Effect Of New Mtor Inhibitor Y31on Cell Proliferation In Adpkd Cysts-lining Epithelial Cell And Its Mechanism

Autosomal dominant polycystic kidney disease (ADPKD) is the most commoninherited kidney disease, the incidence rate is approximately1/1000~1/400,ChineseADPKD patients are about1.5million. About50%of the ADPKD patients eventuallydeveloped into end-stage renal diseaese in the age of60. ADPKD is characterized by theprogressive formation of multiple renal cysts affecting all segments of the renal tubules,eventually destroying the kidney normal structure and function, development to end-stagerenal disease. The pathogenesis of ADPKD is not yet fully been fully elucidated,, and noeffective treatment method is found. Now ADPKD research is mainly concentrated in thecyst formation process of cell proliferation and the molecular mechanism of apoptosis,hoping to find the new therapeutic targets and effective drugs.mT0R (the mammalian target of rapamycin) signaling pathway involved in ADPKDoccurrence and the development process has been recognized. and there are a number ofstudies also showed that the in vitro cells and many kinds of ADPKD model experimentalanimals, mTOR inhibitors rapamycin can inhibit renal tubular epithelial cell proliferationand renal cyst formation. The early clinical trial results also suggest that different stages ofdevelopment of rapamycin in ADPKD showed a treatment effect, and have not yet found asignificant adverse reactions. However, the recent foreign magazine reported the twoclinical trials results, that traditional mTOR inhibitors did not slow the decline inprogressive renal impairment.For this reason, we cooperate with the Chinese academy of sciences to develop a newdrug Y31, a new mTOR inhibitor,its pharmacokinetic and water-soluble are better thanrapamycin. My study was designed to observe the WT9-12cell line changes in cellproliferation, cell cycle, apoptosis in the effect ofY31, and its mechanism. To discusse itsapplication in the clinical treatment ADPKD prospect.Experiments with human ADPKD polycystic kidney disease cyst-lining epithelial cell lines(WT9-12) by Harvard University Professor Zhou Jing gifts. The WT9-12cells were treatedwith different concentrations of Y31culture24,48,72and96h, MTT assay to detect theinhibition ratio, we found a dose and time dependent inhibition of cell growth,and persistedfor3days with an IC50of25μM. Detected by flow cytometry cell cycle and apoptosis, theresults show that WT9-12cells in the concentration of25μmol/L Y31for72h wassignificantly inhibited cell cycle progression, cell cycle was blocked at G0/G1phase, the number of cells in G0/G1phase cells increased by about10%, compared with the controlgroup(P <0.05). When the cell cultured in25μM for72h,Y31also induced cell apoptosis,early apoptosis rate increased by nearly28.5%, compared with the control group (P <0.05).In addition, the Western blot suggest that WT9-12cells in the concentration of25μmol/Lof Y31after72h, the expression of cell cycle regulation protein p21significantlyincreased, but cyclinD1expression decreased.The expression of PARP protein, Reflectcell apoptosis, was also raised. In summary, the new mTOR inhibitor Y31inhibite WT9-12cell proliferation is through block the cell cycle in G0/G1phase and promote cellapoptosis, and tally with flow cytometry technical analysis results.mTOR signaling pathway plays an important role in the ncidence and development ofpolycystic kidney disease, inhibit mTOR signal pathway of polycystic kidney disease canobviously inhibit cysts progress, p70S6K and4E-Bp1is two main downstream substrateof mTOR. WT9-12cells was culture in different concentration of Y31for72h, totalprotein was extracted. Western blot analysis showed that: Y31reduced p-mTOR,p-p70S6K protein expression, had no significant effect on other downstream substrates4E-BP1phosphorylation levels.The form of Cell death are cell necrosis, apoptosis and cell autophagy. mTOR signalingpathway in addition to the regulation of apoptosis, but it is also an important regulator ofautophagy protein. There are already several studies have shown that autophagy increasethe tumor suppressor, effect of excess macrophages caused by autophagic death can killtumor cells. The experiment also examed the important protein of cell autophagy LC3andBeclin1, the results showed that Y31can increase LC3and Beclin1expression, indicatingthat Y31can promote the autophagic cell death, autophagy is another mechanism of Y31inhibit WT9-12cell proliferation.In summary, this study shows that main role of the new mTOR inhibitors Y31suppressthe level of phosphorylation of mTOR and its downstream molecules p70S6K, to block thecell cycle, increase apoptosis, induce cell autophagy, and ultimately suppress kidneycyst-lining epithelial cell proliferation.