Treatment Of Bladder Cancer Cells Research Articles

Parthenolide Induces Apoptosis and Cell Cycle Arrest of Human 5637 Bladder Cancer Cells In Vitro

Parthenolide, the principal component of sesquiterpene lactones present in medical plants such as feverfew (Tanacetum parthenium), has been reported to have anti-tumor activity. In this study, we evaluated the therapeutic potential of parthenolide against bladder cancer and its mechanism of action. Treatment of bladder cancer cells with parthenolide resulted in a significant decrease in cell viability. Parthenolide induced apoptosis through the modulation of Bcl-2 family proteins and poly (ADP-ribose) polymerase degradation. Treatment with parthenolide led to G1 phase cell cycle arrest in 5637 cells by modulation of cyclin D1 and phosphorylated cyclin-dependent kinase 2. Parthenolide also inhibited the invasive ability of bladder cancer cells. These findings suggest that parthenolide could be a novel therapeutic agent for treatment of bladder cancer.

Pazopanib Synergizes with Docetaxel in the Treatment of Bladder Cancer Cells

To investigate the efficacy of pazopanib, both alone and in combination with docetaxel, in bladder cancer cells. Bladder cancer expresses many potential therapeutic targets of biological agents, including the vascular endothelial growth factor receptor (VEGFR). Pazopanib is a small molecule inhibitor of VEGFR-1, -2-3, platelet-derived growth factor receptor (PDGFR), and c-Kit. Using human bladder cancer cells HTB3, HT1376, J82, RT4, CRL1749, T24, Sup, and HTB9, the treatment effect of pazopanib and cytotoxic chemotherapy was assessed using a tetrazolium-based assay. The combinatorial effect of these agents on clonogenic growth was further examined. Western blotting was used to assess changes in relevant downstream targets, including phospho-AKT, phospho-FAK, total AKT, and total FAK. Single-agent pazopanib had modest activity. However, synergy was seen with the combination of docetaxel and pazopanib in these multiple cells lines. J82 and T24 cells were selected for additional clonogenic testing because of their resistance to single-agent docetaxel chemotherapy. 1.25 nM of docetaxel had little effect on clonogenic formation; however, in combination with pazopanib, significant inhibition of colony formation was observed. This combination treatment additionally decreased phospho-AKT, an important mediator of cell survival in all cell lines, whereas phospho-FAK expression was variably affected. The present study demonstrates synergistic efficacy of pazopanib with docetaxel in docetaxel-resistant bladder cancer cells. This work supports future evaluation of pazopanib with docetaxel for the treatment of bladder cancer with the potential of improved efficacy and toxicity.

Resveratrol induces apoptosis and cell cycle arrest of human T24 bladder cancer cells in vitro and inhibits tumor growth in vivo

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.

1,1-Bis(3′-indolyl)-1-(p-chlorophenyl)methane activates the orphan nuclear receptor Nurr1 and inhibits bladder cancer growth

Nurr1 is an orphan nuclear receptor and a member of the nerve growth factor I-B subfamily of transcription factors with no known endogenous ligand or stimulator. We show, for the first time, evidence that Nurr1 is expressed in a panel of 11 human bladder cancer cell lines. A new class of methylene-substituted diindolylmethanes (C-DIM) were screened and 1,1-bis(3'-indolyl)-1-(p-chlorophenyl)methane (DIM-C-pPhCl) activated the ligand-binding domain of Nurr1. Treatment of bladder cancer cells with Nurr1-active C-DIM resulted in decreased cell survival (MTT assay) and induction of cell death pathways, resulting in poly(ADP-ribose) polymerase cleavage and DNA fragmentation. The specificity of the Nurr1-active compound was shown using RNA interference in 253J B-V cells, whereby small interfering RNA against Nurr1 attenuated ligand-dependent activation of Nurr1 and poly(ADP-ribose) polymerase cleavage. Furthermore, activation of Nurr1 resulted in stimulation of tumor necrosis factor-related apoptosis-inducing ligand and small interfering RNA experiments attenuated tumor necrosis factor-related apoptosis-inducing ligand production. In an orthotopic model of human bladder tumors established in nude mice, administration of a Nurr1-active C-DIM suppressed bladder cancer growth. These results identify Nurr1 as a potential target for bladder cancer therapy and also identify a novel agent for activating Nurr1.

MAGE‐A9 mRNA and protein expression in bladder cancer

In a previous analysis, we showed that MAGE-As were the most frequently expressed cancer-testis antigens in human bladder tumours. Here, we further characterized by RT-PCR the expression of this family of genes by analyzing specifically MAGE-A3, -A4, -A8 and -A9 mRNAs in 46 bladder tumours and 10 normal urothelia. We found that they were expressed in 30, 33, 56 and 54% of tumours, respectively. Although MAGE-A8 was the most frequent, its expression was low and was also found in most normal urothelia. The other MAGE-A mRNAs were all tumour-specific but MAGE-A9 mRNA was expressed at a higher level and was two times more frequent in superficial than in invasive tumours. To study the expression of the protein, we produced 2 MAGE-A9-specific monoclonal antibodies (mAbs) presenting no cross-reactivity with other MAGE-A proteins. MAb 14A11, was used to analyse the expression of the antigen in testis and tumour samples by immunohistochemistry. In testis, MAGE-A9 expression was restricted to primary spermatocytes. Most bladder tumours that expressed the MAGE-A9 transcript were positive with mAb 14A11. Staining was heterogeneous but half of the tumours showed over 75% positive cells. Finally, we showed that treatment of bladder cancer cells with the methylation inhibitor, 5-aza-2'-deoxycytidine, alone or in combination with the histone deacetylase inhibitors MS-275 and 4-phenylbutyrate could strongly induce the expression of MAGE-A9. These results show that MAGE-A9 is frequently expressed in superficial bladder cancer and could be a relevant target for immunotherapy or chemoimmunotherapy because its expression can be induced by chemotherapeutic drugs.

ENHANCED SENSITIVITY OF BLADDER CANCER CELLS TO CISPLATIN MEDIATED CYTOTOXICITY AND APOPTOSIS IN VITRO AND IN VIVO BY THE SELECTIVE CYCLOOXYGENASE-2 INHIBITOR JTE-522

Cyclooxygenase-2 (COX-2) is a key inducible enzyme involved in the production of prostaglandins and its inhibitors have been shown to induce apoptosis in various cancer cells. Several anticancer agents also mediate apoptosis and may share the common intracellular pathways leading to apoptosis with COX-2 inhibitors. We reasoned that combination treatment of bladder cancer cells with COX-2 inhibitors and anticancer agents may result in synergistic apoptosis. We examined whether the selective COX-2 inhibitor JTE-522 (4-(4-cyclohexyl-2-methyloxazol-5-yl)-2-fluorobenzenesulfonamide) synergizes with anticancer agents in cytotoxicity and apoptosis against bladder cancer cells in vitro and in vivo. Cytotoxicity was determined by the microculture tetrazolium dye assay. Combination treatment of T24 bladder cancer cells with JTE-522 and cis-diamminedichloroplatinum (II) (CDDP) resulted in a synergistic cytotoxic effect. Synergy achieved in cytotoxicity with JTE-522 and CDDP was shown to be due to apoptosis. Treatment of T24 cells with JTE-522 decreased expression of the anti-apoptotic molecule Bcl-2. The in vivo significant growth inhibitory effect of JTE-522 and CDDP against the T24 line heterotransplanted in SCID mice was also observed. This study demonstrates that combination treatment of bladder cancer cells with the selective COX-2 inhibitor JTE-522 and CDDP results in synergistic cytotoxicity and apoptosis in vitro and in vivo. These findings support the potential clinical application of a combination of JTE-522 and CDDP for the treatment of bladder cancer as a new form of therapy with more selective cytotoxicity and fewer collateral side effects.

Synergistic Cytotoxicity and Apoptosis of JTE-522, a Selective Cyclooxygenase-2 Inhibitor, and 5-fluorouracil Against Bladder Cancer

Cyclooxygenase-2 (COX-2) is a key inducible enzyme involved in the production of prostaglandins that has been shown to induce apoptosis in various cancer cells. Several anticancer agents also mediate apoptosis and may share the common intracellular pathways leading to apoptosis. Since over expression of COX-2 has been demonstrated in bladder cancer, we reasoned that combination treatment with a COX-2 inhibitor and anticancer agents in bladder cancer cells may result in synergistic apoptosis. We examined whether the selective COX-2 inhibitor JTE-522 induces apoptosis in bladder cancer cells and whether JTE-522 may act synergistically with anticancer agents to achieve cytotoxicity and apoptosis in these cells. Cytotoxicity was determined by microculture tetrazolium dye assay. Synergy was assessed by isobolographic analysis. COX-2 mRNA expression was observed in the HT1197 bladder cancer cell line. JTE-522 was cytotoxic in HT1197 cells. Treating HT1197 cells with JTE-522 combined with doxorubicin or mitomycin C did not show synergistic cytotoxicity. However, combination treatment of HT1197 cells with JTE-522 and 5-fluorouracil (5-FU) resulted in a synergistic cytotoxic effect. Synergy was also achieved in the T24 bladder cancer line. Synergistic cytotoxicity was noted irrespective of treatment sequence but the highest percent cytotoxicity was obtained when HT1197 cells were treated with JTE-522 and 5-FU simultaneously. The synergy achieved in cytotoxicity with JTE-522 and 5-FU was shown to be due to apoptosis. The mechanisms responsible for synergistic cytotoxicity and apoptosis was examined. Treating HT1197 cells with 5-FU enhanced expression of the pro-apoptotic molecule Bax, while JTE-522 treatment reduced expression of the anti-apoptotic molecule Bcl-XL, resulting in a significantly higher ratio of Bax-to-Bcl-XL. This study shows that combination treatment of bladder cancer cells with the selective COX-2 inhibitor JTE-522 and 5-FU results in synergistic cytotoxicity and apoptosis due to the enhanced Bax-to-Bcl-XL expression ratio. These findings support the in vivo potential application of a combination of JTE-522 and 5-FU for bladder cancer.

Blockage of IGF-1R signaling sensitizes urinary bladder cancer cells to mitomycin-mediated cytotoxicity

A major problem which is poorly understood in the management of bladder cancer is low sensitivity to chemotherapy and high recurrence after transurethral resection. Insulin-like growth factor 1 receptor (IGF-1R) signaling plays a very important role in progression, invasion and metastasis of bladder cancer cells. In this study, we investigated whether IGF-1R was involved in the growth stimulating activity and drug resistance of bladder cancer cells. The results showed: The mRNAs of IGF-1, IGF-2 and IGF-1R were strongly expressed in serum-free cultured T24 cell line, whereas normal urothelial cells did not express these factors/receptors or only in trace levels; T24 cell responded far better to growth stimulation by IGF-1 than did normal urothelial cells; blockage of IGF1R by antisense oligodeoxynucleotide (ODN) significantly inhibited the growth of T24 cell and enhanced sensitivity and apoptosis of T24 cells to mitomycin (MMC). These results suggested that blockage of IGF-IR signaling might potentially contribute to the treatment of bladder cancer cells which are insensitive to chemotherapy.

Treatment of bladder cancer cells in vitro and in vivo with 2–5A antisense telomerase RNA

Bladder cancer is the most common malignant tumor of the urinary tract. Novel treatment approaches are essential because of the failure of current treatment options to cure a high percentage of patients. Telomerase, a ribonucleoprotein, is detected in almost all bladder cancer, but not in normal bladder tissues. Therefore, telomerase is expected to be a very promising candidate for targeted therapy of bladder cancer. In this study, we synthesized a 19-mer antisense oligonucleotide against the RNA component of human telomerase (hTR) linked to a 2-5A molecule (2-5A-anti-hTR) and investigated its antitumor effect against bladder cancer cells. The 2-5A antisense strategy relies on the recruitment and activation of RNase L at the site of targeted RNA sequence. Here we demonstrate that treatment with 2-5A-anti-hTR reduced the viability of seven bladder cancer cell lines (UM-UC-2, UM-UC-3, UM-UC-6, UM-UC-9, UM-UC-14, RT4 and T24) expressing telomerase activity to 21-55% within 4 days. The cytotoxicity was mainly due to induction of caspase-dependent apoptosis. In contrast, normal fibroblast WI38 cells lacking telomerase activity were resistant to the treatment. Furthermore, treatment of subcutaneous UM-UC-2 tumors in nude mice with 2-5A-anti-hTR significantly suppressed the tumor growth through induction of apoptosis (P < 0.001). These findings may offer a strong support to the feasibility of the 2-5A-anti-hTR treatment for human bladder cancer.

ENHANCED SENSITIVITY OF BLADDER CANCER CELLS TO TUMOR NECROSIS FACTOR RELATED APOPTOSIS INDUCING LIGAND MEDIATED APOPTOSIS BY CISPLATIN AND CARBOPLATIN

The development and acquisition of multiple drug resistance in cancer cells are a consequence of cancer chemotherapy and remain a major obstacle in treatment. Therefore, there is an obvious need for alternative approaches, such as immunotherapy and gene therapy. Tumor necrosis factor related apoptosis inducing ligand (TRAIL) is one of the tumor necrosis factor ligand families and it selectively induces apoptosis against cancer cells. Several cytotoxic anticancer drugs also mediate apoptosis and may share the common intracellular pathways leading to apoptosis. We reasoned that combination treatment of cancer cells with TRAIL and drugs may overcome this resistance. We evaluated whether bladder cancer cells are sensitive to TRAIL mediated cytotoxicity and whether TRAIL may synergize with anticancer agents in cytotoxicity and apoptosis against bladder cancer cells. Cytotoxicity was determined by a 1-day microculture tetrazolium dye assay. Synergy was assessed by isobolographic analysis. Human T24 bladder cancer line was relatively resistant to TRAIL and TRAIL was not cytotoxic against normal bladder cells. Treatment of T24 cells with TRAIL in combination with 5-fluorouracil or mitomycin C did not overcome resistance to these agents. However, treatment of T24 cells with a combination of TRAIL and cisplatin resulted in a synergistic cytotoxic effect. Synergy was also achieved in the cisplatin resistant T24 line (T24/CDDP), 2 other bladder cancer lines and 3 freshly derived bladder cancer cells. The combination of TRAIL and carboplatin resulted in a synergistic cytotoxic effect on T24 cells. However, the combination of TRAIL and trans-diamminedichloroplatinum (II) resulted in an antagonistic cytotoxic effect. The synergy achieved in cytotoxicity with TRAIL and cisplatin was also achieved in apoptosis. Treating T24 cells with cisplatin enhanced the expression of bax but not bcl-2. Incubation of T24 cells with TRAIL increased the intracellular accumulation of cisplatin. This study demonstrates that combination treatment of bladder cancer cells with TRAIL and cisplatin overcomes their resistance. The sensitization obtained with established cisplatin resistant and freshly isolated bladder cancer cells required low subtoxic concentrations of cisplatin, supporting the in vivo potential application of a combination of TRAIL and cisplatin for treating TRAIL resistant and cisplatin resistant bladder cancer.

ADRIAMYCIN-MEDIATED POTENTIATION OF CYTOTOXICITY AGAINST FRESHLY ISOLATED BLADDER CANCER CELLS BY AUTOLOGOUS NON-ACTIVATED PERIPHERAL BLOOD LYMPHOCYTES AND TUMOR INFILTRATING LYMPHOCYTES

Previous studies indicated that cancer patients lack functional anti-tumor cytotoxic lymphocytes. However, anti-tumor cytotoxic lymphocytes may coexist with immunoresistant tumor cells. We reasoned that anti-tumor cytotoxic activity of lymphocytes may be revealed if the tumor cells are sensitized to killing. It has been reported that adriamycin (ADR) exhibits various immunomodulating activities. In the present study, we investigate the effect of ADR on the susceptibility of freshly isolated bladder cancer cells to lysis by autologous non-activated peripheral blood lymphocytes (PBL) and tumor infiltrating lymphocytes (TIL). Cytotoxicity was determined by a 1-day microculture tetrazolium dye assay. Treatment of ADR-resistant fresh bladder cancer cells with ADR at 0.1 microg./ml. or more for 3 hours or more enhanced their susceptibility to lysis by autologous PBL. This ADR-induced enhancement of susceptibility of fresh bladder cancer cells to lysis by PBL was also observed when lymphokine activated killer cells, purified natural killer cells and T lymphocytes were used as effector cells. Furthermore, while cytotoxicity of freshly derived TIL against autologous bladder cancer cells was minimal, significant cytotoxicity was observed with ADR-treated bladder cancer cells. The ADR analogs, epirubicin and pirarubicin, also enhanced the susceptibility of bladder cancer cells to lysis by autologous PBL. Treatment of bladder cancer cells with ADR had no effect on the expression of MHC class I on the cancer cells or the frequency of bladder cancer target cell conjugates to autologous PBL. Treatment of bladder cancer cells with ADR augmented their sensitivity to anti-Fas monoclonal antibody and tumor necrosis factor-a. Pretreatment of effector cells with ADR had no effect on their cytotoxic function. These findings demonstrate that PBL and TIL in patients with bladder cancer exhibit anti-tumor cytotoxic function, but their function is not manifested due to development or acquisition of tumor cell resistance to killing. However, the resistance of bladder cancer cells to killing by cytotoxic lymphocytes is overcome if cancer cells are sensitized by subtoxic concentrations of ADR. These findings suggest that treatment of bladder cancer patients with low doses of ADR may sensitize the cancer cells to killing by autologous circulating and tumor infiltrating lymphocytes and may be a novel immunotherapeutic modality for the treatment of drug-resistant and/or immune-resistant bladder cancer.

Chemoimmunosensitization of the T24 human bladder cancer line to Fas-mediated cytotoxicity and apoptosis by cisplatin and 5-fluorouracil.

Previous studies have demonstrated that cisplatin (CDDP) sensitizes bladder cancer cells to Fas-mediated cytotoxicity and that CDDP also enhances the cytotoxic effect of 5-fluorouracil (5-FU). These agents mediate apoptosis and may share common intracellular pathways leading to cell killing. We reasoned that combination treatment with CDDP, 5-FU and anti-Fas monoclonal antibody (mAb) might overcome the drug-resistance. We investigated whether CDDP, 5-FU and anti-Fas mAb synergize in cytotoxicity and apoptosis against the T24 bladder cancer line. Cytotoxicity was monitored by a one day microculture tetrazolium dye assay. Treatment of T24 cells with anti-Fas mAb in combination with CDDP or 5-FU resulted in a synergistic cytotoxic effect. In addition, combination treatment of T24 cells with CDDP, 5-FU and anti-Fas mAb further enhanced the synergistic cytotoxicity achieved by two agents. The synergy achieved in cytotoxicity with CDDP, 5-FU and anti-Fas mAb was also achieved in apoptosis. The current study demonstrates that combination treatment of bladder cancer cells with CDDP, 5-FU and anti-Fas mAb overcomes their resistance. In addition, the sensitization required low concentrations of CDDP and 5-FU, and thus supporting the potential in vivo application of combination of CDDP, 5-FU and immunotherapy in the treatment of drug-resistant bladder cancer.

Effect of anticancer agents on Fas-mediated cytotoxicity against bladder cancer cells

Fas and Fas ligand play an important role in cytotoxic T lymphocyte-mediated cytotoxicity. Like Fas ligand, anti-Fas monoclonal antibody (mAb) induces apoptosis of cells expressing Fas and mimics tumor necrosis factor-alpha (TNF-alpha) in its cytotoxic activity, but not in regard to other TNF-alpha-mediated activities. Since combination treatment with TNF-alpha and some anticancer chemotherapeutic agents results in synergistic cytotoxicity against various cancer cells, anti-Fas mAb may also synergize with anticancer agents in exerting cytotoxicity. The present study examined this hypothesis using bladder cancer cells. Cytotoxicity was examined by a 1-day microculture tetrazolium dye assay. Treatment of T24 cells with anti-Fas mAb in combination with mitomycin C, methotrexate, or 5-fluorouracil did not overcome their resistance to these agents. However, combination treatment with anti-Fas mAb and adriamycin (ADR) resulted in a synergistic cytotoxic effect on T24 cells, three other bladder cancer lines, and fresh bladder cancer cells derived from four patients. Treatment with ADR enhanced the expression of Fas on T24 cells. The expression of P-glycoprotein was not affected by the antibody-mediated sensitization. This study showed that combination treatment of bladder cancer cells with anti-Fas mAb and ADR can overcome their resistance and that the upregulation of Fas expression by ADR may play a role in the enhanced cytotoxicity.

SENSITIZATION OF HUMAN BLADDER CANCER CELLS TO FAS-MEDIATED CYTOTOXICITY BY CIS-DIAMMINEDICHLOROPLATINUM (II)

Purpose The resistance of bladder cancer to anticancer chemotherapeutic drugs is a major problem. Several immunotherapeutic approaches have been developed to treat drug-resistant tumor cells. The Fas antigen (Fas)-Fas ligand pathway is involved in cytotoxic T lymphocyte and natural killer cell-mediated cytotoxicity. Like the Fas ligand, anti-Fas monoclonal antibody (mAb) induces apoptosis in tumor cells expressing Fas. Several anticancer drugs also mediate apoptosis and may share with Fas common intracellular pathways leading to cell killing. We reasoned that treatment of drug-resistant cancer cells with a combination of anti-Fas mAb and drugs might overcome their resistance. This study has investigated whether anticancer drugs synergize with anti-Fas mAb in cytotoxicity against bladder cancer cells. Materials and Methods Cytotoxicity was determined by a 1-day microculture tetrazolium dye assay. Synergy was assessed by isobolographic analysis. Results Treatment of the T24 human bladder cancer cell line with anti-Fas mAb in combination with 5-fluorouracil, mitomycin C or methotrexate did not overcome resistance to these agents. However, treatment of T24 tumor cells with a combination of anti-Fas mAb and cis-diamminedichloroplatinum (II) (CDDP) resulted in a synergistic cytotoxic effect. In addition, the CDDP-resistant T24 line (T24/CDDP) was sensitive to treatment with a combination of anti-Fas mAb and CDDP. Synergy by combination of anti-Fas mAb and CDDP was also achieved in three other bladder cancer lines and four freshly derived human bladder cancer cells. The combination of anti-Fas mAb and carboplatin also resulted in a synergistic cytotoxic effect on T24 cells; however, the combination of anti-Fas mAb and trans-diamminedichloroplatinum (II) resulted in an additive cytotoxic effect. Treatment with CDDP enhanced the expression of Fas on T24 cells. The synergy achieved in cytotoxicity with anti-Fas mAb and CDDP was also achieved in apoptosis. Incubation of T24 cells with anti-Fas mAb increased the intracellular accumulation of CDDP. Treatment of freshly isolated bladder cancer cells with CDDP enhanced their susceptibility to lysis by autologous lymphocytes. Conclusions This study demonstrates that combination treatment of bladder cancer cells with anti-Fas mAb and CDDP overcomes their resistance. Synergy was achieved with established CDDP-resistant bladder cancer cells and freshly isolated bladder cancer cells. In addition, the sensitization required low concentrations of CDDP, thus supporting the potential in vivo application of combination of CDDP and immunotherapy in the treatment of CDDP- and/or immunotherapy-resistant bladder cancer.