leupeptins and Prostatic-Neoplasms

Androgen receptor (AR) degradation is the primary regulator of androgen receptor activity. This study was designed to investigate the influence of the proteasome on AR protein stability after enzalutamide (Enz) treatment.. Cell counting after treatment was utilized to assess the effect of Enz on cell proliferation. Changes in mRNA levels were evaluated using reverse transcription-polymerase chain reaction (RT-PCR). Proteasome activity was assessed by measurement of the chymotrypsin-like activity of the beta-5 subunit of the proteasome. Changes in protein levels after treatment with Enz, MG132 (MG), bortezomib (Bor), or their combination were assessed using western blot analysis.. Treatment with Enz led to a significant reduction of cell proliferation and AR protein levels. However, AR mRNA levels were unchanged. Inhibition of proteasome activity by MG counteracts the Enz-mediated AR degradation transiently, whereas Bor showed no inhibition of the Enz-mediated AR degradation.. Enz-mediated change in AR stability as an early and essential event after treatment was shown. However, investigations of the ubiquitin/proteasome system indicate involvement of several proteases in the Enz-mediated AR degradation process.

Topics: Antineoplastic Combined Chemotherapy Protocols; Benzamides; Bortezomib; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Humans; Leupeptins; Male; Nitriles; Phenylthiohydantoin; Prostate; Prostatic Neoplasms; Proteasome Endopeptidase Complex; Proteolysis; Receptors, Androgen; RNA, Messenger

Elongation factor for RNA polymerase 2 (ELL2) and ELL associated factor 2 (EAF2) have been reported to have tumor suppressive properties in prostate epithelial cells.. We investigated ELL2 expression in human prostate cancer specimens, and ELL2 protein stability and ubiquitination in prostate cancer cells.. Immunostaining analysis of human prostate cancer specimens was used to determine ELL2 expression in tumor and normal tissues. ELL2 knockdown in prostate cancer cell lines LNCaP and C4-2 was used to compare proliferation and motility. Deletion and site-directed mutagenesis was used to identify amino acid residues in ELL2 that were important for degradation.. ELL2 protein was downregulated in prostate cancer specimens and was up-regulated by androgens in prostate cancer cell lines LNCaP and C4-2. ELL2 knockdown enhanced prostate cancer cell proliferation and motility. ELL2 protein has a short half-life and was stabilized by proteasome inhibitor MG132. Amino acid residues K584 and K599 in ELL2 were important for ELL2 degradation. EAF2 could stabilize ELL2 and inhibited its polyubiquitination.. Our findings provide further evidence that ELL2 is a potential tumor suppressor frequently down-regulated in clinical prostate cancer specimens and provides new insights into regulation of ELL2 protein level by polyubiquitination and EAF2 binding.

Topics: Androgens; Cell Line, Tumor; Cell Movement; Cell Proliferation; Down-Regulation; Gene Knockdown Techniques; HEK293 Cells; Humans; Leupeptins; Male; Prostatic Neoplasms; Protein Stability; Transcription Factors; Transcriptional Elongation Factors; Ubiquitination

Androgen deprivation therapy in prostate cancer is extremely effective; however, due to the continuous expression and/or mutagenesis of androgen receptor (AR), the resistance to antihormonal therapy is a natural progression. Consequently, targeting the AR for degradation offers an alternate approach to overcome this resistance in prostate cancer. In this study, we demonstrate that carnosic acid, a benzenediol diterpene, binds the ligand-binding domain of the AR and degrades the AR via endoplasmic reticulum (ER) stress-mediated proteasomal degradative pathway. In vitro, carnosic acid treatment induced degradation of AR and decreased expression of prostate-specific antigen in human prostate cancer cell lines LNCaP and 22Rv1. Carnosic acid also promoted the expression of ER proteins including BiP and CHOP in a dose-dependent manner. Downregulation of CHOP by small interfering RNA somewhat restored expression of AR suggesting that AR degradation is dependent on ER stress pathway. Future studies will need to evaluate other aspects of the unfolded protein response pathway to characterize the regulation of AR degradation. Furthermore, cotreating cells individually with carnosic acid and proteasome inhibitor (MG-132) and carnosic acid and an ER stress modulator (salubrinal) restored protein levels of AR, suggesting that AR degradation is mediated by ER stress-dependent proteasomal degradation pathway. Degradation of AR and induction of CHOP protein were also evident in vivo along with a 53% reduction in growth of xenograft prostate cancer tumors. In addition, carnosic acid-induced ER stress in prostate cancer cells but not in normal prostate epithelial cells procured from patient biopsies. In conclusion, these data suggest that molecules such as carnosic acid could be further evaluated and optimized as a potential therapeutic alternative to target AR in prostate cancer.

Topics: Abietanes; Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cinnamates; Endoplasmic Reticulum Stress; Gene Expression Regulation, Neoplastic; Humans; Leupeptins; Male; Mice; Prostate-Specific Antigen; Prostatic Neoplasms; Proteolysis; Receptors, Androgen; Thiourea; Transcription Factor CHOP; Unfolded Protein Response; Xenograft Model Antitumor Assays

The proteasome inhibitors Bortezomib (BZM) and MG132 trigger cancer cell death via induction of endoplasmic reticulum (ER) stress and unfolded protein response. Epigallocatechin gallate (EGCG), the most bioactive green tea polyphenol, is known to display strong anticancer properties as it inhibits proteasome activity and induces ER stress. We investigated whether combined delivery of a proteasome inhibitor with EGCG enhances prostate cancer cell death through increased induction of ER stress. Paradoxically, EGCG antagonized BZM cytotoxicity even when used at low concentrations. Conversely, the MG132 dose-response curve was unaffected by co-administration of EGCG. Moreover, apoptosis, proteasome inhibition and ER stress were inhibited in PC3 cells simultaneously treated with BZM and EGCG but not with a combination of MG132 and EGCG; EGCG enhanced autophagy induction in BZM-treated cells only. Autophagy inhibition restored cytotoxicity concomitantly with CHOP and p-eIF2α up-regulation in cells treated with BZM and EGCG. Overall, these findings demonstrate that EGCG antagonizes BZM toxicity by exacerbating the activation of autophagy, which in turn mitigates ER stress and reduces CHOP up-regulation, finally protecting PC3 cells from cell death.

Topics: Antineoplastic Agents; Autophagy; Bortezomib; Catechin; Cell Line, Tumor; Endoplasmic Reticulum Stress; Eukaryotic Initiation Factor-2; Humans; Leupeptins; Male; Microtubule-Associated Proteins; Prostatic Neoplasms; Proteasome Inhibitors; Transcription Factor CHOP; Up-Regulation

Cdc25C is a cell cycle protein of the dual specificity phosphatase family essential for activating the cdk1/Cyclin B1 complex in cells entering into mitosis. Since altered cell cycle is a hallmark of human cancers, we investigated androgen regulation of Cdc25C protein in human prostate cancer (PCa) cells, including androgen-sensitive (AS) LNCaP C-33 cells and androgen-independent (AI) LNCaP C-81 as well as PC-3 cells. In the regular culture condition containing fetal bovine serum (FBS), Cdc25C protein levels were similar in these PCa cells. In a steroid-reduced condition, Cdc25C protein was greatly decreased in AS C-33 cells but not AI C-81 or PC-3 cells. In androgen-treated C-33 cells, the Cdc25C protein level was greatly elevated, following a dose- and a time-dependent manner, correlating with increased cell proliferation. This androgen effect was blocked by Casodex, an androgen receptor blocker. Nevertheless, epidermal growth factor (EGF), a growth stimulator of PCa cells, could only increase Cdc25C protein level by about 1.5-fold. Altered expression of Cdc25C in C-33 cells and PC-3 cells by cDNA and/or shRNA transfection is associated with the corresponding changes of cell growth and Cyclin B1 protein level. Actinomycin D and cycloheximide could only partially block androgen-induced Cdc25C protein level. Treatments with both proteasomal and lysosomal inhibitors resulted in elevated Cdc25C protein levels. Immunoprecipitation revealed that androgens reduced the ubiquitination of Cdc25C proteins. These results show for the first time that Cdc25C protein plays a role in regulating PCa cell growth, and androgen treatments, but not EGF, greatly increase Cdc25C protein levels in AS PCa cells, which is in part by decreasing its degradation. These results can lead to advanced PCa therapy via up-regulating the degradation pathways of Cdc25C protein.

Topics: Androgens; cdc25 Phosphatases; Cell Cycle Proteins; Cell Line, Tumor; Dihydrotestosterone; Humans; Leucine; Leupeptins; Lysosomes; Male; Prostatic Neoplasms; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Receptors, Androgen; Transcriptome; Up-Regulation

Histone deacetylase (HDAC) inhibitors are successful for treatment of advanced cutaneous T-cell lymphoma but only show modest effect in solid tumors. Approaches for HDAC inhibitors to improve activity against solid tumors are necessary.. Sulforhodamine B assay and flow cytometric analysis detected cell proliferation and cell-cycle progression, respectively. Protein expression was determined by Western blotting. Comet assay and DNA end-binding activity of Ku proteins detected DNA damage and DNA repair activity, respectively. siRNA technique was used for knockdown of specific cellular target.. WJ25591 displayed inhibitory activity against HDAC1 and cell proliferation in human hormone-refractory prostate cancers PC-3 and DU-145. WJ25591 caused an arrest of cell-cycle at both G1- and G2-phase and increased protein expressions of p21 and cyclin E, followed by cell apoptosis. WJ25591-induced Bcl-2 down-regulation and activation of caspase-9, -8, and -3, suggesting apoptotic execution through both intrinsic and extrinsic apoptotic pathways. WJ25591 also significantly inhibited DNA repair activity but not directly induced DNA damage. Moreover, the proteasome inhibitor MG-132 dramatically sensitized WJ25591-induced cell apoptosis. The siRNA technique demonstrated that endoplasmic reticulum (ER) stress, in particular CHOP/GADD153 up-regulation, contributed to the synergistic effect.. The data suggest that WJ25591 inhibited HDAC activity, leading to cell-cycle arrest and inhibition of DNA repair. Caspase cascades are subsequently triggered to execute cell apoptosis. MG-132 dramatically sensitizes WJ25591-mediated apoptosis, at least partly, through ER stress response. The data also reveal that combination of HDAC inhibitors and proteasome inhibitors may be a potential strategy against hormone-refractory prostate cancers.

Topics: Antineoplastic Agents; Cell Cycle; Cell Line, Tumor; Drug Synergism; Endoplasmic Reticulum Stress; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Leupeptins; Male; Prostatic Neoplasms; Proteasome Inhibitors

Human steroid sulfatase (STS) plays an important role in regulating the formation of biologically active estrogens and may be a promising target for treating estrogen-mediated carcinogenesis. The molecular mechanism of STS gene expression, however, is still not clear. Growth factors are known to increase STS activity but the changes in STS expression have not been completely understood. To determine whether insulin-like growth factor (IGF)-II can induce STS gene expression, the effects of IGF-II on STS expression were studied in PC-3 human prostate cancer cells. RT-PCR and Western blot analysis showed that IGF-II treatment significantly increased the expression of STS mRNA and protein in concentration- and time-dependent manners. To understand the signaling pathway by which IGF-II induces STS gene expression, the effects of specific PI3-kinase/Akt and NF-κB inhibitors were determined. When the cells were treated with IGF-II and PI3-kinase/Akt inhibitors, such as LY294002, wortmannin, or Akt inhibitor IV, STS expression induced by IGF-II was significantly blocked. Moreover, we found that NF-κB inhibitors, such as MG-132, bortezomib, Bay 11-7082 or Nemo binding domain (NBD) binding peptide, also strongly prevented IGF-II from inducing STS gene expression. We assessed whether IGF-II activates STS promoter activity using transient transfection with a luciferase reporter. IGF-II significantly stimulated STS reporter activity. Furthermore, IGF-II induced expression of 17β-hydroxysteroid dehydrogenase (HSD) 1 and 3, whereas it reduced estrone sulfotransferase (EST) gene expression, causing enhanced estrone and β-estradiol production. Taken together, these results strongly suggest that IGF-II induces STS expression via a PI3-kinase/Akt-NF-κB signaling pathway in PC-3 cells and may induce estrogen production and estrogen-mediated carcinogenesis.

Topics: 17-Hydroxysteroid Dehydrogenases; Androstadienes; Boronic Acids; Bortezomib; Cell Line, Tumor; Chromones; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Humans; Insulin-Like Growth Factor II; Leupeptins; Male; Morpholines; NF-kappa B; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Polymerase Chain Reaction; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Pyrazines; Sequence Analysis, DNA; Signal Transduction; Steryl-Sulfatase; Sulfones; Sulfotransferases; Wortmannin

Expression of clusterin (CLU) closely correlates with the regulation of apoptosis in cancer. Although endoplasmic reticulum (ER) stress-induced upregulation and retrotranslocation of cytoplasmic CLU (presecretory (psCLU) and secreted (sCLU) forms) has been linked to its anti-apoptotic properties, mechanisms mediating these processes remain undefined. Here, we show using human prostate cancer cells that GRP78 (Bip) associates with CLU under ER stress conditions to facilitate its retrotranslocation and redistribution to the mitochondria. Many ER stress inducers, including thapsigargin, MG132 or paclitaxel, increased expression levels of GRP78 and CLU, as well as post-translationally modified hypoglycosylated CLU forms. ER stress increased association between GRP78 and CLU, which led to increased cytoplasmic CLU levels, while reducing sCLU levels secreted into the culture media. GRP78 stabilized CLU protein and its hypoglycosylated forms, in particular after paclitaxel treatment. Moreover, subcellular fractionation and confocal microscopy with CLUGFP indicated that GRP78 increased stress-induced CLU retrotranslocation from the ER with co-localized redistribution to the mitochondria, thereby reducing stress-induced apoptosis by cooperatively stabilizing mitochondrial membrane integrity. GRP78 silencing reduced CLU protein, but not mRNA levels, and enhanced paclitaxel-induced cell apoptosis. Taken together, these findings reveal novel dynamic interactions between GRP78 and CLU under ER stress conditions that govern CLU trafficking and redistribution to the mitochondria, elucidating how GRP78 and CLU cooperatively promote survival during treatment stress in prostate cancer.

Topics: Apoptosis; Cell Line, Tumor; Clusterin; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Heat-Shock Proteins; Humans; Leupeptins; Male; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Membranes; Paclitaxel; Prostatic Neoplasms; Protein Transport; RNA Interference; RNA, Messenger; RNA, Small Interfering; Thapsigargin; Up-Regulation

Green tea polyphenols (GTPs) reactivate epigenetically silenced genes in cancer cells and trigger cell cycle arrest and apoptosis; however, the mechanisms whereby these effects occur are not well understood. We investigated the molecular mechanisms underlying the antiproliferative effects of GTP, which may be similar to those of histone deacetylase (HDAC) inhibitors. Exposure of human prostate cancer LNCaP cells (harboring wild-type p53) and PC-3 cells (lacking p53) with 10-80 μg/ml of GTP for 24 h resulted in dose-dependent inhibition of class I HDAC enzyme activity and its protein expression. GTP treatment causes an accumulation of acetylated histone H3 in total cellular chromatin, resulting in increased accessibility to bind with the promoter sequences of p21/waf1 and Bax, consistent with the effects elicited by an HDAC inhibitor, trichostatin A. GTP treatment also resulted in increased expression of p21/waf1 and Bax at the protein and message levels in these cells. Furthermore, treatment of cells with proteasome inhibitor, MG132 together with GTP prevented degradation of class I HDACs, compared with cells treated with GTP alone, indicating increased proteasomal degradation of class I HDACs by GTP. These alterations were consistent with G(0)-G(1) phase cell cycle arrest and induction of apoptosis in both cell lines. Our findings provide new insight into the mechanisms of GTP action in human prostate cancer cells irrespective of their p53 status and suggest a novel approach to prevention and/or therapy of prostate cancer achieved via HDAC inhibition.

Topics: Acetylation; Apoptosis; bcl-2-Associated X Protein; Cell Cycle Checkpoints; Cell Proliferation; Chromatin; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Leupeptins; Male; Polyphenols; Promoter Regions, Genetic; Prostatic Neoplasms; Proteasome Endopeptidase Complex; Protein Binding; Proteolysis; Tea; Tumor Cells, Cultured; Tumor Suppressor Protein p53

We recently reported that hematological and neurological expressed 1 (HN1) is a ubiquitously expressed, EGF-regulated gene. Expression of HN1 in prostate cell lines down-regulates PI3K-dependent Akt activation. Here, we investigate whether HN1 is regulated by androgens through the putative androgen response elements (AREs) found in its promoter. Knockdown of HN1 expression by siRNA silencing leads to an increase in Akt((S473)) phosphorylation, resulting in the translocation of androgen receptor (AR) to the nucleus; these effects can be abrogated by the non-specific Akt inhibitor LY294002 but not by the ERK inhibitor PD98059. Furthermore, HN1 overexpression correlates with an increase in ubiquitination-mediated degradation (a consequence of the decrease in S213/210 phosphorylation of AR), ultimately resulting in the down-regulation of AR-mediated expression of the KLK3, KLK4, NKX3.1 and STAMP2 genes. We also found that HN1 overexpression suppresses colony formation as well as R1881-mediated growth in LNCaP cells, while it has the opposite effect (increasing colony formation but not proliferation) in PC-3 and DU145 cells. Therefore, we suggest that HN1 maintains a balance between the androgen-regulated nuclear translocation of AR and steady-state Akt phosphorylation, predominantly in the absence of androgens. If so, the balance between cell growth and EGF- and AR-signaling must be tightly regulated by HN1. This work has important implications for prostate cancer research, as AR, EGFR and HN1 are known to be highly expressed in prostate adenocarcinomas.

Topics: Androgens; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Dexamethasone; Estradiol; Gene Expression; Gene Expression Regulation; Glucocorticoids; Humans; Kallikreins; Leupeptins; Male; Metribolone; Microtubule-Associated Proteins; Nerve Tissue Proteins; Nuclear Proteins; Phosphorylation; Prostatic Neoplasms; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Transport; Proteolysis; Proto-Oncogene Proteins c-akt; Receptors, Androgen; Transcriptional Activation

The spindle assembly checkpoint (SAC) is a conserved mechanism that ensures the fidelity of chromosome distribution in mitosis by preventing anaphase onset until the correct bipolar microtubule-kinetochore attachments are formed. Errors in SAC function may contribute to tumorigenesis by inducing numerical chromosome anomalies (aneuploidy). On the other hand, total disruption of SAC can lead to massive genomic imbalance followed by cell death, a phenomena that has therapeutic potency. We performed a cell-based high-throughput screen with a compound library of 2000 bioactives for novel SAC inhibitors and discovered a plant-derived phenolic compound eupatorin (3',5-dihydroxy-4',6,7-trimethoxyflavone) as an anti-mitotic flavonoid. The premature override of the microtubule drug-imposed mitotic arrest by eupatorin is dependent on microtubule-kinetochore attachments but not interkinetochore tension. Aurora B kinase activity, which is essential for maintenance of normal SAC signaling, is diminished by eupatorin in cells and in vitro providing a mechanistic explanation for the observed forced mitotic exit. Eupatorin likely has additional targets since eupatorin treatment of pre-mitotic cells causes spindle anomalies triggering a transient M phase delay followed by impaired cytokinesis and polyploidy. Finally, eupatorin potently induces apoptosis in multiple cancer cell lines and suppresses cancer cell proliferation in organotypic 3D cell culture model.

Topics: Antimitotic Agents; Apoptosis; Aurora Kinase B; Aurora Kinases; Cell Culture Techniques; Cell Proliferation; Centrosome; Flavonoids; HeLa Cells; High-Throughput Screening Assays; Humans; Leupeptins; M Phase Cell Cycle Checkpoints; Male; Microscopy, Fluorescence; Nocodazole; Polyploidy; Prostatic Neoplasms; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Serine-Threonine Kinases; Pyrimidines; Thiones; Time-Lapse Imaging

The androgen receptor (AR) acts as a ligand-dependent transcription factor, whereas mutant AR lacking the C-terminal ligand-binding domain functions in a ligand-independent manner. In the present study we report that the C-terminal truncated AR, which we named AR-NH1 (the N-terminal fragment of AR cleaved in the neighborhood of helix 1 of the ligand-binding domain), is produced in LNCaP prostatic carcinoma cells. The AR-NH1 of ~90 kDa was observed in an androgen-independent LNCaP subline and was further accumulated by the proteasome inhibitor MG132. MG132 treatment caused the accumulation of AR-NH1 even in parent LNCaP cells. AR-NH1 was produced in the absence of ligand or in the presence of the AR antagonist bicalutamide, whereas AR agonists suppressed its production. AR-NH1 was detected with different AR antibodies recognizing amino acid residues 1-20 and 300-316 and was also generated from exogenous AR. Both siRNA-mediated AR knockdown and treatment with a serine protease inhibitor (4-(2-aminoethyl)-benzenesulfonyl fluoride) reduced AR-NH1 levels. According to the predicted cleavage site (between amino acid residues 660-685) and its nuclear localization, it is assumed that AR-NH1 functions as a constitutively active transcription factor. These data suggest that AR-NH1 is produced under hormone therapy and contributes to the development of castration-resistant prostate cancer due to its ligand-independent transcriptional activity.

Topics: Androgen Receptor Antagonists; Androgens; Anilides; Antineoplastic Agents; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Humans; Leupeptins; Male; Nitriles; Prostatic Neoplasms; Receptors, Androgen; RNA Interference; RNA, Small Interfering; Serine Proteinase Inhibitors; Sulfones; Tosyl Compounds

Hsp27 is a stress-activated multifunctional chaperone that inhibits treatment-induced apoptosis and causes treatment resistance in prostate and other cancers. We previously showed that targeted suppression of Hsp27 sensitizes cancer cells to hormone and chemotherapy. However, mechanisms by which Hsp27 confers cell treatment resistance are incompletely defined. Here, we report that Hsp27 protects human prostate cancer cells against proteotoxic stress induced by proteasome inhibition, and that Hsp27 silencing using siRNA or antisense (OGX-427) induced both apoptosis and autophagy through mechanisms involving reduced proteasome activity and induction of endoplasmic reticulum (ER) stress. We found that autophagy activation protected against ER stress-induced cell death, whereas inhibition of autophagy activation following Hsp27 silencing using either pharmacologic inhibitors or atg3 silencing enhanced cell death. Importantly, cotargeting Hsp27 and autophagy by combining OGX-427 with the autophagy inhibitor, chloroquine, significantly delayed PC-3 prostate tumor growth in vivo. These findings identify autophagy as a cytoprotective, stress-induced adaptive pathway, activated following disruption of protein homeostasis and ER stress induced by Hsp27 silencing. Combinatorial cotargeting cytoprotective Hsp27 and autophagy illustrates potential benefits of blocking activation of adaptive pathways to improve treatment outcomes in cancer.

Topics: Animals; Antineoplastic Agents; Apoptosis; Autophagy; Cell Line, Tumor; Chloroquine; Drug Synergism; Endoplasmic Reticulum Stress; Gene Expression; Gene Silencing; HSP27 Heat-Shock Proteins; Humans; Leupeptins; Male; Oligonucleotides, Antisense; Prostatic Neoplasms; Proteasome Endopeptidase Complex; Tumor Burden; Ubiquitin; Xenograft Model Antitumor Assays

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

Isoprenoids are recognized for their ability to suppress carcinogenic processes in vivo and in vitro. We previously established that the isoprenoid, perillyl alcohol, acted mechanistically on translation of specific proteins through modulation of mechanistic target of rapamycin (mTOR) signaling. Telomerase-the enzyme responsible for immortalizing cells through the addition of telomeric repeats-is de-repressed early in an aspiring cancer cell. Here the effects of biologically-relevant concentrations and short incubations (1-16 h) of perillyl alcohol or the mTOR inhibitor, rapamycin, on telomerase activity were examined in prostate cancer cell lines. A rapid suppression of telomerase activity was observed (from ∼65% to >95%) determined by real-time quantitative telomerase repeat amplification protocol and confirmed by polyacrylamide gel-analysis. Using real-time reverse transcriptase-PCR, we demonstrated that human telomerase reverse transcriptase (hTERT) mRNA levels were unaltered. Western blot analysis revealed that hTERT protein levels decreased in response to perillyl alcohol or rapamycin. This decrease was partially blocked by pretreatment with a proteasome inhibitor MG-132, indicating that proteasomal degradation contributed to the loss of hTERT protein. No change in hTERT phosphorylation at Ser824 was observed, indicating the absence of cellular hTERT protein redistribution. These findings provide evidence for a unique link between nutrient- and macrolide-mediated regulation of mTOR and hTERT, a key enzyme that regulates DNA structure and stability.

Topics: Blotting, Western; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Leupeptins; Male; Monoterpenes; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sirolimus; Telomerase; TOR Serine-Threonine Kinases

Androgen receptor (AR) plays a key role in prostate development and carcinogenesis. Increased expression and/or stability of AR is associated with sensitization of prostate cancer cells to low levels of androgens, leading to castration resistance. Hence, understanding the mechanisms regulating AR protein stability is clinically relevant and may lead to new approaches to prevent and/or treat prostate cancer. Using fluorescence microscopy, Western blot, and pulse chase assay, we showed that nuclear export signal (NES)(AR), a nuclear export signal in the ligand binding domain (LBD) of AR, can significantly enhance the degradation of fusion protein constructs in PC3 prostate cancer cells. The half-life of GFP-NES(AR) was less than 3 h, which was 10 times shorter than that of green fluorescent protein (GFP) control. Further analysis showed that NES(AR) can signal for polyubiquitination and that degradation of NES(AR)-containing fusion proteins can be blocked by proteasome inhibitor MG132. Ubiquitination of GFP-AR or GFP-LBD was suppressed in the presence of dihydrotestosterone, which is known to suppress NES(AR) while inducing nuclear localization signal 2 in AR or LBD, suggesting that the export activity of NES(AR) is required for NES(AR)-mediated polyubiquitination. Treatment with MG132 also induced aggresome formation of NES(AR)-containing fusion proteins in perinuclear regions of the transfected PC3 cells, indicating a role for NES(AR) in inducing unfolded protein responses. The above observations suggest that NES(AR) plays a key role in AR ubiquitination and proteasome-dependent degradation in prostate cancer cells.

Topics: Active Transport, Cell Nucleus; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Green Fluorescent Proteins; Humans; Leupeptins; Ligands; Male; Polyubiquitin; Prostatic Neoplasms; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Receptors, Androgen; Signal Transduction; Time Factors; Ubiquitin

A stem cell model of prostate cancer tumourigenesis explains progression to castration resistant prostate cancer (CRPC) and offers novel perspectives in targeting this cancer in its more advanced forms. Androgen receptor (AR) regulated pathways are central mechanisms in progression to CRPC. However, AR was thought to be lacking in prostate stem cell enriched fractions. Potential low levels of AR expression in stem cell enriched cells were investigated and potential direct effects of androgen were examined.. Human prostate stem cell enriched populations, based on high α(2)β(1) integrin expression (α(2)β(1)(hi)), were selected from primary human prostate tissue in men undergoing transurethral prostatectomy or cystoprostatectomy. Effects on differentiation were assayed with flow cytometry using differentiation-specific markers.. Low levels of AR were demonstrable in α(2)β(1)(hi) cells following inhibition of the proteasome using MG132. Furthermore, a direct effect of androgen was shown in stabilising/inducing AR expression. Androgen treatment of α(2)β(1)(hi) cells was associated with the induction of differentiation using a number of differentiation-specific markers (prostatic acid phosphatase, cytokeratin 18 and AR) with increases ranging from 49% to 67% (p<0.05). These effects were blocked with the AR-specific inhibitor bicalutamide (p<0.05). These data support a role of direct androgen activity on stem cell enriched cells in the prostate and the implications of these findings are discussed.

Topics: Aged; Aged, 80 and over; Androgen Antagonists; Androgen Receptor Antagonists; Androgens; Anilides; Antigens, Differentiation; Biomarkers, Tumor; Cell Transformation, Neoplastic; Cysteine Proteinase Inhibitors; Epithelial Cells; Humans; Leupeptins; Male; Metribolone; Middle Aged; Neoplastic Stem Cells; Nitriles; Prostatic Neoplasms; Receptors, Androgen; Testosterone Congeners; Tosyl Compounds

Clusterin is a stress-activated, cytoprotective chaperone that confers broad-spectrum treatment resistance in cancer. However, the molecular mechanisms mediating CLU transcription following anticancer treatment stress remain incompletely defined. We report that Y-box binding protein-1 (YB-1) directly binds to CLU promoter regions to transcriptionally regulate clusterin expression. In response to endoplasmic reticulum stress inducers, including paclitaxel, YB-1 is translocated to the nucleus to transactivate clusterin. Furthermore, higher levels of activated YB-1 and clusterin are seen in taxane-resistant, compared with parental, prostate cancer cells. Knockdown of either YB-1 or clusterin sensitized prostate cancer cells to paclitaxel, whereas their overexpression increased resistance to taxane. Clusterin overexpression rescued cells from increased paclitaxel-induced apoptosis following YB-1 knockdown; in contrast, however, YB-1 overexpression did not rescue cells from increased paclitaxel-induced apoptosis following clusterin knockdown. Collectively, these data indicate that YB-1 transactivation of clusterin in response to stress is a critical mediator of paclitaxel resistance in prostate cancer.

Topics: Apoptosis; Benzoquinones; Bridged-Ring Compounds; Cell Line, Tumor; Cell Proliferation; Clusterin; Drug Resistance, Neoplasm; Endoplasmic Reticulum Stress; Gene Expression Regulation, Neoplastic; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Leupeptins; Male; Paclitaxel; Promoter Regions, Genetic; Prostatic Neoplasms; Protein Binding; RNA, Small Interfering; Taxoids; Transcriptional Activation; Y-Box-Binding Protein 1

We have reported that clioquinol alters lysosome integrity, inhibits nuclear factor kappa B (NF-kappaB) activity, and induces apoptosis in human cancer cells. The present study investigated whether clioquinol targets both pathways dependently or independently in human prostate cancer DU 145 cells. Clioquinol inhibited NF-kappaB activity, an effect being more pronounced in the presence of zinc. This inhibition was mediated through a reduced nuclear level of p65, the most frequently detected NF-kappaB subunit. Clioquinol also induced alterations of lysosome permeability in a zinc concentration-dependent manner. Pretreatment of the cells with ammonium, a lysosome protection agent, attenuated clioquinol-induced disruption of the lysosomes, yet ammonium had no effect on clioquinol-induced inhibition of NF-kappaB signaling. MG132, an established NF-kappaB inhibitor, suppressed NF-kappaB activity without causing alterations of lysosome permeability. These findings indicate that clioquinol targets NF-kappaB and lysosome pathways independently, favoring further development of clioquinol as a novel anticancer agent.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Membrane Permeability; Clioquinol; Humans; Leupeptins; Lysosomes; Male; NF-kappa B; Prostatic Neoplasms; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Quaternary Ammonium Compounds; Signal Transduction; Zinc

This study was to investigate the effect of phenethyl isothiocyanate (PEITC), a constituent of many edible cruciferous vegetables, on the expression of alpha- and beta-tubulins, which are the main components of microtubules in prostate cancer cells. Flow cytometry, light microscopy and western blot were used to study the cell cycle distribution, morphology changes and the expression of alpha- and beta-tubulins in prostate cancer cells treated with PEITC. The results showed that PEITC-induced G2-M cell phase arrest and inhibited the expression of alpha- and beta-tubulin proteins in a number of human prostatic carcinoma cell lines. Further, it is showed that this inhibitory effect could be reversed by antioxidant N-acetyl cysteine and proteasome inhibitor MG132. Finally, it is concluded that PEITC inhibited the expression of alpha- and beta-tubulins in prostate cancer cells, which is at least related to the oxygen reaction species and protein degradation.

Topics: Anticarcinogenic Agents; Antioxidants; Blotting, Western; Cell Cycle; Cell Line, Tumor; Cysteine; Flow Cytometry; G2 Phase; Humans; Isothiocyanates; Leupeptins; Male; Prostatic Neoplasms; Protein Isoforms; Tubulin

The Clusterin (CLU) gene produces different forms of protein products, which vary in their biological properties and distribution within the cell. Both the extra- and intracellular CLU forms regulate cell proliferation and apoptosis. Dis-regulation of CLU expression occurs in many cancer types, including prostate cancer. The role that CLU plays in tumorigenesis is still unclear. We found that CLU over-expression inhibited cell proliferation and induced apoptosis in prostate cancer cells. Here we show that depletion of CLU affects the growth of PC-3 prostate cancer cells. Following siRNA targeting all CLU mRNA variants, all protein products quickly disappeared, inducing cell cycle progression and higher expression of specific proliferation markers (i.e., H3 mRNA, PCNA, and cyclins A, B1, and D) as detected by RT-qPCR and Western blot. Quite surprisingly, we also found that the turnover of CLU protein is very rapid and tightly regulated by ubiquitin-proteasome mediated degradation. Inhibition of protein synthesis by cycloheximide showed that CLU half-life is less than 2 h. CLU protein products were found poly-ubiquitinated by co-immuniprecipitation. Proteasome inhibition by MG132 caused stabilization and accumulation of all CLU protein products, including the nuclear form of CLU (nCLU), and committing cells to caspase-dependent death. In conclusion, proteasome inhibition may induce prostate cancer cell death through accumulation of nCLU, a potential tumor suppressor factor.

Topics: Animals; Apoptosis; Cell Cycle; Cell Proliferation; Clusterin; Cysteine Proteinase Inhibitors; Gene Silencing; Humans; Leupeptins; Male; Polyubiquitin; Prostatic Neoplasms; Proteasome Endopeptidase Complex; Protein Isoforms; Protein Stability; Rats; RNA, Small Interfering; Tumor Cells, Cultured

The novel proteasome inhibitor NPI-0052 has been shown to sensitize tumor cells to apoptosis by various chemotherapeutic drugs and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), although the mechanisms involved are not clear. We hypothesized that NPI-0052-mediated sensitization may result from NF-kappaB inhibition and downstream modulation of the metastasis inducer Snail and the metastasis suppressor/immunosurveillance cancer gene product Raf-1 kinase inhibitory protein (RKIP). Human prostate cancer cell lines were used as models, as they express different levels of these proteins. We show that NPI-0052 inhibits both NF-kappaB and Snail and induces RKIP expression, thus resulting in cell sensitization to CDDP and TRAIL. The direct role of NF-kappaB inhibition in sensitization was corroborated with the NF-kappaB inhibitor DHMEQ, which mimicked NPI-0052 in sensitization and inhibition of Snail and induction of RKIP. The direct role of Snail inhibition by NPI-0052 in sensitization was shown with Snail small interfering RNA, which reversed resistance and induced RKIP. Likewise, the direct role of RKIP induction in sensitization was revealed by both overexpression of RKIP (mimicking NPI-0052) and RKIP small interfering RNA that inhibited NPI-0052-mediated sensitization. These findings show that NPI-0052 modifies the NF-kappaB-Snail-RKIP circuitry in tumor cells and results in downstream inhibition of antiapoptotic gene products and chemoimmunosensitization. The findings also identified Snail and RKIP as targets for reversal of resistance.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; Blotting, Western; Boronic Acids; Bortezomib; Cisplatin; Humans; Lactones; Leupeptins; Male; Melanoma; Membrane Potential, Mitochondrial; NF-kappa B; Phosphatidylethanolamine Binding Protein; Prostatic Neoplasms; Proteasome Inhibitors; Proto-Oncogene Proteins c-raf; Pyrazines; Pyrroles; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Snail Family Transcription Factors; TNF-Related Apoptosis-Inducing Ligand; Transcription Factors; Transfection; Tumor Cells, Cultured

Proteasome inhibitors are known to suppress the proteasome-mediated degradation of IkappaBalpha in stimulated cells. This results in the cytoplasmic retention of NFkappaB and its reduced nuclear transcriptional activity. In this study, we show that in the metastatic prostate cancer cells, the proteasome inhibitors exhibit a novel, previously unrecognized effect: they increase the cellular levels of IkappaBalpha, which then translocates to the nucleus, associates with the nuclear p65 NFkappaB, thus inhibiting the constitutive NFkappaB DNA binding activity and inducing apoptosis. The proteasome inhibition-induced nuclear translocation of IkappaBalpha is dependent on de novo protein synthesis, occurs also in other cell types, and does not require IkappaBalpha phosphorylation on Ser-32. Since NFkappaB activity is constitutively increased in many human cancers as well as in inflammatory disorders, the proteasome inhibition-induced nuclear translocation of IkappaBalpha could thus provide a new therapeutic strategy aimed at the specific inhibition of NFkappaB activity by the nuclear IkappaBalpha.

Topics: Apoptosis; Cell Line, Tumor; Cell Nucleus; Cysteine Proteinase Inhibitors; Humans; I-kappa B Proteins; Leupeptins; Male; NF-KappaB Inhibitor alpha; Prostatic Neoplasms; Proteasome Endopeptidase Complex; Protein Transport

Experimental data suggest therapeutic advantage from selective disruption of the hypoxia response. We recently found that the proteasome inhibitor bortezomib decreases tumor carbonic anhydrase IX (CAIX) expression in colon cancer patients and herein report a companion laboratory study to test if this effect was the result of hypoxia-inducible factor (HIF) inhibition. Human cervical (SiHa and Me180) and colon (RKO) carcinoma cell lines were treated with bortezomib or the structurally unrelated proteasome inhibitor MG132 in normoxic and hypoxic conditions in vitro. Two different in vivo experiments investigated bortezomib effects after single dose (2 mg/kg, 24 h) or longer exposure in severe combined immunodeficient mice bearing SiHa xenografts. Treatment with either drug produced accumulation of HIF-1alpha in vitro but strongly inhibited the production of CAIX and vascular endothelial growth factor (VEGF) under hypoxia. This correlated with more than 10-fold reduction in HIF-1 transcriptional activity under hypoxic conditions. A similar effect of bortezomib was seen in vivo, using the nitroimidazole probe EF5 to define regions of tumor hypoxia and a triple immunofluorescence technique to measure the spatial distributions of HIF-1alpha and CAIX. Plasma VEGF levels decreased by approximately 90% during treatment with bortezomib, indicating that this agent can potently inhibit the hypoxia response in tumors.

Topics: Antigens, Neoplasm; Antineoplastic Agents; Boronic Acids; Bortezomib; Carbonic Anhydrase IX; Carbonic Anhydrases; Carcinoma, Squamous Cell; Caspase 3; Cell Hypoxia; Cell Line, Tumor; Cell Nucleus; Colonic Neoplasms; E1A-Associated p300 Protein; Enzyme Activation; Female; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Leupeptins; Male; Prostatic Neoplasms; Protease Inhibitors; Protein Binding; Pyrazines; Uterine Cervical Neoplasms; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent because it induces apoptosis in cancer cells but not in normal cells. Unfortunately, some cancer cells develop resistance to TRAIL-induced apoptosis. Therefore, it is clinically relevant to determine the molecular mechanisms that differentiate between TRAIL-sensitive and TRAIL-resistant tumors. Previously, we have shown that the antiapoptotic molecule cellular-FLICE-inhibitory protein long isoform [c-FLIP(L)] is necessary and sufficient to maintain resistance to TRAIL-induced apoptosis. We have found that c-FLIP(L) is transcriptionally regulated by the activator protein-1 (AP-1) family member protein c-Fos. Here, we report that MG-132, a small-molecule inhibitor of the proteasome, sensitizes TRAIL-resistant prostate cancer cells by inducing c-Fos and repressing c-FLIP(L). c-Fos, which is activated by MG-132, negatively regulates c-FLIP(L) by direct binding to the putative promoter region of the c-FLIP(L) gene. In addition to activating c-Fos, MG-132 activates another AP-1 family member, c-Jun. We show that c-Fos heterodimerizes with c-Jun to repress transcription of c-FLIP(L). Therefore, MG-132 sensitizes TRAIL-resistant prostate cancer cells by activating the AP-1 family members c-Fos and c-Jun, which, in turn, repress the antiapoptotic molecule c-FLIP(L).

Topics: Antineoplastic Combined Chemotherapy Protocols; CASP8 and FADD-Like Apoptosis Regulating Protein; Cell Nucleus; Drug Resistance, Neoplasm; Drug Synergism; Humans; Leupeptins; Male; Models, Biological; Promoter Regions, Genetic; Prostatic Neoplasms; Protein Binding; Protein Transport; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; TNF-Related Apoptosis-Inducing Ligand; Transcription Factor AP-1; Tumor Cells, Cultured

Proteasome inhibitors are known to induce apoptosis in a variety of cancer cells. On the other hand, maspin, a non-inhibitory serine protease inhibitor, is shown to sensitize cancer cells to therapeutic agents that induce apoptosis. We examined the consequence of maspin expression in prostate cancer cells targeted for treatment with various proteasome inhibitors. We observed that proteasome inhibitors induced apoptosis more effectively in maspin transfected human prostate cancer DU145 cells than in control cells. Interestingly, increased apoptosis in these cells was associated with a significant induction of maspin expression. MG-132, a proteasome inhibitor, induced endogenous and ectopic [cytomegalovirus promoter (CMV)-driven] maspin expression, and maspin siRNA attenuated MG-132-induced apoptosis. Proteasome inhibitor-induced maspin expression was inhibited by actinomycin D (Act D) and cyclohexamide (CHX), and by the inhibitors of p38MAPK, but not ERK1/2 or NF-kappaB. Electrophoretic mobility-shift assay (EMSA) and promoter-reporter activity analyses suggested that p38MAPK activated transcription factor AP-1 is responsible for proteasome inhibitor-induced maspin expression. Taken together, these observations demonstrate that proteasome inhibitors induce maspin expression by activating p38MAPK pathway, and that maspin thus expressed, in turn, augments proteasome inhibitor-induced apoptosis in prostate cancer cells. Our results suggest that gene therapy involving ectopic maspin expression may dramatically improve the efficacy of proteasome inhibitors for the treatment of prostate cancer.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Cytomegalovirus; Dose-Response Relationship, Drug; Genetic Vectors; Humans; Imidazoles; Leupeptins; Luteolin; Male; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Promoter Regions, Genetic; Prostatic Neoplasms; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Kinase Inhibitors; Pyridines; RNA Interference; RNA, Messenger; RNA, Small Interfering; Serpins; Transcription Factor AP-1; Transcription, Genetic; Transfection

Abnormal differentiation in epithelial stem cells or their immediate proliferative progeny, the transiently amplifying population (TAP), may explain malignant pathogenesis in the human prostate. These models are of particular importance as differing sensitivities to androgen among epithelial cell subpopulations during differentiation are recognised and may account for progression to androgen independent prostate cancer. Androgens are crucial in driving terminal differentiation and their indirect effects via growth factors from adjacent androgen responsive stroma are becoming better characterised. However, direct effects of androgen on immature cells in the context of a prostate stem cell model have not been investigated in detail and are studied in this work. In alpha2beta1hi stem cell enriched basal cells, androgen analogue R1881 directly promoted differentiation by the induction of differentiation-specific markers CK18, androgen receptor (AR), PSA and PAP. Furthermore, treatment with androgen down-regulated alpha2beta1 integrin expression, which is implicated in the maintenance of the immature basal cell phenotype. The alpha2beta1hi cells were previously demonstrated to lack AR expression and the direct effects of androgen were confirmed by inhibition using the anti-androgen bicalutamide. AR protein expression in alpha2beta1hi cells became detectable when its degradation was repressed by the proteosomal inhibitor MG132. Stratifying the alpha2beta1hi cells into stem (CD133(+)) and transient amplifying population (TAP) (CD133(-)) subpopulations, AR mRNA expression was found to be restricted to the CD133(-) (TAP) cells. The presence of a functional AR in the TAP, an androgen independent subpopulation for survival, may have particular clinical significance in hormone resistant prostate cancer, where both the selection of immature cells and functioning AR regulated pathways are involved.

Topics: AC133 Antigen; Acid Phosphatase; Aged; Aged, 80 and over; Androgen Antagonists; Anilides; Antigens, CD; Cell Differentiation; Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Epithelial Cells; Fibroblast Growth Factor 7; Glycoproteins; Humans; Integrin alpha2beta1; Keratin-18; Leupeptins; Male; Metribolone; Middle Aged; Neoplastic Stem Cells; Nitriles; Peptides; Phenotype; Prostate-Specific Antigen; Prostatic Neoplasms; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Tyrosine Phosphatases; Receptors, Androgen; RNA, Messenger; Signal Transduction; Testosterone Congeners; Tosyl Compounds

The proteasome-mediated protein degradation is critical for regulation of a variety of cellular processes, including cell cycle, cell death, differentiation and immune response. Proteasome inhibitors have recently been shown to be potent anti-cancer agents against a variety of cancer cells. Our study demonstrated that proteasome inhibitor MG132 (carbobenzoxy-L-leucyle-L-leucyl-L-leucinal) was a potent death-inducing agent for PC3 prostate cancer cells. MG132-induced cell death was partially inhibited by pan-caspase inhibitor zAVD-fmk and translational inhibitor cycloheximide. To understand the signaling pathways of proteasome inhibitor-induced cell death, we performed gene profiling study using Affymetrix human DNA microarrays to identify the genes whose expression was affected by proteasome inhibitor MG132 in PC3 cells. The genes with more than threefold increased expression induced by MG132 were functionally categorized into the following groups: heat shock and chaperone proteins, ubiquitination and protein degradation, transcription/translation factors, cell death and cell cycle arrest, signaling molecules and enzymes, and secreted cytokines. Among them, heat shock proteins and anti-oxidant enzymes may promote cell survival, while pro-death proteins such as GADD45B and STK17a may promote cell death. Interestingly, expression of a few autophagic genes was elevated by MG132 treatment. Furthermore, autophagy inhibitor 3-methyladenine partially inhibited MG132-induced cell death, indicating that autophagic cell death may contribute to MG132-induced cell death. Taken together, our results demonstrated that proteasome inhibition elicits activation of multiple signaling pathways in prostate cancer cells.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Autophagy; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cycloheximide; Cysteine Proteinase Inhibitors; Cytokines; Dose-Response Relationship, Drug; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Heat-Shock Proteins; Humans; Intercellular Signaling Peptides and Proteins; Leupeptins; Male; Microscopy, Electron; Molecular Chaperones; Oligonucleotide Array Sequence Analysis; Prostatic Neoplasms; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Synthesis Inhibitors; Signal Transduction

Histone deacetylase inhibitors (HDACI) are potential therapeutic agents that inhibit tumor cell growth and survival. Although there are several publications regarding the effects of HDACIs on prostate cancer cell growth, their mechanism(s) of action remains undefined. We treated several human prostate cancer cell lines with the HDACI trichostatin A and found that trichostatin A induced cell death in androgen receptor (AR)-positive cell lines to higher extent compared with AR-negative cell lines. We then discovered that trichostatin A and other HDACIs suppressed AR gene expression in prostate cancer cell lines as well as in AR-positive breast carcinoma cells and in mouse prostate. Trichostatin A also induced caspase activation, but trichostatin A-induced AR suppression and cell death were caspase independent. In addition, we found that doxorubicin inhibited AR expression, and p21 protein completely disappeared after simultaneous treatment with trichostatin A and doxorubicin. This effect may be attributed to the induction of protease activity under simultaneous treatment with these two agents. Further, simultaneous treatment with trichostatin A and doxorubicin increased cell death in AR-positive cells even after culturing in steroid-free conditions. The protease/proteasome inhibitor MG132 protected AR and p21 from the effects of trichostatin A and doxorubicin and inhibited trichostatin A-induced cell death in AR-positive prostate cells. Taken together, our data suggest that the main mechanism of trichostatin A-induced cell death in AR-positive prostate cancer is inhibition of AR gene expression. The synergistic effect of simultaneous treatment with trichostatin A and doxorubicin is mediated via inhibition of AR expression, induction of protease activity, increased expression of p53, and proteolysis of p21.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Blotting, Western; Breast Neoplasms; Caspases; Cyclin-Dependent Kinase Inhibitor p21; Cysteine Proteinase Inhibitors; Doxorubicin; Drug Synergism; Drug Therapy, Combination; Enzyme Activation; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Leupeptins; Luciferases; Male; Mice; Promoter Regions, Genetic; Prostate; Prostatic Neoplasms; Receptors, Androgen; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured; Tumor Suppressor Protein p53

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 purpose of this study is to evaluate the utility of MG-132, a broad spectrum proteasome inhibitor, to selectively enhance radiation sensitivity in prostate cancer without affecting normal surrounding urothelial tissue. PC3 prostate cancer cells and normal URO-tsa bladder epithelial cells were treated with or without MG-132 and exposed to 0, 2, 4, or 6 Gy radiation. Cell viability and clonogenic survival assays were performed, and nuclear factor kappa-B (NF-kappaB) activity was evaluated with electrophoretic mobility shift assay (EMSA). MG-132 was associated with decreased cell viability (between 24% and 33%) and clonogenic survival (between 71% and 88%) alone and in combination with radiation in PC3 cells. MG-132 had no effect on cell viability or clonogenic survival following radiation in URO-tsa cells. Constitutive and radiation-induced NF-kappaB binding activity was higher in PC3 cells compared with URO-tsa cells. Furthermore, MG-132 at concentrations associated with reductions in cell viability and clongenic survival inhibited NF-kappaB binding activity in PC3 cells with no effect in URO-tsa cells. These results provide strong evidence that proteasome inhibition and concomitant NF-kappaB inhibition can be used to selectively enhance tumor radiation sensitivity in prostate cancer without affecting normal surrounding bladder tissue.

Topics: Apoptosis; Colony-Forming Units Assay; Cysteine Proteinase Inhibitors; Humans; I-kappa B Kinase; Leupeptins; Male; NF-kappa B; Prostatic Neoplasms; Proteasome Inhibitors; Radiation Tolerance; Radiation-Sensitizing Agents; Tumor Cells, Cultured

Prostate cancer usually progresses to androgen refractory after an initial anti-androgen treatment. The androgen receptor (AR) is a pivotal factor for the androgen-mediated growth and maintenance of the prostate. Abnormality of the AR, such as overexpression has been postulated to be related to the hormone independent growth of the cancer. Although we previously demonstrated that the AR expression could be modulated by isothiocyanates, which are natural constituents of cruciferous vegetables, the mechanism, however, remained to be clarified. We have since investigated the mechanism of phenethyl isothiocyanate (PEITC) in AR regulation.. A human androgen dependent prostate cancer cell line LNCaP (AD) and its sub-line LNCaP (AI), i.e. androgen independent but overexpressing AR, were exposed to PEITC. The effects of PEITC on cell growth and AR expression/transcription were analyzed with MTT assay, real-time PCR and western blotting. The AR promoter activity was analyzed with the reporter activity after transfection with pAR-luc. The effects on Sp1, the major transcription factor of the AR, were tested with Sp1-luc activity, western blotting and electrophoretic mobility shift assay.. PEITC induced a significant growth inhibition, with equal IC(50), in both AD and AI cells. The AR present in both cells was repressed as demonstrated with real-time PCR and western blot. PEITC mediates dual effects at transcriptional and post-translational levels to regulate the AR. At transcriptional level the AR level was reduced via inhibition of the transcription factor Sp1, and at post-translational level by accelerating protein degradation.. PEITC represses AR transcription and expression, and mediates growth arrest in androgen dependent and independent prostate cancer cells. With the AR modulation and growth attenuation, PEITC and possibly other isothiocyanates, may prevent and inhibit hormone sensitive and refractory prostate cancer.

Topics: Androgen Receptor Antagonists; Anticarcinogenic Agents; Cell Line, Tumor; Cycloheximide; Humans; Isothiocyanates; Leupeptins; Male; Neoplasms, Hormone-Dependent; Prostatic Neoplasms; Receptors, Androgen; Sp1 Transcription Factor; Transcription, Genetic

Heat shock protein 70 (HSP70) is released from tumour cells and stimulates a potent anti-tumour immune response.. This study examined the role of hyperthermia, including heating conditions from the fever range, the hyperthermia range and the thermal ablation range, in HSP70 release from prostate carcinoma cells. It has observed HSP70 release from human prostate carcinoma cell lines (PC-3 and LNCaP) treated with hyperthermia.. The effects of hyperthermia were complex and appeared to involve at least two mechanisms for HSP70 release. Hyperthermia at 40 degrees C strongly stimulated HSP70 release by an active secretion pathway. However, as temperatures were increased, this rapid secretion pathway became progressively inhibited and by a temperature of 55 degrees C, active secretion was abolished. However, when cells exposed to these heating conditions were allowed to recover at 37 degrees C for 24 h after heating, HSP70 release was observed at the high ablation temperature range and this appeared to be related to a concomitant damage to the plasma membrane.. Thus, at least two mechanisms contribute to HSP70 release during hyperthermia and the relative contribution from each pathway depends on the temperature conditions.

Topics: Cell Line, Tumor; Cell Membrane; Cell Membrane Permeability; Enzyme-Linked Immunosorbent Assay; Hot Temperature; HSP70 Heat-Shock Proteins; Humans; Hyperthermia, Induced; Leupeptins; Male; Prostatic Neoplasms; Proteasome Inhibitors; Trypan Blue

Combined treatment with a proteasome inhibitor and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising strategy for cancer therapy. Proteasome inhibitors induce the expression of death receptor 5 (DR5), a receptor for TRAIL, and sensitize cancer cells to TRAIL-induced apoptosis; however, the molecular mechanism of DR5 up-regulation has not been elucidated. In this study, we report that CCAAT/enhancer-binding protein homologous protein (CHOP) is a regulator of DR5 induction by proteasome inhibitor MG132. MG132 induced DR5 expression at a protein and mRNA level in prostate cancer DU145 cells. Furthermore, MG132 increased DR5 promoter activity. Using a series of deletion mutant plasmids containing DR5 promoters of various sizes, we found that MG132 stimulated the promoter activity via the region of -289 to -253. This region contained a CHOP-binding site. Site-directed mutation of the site abrogated the promoter activity enhanced by MG132. An electrophoretic mobility shift assay showed that CHOP directly bound to the MG132-responsive site on the DR5 promoter. Expression of the CHOP protein was increased with MG132 along with DR5 up-regulation. Furthermore, CHOP small interfering RNA attenuated the DR5 up-regulation due to MG132. These results indicate that the proteasome inhibitor MG132 induces DR5 expression through CHOP up-regulation.

Topics: Apoptosis; Apoptosis Regulatory Proteins; CCAAT-Enhancer-Binding Proteins; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Humans; Leupeptins; Male; Membrane Glycoproteins; Mutagenesis, Site-Directed; Promoter Regions, Genetic; Prostatic Neoplasms; Proteasome Inhibitors; Receptors, TNF-Related Apoptosis-Inducing Ligand; Receptors, Tumor Necrosis Factor; RNA, Messenger; TNF-Related Apoptosis-Inducing Ligand; Transcription Factor CHOP; Transcription Factors; Tumor Necrosis Factor-alpha; Up-Regulation

The proteasome inhibitor Velcade (bortezomib/PS-341) has been shown to block the targeted proteolytic degradation of short-lived proteins that are involved in cell maintenance, growth, division, and death, advocating the use of proteasomal inhibitors as therapeutic agents. Although many studies focused on the use of one proteasomal inhibitor for therapy, we hypothesized that the combination of proteasome inhibitors Lactacystin (AG Scientific, Inc., San Diego CA) and MG132 (Biomol International, Plymouth Meeting, PA) may be more effective in inducing apoptosis. Additionally, this regimen would enable the use of sublethal doses of individual drugs, thus reducing adverse effects. Results indicate a significant increase in apoptosis when LNCaP prostate cancer cells were treated with increasing levels of Lactacystin, MG132, or a combination of sublethal doses of these two inhibitors. Furthermore, induction in apoptosis coincided with a significant loss of IKKalpha, IKKbeta, and IKKgamma proteins and NFkappaB activity. In addition to describing effective therapeutic agents, we provide a model system to facilitate the investigation of the mechanism of action of these drugs and their effects on the IKK-NFkappaB axis.

Topics: Acetylcysteine; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cysteine Proteinase Inhibitors; Drug Synergism; Humans; I-kappa B Kinase; Leupeptins; Male; NF-kappa B; Prostatic Neoplasms; Proteasome Inhibitors; Tumor Cells, Cultured

The serine/threonine kinase Raf-1 is a major regulator of the mitogen activated protein kinase (MAPK) pathway, which has been associated with the progression of prostate cancer to the more advanced and androgen-independent disease. Cdc25A phosphatase has been implicated in the regulation of Raf-1 and the MAPK pathway.. We used a novel and potent Cdc25A inhibitor, 2,3-bis-[2-hydroxyethylsulfonyl]-[1,4] naphthoquinone (NSC 95397), and its congener (2-mercaptoethanol)-3-methyl-1, 4-naphthoquinone (NSC 672121) to study the role of Cdc25A on the MAPK pathway in human prostate cancer cells.. We found Raf-1 physically interacted with Cdc25A in PC-3 and LNCap cells and inhibitors of Cdc25A induced both extracellular signal-regulated kinase (Erk) activation and Raf-1 tyrosine phosphorylation. NSC 95397 attenuated Cdc25A and Raf-1 interactions due to accelerated degradation of Cdc25A, which was mediated by proteasome degradation. The MAPK kinase (MEK) inhibitor U0126 completely inhibited Erk activation by NSC 95397 and NSC 672121.. These results indicate Cdc25A phosphatase regulates Raf-1/MEK/Erk kinase activation in human prostate cancer cells.

Topics: Blotting, Western; Butadienes; cdc25 Phosphatases; Cell Line, Tumor; Enzyme Inhibitors; Humans; Leupeptins; Male; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Naphthoquinones; Nitriles; Phosphorylation; Prostatic Neoplasms; Proto-Oncogene Proteins c-raf

The 26S proteasome is a ubiquitin-dependent proteolytic system that has been implicated in the regulation of cell cycle progression and apoptosis. We investigated the effects of the proteasome inhibitors MG115 and PSI alone or in combination with different concentrations of the antiandrogen hydroxyflutamide on the cellular proliferation, apoptosis and viability of 10 prostatic adenocarcinoma cell cultures. Treatment with both proteasome inhibitors resulted in apoptosis induction, whereas the combinations with hydroxyflutamide generally did not, with the exception of MG115 combined with 10(-7) M hydroxyflutamide. MG115 caused a significant decrease in cellular proliferation, as did the combinations of both proteasome inhibitors with hydroxyflutamide, whereas hydroxyflutamide alone was only effective at a concentration of 10(-5) M. Cellular viability was significantly reduced when both proteasome inhibitors were combined with 10(-5) M hydroxyflutamide. Although the results varied among different cell lines, we conclude that proteasome inhibitors are able to induce apoptosis and reduce cellular proliferation. They might prove effective as antineoplastic substances in prostatic adenocarcinoma alone or in combination with antiandrogens.

Topics: Adenocarcinoma; Androgen Antagonists; Apoptosis; Cell Division; Cell Survival; Cysteine Proteinase Inhibitors; Drug Interactions; Flutamide; Humans; Leupeptins; Male; Oligopeptides; Prostatic Neoplasms; Protease Inhibitors; Tumor Cells, Cultured

The androgen receptor (AR) is a member of the nuclear receptor superfamily that requires the action of molecular chaperones for folding and hormone binding. C-terminal Hsp-interacting protein (Chip) is a cochaperone that interacts with Hsp70 and Hsp90 molecular chaperones via a tetratricopeptide domain and inhibits chaperone-dependent protein folding in vitro. Chip also stimulates protein degradation by acting as an E3 ubiquitin ligase via a modified ring finger domain called a U box. We analyzed whether Chip affected AR levels using a transient transfection strategy. Chip overexpression led to a large decrease in AR steady state levels and increased levels of AR ubiquitinylation. However, Chip effects were not fully reversed by proteasome inhibitors, suggesting that mechanisms alternative to or in addition to proteasome-mediated degradation were involved. This hypothesis was supported by the finding that Chip overexpression reduced the rate of AR degradation, consistent with an effect on AR folding, perhaps leading to aggregation. The possibility that Chip affected AR folding was further supported by the finding that the effects of exogenous Chip were reproduced by a mutant lacking the U box. These results are discussed in terms of the role played by molecular chaperones in AR biogenesis.

Topics: Acetylcysteine; Benzoquinones; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dihydrotestosterone; Gene Expression; HeLa Cells; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Leupeptins; Ligases; Male; Molecular Chaperones; Multienzyme Complexes; Prostatic Neoplasms; Proteasome Endopeptidase Complex; Quinones; Receptors, Androgen; Receptors, Estrogen; Receptors, Glucocorticoid; Tumor Cells, Cultured; Ubiquitin; Ubiquitin-Protein Ligases

We analyzed gene expression of MBD1, MBD2, MBD3, MBD4, and MeCP2 and protein expression of MBD1, MBD2, and MeCP2 in prostate cancer cell lines, benign prostate epithelium (BPH-1) cell line, 49 BPH tissues, and 46 prostate cancer tissues. The results of this study demonstrate that MBD2 gene is expressed in all samples and MeCP2 gene is expressed in all cancer cell lines but not in BPH-1 cell line. However, there was no protein expression for MBD2 and MeCP2 in cancer cell lines and cancer tissues. For CXXC sequence containing MBD1, both protein and mRNA were expressed in cancer cell lines, cancer tissues, BPH-1 cell line, and BPH tissues. We observed that, in BPH tissues and low-grade cancer tissues, MBD1 protein expression was very high and gradually decreased with increase of cancer grade. Treatment of cancer cell lines with proteasome inhibitor (MG-132) did not restore expression of MBD2 and MeCP2 proteins. When prostate cancer cell lines were treated with hypomethylating agent, 5-aza-2(')-deoxycytidine (DNMT inhibitor), HDAC1 and HDAC2 expression was decreased. This is the first report demonstrating that CXXC sequence containing MBD1 is overexpressed and can be the major factor of hypermethylated chromatin segments through HDAC1/2 translocation and histone deacetylation in human prostate cancer.

Topics: Amino Acid Sequence; Antineoplastic Agents; Azacitidine; Cell Line; Chromosomal Proteins, Non-Histone; CpG Islands; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Histone Deacetylases; Humans; Leupeptins; Male; Methyl-CpG-Binding Protein 2; Prostatic Neoplasms; Repressor Proteins; RNA, Messenger; Transcription Factors

The interleukin-10 (IL-10) activation of Janus kinase (JAK) family members (JAK1/TYK2) and IL-10E1 is subsequently inactivated by approximately 3-4 h in primary prostate tumor lines. We examined the effect of proteasome inhibition on IL-10 activation of the IL-10E1 pathway following stimulation of HPCA-10a cells. Treatment of HPCA-10a cells with the proteasome inhibitor, N-acetyl-L-leucinyl-L-leucinyl-norleucinal (LLnL), led to stable tyrosine phosphorylation of the IL-10 receptor and IL-10E1 following stimulation. Further investigation showed that these stable phosphorylation events were the result of prolonged activation of JAK1 and TYK2 plus IL-10E1. IL-10E1 signaling normally induced the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and LLnL treatment of the HPCA-10a and HPCA-10c cells significantly enhanced IL-10 induction of TIMP-1 levels to block tumor cell invasion in modified Boyden chamber invasion assays. These observations were confirmed using pharmacologic inhibitors by Western blot and ELISAs. In the presence of LLnL, stable phosphorylation of IL-10E1 and induction of TIMP-1 was abrogated if the tyrosine kinase inhibitor, staurosporine, was added. The effect of staurosporine on IL-10E1 phosphorylation and TIMP-1 could be overcome if the phosphatase inhibitor, vanadate, was also added, suggesting that phosphorylated IL-10E1 could be stabilized by phosphatase, but not by proteasome inhibition. These observations are consistent with the hypothesis that proteasome-mediated protein degradation can modulate the activity of the IL-10E1 pathway and TIMP-1 induction by regulating the deactivation of JAK1/TYK2.

Topics: Antibodies; Cell Line, Tumor; Electrophoretic Mobility Shift Assay; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Humans; Interleukin-10; Leupeptins; Male; Phosphorylation; Phosphotyrosine; Prostatic Neoplasms; Protease Inhibitors; Proto-Oncogene Proteins c-rel; Signal Transduction; Staurosporine; Tissue Inhibitor of Metalloproteinase-1

Mortality and morbidity of prostate cancer result from extracapsular invasion and metastasis. This tumor progression depends on active cell motility. Previous studies have shown that calpain-regulated rear detachment enabling forward locomotion is required for cell migration initiated by growth factor and adhesion receptors. Therefore, we asked whether calpain would be a target for limiting tumor progression, using as our model the PA DU-145 human prostate carcinoma cell line and a highly invasive subline, wild-type DU-145, derived from it. In vitro, the calpain-specific inhibitor CI-I (ALLN) and the preferential-but-less-specific inhibitor leupeptin decreased transmigration of both cell lines across a Matrigel barrier. These calpain inhibitors limited epidermal growth factor-induced motility but did not alter the growth rate of the tumor cells, as expected. Antisense down-regulation of the growth factor-activated calpain-2 (m-calpain) isoform also reduced transmigration and cell motility. These in vitro findings were then buttressed by in vivo studies, in which i.p. DU-145 tumor xenografts were treated with leupeptin. Tumor invasion into the diaphragm was reduced by leupeptin treatment for both the PA and wild-type DU-145 cells (from 1.7 to 0.78 for the parental line and 2.3 to 1.2 for the invasive derivative, respectively). Tumor cells of both types engineered to express calpain-2 antisense constructs also demonstrated a similar 50% reduced invasiveness in vivo. Finally, we found by gene expression survey of 53 human prostate tumors and 23 normal prostates that calpain was not up-regulated in relationship to invasiveness or metastatic activity, consistent with expectation from the biological role of this effector. Taken together, these results strongly suggest that epigenetic activation of calpain plays an important role in the invasion of human prostate cancer and that it can be targeted to reduce tumor progression.

Topics: Animals; Calpain; Cell Movement; Cysteine Proteinase Inhibitors; Down-Regulation; ErbB Receptors; Glycoproteins; Humans; Leupeptins; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Oligonucleotides, Antisense; Prostatic Neoplasms; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Platelet-type arachidonate 12-lipoxygenase (12-LOX) is highly expressed in many types of cancers and plays an important role in cancer pathophysiology. Arachidonic acid metabolism by 12-LOX results in the stable end product 12(S)-hydroxy eicosatetraenoic acid (12(S)-HETE), which is a signaling molecule with effects on cell proliferation, motility, invasiveness, angiogenesis, and inhibition of apoptosis. The myriad biological activities manifested by 12(S)-HETE appear to be mediated, at least in part, by the activation of NF-kappaB. Overexpression of the 12-LOX in PC-3 prostate cancer cells resulted in the constitutive activation of the transcription factor. The enzymatic product of arachidonic acid metabolism, 12(S)-HETE, mediates the activation of NF-kappaB by the 12-LOX. 12(S)-HETE treatment of PC-3 cells induced the degradation of IkappaB by the S6 proteasomal pathway and the activated NF-kappaB translocated to the nucleus causing kappaB-induced transcription. Specificity of the NF-kappaB activation by 12(S)-HETE was established by the use of a 12-LOX-specific inhibitor and 13(S)-HODE, a known 12(S)-HETE antagonist. Considering the known involvement of MAP kinase pathway in NF-kappaB activation and that of 12(S)-HETE in MAP kinase pathway, 12-LOX present in prostate cancer tissues may contribute to the constitutive activation of NF-kappaB in prostate cancer cells.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Antineoplastic Agents; Arachidonate 12-Lipoxygenase; Arachidonic Acid; Blood Platelets; Gene Expression Regulation, Neoplastic; Humans; I-kappa B Proteins; Leupeptins; Linoleic Acids; Lipoxygenase Inhibitors; Male; NF-kappa B; Prostatic Neoplasms; Transcription Factors; Transfection; Tumor Cells, Cultured

The standard therapy for advanced prostate cancer is androgen ablation. Despite transitory responses, hormonally treated patients ultimately relapse with androgen-independent disease that is resistant to further hormonal manipulation and cytotoxic chemotherapy. To develop an additional approach to the treatment of advanced prostate cancer, we have been studying the signal transductions controlling the growth of human androgen-independent prostate carcinoma cell lines. We report here that elevation of intracellular cAMP markedly inhibits the growth of the hormone-refractory cell line PC-3. To examine the mechanism of cAMP action in PC-3 cells, we tested the effect of the cAMP analog dibutyryl cAMP (Bt2-cAMP) on the regulation of the potent negative growth factor transforming growth factor beta (TGF-beta). Bt2-cAMP selectively induced the secretion of TGF-beta 2 and not TGF-beta 1 by PC-3 cells. This TGF-beta 2 was shown to be bioactive by using the CCL-64 mink lung cell assay. TGF-beta 1 was not activated despite being present at 3-fold higher concentrations than TGF-beta 2. Northern analysis showed that Bt2-cAMP induced an increase in the five characteristic TGF-beta 2 transcripts and had no effect on the level of TGF-beta 1 or TGF-beta 3 transcripts. TGF-beta 2 induction was only weakly enhanced by cycloheximide and was completely inhibited by actinomycin D. These data show that Bt2-cAMP induces the expression of active TGF-beta 2 by PC-3 prostate carcinoma cells, suggesting a new approach to the treatment of prostate cancer and a new molecular mechanism of cAMP action.

Topics: 1-Methyl-3-isobutylxanthine; Aprotinin; Blotting, Northern; Bucladesine; Cell Division; Cell Line; Cyclic AMP; Cycloheximide; Dactinomycin; DNA Replication; Gene Expression Regulation, Neoplastic; Humans; Kinetics; Leupeptins; Male; Pentoxifylline; Phenylmethylsulfonyl Fluoride; Prostatic Neoplasms; RNA, Neoplasm; Theophylline; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured