stilbenes and Urinary-Bladder-Neoplasms

To provide an overview of the scientific and clinical studies underlying the most common vitamin and herbal preparations used in prostate and bladder cancer and evaluate the evidence behind them. A literature search was undertaken on PubMed using various keywords relating to the use of complementary and alternative medicine (CAM) in prostate and bladder cancer.Vitamin E and selenium supplementation can potentially have adverse effects by increasing the risk of prostate cancer. Initial clinical studies of pomegranate and green tea, investigating their chemotherapeutic properties in prostate and bladder cancer have yielded encouraging results. Curcumin, resveratrol, and silibinin have potential anticancer properties through multiple molecular targets; their clinical effectiveness in prostate and bladder cancer is yet to be evaluated. Zyflamend, like PC-SPES, is a combined CAM therapy used in prostate cancer. Acupuncture is popular among patients experiencing hot flushes who are receiving androgen-deprivation therapy for prostate cancer. Conclusive evidence for the use of CAM in prostate and bladder cancer is lacking and not without risk.

Topics: Acupuncture; Antineoplastic Agents; Antioxidants; Camellia sinensis; Complementary Therapies; Drugs, Chinese Herbal; Female; Humans; Male; Plant Extracts; Prostatic Neoplasms; Resveratrol; Selenium; Silybin; Silymarin; Stilbenes; Urinary Bladder Neoplasms; Vitamin E; Vitamins

Although our previous studies have identified that isorhapontigenin (ISO) is able to initiate autophagy in human bladder cancer (BC) cells by activating JNK/C-Jun/SESN2 axis and possesses an inhibitory effect on BC cell growth, association of autophagy directly with inhibition of BC invasion has never been explored. Also, upstream cascade responsible for ISO activating JNK remains unknown. Thus, we explored both important questions in the current study and discovered that ISO treatment initiated RAC1 protein translation, and its downstream kinase MKK7/JNK phosphorylation/activation, and in turn promoted autophagic responses in human BC cells. Inhibition of autophagy abolished ISO inhibition of BC invasion, revealing that autophagy inhibition was crucial for ISO inhibition of BC invasion. Consistently, knockout of RAC1 also attenuated induction of autophagy and inhibition of BC invasion by ISO treatment. Mechanistic studies showed that upregulation of RAC1 translation was due to ISO inhibition of miR-365a transcription, which reduced miR-365a binding to the 3'-UTR of RAC1 mRNA. Further study indicated that inhibition of miR-365a transcription was caused by downregulation of its transcription factor SOX2, while ISO-promoted Dicer protein translation increased miR-145 maturation, and consequently downregulating SOX2 expression. These findings not only provide a novel insight into the understanding association of autophagy induction with BC invasion inhibition by ISO, but also identify an upstream regulatory cascade, Dicer/miR145/SOX2/miR365a/RAC1, leading to MKK7/JNKs activation and autophagy induction.

Topics: 3' Untranslated Regions; Autophagy; Cell Line, Tumor; DEAD-box RNA Helicases; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Nuclear Proteins; Phosphorylation; Protein Biosynthesis; rac1 GTP-Binding Protein; Ribonuclease III; Sestrins; SOXB1 Transcription Factors; Stilbenes; Urinary Bladder Neoplasms

Epithelial mesenchymal transition (EMT) is highly correlated with metastasis during cancer development. Although previous studies have revealed that ISO is able to inhibit cancer cell invasion and stem-cell properties, little is known about the effects of ISO on EMT markers. The present study explores the potential regulation of ISO on EMT, leading to the inhibition of migration and invasion of bladder cancer cells. We found that ISO inhibited Vimentin, one of the EMT markers, in the invasive bladder cancer cell lines U5637 and T24T. ISO reduced Vimentin protein level by increasing the expression of METTL14. On the other hand, ISO upregulated the METTL14 mRNA by activating the transcription factor FOXO3a. The results demonstrate that ISO inhibits invasion by affecting the EMT marker and offer a novel insight into understanding the upregulation of METTL14 by ISO.

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Forkhead Box Protein O3; Gene Expression Regulation, Neoplastic; Humans; Methyltransferases; Neoplasm Invasiveness; Signal Transduction; Stilbenes; Urinary Bladder Neoplasms; Vimentin

This study was aimed to investigate the effect of piceatannol (PIC) on the proliferation and apoptosis of bladder cancer cell line EJ, and the underlying mechanism.   Bladder cancer cell line EJ was incubated with different concentrations of PIC, and CCK-8 method was used to determine the effect of the treatment on cell proliferation. The effect of PIC on cell cycle, apoptosis and the expressions of related signal pathway proteins were determined using Western blotting. Flow cytometry showed that PIC inhibited the proliferation of EJ cells in a concentration- and time-dependent fashion. Moreover, EJ cells were significantly blocked in G0/G1 phase, when compared with the blank control group (p < 0.05). In addition, PIC enhanced apoptosis of EJ cells in a concentration-dependent manner (p < 0.05). Results from western blotting showed that, compared with the control group, PIC upregulated the protein expression of PTEN, but downregulated Akt protein phosphorylation, relative to control cells. PIC significantly inhibits the proliferation of EJ cells and enhances their apoptosis through a mechanism related to the activation of PTEN/Akt signaling pathway.

Topics: Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Humans; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Signal Transduction; Stilbenes; Urinary Bladder Neoplasms

Cancer stem cells (CSC) are highly associated with poor prognosis in cancer patients. Our previous studies report that isorhapontigenin (ISO) down-regulates SOX2-mediated cyclin D1 induction and stem-like cell properties in glioma stem-like cells. The present study revealed that ISO could inhibit stem cell-like phenotypes and invasivity of human bladder cancer (BC) by specific attenuation of expression of CD44 but not SOX-2, at both the protein transcription and degradation levels. On one hand, ISO inhibited cd44 mRNA expression through decreases in Sp1 direct binding to its promoter region-binding site, resulting in attenuation of its transcription. On the other hand, ISO also down-regulated USP28 expression, which in turn reduced CD44 protein stability. Further studies showed that ISO treatment induced miR-4295, which specific bound to 3'-UTR activity of usp28 mRNA and inhibited its translation and expression, while miR-4295 induction was mediated by increased Dicer protein to enhance miR-4295 maturation upon ISO treatment. Our results provide the first evidence that ISO has a profound inhibitory effect on human BC stem cell-like phenotypes and invasivity through the mechanisms distinct from those previously noted in glioma stem-like cells.

Topics: 3' Untranslated Regions; Binding Sites; Cell Line, Tumor; Cyclin D1; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Hyaluronan Receptors; MicroRNAs; Neoplastic Stem Cells; Promoter Regions, Genetic; RNA, Messenger; SOXB1 Transcription Factors; Stem Cells; Stilbenes; Transcription, Genetic; Ubiquitin Thiolesterase; Urinary Bladder Neoplasms

There are few approved drugs available for the treatment of muscle-invasive bladder cancer (MIBC). Recently, we have demonstrated that isorhapontigenin (ISO), a new derivative isolated from the Chinese herb Gnetum cleistostachyum, effectively induces cell-cycle arrest at the G0/G1 phase and inhibits anchorage-independent cell growth through the miR-137/Sp1/cyclin D1 axis in human MIBC cells. Herein, we found that treatment of bladder cancer (BC) cells with ISO resulted in a significant upregulation of p27, which was also observed in ISO-treated mouse BCs that were induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). Importantly, knockdown of p27 caused a decline in the ISO-induced G0-G1 growth arrest and reversed ISO suppression of anchorage-independent growth in BC cells. Mechanistic studies revealed that ISO promoted p27 expression at mRNA transcription level through increasing direct binding of forkhead box class O1 (FOXO1) to its promoter, while knockdown of FOXO1 attenuated ISO inhibition of BC cell growth. On the other hand, ISO upregulated the 3'-untranslated region (3'-UTR) activity of p27, which was accompanied by a reduction of miR-182 expression. In line with these observations, ectopic expression of miR-182 significantly blocked p27 3'-UTR activity, whereas mutation of the miR-182-binding site at p27 mRNA 3'-UTR effectively reversed this inhibition. Accordingly, ectopic expression of miR-182 also attenuated ISO upregulation of p27 expression and impaired ISO inhibition of BC cell growth. Our results not only provide novel insight into understanding of the underlying mechanism related to regulation of MIBC cell growth but also identify new roles and mechanisms underlying ISO inhibition of BC cell growth.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p27; Forkhead Box Protein O1; Gene Expression Regulation, Neoplastic; Humans; Mice; MicroRNAs; RNA Processing, Post-Transcriptional; Stilbenes; Transcriptional Activation; Up-Regulation; Urinary Bladder Neoplasms

Loss of TSC1 function, a crucial negative regulator of mTOR signaling, is a common alteration in bladder cancer. Mutations in other members of the PI3K pathway, leading to mTOR activation, are also found in bladder cancer. This provides rationale for targeting mTOR for treatment of bladder cancer characterized by TSC1 mutations and/or mTOR activation. In this study, we asked whether combination treatment with rapamycin and resveratrol could be effective in concurrently inhibiting mTOR and PI3K signaling and inducing cell death in bladder cancer cells. In combination with rapamycin, resveratrol was able to block rapamycin-induced Akt activation, while maintaining mTOR pathway inhibition. In addition, combination treatment with rapamycin and resveratrol induced cell death specifically in TSC1

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line; Cell Movement; Cell Proliferation; Embryo, Mammalian; Enzyme Activation; Fibroblasts; Humans; Mechanistic Target of Rapamycin Complex 1; Mice; Multiprotein Complexes; Proto-Oncogene Proteins c-akt; Resveratrol; Signal Transduction; Sirolimus; Stilbenes; TOR Serine-Threonine Kinases; Tuberous Sclerosis Complex 1 Protein; Tumor Suppressor Proteins; Urinary Bladder Neoplasms

Resveratrol is a polyphenolic compound, which has been revealed to induce apoptosis in numerous human cancer cells; however, the effects of resveratrol on the migration and invasion of human bladder cancer cells have not been reported. The present study aimed to evaluate the anti‑metastatic potential of resveratrol against bladder cancer and its mechanism of action. The results indicated that resveratrol inhibits the adhesion, migration and invasion of bladder cancer cells in a dose‑dependent manner. Resveratrol was shown to significantly inhibit the expression and secretion of matrix metalloproteinase (MMP)‑2 and MMP‑9 in bladder cancer cells. Furthermore, resveratrol suppressed the phosphorylation of c‑Jun N‑terminal kinase and extracellular signal‑regulated protein kinase. In conclusion, the present study is the first, to the best of our knowledge, to demonstrate that resveratrol may be considered a novel anticancer agent for the treatment of bladder cancer via the inhibition of migration and invasion.

Topics: Antineoplastic Agents, Phytogenic; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Proliferation; Humans; JNK Mitogen-Activated Protein Kinases; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Phosphorylation; Resveratrol; Stilbenes; Urinary Bladder; Urinary Bladder Neoplasms

Multidrug resistance (MDR) is a significant barrier to the effective treatment of bladder cancer. In order to improve the management of bladder cancer, it is crucial to identify strategies that may reverse MDR. The effects of three herbal medicines, ginsenoside Rh2, (‑)‑epigallocatechin gallate (EGCG) and resveratrol (RES) on bladder cancer were determined. The effect of these three herbal medicines against the drug resistance in adriamycin (ADM)‑resistant pumc‑91 cells (pumc‑91/ADM) was assessed using the Cell Counting Kit‑8 cell proliferation assay system. Cell cycle distribution analysis was performed using flow cytometry following treatment with RES. The mRNA and protein expression levels of multidrug resistance protein 1 (MRP1), lung resistance protein (LRP), glutathione S‑transferase (GST), B cell leukemia/lymphoma‑2 (BCL‑2) and topoisomerase‑II (Topo‑II) were evaluated using reverse transcription‑quantitative polymerase chain reaction and immunofluorescence, respectively. RES enhanced the cytotoxicity of anticancer agents on pumc‑91/ADM cells; however, Rh2 and EGCG were unable to induce a similar effect. Additionally, RES treatment led to S phase cell cycle arrest accompanied by a decrease in the number of cells in the G1 phase. A significant decrease of MRP1, LRP, GST, BCL‑2 levels and an increase of Topo‑II levels were observed in RES groups compared with the control group. RES effectively reversed ADM resistance in pumc‑91/ADM cells and the underlying molecular mechanism may be associated with the alteration of MRP1, LRP, GST, BCL‑2 and Topo‑II expression levels. Therefore, RES may be a potential candidate for reversing drug resistance in bladder cancer chemotherapy.

Topics: Antineoplastic Agents; Biomarkers; Cell Cycle; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Drugs, Chinese Herbal; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Resveratrol; RNA, Messenger; Stilbenes; Urinary Bladder Neoplasms

Resveratrol (RV) is a natural polyphenol compound with a wide range of activities, including inhibition of human bladder cancer (HBC) cell growth. Because RV is rapidly metabolized and has poor bioavailability, it is unclear whether the antitumor activity is due to RV or its metabolites. We therefore used liquid chromatography-mass spectroscopy, qRT-PCR, immunocytochemistry and western blotting to evaluate the metabolic profile and biotransformation of RV in the T24 and EJ HBC cell lines. Both T24 and EJ cells generated the same RV metabolite, RV monosulfate (RVS), and both exhibited upregulation of the RV-associated metabolic enzyme SULT1A1 (sulfotransferase). Despite these similarities, T24 cells were more sensitive to RV than EJ cells, yet T24 cells exhibited no sensitivity to an RVS mixture (84.13% RVS). Primary rat bladder epithelial cells showed no adverse effects when exposed to a therapeutic dose (100 μM) of RV. The differences in RV sensitivity between the two HBC cell lines did not reflect differences in the RV metabolic profile or SULT1A1 expression. Because RV exhibited stronger antitumor activity and better safety than RVS, we conclude that RV has significant therapeutic potential for HBC treatment, provided individual differences are considered during clinical research and application.

Topics: Animals; Antineoplastic Agents, Phytogenic; Arylsulfotransferase; Cell Line, Tumor; Cell Proliferation; Humans; Rats; Resveratrol; Stilbenes; Urinary Bladder Neoplasms

Our recent studies found that isorhapontigenin (ISO) showed a significant inhibitory effect on human bladder cancer cell growth, accompanied with cell-cycle G0-G1 arrest as well as downregulation of Cyclin D1 expression at transcriptional level via inhibition of Sp1 transactivation in bladder cancer cells. In the current study, the potential ISO inhibition of bladder tumor formation has been explored in a xenograft nude mouse model, and the molecular mechanisms underlying ISO inhibition of Sp1 expression and anticancer activities have been elucidated both in vitro and in vivo. Moreover, the studies demonstrated that ISO treatment induced the expression of miR-137, which in turn suppressed Sp1 protein translation by directly targeting Sp1 mRNA 3'-untranslated region (UTR). Similar to ISO treatment, ectopic expression of miR-137 alone led to G0-G1 cell growth arrest and inhibition of anchorage-independent growth in human bladder cancer cells, which could be completely reversed by overexpression of GFP-Sp1. The inhibition of miR-137 expression attenuated ISO-induced inhibition of Sp1/Cyclin D1 expression, induction of G0-G1 cell growth arrest, and suppression of cell anchorage-independent growth. Taken together, our studies have demonstrated that miR-137 induction by ISO targets Sp1 mRNA 3'-UTR and inhibits Sp1 protein translation, which consequently results in reduction of Cyclin D1 expression, induction of G0-G1 growth arrest, and inhibition of anchorage-independent growth in vitro and in vivo. Our results have provided novel insights into understanding the anticancer activity of ISO in the therapy of human bladder cancer.

Topics: 3' Untranslated Regions; Animals; Base Sequence; Binding Sites; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Ectopic Gene Expression; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; Mice; MicroRNAs; Protein Biosynthesis; RNA Interference; RNA, Messenger; Sp1 Transcription Factor; Stilbenes; Tumor Burden; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays

Although our most recent studies have identified Isorhapontigenin (ISO), a novel derivative of stilbene that isolated from a Chinese herb Gnetum cleistostachyum, for its inhibition of human bladder cancer growth, nothing is known whether ISO possesses an inhibitory effect on bladder cancer invasion. Thus, we addressed this important question in current study and discovered that ISO treatment could inhibit mouse-invasive bladder cancer development following bladder carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) exposure in vivo We also found that ISO suppressed human bladder cancer cell invasion accompanied by upregulation of the forkhead box class O 1 (FOXO1) mRNA transcription in vitro Accordingly, FOXO1 was profoundly downregulated in human bladder cancer tissues and was negatively correlated with bladder cancer invasion. Forced expression of FOXO1 specifically suppressed high-grade human bladder cancer cell invasion, whereas knockdown of FOXO1 promoted noninvasive bladder cancer cells becoming invasive bladder cancer cells. Moreover, knockout of FOXO1 significantly increased bladder cancer cell invasion and abolished the ISO inhibition of invasion in human bladder cancer cells. Further studies showed that the inhibition of Signal transducer and activator of transcription 1 (STAT1) phosphorylation at Tyr701 was crucial for ISO upregulation of FOXO1 transcription. Furthermore, this study revealed that metalloproteinase-2 (MMP-2) was a FOXO1 downstream effector, which was also supported by data obtained from mouse model of ISO inhibition BBN-induced mouse-invasive bladder cancer formation. These findings not only provide a novel insight into the understanding of mechanism of bladder cancer's propensity to invasion, but also identify a new role and mechanisms underlying the natural compound ISO that specifically suppresses such bladder cancer invasion through targeting the STAT1-FOXO1-MMP-2 axis. Cancer Prev Res; 9(7); 567-80. ©2016 AACR.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Movement; Forkhead Box Protein O1; Humans; Interferon-Stimulated Gene Factor 3; Matrix Metalloproteinase 2; Mice; Mice, Inbred C57BL; Neoplasm Invasiveness; Random Allocation; Signal Transduction; Stilbenes; Urinary Bladder Neoplasms

Isorhapontigenin (ISO) is a new derivative of stilbene isolated from the Chinese herb Gnetum cleistostachyum. Our recent studies have revealed that ISO treatment at doses ranging from 20 to 80 μM triggers apoptosis in multiple human cancer cell lines. In the present study, we evaluated the potential effect of ISO on autophagy induction. We found that ISO treatment at sublethal doses induced autophagy effectively in human bladder cancer cells, which contributed to the inhibition of anchorage-independent growth of cancer cells. In addition, our studies revealed that ISO-mediated autophagy induction occurred in a SESN2 (sestrin 2)-dependent and BECN1 (Beclin 1, autophagy related)-independent manner. Furthermore, we identified that ISO treatment induced SESN2 expression via a MAPK8/JNK1 (mitogen-activated protein kinase 8)/JUN-dependent mechanism, in which ISO triggered MAPK8-dependent JUN activation and facilitated the binding of JUN to a consensus AP-1 binding site in the SESN2 promoter region, thereby led to a significant transcriptional induction of SESN2. Importantly, we found that SESN2 expression was dramatically downregulated or even lost in human bladder cancer tissues as compared to their paired adjacent normal tissues. Collectively, our results demonstrate that ISO treatment induces autophagy and inhibits bladder cancer growth through MAPK8-JUN-dependent transcriptional induction of SESN2, which provides a novel mechanistic insight into understanding the inhibitory effect of ISO on bladder cancers and suggests that ISO might act as a promising preventive and/or therapeutic drug against human bladder cancer.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Cell Line, Tumor; Down-Regulation; Drug Design; Drugs, Chinese Herbal; Gene Expression Regulation, Neoplastic; Gnetum; HeLa Cells; Humans; Microscopy, Fluorescence; Mitogen-Activated Protein Kinase 8; Nuclear Proteins; Plant Extracts; Promoter Regions, Genetic; Stilbenes; Transcription Factor AP-1; Urinary Bladder Neoplasms

Resveratrol, an edible polyphenolic phytoalexin present in grapes and red wine, has been reported to inhibit proliferation and induce apoptosis of bladder cancer cells. In the present study, the molecular mechanisms of resveratrol on human bladder cancer cell apoptosis was examined. The effect of resveratrol on the viability and apoptosis of T24 and 5637 cells was measured using an MTT assay and flow cytometric analysis, respectively. Next, the effect of resveratrol on miR‑21 expression was detected by real‑time PCR. The expression of phospho‑Akt and Bcl‑2 following treatment with resveratrol or the downregulation of miR‑21 expression were also measured. Resveratrol induced the cytotoxicity and apoptosis of T24 and 5637 cells in a dose‑dependent manner. Resveratrol decreased the expression of miR‑21, the level of phospho‑Akt and Bcl‑2 protein expression. In addition, the downregulation of miR‑21 expression inhibited the level of phospho‑Akt and Bcl‑2 expression. Insulin‑like growth factor‑1 was able to reverse the effect of the miR‑21 inhibitor on Bcl‑2 expression and apoptosis in T24 and 5637 cells. Notably, overexpression of miR‑21 expression was able to restore the inhibition of Akt activity, downregulation of Bcl‑2 expression and apoptosis induced by resveratrol. Collectively, data revealed that the effect of resveratrol on bladder cancer cell apoptosis was due to miR‑21 regulation of the Akt/Bcl‑2 signaling pathway.

Topics: Apoptosis; Cell Line, Tumor; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Phosphorylation; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Resveratrol; Signal Transduction; Stilbenes; Urinary Bladder Neoplasms

Conventional adjuvant chemotherapies for bladder transitional cell carcinomas (TCCs) may cause strong systemic toxicity and local irritation. Non-toxic resveratrol inhibits TCC cell growth but its feasibility in clinical management of TCCs remains obscure. This study aimed to evaluate the safety and anti-TCC efficacy of resveratrol, using the experimental models closer to the clinical treatment condition. Human TCC EJ cells were exposed to 100 µM, 150 µM and 200 µM resveratrol respectively for 1 hour and 2 hours to mimic intravesical drug instillation and the cell responses were analyzed by multiple experimental approaches. An orthotopic TCC nude mouse model was established by injecting EJ cells into the sub-urothelial layer and used for short-term intravesical resveratrol instillation. The safety of resveratrol instillation was evaluated and compared with that of MCC. The results revealed that 2 h 150 µM or 200 µM resveratrol treatment leaded to remarkable S phase arrest and apoptosis at 72 h time-point, accompanied with attenuated phosphorylation, nuclear translocation and transcription of STAT3, down-regulation of STAT3 downstream genes (survivin, cyclinD1, c-Myc and VEGF) and nuclear translocations of Sirt1 and p53. The importance of STAT3 signaling in cell growth was confirmed by treating EJ cells with JAK2 inhibitor tyrphostin AG490. The efficacy and safety of resveratrol instillation were proved by the findings from nude mouse orthotopic xenograft models, because this treatment caused growth suppression, distinctive apoptosis and STAT3 inactivation of the transplanted tumors without affecting normal urothelium. Our results thus suggest for the first time the practical values of resveratrol as a safe and effective agent in the post-operative treatment of TCCs.

Topics: Active Transport, Cell Nucleus; Analysis of Variance; Animals; Apoptosis; Blotting, Western; Carcinoma, Transitional Cell; Cell Line, Tumor; Cell Proliferation; DNA Primers; Dose-Response Relationship, Drug; Flow Cytometry; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Mice; Mice, Nude; Phosphorylation; Resveratrol; Reverse Transcriptase Polymerase Chain Reaction; S Phase Cell Cycle Checkpoints; STAT3 Transcription Factor; Stilbenes; Tyrphostins; Urinary Bladder Neoplasms

Isorhapontigenin (ISO) is a new derivative of stilbene compound that was isolated from the Chinese herb Gnetum Cleistostachyum and has been used for treatment of bladder cancers for centuries. In our current studies, we have explored the potential inhibitory effect and molecular mechanisms underlying isorhapontigenin anticancer effects on anchorage-independent growth of human bladder cancer cell lines. We found that isorhapontigenin showed a significant inhibitory effect on human bladder cancer cell growth and was accompanied with related cell cycle G(0)-G(1) arrest as well as downregulation of cyclin D1 expression at the transcriptional level in UMUC3 and RT112 cells. Further studies identified that isorhapontigenin downregulated cyclin D1 gene transcription via inhibition of specific protein 1 (SP1) transactivation. Moreover, ectopic expression of GFP-cyclin D1 rendered UMUC3 cells resistant to induction of cell-cycle G(0)-G(1) arrest and inhibition of cancer cell anchorage-independent growth by isorhapontigenin treatment. Together, our studies show that isorhapontigenin is an active compound that mediates Gnetum Cleistostachyum's induction of cell-cycle G(0)-G(1) arrest and inhibition of cancer cell anchorage-independent growth through downregulating SP1/cyclin D1 axis in bladder cancer cells. Our studies provide a novel insight into understanding the anticancer activity of the Chinese herb Gnetum Cleistostachyum and its isolate isorhapontigenin.

Topics: Animals; Antineoplastic Agents, Phytogenic; Binding Sites; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Disease Models, Animal; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Promoter Regions, Genetic; Sp1 Transcription Factor; Stilbenes; Transcription, Genetic; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays

Over 6 million people die annually in the world because of cancer. Several groups are focused on studying cancer chemoprevention approaches. Resveratrol, a polyphenol, at high dosages, has been reported as antitumor and chemopreventive. However, it has a dose-dependent effect on cell death, even on some cancer cells.. Our aim was to investigate this dose-dependent effect on human bladder carcinoma ECV304 cells during oxidative stress condition.. For this purpose, ECV304 cells incubated with different Resveratrol concentrations were analyzed as for their metabolic rate, membrane permeability, DNA fragmentation, anti/proapoptotic protein levels and phosphatidylserine exposure after oxidative stress.. Resveratrol induced cell death at high concentrations (>20 μM), but not at low ones (0.1-20 μM). Pretreatment with 2.5 μM protected the cells from oxidative damage, whereas 50 μM intensified the cell death and significantly increased Bad/Bcl-2 ratio (proapoptotic/antiapoptotic proteins). Resveratrol was able to modulate NO and PGE(2) secretion and performed an anti-adhesion activity of neutrophils on PMA-activated ECV304 cells.. Resveratrol at high doses induces cell death of ECV304 cells whereas low doses induce protection. Modulation of Bcl-2 protein induced by Resveratrol could be mediating this effect. This information about the role of Resveratrol on cancer alerts us about its dose-dependent effects and could lead the design of future chemoprevention strategies.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; bcl-Associated Death Protein; Cell Line, Tumor; Dinoprostone; Dose-Response Relationship, Drug; Humans; Neutrophils; Nitric Oxide; Oxidative Stress; Phytotherapy; Plant Extracts; Proto-Oncogene Proteins c-bcl-2; Resveratrol; Stilbenes; Tetradecanoylphorbol Acetate; Urinary Bladder Neoplasms

Resveratrol shows chemopreventive activity in a variety of human cancers by targeting mitochondria and triggering apoptosis. The purpose of this study was to investigate the antitumor action of resveratrol in bladder cancer and its underlying mechanism.. Using two different bladder cell lines, BTT739 and T24, the cytotoxicity of resveratrol were determined by MTT assay. The apoptosis induced by resveratrol was assayed by transferase dUTP nick end labeling staining. To show whether the mitochondrial dysfunction involved in the effects of resveratrol, mitochondrial function was detected by mitochondrial membrane potential, reactive oxygen species production and adenosine 5'-triphosphate content. In addition, the markers of apoptosis in the intrinsic mitochondrial-dependent pathway were analyzed by the release of cytochrome c and the activities of caspase-9 and caspase-3.. Resveratrol effectively decreased cell viability and induced apoptosis in a concentration- and time-dependent manner. In addition, resveratrol significantly disrupted the mitochondrial membrane potential in both intact cells and isolated mitochondria. Resveratrol also increased reactive oxygen species production and reduced adenosine 5'-triphosphate concentrations. Western blot analysis showed that resveratrol provoked the release of cytochrome c from mitochondria to the cytosol. Furthermore, resveratrol significantly promoted the activation of caspase-9 and caspase-3.. These findings suggest that resveratrol efficiently triggers apoptosis in bladder cancer cells through the intrinsic mitochondrial-dependent pathway, which is associated with mitochondrial dysfunction. Resveratrol might have great pharmacological promise in the treatment of bladder cancer.

Topics: Adenosine Triphosphate; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma; Caspase 3; Caspase 9; Cell Line, Tumor; Cytochromes c; Drug Screening Assays, Antitumor; Enzyme Activation; Humans; Membrane Potential, Mitochondrial; Mice; Reactive Oxygen Species; Resveratrol; Stilbenes; Urinary Bladder Neoplasms

Although the Chinese herb Gnetum cleistostachyum has been used as a remedy for cancers for hundred years, the active compounds and molecular mechanisms underlying its anti-cancer activity have not been explored. Recently a new derivative of stilbene compound, isorhapontigenin (ISO), was isolated from this Chinese herb. In the present study, we examined the potential of ISO in anti-cancer activity and the mechanisms involved in human cancer cell lines. We found that ISO exhibited significant inhibitory effects on human bladder cancer cell growth that was accompanied by marked apoptotic induction as well as down-regulation of the X-linked inhibitor of apoptosis protein (XIAP). Further studies have shown that ISO down-regulation of XIAP protein expression was only observed in endogenous XIAP, but not in constitutionally exogenously expressed XIAP in the same cells, excluding the possibility of ISO regulating XIAP expression at the level of protein degradation. We also identified that ISO down-regulated XIAP gene transcription via inhibition of Sp1 transactivation. There was no significant effect of ISO on apoptosis and colony formation of cells transfected with exogenous HA-tagged XIAP. Collectively, current studies, for the first time to the best of our knowledge, identify ISO as a major active compound for the anti-cancer activity of G. cleistostachyum by down-regulation of XIAP expression and induction of apoptosis through specific targeting of a SP1 pathway, and cast new light on the treatment of the cancer patients with XIAP overexpression.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Cell Line, Tumor; Down-Regulation; Drug Screening Assays, Antitumor; Drugs, Chinese Herbal; Gene Expression Regulation, Neoplastic; Gnetum; Humans; Neoplasm Proteins; Proteolysis; Stilbenes; Urinary Bladder Neoplasms; X-Linked Inhibitor of Apoptosis Protein

Resveratrol, a naturally occurring polyphenolic antioxidant compound present in grapes and red wine, has been reported to hold various biochemical responses. In this preliminary study, we evaluate the chemopreventive potential of resveratrol against bladder cancer and its mechanism of action. Treatment of bladder cancer cells with resveratrol resulted in a significant decrease in cell viability. Resveratrol induced apoptosis through the modulation of Bcl-2 family proteins and activation of caspase 9 and caspase 3 followed by poly(ADP-ribose) polymerase degradation. Treatment with resveratrol led to G(1) phase cell cycle arrest in T24 cells by activation of p21 and downregulation of cyclin D1, cyclin-dependent kinase 4, and phosphorylated Rb. Resveratrol also inhibited the phosphorylation of Akt, whereas the phosphorylation of p38 MAPK was enhanced. In addition, resveratrol treatment decreased the expression of vascular endothelial growth factor and fibroblast growth factor-2, which might contribute to the inhibition of tumor growth on the bladder cancer xenograft model. These findings suggest that reveratrol could be an important chemoprevention agent for bladder cancer.

Topics: Animals; Anticarcinogenic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; G1 Phase; Humans; Male; Mice; Mice, Inbred BALB C; p38 Mitogen-Activated Protein Kinases; Resveratrol; Stilbenes; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays

We optimized agent testing in an in vivo bladder cancer model and determined the most sensitive, relevant protocol to test efficacy in clinical prevention trials.. Female Fischer-344 rats (Harlan) were treated with the bladder carcinogen OH-BBN (TCI America, Portland, Oregon) for 8 weeks. Rats were treated with naproxen (400 mg/kg diet), aspirin (Sigma(R)) (300 or 3,000 mg/kg diet), Iressa(R) (10 mg/kg gefitinib body weight daily) or resveratrol (1,000 mg/kg diet) using 1 of 3 protocols, including treatment beginning 1) 1 week after OH-BBN and continuing for 7 months, 2) 3 months after OH-BBN after microscopic lesions already existed and continuing for 3 months, and 3) 1 week after OH-BBN and continuing for 4 months. In protocols 1 and 2 bladder lesion weight and large tumors were primary end points, and in protocol 3 microscopic cancer was the end point.. Using protocol 1 naproxen, Iressa, resveratrol, and low and high dose aspirin altered the formation of large bladder tumors by 87% (decreased), 90% (decreased), 3% (increased), 6% (decreased) and 60% (decreased), respectively. Using protocol 2 Iressa and naproxen were also highly effective. Protocol 3 evaluation revealed that only Iressa caused a significant decrease in microscopic bladder cancers (63%).. Initiating treatment after OH-BBN or when bladder lesions already existed showed naproxen and Iressa to be effective in preventing formation of large cancers. Low dose aspirin and resveratrol were ineffective. In protocol 3, in which microscopic lesions were the end point, only Iressa was effective. Thus, an established cancer end point appears preferable. Naproxen, which has an excellent cardiovascular profile, or epidermal growth factor receptor inhibitors may be effective in an adjuvant setting.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents; Aspirin; Cyclooxygenase Inhibitors; Disease Models, Animal; Drug Screening Assays, Antitumor; Female; Gefitinib; Naproxen; Quinazolines; Rats; Rats, Inbred F344; Research Design; Resveratrol; Stilbenes; Urinary Bladder Neoplasms

Bladder cancer is one of the most common malignancies in the world. The majority of bladder cancer deaths are due to unresectable lesions that are resistant to chemotherapy. Pterostilbene (PT), a naturally occurring phytoalexin, possesses a variety of pharmacologic activities, including antioxidant, cancer prevention activity and cytotoxicity to many cancers. We found that PT effectively inhibits the growth of sensitive and chemoresistant human bladder cancer cells by inducing cell cycle arrest, autophagy and apoptosis. Down-regulations of Cyclin A, B and D1 and pRB are the results of PT-induced cell cycle arrest.. Autophagy occurred at an early stage and was observed through the formation of acidic vesicular organelles (the marker for autophagy) and microtubule-associated protein 1 light chain 3-II production. Apoptosis occurred at a later stage and was detected by Annexin V and 4',6-diamidino-2-phenylindole staining. PT-induced autophagy was triggered by the inhibition of active human protein kinase/the mammalian TOR/p70S6K pathway and activation of extracellular signal-regulated kinase pathway. Inhibition of autophagy by pretreatment with 3-methyladenine, bafilomycin A1, Beclin 1 or extracellular signal-regulated kinase short hairpin RNA enhanced PT-triggered apoptosis.. This is the first study to demonstrate that PT causes autophagy in cancer cells and suggests that PT could serve as a new and promising agent for the treatment of sensitive and chemoresistant bladder cancer cells.

Topics: Adenine; Apoptosis; Autophagy; Cell Cycle; Cell Line, Tumor; Down-Regulation; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Microtubule-Associated Proteins; Mitogen-Activated Protein Kinases; Signal Transduction; Stilbenes; Urinary Bladder Neoplasms

Bladder cancer is a highly recurrent cancer after intravesical therapy, so new drugs are needed to treat this cancer. Hence, we investigated the anti-cancer activity of combretastatin A-4 (CA-4), an anti-tubulin agent, in human bladder cancer cells and in a murine orthotopic bladder tumour model.. Cytotoxicity of CA-4 was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, propidium iodide (PI) staining assay and clonogenic survival assay. In vivo microtubule assembly assay, cell cycle analyses, Western blot and cell migration assay were used to study the mechanism of CA-4. The effect of intravesical CA-4 therapy on the development of tumours was studied in the murine orthotopic bladder tumour model.. CA-4 inhibited microtubule polymerization in vivo. Cytotoxic IC(50) values of CA-4 in human bladder cancer cells were below 4 nM. Analyses of cell-cycle distribution showed CA-4 obviously induced G(2)-M phase arrest with sub-G(1) formation. The analyses of apoptosis showed that CA-4 induced caspase-3 activation and decreased BubR1 and Bub3 in cancer cells. In addition to apoptosis, CA-4 was also found to induce the formation of multinucleated cells. CA-4 had a significantly reduced cell migration in vitro. Importantly, the in vivo study revealed that intravesical CA-4 therapy retarded the development of murine bladder tumours.. These data demonstrate that CA-4 kills bladder cancer cells by inducing apoptosis and mitotic catastrophe. It inhibited cell migration in vitro and tumour growth in vivo. Hence, CA-4 intravesical therapy could provide another strategy for treating superficial bladder cancers.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Dose-Response Relationship, Drug; Female; Humans; Inhibitory Concentration 50; Mice; Mice, Inbred C57BL; Microtubules; Neoplasm Invasiveness; Phosphorylation; Proto-Oncogene Proteins c-akt; Stilbenes; Time Factors; Tubulin Modulators; Tumor Burden; Urinary Bladder Neoplasms

Piceatannol (3,3',4,5'-Tetrahydroxy-trans-stilbene) is a polyphenol present in grapes and wine. Piceatannol is a protein kinase inhibitor that modifies multiple cellular targets exerting immunosuppressive, antileukemic, and antitumorigenic activities in several cell lines and animal models. In this study, the antiproliferative activity of piceatannol was investigated. The results showed that piceatannol inhibited the proliferation of T24 and HT1376 human bladder cancer cells by blocking cell cycle progression in the G0/G1 phase and inducing apoptosis. ELISA showed that the G0/G1 phase arrest is due to an increased in the expression of p21/WAF1. An enhancement in Fas/APO-1 and membrane-bound Fas ligand (mFasL) might be responsible for the apoptotic effect induced by piceatannol. Our study reports the novel finding, that the induction of p21/WAF1 and activity of the Fas/mFasL apoptotic system may participate in the antiproliferative activity of piceatannol in T24 and HT1376 cells.

Topics: Apoptosis; Cell Cycle; Cell Division; Cell Line, Tumor; Cell Membrane; Fas Ligand Protein; Humans; Protein-Tyrosine Kinases; Stilbenes; Tumor Suppressor Protein p53; Urinary Bladder Neoplasms; Vitis; Wine

Combretastatin A4 phosphate (CA4P) causes rapid disruption of the tumor vasculature and is currently being evaluated for antivascular therapy. We describe the initial results obtained with a noninvasive multiparametric magnetic resonance imaging (MRI) approach to assess the early effects of CA4P on rat bladder tumors implanted on nude mice. MRI (4.7 T) comprised a fast spin-echo sequence for growth curve assessment; a multislice multiecho sequence for T2 measurement before, 15 minutes after, and 24 hours after CA4P (100 mg/kg); and a fast T2w* gradient-echo sequence to assess MR signal modification under carbogen breathing before, 35 minutes after, and 24 hours after CA4P. The tumor fraction with increased T2w* signal intensity under carbogen (T+) was used to quantify CA4P effect on functional vasculature. CA4P slowed tumor growth over 24 hours and accelerated necrosis development. T+ decrease was observed already at 35 minutes post-CA4P. Early T2 increase was observed in regions becoming necrotic at 24 hours post-CA4P, as confirmed by high T2 and histology. These regions exhibited, under carbogen, a switch from T2w* signal increase before CA4P to a decrease postCA4P. The combination of carbogen-based functional MRI and T2 measurement may be useful for the early follow-up of antivascular therapy without the administration of contrast agents.

Topics: Animals; Antineoplastic Agents, Phytogenic; Carbon Dioxide; Contrast Media; Magnetic Resonance Imaging; Mice; Mice, Nude; Neoplasm Transplantation; Neovascularization, Pathologic; Oxygen; Radiation-Sensitizing Agents; Rats; Stilbenes; Time Factors; Urinary Bladder Neoplasms

A functional vascular network is essential for the survival, growth and spread of solid tumours, making blood vessels a key target for therapeutic strategies. Combretastatin A-4 phosphate (CA-4-P) is a tubulin-depolymerising agent in Phase II clinical trials as a vascular disrupting agent. Not much is known of the molecular effect of CA-4-P under tumour conditions. The tumour microenvironment differs markedly from that in normal tissue, specifically with respect to oxygenation (hypoxia). Gene regulation under tumour conditions is governed by hypoxia inducible factor 1 (HIF-1), controlling angiogenic and metastatic pathways.. We investigated the effect of CA-4-P on factors of the upstream and downstream signalling pathway of HIF-1 in vitro.. CA-4-P treatment under hypoxia tended to reduce HIF-1 accumulation in a concentration-dependent manner, an effect which was more prominent in endothelial cells than in cancer cell lines. Conversely, CA-4-P increased HIF-1 accumulation under aerobic conditions in vitro. At these concentrations of CA-4-P under aerobic conditions, nuclear factor kappaB was activated via the small GTPase RhoA, and expression of the HIF-1 downstream angiogenic effector gene, vascular endothelial growth factor (VEGF-A), was increased.. Our findings advance the understanding of signal transduction pathways involved in the actions of the anti-vascular agent CA-4-P.

Topics: Antineoplastic Agents, Phytogenic; Carcinoma; Colonic Neoplasms; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Humans; Hypoxia-Inducible Factor 1; Signal Transduction; Stilbenes; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Topics: Animals; Azo Compounds; Carcinogens; Coloring Agents; Cricetinae; Dogs; Furans; Hydrocarbons; Hyperplasia; Mice; Neoplasms; Neoplasms, Experimental; Pharmacology; Rats; Research; Stilbenes; Urinary Bladder Diseases; Urinary Bladder Neoplasms