| Objective:To investigate the effects of mellitin on cell proliferation and apoptosis of gastric adenocarcinoma SGC-7901 cells and their probable mechanism involved,aiming at offering credible academic and experimental bases for clinnical use.Method:SGC-7901 cells were treated with different concentrations (1,2,4,8,16 and 32μg/mL)of mellitin for various periods(12,24,36,48 and 72h).(1)The cell proliferation was examined by MTT assay. (2)Light microscope was use to observe morphologic changes.(3)Hoechesst 33258 was used to examine the nuclear changes by fluorescence microscope.(4)Ultramicro structure was observed through transmission electron microscope (TEM).(5)Gel electrophoresis was used to observe the typical DNA ladder. (6) Apoptosis and cell cycle were determined by flow cytometry (FCM). (7) Expressions ofBcl-2,Bax proteins in gastric cancer cells were measured by immunohistochemistry. (8) RT-PCR was used to detect the mRNA transcriptions of Bcl-2, Bax,P53 DR4 and DR5.Results:(1)Melltin inhibited the growth of gastric adenocarcinoma SGC-7901 cells in time-and concentration-dependent mannerswith IC50 value of 2.43μg/mL (treatment with mellitin after 48h) The difference of inhibition rate between test group and control group was obvious(P<0.05).(2)Under a ligter microscope,cells were decreased,turned small and round,lost their preperty of adherent growth.(3) When treated with 8μg/mL mellitin for 48h through fluorescence microscope,SGC7901 cells showed the typical apoptotic morphologic changes including chromatin clumping, nuclear fragmentation, and chromatin condensation. (4) The apoptotic morphology, such as karyopycnosis and apoptotic body, could be observed by TEM in mellitin groups. (5) The 4,8μg/mL mellitin groups produced characteristic DNA ladder on DNA gel, but not in the control group.(6) And a tipical subdiploid peak before phase G0/G1 was detected by flow cytometry. The rates of apoptosis were correlated with the concentration of Mellitin, after treatment with2,4,8μg/mL of Mellitin for 48 h,the cell apoptotic rates were respectively 5.69±0.74%,10.58±1.32% and 29.57±1.58%, (7)Compared with control group, the expression of Bcl-2 decreased distinctly and the rate of expression decresed also in the test groups,and the expression of Bax increased distinctly and the rate of expression incresed also in the test groups,the difference was significant (P<0.05) (8) The expression of Bax mRNA obviously decreased respectively.,the expression of Bcl-2 mRNA obviously increased respectively and Bax/Bcl-2 ratio was decreased. The expression of P53,DR4 and DR5 increased respectively.Conclusion:Mellitin can induce apoptosis and inhibit proliferation of gastric adenocarcinoma SGC-7901 cells and display effect in a dose-dependent manner by regulating expression of Bcl-2,Bax,P53 genes.
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