Knockdown of BRCA2 enhances cisplatin and cisplatin-induced autophagy in ovarian cancer cells

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   Figure 1

   Correlation between the BRCA2 expression level in cancer tissues and the clinical outcome in ovarian cancer patients. Representative immunohistochemical images of BRCA2 expression in cancer tissues (A); the scale was 100 μm. Scores of the BRCA2 level in platinum-resistant and -sensitive cancer (B): resistant cancer had a higher BRCA2 level. A Kaplan–Meier analysis of progression-free survival (C) and platinum-free duration (D): cases with a low BRCA2 level in cancer tissues had longer progression-free survival and platinum-free duration. ^▲P < 0.05.
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   Figure 2

   Effects of silencing BRCA2 on the expression of RAD51 and on DNA repair. Basal level of the BRCA2 protein in 5 cell lines (A and B): a higher level was noted in ES-2 and CAOV-3, and therefore these two cell lines were employed for silencing trials. Silencing BRCA2 decreased the level of BRCA2 and RAD51 proteins in CAOV-3 (C, D and E) and ES-2 (F, G and H) cells: CDDP induced upregulation of RAD51, which was suppressed after silencing BRCA2. DSB detected with the neutral comet assay after CDDP exposure (4 μM) in CAOV-3 (I and J) and ES-2 (K and L) cells: silencing BRCA2 decreased DNA repair, increasing the percentage of comet-formed cells. RAD51 foci after CDDP exposure (8 μM) in CAOV-3 (M and N) and ES-2 (O and P) cells: CDDP induced the formation of RAD51 foci, which was suppressed in BRCA2-silenced cells. Images were captured under 200× field. Values were mean ± standard deviation for 3 independent experiments. ^▲P < 0.05.
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   Figure 3

   Silencing BRCA2 enhanced the action of CDDP in vitro. Survival percentages after CDDP exposure in CAOV-3 (A) and ES-2 (B) cells: a lower survival fraction occurred in BRCA2-silenced cells. Clone-forming assay in CAOV-3 (C and D) and ES-2 (E and F) cells: less colony number occurred after silencing BRCA2. Values were mean ± standard deviation for 3 independent experiments. ^▲P < 0.05.
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   Figure 4

   Silencing BRCA2 enhanced the anticancer effect of CDDP on transplanted CAOV-3 tumors in mice (n = 5). Gross morphology of tumors (A). Tumor volume and mass (B and C): CDDP treatment led to the smallest size in BRCA2-silenced tumors. BRCA2 and RAD51 proteins in tumor tissues (D and E): RAD51 was upregulated after CDDP treatment, which was suppressed after silencing BRCA2; the scale was 50 μm. Values were mean ± standard deviation. ^▲P < 0.05.
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   Figure 5

   Silencing BRCA2 enhanced CDDP-induced autophagy. Autophagy detected by the LC3-II accumulation in CAOV-3 (A) and ES-2 (B) cells: a higher level was noted in BRCA2-silenced cells. The autophagy flux in CAOV-3 cells observed using the tandem mRFP-GFP fluorescence assay (C and D): both yellow (RFP⁺GFP⁺) and red (RFP⁺GFP⁻) dots were increased after CDDP exposure, and the number of yellow dots was less than that of red dots, demonstrating the occurrence of autophagy flux; a remarkable increase in yellow dots occurred when using CQ, demonstrating the blockage of fusion of autophagosomes and autolysomes. Images were captured under 600× field. Values were mean ± standard deviation for 3 independent experiments. ^▲P < 0.05.
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   Figure 6

   CQ enhanced the action of CDDP in BRCA2-silenced cells in vitro. DNA break detected with the neutral comet assay in CAOV-3 (A) and ES-2 (B) cells: DNA repair was suppressed by silencing BRCA2, which was further exacerbated when adding CQ. RAD51 foci in CAOV-3 (C) and ES-2 (D) cells: the foci number was decreased in BRCA2-silenced cells, and adding CQ led to less foci. Percentages of dead cells in CAOV-3 (E) and ES-2 (F) cells: silencing BRCA2 increased the cell death fraction, with the highest value noted when adding CQ. Apoptosis in CAOV-3 (G and H) and ES-2 (G and I) cells: CDDP-induced apoptosis was enhanced after silencing BRCA2 , and CQ further increased the percentage of apoptotic cells. Values were mean ± standard deviation for 3 independent experiments. ^▲P < 0.05.
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   Figure 7

   CAOV-3 cells’ response to CDDP after inhibiting ATG7. BRCA2 and ATG7 proteins (A): levels were decreased after transferring siRNA. Autophagy detected by the LC3 assay (B): silencing BRCA2 enhanced CDDP-induced autophagy, increasing the LC3-II level; this was inhibited after knockdown of ATG7. Cell death percentages after CDDP exposure (4 μM) (C): knockdown of ATG7 enhanced CDDP in BRCA2-silenced cells. Values were mean ± standard deviation for 3 independent experiments. ^▲P < 0.05.
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   Figure 8

   CQ enhanced the anticancer effect of CDDP against BRCA2-silenced CAOV-3 tumors in mice (n = 5). Gross morphology of tumors (A). Tumor volume (B) and mass (C): CDDP treatment reduced the tumor, with the smallest tumor when combining CDDP and CQ. Pathological examinations of tumor xenografts (D); the scale was 50 μm. Autophagy detected by LC3-II accumulation in tumors (E): a higher level was detected in group CDDP + CQ. Values were mean ± standard deviation. ^▲P < 0.05.

• © 2018 Society for Endocrinology