Ionizing radiation sensitizes breast cancer cells to Bcl-2 inhibitor, ABT-737, through regulating Mcl-1

Abstract

Breast-conserving surgery followed by radiation therapy has become the standard of care for early stage breast cancer. However, there are some patients that develop a local failure. We have previously shown that Bcl-2 overexpression was associated with an increased risk of local recurrence in patients with early stage breast cancer. The purpose of this study was to explore an approach to overcome radiation resistance by targeting pro-survival Bcl-2 family proteins in breast cancer cells. The breast cancer cell lines MCF-7, ZR-75-1 and MDA-MB231 were used in this study. siRNAs were employed to silence myeloid cell leukemia 1 (Mcl-1). A small molecule inhibitor of Bcl-2, ABT-737, was used to target anti-apoptotic Bcl-2 family proteins. Apoptosis was identified by FITC Annexin V, PI staining and Western blot analysis. The sensitivity to ionizing radiation and ABT-737 were measured by clonogenic assays. The effect of radiation and ABT-737 was also tested in a MCF-7 xenograft mouse model. Our data demonstrate that the combination of ABT-737 and radiation-induced apoptosis had an inhibitory effect on breast cancer cell proliferation. However, treatment with ABT-737 resulted in elevated Mcl-1 in breast cancer cell lines. Targeting Mcl-1 by siRNA sensitized MCF-7 cells to ABT-737. We revealed that radiation blunted Mcl-1 elevation induced by ABT-737, and that radiation downregulated Mcl-1 by promoting its degradation. Our results indicate that radiation and ABT-737 exert a synergistic effect on breast cancer cell lines through downregulating Mcl-1 and activating the bak-apoptotic pathway. These results support the combination of radiation and pro-survival Bcl-2 family inhibitor as a potential novel therapeutic strategy in the local-regional management of breast cancer.

FIG. 1

The combination of radiation and ABT-737 enhanced the sensitivity to ABT-737 or radiation in breast cancer cells. Panel A: MCF-7 cells were plated in 100 mm cell culture dishes and irradiated with 8 Gy in the presence or absence of 5 μM of ABT-737 in MCF-7 cells. The cells were examined 48 h after irradiation under an inverted microscope. Panel B: MCF-7 cells were plated at 2.5 × 10⁴ per well in 24-well tissue culture plates. Viable cells were counted 48 h after irradiation by trypan blue exclusion using a Vi-CELL cell viability analyzer. Panel C: MCF-7 cells and panel D: ZR-75-1 cells were plated in 100 cell culture dishes and irradiated with 4 Gy in the presence or absence of 2.5 μM of ABT-737 for 14 days. At the end of incubation, cells were stained with 1% methylene blue in 50% methanol and colonies were counted. The results shown are the representative of three independent experiments.

FIG. 2

The effect of radiation and ABT-737 on apoptosis in MCF-7 cells. MCF-7 cells were treated with ABT-737 (5 μM) for 24 h and then irradiated with 8 Gy. The cells were harvested at 4, 24 and 48 h after irradiation. Apoptosis was determined by measuring the apoptotic marker, cleaved PARP (panel A), by Western blot assays. The combination of radiation and ABT-737 increased Bak (panel B) protein levels in MCF-7 cells. Bands were quantified using Scion Image software. Error bars (SD) are calculated from three independent experiments. Panel C: Apoptotic cells stained with Annexin-V detected by flow cytometry after treatment with ABT-737 (5 μM) and 8 Gy of radiation (48 h). The results shown are the representative of three independent experiments.

FIG. 3

The effect of Mcl-1 siRNA on cell viability in MCF-7 cells. Panel A: MCF-7 cells were transfected with Mcl-1 siRNA for 48 h. Mcl-1 and α-tubulin were detected by Western blot assays. At 24 h after Mcl-1 siRNA transfection, MCF-7 cells were plated in 100 mm dishes for clonogenic assays. Panel B: Cells were treated with 0.63, 1.25, 2.5 and 5 μM of ABT-737. Panel C: Cells were treated with 2, 4, 6 and 8 Gy of radiation. Fourteen days later, cells were stained with 1% methylene blue in 50% methanol, and colonies were counted. The results shown are the representative of three independent experiments. NT: nontargeting control siRNA. *P < 0.05; **P < 0.01.

FIG. 4

The effect of radiation and ABT-737 on Mcl-1 in MCF-7, MDA-MB 231 and ZR-75-1 cells. Panel A: ABT-737 upregulated Mcl-1 protein levels in MCF-7 cells. MCF-7 cells were collected after treatment with various concentrations of ABT-737 for 24 h. Panel B: MCF-7, MDA-MB 231 and ZR-75-1 cells were treated with ABT-737 (5 μM) for 24 h. Cells were then irradiated with 8 Gy and harvested at 30 min after exposure for Western blot assays. Bands were quantified using Scion Image software. Error bars (SD) are calculated from three independent experiments.

FIG. 5

Radiation regulates degradation of Mcl-1 in MCF-7 cells. MCF-7 cells were treated with ABT-737 (5 μM) for 24 h in the presence or absence of MG132 (4 h). Cells were then irradiated with 8 Gy and harvested at 30 min after exposure for Western blot assays. Bands were quantified using Scion Image software. Error bars (S.D.) are calculated from three independent experiments.

FIG. 6

Effect of ABT-737 and radiation on a xenograft mouse model. MCF-7 cells were subcutaneously injected in the thigh of ovariectomized female nude mice with a 90 day release 17-estradiol pellet. ABT-737 and/or radiation (3 Gy, 4 days) was administrated once tumors reached an average volume of about 300 mm³. *P < 0.05, compared with radiation alone and ABT-737 alone, respectively.