ML216-Induced BLM Helicase Inhibition Sensitizes PCa Cells to the DNA-Crosslinking Agent Cisplatin

Abstract

Using standard DNA-damaging medicines with DNA repair inhibitors is a promising anticancer tool to achieve better therapeutic responses and reduce therapy-related side effects. Cell viability assay, neutral comet assay, western blotting (WB), and cell cycle and apoptosis analysis were used to determine the synergistic effect and mechanism of ML216, a Bloom syndrome protein (BLM) helicase inhibitor, and cisplatin (CDDP), a DNA-crosslinking agent, in PCa cells. Based on the online database research, our findings revealed that BLM was substantially expressed in PCa, which is associated with a bad prognosis for PCa patients. The combination of ML216 and CDDP improved the antiproliferative properties of three PCa cell lines. As indicated by the increased production of γH2AX and caspase-3 cleavage, ML216 significantly reduced the DNA damage-induced high expression of BLM, making PC3 more susceptible to apoptosis and DNA damage caused by CDDP. Furthermore, the combination of ML216 and CDDP increased p-Chk1 and p-Chk2 expression. The DNA damage may have triggered the ATR-Chk1 and ATM-Chk2 pathways simultaneously. Our results demonstrated that ML216 and CDDP combination therapy exhibited synergistic effects, and combination chemotherapy could be a novel anticancer tactic.

Keywords: BLM inhibitor; CDDP; ML216; combined therapy.

Figure 1

Expression of BLM in PCa. (A) In the TCGA database, the expression of BLM transcripts (n = 497) was significantly higher than that of normal tissue (n = 52) in PCa (*** p < 0.001). (B) The western blotting was used to analyze BLM protein expression levels in PCa cell lines. (C) The IF was used to analyze BLM protein levels in PCa cell lines. Scale bars: 50 μm. (D) The comparison of DFS between the high BLM expression group (n = 244) and low BLM expression group (n = 242) (p = 0.00065); n: the number of patients. (E) Comparison of overall survival in the indicated groups (p > 0.05).

Figure 2

ML216 sensitizes CDDP-induced cytotoxicity in PCa cells. (A–C) Inhibition ratios in prostate cells following 48 h treatment: for PC3 cells, ML216 IC₅₀−55.56 μmol/L and CDDP IC₅₀−7.43 μmol/L (A); for LNcap cells, ML216 IC₅₀−58.94 μmol/L and CDDP IC₅₀−8.43 μmol/L (B); for 22RV1 cells, ML216 IC₅₀−51.18 μmol/L and CDDP IC₅₀−6.83 μmol/L (C); for WPMY-1 cells, ML216 IC₅₀−92.52 μmol/L and CDDP IC₅₀−6.06 μmol/L (D). (E–H) MTT assay was conducted in PC3, LNCap, 22RV1 and WPMY-1 cells treated with CDDP and ML216 for 48 h. Inhibition ratio curves were shown, respectively.

Figure 3

ML216 enhances CDDP-induced DSBs by inhibiting BLM in PC3 cells (A) PC3 cells were co-treated with CDDP (1 μM) and ML216 (10 μM) for 48 h, and WB examined BLM and γH2AX. GAPDH was used as a loading control. (B) Detection of γH2AX foci (green) 48 h after ML216 (10 μM) and CDDP (1 μM) treatment. Scale bars: 50 μm. (C) Comet assay of PC3 cells treated with ML216 and CDDP for 48 h. Scale bars: 50 μm. (D) Quantifying %DNA in tails in ML216 and CDDP-treated groups compared with the DMSO-treated group (control group). Quantification of 5 fields with 100 cells in total. (*** p < 0.001).

Figure 4

ML216 hypersensitizes CDDP-induced apoptosis in PC3 cells. (A) Cell apoptosis analysis on PC3 cells for control, ML216 (10 μM), CDDP (1 μM), or both treatments. (B) Quantitative analysis of apoptosis. (** p < 0.01, *** p < 0.001) (C) Protein levels of cleavage caspase 3. PC3 cells were treated with ML216, CDDP, or both for 48 h. GAPDH was used as a loading control.

Figure 5

The combination of ML216 and CDDP activates ATM/Chk2 and ATR/Chk1 signaling. (A) Flow cytometry was used to detect the cell cycle distribution of PC3 cells after 48 h of treatment with ML216 (10 μM), CDDP (1 μM), or both. (B) The percentage of PC3 cells in G0/G1, S, and G2/M phases. The data were presented as mean ± SD. (** p < 0.01, *** p < 0.001) (C) The WB was used to analyze the protein levels of p-Chk1 and p-Chk2 in PC3 cells; GAPDH was used as a loading control.