Abstract:
The purposes of this study were to examine the effects of trifluoperazine on cisplatin-induced cell death and types of cell death in H460 (human non-small cell lung cancer) cells and their resistant counterparts; cisplatin-induced resistant H460/cis cells and Bcl-2 transfected RALP cells. Moreover, the possible mechanisms of trifluoperazine in modulating cisplatin-induced cell death were evaluated. The cisplatin-induced resistant clone (H460/cis) of lung carcinoma H460 cells was established by exposing the cells with gradually increasing concentrations of cisplatin until chemoresistance acquisition, measured by MTT assay. The apoptotic defect clone (RALP) was generated by transfecting H460 cells with Bcl-2 enforced-expression plasmids. The selected RALP clone no.1 and 6 showed the different level of Bcl-2 protein expression determined by Western blot analysis. H460/cis, RALP1and RALP6 cells showed significantly resistance to cisplatin-induced cytotoxicity. Then, all cells were co-treated with sub-toxic concentrations of trifluoperazine (1–5 µM) and toxic concentrations of cisplatin (20-50 µM). The results indicated that treatment with trifluoperazine could significantly sensitize H460/cis, RALP1 and RALP6 cells to cisplatin-induced cell death. This sensitization did not affect apoptosis or necrosis induction, determined by cell counter-staining with Hoechst/PI and comet assay. Interestingly, it was well correlated with the increasing incidence of autophagy formation detected by acridine orange staining and the induction of an autophagy marker quantitated by Western blot analysis. Nonetheless, this resistance reversal effect showed the selectivity due to not cause more toxicity in sensitive H460 cells. The mechanisms of trifluoperazine in potentiating the effect of cisplatin were partially through the induction of autophagy, since 3-methyladenine, a specific autophagy inhibitor of class III phosphatidylinositol 3 kinase, could reverse its effects. Furthermore, the level of Bcl-2 protein was diminished in cisplatin-trifluoperazine co-treatment. These suggested that trifluoperazine could sensitize the chemoresistance of non-small cell lung cancers, both acquired resistance and Bcl-2 overexpression through the induction of autophagic cell death and lowering the level of Bcl-2 proteins. In conclusion, this study provided the novel sensitizing effects of trifluoperazine in cisplatin-induced cell death in cisplatin-induced acquired resistance and Bcl-2-mediated apoptosis resistance, which might facilitate the development of new strategy to overcome the chemotherapeutic resistance cancers.
