ISP-1 And Its Derivative FTY720 Induce Apoptosis And Gene Expression Profiles Of Cell Cycle Regulatory Proteins In Glomerular Mesangial Cell

Objective ISP-1( (2S,3R,4R)-(E)-2-amino-3,4-dihydro xyl-2-hydro- xymethyl- 14-oxoeicos-6-enoic acid) is myriocin,newly isolated from Cordyceps sinensis, a Chinese herb traditionally used in the treatment of renal diseases. FTY720(2-amino-2-[2-(4-octyophenyl) ethyl ]-1 ,3-propanediol hydrochloride)is from ISP-1 by chemical modification. Both of them are immunosuppressive substance. The present study was aimed to observe the effects of ISP-1 and FTY720 on glomerular mesangial cell(GMC) apoptosis and on gene expression profiles of cell cycle regulatory proteins.Methods Rat GMC were cultured with 10μmol/L, 50μmol/L, 100μmol/L ISP-1 and 10μmol/L, 20μmol/L FTY720 for 6h, 12h, 24h, 48h. Apoptosis was evaluated through MTT array, Propidium iodide staining and flow cytometry, Hoechst33258/PI fluorescence stainig and DNA fragmentation analysis. Gene expression profiles of cell cycle regulatory proteins were detected by SuperArray Real-Time PCR microarray analysis. The expression of Bax and Bcl-2 proteins were investigated by Westernblot. Results ISP-1 could significantly induce GMC apoptosis in a time-dependent and dose-dependent manner, which was most prominent with 100μmol/L ISP-1 after 48h; and FTY720 could also significantly induce GMC apoptosis in a time-dependent and dose-dependent manner. MTT array showed GMC proliferation was inhibited in a time and dose-dependent manner by ISP-1 and FTY720。Apoptotic bodies of GMC were observed by Hoechst /PI fluorescence staining, hypolipoid DNA peak, and a typical ladder patterns were identified in DNA electrophoresis. SuperArray Real-Time PCR microarray analysis revealed that 100μmol/L ISP-1 could up-regulate the expression of genes involved in DNA damage, apoptosis and cell cycle regulation, such as Rad51, Atm, Brca1, caspase3, cyclinA2, cyclinC, chek1, cyclinB1, cyclinB2, cyclinD2, cyclinF, Cdc25a, P27 and etc. 20μmol/L FTY720 could up-regulate the expression of genes involved in DNA damage, apoptosis and cell cycle regulation, such as GMC Dnajc2,RGD1562436_predicted,LOC688900,Atm,Gadd45a,cyclinA1,cyclinD2,cyclinE1,Grp132-predice,LOC307231,LOC367976,LOC686524,Mki67_predicted,Ppp2r3a,Trp63. Westernblot showed that 100μmol/L ISP-1 significantly up-regulate Bax protein expression and down-regulate Bcl-2 protein expression respectively.Conclusion Both ISP-1 and FTY720 could induce GMC apoptosis in a time and dose-dependent manner, propabably through influence gene expression of cell cycle regulatory proteins and apoptosis related proteins.