Investigation For The Killing Activity Of PTD-BCR/ABL SH3 Fusion Protein To Hepatocellular Carcinoma

There are two kinds of primary malignant liver tumours, hepatocellular carcinoma (HCC) and cholangiocarcinoma. Hepatocellular carcinoma is 10 times more frequent than cholangiocarcinoma. The overall incidence is estimated to be 1 million cases per year with a wide geographic variability. China is a countary with high risk of HCC, 45 percent of the new cases of the world are occurred in China approximately. There also appears to be a clear association with cirrhosis with an annual risk of developing HCC in such cases of 1% to 6%. Until now, the main treatments include surgical resection, liver transplantation, cryoablation, radiofrequency (RF), microwave therapy, etc. But the effect of single therapy mode is usually not good enough, onetreatment always combine with others.The thought of inducing tumor cell apoptosis will lead to the emergence of novel antitumor therapy with high efficacy and low toxicity and inflammatory reaction.SH3 domain belongs to Src homology domain which exists in protein-tyrosine kinase and its downstream proteins. Protein-tyrosine kinases include both SH2 and SH3 domains participate in signal net, which alsocontains signal transduction of small G protein and phosphocreatine second messenger. Because SH3 domain is involved with growth-adapting signal transduction, it attracts many attention of being applied in targeted anti-proliferation drugs.PTD derives from tat of HIV-1 can translocate across cell membrane and get into cytoplasma directly effiently and promptly. When fused with some effectors it can lead these factors into cytoplasma, which can improve the cytotoxity of them.In this paper, hepatocellular carcinoma cells were co-cultured with PTD-BCR/ABL SH3 fusion protein. We wanted to know whether this fusion protein could induce stereo-tumor apoptosis and discuss the mechanism of apoptosis.After hepatocellular carcinoma cells were co-cultured with PTD-BCR/ABL SH3 fusion protein, most fusion protein was localized in nuclei, and little in cytoplasma. After co-ccultured 4h some cells shrinked, and after 24h 80% died by immunocytochemistry staining and light microscope observation. The results of cell counting and MTT assay in expriment group showed that the cell growth was inhibited, and when the protein's concentration >0.14mg/ml, the higher protein concentration was given, the more inhibition cells suffered. It was dosage and time dependent. But when protein concentration declined to 0.08mg/ml, cell groethed again after 36h, which indicated this dosage couldn't inhibit cell growth effectively. Electron microscopy analyses displayed shrinkage nuclei, chroatin condensation, plasma membrane bledding and chromatin breakage fragments and apoptotic bodies.We next tested antitumor activity of PTD-BCR/ABL SH3 fusion protein in nude mouse models. It was observed that tumor local administration of PTD-BCR/ABL SH3 fusion protein suppressed HHCC tumor. PTD-BCR/ABL SH3 fusion protein was administrated into the nude micewith HHCC xenograft. The growth rate of tumor of treament group was distinctly slower than that of control group, and survival time of treament group mice was longer than that of control group. Tumor cells of control group growth actively and showed pathologic karyokinestic image, and treament group showed many acidophilia bodys. While in other tissues, such as heart, liver, spleen, lung, kidney displayed normal morphology.In summary, PTD-BCR/ABL SH3 fusion protein can enter into hepatocellular carcinoma cells and induce these cells apoptosis, showes high cytotoxity, and in vivo it can suppress tumor growth and prolong nude mice' life span. Therefore the present study probably provides laboratory data for the application of PTD-BCR/ABL SH3 fusion protein into interventional therapy of hepatocellular carcinoma.