereas it is mainly involved in proteasome inhibitior-mediated cell cycle arrest and apoptosis through activating the Raf-1/p38 pathway. Furthermore, proteasome inhibitors may be effective drugs for advanced prostate cancers overexpressing FLIP.

Topics: Apoptosis; beta Catenin; CASP8 and FADD-Like Apoptosis Regulating Protein; Cell Cycle; Cell Line, Tumor; Cell Survival; Cyclin D1; Cysteine Proteinase Inhibitors; Humans; Immunohistochemistry; Immunoprecipitation; Intracellular Signaling Peptides and Proteins; Leupeptins; Male; Prostatic Neoplasms; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins c-raf; Reverse Transcriptase Polymerase Chain Reaction

The G1 regulators of the cell cycle, cyclin D(1) and D(3), have been implicated in the regulation of Schwann cell proliferation and differentiation. The purpose of this study is to evaluate cyclin D(1) and D(3) protein expression and the corresponding clinical characteristics of vestibular schwannomas.. Tissue sections of 15 sporadic vestibular schwannomas were prepared. Immunohistochemical analysis of the vestibular schwannomas was performed with anticyclin D(1) and anticyclin D(3) antibodies. The immunoreactivity was evaluated in comparison with adjacent vestibular nerves. Tissue sections of breast carcinoma and prostate carcinoma were used as positive controls for cyclin D(1) and D(3) staining, respectively. Patient demographics, tumor characteristics, and cyclin D expression were reviewed, and statistical analysis was performed.. While the breast carcinoma control expressed abundant cyclin D(1) protein, none of the 15 vestibular schwannomas showed detectable cyclin D(1) staining. In contrast, seven of 15 vestibular schwannomas stained positive for the cyclin D(3) protein. Cyclin D(3) staining was taken up in the nucleus of schwannoma tumor cells in greater proportion than Schwann cells of adjacent vestibular nerve. Although sample size was small, no significant difference in the average age of presentation, tumor size, and male to female ratios for the cyclin D(3)(+) or cyclin D(3)(-) groups was found.. The Cyclin D(1) protein does not appear to play a prominent role in promoting cell cycle progression in vestibular schwannomas. In contrast, cyclin D(3) expression was seen in nearly half of the tumors examined, suggesting that it may have a growth-promoting role in some schwannomas. Further studies are needed to define its cellular mechanism.

Topics: Antibodies, Neoplasm; Biomarkers, Tumor; Breast Neoplasms; Carcinoma; Cyclin D1; Cyclin D3; Cyclins; Diagnosis, Differential; Female; Humans; Immunohistochemistry; Male; Neuroma, Acoustic; Prostatic Neoplasms

Zanthoxyli Fructus belongs to the family of oranges and is used as a seasoning in Asian countries including Japan. This study found that a water extract of Zanthoxyli Fructus possessed anti-tumor activity against a wide variety of cancer cells including those from prostate (LNCaP, DU145, PC-3), breast (MCF-7, T47D, MDA-MB231), lung (NCI-H460, -H520), as well as leukemia (HL-60, NB4, Jurkat) in vitro, as measured by the trypan blue exclusion test. Importantly, Zanthoxyli Fructus slowed the proliferation of LNCaP, DU145, and MDA-MB231 cells present as xenografts in BALB/c nude mice without adverse effects. Further studies explored the molecular mechanism by which Zanthoxyli Fructus inhibited the proliferation of androgen-dependent human prostate cancer LNCaP cells because Zanthoxyli Fructus possessed the strongest anti-tumor activity against these cells. Zanthoxyli Fructus blocked androgen receptor (AR) signaling in conjunction with down-regulation of nuclear levels of AR and induced apoptosis of these cells, as measured by the reporter assay, Western blot analysis, and TUNEL assay, respectively. As expected, Zanthoxyli Fructus also decreased the level of the AR-target molecule, prostate-specific antigen in these cells. Furthermore, Zanthoxyli Fructus inhibited AKT kinase and down-regulated levels of cyclin D1 protein, as measured by the AKT kinase assay with GSK-3alpha/beta as a substrate and Western blot analysis, respectively. Taken together, Zanthoxyli Fructus might be useful as an adjunctive therapeutic agent for the treatment of individuals with a variety of cancer types.

Topics: Androgen Receptor Antagonists; Animals; Apoptosis; Breast Neoplasms; Cell Growth Processes; Cyclin D1; Down-Regulation; HL-60 Cells; Humans; Jurkat Cells; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasms, Hormone-Dependent; Phosphorylation; Plant Extracts; Promoter Regions, Genetic; Prostate-Specific Antigen; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Receptors, Androgen; Signal Transduction; Transfection; Zanthoxylum

Accumulating evidence suggests that the androgen receptor (AR) may play an important role in the development and progression of prostate cancer. To find new, useful compounds that effectively may attenuate the function of AR in prostate cancer cells, the authors investigated the effect of gum mastic, a natural resin, on AR activity. An androgen-responsive prostate cancer cell line LNCaP was used as a model for this study. Gene transfer, reverse transcriptase-polymerase chain reaction analysis, electrophoretic mobility shift assay, and Western blot analysis were used to test the effect of gum mastic on the expression and function of the AR. To demonstrate the inhibitory effect of gum mastic on the function of the AR, the expression of androgen-regulated genes, including prostate-specific antigen (PSA), human kallikrein 2 (hK2), and NKX3.1 were measured. In addition, transient transfection assays with the PSA promoter and the AR promoter also were used to test the effects of mastic. The results showed that gum mastic inhibited the expression of the AR at the transcriptional level, resulting in the down-regulation of both AR messenger RNA and protein levels. Therefore, the function of the AR was inhibited, as reflected by the reduced expression of NKX3.1 and PSA and by androgen-stimulated growth. Because gum mastic exhibited a strong in vitro potency to attenuate the expression and function of the AR, further investigation will be required to determine whether this naturally occurring substance has in vivo potency to inhibit prostate cancer development.

Topics: Androgens; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Disease Progression; Electrophoretic Mobility Shift Assay; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Male; Mastic Resin; Neoplasms, Hormone-Dependent; Promoter Regions, Genetic; Prostate-Specific Antigen; Prostatic Neoplasms; Protein Binding; Receptors, Androgen; Resins, Plant; Reverse Transcriptase Polymerase Chain Reaction; Tissue Kallikreins; Transcription Factors; Transcription, Genetic

Cyclooxygenase-2 (COX-2) plays an important role in tumor development and progression. Inconsistent reports on the expression of COX-2 in early versus advanced prostate cancer raised the question on whether COX-2 inhibition affects prostate carcinogenesis. Evidence from recent studies indicates that prostate carcinogenesis depends on the altered expression of several factors including androgen receptor signaling, proinflammatory, and cell cycle regulatory genes. Very often, the outcome of androgen ablation treatment is not effective and, eventually, the cancer becomes androgen independent followed by activation of several survival genes and transcription factors. Most importantly, the extent of the influence of COX-2 on the regulation of the androgen receptor, cyclin D1, and other factors involved in cancer growth is not known. Using RNA interference-mediated COX-2 inhibition in metastatic prostate cancer cells, this study has shown that the silencing of COX-2 at the mRNA level can induce cell growth arrest and down-regulate androgen receptor and cyclin D1. We have further shown for the first time that COX-2 knockdown prostate cancer cells depict morphologic changes associated with enhanced expression of differentiation markers, particularly the neuronal protein synaptophysin along with activation of p21((Waf1/Cip1)) and p27((Kip1)). In summary, our findings determined the role of COX-2 in prostate carcinogenesis and its control on COX-2-independent targets. Second, abrogation of COX-2 and activation of synaptophysin provide evidence for the control of COX-2 on the expression of a neuronal protein. Finally, our findings provide evidence of COX-2-independent targets promoting cell growth arrest and differentiation in cells lacking COX-2 expression at the mRNA level.

Topics: Animals; Cell Cycle; Cell Differentiation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclooxygenase 2; Cyclooxygenase Inhibitors; Humans; Male; Mice; Mice, Inbred C57BL; Prostatic Neoplasms; Receptors, Androgen; RNA Interference; RNA, Small Interfering; Synaptophysin; Transfection; Tumor Cells, Cultured

Prostate cancer is one of the most frequently diagnosed cancers in human males. Progression of these tumors is facilitated by autocrine/paracrine growth factors which activate critical signaling cascades that promote prostate cancer cell growth, survival and migration. Among these, Ras pathways have a major role. Here we examined the effect of the Ras inhibitor S-trans, trans-farnesylthiosalicylic acid (FTS), on growth and viability of androgen-dependent and androgen-independent prostate cancer cells. FTS downregulated Ras, inhibited signaling to Akt and reduced the levels of cell-cycle regulatory proteins including cyclin D1, p-RB, E2F-1 and cdc42 in LNCaP and PC3 cells. Consequently the anchorage-dependent and anchorage-independent growth of LNCaP and PC3 cells were inhibited. FTS also induced apoptotic cell death which was inhibited by the broad-spectrum caspases inhibitor, Boc-asp-FMK. Our study demonstrated that androgen-dependent and androgen-independent prostate cancer cells require active Ras for growth and survival. Ras inhibition by FTS results in growth arrest and cell death. FTS may be qualified as a potential agent for the treatment of prostate cancer.

Topics: Amino Acid Chloromethyl Ketones; Androgens; Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin D1; Dose-Response Relationship, Drug; Down-Regulation; E2F1 Transcription Factor; Farnesol; Humans; Male; Myotonin-Protein Kinase; Prostatic Neoplasms; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-akt; ras Proteins; Salicylates; Serine Proteinase Inhibitors; Signal Transduction

To observe the effect of KLF6 on prostate cancer cell line PC-3 by transgenic method.. We obtained KLF6 cDNA by RT-PCR method from the liver cell, transfected plasmid pEGFP-C, recombinated with KLF6 into PC-3 cells, and used them as a transfection group and a control group. MTT, flow cytometer and immunocytochemical methods were used to observe the effect of anti-oncogene wild type KLF6 on prostate cancer cell line PC-3 by transgenic method for 48 hours.. After transfected into PC-3 cells, KLF6 enhanced growth suppression, (30.0 +/- 5.4)% in the transfection group and 0% in the control, P < 0.01, apoptosis, (24.3 +/- 2.3)% in the transfection group and (5.2 +/- 0.7)% in the control, P < 0.01, the down-regulation of the expression of cyclin D1, (25.3 +/- 3.7)% in the transfection group and (38.5 +/- 4.6)% in the control, P < 0.05 and Bcl-2, (18.7 +/- 3.2)% in the transfection group, and (41.8 +/- 5.9)% in the control, P < 0.01 in PC-3 cells. It also decreased the ratio of the cell phase G2/M, increased the ratio of G0/G1 from (58.6 +/- 7.3)% in the control to (80.0 +/- 9.8)% in the transfection group, P < 0.05.. PC-3 cells transfected with wild type KLF6 can enhance its growth suppression and apoptosis. It shows great potential for the gene therapy of androgen-independent carcinoma of the prostate.

Topics: Apoptosis; Cell Cycle; Cell Line, Tumor; Cyclin D1; Down-Regulation; Flow Cytometry; Humans; Immunohistochemistry; Kruppel-Like Factor 6; Kruppel-Like Transcription Factors; Male; Prostatic Neoplasms; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; Transfection

Co-factor ACTR is frequently overexpressed and/or amplified in multiple types of tumors. The mechanism of its function in prostate cancer (CaP) is still unclear.. The effects of ACTR and androgen receptor (AR) depletion on cell proliferation and gene expression and their functions were analyzed in a panel of androgen-dependent and -independent CaP cells and CWR22 xenograft.. ACTR and AR, but not TIF2, are required for proliferation of androgen-dependent and -independent cells, and for tumor growth. While AR depletion inhibited the expression of cyclin D1, cyclin B, and cdc2, ACTR depletion reduced the expression of cyclin E and cdk2. In response to serum stimulation, AR and ACTR are recruited to the corresponding target gene promoters to activate their expression in androgen-independent manner.. These findings suggest that AR and ACTR may play important roles in androgen ablation resistance by controlling key cell cycle gene expression.

Topics: Androgens; Animals; CDC2-CDC28 Kinases; Cell Cycle Proteins; Cell Division; Cell Line, Tumor; Cyclin B; Cyclin D1; Cyclin E; Gene Expression Regulation, Neoplastic; Histone Acetyltransferases; Humans; Male; Mice; Neoplasm Transplantation; Nuclear Receptor Coactivator 2; Nuclear Receptor Coactivator 3; Promoter Regions, Genetic; Prostatic Neoplasms; Receptors, Androgen; Trans-Activators; Transplantation, Heterologous

Prostate cancer almost exclusively metastasizes to skeletal sites, indicating that the bone provides a favorable microenvironment for its localization and progression. A natural yet understudied factor in bone that could facilitate tumor localization is elevated extracellular calcium ([Ca2+]o). The present study found that elevated [Ca2+]o (2.5 mmol/L) enhanced proliferation of skeletal metastatic prostate cell lines (PC-3 and C4-2B), but not the nonskeletal metastatic, epithelial-derived prostate cell line LNCaP. The proliferative effect of elevated [Ca2+]o was associated with higher expression of the calcium-sensing receptor (CaSR), a heterotrimeric G-protein-coupled receptor that is the predominant cell-surface sensor for [Ca2+]o. Knockdown of the CaSR via RNA interference reduced cell proliferation in vitro and metastatic progression in vivo. CaSR signaling in PC-3 cells was evaluated by measuring the elevated [Ca2+]o-dependent inhibition of cyclic AMP accumulation, induced by either prostaglandin E2 or forskolin. Elevated [Ca2+]o stabilized expression of cyclin D1, a protein required for cell cycle transition. Furthermore, elevated [Ca2+]o triggered activation of the Akt signaling pathway and enhanced PC-3 cell attachment. Both pertussis toxin (a G-protein inhibitor) and LY294002 (an inhibitor of Akt signaling) reduced cell attachment. These data suggest that elevated [Ca2+]o following increased bone remodeling could facilitate metastatic localization of prostate cancer via the CaSR and the Akt signaling pathway. Taken together, [Ca2+]o is a candidate mediator of prostate cancer bone metastasis.

Topics: Bone Neoplasms; Calcium; Calcium Signaling; Cell Adhesion; Cell Growth Processes; Cell Line, Tumor; Cyclic AMP; Cyclin D1; Humans; Male; Neomycin; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Receptors, Calcium-Sensing; RNA Interference; Transfection

DACH-Ac-Pt [(1R,2R-diaminocyclohexane)-(trans-diacetato)-(dichloro)-platinum(IV)] is a novel cisplatin (CDDP) analog, and we have evaluated its potential activity in human prostate cancers.. Cytotoxic, biochemical pharmacologic, cell cycle, and Western blot evaluations were conducted with platinum agents to assess the role of p53 genotype and androgen-dependence status on cellular response.. CDDP and DACH-Ac-Pt were equiactive against mutant p53 and androgen-independent DU-145 or PC-3 tumor cells. In wild-type p53 cells, CDDP was threefold more potent against androgen-dependent LNCaP than isogenic androgen-independent LNCaP-LN3 cells. However, the analog was equipotent in these two wild-type p53 tumor models. The greater potency of DACH-Ac-Pt than CDDP in wild-type p53 cells was not due to increased cellular drug uptake or increased adduct levels, but correlated with a lower tolerance to DNA damage. The analog also activated the p53-p21(WAF1/CIP1) signal transduction pathway more efficiently in LNCaP and LNCaP-LN3 cells, and this induced G(1)-phase cell-cycle arrest. CDDP, in contrast, activated this pathway efficiently in LNCaP cells only. In addition, and compared to CDDP, DACH-Ac-Pt was more effective in inducing Bax and increasing the Bax/Bcl-2 ratios in both the tumor models.. DACH-Ac-Pt is highly effective against wild-type p53 LNCaP and its LN3 variant, and this activity is androgen-independent. The differential induction of p21(WAF1/CIP1) and increase in Bax/Bcl-2 ratios with CDDP and DACH-Ac-Pt in LNCaP-LN3 cells appear to be linked to the relative activity of the two agents against this model.

Topics: Androgens; Antineoplastic Agents; bcl-2-Associated X Protein; Cell Cycle; Cell Line, Tumor; Cisplatin; Cyclin D1; DNA Adducts; Genes, p53; Humans; Male; Neoplasm Metastasis; Organoplatinum Compounds; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Signal Transduction

The androgen receptor (AR) is a member of the nuclear receptor superfamily, the activity of which is critical for the development and progression of prostate cancer. We and others have previously demonstrated that cyclin D1 is a potent corepressor of the AR. Although cyclin D1 is suspected to recruit histone deacetylases to the AR complex, previous studies have demonstrated that this activity alone is insufficient for cyclin D1 function. Here, we uncover a novel, secondary means of cyclin D1-mediated repression, through modulation of AR amino-carboxy terminal interactions. We show that cyclin D1 predominantly binds the N-terminal domain of the AR, dependent on the AR 23FxxLF27 motif. Through this motif, cyclin D1 abrogates the ability of the AR N-terminal domain to interact with the C terminus. Secondary amino-terminal domain sites capable of fostering interaction with the C terminus were refractory to cyclin D1 action, indicating that the ability of cyclin D1 to modulate AR amino-carboxy terminal interactions is specific to 23FxxLF27. Deletion of the N-terminal cyclin D1 binding site severely compromised AR activity (due to loss of FxxLF) but unmasked a repressor action through interaction with the AR C terminus. In summary, these data reveal novel, unexpected mechanisms of cyclin D1 activity and demonstrate that this function of cyclin D1 is critical for AR modulation.

Topics: Adenocarcinoma; Amino Acid Motifs; Binding Sites; Cell Line, Tumor; Cyclin D1; Dose-Response Relationship, Drug; Genes, Reporter; Humans; Immunoblotting; Immunoprecipitation; Male; Plasmids; Prostatic Neoplasms; Protein Binding; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Receptors, Androgen; Transcription, Genetic; Two-Hybrid System Techniques

Regulation of nuclear receptor activity is the focus of numerous ongoing studies to develop novel therapies for the treatment of hormone-related cancer. Although cyclin D1 functions to control the activity of several nuclear receptors, the region(s) of the protein responsible for such transcriptional comodulation remain poorly defined. Herein, we map the region of cyclin D1 required for binding and repression of the androgen receptor (AR) to a central, exclusively alpha-helical domain. Deletion of this domain disrupted AR binding and corepressor activity. Further investigations showed that this domain is sufficient for AR interaction and possesses the ability to bind histone deacetylase 3. Strikingly, overexpression of this repressor region attenuates cell cycle progression in prostatic adenocarcinoma cells. The requirement of this domain for nuclear receptor repression was conserved with respect to thyroid hormone receptor beta-1, whereas cyclin D1 activation of the estrogen receptor occurred independently of the central region. Together, these data identify a minimal repression module within cyclin D1 and demonstrate that the coactivator and corepressor functions of cyclin D1 are distinct. In addition, our data suggest that properties of the cyclin D1 central domain could be exploited to develop novel prostate cancer therapeutics.

Topics: Amino Acid Sequence; Animals; Chlorocebus aethiops; COS Cells; Cyclin D1; Flow Cytometry; Genes, Reporter; Humans; Male; Models, Molecular; Molecular Sequence Data; Plasmids; Prostatic Neoplasms; Protein Conformation; Receptors, Androgen; Sequence Deletion; Transfection

An important factor implicated in tumor cell predisposition for invasion and metastasis is the malignancy-related upregulation of urokinase plasminogen activator receptor (uPAR). uPAR signals by activating different tyrosine kinases in different cells. We examined the effects of inhibiting uPAR signaling by inhibition of uPAR expression with antisense oligonucleotides (aODNs) in PC3 human prostate cancer cells and evaluated aODN effect in a mouse model of prostate cancer bone metastasis. Following uPAR aODN treatment, PC3 cells exhibited a strong decrease in uPAR expression, evaluated by flow cytometry and by polymerase chain reaction, and of FAK/JNK/Jun phosphorylation. The synthesis of cyclins A, B, D1 and D3 was inhibited, as shown by Western blotting, flow cytometry and polymerase chain reaction, and PC3 cells accumulated in the G2 phase of the cell cycle. PC3 cells' adhesion was unaffected, while proliferation and invasion of Matrigel were impaired. A total of 60 mice were subjected to intracardiac injection of PC3 cells and were randomly assigned to three groups: aODN (treated with 0.5 mg intraperitoneum/mouse/day), dODN (treated with the same amounts of a degenerated ODN) and control (injected with a saline solution). At 28 days after heart injection, mice were subjected to a digital scan of total body radiography, which revealed 80% reduction in mice affected by bone metastasis. The use of uPAR aODNs produced a substantial prophylactic effect against prostate cancer bone metastasis, which has to be ascribed to downregulation of uPAR expression.

Topics: Animals; Bone Neoplasms; Cell Adhesion; Cell Line, Tumor; Cell Proliferation; Cyclin A; Cyclin B; Cyclin B1; Cyclin D1; Cyclin D3; Cyclins; Extracellular Matrix; Genetic Therapy; Humans; Injections; Male; Mice; Mice, Nude; Neoplasm Transplantation; Oligonucleotides, Antisense; Prostatic Neoplasms; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Signal Transduction

Expression of the p27kip1 cell cycle inhibitor is downregulated in a wide range of carcinomas, yet it is rarely inactivated completely. Our recent studies of a mouse model of prostate carcinogenesis have revealed that cancer progression is enhanced by a two-fold reduction in p27kip1 gene dosage, but is unexpectedly inhibited by further decrease in p27kip1 activity. This paradoxical finding may explain the unusual features of p27kip1 downregulation in human cancer, and also suggests a potential route for therapeutic intervention.

Topics: Animals; Cell Cycle; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Disease Models, Animal; Disease Progression; Down-Regulation; Gene Dosage; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Mice, Transgenic; Mutation; Phenotype; Prostatic Neoplasms

Raf-1, a protein serine-threonine kinase, plays a critical role in mitogen-activated protein kinase kinase (MKK/MEK)- mitogen-activated protein kinase (extracellular signal-regulated kinase) (MAPK/ERK) pathways. We show here that systemically delivered novel cationic cardiolipin liposomes (NeoPhectin-AT) containing a small interfering RNA (siRNA) against Raf-1 silence the expression of Raf-1 in tumor tissues and inhibit tumor growth in xenograft model of human prostate cancer. The knockdown of Raf-1 expression by siRNA is also associated with down-regulation of cyclin D1 expression in vivo.

Topics: Cardiolipins; Cations; Cyclin D1; Down-Regulation; Gene Silencing; Humans; Liposomes; Male; Prostatic Neoplasms; Proto-Oncogene Proteins c-raf; RNA Interference; RNA, Small Interfering; Transplantation, Heterologous; Tumor Cells, Cultured

Cyclin-dependent kinase 6 (CDK6) binds to and is activated by cyclin D1 and thereby enhances the transition of cells through the G1 phase of the cell cycle. The present study indicates that, in human prostate cancer cells, CDK6 can also bind to the androgen receptor (AR) and stimulate its transcriptional activity in the presence of dihydrotestosterone. This effect of CDK6 does not require its kinase activity and is inhibited by cyclin D1 and p16INK4a. The T877A mutant of the AR frequently found in advanced cases of prostate cancer displays an exaggerated stimulation of transcriptional activity by CDK6. Androgen-sensitive LNCaP prostate cancer cells engineered to stably overexpress CDK6 display increased expression of the prostate-specific antigen and enhanced growth in the presence of dihydrotestosterone. CDK6 is present in the chromatin structure of these cells in association with the AR and the promoter region of the prostate-specific antigen gene. These findings suggest that CDK6 may play an important role in the development and/or progression of a subset of human prostate cancers by stimulating the activity of the AR.

Topics: Base Sequence; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinases; DNA, Neoplasm; Gene Expression; Humans; Male; Point Mutation; Prostatic Neoplasms; Receptors, Androgen; Signal Transduction; Transcription, Genetic; Transfection; Trinucleotide Repeats

BCL-2 overexpression occurs in many cancer types and is associated with chemoresistance and radioresistance. The mechanisms responsible for its aberrant expression are thought to be transcriptionally mediated but remain unclear. We examined the cell type-specific mechanism of BCL-2 gene transcription in various solid organ malignancies.. Regulation of BCL-2 gene transcription was examined in seven different human cancer cell lines including two pancreatic (MIA-PaCa-2, PANC-1), two prostate (LNCaP, PC-3), two lung (Calu-1, A549) and one breast (MCF-7) cancer cell line. Cells were treated with inhibitors of phosphatidylinositol-3 kinase (PI3K), MEK/ERK, and p38MAPK. The effect of mutation of a NF-kappaB site in the BCL-2 promoter was determined, as was the effect of inhibition of NF-kappaB function using a 26S proteasome inhibitor (bortezomib) on both BCL-2 transcription and induction of apoptosis.. BCL-2 expression varied both between and within tumor types; four of seven cell lines demonstrated high BCL-2 levels (MIA-PaCa-2, PC-3, Calu-1 and MCF-7). No signaling pathway was uniformly responsible for overexpression of BCL-2; however, mutation of the NF-kappaB site decreased BCL-2 promoter activity in all cell lines. Inhibition of NF-kappaB activity decreased BCL-2 protein levels independently of the signaling pathway involved in transcriptional activation of the BCL-2 gene.. Diverse signaling pathways variably regulate BCL-2 gene expression in a cell type-specific fashion. Therapy to decrease BCL-2 levels in various human cancers would be more broadly applicable if targeted to transcriptional activation rather than signal transduction cascades. Finally, the apoptotic efficacy of proteasome inhibition with bortezomib paralleled the ability to inhibit NF-kappaB activity and decrease BCL-2 levels.

Topics: Boronic Acids; Bortezomib; Breast Neoplasms; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Male; NF-kappa B; Pancreatic Neoplasms; Prostatic Neoplasms; Protease Inhibitors; Pyrazines; Signal Transduction; Transcription, Genetic; Tumor Cells, Cultured

Recent studies indicate that natural isothiocyanates, such as sulforaphane (SFN) and phenethyl isothiocyanate (PEITC) possess strong antitumor activities in vitro and in vivo. The nuclear factor kappa B (NF-kappaB) is believed to play an important role in cancer chemoprevention due to its involvement in tumor cell growth, proliferation, angiogenesis, invasion, apoptosis, and survival. In this study, we investigated the effects and the molecular mechanisms of SFN and PEITC on NF-kappaB transcriptional activation and NF-kappaB-regulated gene expression in human prostate cancer PC-3 C4 cells. Treatment with SFN (20 and 30 microM) and PEITC (5 and 7.5 microM) significantly inhibited NF-kappaB transcriptional activity, nuclear transloction of p65, and gene expression of NF-kappaB-regulated VEGF, cylcin D1, and Bcl-X(L) in PC-3 C4 cells. To further elucidate the mechanism, we utilized the dominant-negative mutant of inhibitor of NF-kappaB alpha (IkappaBalpha) (SR-IkappaBalpha). Analogous to treatments with SFN and PEITC, SR-IkappaBalpha also strongly inhibited NF-kappaB transcriptional activity as well as VEGF, cylcin D1, and Bcl-X(L) expression. Furthermore, SFN and PEITC also inhibited the basal and UVC-induced phosphorylation of IkappaBalpha and blocked UVC-induced IkappaBalpha degradation in PC-3 C4 cells. In examining the upstream signaling, we found that the dominant-negative mutant of IKKbeta (dnIKKbeta) possessed inhibitory effects similar to SFN and PEITC on NF-kappaB, VEGF, cylcin D1, Bcl-X(L) as well as IkappaBalpha phosphorylation. In addition, treatment with SFN and PEITC potently inhibited phosphorylation of both IKKbeta and IKKalpha and significantly inhibited the in vitro phosphorylation of IkappaBalpha mediated by IKKbeta. Taken together, these results suggest that the inhibition of SFN and PEITC on NF-kappaB transcriptional activation as well as NF-kappaB-regulated VEGF, cyclin D1, and Bcl-X(L) gene expression is mainly mediated through the inhibition of IKK phosphorylation, particularly IKKbeta, and the inhibition of IkappaBalpha phosphorylation and degradation, as well as the decrease of nuclear translocation of p65 in PC-3 cells.

Topics: Anticarcinogenic Agents; bcl-X Protein; Cell Line, Tumor; Cell Survival; Cyclin D1; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; I-kappa B Kinase; I-kappa B Proteins; Isothiocyanates; Male; NF-kappa B; NF-KappaB Inhibitor alpha; Phosphorylation; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-bcl-2; Sulfoxides; Thiocyanates; Transcription, Genetic; Vascular Endothelial Growth Factor A

We characterized interleukin-8 (IL-8) and IL-8 receptor expression (CXCR1 and CXCR2) in prostate cancer to address their significance to this disease.. Immunohistochemistry was conducted on 40 cases of human prostate biopsy containing histologically normal and neoplastic tissue, excised from patients with locally confined or invasive androgen-dependent prostate cancer, and 10 cases of transurethral resection of the prostate material from patients with androgen-independent disease.. Weak to moderate IL-8 expression was strictly localized to the apical membrane of normal prostate epithelium. In contrast, membranous expression of IL-8, CXCR1, and CXCR2 was nonapical in cancer cells of Gleason pattern 3 and 4, whereas circumferential expression was present in Gleason pattern 5 and androgen-independent prostate cancer. Each of IL-8, CXCR1, and CXCR2 were also increasingly localized to the cytoplasm of cancer cells in correlation with advancing stage of disease. Cytoplasmic expression (but not apical membrane expression) of IL-8 in Gleason pattern 3 and 4 cancer correlated with Ki-67 expression (R = 0.79; P < 0.001), cyclin D1 expression (R = 0.79; P < 0.001), and microvessel density (R = 0.81; P < 0.001). In vitro studies on androgen-independent PC3 cells confirmed the mitogenic activity of IL-8, increasing the rate of cell proliferation through activation of both CXCR1 and CXCR2 receptors.. We propose that the concurrent increase in IL-8 and IL-8 receptor expression in human prostate cancer induces autocrine signaling that may be functionally significant in initiating and promoting the progression of prostate cancer by underpinning cell proliferation and angiogenesis.

Topics: Aged; Androgens; Biomarkers, Tumor; Biopsy; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cytoplasm; Humans; Immunohistochemistry; Interleukin-8; Male; Neovascularization, Pathologic; Prostate; Prostatic Neoplasms; Receptors, Interleukin-8A; Receptors, Interleukin-8B

We investigated the effects of androgen receptor (AR) down regulation with a small interference RNA molecule (siRNA_AR(start)) on androgen sensitive LNCaP and androgen independent LNCaPabl prostate cancer cells, the latter representing an in vitro model for the development of therapy resistance in prostate cancer. Although LNCaPabl cells express increased levels of AR in comparison with androgen sensitive LNCaP cells, the protein was significantly down regulated in response to siRNA_AR(start) treatment. This AR down regulation resulted in a marked cell growth inhibition in both cell lines. By contrast, DU-145 prostate cancer cells, which lack AR expression, were not inhibited by the siRNA_AR(start). In consequence to AR down regulation, both cell lines, LNCaP and LNCaPabl, shared a highly similar gene expression profile in terms of major changes in cell cycle regulatory genes. The cell cycle inhibitor p21(Waf1/Cip1) as well as cyclin D1 were significantly up regulated by siRNA_AR(start) treatment, considering a switch in cyclin expression towards cell cycle retardation. Control molecules had moderate effects on cell proliferation and gene expression, respectively. In summary, we found that AR inhibition with siRNA induces cell growth retardation in androgen sensitive as well as in androgen independent prostate cancer cells and thus may represent an interesting approach to combat hormone-refractory prostate cancer.

Topics: Androgen Receptor Antagonists; Androgens; Cell Cycle Proteins; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Male; Prostatic Neoplasms; Receptors, Androgen; RNA Interference; RNA, Small Interfering; Transfection; Tumor Cells, Cultured; Up-Regulation

Silymarin has been shown to be a potent anticarcinogenic agent. Here, we investigated the modifying effects of dietary feeding with a naturally occurring polyphenolic antioxidant flavonoid silymarin on 3,2'-dimethyl-4-aminobiphenyl (DMAB)-induced prostatic carcinogenesis in male F344 rats.. Male F344 rats were given s.c. injections of DMAB (25 mg/kg body weight) every other week for 20 weeks. They also received the experimental diet containing 100 or 500 ppm silymarin for 40 weeks, starting 1 week after the last dosing of DMAB. All of the rats were sacrificed 60 weeks after the start of the experiment. Histopathology and immunohistochemistry for proliferative cell nuclear antigen, cyclin D1, and apoptotic indices were done in the prostatic lesions, including invasive adenocarcinomas, intraepithelial neoplasms, and nonlesional glands.. Dietary feeding with 500 ppm silymarin significantly inhibited the incidence of prostatic adenocarcinoma when compared with the DMAB-alone group (17.6% versus 50.0%, P < 0.05). The proliferative cell nuclear antigen- and cyclin D1-positive indices in adenocarcinomas, prostatic intraepithelial neoplasm, and nonlesional glands in rats treated with DMAB and silymarin were slightly lower than that of the DMAB-alone group. Also, dietary administration of silymarin increased apoptotic index in prostatic adenocarcinoma by measuring immunohistochemically positive nuclei for ssDNA.. Our results indicate that silymarin exerts chemopreventive ability against chemically induced prostatic carcinogenesis through apoptosis induction and modification of cell proliferation.

Topics: Adenocarcinoma; Aminobiphenyl Compounds; Animals; Antioxidants; Body Weight; Cyclin D1; Dietary Supplements; Immunohistochemistry; Liver; Male; Organ Size; Proliferating Cell Nuclear Antigen; Prostate; Prostatic Neoplasms; Rats; Rats, Inbred F344; Silymarin; Testis; Treatment Outcome

The cyclin D1 gene is frequently overexpressed in human breast cancer and is capable of inducing mammary tumorigenesis when overexpressed in transgenic mice. The BRCA1 breast tumor susceptibility gene product inhibits breast cancer cellular growth and the activity of several transcription factors. Herein, cyclin D1 antagonized BRCA1-mediated repression of estrogen receptor alpha (ERalpha)-dependent gene expression. Cyclin D1 repression of BRCA1 function was mediated independently of its cyclin-dependent kinase, retinoblastoma protein, or p160 (SRC-1) functions in human breast and prostate cancer cells. In vitro, cyclin D1 competed with BRCA1 for ERalpha binding. Cyclin D1 and BRCA1 were both capable of binding ERalpha in a common region of the ERalpha hinge domain. A novel domain of cyclin D1, predicted to form a helix-loop-helix structure, was required for binding to ERalpha and for rescue of BRCA1-mediated ERalpha transcriptional repression. In chromatin immunoprecipitation assays, 17beta-estradiol (E2) enhanced ERalpha and cyclin D1 recruitment to an estrogen response element (ERE). Cyclin D1 expression enhanced ERalpha recruitment to an ERE. E2 reduced BRCA1 recruitment and BRCA1 expression inhibited E2-induced ERalpha recruitment at 12 hours. Cyclin D1 expression antagonized BRCA1 inhibition of ERalpha recruitment to an ERE, providing a mechanism by which cyclin D1 antagonizes BRCA1 function at an ERE. As cyclin D1 abundance is regulated by oncogenic and mitogenic signals, the antagonism of the BRCA1-mediated ERalpha repression by cyclin D1 may contribute to the selective induction of BRCA1-regulated target genes.

Topics: Binding, Competitive; Breast Neoplasms; Carrier Proteins; Cyclin D1; Estradiol; Estrogen Receptor alpha; Female; Genes, BRCA1; Humans; Male; Membrane Proteins; Presenilin-2; Promoter Regions, Genetic; Prostatic Neoplasms; Protein Structure, Tertiary; Response Elements; Transcriptional Activation; Transfection; Ubiquitin-Protein Ligases

Signal transducer and activator of transcription 3 (Stat3) is constitutively activated in a variety of cancers and it is a common feature of prostate cancer. Thus, Stat3 represents a promising molecular target for tumor therapy. We applied a DNA vector-based Stat3-specific RNA interference approach to block Stat3 signaling and to evaluate the biological consequences of Stat3 down-modulation on tumor growth using a mouse model.. To investigate the therapeutic potential of blocking Stat3 in cancer cells, three small interfering RNAs (siRNA; Stat3-1, Stat3-2, and Stat3-3) specific for different target sites on Stat3 mRNA were designed and used with a DNA vector-based RNA interference approach expressing short hairpin RNAs to knockdown Stat3 expression in human prostate cancer cells in vitro as well as in vivo.. Of the three equivalently expressed siRNAs, only Stat3-3 and Stat3-2, which target the region coding for the SH2 domain and the coiled-coil domain, respectively, strongly suppressed the expression of Stat3 in PC3 and LNCaP cells. The Stat3-1 siRNA, which targeted the DNA-binding domain, exerted no effect on Stat3 expression, indicating that the gene silencing efficiency of siRNA may be dependent on the local structure of Stat3 mRNA. The Stat3 siRNAs down-regulated the expression of Bcl-2 (an anti-apoptotic protein), and cyclin D1 and c-Myc (cell growth activators) in prostate cancer cells. Inhibition of Stat3 and its related genes was accompanied by growth suppression and induction of apoptosis in cancer cells in vitro and in tumors implanted in nude mice.. These data indicate that Stat3 signaling is a promising molecular target for prostate cancer therapy and that vector-based Stat3 siRNA may be useful as a therapeutic agent for treatment of prostate cancer.

Topics: Animals; Apoptosis; Base Pairing; Base Sequence; Cell Cycle; Cell Proliferation; Cyclin D1; DNA-Binding Proteins; Down-Regulation; Genetic Vectors; Humans; Male; Mice; Mice, Nude; Molecular Sequence Data; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; RNA, Small Interfering; STAT3 Transcription Factor; Trans-Activators; Tumor Cells, Cultured

Our previous studies indicate that the activation of mitogen-activated protein kinase (MAPK) pathway is involved in bombesin-induced cell proliferation in prostate cancer cells. Cyclin D1 is a critical regulator involved in cell cycle progression through the G1 phase into the S phase, thereby contributing to cell proliferation. Mostly, mitogen-stimulated expression of cyclin D1 is attributed to the extracellular signal-regulated kinase (ERK) activation. Here, we found that bombesin induced human cyclin D1 expression on both mRNA and protein levels in DU-145 prostate cancer cells. Mutational analyses showed that bombesin-enhanced cyclin D1 transcription required the binding of nuclear proteins to the -143 to -105 region of the human cyclin D1 promoter, which contains binding sites for transcription factors Sp-1 and early growth response protein (Egr-1). Do novo protein synthesis was requisite for bombesin-induced cyclin D1 expression. Further studies showed Egr-1 was induced upon bombesin stimulation. The induction of Egr-1 expression and its binding to the cyclin D1 promoter were essential for bombesin-enhanced cyclin D1 transcription. Inhibition of MAPK pathway with either the MEK1 inhibitor PD98059 or a dominant-negative Ras mutant, RasN17, abolished bombesin-induced cyclin D1 activation. Taken together, bombesin-induced cyclin D1 expression in prostate cancer cells is mediated by Egr-1 activation and the interaction of Egr-1 with the Egr-1/Sp1 motif of the cyclin D1 promoter through the activation of MAPK pathway. These findings represent a novel mechanism of bombesin-dependent stimulation of mitogenesis by regulating directly the cell cycle in prostate cancer.

Topics: Bombesin; Cell Line, Tumor; Cyclin D1; Early Growth Response Protein 1; Humans; Male; MAP Kinase Signaling System; Promoter Regions, Genetic; Prostatic Neoplasms; Protein Binding; RNA, Messenger; Transcriptional Activation; Transfection

The development and progression of epithelial cancers are the result of an imbalance in signals promoting and inhibiting cellular proliferation and apoptosis. The aim of this study is to evaluate the expression of cell-cycle and apoptosis regulators and correlate them with clinical outcome in the most frequent carcinomas, in order to establish common prognostic biomarkers independent of cancer origin. Using tissue microarrays (TMAs), we have analysed the immuno-expression of Ki-67, Bcl-2, Bax, cyclin D1, cyclin D3, CDK1, CDK2, CDK6, p16, p21, and p27 in a series of 205 carcinomas of the large bowel, breast, lung and prostate (80, 73, 37 and 15 cases, respectively). By univariate analysis, positivity for p27, p16 and Bcl-2 was associated with better overall survival (P<0.0135, P<0.0442 and P<0.0001, respectively). The risk of mortality was 2.3-fold greater in patients without Bcl-2 expression. TMA immunohistochemical analysis identified a subset of epithelial cancers with overlapping alterations in cell-cycle checkpoints, apoptosis regulators and tumour suppressor pathways. We found that in most common epithelial cancers, regardless of origin, Bcl-2 appears to be the key biological factor influencing clinical behaviour.

Topics: Adult; Analysis of Variance; bcl-2-Associated X Protein; Biomarkers, Tumor; Breast Neoplasms; Cell Cycle Proteins; Cyclin D1; Cyclin D3; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cyclins; Female; Humans; Immunohistochemistry; Intestinal Neoplasms; Ki-67 Antigen; Lung Neoplasms; Male; Middle Aged; Neoplasms; Prognosis; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Survival Analysis; Tissue Array Analysis

To evaluate the antiproliferative activity of contragestazol (DL111-IT) on the human prostate cancer cell line PC3 in vitro and in vivo and to elucidate its potential molecular mechanisms.. The cell killing ability of DL111-IT was measured by the 3-(4,5-dimethylthia-zol,2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent assay method and the tumor xenograft model. The cell cycle was analyzed by flow cytometry and protein expression, including retinoblastoma (pRb), cyclin-dependent kinase 4 (CDK4) and cyclin D1, was detected by Western blotting.. DL111-IT exhibited high efficiency on cell growth inhibition of the human androgen-independent prostate cancer cell line PC3. The drug concentration that yielded 50% cell inhibition (IC50 value) was 9.9 mg/mL. In the PC3 tumor xenograft study, DL111-IT (1.25 mg/kg-20.0 mg/kg) given once a day for 10 days significantly inhibited tumor growth, with the inhibition rate ranging from 21% to 50%. Flow cytometric analysis indicated that DL111-IT could cause G1 arrest in the PC3 cell line, but not apoptosis. DL111-IT enhanced pRb expression and down-regulated CDK4 and cyclin D1 expression, suggesting that cell cycle regulation might contribute to the anticancer property of DL111-IT.. DL111-IT inhibits the proliferation of human androgen-independent prostate cancer cell line PC3 in vitro and in vivo by a cell cycle regulation pathway.

Topics: Androgens; Animals; Cell Division; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase 4; Dose-Response Relationship, Drug; Female; G1 Phase; Humans; Immunosuppressive Agents; In Vitro Techniques; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Prostatic Neoplasms; Resting Phase, Cell Cycle; Retinoblastoma Protein; Transplantation, Heterologous; Triazoles

We have evaluated cell survival, apoptosis, and cell cycle responses in a panel of DNA mismatch repair (MMR)-deficient colon and prostate cancer cell lines after alkylation and UV-C damage. We show that although these MMR-deficient cells tolerate alkylation damage, they are as sensitive to UV-C-induced damage as are the MMR-proficient cells. MMR-proficient cells arrest in the S-G2 phase of the cell cycle and initiate apoptosis following alkylation damage, whereas MMR-deficient cells continue proliferation. However, two prostate cancer cell lines that are MMR-deficient surprisingly arrest transiently in S-G2 after alkylation damage. Progression through G1 phase initially depends on the expression of one or more of the D-type cyclins (D1, D2, and/or D3). Analysis of cyclin D1 expression shows an initial MMR-independent decrease in the protein level after alkylation as well as UV-C damage. At later time points, however, only DNA damage-arrested cells showed decreased cyclin D1 levels irrespective of MMR status, indicating that reduced cyclin D1 could be a result of a smaller fraction of cells being in G1 phase rather than a result of an intact MMR system. Finally, we show that cyclin D1 is degraded by the proteasome in response to alkylation damage.

Topics: Alkylating Agents; Alkylation; Apoptosis; Base Pair Mismatch; Cell Cycle; Cell Line, Tumor; Cell Survival; Colonic Neoplasms; Cyclin D1; Cysteine Endopeptidases; DNA Damage; DNA Methylation; DNA Repair; DNA, Neoplasm; Humans; Male; Methylnitronitrosoguanidine; Multienzyme Complexes; Prostatic Neoplasms; Proteasome Endopeptidase Complex; Time Factors; Ultraviolet Rays

It is known that gamma-tocopherol inhibits human prostate cancer cell proliferation via down-regulation of cyclin-related signalling but tocopherol and tocotrienol metabolites with a shortened phytyl chain, carboxyethyl hydroxychromans, were not previously investigated as anti-proliferative agents. In this study, the effect of the two main tocopherols, namely, alpha-tocopherol and gamma-tocopherol, and their corresponding metabolites (alpha- and gamma-carboxyethyl hydroxychromans) was studied on proliferation and cyclin D1 expression of the prostate cancer cell line PC-3. The hydrosoluble vitamin E analogues Trolox and alpha-tocopherol succinate were also tested. The most effective inhibitors of PC-3 proliferation were gamma-tocopherol and gamma-carboxyethyl hydroxychroman. Their effect was discernable at 1 microM and reached a plateau at concentrations > or = 10 microM with maximal inhibition values ranging between 70 and 82%. alpha-Tocopherol, alpha-carboxyethyl hydroxychroman, and the analogue Trolox were much less effective; a weak effect was observed for concentrations < or = 10 microM and a maximal inhibition of less than 45% was found at 50 microM concentration. PC-3 cells showed higher inhibition, particularly by the gamma derivatives, than HTB-82 and HECV cells. Tocopherols and carboxyethyl hydroxychromans exerted an inhibitory effect on cyclin D1 expression parallel to the retardation of cell growth. gamma-Carboxyethyl hydroxychroman and gamma-tocopherol showed effects also upstream of the cyclin modulation. Furthermore, the inhibition of cyclin D1 expression by gamma-carboxyethyl hydroxychroman was competed for by alpha-carboxyethyl hydroxychroman. In conclusion, this study shows that carboxyethyl hydroxychroman metabolites are as effective as their vitamin precursors to inhibit PC-3 growth by specific down-regulation of cyclin expression, with the gamma forms being the most effective ones. Although the inhibition of PC-3 cell growth and diminution of cyclin expression are clearly visible, more subtle mechanistic effects of tocopherols and their corresponding carboxyethyl hydroxychroman metabolites deserve further investigations.

Topics: alpha-Tocopherol; Antioxidants; Cell Division; Chromans; Cyclin D1; Dose-Response Relationship, Drug; gamma-Tocopherol; Humans; Male; Propionates; Prostatic Neoplasms

Curcumin (Diferuloylmethane) is a major chemical component of turmeric (curcuma longa) and is used as a spice to give a specific flavor and yellow color in Asian food. Curcumin exhibits growth inhibitory effects in a broad range of tumors as well as in TPA-induced skin tumors in mice. This study was undertaken to investigate the radiosensitizing effects of curcumin in p53 mutant prostate cancer cell line PC-3. Compared to cells that were irradiated alone (SF(2)=0.635; D(0)=231 cGy), curcumin at 2 and 4 microM concentrations in combination with radiation showed significant enhancement to radiation-induced clonogenic inhibition (SF(2)=0.224: D(0)=97 cGy and SF(2)=0.080: D(0)=38 cGy) and apoptosis. It has been reported that curcumin inhibits TNF-alpha-induced NFkappaB activity that is essential for Bcl-2 protein induction. In PC-3 cells, radiation upregulated TNF-alpha protein leading to an increase in NFkappaB activity resulting in the induction of Bcl-2 protein. However, curcumin in combination with radiation treated showed inhibition of TNF-alpha-mediated NFkappaB activity resulting in bcl-2 protein downregulation. Bax protein levels remained constant in these cells after radiation or curcumin plus radiation treatments. However, the downregulation of Bcl-2 and no changes in Bax protein levels in curcumin plus radiation-treated PC-3 cells, together, altered the Bcl2 : Bax ratio and this caused the enhanced radiosensitization effect. In addition, significant activation of cytochrome c and caspase-9 and -3 were observed in curcumin plus radiation treatments. Together, these mechanisms strongly suggest that the natural compound curcumin is a potent radiosesitizer, and it acts by overcoming the effects of radiation-induced prosurvival gene expression in prostate cancer.

Topics: Apoptosis; Caspases; Cell Line, Tumor; Combined Modality Therapy; Curcumin; Cyclin D1; Cytochromes c; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Gene Expression Regulation, Neoplastic; Humans; Male; NF-kappa B; Prostatic Neoplasms; Radiation Tolerance; Radiation-Sensitizing Agents; Radiation, Ionizing; Tumor Necrosis Factor-alpha

Development of androgen independence and resistance to apoptosis in prostate cancer are often correlated with high levels of serum tumor necrosis factor (TNF)-alpha in these patients. The loss of sensitivity to TNF-alpha-induced apoptosis in androgen-insensitive prostate carcinoma cells is due in part to constitutive activation of Rel/nuclear factor (NF)-kappaB transcription factors that regulate several cell survival and antiapoptotic genes. Our previous studies have demonstrated growth inhibitory and apoptotic effects of apigenin, a common plant flavonoid, in a variety of human prostate carcinoma cells. Here we examined whether apigenin is effective in inhibiting NF-kappaB expression in androgen-insensitive human prostate carcinoma cells exhibiting high constitutive levels of NF-kappaB.. Using androgen-insensitive human prostate carcinoma PC-3 cells, the effect of apigenin was assessed on NF-kappaB activation by electrophoretic mobility shift assay and reporter gene assay. Expression of NF-kappaB subunits p65 and p50, IkappaBalpha, p-IkappaBalpha, in-beads kinase assay and NF-kappaB-regulated genes were determined by Western blot analysis. Apoptosis was determined by annexin V/propidium iodide staining after fluorescence-activated cell-sorting analysis.. Treatment of cells with 10-40- micro M doses of apigenin inhibited DNA binding and reduced nuclear levels of the p65 and p50 subunits of NF-kappaB. Apigenin inhibited IkappaBalpha degradation and IkappaBalpha phosphorylation and significantly decreased IKKalpha kinase activity. Apigenin also inhibited TNF-alpha-induced activation of NF-kappaB via the IkappaBalpha pathway, thereby sensitizing the cells to TNF-alpha-induced apoptosis. The inhibition of NF-kappaB activation correlated with a decreased expression of NF-kappaB-dependent reporter gene and suppressed expression of NF-kappaB-regulated genes [specifically, Bcl2, cyclin D1, cyclooxygenase-2, matrix metalloproteinase 9, nitric oxide synthase-2 (NOS-2), and vascular endothelial growth factor].. Our results indicate that inhibition of NF-kappaB by apigenin may lead to prostate cancer suppression by transcriptional repression of NF-kappaB-responsive genes as well as selective sensitization of prostate carcinoma cells to TNF-alpha-induced apoptosis.

Topics: Apigenin; Apoptosis; Biological Transport; Cell Line, Tumor; Cell Nucleus; Cyclin D1; Cyclooxygenase 2; Dose-Response Relationship, Drug; Down-Regulation; Flavonoids; Gene Expression Regulation, Neoplastic; Humans; I-kappa B Kinase; I-kappa B Proteins; Isoenzymes; Luciferases; Male; Matrix Metalloproteinase 9; Membrane Proteins; NF-kappa B; NF-kappa B p50 Subunit; NF-KappaB Inhibitor alpha; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Phosphorylation; Prostaglandin-Endoperoxide Synthases; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-bcl-2; Recombinant Fusion Proteins; Transcription Factor RelA; Transfection; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A

R-etodolac, a nonsteroidal anti-inflammatory drug, inhibits the progression of CWRSA6 androgen-independent and LuCaP-35 androgen-dependent prostate cancer xenograft growth through downregulation of cyclin D1 expression via the PPARgamma pathway. PPARgamma protein degradation, observed post-R-etodolac treatment, resulted from phospho-MAP kinase (p44/42) induction by R-etodolac negatively regulating PPARgamma function. Negative regulation of PPARgamma was overcome by a combination regimen of R-etodolac with the HER-kinase axis inhibitor, rhuMab 2C4, which demonstrated an additive antitumor effect. We further show that the inhibition of HER-kinase activity by rhuMab 2C4 is sufficient to inhibit PPARgamma protein degradation. This study introduces a novel concept of an in vivo crosstalk between the HER-kinase axis and PPARgamma pathways, ultimately leading to negative regulation of PPARgamma activity and tumor growth inhibition.

Topics: Animals; Antineoplastic Agents; Azo Compounds; Blotting, Western; CD36 Antigens; Cell Line; Cell Line, Tumor; Coloring Agents; Cyclin D1; Down-Regulation; Enzyme Activation; ErbB Receptors; Etodolac; Flow Cytometry; Humans; Lipid Metabolism; Male; MAP Kinase Signaling System; Mice; Mice, Nude; Mitogen-Activated Protein Kinases; Models, Biological; Models, Chemical; Monocytes; Neoplasm Transplantation; NIH 3T3 Cells; Prostatic Neoplasms; Protein Isoforms; Receptors, Cytoplasmic and Nuclear; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Transcription Factors; Transcriptional Activation; Transfection

In human prostate cancer, the frequent down-regulation of p27(kip1) protein expression is correlated with poor clinical outcome, yet p27(kip1) rarely undergoes mutational inactivation. Here, we investigate the consequences of reducing or eliminating p27(kip1) function for prostate carcinogenesis in the context of a mouse modeling lacking the Nkx3.1 homeobox gene and the Pten tumor suppressor. Unexpectedly, we find that triple mutant mice heterozygous for a p27(kip1) null allele (Nkx3.1(+/- or -/-); Pten(+/-); p27(+/-)) display enhanced prostate carcinogenesis, whereas mice that are homozygous null for p27(kip1) (Nkx3.1(+/- or -/-); Pten(+/-); p27(-/-)) show inhibition of cancer progression. Expression profiling reveals that Cyclin D1 is highly up-regulated in compound p27(kip1) heterozygotes, but is down-regulated in the compound p27(kip1) homozygous mutants. Using RNA interference in prostate cancer cell lines with distinct p27(kip1) gene doses, we show that prostate tumorigenicity depends on levels of p27(kip1) and that the consequences of p27(kip1) gene dosage can be attributed, in part, to altered levels of Cyclin D1. Our findings suggest that p27(kip1) possesses dosage-sensitive positive as well as negative modulatory roles in prostate cancer progression.

Topics: Animals; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Disease Models, Animal; Disease Progression; Gene Dosage; Gene Expression Regulation, Neoplastic; Genotype; Homeodomain Proteins; Male; Mice; Mice, Knockout; Mice, Mutant Strains; Prostatic Neoplasms; Protein Tyrosine Phosphatases; PTEN Phosphohydrolase; Transcription Factors; Tumor Suppressor Proteins

CCND1 mRNA is alternatively spliced to produce 2 transcripts, and the splicing pattern may be modulated by a frequent A870G single-nucleotide polymorphism within the conserved splice donor site of exon 4. Several studies have suggested a significant association between the CCND1 genotype and onset or progression of various cancers. To investigate the correlation of the polymorphism with genetic susceptibility to PCa and its disease status, we examined the polymorphism in 214 cases of PCa, 234 cases of BPH and 254 male controls. The CCND1 A allele was more frequently observed in the PCa group (p = 0.015) and the BPH group (p = 0.018) than the control group. Men with the AA genotype had an increased risk of PCa (aOR = 1.93, 95% CI 1.13-3.30, p = 0.016) and BPH (aOR = 1.84, 95% CI 1.09-3.09, p = 0.023) compared to those with the GG genotype. No significant association was observed when men with the AG genotype were compared to those with the GG genotype (PCa: aOR = 1.00, 95% CI 0.65-1.54, BPH: aOR = 0.91, 95% CI 0.60-1.39). The risk of PCa associated with the AA genotype appeared to be stronger in men aged 73 years or younger (aOR = 2.89, 95% CI 1.38-6.01, p = 0.005), whereas no association was found in men older than 73 years (aOR = 1.02, 95% CI 0.44-2.34). No significant difference in genotype frequency was found among patients with low-, intermediate- and high-grade tumors (p = 0.730) or between patients with localized and metastatic PCa (p = 0.679). However, in patients with high-grade or metastatic PCa, a significantly increased risk associated with the AA genotype compared to controls was observed, while no significant results were found in those with low/intermediate or localized PCa. The A allele of the CCND1 A870G polymorphism was recessively associated with susceptibility to PCa and BPH in a Japanese population, giving a 2-fold increased risk of PCa and BPH in men with the AA genotype compared to those with the GG genotype. Although the risk of PCa associated with the AA genotype appeared to contribute especially to men aged 73 years or younger and the A allele may be associated with disease status of PCa, these conjectures require validation in future studies on a larger number of subjects.

Topics: Adenocarcinoma; Aged; Alternative Splicing; Case-Control Studies; Cyclin D1; Dinucleotide Repeats; Genetic Predisposition to Disease; Genotype; Humans; Male; Polymorphism, Genetic; Prostatic Hyperplasia; Prostatic Neoplasms; Risk Factors

Because ErbB-2 receptor is involved in hormone-independency for growth and metastasis of prostate cancer cells, the aim was to investigate the effects of quercetin on ErbB-2 and ErbB-3 expression and its critical components such as MAP kinase and PI-3 kinase. Hemocytometric counts and [3H]-thymidine incorporation were used to determine the effects of quercetin, EGF and TGF-alpha on cell proliferation and DNA synthesis in PC-3 and LnCap cells. Changes in ErbB-2, ErbB-3 and components of MAPK and PI-3K pathways were analyzed by Western blot analysis. Treatment of PC-3 and LnCap cells with quercetin resulted in a dose-dependent growth inhibition. The rate of DNA synthesis was decreased by 40, 55 and 65% on treatment with 14.5, 29.0 and 58.0 microM of quercetin, respectively. Concomitantly, these treatments led to a dose-dependent decrease in ErbB-2, ErbB-3 and their basal autophosphorylation levels as compared to controls. Cyclin D1 expression and basal phosphorylation of c-Raf, MAPK, Elk-1 and Akt-1 in PC-3 cells was also inhibited by quercetin treatment. Co-treating PC-3 cells with quercetin significantly attenuated EGF- and TGF-alpha-induced growth and phosphorylation of ErbB-2, ErbB-3, c-Raf, MAPK kinase 1/2 (MEK1/2), MAPK, Elk-1 and Akt-1. Since ErbB receptor is important for growth, metastasis and drug resistance, inhibition of ErbB-2 and ErbB-3 by pharmacological doses of quercetin may provide a new approach for treatment of prostate cancers.

Topics: Blotting, Western; Cell Division; Cell Line, Tumor; Cyclin D1; DNA; Dose-Response Relationship, Drug; Epidermal Growth Factor; Humans; Male; MAP Kinase Signaling System; Phosphatidylinositol 3-Kinases; Phosphorylation; Prostatic Neoplasms; Proto-Oncogene Proteins c-raf; Quercetin; Receptor, ErbB-2; Receptor, ErbB-3; Transforming Growth Factor alpha

Cdc37 is a co-chaperone protein that recruits several immature client kinases to Hsp90 for proper folding. Cdc37 up-regulation is a common early event in localized human prostate cancer. Although targeted overexpression in mice leads to prostate epithelial cell hyperplasia, the effect of Cdc37 dysregulation in human prostate cells is unclear. In this study, we examine the role of Cdc37 in the growth regulation of normal prostate epithelial cells using a unique human model system. We demonstrate that Cdc37 overexpression drives proliferation, whereas loss of Cdc37 function arrests growth and leads to apoptosis. With increased Cdc37 expression, molecular analysis of Cdc37 client pathways demonstrates enhanced Raf-1 activity, greater Cdk4 levels, and reduced expression of the cyclin-dependent kinase inhibitor p16/CDKN2. To further investigate these downstream pathways, enhanced Raf-1 or Cdk4 activities were selectively induced in human prostate epithelial cells. Raf-1 activation inhibited proliferation and generated an enlarged, flattened morphology. Induction of Cdk4 activity using cyclin D1 overexpression, however, was sufficient to promote proliferation. These data indicate that Cdc37 induces proliferation and is critical for survival in human prostate epithelial cells. These alterations in cell division and survival may be important in the development and progression of early prostate cancer.

Topics: Apoptosis; Bromodeoxyuridine; Cell Cycle Proteins; Cell Division; Cell Survival; Chaperonins; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Disease Progression; Drosophila Proteins; Enzyme Activation; Epithelial Cells; Humans; Male; Molecular Chaperones; Prostate; Prostatic Neoplasms; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-raf

Androgen receptor (AR) activity is required for prostate growth, differentiation, and secretion. Deregulation of AR activity results in inappropriate mitogenic signaling and is thought to contribute both to the initiation and progression of prostate cancers. Cyclin D1 functions as a strong AR corepressor by directly interacting with and inhibiting receptor activity. However, the extent to which cyclin D1 functions to inhibit AR activity under conditions associated with cancer progression has not been determined. We now demonstrate that cyclin D1 action is conserved in multiple tumor cell backgrounds, inhibiting AR-dependent gene activation in breast, bladder, and androgen-independent prostatic adenocarcinoma cell lines. In androgen-dependent prostatic adenocarcinomas, cyclin D1 effectively muted androgen-stimulated target gene expression in a manner analogous to dominant negative ARs. The ability of cyclin D1 to inhibit AR activity was conserved with regard to target promoter, repressing transactivation from mouse mammary tumor virus, probasin, and prostate-specific antigen promoters. Inappropriate, nonligand AR activation, postulated to act through regulation of receptor phosphorylation, was also sensitive to cyclin D1 regulation. Moreover, we show that several phosphorylation site mutants of the AR were equally inhibited by cyclin D1 as compared with the wild-type receptor. Given these data establishing the potency of cyclin D1-mediated repression, we evaluated the ability of cyclin D1 to inhibit tumor-derived AR alleles and polymorphisms associated with tumor progression and increased prostate cancer risk. We demonstrate that the AR alleles and polymorphisms tested respond completely to cyclin D1 corepressor activity. In addition, activation of a common tumor-derived AR allele by 17 beta-estradiol and progesterone was inhibited through ectopic expression of cyclin D1. Taken together, these data establish the potency of cyclin D1 as an AR corepressor and provide support for additional studies examining the efficacy of developing novel prostate cancer therapies for both androgen-dependent and -independent tumors.

Topics: Adenocarcinoma; Androgen Receptor Antagonists; Binding Sites; Cell Line; Cyclin D1; DNA; Humans; Male; Neoplasms, Hormone-Dependent; Phosphorylation; Promoter Regions, Genetic; Prostatic Neoplasms; Receptors, Androgen; Repressor Proteins; Transcriptional Activation

Topics: Aged; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Cyclin D1; Humans; Lymphoma, Mantle-Cell; Male; Prostatic Neoplasms; Tomography, X-Ray Computed

Potential modulatory effects of melatonin on the proliferation of androgen-sensitive LNCaP and androgen-insensitive PC-3 and DU 145 prostate cancer cells were reported recently. In this study, we investigated the effects of combined melatonin and castration on LNCaP tumor growth in vivo, the interactions between melatonin and epidermal growth factor (EGF) on LNCaP cell proliferation, and melatonin actions on the proliferation of PC-3 and DU 145 cells.. Tumor development and growth in castrated nude mice inoculated with LNCaP cells or in intact animals inoculated with DU 145 cells, with or without daily melatonin treatment, were monitored by observation and caliper measurement. MT(1) receptor expression in native or transfected prostate cancer cell lines was examined by immunocytochemistry or 2-[(125)I]iodomelatonin binding. Cyclin D1 expression in LNCaP cells was assessed by Western blotting, and cell proliferation was measured by thymidine incorporation and/or cell count.. Melatonin treatment was associated with further decreases in LNCaP tumor incidence and growth rate in castrated nude mice. Melatonin and 2-iodomelatonin (a melatonin receptor agonist) attenuated EGF-stimulated increases in LNCaP cell proliferation and cyclin D1 levels. Melatonin had no effect on the proliferation or growth of MT(1) receptor-expressing DU 145 cells, and of PC-3 cells in which MT(1) receptor protein was undetectable. The proliferation of transfected PC-3 cells expressing MT(1) receptor was unaffected by 2-iodomelatonin.. Together with previous data, the present results indicate synergistic action of melatonin and castration in inhibiting the growth of androgen-sensitive LNCaP tumor. Androgen-sensitive prostate cancer cell proliferation may be modulated by opposite changes in cyclin D1 levels induced by activated MT(1) and EGF receptors. In androgen-insensitive prostate cancer cells, MT(1) receptor-mediated signal transduction may become defective not only through changes in membrane receptor protein expression and/or functions, but also by means of alterations in downstream postreceptor signaling events.

Topics: Androgens; Animals; Cell Division; Cyclin D1; Epidermal Growth Factor; ErbB Receptors; Humans; Male; Melatonin; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Orchiectomy; Prostatic Neoplasms; Receptors, Cell Surface; Receptors, Cytoplasmic and Nuclear; Receptors, Melatonin; Transplantation, Heterologous; Tumor Cells, Cultured

Whereas hydroxyflutamide (HF) has been used as an antiandrogen to block androgen-stimulated prostate tumor growth, the antiandrogen withdrawal syndrome that allows antiandrogens to stimulate prostate tumor growth still occurs in many patients treated with androgen ablation therapy. This was previously explained by mutations in the androgen receptor (AR) and/or modulation from AR coregulators, so that HF becomes an AR agonist. Using immunohistochemical analysis, we analyzed four prostate cancer patients undergoing androgen ablation therapy with flutamide and compared their phospho-extracellular signal-regulated kinase 1/2 levels in prostate cancer biopsies before receiving HF and after experiencing disease progression while taking HF. We found a significant increase of activated mitogen-activated protein (MAP) kinase in prostate tumors from patients receiving HF during androgen ablation therapy. In vitro studies showed that HF induced a rapid activation of the Ras/MAP kinase pathway in human prostate cancer DU145 cells which lack the AR, as well as in PC-3AR2 and CWR22 cells which express the AR. Cycloheximide failed to inhibit this activation, but both AG1478, an inhibitor of the epidermal growth factor receptor (EGF-R), and an EGF-R-neutralizing antibody blocked this HF-mediated activation of MAP kinase, which suggests that the activation of Ras/MAP kinase by HF is a membrane-initiated, non-AR-mediated, and nongenomic action. The consequence of this activation may result in increasing cell proliferation and cyclin D1 expression. This raises a concern for using HF in the complete-androgen-ablation therapy in prostate cancer treatment and provides a possible pathway that might contribute to the HF withdrawal syndrome.

Topics: Androgen Antagonists; Cell Division; Cyclin D1; Enzyme Activation; ErbB Receptors; Flutamide; Humans; Male; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Neoplasms, Hormone-Dependent; Phosphorylation; Prostatic Neoplasms; Proto-Oncogene Proteins c-raf; Quinazolines; ras Proteins; Receptors, Androgen; Substance Withdrawal Syndrome; Tyrphostins

Tea polyphenols have been proposed as potential chemopreventive agents against prostate cancer, primarily because of their high intake by populations with reduced cancer incidence and their reported ability to inhibit proliferation and increase apoptosis in prostate cancer cells in culture. Insulin-like growth factor-I (IGF-I) has been implicated as a risk factor for the development of prostate cancer by epidemiological studies and has been shown to be causative in animal models. One of the primary signal transduction pathways activated by IGF-I binding to its receptor is the Akt pathway. We determined that phosphorylated Akt levels are very low in serum-starved human normal prostate epithelial cells (PrEC) and Du145 prostate carcinoma cells, and that treatment of these cells with IGF-I results in a rapid and sustained phosphorylation of Akt. Pre-treatment of PrEC and Du145 cells with doses as low as 20 microg/ml of a mixture of black tea polyphenols (BTP) substantially reduced IGF-I-mediated Akt phosphorylation. This effect of BTP appears to be due partially to the reduced autophosphorylation of IGF-I receptor-1 in BTP-treated cells. BTP pre-treatment also decreased downstream effects of Akt activation including phosphorylation of glycerol synthase kinase-3, increased cyclin D1 protein levels and increased DNA synthesis. Our results indicate that polyphenols from black tea inhibit the IGF-I signal transduction pathway, which has been linked to increased prostate cancer incidence in human populations and, therefore, provide further support for the potential of BTP to prevent prostate cancer.

Topics: Calcium-Calmodulin-Dependent Protein Kinases; Cell Division; Cell Line; Cyclin D1; DNA; Epithelial Cells; Flavonoids; Flow Cytometry; Glycogen Synthase Kinase 3; Humans; Insulin-Like Growth Factor I; Male; Phenols; Phosphorylation; Polymers; Polyphenols; Prostate; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Receptor, IGF Type 1; Signal Transduction; Tea; Tumor Cells, Cultured

The relation between the consumption of cruciferous vegetables and reduced prostate cancer occurrence has been documented, although the responsible phytochemicals are unknown. The effects of sulforaphane (SFN) which occurs as the precursor glucosinolate in broccoli and other cruciferous vegetables, and its metabolite N-acetylcysteine conjugate (SFN-NAC) on prostate cancer cells were investigated. SFN and SFN-NAC were analyzed with the androgen-dependent human prostate cancer LNCaP cell line model. Cell growth and apoptosis were determined with the expression of androgen receptor and prostate specific antigen, DNA synthesis, cell cycle progression, DNA strand breaks and caspase activation to ascertain the effects and mechanism. SFN and SFN-NAC were demonstrated for the first time to mediate a dose-dependent apoptosis and growth arrest in the prostate cancer cells. Caspases were activated and DNA strand breaks were detected in apoptotic cells. The expression of phosphorylated and dephosphorylated androgen receptors, and the production of prostate specific antigen were attenuated. The expression of cyclin D1 and DNA synthesis were inhibited along with G1 cell cycle block, causing decreased cell density and growth. SFN and its metabolite SFN-NAC have similar activities to induce growth arrest and apoptosis, indicating that the effects of SFN are maintained through the metabolic processes. SFN as a dietary component of cruciferous vegetables active in the prevention of prostate cancer is discussed.

Topics: Anticarcinogenic Agents; Apoptosis; Cell Cycle; Cell Division; Cyclin D1; Dose-Response Relationship, Drug; Humans; Isothiocyanates; Male; Prostate-Specific Antigen; Prostatic Neoplasms; Sulfoxides; Thiocyanates; Tumor Cells, Cultured

The androgen receptor (AR) is a ligand-regulated member of the nuclear receptor superfamily. The cyclin D1 gene product, which encodes the regulatory subunit of holoenzymes that phosphorylate the retinoblastoma protein (pRB), promotes cellular proliferation and inhibits cellular differentiation in several different cell types. Herein the cyclin D1 gene product inhibited ligand-induced AR- enhancer function through a pRB-independent mechanism requiring the cyclin D1 carboxyl terminus. The histone acetyltransferase activity of P/CAF (p300/CBP associated factor) rescued cyclin D1-mediated AR trans-repression. Cyclin D1 and the AR both bound to similar domains of P/CAF, and cyclin D1 displaced binding of the AR to P/CAF in vitro. These studies suggest cyclin D1 binding to the AR may repress ligand-dependent AR activity by directly competing for P/CAF binding.

Topics: Acetyltransferases; Amino Acid Sequence; Androgen Receptor Antagonists; Blotting, Western; Cell Cycle Proteins; Cyclin D1; Histone Acetyltransferases; Humans; Ligands; Male; Molecular Sequence Data; Mutation; p300-CBP Transcription Factors; Prostatic Neoplasms; Receptors, Androgen; Sequence Alignment; Signal Transduction; Transcription Factors; Tumor Cells, Cultured

We report that all- trans retinoic acid (ATRA) enhanced the toxicity of docetaxel against DU145 and LNCaP prostate cancer cells, and that the nature of the interaction between ATRA and docetaxel was highly synergistic. Docetaxel-induced apoptotic cell death was associated with phosphorylation and hence inactivation of Bcl-2. ATRA enhanced docetaxel-induced apoptosis and combined treatment with ATRA and docetaxel resulted in down-regulation of Bcl-2. Docetaxel caused phosphorylation and hence inactivation of cdc2 kinase result ing in G2/M arrest. ATRA inhibited docetaxel-induced phosphorylation of cdc2 resulting in activation of cdc2 kinase and partial reversal of the G2/M arrest. ATRA also inhibited docetaxel-induced activation of MAPK indicating that the effects of docetaxel and ATRA on cdc2 phosphorylation are dependent on MAPK. We conclude that ATRA synergistically enhances docetaxel toxicity by down-regulating Bcl-2 expression and partially reverses the docetaxel-induced G2/M arrest by inhibiting docetaxel-induced cdc2 phosphorylation in a pathway that is dependent on MAPK.

Topics: Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Apoptosis; Cell Cycle; Cyclin D1; Docetaxel; Down-Regulation; Drug Interactions; Humans; Male; Paclitaxel; Prostatic Neoplasms; Taxoids; Tretinoin; Tumor Cells, Cultured

Prostate cancer is one of the most common cancers in men and it is the second leading cause of cancer related death in men in the United States. Recent dietary and epidemiological studies have suggested the benefit of dietary intake of fruits and vegetables in lowering the incidence of prostate cancer. A diet rich in fruits and vegetables provides phytochemicals, particularly indole-3-carbinol (I3C), which may be responsible for the prevention of many types of cancer, including hormone-related cancers such as prostate. Studies to elucidate the role and the molecular mechanism(s) of action of I3C in prostate cancer, however, have not been conducted. In the current study, we investigated whether I3C had any effect against prostate cancer cells and, if so, attempts were made to identify the potential molecular mechanism(s) by which I3C elicits its biological effects on prostate cancer cells. Here we report for the first time that I3C inhibits the growth of PC-3 prostate cancer cells. Induction of G1 cell cycle arrest was also observed in PC-3 cells treated with I3C, which may be due to the observed effects of I3C in the up-regulation of p21(WAF1) and p27(Kip1) CDK inhibitors, followed by their association with cyclin D1 and E and down-regulation of CDK6 protein kinase levels and activity. The induction of p21(WAF1) appears to be transcriptionally upregulated and independent of the p53 responsive element. In addition, I3C inhibited the hyperpohosphorylation of the Retinoblastoma (Rb) protein in PC-3 cells. Induction of apoptosis was also observed in this cell line when treated with I3C, as measured by DNA laddering and poly (ADP-ribose) polymersae (PARP) cleavage. We also found an up-regulation of Bax, and down-regulation of Bcl-2 in I3C-treated cells. These effects may also be mediated by the down-regulation of NF-kappaB observed in I3C treated PC-3 cells. From these results, we conclude that I3C inhibits the growth of PC-3 prostate cancer cells by inducing G1 cell cycle arrest leading to apoptosis, and regulates the expression of apoptosis-related genes. These findings suggest that I3C may be an effective chemopreventive or therapeutic agent against prostate cancer.

Topics: Anticarcinogenic Agents; Apoptosis; bcl-2-Associated X Protein; Cell Cycle Proteins; Cell Division; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cyclins; Down-Regulation; G1 Phase; Humans; Indoles; Male; Microtubule-Associated Proteins; NF-kappa B; Phosphorylation; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Retinoblastoma Protein; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

Cyclin D1 is a key regulator of the G1 phase progression of the cell cycle. There is increasing evidence that deregulated cyclin D1 expression is implicated in tumorigenesis and tumor progression in certain neoplasms. Recently, it has been reported that cyclin D1 overexpression might be related to the evolution of androgen-independent disease in prostate cancer. This study was conducted to investigate patterns of cyclin D1 expression in prostate cancer samples representing different points in the natural history and treatment evolution of the disease. Association with clinical outcomes was also explored. Using immunohistochemistry, 86 radical prostatectomy specimens (53 naive and 33 after androgen deprivation) and 22 androgen-independent bone metastases were studied. We examined the difference in cyclin D1 expression in primary versus metastatic cases. In addition, we examined the association in primary cases between cyclin D1 expression and clinicopathological parameters of poor clinical outcome, including time to prostate-specific antigen relapse and Ki67 proliferative index. Cyclin D1-positive phenotype, defined as identification of positive immunoreactivity in the nuclei of > or =20% of tumor cells, was observed in 10 of 86 (11%) primary cases compared with 15 of 22 (68%) androgen-independent bone metastases (P = 0.001). There was no correlation between cyclin D1 overexpression and either Gleason score, neo-adjuvant hormone treatment, or prostate-specific antigen relapse We observed a statistical association between cyclin D1 overexpression and high Ki67 proliferative index, defined as > or =20% of positive tumor cells (P = 0.02). These data support the hypothesis that cyclin D1 overexpression may represent an oncogenic event in androgen-independent metastatic prostate cancer to the bone.

Topics: Cohort Studies; Cyclin D1; Humans; Immunohistochemistry; Ki-67 Antigen; Male; Neoplasm Metastasis; Prostate-Specific Antigen; Prostatic Neoplasms

Although insulin-like growth factor (IGF) binding protein-5 (IGFBP-5) is highly up-regulated in normal and malignant prostate tissues after androgen withdrawal, its functional role in castration-induced apoptosis and androgen-independent progression remains undefined. To analyze the functional significance of IGFBP-5 overexpression in IGF-I-mediated mitogenesis and progression to androgen-independence, IGFBP-5-overexpressing human androgen-dependent LNCaP prostate cancer cells were generated by stable transfection. The growth rates of IGFBP-5-transfected LNCaP cells were significantly faster, compared with either the parental or vector-only transfected LNCaP cells in both the presence and absence ofdihydrotestosterone. IGFBP-5-induced increases in LNCaP cell proliferation occurs through both IGF-I-dependent and -independent pathways, with corresponding increases in the cyclin D1 messenger RNA expression and the fraction of cells in S + G2/M phases of the cell cycle. Changes in Akt/protein kinase B, a downstream component of phosphatidylinositol 3'-kinase (PI3K) pathway, in the LNCaP sublines also paralleled changes in their growth rates. Although treatment with a PI3K inhibitor induced apoptosis in both control and IGFBP-5-overexpressing LNCaP cells, this PI3K inhibitor-induced apoptosis was prevented by exogenous IGF-I treatment only in IGFBP-5 transfectants, suggesting that IGFBP-5 overexpression can potentiate the antiapoptotic effects of IGF-I. Furthermore, tumor growth and serum prostate-specific antigen levels increased several fold faster in mice bearing IGFBP-5-transfected LNCaP tumors after castration, despite having similar tumor incidence and tumor growth rates with controls when grown in intact mice before castration. Collectively, these data suggest that IGFBP-5 overexpression in prostate cancer cells after castration is an adaptive cell survival mechanism that helps potentiate the antiapoptotic and mitogenic effects of IGF-I, thereby accelerating progression to androgen independence through activation of the PI3K-Akt/ protein kinase B signaling pathway.

Topics: Androgens; Apoptosis; Cell Division; Cyclin D1; Dihydrotestosterone; Enzyme Activation; Enzyme Inhibitors; Gene Expression; Humans; Insulin-Like Growth Factor Binding Protein 5; Insulin-Like Growth Factor I; Male; Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Prostate-Specific Antigen; Prostatic Neoplasms; RNA, Messenger; Transfection; Tumor Cells, Cultured

There is currently no effective treatment for recurrent hormone refractory carcinomas of the prostate gland. An understanding of the underlying mechanisms responsible for the progression of these lesions is likely to be important for the development of new therapeutic approaches. Recently, it has been suggested that the transition to a hormone independent state is accompanied by increased proliferation and bcl-2 gene expression, as well as by a decreased apoptotic state.. To investigate the possible role of Bcl-2 and other cell cycle regulating proteins in the development of prostatic tumours.. Immunohistochemistry was used to study the relation between the expression of Bcl-2 and the androgen receptor, as well as p21WAF1/CIP1 (p21), and cyclin D1 status, in a series of 89 prostate cancer samples taken before androgen withdrawal treatment.. Androgen receptor negative tumours expressed significantly higher amounts of Bcl-2 than those prostate carcinomas with low/medium androgen receptor values. However, in tumours expressing the highest amounts of androgen receptor, Bcl-2 expression was also high. A significant positive relation between Bcl-2 and p21 expression, as well as an inverse relation between Bcl-2 and cyclin D1 expression, was noted. Androgen receptor positive samples also expressed significantly higher amounts of cyclin D1.. These results suggest that p21 and cyclin D1 expression in prostatic cancer might be modulated by Bcl-2 and by androgens and in turn this could be relevant to the progression of prostatic cancer.

Topics: Cyclin D1; Genes, bcl-2; Humans; Male; Neoplasms, Hormone-Dependent; Phosphotransferases; Prostatic Neoplasms; Receptors, Androgen

Genetic mechanisms leading to androgen-independent growth in advanced prostatic carcinomas (PC) are still poorly understood. Analysis of genes potentially involved in the regulation of tumor cell proliferation and apoptosis might confer better insight into this process and might lead to improved therapeutic strategies. Fluorescence in situ hybridization (FISH) analysis of dissociated nuclei with DNA probes for MYC (8q24)/#8, cyclin D1 gene (CCND1; 11q13)/#11, ERBB2 (17q13)/#17, the androgen receptor gene (AR; Xq12)/#X, and the retinoblastoma gene (RB; 13q14) was applied to formalin-fixed tissue from 63 patients with advanced PC after androgen deprivation therapy (ADT); matched tumor tissue before ADT was also available in 22 of these cases. The cut-points used were: "increased copy number," > or = 30% of all nuclei with increased FISH signals (centromere and/or gene); "amplification," > or = 15% of nuclei with "increased gene copy number." CCND1 and MYC gene "amplifications" were present before ADT in 25% and 33% of the cases, respectively; the frequency of these "amplifications" increased to 37% and 57% after ADT. Loss of the RB gene was nearly four times more frequent after ADT than before therapy (22% versus 6%). AR and ERBB2 gene "amplifications" occurred only after ADT in 36% and 30% of cases, respectively. With the exception of the AR gene, the copy number increase was low. After treatment, MYC and AR gene "amplifications" correlated with the proliferation rate (Ki-67/MIB1 index; p = 0.01 and p = 0.04), whereas ERBB2 "amplifications" were associated with increased apoptotic index (PCD/TUNEL; p = 0.016). However, no correlation between FISH results and clinical follow-up could be established. FISH analysis of genes putatively involved in PC progression revealed characteristic patterns of aberrations in advanced PC before and after ADT. Distinct changes in gene copy number before and after therapy suggests possible involvement of these genes in the escape from androgen control.

Topics: Aged; Androgen Antagonists; Cyclin D1; Gene Dosage; Genes, erbB-2; Genes, myc; Genes, Retinoblastoma; Humans; In Situ Hybridization, Fluorescence; Ki-67 Antigen; Male; Middle Aged; Prostatic Neoplasms; Receptors, Androgen

To assess several molecular markers (detected by immunohistochemistry, IHC) to determine whether they can be used to improve the prognostic value of histological grade alone in predicting the behaviour of prostate cancer.. Tumour tissue was retrieved from 156 men in whom tumour grade, stage and survival were known. The outcome measures were: (i) local stage (T-stage, organ-confined vs extraprostatic); (ii) metastatic status (M-stage, bone metastasis vs no bone metastasis); and (iii) survival. The IHC markers used were chosen to provide a broad representation of various aspects of tumour biology, i.e. the androgen receptor (AR) and oestrogen receptor (ER), adhesion molecules (E-cadherin), proliferation markers (MIB-1), tumour-suppressor genes (TP53 and the retinoblastoma gene product, Rb) and other novel cancer-related proteins (cyclin D1 and the breast cancer susceptibility gene product, BRCA2). All factors were assessed using logistic regression and Cox proportional-hazards survival models for predictive value, after adjusting for effects.. MIB-1, ER, cyclin D1 and E-cadherin all showed close statistically significant univariate associations with histological grade. Univariate analysis also identified close statistically significant associations between T-stage and both MIB-1 and E-cadherin. Likewise, there were close univariate associations for both M-stage and survival, and MIB-1, cyclin D1 and ER. Logistic regression modelling identified MIB-1, cyclin D1 and ER as statistically significant predictors of M-stage and, once MIB-1 was entered into the model, the effects of grade no longer made a significant contribution. MIB-1 was a significant predictor for T-stage, but the effects of grade remained significant in this model. Cox proportional-hazards modelling identified MIB-1, cyclin D1 and ER as being statistically significant predictors of survival, after adjusting for grade. After adjusting for both grade and MIB-1, the effects of cyclin D1 and ER were no longer statistically significant. Excess MIB-1, cyclin D1 or ER expression tended to be present within the most poorly differentiated and advanced-stage lesions; this provides an inherent instability to the models described. TP53, Rb, AR and BRCA2 were of limited prognostic value.. MIB-1, ER and cyclin D1 provide prognostic information that is clearly independent of grade. However, their true clinical value is probably limited because they are expressed mainly in the most advanced lesions. Nevertheless, MIB-1 expression is of sufficient value to warrant inclusion in future prognostic models. Furthermore, the expression of cyclin D1 and ER may reflect aspects of tumour biology that individually are worthy of further investigation. However, none of the IHC markers used in this study can be recommended for use in routine histological preparations.

Topics: Adult; Aged; Aged, 80 and over; Analysis of Variance; Antigens, Nuclear; Biomarkers, Tumor; Cyclin D1; Humans; Immunohistochemistry; Ki-67 Antigen; Logistic Models; Male; Middle Aged; Neoplasm Staging; Nuclear Proteins; Prostatic Neoplasms; Survival Analysis

Overexpression of cyclin D1 has been documented in a number of human cancers. Increased expression of cyclin D1 can contribute to cellular transformation and abnormal proliferation.. Quantitative RT-PCR and/or Western blot analysis were used to determine the level of cyclin D1 expression in 96 human prostate tumors, 15 benign prostate hyperplasias, 4 prostate cancer cell lines, and 3 xenografts.. Our results demonstrate that 4.2% of the prostate tumors examined overexpressed cyclin D1 transcripts. In the cell lines, expression was normal, with the exception that reduced levels of cyclin D1 transcript and protein were observed in the DU145 cell line, as expected from cells with mutant RB. Normal levels of cyclin D1 were found in all xenograft tumors and BPH specimens examined.. These data show that overexpression of cyclin D1 occurs rarely in human prostate tumors. However, when overexpression of cyclin D1 does occur, it may identify a subset of tumors with a different molecular biology.

Topics: Blotting, Southern; Blotting, Western; Cyclin D1; DNA Primers; DNA, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Male; Prostatic Hyperplasia; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; Tumor Cells, Cultured

Par-4 is a widely expressed protein that sensitizes both prostatic and non-prostatic cells to apoptosis. Constitutive- or regulated- overexpression of Par-4 caused a reduction in the levels of the anti-apoptotic protein Bcl-2. Replenishment of Bcl-2 levels abrogated susceptibility to Par-4-dependent apoptosis, suggesting that Par-4-mediated apoptosis requires downmodulation of Bcl-2 levels. The inverse correlation between Par-4 and Bcl-2 expression was recapitulated in human prostate tumors. Par-4 but not Bcl-2 was detected in the secretory epithelium of benign prostatic tumors and in primary and metastatic prostate cancers that are apt to undergo apoptosis. Moreover, xenografts of human, androgen-dependent CWR22 tumors showed Par-4 but not Bcl-2 expression. By contrast, androgen-independent CWR22R tumors derived from the CWR22 xenografts showed mutually exclusive expression patterns of Par-4 and Bcl-2. These findings suggest a mechanism by which Par-4 may sensitize prostate tumor cells to apoptosis.

Topics: 3T3 Cells; Animals; Apoptosis; Apoptosis Regulatory Proteins; Carrier Proteins; Cyclin D1; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Male; Mice; Prostatic Neoplasms

Prostate cancer development and progression is driven by the accumulation of genetic changes, the nature of which remains incompletely understood To facilitate high-throughput analysis of molecular events taking place in primary, recurrent, and metastat prostate cancer, we constructed a tissue microarray containing small 0.6-mm cylindrical samples acquired from 371 formalin-fixed blocks, including benign prostatic hyperplasia (n = 32) and primary tumors (n = 223), as well as both locally recurrent tumors (n = 54) and metastases (n = 62) from patients with hormone-refractory disease. Fluorescence in situ hybridization (FISH) was applied to the analysis of consecutive tissue microarray sections with probes for five different genes. High-level (> or =3X) amplifications were very rare (<2%) in primary prostate cancers However, in metastases from patients with hormone-refractory disease, amplification of the androgen receptor gene was seen in 22%, MYC in 11%, and Cyclin-D1 in 5% of the cases. In specimens from locally recurrent tumors, the corresponding percentages were 23, 4, and 8%. ERBB2 and NMYC amplifications were never detected at any stage of prostate cancer progression. In conclusion, FISH to tissue microarray sections enables high-throughput analysis of genetic alterations contributing to cancer development and progression. Our results implicate a role for amplification of androgen receptor in hormonal therapy failure and that of MYC in the metastatic progression of human prostate cancer.

Topics: Cyclin D1; Gene Amplification; Genes, erbB-2; Genes, myc; Humans; In Situ Hybridization, Fluorescence; Male; Prostatic Neoplasms; Receptors, Androgen

The cyclin D1 gene is amplified and/or overexpressed in several types of human cancer, including cancers of the breast, esophagus, head, and neck. However, the role of cyclin D1 in prostate cancer has not been previously studied in detail.. Six human prostate cancer cell lines and cultures of normal human prostate cells were examined by Western and Northern blot analyses for levels of expression of the cyclin D1 protein and mRNA, respectively. Southern blot analyses were performed to examine possible amplification of this gene. Immunostaining for cyclin D1 was performed on 50 primary prostate cancer samples.. Cyclin D1 protein was expressed at relatively high levels in all of the six human prostate cancer cell lines examined, but was not detected in the cultures of normal human prostate cells. The ALVA 41 cell line expressed the highest level of this protein. Relatively high levels of cyclin D1 mRNA were also found in all of the prostate cancer cell lines. Nevertheless, none of these cell lines revealed amplification of the cyclin D1 gene. Twelve of the 50 primary prostate cancer samples (24%) revealed regions of moderate to strongly positive staining for cyclin D1.. The increased expression of cyclin D1 in several prostate cancer cell lines and in a subset of primary prostate cancer samples suggests that further studies on the expression of this gene and related genes may be of interest in understanding the pathogenesis of prostate cancer.

Topics: Blotting, Northern; Blotting, Southern; Blotting, Western; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Male; Prostatic Neoplasms; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured

The human prostate carcinoma cell line, LNCaP, proliferates under stimulation by a limited number of mitogenic signals, which include members of the growth factor and steroid hormone families. Androgens and epidermal growth factor (EGF) are among the LNCaP cell mitogens. We tested the hypothesis that these mitogens stimulate LNCaP cell proliferation at least in part through the induction of cyclin D1, a protein requisite for cell cycle progression, which is expressed in the G1 phase of the cell cycle.. LNCaP cells were grown in serum-free medium with 10 ng/ml or 100 ng/ml EGF, 0.1 nM or 1.0 nM mibolerone (a potent androgen agonist), or vehicle (distilled water or 0.01% ethanol). Expression of cyclin D, mRNA, and protein were assessed by Northern and Western blot analyses. Transcription regulation was assessed by nuclear runoff assay.. Western analyses demonstrated that EGF stimulated cyclin D1 protein expression 4-fold over 12 hr. Northern analyses showed a 4-fold increase in mRNA expression, peaking within 4 hr of EGF stimulation. There were no effects on cyclin D1 protein or mRNA expression with mibolerone treatments. We further explored the mechanism of cyclin D1 induction. LNCaP cells stimulated for 1 hr with EGF demonstrated a 2-fold increase in cyclin D1 message, as assayed by nuclear runoff transcription assay. In addition, we demonstrated the involvement of the protein kinase C pathway in mediating the EGF induction of cyclin D1.. We conclude that one of the mechanisms by which growth factors such as EGF may stimulate prostate cell proliferation is through the direct induction of cyclin proteins, which are necessary for entry of cells into mitosis.

Topics: Blotting, Northern; Blotting, Western; Cyclin D1; Epidermal Growth Factor; Flow Cytometry; Humans; Male; Prostatic Neoplasms; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured

Deregulated expression of cyclin D1 has been found in several types of human tumors. In order to investigate factors involved in human prostate cancer progression, we studied the effects of cyclin D1 overexpression on human prostate cancer cell proliferation and tumorigenicity by transfecting LNCaP cells with a retroviral vector containing human cyclin D1 cDNA. When compared to the parental and control-vector transfected LNCaP cells, these cyclin D1-transfected cells had more cells in S-phase and lower growth factor requirements. Furthermore, these cells grew more in androgen-free medium. We also detected higher levels of Rb phosphorylation and E2F-1 protein levels in LNCaP/cyclin D1 cells than that in the parental and vector control cells in medium with or without androgen. Cyclin D1 transfected clones formed tumors more rapidly than control and parental cells. These tumors were refractory to the androgen-ablation treatment by castration, whereas tumors from parental and vector-control LNCaP cells regressed within 4 weeks after castration. These results suggest that overexpression of cyclin D1 changes the growth properties, increases tumorigenicity and decreases the requirement for androgen stimulation in LNCaP cells both in vitro and in vivo.

Topics: Androgens; Animals; Cell Division; Cyclin D1; Humans; Male; Mice; Mice, Nude; Prostatic Neoplasms; RNA, Messenger; Tumor Cells, Cultured

A series of 66 prostate cancer samples were studied immunohistochemically for expression of p53, p21, a cyclin-dependent kinase inhibitor regulated by p53, and cyclin D1, a cell cycle regulatory protein. Twenty samples (30%) showed positive staining for p53, 14 (21%) for p21, and 20 (30%) for cyclin D1. p53 expression was correlated with a high Gleason score while p21 and cyclin D1 did not demonstrate any clear clinicopathological factors. A positive correlation between p53 and p21 expression was observed, however these samples with both positive immunoreactivity had no p53 mutation, suggesting the possibility that p53 may be wild-type and induce p21 expression, and/or p21 is likely to be induced by a p53-independent pathway.

Topics: Adenocarcinoma; Aged; Aged, 80 and over; Biomarkers, Tumor; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA, Neoplasm; Humans; Immunoenzyme Techniques; Male; Middle Aged; Prostatic Neoplasms; Retrospective Studies; Tumor Suppressor Protein p53

Cyclin-dependent kinases (CDK) and cyclins constitute the subunits of the maturation-promoting factor that controls the process of cell division. High levels of these proteins have been reported in human malignancies of the stomach, colon, breast, and lung, and have been implicated in aberrant cell division and dysregulated tumor growth.. p34cdc2 CDK and cyclin D1 (D1) protein expression were evaluated in 140 radical prostatectomy specimens harboring adenocarcinoma (PAC), using the respective monoclonal antibodies on archival tissue sections. In each case, slides stained with hematoxylin and eosin were examined for evaluation of Gleason's grade and pathologic stage. The DNA content of the tumors was determined by the Feulgen method with the CAS200 Image Analyzer (Cell Analysis Systems, Lombard, IL). Nuclear immunoreactivity for the two proteins was semiquantitatively scored, and results were correlated with Gleason's grade, stage, ploidy, metastatic status, and disease recurrence after radical prostatectomy.. p34cdc2 was expressed in 84 of 140 PACs (60%) and correlated with high Gleason's grade (P = 0.0001), advanced pathologic stage (P = 0.01), nondiploid DNA content (P = 0.0001), and metastases (P = 0.04). On multivariate analysis using the Cox proportional hazards model, p34cdc2 immunoreactivity (P = 0.0001) and high Gleason's grade (P = 0.01) each independently predicted disease recurrence. When tumors were of low Gleason's grade and lacked p34cdc2 expression, 4 of 39 PACs (10%) recurred, as compared with 18 of 47 (38%) that recurred when tumors were of high Gleason's grade and expressed p34cdc2 protein. D1 was positive in 31 of 140 PACs (22%) and showed a trend (P = 0.07) of high Gleason's grade, but it did not reach statistical significance with any of the prognostic variables. In the majority of PACs expressing both p34cdc2 and D1 proteins, the adjacent benign prostate acini showed focal, scattered nuclear positivity of the basal and secretory epithelial cells.. p34cdc2 is expressed in a majority of PACs and correlates with high Gleason's grade, advanced pathologic stage, nondiploid DNA content, and metastases. On multivariate analyses high Gleason's grade and p34cdc2 immunoreactivity predict disease recurrence independently of the pathologic stage. Thus, p34cdc2 appears to play a critical role in the evolution, proliferation, and spread of PACs and may be of prognostic value when applied to initial prostate tissue samples taken by needle biopsy.

Topics: Adenocarcinoma; Aged; Aged, 80 and over; Biomarkers, Tumor; CDC2 Protein Kinase; Cyclin D1; Cyclins; DNA, Neoplasm; Humans; Immunohistochemistry; Lymphatic Metastasis; Male; Middle Aged; Multivariate Analysis; Oncogene Proteins; Ploidies; Prognosis; Proportional Hazards Models; Prostatic Neoplasms; Survival Rate

Cyclin D1, a critical positive regulator of G1 progression, has been implicated in the pathogenesis of certain cancers. Regulation of cyclin D1 occurs at the transcriptional and posttranscriptional level. Here we present evidence that cyclin D1 levels are regulated at the posttranscriptional level by the Ca2+-activated protease calpain. Serum starvation of NIH 3T3 cells resulted in rapid loss of cyclin D1 protein that was completely reversible by calpain inhibitors. Actinomycin D and lovastatin induced rapid loss of cyclin D1 in prostate and breast cancer cells that was reversible by calpain inhibitors and not by phenylmethylsulfonyl fluoride, caspase inhibitors, or lactacystin, a specific inhibitor of the 26 S proteasome. Treatment of intact NIH 3T3, prostate, and breast cancer cells with a calpain inhibitor dramatically increased the half-life of cyclin D1 protein. Addition of purified calpain to PC-3-M lysates resulted in Ca2+-dependent cyclin D1 degradation. Transient expression of the calpain inhibitor calpastatin increased cyclin D1 protein in serum-starved NIH 3T3 cells. Cyclins A, E, and B1 have been reported to be regulated by proteasome-associated proteolysis. The data presented here implicate calpain in cyclin D1 posttranslational regulation.

Topics: 3T3 Cells; Acetylcysteine; Animals; Calpain; Cyclin B; Cyclin B1; Cyclin D1; Cysteine Proteinase Inhibitors; Dactinomycin; Half-Life; Humans; Male; Mice; Prostatic Neoplasms; Protein Synthesis Inhibitors; Tumor Cells, Cultured