Effects of ABT-737 combined with irradiation treatment on uterine cervical cancer cells

Abstract

The aim of the present study was to examine the role of ABT-737, an inhibitor of B-cell lymphoma 2 (Bcl-2), in enhancing the effect of irradiation on uterine cervical cancer. Based on The Cancer Genomic Atlas (TCGA), Bcl-2 mRNA expression was associated with the Tumor-Node-Metastasis stage of cervical cancer. Therefore, it was hypothesized that Bcl-2 inhibition may decrease the progression of cervical cancer. ABT-737 was added to irradiation treatment to evaluate its effectiveness in inhibiting cancer cell progression. SiHa and CaSki cervical cancer cells were selected for in vitro assays. Patients with advanced stage III uterine cancer had slightly increased mRNA expression levels of Bcl-2 compared with patients with stage I cancer, although the difference was not significant. ABT-737 and radiation administration induced a synergistic cytotoxic effect based on the MTT assay and flow cytometry results, where an increase in apoptosis was observed. The apoptotic percentages were significantly increased in the cells treated with a combination of ABT-737 and irradiation. Loss of mitochondrial membrane potential and gain of reactive oxygen species (ROS) were detected by flow cytometry in CaSki and SiHa cells treated with ABT-737 and radiation. Additionally, the protein expression levels of the cleaved forms of poly ADP ribose polymerase and caspase-7 were increased following the combined treatment. In conclusion, ABT-737 and irradiation may induce apoptosis via loss of mitochondrial membrane potential and a ROS-dependent apoptotic pathway in CaSki and SiHa cells. The present study indicates that ABT-737 may be a potential irradiation adjuvant when treating cervical cancer.

Keywords: ABT-73; B-cell lymphoma 2; apoptosis; irradiation; mitochondrial membrane potential; uterine cervical cancer.

Figure 1.

mRNA expression levels of Bcl-2 among cancer tissues of the uterine cervix in stages I–IV based on The Cancer Genome Atlas data. There was no significant association between Bcl-2 expression levels and cervical cancer stage. Bcl-2, B-cell lymphoma 2.

Figure 2.

Effect of ABT-737 and irradiation on cell viability in CaSki and SiHa uterine cervical cancer cells after 48 h. (A) CaSki and (B) SiHa cells were treated with different concentrations of ABT-737 and radiation for 48 h. Cell viability was analyzed by an MTT assay. Significant differences vs. untreated control (ABT-737 0 µM/irradiation 0 Gy) were determined by Dunnett's test and are indicated by *P<0.05, **P<0.01 and ***P<0.001.

Figure 3.

Effect of ABT-737 combined with irradiation on the MMP in CaSki and SiHa cells. (A) Fluorescent images and (B) flow cytometry of CaSki cells treated with ABT-737. (C) Fluorescent images and (D) flow cytometry of CaSki cells treated with ABT-737. Cells were stained with JC-1 dye to detect the MMP by (A and C) fluorescence microscopy and (B and D) flow cytometry. Changes in MMP were assessed by the intensity of red fluorescence (R2) and green fluorescence (R1 or R3). The arrows signify the apoptotic cells that show green fluorescence. Results are representative of three independent experiments. MMP, mitochondrial membrane potential; FL, fluorescence.

Figure 4.

Effect of ABT-737 and irradiation on apoptosis in CaSki and SiHa cells. (A) CaSki and (B) SiHa cells were treated with ABT-737 combined with irradiation for 48 h. Cells were stained by Annexin V-FITC and PI. Annexin V-FITC positive (early apoptosis) and Annexin V-FITC/PI positive (late apoptosis) were quantified as apoptotic cells. (C) CaSki and (D) SiHa cells were treated with ABT-737 combined with irradiation for 48 h. Cleaved PARP and cleaved caspase-7 were detected by western blotting. β-actin was used as a loading control. FITC, fluorescein isothiocyanate; PI, propidium iodide; PARP, poly ADP ribose polymerase; FL, fluorescence.

Figure 5.

Effect of ABT-737 combined with irradiation on ROS production in CaSki and SiHa cells. (A) CaSki and (B) SiHa cells were treated with ABT-737 combined with irradiation for 48 h. Cells were stained with 2′,7′-dichlorodihydrofluorescein diacetate dye to detect ROS by flow cytometry. Results are representative of three independent experiments. ROS, reactive oxygen species.

Figure 6.

Effect of ABT-737 and irradiation on the cell cycle in CaSki and SiHa cells. (A) CaSki and (B) SiHa cells were treated by ABT-737 combined with irradiation for 48 h. The cells were stained with propidium iodide dye to detect the cell cycle by flow cytometry. (C) CaSki and (D) SiHa cells were treated with ABT-737 combined with irradiation for 48 h. pRB, RB, CDK6 and TS protein expression were assessed by western blot. β-actin was a loading control. RB, retinoblastoma; p, phosphorylated; CDK6, cyclin-dependent kinase-6; TS, thymidylate synthase; FL, fluorescence.