639. Combination Therapy of Bufalin and rAd-p53 for the Treatment of Hepatocellular Carcinoma In Vivo

Abstract

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death worldwide, yet effective therapeutic options for advanced HCC are limited. Recombinant adenovirus-p53 (rAd-p53) was the first commercial gene therapy product in the world, which was shown to be effective in treating various cancer types. Bufalin, a bioactive monomeric compound in toad venom that belongs to Traditional Chinese Medicine (TCM), is also a commercial antitumor drug for HCC. Thus, we wished to investigate the possibility of combining the therapeutic effects of rAdv-p53 and of bufalin, both in vitro and in vivo. We previously observed that bufalin, at as low a concentration as 40 nM, significantly enhanced rAd vector-mediated transgene expression, not only a reporter gene, EGFP, but also a therapeutic gene, p53, in various HCC cell lines. The combination of rAd-p53 and bufalin strongly inhibited the proliferation of HCC cell lines in vitro. The combined therapeutic effect was more effective than either treatment alone, both in a time-dependent and a dose-dependent manner. Additional mechanistic studies suggested that bufalin increased the rAd vector-medicated p53 gene expression, which induced apoptosis through the Bcl-2-PARP pathway, and eventually resulted the inhibition of proliferation. In the current studies, we evaluated the therapeutic effects of the combination of rAd-p53 and bufalin in a murine HCC xenograft model in vivo. HCC tumor-bearing nude mice were divided into 5 groups (n=6): i) PBS treatment as a negative control; ii) rAd-p53 treatment; iii) bufalin treatment; iv) rAd-null + bufalin treatment; and v) rAd-p53 + bufalin treatment. Bufalin was administered intra-peritoneally, whilst rAd vectors were injected intra-tumorally. Post-treatment, we observed a statistically significant suppression of tumor growth resulting from administration of a combination of rAd-p53 and bufalin, in comparison with other groups. Survival was also prolonged significantly in the combination group. H&E staining of subcutaneous tumor sections showed that most necrotic regions were visible in the combination group. In addition, most apoptotic cells were localized in tumor tissues from the combination group, as determined by immunohistochemical staining. Furthermore, in tumor tissues from the rAd-p53 vector-treated group, cancer cells around the necrotic areas were positive for p53 protein expression, and co-treatment with bufalin resulted in a significant increase in p53 protein expression. These studies suggest that rAd-p53 + bufalin combination therapy may be a useful strategy for targeting malignant tumors.