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 β‐1 (TGFβ‐1) levels in tamoxifen‐conditioned culture medium were determined with enzyme‐linked immunosorbant assay (ELISA). Results  Tamoxifen at concentrations ≥30 μm 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 ≥40 μm and to HTB9 at ≥30 μm. 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 μm down‐regulated the secretion of TGFβ‐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 TGFβ‐1 in a breast cancer model, suggesting that there may be a different mechanism of response to TGFβ‐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 TGFβ‐1 or the function of mdr‐1 . Although cytotoxic levels of tamoxifen (>50 μm) can be achieved easily in the intravesical model, further study is necessary before tamoxifen can be used clinically in intravesical chemotherapy.