Expression of MDR-1 gene in transitional cell carcinoma and its correlation with chemotherapy response.

PURPOSE: Expression of the mdr-1 gene has been correlated with the chemoresistance mechanisms of some cancer models. In the present study, we tried to evaluate mdr-1 gene expression in transitional cell carcinoma (TCC) in both cultured cells and clinical tumors. The expression status of mdr-1 was further correlated with the response to chemotherapy in both systemic and intravesical models. MATERIALS AND METHODS: We evaluated mdr-1 expression levels by reverse transcription polymerase chain reaction and Southern blotting (RT-PCR-SB) and the activity of P-glycoprotein (P-gp) by flow cytometric rhodamine-123 retention and efflux study in 10 TCC cell lines, 2 doxorubicin-resistant sublines (RLs), T24/A and NTUB1/A, and 2 cisplatin-RLs, T24/P and NTUB1/P. Eighty-eight clinical tumors with their benign counterparts were also assayed by RT-PCR-SB to determine mdr-1 expression status. Of the 88 TCC cases, 28 were treated with systemic and 60 with intravesical chemotherapy. Response to chemotherapy in either form was correlated with mdr-1 expression status. RESULTS: By RT-PCR-SB, mdr-1 expression signals were observed in only 2 of the 10 TCC cell lines; only 1 of these had a strong signal and active P-gp function. The other, bearing a weak signal, was negative for active P-gp function. All of the 4 RLs studied showed elevated mdr-1 transcript levels as compared with their mdr-1 negative parental cell lines. Doxorubicin-RLs showed much stronger expression signals than cisplatin-RLs. Active P-gp functions were observed in the 2 doxorubicin-RLs but not in the 2 cisplatin-RLs. The efflux of rhodamine-123 in cells with active P-gp function can be significantly inhibited by 10 microM. verapamil. Of the 88 clinical tumors, 62 (70.5%) were positive and 26 (29.5%) were negative for mdr-1 expression by RT-PCR-SB. All benign counterparts of the 88 tumors were positive for mdr-1 expression. However, no differences in chemotherapy responses were found between the positive and negative mdr-1 expression groups in either systemic chemotherapy (p = 0.32, one-tailed Fisher's exact test) or intravesical chemotherapy (p = 0.52, Cox-Mantel log rank test). CONCLUSIONS: Expression of mdr-1 was not commonly seen in TCC cell lines but can be significantly induced by chronic exposure to doxorubicin. Benign transitional cell epithelia seemed to universally express the mdr-1 gene. However, clinical TCCs lost mdr-1 transcript expressions in about 30% of cases. Most important, it appeared that mdr-1 expression status did not correlate with the response to chemotherapy in either systemic or intravesical models.

Combined cytotoxic effects of tamoxifen and chemotherapeutic agents on bladder cancer cells: a potential use in intravesical chemotherapy.

OBJECTIVE: To evaluate whether tamoxifen enhances the cytotoxicity of chemotherapeutic agents on bladder cancer cells, and the possible mechanism(s) of action. MATERIALS AND METHODS: The in vitro inhibition of cell growth was examined in a model simulating intravesical chemotherapy using two bladder cancer cell lines (TSGH-8301, HTB9) and three commonly used intravesical cytotoxic agents (doxorubicin, mitomycin C, and thiotepa) in the presence or absence of tamoxifen or verapamil as modulators. The expression of the multi-drug resistance-related gene mdr-1 was evaluated by reverse-transcription polymerase chain reaction and Southern blotting (RT-PCR-SB) to determine its transcript level, by flow cytometric analysis of the P-glycoprotein (P-gp) product level with C-219 monoclonal antibody and by the rhodamine-123 retention and efflux assay for P-gp activity. Transforming growth factor beta-1 (TGF beta-1) levels in tamoxifen-conditioned culture medium were determined with enzyme-linked immunosorbant assay (ELISA). RESULTS: Tamoxifen at concentrations > or = 30 microM significantly enhanced the cytotoxicity of the three chemotherapeutic agents to both cell lines, as shown by a marked reduction in the drug concentration which inhibited growth by 50% (IC50). The enhancement of cytotoxicity was significantly dependent on the concentration of tamoxifen. However, tamoxifen alone caused significant toxic effects to TSGH-8301 at > or = 40 microM and to HTB9 at > or = 30 microM. Median-effect analysis showed additive or less-than-additive combination effects between tamoxifen and chemotherapeutic agents and only a minimal synergism in a narrow range of maximal cytotoxicity (fraction affected > 0.9). Thus, the reduction of IC50s by tamoxifen was mostly because it was cytotoxic to the bladder cancer cells used. No enhancement of cytotoxicity was observed in verapamil-modulated cells. Transcripts of mdr-1 could not be detected by RT-PCR-SB, nor was P-gp detected by flow cytometric analysis in the two cell lines. Furthermore, no active P-gp function was detected by the rhodamine-123 retention and efflux study, indicating that the primary chemoresistance mechanisms of the two cell lines were not mediated by mdr-1, nor could tamoxifen or verapamil act through modulation of the mdr-1 pathway in the two cell lines. Tamoxifen at 3 and 10 microM down-regulated the secretion of TGF beta-1 from TSGH-8301 in a concentration-dependent manner, in contrast to the findings that tamoxifen was cytotoxic to the bladder cancer cells used and that tamoxifen up-regulated TGF beta-1 in a breast cancer model, suggesting that there may be a different mechanism of response to TGF beta-1 in these bladder cancer cells. CONCLUSION: Tamoxifen enhanced the cytotoxicity of chemotherapeutic agents largely through its toxic effects on the bladder cancer cells. The mode of action of tamoxifen was not through the regulation of TGF beta-1 or the function of mdr-1. Although cytotoxic levels of tamoxifen (> 50 microM) can be achieved easily in the intravesical model, further study is necessary before tamoxifen can be used clinically in intravesical chemotherapy.