Therapeutic targeting of liver cancer with a recombinant DNA vaccine containing the hemagglutinin-neuraminidase gene of Newcastle disease virus via apoptotic-dependent pathways

Abstract

A total of ~38.6 million mortalities occur due to liver cancer annually, worldwide. Although a variety of therapeutic methods are available, the efficacy of treatment at present is extremely limited due to an increased risk of malignancy and inherently poor prognosis of liver cancer. Gene therapy is considered a promising option, and has shown notable potential for the comprehensive therapy of liver cancer, in keeping with advances that have been made in the development of cancer molecular biology. The present study aimed to investigate the synergistic effects of the abilities of the hemagglutinin neuraminidase protein of Newcastle disease virus (NDV), the pro-apoptotic factor apoptin from chicken anaemia virus, and the interferon-γ inducer interleukin-18 (IL-18) in antagonizing liver cancer. Therefore, a recombinant DNA plasmid expressing the three exogenous genes, VP3, IL-18 and hemagglutinin neuraminidase (HN), was constructed. Flow cytometry, acridine orange/ethidium bromide staining and analysis of caspase-3 activity were performed in H22 cell lines transfected with the recombinant DNA plasmid. In addition, 6-week-old C57BL/6 mice were used to establish a H22 hepatoma-bearing mouse model. Mice tumor tissue was analyzed by immunohistochemistry and scanning electron microscopy. The results of the present study revealed that the recombinant DNA vaccine containing the VP3, IL-18 and HN genes inhibited cell proliferation and induced autophagy via the mitochondrial pathway in vivo and in vitro.

Keywords: Newcastle disease virus; apoptosis; gene therapy; hemagglutinin neuraminidase gene; hepatocellular carcinoma; recombinant plasmid.

Figure 1.

The pIRVP3IL-18HN recombinant plasmid promoted apoptosis of H22 hepatoma cells. (A) Western blot analysis of the expression of pIRVP3IL-18HN in hepatoma cells. (B) Cytotoxic effects of pIRVP3IL-18HN on H22 cells were time- and concentration-dependent. (C) Observation of the apoptotic morphology of H22 hepatoma cells at 72 h after transfection with (Ca) pIRESneo and (Cb) pIRVP3IL-18HN plasmids under a light microscope at ×200 magnification and by (Cc and Cd) transmission electron microscopy at ×16,000 magnification. (D) Modifications observed in H22 hepatoma cells that were stained with acridine orange/ethidium bromide as observed under fluorescence microscopy at (Da and Db) 48 and (Dc and Dd) 72 h after transfection with pIRESneo and pIRVP3IL-18HN. (E) Flow cytometric analysis of H22 cell cycle at 72 h after transfection with pIRESneo and pIRVP3IL-18HN revealed that pIRVP3IL-18HN arrested the cell cycle at S phase. *P<0.05. IL-18, interleukin 18; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HN, hemagglutinin neuraminidase.

Figure 2.

pIRVP3IL-18HN recombinant plasmid promoted apoptosis of H22 cells via the mitochondrial pathway. (A) Assessment of mitochondrial membrane potential in H22 cells by flow cytometry at 72 h after transfection with the pIRESneo and pIRVP3IL-18HN plasmids revealed that pIRVP3IL-18HN downregulated the mitochondrial membrane potential in H22 cells. (B) Measurement of reactive oxygen species in H22 cells by flow cytometry at 72 h after transfection with pIRESneo and pIRVP3IL-18HN plasmids demonstrated that pIRVP3IL-18HN enhanced ROS production leading to increased tumor cell apoptosis. (C) Detection of cytochrome c levels in H22 cells by western blot analysis subsequent to transfection with the pIRVP3IL-18HN plasmid. (D) Examination of caspase-3 activity in H22 cells by enzyme-linked immunosorbent assay at 72 h after pIRESneo and pIRVP3IL-18HN plasmid transfection revealed that casapase-3 activity was significantly increased in pIRVP3IL-18HN-transfected cells compared with pIRESneo-transfected cells. *P=0.018. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 3.

Tumor suppressive effects of pIRVP3IL-18HN in vivo. (A) The growth curve of H22 tumors upon treatment with the recombinant plasmid carrying the HN, VP3 and IL-18 genes. (B) Comparison of tumor inhibition rates on treatment with various recombinant plasmids revealed that pIRVP3IL-18HN-transfection resulted in the slowest tumor growth rate. (C) Imaging of the femurs of H22 hepatoma-bearing mice. (D) Histopathological analysis of the tumor tissue biopsy following transfection with pIRESneo and pIRVP3IL-18HN using a light microscope at (Da and Db) ×100 and (Dc and Dd) ×400 magnification. (Ea) Normal structure of the tumorigenic nuclei in PBS group and (Eb) chromatin margination, (Ec) mitochondrial swelling and (Ed) apoptotic body formation subsequent to transfection with pIRVP3IL-18HN, as observed in tumor tissues under an electron microscope that was set at a ×16,000 magnification. *P<0.05; **P<0.01. PBS, phosphate-buffered saline.