The Study Of Inhibition Effect And Adjustment Mechanism Of Allicin To The G₂/M Phase Of SGC-7901 And BGC-823 Cell

PrefaceCell cycle is defined that a period from one cell division finish to the next time which includes four phases: G₁ phase, S phase , G₂ phase and M phase. But G₀ is a dormancy of the cell. There are three points which control the cell , which include G₀ - G₁, G₁ - S and G₂ - M. Especially G₁ - S and G₂ - M are more important. Control point G₁ - S is the driving mechanism of the cell cycle and can prevent the injury DNA cell from being into the S phase. During the inhibition of G₁ phase , there are two aspect mechanisms; PRb and P53. G₂ - M phase is the checking and controlling mechanism of the cell cycle. It's function is making sure the copy of the cell accurately. Control point G₂ can prevent the cell of the injury DNA and unfinished copy DNA to M phase . The transition from G₂ phase to M interferes with the cell division , which consist of sytoplasm and nucleus DNA division and finish the transition from one cell to two cells. During the carcinoma cell, the cell division will increase by index , therefore, G₂ - M phase inhibition is more important. Now G₂/M phase inhibition mechanism is not clear. In eucaryotic cell, normal cell G2/M is adjusted and controlled by the compound of cyclin B and P34cdc2, which is cycle protein dependant kinase. Cyclin B is adjustable subunit. On the late of phase G2, cy-clinB and P34cdc2 form MPF, then Thrl4 and Tyt15 on N side of P34cdc2 de-phosphorylate, Thr161 phosphorylate and make P34cdc2 have kinase activity, and induce a series of substrate level phosphorylation . Finally it lead to mem-brane of nucleus break up and coacervation of chromoplasm. The research shows that G2/M phase inhibition accesses are probably related to the following approaches : (1) It's sure that some P53 aim genes induce G2 phase inhibition . Cdc2 is necessary gene in this process. (2)p53 can induce G2 phase inhibition by unde-pendant cdc2. (3) Bel -2 phosphorylation can lead to G2 - M phase inhibition. (4)Cyclin G is probably related to the G2 -M check and control point. (5)P15 can not only inhibit G2 to M ,but also over - express of PI 5 can inhibit G2 to M.Allicin is diallyl trisulfide by chemistry and is a kind of compound which is separated from the corn of the garlic. Allicin has many biological functions, such as anti - fungal, antibacterial function and decreasing blood pressure and preventing from and treating arteriosclerosis and also has a better function of an-ticancer and preventing from cancer. The mechanism of Allicinvs antitumor is as the following : it can impede the compound of carcinogen and can anti - mutate; kill directly the tumor cells; inhibit proliferation of tumor cells; induce the arrest of the cell cycle and apoptosis and differentiation of tumor cells; adjust human body immunological function ; increase sensitivity and prevent drug - resistant for some anticarcinogen . The research which is made by Filomeni et al has showed that Allicin can make the SH - SY5Y stop at G2/M phase . The study made by Wu et al has come out that Allicin can make J5cancer cell inhibit at G2/M phase and has indicated obvious time and dose dependant. The study made by Wong et al about OSC3 from Allicin has showed that OSC3 can inhibit Human Leukemia cell to stop at G2/M phase. There are many research on the mechanism of Allicin"s antitumor function which can be studied by many kinds of methods.There are lots of records on Allicin inhibition to tumor cell cycle and make it on G2/M phase in China and all over the world. But we still continue to take time to do research. How Allicin make the tumor cell stop at G2/M phase on mechanism still is a question.The test is planed to observe inhibition function which Allicin act the tumor cell cycle. We plan to put the Allicin on SGC -7901 and BGC -823 which are on the human stomach. We study inhibition function about Allicin to tumor cells and infection about Allicin to tumor cells cycle by MTT method and flow cytome-try. It detects the expression level of cdc2 and cyclinB during G2/M phase of the cells by SP immunohistochemistry and explores the mechanism about Allicin to tumor cell cycle inhibition.MethodSGC -7901 and BGC -823 are developed in the culture box, taking logarithm about cell which is developing. We check cell proliferation inhibition and IC50 of Allicin by MTT method . The abscissa is Allicin consistency, and vertical line is cell inhibitive rate, we can draw a chart about cell proliferation inhibition, and chech IC50. The formula is that cell proliferation inhibition rate = (1 - light density of test group/light density of control group) x 100%. It can detect the infection to the cell cycle of SGC —7901 and BGC -823. we can detect cdc2 and cyclinB protein expression by immunohistochemistey method and set control group. We can see the tumor cells both with Allicin and without Allicin under microscope. The standard of judgment is that clear brown - yellow particle seen at nucleus and sytoplasm is positive cell.ResultAllicin has obvious inhibitive function to both SGC -7901 and BGC -823. The IC50 of 72h is 23ug/ml and 35ug/ml respectively. These cells are treated by Allicin at the consistency of 23ug/ml and 35ug/ml for 24h and 48h assayed by flow cytometey compared with the control group . The percentage of G0/G1 phase of the cells decreases and that of G2/M phase increases significantly in the Allicin treated group (P<0.01). It can indicate that Allicin causes arrest of gastric cancer cell in G2/M phase. Both SGC -7901 and BGC -823 without Allicin treatment, cdc2 and cyclin B are positive. To SGC -7901 with 23ug/ml of Allicin treatment, Cdc2 positive expression rate is 87. 2% , cyclinB is 59. 3%. To BGC -823 with 35ug/ml of Allicin treatment, Cdc2 positive expression rate is 84.4% ,cyclinB is 62. 8% (P <0. 01) .