| Mitotic Arrest Induced by Allicin in SGC-7901 Cell Line and Its Possible MechanismsPrefaceGarlic is the bulb of onion genus lily family plant. Epidemiological studies have in -dicated that garlic has some remarkable disease prevention and resistant effects. The effective constituents of garlic mainly contain some organosulfur compounds. Allicin whose chemical name is diallyl trisulfide, is a typical component of them. Allicin has many biologic activities to resist the diseases such as anti-infection, kata- choleste -rol, angio -sclerosis prevention and enhancing the antioxidation and detoxification. Recently, many researches demonstrate that allicin has shown some satisfactory anticancer ef -fects. There are several mechanisms contributed to its antineoplastic effects. Causing tu -mor cell cycle arrest was an important way among them. We previously demonstrated that allicin can induce mitotic arrest in human gastric cell line SGC-7901 and MGC-803. but the accurate mechanism of mitotic arrest causing by allicin has not been established. Vincristine as a M phase specific antitumor drug, can block tumor cell cycle in M phase, by destroying the stabilization of microtubule dynamics, encouraging the deploymerisition of microtubule and interfering the assembly of mitotic spindle. The p34cdc2/cyclin B1 complex which was also called M-phase promoting factor (MPF) is a regulator to the stabilization of microtubule dynamics, by causing phosphorylation of the microtubu le-associated proteins (MAPs). Is allicin similar to vincristine, can also destroy the stabilization of microtubule dynamics, and promote the deploymerisition of microtubule? Is the mitotic arrest causing by them regulated by MPF? In this study, we have focused our attention on answering these questions ObjectiveWe used immunofluorescence and confocal laser microscope technique to determine the effect of allicin on inducing mitotic arrest in human gastric cell line SGC-7901, and its possible mechanisms.MethodThe SGC-7901 cells were treated with allicin or vincristine. Proliferation inhibitory rate was detected by MTT colormetric assay. The immunofluorescence staining method was used. Briefly, exponentially growing cells were plated into 6-well microplates and were treated with IC50 dose allicin or vincristine for 24h or 72h. After being stained by anti-αtubulin or anti- cyclin B1, the cells were observed and photographed under the confocal laser scanning microscope. PBS was used as negative control. The mitotic arrest was confirmed by counting the percentage of mitotic cells in SGC7901 cells treated with allicin or vincristine and observing the morphologic changes of the microtubule structure and the location of cyclin B1. Further more, Leica confocal software was used to quantitately analyze the expression of cyclin B1. The experiment was repeated for 3 times. The average fluorescence intensity was considered to be the expression level of the protein.Result1. The SGC-7901 cells were inhibited after exposure to allicin for 24 hr, the IC50 was 7.2μg/ml and for 72 hr, the IC50 was 20μg/ml; When exposure to vincristine for 24 hr, the IC50 was 0.29μg/ml and for 72 hr, the IC50 was 0.059μg/ml.2. After being treated with allicin or vincristine the percentage of mitotic cells in SGC 7901 cells reached 30.61％and 34.92％respectively, they were significantly exceed to the control, which was only 4.69％. The cells treated with allicin or vincristine became more shrunken and nepheloid, the microtubule networks in them disappeared. Contrastly, the control cell exhibited an intact microtubule network. The cyclin B1 was mainly located in the cytoplasm in normal conditions, after exposure to allicin or vincristine it was expressed more significantly and concentrated in the nucleus.3. The fluorescence intensity of the cyclin B1 protein in cells treated with allicin or vincristine was much more significant than control cell's. All these changes caused by allicin were similar to that vincristine did.ConclusionSimilar to vincristine, the mechanism of allicin -induced mitotic arrest in human gastric cell line SGC-7901 was due to that it elevated the expression of p34cdc2/cyclin B1 and enhanced its function in causing phosphorylation of the microtubule-associated proteins (MAPs). Allicin might thus be a new leading compound for designing novel anticancer drugs.
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