Selective Inhibition Of T Cell Protein Tyrosine Phosphatase And The Cellular Effect By A Binuclear Copper (Ⅱ) Complex

Protein tyrosine phosphatases (PTPs) could widely affect cell process, which contains cell proliferation, differentiation, migration, apoptosis and immune process. Some diseases such as diabetes, cancer, obesity, rheumatoid, immune dysfunction are associated with the abnormal expression of the PTPs. The inhibition of the activity of PTPs can improve cell phosphorylation level with regulating cell signaling transduction. T cell protein tyrosine phosphatase (TCPTP) is an intracellular non-receptor protein tyrosine phosphatase. In recent years the research of TCPTP inhibitor got widespread attention because of its important role in the control of inflammation and the regulation of tumor. It was shown that copper complexes had efficient inhibition activity on PTP. Therefore the investigations of anti-cancer activity and mechanism of copper complexes targeting TCPTP provide a theoretical basis for the future development of new copper complexes as anti-cancer drugs.We investigated the selective inhibition of TCPTP and the effect on proliferation and apoptosis over four human tumor cells by a binuclear copper complex,[Cu2(μ-IDA)(phen)3(NO3)]NO3·4H2O (phen=1,10-phen-anthroline, H2IDA=iminodiacetic acid)(1),based on the results that complex1could effectively and selectively inhibit recombinant TCPTP in vitro. Furthermore we initially established xenograft model to investigate the antitumor activity and inhibition of TCPTP in vivo of complex1. Main results were obtained as follow:1. The viability of four human tumor cells containing of breast cancer cells (MCF7), cervical cancer cells (Hela), liver cancer the cells (HepG2) and glioma cells (U251) were detected by MTT staining. Complex1selectively and efficiently inhibited the proliferation of four tumor cells with time and concentration dependent models. The inhibition of Hela proliferation was the strongest and in contrast the inhibition of human glioma cells proliferation was weakest among the four cell lines. Copper ion had almost no effect on the inhibition of tumor cell proliferation. The ligands could effect on the inhibition of cell proliferation but significantly weaker than that of complex1.2. Flow cytometry and Annexin V-PI double staining were used to detect the ability of tumor cells apoptosis inducing by complex1.The results showed that complex1selectively induced apoptosis with concentration dependent model after the cells were treated with12h. At the concentration of10μM, the late apoptosis percentage of MCF7cells reached as high as89.5%while the late apoptosis percentage of U251is only49.5%. Under the same concentration and time, copper ion and ligands had no apoptosis-inducing effect on four tumor cells.3. Western blot detecting the phosphorylation levels of PTP substrates indicated that complex1efficiently and selectively inhibited the activity of TCPTP in four tumor cells. After treated7hours with10μM complex1, phosphorylation levels of TCPTP substrates on MCF7were significantly increased, while phosphorylation level of PTP1B substrate had no obvious tendency. The same phenomenon on U251cells appeared when treated with5μM complex1after7hours. The level of EGFR expression increased while the level of ubiquitin expression had no change substantially. Therefore, complex1could inhibit cell proliferation and induce apoptosis of four tumor cells through selectively inhibiting TCPTP activity rather than inhibiting proteasome. In addition complex1may impact other sites and then effect the expression of EGFR.4. We initially built MCF7xenograft tumor model in nude mice to study complex1’s anti-tumor activity and inhibition activity of TCPTP from tissue.The results initially indicate that complex1had anti-tumor effect in vivo.Complex1could inhibit TCPTP activity in tissue and affect the expression of EGFR.