Synergistic effects of cisplatin and proteasome inhibitor bortezomib on human bladder cancer cells

Abstract

The proteasome inhibitor bortezomib is a promising novel agent in bladder cancer therapy; however, inducible cytoprotective mechanisms may limit its potential efficacy. To date, the cellular and molecular effects of proteasome inhibitors on bladder cancer cells have been poorly characterized. Despite the consistent rate of initial responses, cisplatin treatment typically results in the development of chemoresistance, leading to therapeutic failure. Therefore, the present study aimed to characterize the molecular mechanisms underlying the anti-proliferative effects of cisplatin and bortezomib combination therapy on the human T24 bladder cancer cell line, by analyzing the protein expression levels of apoptotic genes. Cytotoxic effects were measured using a water-soluble tetrazolium salt-1 assay, and the apoptosis-associated molecules were examined using western blot analysis and ELISA. It was observed that combined administration of cisplatin and bortezomib induced upregulation of caspase-3, -8 and -9, B-cell lymphoma-2 (Bcl-2)-like 11 and Bcl-2-interacting killer, but downregulated Bcl-2 and Bcl-extra large protein expression levels in T24 cells in a dose-dependent manner. Furthermore, enhanced protein expression of caspase-8 and -9, in line with the significantly increased caspase-3 activation, was detected when the cells were treated with a combination of cisplatin and bortezomib, compared with that of either agent alone. Bortezomib appeared to synergize with cisplatin to promote apoptosis via the extrinsic and intrinsic apoptotic pathways. Taken together, the results of the current study provide the preclinical framework for additional evaluation of the effects of combining bortezomib with other agents to induce apoptosis in bladder cancer cells.

Keywords: apoptosis; bladder cancer cells; bortezomib; cisplatin.

Figure 1.

Combined treatment with cisplatin and bortezomib inhibits cell proliferation of T24 cells. Graphical representation of water soluble tetrazolium salt assay, indicating percentage change in cell viability in T24 cells treated with (A) cisplatin (0, 0.5, 1, 2.5, 5, 10 and 20 mM), (B) bortezomib (0, 1, 5, 7.5, 10, 25, 50, 100, 200 and 300 nM) and (C) cisplatin plus bortezomib for 24 h. Data points represent the mean ± standard deviation of triplicate experiments. *IC₅₀ value. C, cisplatin; B, bortezomib.

Figure 2.

Combined cisplatin and bortezomib treatment enhances casp-3, -8 and -9 activity. (A) Expression levels of effector casp-3 protein, determined by ELISA. Values are expressed as the mean ± standard deviation of triplicate experiments. (B) Expression levels of initiator apoptotic caspases, detected by western blotting. Casp, caspase; C, control; cis, cisplatin; Bor, bortezomib.

Figure 3.

Combined treatment downregulates the expression of anti-apoptotic proteins and enhances the expression of pro-apoptotic proteins. Western blot analysis results of (A) anti-apoptotic and (B) pro-apoptotic protein expression 24 h following cis, bor and cis plus bortezomib combination treatment. C, control; cis, cisplatin; bor, bortezomib; Bcl-2, B-cell lymphoma-2; Bcl-xL, B-cell lyphoma-extra large; Bim, B-cell lyphoma-2-like 11; Bik, Bcl-2-interacting killer.