Study On Src Homology Domain As A New Anti-cancer Target

Background/Aims:With the development of life science, many basic processes of malignant tumors had been revealed, including signal transduction, apoptosis, angiogenesis, and interaction of cell and extracellular matrix in these years. All of These progresses provided new methods to develop anti-tumor medicines. Many studies had proved that protein tyrosine kinase (PTK) participated in signal transduction of occurence, development, metastasis in many malignant tumors. PTKs interact with a diverse group of signaling molecules that share common structural elements known as Src homology domain--SH2 and SH3. As a result, SH2 and SH3 take part in signal transduction by mediating interaction of PTKs and phosphorylated tyrosine (pTyr) residue. Because of the key roles of SH2 and SH3 in PTK signaling, Src homology domains have been extensively studied, with the aim of targeting the proliferation signal. Based on this background, we used PTD-BCR/ABL SH3 fusion protein to nude mice bearing HepG-2 tumors to investigate inhibitory activity to tumor growth and life span. By the similar way of constructing PTD-BCR/ABL SH3 fusion protein, we subcloned Grb2-SH2 cDNA into expression vector, expressed and purified a new fusion protein named after His-PTD-Grb2-SH2 which also contained protein transduction domain (PTD) -the sequence is TAT48-60 (GRKKRRQRRRPPQ). His-PTD-Grb2-SH2 had been used in vitro to investigate its biological behavior. We hope these works could provide new experimental prooves to Study on Src homology domain as a new anti-cancer target.Methods:HepG-2 (1×106) cells were injected subcutaneously into the right hind flank of mice to built Tumor growth model. PTD-BCR/ABL SH3 fusion protein had been injected by tumors'side to investigate inhibitory activity to tumor growth and life span. Based on the way of constructing PTD-BCR/ABL SH3 fusion protein, we subcloned Grb2-SH2 cDNA into expression vector pET-16b-PTD. We expressed and purified a new fusion protein named after His-PTD-Grb2-SH2 which contained protein transduction domain(PTD) and Grb2-SH2 domain. After identificated by Western-blot, this fusion protein has been studied in vitro.we used immunofluorescence to see whether the new protein has the ablility to go through the living cells'membrane. Later we continued with morphology research by light microscope observation. MTT assay has also been used to draw a growth curve and to analyze inhibition ratio.Results:PTD-BCR/ABL SH3 protein showed highly cytotoxity to HepG-2 tumors in vivo. The growth speed of tumors in treatment group was distinctly slower than that of control group, and survival time of mice in treatment group mice was longer than that of the control group. Tumors'growth had been inhibited in the treatment group. In the study on SH2 domain, we successfully obtained the recombined expression vector contain Grb2-SH2 sequence which had been identificated by Restriction mapping and Sequencing. After expressed, purified and identificated by Western-blot, we finally obtained a novel fusion protein named after His-PTD-Grb2-SH2. Immunofluorescence showed the protein can successfully go through the membrances of breast cancer cell line SKBR-3 and gastric cancer cell line SGC-7901. Growth curves and inhibition ratios drawed by MTT assay showed that this fusion protein had significant cytotoxity to these two cell lines.Conclusion:During the process of this Study on Src homology domain as a new anti-cancer target, we adopt a new method by transducting the Src homology domain -SH2 and SH3 into cells and use them to compete against other proteins for the binding sites of substrates and inhibit their functions. PTD-BCR/ABL SH3 fusion protein has showed therapeutic effects to HepG-2 tumor in vivo. The novel fusion protein His-PTD-Grb2-SH2 showed the ability to pass through cell membrances and killing effect to cancer cells in vitro.All these works provide new experimental prooves to further investigation.