Synergistic effects of bloom helicase (BLM) inhibitor AO/854 with cisplatin in prostate cancer

Abstract

To determine the synergistic effect and mechanism of AO/854, a new Bloom syndrome protein (BLM) helicase inhibitor, and cisplatin (CDDP), a DNA-crosslinking agent, cell viability assays, neutral comet assays, and Western blotting (WB) were performed on prostate cancer (PCa) cells. According to our findings, combining AO/854 and CDDP enhanced the antiproliferative capabilities of PC3 cell lines. As evidenced by the upregulation of γH2AX, cleaved caspase-3/caspase-3, and BAX/Bcl-2, AO/854 dramatically increased PC3 apoptosis and DNA damage induced by CDDP. Furthermore, combining AO/854 and CDDP synergistically inhibited PC3 cell migration and invasion. In addition, AO/854 inhibited CDDP-induced S-phase cell-cycle arrest in PC3 cells while enhancing G2/M-phase cell-cycle arrest. In vivo, the antitumor efficacy of the combination therapy group was greater than that of the groups treated with AO/854 or CDDP alone. Our findings indicate that synergistic chemotherapy with AO/854 and CDDP may be a novel anticancer strategy for PCa.

Keywords: AO/854; BLM; CDDP; Combined therapy.

Fig. 1

Levels of BLM in PC3 cells are inversely associated with sensitivity to CDDP. (A) WB analysis of the protein expression of BLM increased or decreased after BLM overexpression or RNAi. Original blots are presented in Figs. S1-S3. (B) IF analysis of the protein expression of BLM increased or decreased after BLM overexpression or RNAi. (C) Overexpression of BLM reduced the sensitivity of PC3 cells to CDDP. (D) Interfering with sh BLM increased the sensitivity of PC3 cells to CDDP. The presented results were representative of experiments repeated at least three times. Data were represented as mean ± SD. An unpaired t-test was used to determine statistically significant differences. (**P < 0.01, ***P < 0.001).

Fig. 2

AO/854 sensitizes CDDP-induced cytotoxicity in PC3, 22RV1 and LNCap cell lines. (A) Combined effect of AO/854 and CDDP on cell proliferation inhibition in PC3 cells. (B) The combined effect of AO/854 and CDDP on cell proliferation inhibition in 22RV1 cells. (C) The combined effect of AO/854 and CDDP on cell proliferation inhibition in LNCap cells. The presented results were representative of experiments repeated at least three times. Data were represented as mean ± SD.

Fig. 3

The EdU assay revealed that the AO/854 and CDDP combination had a stronger inhibitory capacity on cell proliferation in PC3 cells. (A) The results of fluorescence microscopy. Scale bars: 50 μm. (B) The results of the statistical analysis of EdU-positive cells. The ratio of EdU-positive cells was positively correlated with cell proliferation ability. Data are represented as mean ± SD (*P < 0.05, ***P < 0.001).

Fig. 4

The combination of AO/854 and CDDP could effectively suppress the ability of metastasis and invasion in PC3 cells. (A-B) Inhibitory effects of AO/854 (2.5 µmol/L) and CDDP (2.5 µmol/L) on the migration activity of PC3 cells. Scale bars: 50 μm. (C-D) Inhibitory effects of AO/854 (2.5 µmol/L) and CDDP (2.5 µmol/L) on invasion activity of PC3 cells. Scale bars: 50 μm. (E-F) The results of WB for E-cadherin, N-cadherin, and vimentin. PC3 cells were treated with AO/854 (2.5 µmol/L) and CDDP (2.5 µmol/L) for 48 h. GAPDH was examined as a loading control. The presented results were representative of experiments repeated at least three times. Data were represented as mean ± SD (* P < 0.05, ** P < 0.01, ***P < 0.001). Original blots are presented in Figs. S4–S6.

Fig. 5

AO/854 enhances CDDP-induced DSBs in PC3 cells (A) Protein levels of γH2AX as detected by WB after being treated with AO/854 (2.5 µmol/L) and CDDP (2.5 µmol/L) for 48 h. GAPDH was examined as a loading control. Original blots are presented in Figs. S7-S9. (B) Detection of γH2AX foci (green) after CDDP (2.5 µmol/L) and AO/854 (2.5 µmol/L) treatment. Scale bars: 50 μm. (C-D) The results of comet assay of PC3 cells. Scale bars: 50 μm. The presented results were representative of experiments repeated at least three times. Data were represented as mean ± SD (* P < 0.05, ***P < 0.001).

Fig. 6

(A-B) AO/854 abrogated CDDP-induced S cell cycle arrest and enhanced G2/M cell cycle arrest in PC3 cells. The presented results were representative of experiments repeated at least three times. Data were represented as mean ± SD ( ** P < 0.01, ***P < 0.001).

Fig. 7

AO/854 hypersensitizes CDDP-induced apoptosis in PC3 cells. (A-B) Cell apoptosis analysis on PC3 cells for control, CDDP (2.5 µmol/L), AO/854 (2.5 µmol/L), or both treatments. Q1 shows the necrotic cells, Q2 shows the late apoptotic cells, Q3 shows intact cells and Q4 shows the early apoptotic cells. (C) Protein levels of BAX, Bcl–2 and cleavage caspase-3. Original blots are presented in Figs. S10-S15. (D) The ratio of BAX/Bcl-2. (E) The ratio of cleavage caspase-3/caspase-3. PC3 cells were treated with AO/854 (2.5 µmol/L), CDDP (2.5 µmol/L), or both for 48 h. GAPDH was used as a loading control. The presented results are representative of experiments repeated three times. Data are represented as mean ± SD (* P < 0.05, ** P < 0.01).

Fig. 8

AO/854 and CDDP suppressed tumor growth in the PC3 xenograft model (n = 6). (A) Representative images of PC3 xenograft tumors after CDDP (2 mg/kg, once every two days), AO/854 (2 mg/kg, once every two days), or both treatments. (B) Quantification of tumor weight. (**P < 0.01, ***P < 0.001). (C) The curve of changes in tumor volume. (D) Representative images of immunohistochemical staining analyses of γH2AX in tumor tissues. Note: Tumor data for the control, AO/854 groups have been published previously³⁰.

Fig. 9

Schematic illustration depicting the synergistic effects of BLM inhibitor AO/854 with cisplatin in PCa.