Antitumor effect of hypoxia response element chimeric tumor specific promoter regulated oncolytic adenovirus on renal cell carcinoma

Abstract

Objective To investigate the effect of chimeric promoter composed of hypoxia response element (HRE) and tumor specific cell proliferation-associated nuclear antigen (Ki-67) promoter on the replication ability of oncolytic adenovirus and its cytotoxic activity under hypoxic conditions. Methods The HRE sequence, as an enhancer, was inserted upstream of the Ki-67 promoter and, at the same time carried the reporter gene enhanced green fluorescent protein (EGFP) to construct oncolytic adenovirus Ad-HRE-Ki-67-EGFP, and the titer of adenoviruses was measured by the tissue culture infective dose (TCID50). Normal renal epithelial cells HK-2 and renal cancer cells OSRC-2 and ACHN were infected with two kinds of viruses under normoxic (21% O2) or hypoxic (1% O2) conditions respectively for 24 h, and the virus tumor-targeting was detected by fluorescence microscopy and flow cytometry. Western blotting was performed to detect the expression of hypoxia-related protein hypoxia-inducible factor-1 alpha (HIF-1α), vascular endothelial growth factor (VEGF) and the expression of E1A protein. Cell counting kit-8 (CCK-8) assay was used to detect the cytotoxity of virus on cells. Results The recombinant oncolytic virus Ad-HRE-Ki67-EGFP was successfully constructed. Under normoxic conditions, the efficiency of ACHN and OSRC-2 infection by chimeric HRE oncolytic adenovirus in renal cancer cells was (47.5±2.6)% and (35.2±3.4)%, while that in hypoxic conditions was (86.4±3.4)% and (73.5±2.3)%, indicating that the infection efficiency was higher under hypoxic conditions (ACHN: t=15.760; OSRC-2: t=16.190; P<0.01); Normal renal tubular epithelial cells HK-2 were infected with Ad-Ki-67-EGFP or Ad-HRE-Ki-67-EGFP under normoxic conditions. The infection efficiency was (22.4±3.1)% and (22.9±2.2)% respectively, compared with that of ACHN and OSRC-2 cells [ACHN: (68.4±3.3)% and (72.1±2.9)%; OSRC-2: (56.1±3.1)% and (61.4±2.3)%], the difference was statistically significant (Ad-Ki-67-EGFP: t=17.650, 13.390; Ad-HRE-Ki-67-EGFP: t=23.680, 21.430, P<0.01), indicating that the two adenoviruses have a targeting effect on renal cancer cells. Western blotting data showed that the expression of HIF-1α, VEGF and p53 protein were increased under hypoxic conditions and was time-dependent, indicating that hypoxia can regulate the transcription and translation of HIF-1α, VEGF and p53 proteins. Under hypoxic conditions, the expression level of E1A protein in OSRC-2 and ACHN infected with Ad-HRE-Ki67-EGFP was significantly higher than that after Ad-Ki67-EGFP infection. It indicated that HRE can enhance the replication ability of virus under hypoxic conditions. CCK-8 results showed that when the multiple infection (MOI) values were 20, 100, the survival rate of the Ad-HRE-Ki-67-EGFP-normoxia virus group in ACHN and OSRC-2 cells was (73.4±2.0)%, (56.4±1.5)% and (79.9±1.8)%, (61.3±2.7)%, the Ad-HRE-Ki-67-EGFP-hypoxia virus group was (63.1±2.0)%, (31.6±2.1)% and (68.1±2.6)%, (35.8±3.1)%, the Ad-HRE-Ki-67-EGFP-hypoxia virus group showed a stronger proliferation inhibitory effect [MOI (20): t=6.328, 6.441; MOI (100): t=16.410, 10.790, P<0.01]. Conclusion Under hypoxic conditions, chimeric HRE sequence recombinant oncolytic adenovirus has stronger replication ability and cell killing effect in renal carcinoma cells. Key words: Renal cell carcinoma; Oncolytic adenovirus; Hypoxia response element; Hypoxia