Study Of The Inhibition On HepG2 Cell Proliferation And The Related Mechanism Of α-pinene

1 Purpose and Significance α-pinene is one colorless and transparent fluid of, which Contain special double-loop structure of double bond and have higher reaction activity and diversity. Rec-ent years, it has got more and more attention and rapid development about pharmacologic action of α-pinene.Some scholars thought that the tumor volume of tumor-burdened mice is significantly decreased than the control group in the circumstance ric-h of α-pinene. It explained that α-pinene has inhibitory effect on the growth of tu-mor cells. Although there have some reports about the anti-tumor effect of α-pinene, but the mechanism is still unclear, to explore the anti-tumor effect of α-pinene and its related mechanism, we established a study of the inhibition of cancer cell proliferation and the related mechanism of α-pinene, to confirm the molecular pathways in the process of anti-tumor effect of α-pinene.α-pinene mainly isolated from pinaceae plant pine oil, turpentine oil content is very rich in our country. In order to make more valuable use of abundant resources. The rsearch of pharmacological action of α-pinene has a broad prospect, expecially the deeply research on the antitumor mechanism of it, make for extension of utilization of α-pinene.The object of this study is research inhibition of tumor cell proliferation of α-pinene firstly, and on the basic of it, to explore the effect on tumor cell cycle arrest of α-pinene, further explore the related mechanism of antitumor. The results of this study will provide tha basis for further study of α-pinene. This study used tumor cells as the research object, to explored the effect of α-pinene on tumor cells,aiming at provide theoretical andexperimental basis of the development and expan-ding applications of the antitumor drugs.2 Methods 2.1 Screening of the inhibition effect of α-pinene on tumor cells This study selected liver cancer Hep G2 cells, glioma C6 cells of rat and gastric carcinoma SGC-7901 cells for experimental, using different levels of α-pinene effect 12 h and 24 h on tumor cells, respectively. Detection of inhibition rate of tumor cells after α-pinene by using MTT-test, then drawing growth-inhibitory curves; determination of changing number effect α-pinene on tumor cells by using crystal violet staining method, and Chosen the most effective of α-pinene on tumor cells for subsequent research of cell cycle and the related mechanism.2.2 Detection of the influence of cell cycle effect of Hep G2 cells after treated with α-pinene Guided by the previous studies, selected Hep G2 cells as experimental subject, using different levels of α-pinene effect 12 h and 24 h on tumor cells, respectively. Changes in cell morphology were observed by optical microscope, by using flow cytometry to Detection of the cell cycle phase distribution of Hep G2 cells after treated with α-pinene.2.3 Detection of the influence of the foci of γH2AX of Hep G2 cells after treated with α-pinene To explore the inhibition mechanism of α-pinene on Hep G2 cells, observation on the effect of tumor cell DNA damage firstly. On the basis of previous experiments, researching the change of γH2AX that linked to DNA damage for further study, and using immunofluorescent staining assay to detect the expression of the γH2AX foci in cell nucleus after treated with α-pinene.2.4 Detection of the influence of the expression of related proteins of Hep G2 cells after treated with α-pinene Detection of the cell cycle related protein expression after α-pinene effects on Hep G2 cells by applying western blotting, including ATM, phospho-ATM(S1981), ATR, phosphoATR(S1981), H2 AX, γH2AX(S139), Chk1, p-Chk1(S280), Chk2, p-Chk2(S345), Cdc25 A, p-Cdc25A(S76), Cdk2, p-Cdk2(T160) and Cyclin A. Through expression of the cell cyclerelated proteins,to discuss the specific construction path of DNA damage mechanism of Hep G2 cells.3 Results 3.1 Detection of the inhibition effect of α-pinene on tumor cells MTT-test results have shown that, α-pinene have a stronger inhibitory effect on three kinds of tumor cells(Hep G2, C6 and SGC-7901cells) in vitro, and the inhibition rate assumes concentration and time dependence; crystal violet staining results have shown that, the higher the drug concentration, the less adherent cells, and the more shallow color is. The value of IC50 are 0.76, 0.92, 1.09, respectively. Through comparison, screening out Hep G2 cells of the best effect for subsequent tests.3.2 Detection of the influence of the cell cycle phase distribution of Hep G2 cells after treated with α-pinene The flow cytometry results have shown that, α-pinene(Concentrations were0.5,1, 2μg/ml) could induced the cell cycle arrest in S phase in Hep G2 cells, and the percentage of S phase distribution increased with the increase of drug concentration.3.3 Detection of the influence of the foci of γH2AX of Hep G2 cells after treated with α-pinene The immunofluorescent staining assay showed that, α-pinene could increase the generation of γH2AX foci in aconcentration dependent manner in Hep G2 cell nucleus. When the concentrations of α-pinene were 1μg/ml and 2μg/ml,the positive percent of γH2AX foci were 39% and 81%,the expression of γH2AX is the mark of DNA damage,this illustrated that when the concentrations of α-pinene were 2μg/ml, caused significant DNA damage of Hep G2 cells.3.4 Detection of the influence of the expression of related proteins of Hep G2 cells after treated with α-pinene The results indicated that, When the concentrations were 0.5, 1 and 2μg/ml, α-pinene have the capability of activating ATM, ATR and H2 AX by phosphorylating them, the expression ratio of p-ATM were:1.1, 1.6, 1.8, 2.1; p-ATR were:1.6, 1.8, 1.9, 2.0; γH2AX were:0.5, 0.9, 1.4, 2.0; could increase the amount of expression of p-Chk1, p-Chk2, p-Cdc25 A,p-Cdk2, and the quantity of expression increased with α-pinene concentration;the expression ratio of p-Chk1 were: 0.5, 0.8, 0.9, 1.2;p-Chk2 were:1.6, 1.9, 2.2, 2.9; p-Cdc25 A were:0.7, 1.2, 2.5, 2.9; p-Cdk2 were:0.7, 2.3, 2.7, 3.2; while there has little influence of the expression of Cyclin A, and inhibit the expression of Cdc25 A.4 Conclusions 4.1 Through the screening of the inhibition effect of alpha pinene on Hep G2, C6 and SGC-7901 three kinds of tumor cells,and turned out that more sensitive to Hep G2 cells, the value of IC50 is 0.76μg/ml.4.2 The flow cytometry showed that α-pinene could make the cell cycle arrest of Hep G2 cells, and the more drug level,the more number cells in S phase, to play the role of anti-tumor.4.3 The mechanism of anti-tumor role of α-pinene is by phosphorylating a series of cell cycle proteins, like as p-ATM, p-ATR, p-Chk1, p-Chk2, p-Cdc25 A and p-Cdk2 etc. It was initially thought by inducing DNA damage response, activating cell cycle checkpoints, impacting cell cycle progress to exert anti-tumor effect.