ABT-737 synergizes with Bortezomib to kill melanoma cells

Abstract

The BH3 mimetic ABT-737 is a potent inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-X(L), and Bcl-w. The Bcl-2 family modulates sensitivity to anticancer drugs in many cancers, including melanomas. In this study, we examined whether ABT-737 is effective in killing melanoma cells either alone or in combination with a proteasome inhibitor already in clinical use (Bortezomib) in vitro and in vivo, and further evaluated the mechanisms of action. Results showed that ABT-737 alone induced modest cytotoxicity in melanoma cells, but only at higher doses. Knock-down of the anti-apoptotic proteins Bcl-2, Bcl-X(L), or Mcl-1 with siRNAs demonstrated that Mcl-1 is the critical mediator of melanoma's resistance to ABT-737 treatment. However, ABT-737 displayed strong synergistic lethality when combined with Bortezomib. Immunoblot analyses demonstrated that Bortezomib increased expression of Noxa, a pro-apoptotic Bcl-2 member that antagonizes Mcl-1. Additionally, siRNA-mediated inhibition of Noxa expression protected melanoma cells from cytotoxicity induced by the combination treatment. These results demonstrate that Bortezomib synergizes with ABT-737 by neutralizing Mcl-1's function via increased levels of Noxa. In a xenograft mouse model, although drug doses were limited due to toxicity, ABT-737 or Bortezomib slowed melanoma tumor growth compared to the control, and the drug combination significantly decreased growth compared to either drug alone. These data imply that less toxic drugs fulfilling a function similar to Bortezomib to neutralize Mcl-1 are promising candidates for combination with ABT-737 for treating melanomas.

Keywords: ABT-737; Bcl-2 inhibitor; Bortezomib; Mcl-1; Noxa; Proteasome inhibitor; melanoma.

Fig. 1.. ABT-737 alone has only a small killing effect on melanoma cells, and Mcl-1 is the main mediator of resistance.

MTS assays (A) of WM852c, A375, and 1205Lu cells show only a small killing effect from ABT-737 after 48 h treatments. The control for each cell line was set to 100%. Annexin V Assays (B,D) and Immunoblots (C,E) were performed with melanoma cells transfected with indicated siRNAs. Cells were then treated for 24 h with DMSO control or 3.3 µM ABT-737, 24 h post-transfection. Total Annexin V-positive cells were averaged±s.e.m. for WM852c cells (B) and A375 cells (D), representing two or three independent experiments, respectively. * One-Way ANOVA; p = 0.001; ** p = 0.0004. Remaining WM852c cells (C) and A375 cells (E) from Annexin V assays were lysed for immunoblot analyses.

Fig. 2.. ABT-737 combined with Bortezomib synergistically kills melanoma cells.

For MTS viability assays, WM852c (A) and A375 (B) melanoma cells were treated with indicated compounds for 48 h. Results represent at least three independent experiments. For Annexin V assays (C), A375 and WM852c cells were seeded 24 h prior to treatment with indicated compounds for another 24 h. Total Annexin V-positive cells were averaged±s.e.m., representing three independent experiments. *One-Way ANOVA; Tukey's post-hoc test; p<0.001 (individual Bortezomib treatments compared to combination treatment with ABT-737).

Fig. 3.. Combination of Bortezomib with ABT-737 increases the Noxa/Mcl-1 protein ratio and induces Noxa-dependent apoptosis.

Immunoblots (A) show changes in Bcl-2 family member proteins upon treatments. Numbers at the bottom indicate relative Noxa/Mcl-1 ratios. Cells were treated with vehicle control (‘DMSO’), 1.1 µM ABT-737 (‘ABT’), 15 nM Bortezomib (‘Bort.’), or a combination of both drugs (‘Combo’) for 24 h before being lysed. Annexin V Assays (B) and Immunoblots (C,D) were performed with A375 and WM852c cells transfected with indicated siRNAs. 24 h post-transfection, cells were then treated for 18 h with the indicated compounds before being harvested for analyses. Total Annexin V-positive WM852c or A375 cells were averaged±s.e.m., representing three independent experiments. One-Way ANOVA; * p<0.05; ** p<0.01.

Fig. 4.. ABT-737 and Bortezomib reduce tumor growth in a mouse model.

Tumors were grown in nude mice by injecting 1205Lu cells subcutaneously. Mice were divided into four groups: (1) vehicle only, (2) ABT-737, (3) Bortezomib, (4) both drugs. Kaplan-Meier curves are based on tumor doubling times over the course of the experiment. Both ABT-737 and Bortezomib treatment groups were significantly different from the control (p = 0.005 and 0.014, respectively). The combination group was significantly different from the control (p<0.0001), ABT-737 (p = 0.008) and Bortezomib groups (p = 0.0173).