Dual‑regulated oncolytic adenovirus carrying ERCC1‑siRNA gene possesses potent antitumor effect on ovarian cancer cells

Abstract

Ovarian cancer is a multifactorial and deadly disease. Despite significant advancements in ovarian cancer therapy, its incidence is on the rise and the molecular mechanisms underlying ovarian cancer invasiveness, metastasis and drug resistance remain largely elusive, resulting in poor prognosis. Oncolytic viruses armed with therapeutic transgenes of interest offer an attractive alternative to chemical drugs, which often face innate and acquired drug resistance. The present study constructed a novel oncolytic adenovirus carrying ERCC1 short interfering (si)RNA, regulated by hTERT and HIF promoters, termed Ad‑siERCC1. The findings demonstrated that this oncolytic adenovirus effectively inhibits the proliferation, migration and invasion of ovarian cancer cells. Furthermore, the downregulation of ERCC1 expression by siRNA ameliorates drug resistance to cisplatin (DDP) chemotherapy. It was found that Ad‑siERCC1 blocks the cell cycle in the G₁ phase and enhances apoptosis through the PI3K/AKT‑caspase‑3 signaling pathways in SKOV3 cells. The results of the present study highlighted the critical effect of oncolytic virus Ad‑siERCC1 in inhibiting the survival of ovarian cancer cells and increasing chemotherapy sensitivity to DDP. These findings underscore the potent antitumor effect of Ad‑siERCC1 on ovarian cancers in vivo.

Keywords: ERCC1; adenovirus; hTERT/HIF; ovarian cancer; proliferation.

Figure 1.

Schematic diagram of the recombinant adenovirus vectors and the inhibitory effect of the recombinant adenovirus on SKOV3 cells. (A-B) Vector plasmid carrying ERCC1-siRNA gene. (C) The recombinant adenovirus can inflect and kill tumor cells. Scale bars, left panels, 600 µm; right panels, 400 µm. si, short interfering; NC, negative control.

Figure 2.

Infection and inhibitory effect of the recombinant adenovirus on SKOV3 cells. (A) The recombinant adenovirus can infect cells effectively. The most efficient MOI=0. Scale bars, 600 µm. The recombinant adenovirus reduced the (B) mRNA and (C) protein levels of ERCC1 on SKOV3 cells (n=4 in each group). (D) The recombinant adenovirus decreased the cell viability (n=3 in each group). Data are shown as the mean ± SEM. ***P<0.001 vs. corresponding controls. MOI, multiplicity of infection; NC, negative control.

Figure 3.

The recombinant adenovirus expressing ERCC1 siRNA significantly enhance chemosensitivity to DDP on SKOV3 cells. (A) DDP decreased the cell viability (n=4 in each group). (B) Recombinant adenovirus was more effective in cell mortality than DDP treatments (n=4 in each group). (C) Combined with DDP, the cell viability was lower in cancer cells transduced with Ad-siERCC1 compared with those transduced with Ad-NC (n=4 in each group). (D-E) Cell block in G₁ phase was more obvious in Ad-siERCC1 combined with DDP (n=3 in each group). Data are shown as the mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 vs. corresponding controls; ^##P<0.01 vs. DDP groups; ^&P<0.05 vs. DDP combined with Ad-NC groups. si, short interfering; DDP, cisplatin; NC, negative control.

Figure 4.

Effects of the recombinant adenovirus on SKOV3 cells migration and invasion. (A) Representative images of cells scratch assay. Scale bars, 400 µm. (B) Cells treated with Ad-siERCC1 combined with DDP showed the lowest migration (n=5 in each group). (C) Representative images of cells, Transwell assay. Scale bars, 400 µm. (D) Suppression of SKOV3 cells invasion after treatment with DDP and Ad-siERCC1 (n=5 in each group). All measurements were performed in triplicate. Data are shown as the mean ± SEM. *P<0.05, **P<0.01 vs. corresponding controls; ^#P<0.05 vs. DDP groups, ^&P<0.05 vs. DDP combined with Ad-NC groups. si, short interfering; DDP, cisplatin; NC, negative control.

Figure 5.

Recombinant adenovirus enhancing apoptosis through PI3K/AKT-caspase-3 ameliorates resistance to DDP on SKOV3 cells. (A) The representative images of the Annexin V-APC staining assay. (B) Recombinant adenovirus and/or DDP promoted the apoptosis of ovarian cancer cells and the apoptosis rate in combination group was significantly higher than that in DDP group (n=4 in each group). (C) PI3K was significantly suppressed by recombinant adenovirus and/or DDP and Ad-siERCC1 could greatly enhanced the effect of DDP (n=4 in each group). (D) AKT was significantly suppressed by recombinant adenovirus and (or) DDP and Ad-siERCC1 could greatly enhanced the effect of DDP (n=4 in each group). (E) Procaspase-3 was significantly reduced after recombinant adenovirus and/or DDP incubation, whereas cleaved caspase-3 was considerably increased (n=4 in each group). Data are shown as the mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 vs. corresponding controls; ^#P<0.05, ^##P<0.01, ^###P<0.001 vs. DDP groups, ^&P<0.05, ^&&&P<0.001 vs. DDP combined with Ad-NC groups. DDP, cisplatin; si, short interfering; NC, negative control; p-. phosphorylated.