An oncolytic adenovirus regulated by a radiation-inducible promoter selectively mediates hSulf-1 gene expression and mutually reinforces antitumor activity of I131-metuximab in hepatocellular carcinoma

Abstract

Gene therapy and antibody approaches are crucial auxiliary strategies for hepatocellular carcinoma (HCC) treatment. Previously, we established a survivin promoter-regulated oncolytic adenovirus that has inhibitory effect on HCC growth. The human sulfatase-1 (hSulf-1) gene can suppress the growth factor signaling pathways, then inhibit the proliferation of cancer cells and enhance cellular sensitivity to radiotherapy and chemotherapy. I(131)-metuximab (I(131)-mab) is a monoclonal anti-HCC antibody that conjugated to I(131) and specifically recognizes the HAb18G/CD147 antigen on HCC cells. To integrate the oncolytic adenovirus-based gene therapy and the I(131)-mab-based radioimmunotherapy, this study combined the CArG element of early growth response-l (Egr-l) gene with the survivin promoter to construct a radiation-inducible enhanced promoter, which was used to recombine a radiation-inducible oncolytic adenovirus as hSulf-1 gene vector. When I(131)-mab was incorporated into the treatment regimen, not only could the antibody produce radioimmunotherapeutic effect, but the I(131) radiation was able to further boost adenoviral proliferation. We demonstrated that the CArG-enhanced survivin promoter markedly improved the proliferative activity of the oncolytic adenovirus in HCC cells, thereby augmenting hSulf-1 expression and inducing cancer cell apoptosis. This novel strategy that involved multiple, synergistic mechanisms, including oncolytic therapy, gene therapy and radioimmunotherapy, was demonstrated to exert an excellent anti-cancer outcome, which will be a promising approach in HCC treatment.

Figure 1

Relative activity of the radiation‐inducible enhanced survivin promoter examined by luciferase assay. Normal and HCC cells were seeded into 24‐well plates at a concentration of 105 cells/well and transfected with luciferase plasmids containing the wild‐type survivin promoter (Surp) or the radiation‐inducible enhanced survivin promoter (eSurp), without (A) or with (B) adding I131‐mab into medium. The harvested cells were examined by dual‐luciferase reporter assay to determine the luciferase expression. The results from pGL3‐Control which contains cytomegaoviyns (CMV) promoter were used to normalize the relative activities of promoters in pGL3‐Surp and pGL3‐eSurp; *p < 0.05; **p < 0.01.

Figure 2

Tumor‐selective replication of oncolytic adenoviruses carrying the EGFP gene in HCC cells. L02 and MHCC97H cells in 24‐well plates at 1 × 105 cells/well were infected with the indicated adenoviruses expressing EGFP at an MOI of 1 pfu/cell. Replicates of virus‐infected cells was added I131‐mab at 10 μCi/well. The EGFP‐positive cells were observed under a fluorescence microscope.

Figure 3

Tumor‐selective replication of oncolytic adenoviruses carrying the hSulf‐1 gene in HCC cells. Normal and HCC cells in 24‐well plates at 1 × 105 cells/well were infected with the indicated adenoviruses expressing hSulf‐1 at an MOI of 1 pfu/cell. Replicates of virus‐infected cells was added I131‐mab at 10 μCi/well. Expression of E1a and hSulf‐1 were examined by Western blotting. Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) was used as a loading control. Densitometry analysis was performed to show E1a and hSulf‐1 expression levels, normalized with GAPDH density; *p < 0.05; **p < 0.01.

Figure 4

Selectively killing effect of hSulf‐1‐expressing oncolytic adenovirus combined with I131‐mab on HCC cells. Normal and HCC cells in 96‐well plates at 1 × 104 cells/well were inoculated with the indicated adenoviruses at different MOIs or/and supplemented with I131‐mab at different concentrations. Then all replicates of cells were detected by MTT assay. The absorbance values were determined at wavelength 570 nm with a reference of 655 nm. Cell viability was showed with the survival curves. (A), Cells were individually treated with adenoviruses or I131‐mab. (B), Cells were jointly treated with adenoviruses and I131‐mab; *p < 0.05; **p < 0.01.

Figure 5

Inhibitory effect of the indicated oncolytic adenoviruses with or without I131‐mab on HCC xenografts in nude mice. MHCC97H cells (1 × 106) were subcutaneously implanted into BALB/C nude mice to establish xenograft tumors, followed by treatments of intratumorally injections of the indicated adenoviruses together with or without intraperitoneally injections of I131‐mab. The tumor sizes at the indicated time points were measured and calculated using the formula “maximum diameter × minimum diameter2 × 0.5”; *p < 0.05; **p < 0.01.

Figure 6

Adenovirus‐mediated expression of E1a and hSulf‐1 and adenovirus‐induced apoptosis in HCC xenograft tumor cells. The formalin‐fixed, paraffin‐embedded sections of HCC xenograft specimens were stained by H&E staining to show cancer tissue necrosis, stained by immunohistochemistry to show E1a and hSulf‐1 expression, and stained by TUNEL method to show cell apoptosis.