Mechanism Of Tanshinone IIA Inducing Autophagy And Apoptosis In Human Hepatocarcinoma HepG2Cells

Objective: To observe the effects of Tanshinone IIA (Tan IIA) onthe proliferation, apoptosis, cell cycle, autophagy andPI3K/AKT/mTOR signal transduction pathway of human hepatoma cellline HepG2. Methods: The effect of different concentration Tan IIA onthe proliferation of HepG2cells at different time was detected by MTTassay, and tumor inhibition rate and IC50was calculated. Heochest33258staining method and flow cytometry (FCM) analysis was used todetect apoptosis and cell cycle of HepG2cells. The autophagyphenomenon of HepG2cells was observed by fluorescencemicroscopy after immunofluorescence staining; the expression ofautophagy related molecules LC3B and Beclin1was analyzed by FCM.Real-time fluorescence quantitative PCR assay was used to detect themRNA expression of PI3K/AKT/mTOR signal transduction pathwayrelated molecules (TSC1/TSC2, PI3K, AKT, mTOR) in HepG2cells treated with different dose Tan IIA for48h. Results: The result ofMTT assay shows that:Tan IIA has significant inhibitory effect on theproliferation of human hepatoma HepG2cells, and the inhibitory effectwas largely dependent of time and concentration, compared with thecontrol group, among groups was statistically significant(P <0.05).Theresult observed under inverted fluorescent microscope show: Tan IIA hasinhibitory effect on the growth of human hepatocellular carcinoma cellsHepG2; Compared with the control group, the experimentalgroup increased cell apoptosis, chromatin condensation, formation ofapoptotic bodies, and the phenomenon increased with the increase ofdrug concentration，which the most obvious is100μmol/L concentrationgroup cells. FCM method shows that: Compared with control group,HepG2cells induced by Tan IIA with different dose10μmol/L、30μmol/L,100μmol/L, with the increase of drug concentration, are more inG0/G1phrase and less in S phrase after48h; The apoptosis rate of controlgroup,10μmol/L、30μmol/L、100μmol/L group is respectively(1.10±0.62)%、(21.50±1.87)%、(25.40±1.30)%、(44.20±4.25)%, andapoptosis cells was increased with the increase of drug concentration, thecontrol group and between groups was statisticallysignificant(P<0.05).The result observed under inverted fluorescentmicroscope show that: compared to negative control group， thefluorescent intensity(IOD) of LC3B-AF555decreases with the increasing of drug concentration in HepG2cells treated by Tan IIA after48h, andthe phenomenon of100μmol/L group is the most obvious group. FCMmethod shows that: The expressive trend of LC3B and Beclin1isaccordant, compared with control group, with the increase of drugconcentration, the expression level of LC3B and Beclin1also increases,and has statistical significance (P <0.05).From the picture of the trendline about apoptosis rate and expression rate of LC3B and Beclin1, wecan see autophagy and apoptosis is inversely related.Whenautophagic activity is in high situation, it can significantly inhibit the cellapoptosis. Tan IIA can inhibit autophagy and then promote tumor cellapoptosis. Real-time fluorescent quantitative PCR results showed thatwith increasing the concentration of drug, the mRNA expression level ofPI3K, and AKT, mTOR decreases, while the mRNA expression level ofTSC1and TSC2increases, and the control group and the expression levelof different between drug concentration groups have statisticalsignificance(P<0.05). Conclusion: Tan IIA can inhibit the proliferationand autophagy of HepG2cells, and induce HepG2cells apoptosis.Apoptosis and autophagy negatively correlated, autophagy caninhibit cell apoptosis of cancer cells, while Tan IIA can inhibit autophagyand apoptosis. These are relative with the inhibitory effect ofPI3K/AKT/mTOR signal transduction pathway result from Tan IIA.