Effects Of Wortmannin Combined With Mitomycin C On HepG-2 Cells

Background and Objective: abnormal actiavition of phosphatidy-linositol-3-kinase(PI3K)/protein kinase B(Akt) signaling pathway is closely related to malignant tranformation of cells .this study was to investigate the effects of a PI3K inhibitor Wortmannin combined use of mitomycin C on the proliferation,apoptosis and expressions of related Cyclin D1,Bad genes ,and to explore the role of PI3K/Akt signaling pathway in the regulation of cell apoptosis in liver cancer.Methods:①Growth inhibition ratios of HepG-2 cells treated by different concentration wortmannin , mitomycin C alone or both of them at different times were detected by MTT assay.②The nucleus apoptosis of HepG-2 cells treated by wortmannin(200 nmol/L) mito-mycin C(30 nmol/L) alone or both of them(200+ 30 nmol/L)for 24 hours were examined by Hochest 33258.③The change of cell cycle and apoptosis in HepG-2 cells treated by wortmannin(200 nmol/L) , mitomycin C(30 nmol/L) alone or both of them(200+ 30 nmol/L)for 24 hours were detected by flowcytometry (FCM).④expression of PI3K/Akt signaling transduction pathway related genes and proteins of HepG-2 cells treated by wortmannin(200 nmol/L) , mitomycin C(30 nmol/L) alone or both of them(200+ 30 nmol/L)for 24 hours were tested by reverse transcription-polymerase chain reaction (RT- PCR) and Western Blot assay .Results:①Both Wortmannin and mitomycin C could inhibit growth of HepG-2 cells, show a concentration-dependent and time-dependent relationship. IC50 of Wortmannin and IC50 of mitomycin C are 8.57 and 1.85 more than Combined use of them.②HepG-2 cells treated by wortmannin, mitomycin C alone or both of them for 24 hours. FCM show : they were prohibited at G0/G1 phrase , G2/M phrase, G0/G1 phrase . respectively .apoptosis rates of HepG-2 cells increased from (3.23±1.32)% to (9.17±1.78)%,(9.00±2.01)% and(14.82±1.36)%. respectively (P<0.01).③HepG-2 cells treated by wortmannin, mitomycin C alone for 24 hours.typical changes in apoptotic morphology of HepG-2 cells were observed by fluorescence microscopy .after combined use of them,more notable changes in apoptotic morphology of HepG-2 cells could be observed.④HepG-2 cells treated by wortmannin, mitomycin C alone for 24 hours .activation of p-Akt was inhibited,mRNA and protein levels of Cyclin D1 decreased,but Bad increased(P<0.05). after combined use of them, mRNA and protein levels of p-Akt, Cyclin D1, Bad change more notablely(P<0.05).Conclusion:①the growth and proliferation of HepG-2 cells treated by wortmannin, mitomycin C respectively could be inhibited , more notable inhibition of growth and proliferation could be examined after combined use of them,②apoptosis of HepG-2 cells could be induced by wortmannin , mitomycin C respectively , apoptosis was induced more noticeablely after combined use of them.③One of the mechanism of inhibiting growth and prolifiration of HepG-2 cells was decreased mRNA and protein of Cyclin D1 by wortmannin and mitomycin C; one of the mechanism of inducing apoptosis of HepG-2 cells was increased mRNA and protein of Bad.④wortmannin and mitomycin C inhibit growth ,prolifiration ,and induce apoptosis of HepG-2 cells properly via blocking PI3K/Akt signaling pathway.