Primary Mechanism Study Of Cell Growth Promoted By Microcystin LR

Microcystin LR, produced by bloom-forming cyanobacteria which resulted from eutrophication, is a kind of strong toxin, with average 50ug/kg LD50 to mouse. It can extensively contaminate animals and plants and be hardly degradated by normal methods .Our ecological environment and human health have been under threading seriously by MCLR, with the rapid increasing water pollution today. WHO has ruled the highest permited concentration of MC in our drinking water is lug/L. MCLR is a kind of potent heptapeptide hapatotoxins, owing a base-cyclic seven peptides structure. It inhibits protein phosphatases PP1 and PP2A exclusively. Thereby, it influences the regulation of cellular protein phosphorylation. Studies have found that inhibition of PP1 and PP2A has a dualistic effect on cells exposed to microcystin LR, with both apoptosis and increased cellular proliferation being reported. It can alter the expression level of bcl-2, p53, c-myc, bax, fos, jun and injury mitochondrion to induce hepatic cell apoptosis in higher concentrations. However it also can promote cell growth in lower dose. So MCLR possesses a potent tumor-promoting activity. Now studies on its tumor-promoting mechanism suggest that there are at least two pathway:gene damage and intervention on DNA repairing. But its further mechanism still unknowm. In a word, we need do further study to explain the tumor-promoting mechanism.mTOR complex 1(mTORCl) includes mTOR, a regulatory associated protein of mTOR(RAPTOR) and mammalian LST8/G-proteinβ-subunit like protein(mLST8/GβL). MTORC1 acts as a central regulator of cell growth and proliferation by acting S6 Kinase, which in turn regulates protein synthesis and allows progression from G1 to S phage of the cell cycle. mTOR pathway is stimulated by insulin ,growth factor,serum,amino acid and so on. Now experiments show that activation of mTOR pathway in HCC ranges from 15 to 41%, and mTOR inhibitors show antineoplasic activity in experimental models of HCC. So mTOR signaling acts a pivotal role in hepatocellular carcinoma. Theoretically PP1 and PP2A also can influence mTOR pathway by phosphorylation on Ser/Thr residue. And we found that MCLR and mTOR pathway all have relation to HCC.Here we focus on the impact of MCLR on HL7702,HepG2 in different concentration and time gradients and its carcinogenesis promoting effect through SD rat model. Then we test whether MCLR interacts with mTOR pathway. First, we used MCLR treated HL7702,HepG2 in different conditions and investigated the effect of MCLR promoting cell growth using MTT method to detection. Then tested the expression level of mTOR and S6K by western blot method. Second, we established a cancer-promoting rat model by MCLR to observe its long time chronic effect by HE stain and GSTPi immunohistochemistry. This model refers to hepatectomy to cut off approximately 70% liver and intraperitoneal injection of DEN and MCLR periodically. We extract proteins from liver and tested the expression level of mTOR and S6K by western blot method.Finally, our in vitro studies suggest that 0.39ug/kg MCLR can promote HL7702 and HepG2 growth up by nearly 20% cultured for 24h. In vivo rat model, MCLR shows its strong promoting-carcinogenesis effect with lug/kg as the best dose. Our studies initially indicate that MCLR can alter the phosphorylation level of mTOR and S6K in vivo and in vitro. So it paves a new way to explore the mechanism of HCC by investigating the interaction between MCLR and mTOR pathway.