Targeted Gene Therapy for Mutated H-ras and C-raf.1 Genes Exhibited Variable Efficacy in Inhibiting Hepatocellular Carcinoma in Rats: A Mechanistic Approach.

OBJECTIVE: Mutated H-ras or c-raf.1 gene expressions are often associated with HCC development and progression. Using intravenous administration of respective backbone modified antisense oligomer (ASO) in chemically induced HCC rats, we tried to understand the efficacy and mechanistic intervention of the treatments.

METHODS: ASO-mediated in vitro cell cytotoxicity, cellular apoptosis, and alteration of mitochondrial membrane potential (MMP), and their hepatic accumulation in HCC rats, H-ras and c-raf.1 gene and protein expression, immunohistochemical evaluation of various proteins, caspase 3/9 activities, hepatic cytosolic alanine aminotransferase and microsomal aspartate aminotransferase assays were carried out.

RESULTS: Nanosize and well-dissociated ASOs treated against mutated H-ras and c-raf.1 showed variable IC50 values in human HCC cells and had no significant toxicity at the experimental dose range in normal human liver cells. Labeled ASOs were distributed well in normal rat liver. ASO treatments showed increased apoptosis and loss of MMP in HepG2 and Huh7 cells. ASOs remarkably altered hepatic focal lesion counts and inhibited tumor incidences. ASO treatments inhibited respective gene expression, increased p53 protein expression, and reduced Hep Par I and HSP70 protein expressions in HCC rat liver. ASO treatments variably induced caspase-3 and -9 protein levels in HCC rats. Hepatic marker enzymes did not alter upon ASO administration in normal rats and improved towards normal levels in carcinogen-treated rats.

CONCLUSIONS: ASO treatment against mutated H-ras showed better therapeutic efficacy than ASO treatment against c-raf.1. Thus, blocking mutated H-ras than c-raf.1 might appreciably influence HCC inhibition in rats.