Interferon alpha antagonizes the anti-hepatoma activity of the oncolytic virus M1 by stimulating anti-viral immunity

Abstract

Alpha virus M1 is an oncolytic virus that targets zinc-finger antiviral protein (ZAP)-defective cancer cells, and may be useful for treatment of hepatocellular carcinoma (HCC). Most of HCC patients have hepatitis and need long-term antiviral medication. Thus, it is necessary to clarify whether anti-virus medicines influence oncolytic effect of M1. We examined the effect of drugs used to treat hepatitis B/C on M1-mediated oncolysis in vitro and in vivo. Interferon (IFN)-α induces expression of antiviral IFN-stimulated genes (ISGs) in HCC cells with moderate sensitivity to M1 virus. This leads to reduced replication of M1, and blocking of M1-mediated apoptosis. The antagonistic effect of IFN-α is positively related with the expressive level of ISGs. We also examined a population of 147 HCC patients. A total of 107 patients (73%) had low ZAP expression in liver tissues relative to adjacent tissues. Among these 107 patients, 77% were positive for hepatitis B and 2% were positive for hepatitis C. A combination of M1 virus and IFN should be avoided in those patients with HBV or HCV infection, of who ZAP expression is low but ISGs expression is moderate. In conclusion, this study provides a basis for anti-viral regimens for HCC patients with hepatitis B or C who are given oncolytic virus M1.

Keywords: ZAP; hepatocellular carcinoma; interferon; interferon-stimulated genes; oncolytic virus M1 virus.

Figure 1. Common anti-virus chemicals for hepatitis combined with M1 virus don't antagonize the oncolytic effect in HCC cells

(A) Cells were infected with (MOI = 10) M1 and cell viabilities were determined 48 hours post infection. For each cell line, the percentages of cell viabilities are color-coded by quartile. (B) Schematic representation of cell experimental process. (C, D) The indicated liver cancer cell lines—Hep-3B, Huh-7 and PLC were treated with or without 5 types anti-hepatitis B virus drugs (C), anti-hepatitis C virus drugs (D) and Ribavirin (D) with the concentration of 100 μM, and M1 virus (MOI = 10) for 72 hours. Following 72 hours, cell viabilities were determined by MTT assay (mean ± SD). N.S. Not significant.

Figure 2. IFN-α inhibits the oncolytic effect of M1 in mid-sensitive HCC cells

(A, B) INF α-2a (A) and INF α-2b (B) cancels the oncolytic effect of M1 in mid-sensitive hepatoma cells in Huh-7 and sk-hep-1, but don't antagonize in Hep-3B and PLC. Cells were treated with INF α-2a and INF α-2b with the concentration of 10U/ml, 10²U/ml and 10³U/ml. Cell viabilities were determined by MTT assay (mean ± SD). (C, D) Huh-7 (C )and Hep-3B (D) Cells were pretreated or non-pretreated with 10³IU/ml INF α-2a for 1 hour, then, cells were infected with 10 moi M1 virus for 72 hours (Scale bars: 100 μm.). (E) Huh-7, sk-hep-1and Hep-3B cells treated with combined anti-hepatitis therapy for 1 hour, and 10moi M1 virus for 72h. Following 72 hours, cell viabilities were determined by MTT assay (mean ± SD). CTL, control; N.S. Not significant. *P < 0.05; **P < 0.01, ***P < 0.001.

Figure 3. IFN-α activates M1 virus-induced antiviral factor expression and depresses the replication of M1 virus thus leading to the inhibition of cell apoptosis in mid-sensitive HCC cells

(A) Huh-7 and Hep-3B cells were infected with M1 virus (10 PFU/cell) in the presence or absence of IFN a-2a (10³IU/ml), and IFNB, IFIH1, IRF3, IRF7, IFIT1 and ZAP mRNA levels were quantified by reverse transcription-polymerase chain reaction at 12 hours after M1 infection (mean ± SD). Fold-expression of genes was normalized to β-actin. (B) Viral titer determination in Huh-7 and Hep-3B lines (mean ± SD). (C) Huh-7 and Hep-3B cells were treated with M1 (MOI = 0.01 pfu per cell) for 24 h. The levels of viral genomic RNA and endogenous control β-actin were analyzed by qRTPCR. (mean ± SD) *P < 0.05, **P < 0.01, ***P< 0.001. (D) Western blots showing the expression of viral proteins E1 and NS3 24 hours post infection. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (E) INF α-2a would inhibit apoptosis of cancer cells which M1 causes in Huh-7 cell line, but not in Hep-3B cell line, Huh-7 and Hep-3B cells were treated with 1moi M1 (0.01moi M1 for Hep-3B), ETV, IFN-α or M1 plus anti-hepatitis virus drugs for 72 hours, then cells were stained by hochst33342 and photographed. (F) Expression of cleaved-caspase-3. Huh-7 Cells were treated with 1moi M1, IFN-α or M1/IFN-α combination for 12, 24, 36 and 48 hours, western blotting was performed to detect the candidate proteins. (G) Expression of Cleaved-Caspase-3. Hep-3B Cells were treated with 0.01moi M1 or M1/ETV, DCV, RBV, IFN-α combination for 48 hours, western blotting was performed to detect the candidate proteins. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 4. IFN-α attenuates anti-tumor activity of M1 virus invivo subcutaneous Huh-7tumors

(A) Nude mice (NU/NU) bearing subcutaneous Huh-7 tumors were treated with vehicle ETV (75 μg/kg/day, i.p.), DAC (15 mg/kg/day, i.p.), RBV (15 mg/kg/day, i.p.) IFN-α (35μg/kg/week, s.c.), M1 virus (8.7 × 10⁷ PFU/day, i.v.), M1 virus and anti-hepatitis virus drugs. i.p.intraperitoneal injection, i.v., intravenously injection (tail vein), s.c. subcutaneous injection, PFU, plaque forming unit.(B and C)Body weight (B) and Tumor growth (C) of tumor-bearing mice. Data are shown in means ± SDs. N.S. Not significant. *P < 0.05, compared with the combination group. (D) At experimental endpoints, mice were anesthetized and sacrificed. Tumors weresubsequently dissected and photographed. (E)Intratumoral expression of Ki-67 and Cleaved-Caspase-3. (F) Immunohistochemistry was performed to analyze the expression of Ki-67 and Cleaved-Caspase-3. Relative protein expressions were quantified with Image-Pro Plus 6.0 (IPP 6.0) N.S., not significant. *P < 0.05.

Figure 5. IFN-α attenuates anti-tumor activity of M1 virus in vivo subcutaneous Hep-3B tumors

(A) Nude mice (NU/NU) bearing subcutaneous Hep-3B tumors were treated with vehicle, ETV (75 μg/kg/day, i.p.), DAC (15 mg/kg/day, i.p.), RBV (15 mg/kg/day, i.p.) IFN-α (35μg/kg/week, s.c.), M1 virus (2.7 × 10⁶ PFU/day, i.v.), M1 virus and anti-hepatitis virus drugs. i.p. intraperitoneal injection, i.v., intravenously injection (tail vein), s.c. subcutaneous injection, PFU, plaque forming unit. (B and C) Body weight (B) and Tumor growth (C) of tumor-bearing mice. Data are shown in means ± SDs. N.S. Not significant. *P < 0.05, compared with the combination group. (D) At experimental endpoints, mice were anesthetized and sacrificed. Tumors were subsequently dissected and photographed. (E) Intratumoral expression of Ki-67 and Cleaved-Caspase-3. (F) Immunohistochemistry was performed to analyze the expression of Ki-67 and Cleaved-Caspase-3. Relative protein expressions were quantified with Image-Pro Plus 6.0 (IPP 6.0) N.S., not significant. *P < 0.05.

Figure 6. Clinical investigation on patients with negative ZAP and positive HBV/HCV

(A) Representative cores of ZAP immunostaining in TMA. Higher magnification shown inthe box. (Scale bars: 50 μm.) N, nonneoplastic; T, tumor. (B) Statistical analysis of the expression of ZAP of tumor and serum HBsAg and HCVRNA of patients.