New potential anti-cancer agents synergize with bortezomib and ABT-737 against prostate cancer

Abstract

Background: We previously described the identification of a transcriptional inhibitor ARC and FoxM1 inhibitors, thiazole antibiotics, Siomycin A and thiostrepton that were able to induce potent p53-independent apoptosis in cancer cell lines of different origin. Here, we report the characterization of these drugs individually or in combination with ABT-737 and bortezomib on a panel of prostate cancer cell lines.

Methods: DU 145, LNCaP and PC-3 prostate cancer cells were treated with ARC, Siomycin A and thiostrepton to evaluate their activity as single agents or in combination with ABT-737 and bortezomib to measure their synergistic potential in anti-proliferative and cell cycle assays. Chou-Talalay method was used to quantitate the synergistic interaction. Western blot method was used to determine Mcl-1 and FoxM1 expression and caspase-3 cleavage.

Results: We show that ARC inhibited the viability of prostate cancer cells and induced apoptosis in low nanomolar concentration. It potently downregulated the expression of Mcl-1 and showed synergistic combination effect with Bcl-2 inhibitor ABT-737. Thiazole antibiotics, Siomycin A and thiostrepton inhibited growth, FoxM1 expression and induced cell death in prostate cancer cells in low micromolar concentrations. In addition, thiostrepton and ARC synergistically induced apoptosis in prostate cancer cells following combination treatment with proteasome inhibitor bortezomib. Furthermore, we found that all tested drug combinations were able to induce apoptosis selectively in transformed, but not normal cells of the same origin.

Conclusions: Based on their in vitro activity as single or combination agents, ARC, Siomycin A and thiostrepton represent potential candidates for drug development against prostate cancer.

Figure 1. TAs. induce apoptosis and inhibit cell growth of PC cells

A. Growth inhibition curves of Siomycin A and thiostrepton in PC cells. Mid log LNCaP, PC-3 and DU145 cells were treated with DMSO or various concentrations of Siomycin A or thiostrepton (8.33, 5, 2.5, 1.25, 0.625 and 0.32 µM) for 72 hours and IC₅₀ values were determined by using MTT cell viability assay as described in materials and methods. The values shown are mean ± SD for three separate experiments. IC₅₀ values were 2.1±0.1, 3.1±0.5(LNCaP), 2.5±0.7, 2.9±0.2 (PC-3) and 1.8±0.4, 2.2±0.3(DU145) µM against Siomycin-A and thiostrepton, respectively. B. Thiazole antibiotics induce p53-independent apoptosis in PC cells. Siomycin A and thiostrepton downregulate FoxM1 expression in LNCaP, p53shRNA-LNCaP, PC-3 and DU145 PC cells and induce apoptosis as indicated by appearance of cleaved caspase-3. DU145, PC-3, LNCaP and p53shRNA-LNCaP cells were treated with 5µM of Siomycin A and thiostrepton for 48 hours, lysed and subjected to immunoblotting analysis with antibodies against FoxM1, cleaved caspase-3, p53 and β-actin. C. Quantitation of TA-induced apoptosis by flow cytometry. DU 145 cells were treated with 5µM of Siomycin A and thiostrepton for 48 hours, stained with annexin V-PE and 7-AAD and analyzed by flow cytometry. The values shown are mean of at least two experiments.

Figure 2. Evaluation of effects of ARC on PC cells

A. Growth inhibition curves of ARC in PC cells. ARC inhibits the proliferation of PC cell lines. Mid-log cells were treated with various concentrations of ARC (10, 2, 0.4, 0.08, 0.02, 0.004 µM) for 72 hours and IC₅₀ values were determined as described in materials and methods. IC₅₀ values of ARC were calculated to be 0.05±0.01(LNCaP), 0.08±0.02(PC-3) and 0.03±0.01(DU-145) µM respectively. The values were measured as mean ± SD for three separate experiments. B. ARC inhibits the expression of anti-apoptotic protein Mcl-1 and induces apoptosis in PC cell lines. The cells were treated with increasing concentrations of ARC as indicated for 48 hours, lysed and subjected to immunoblotting analysis with antibodies against Mcl-1, cleaved caspase-3 and β-actin. C. Induction of apoptosis by ARC in PC cells confirmed by annexin V-PE/7-AAD staining. PC cell lines were grown at a density of 50% confluence in 100-mm culture dishes and were treated with varying concentrations of the drugs for 48 hours, stained with annexin V-PE /7-AAD and analyzed by flow cytometry. The values shown are average of at least two experiments.

Figure 3. ARC and thiostrepton synergize with ABT-737 and Bortezomib in induction of apoptosis in human PC cells

A. Immunoblotting analysis of cell death induced by combinations of drugs in DU145 cells. DU145 cell line was treated with DMSO, 25 nM ARC, 1.5 µM thiostrepton, 350 nM ABT-737, 7.5 nM bortezomib or combination of 25 nM ARC and 350 nM ABT-737 or 25nM ARC and 7.5 nM bortezomib or 1.5 µM thiostrepton and 7.5 nM bortezomib respectively for 48 hrs and apoptosis was assessed by immunoblotting for cleaved caspase-3 and β-actin. B. Immunoblotting analysis of cell death induced by combinations of drugs in PC-3 cells. PC-3 cells were treated with 100 nM ARC, 1.5 µM thiostrepton, 350 nM ABT-737, 7.5 nM bortezomib or combination of 100nM ARC and 350 nM ABT-737 or 100 nM ARC and 7.5 nM bortezomib or 1.5 µM thiostrepton and 7.5 nM bortezomib respectively for 48hrs and apoptosis was assessed by immunoblotting for cleaved caspase-3 and β-actin. C. Flow cytometry analysis of cell death induced by combinations of drugs in DU145 cells. Synergistic induction of apoptosis was measured by treating DU145 cells with DMSO (control), single agents (25 nM ARC, 1.5 µM thiostrepton, 350 nM ABT-737, 7.5 nM bortezomib) and their combinations (25nM ARC and 350 nM ABT-737 or 25nM ARC and 7.5 nM bortezomib or 1.5 µM thiostrepton and 7.5 nM bortezomib) for 48 hrs followed by staining the cells with annexin V-PE and 7-AAD and analysis by flow cytometry. Percent of cells undergoing apoptosis are shown as mean ± S.D of three experiments. D. Immunoblotting analysis of cell death induced by combinations of drugs in normal and immortalized BJ cells. Wt-BJ and Imm-BJ cell lines was treated with DMSO, 1.5 µM ARC, 1.5 µM thiostrepton, 350 nM ABT-737, 10 nM bortezomib or combination of 1.5 µM ARC and 350 nM ABT-737 or 1.5 µM ARC and 10 nM bortezomib or 1.5 µM thiostrepton and 10 nM bortezomib respectively for 24 hrs and apoptosis was assessed by immunoblotting for cleaved caspase-3 and PARP with β-actin as a control.