Efficacy of perifosine alone and in combination with sorafenib in an HrasG12V plus shp53 transgenic mouse model of hepatocellular carcinoma generated by hydrodynamic injection

Abstract

Backgrounds & Aims: For patients with advanced hepatocellular carcinoma (HCC), sorafenib is the only systemic drug to show survival benefit. However, considering that the response rate of sorafenib is relatively low, novel therapeutic strategies are needed to improve survival in patients with HCC. Perifosine has shown antitumor activity by inhibition of Akt phosphorylation in many advanced solid tumors. This study investigated the effect of perifosine alone and in combination with sorafenib in an HrasG12V plus short-hairpin RNA downregulating p53 (shp53) transgenic mouse model of HCC.Methods: The mouse model of HCC was generated by hydrodynamic injection of transgenes HrasG12V/shp53, and the mice were treated with perifosine alone and in combination with sorafenib to evaluate effects of drugs on tumor growth and survival. Tumor cell proliferation and tumor angiogenesis were evaluated by immunohistochemical analysis of Ki-67 and CD31, respectively. Tumor cell apoptosis was detected by using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Levels of key enzymes in the PI3K/Akt, Ras/Raf/MAPK, and caspase pathways were evaluated by western blot analysis, and levels of vascular endothelial growth factor (VEGF) were determined by immunohistochemistry and western blot analysis.Results: Treatment with perifosine for 5 weeks alone and in combination with sorafenib, strongly inhibited tumor growth and increased survival. Perifosine inhibited HCC cell proliferation, induced apoptosis, and decreased tumor angiogenesis. Furthermore, its combination with sorafenib enhanced these effects. In addition, Akt phosphorylation was decreased by perifosine and further decreased by combination treatment. Although perifosine alone did not appear to activate the caspase pathway, combination treatment increased the cleavage of caspase-3, caspase-9, and

poly (ADP-ribose) polymerase (PARP). Perifosine did not affect VEGF levels, as assessed by immunohistochemistry and western blot analysis. Conclusion: Perifosine alone and in combination with sorafenib showed antitumor effects in an HrasG12V plus shp53 transgenic mouse model of HCC. The preclinical effect of current study represents a strong rationale for clinical trials using perifosine alone and in combination with sorafenib in the treatment of HCC patients.