Experimental Study On Anti-tumor Effect Of Indomethacin And Genistein On The Human Gastric Cancer Cells

Objective: To study the effect of indomethacin and genistein in inducing growth inhibition and apoptosis on the human gastric cancer cell lines BGC-823, and provide the theoretic basis for gastric cancer therapy. Methods: BGC-823 cells were cultured in the environment of 37℃, 5% CO2 with RPMI-1640 medium, which was supplemented with 10% fetal bovine serum, 100 units/ml penicillin and 100ug/ml streptomycin.The experiment included comparison groups, drugs groups and combined groups. Comparison groups consitituted with RPMI-1640 medium and DMSO; drugs groups were different concentration drugs such as IND 12.5 μg/ml ,25μg/ml ,50μg/ml ,100μg/ml;Gen 2.5μg/ml,5.0 μg/ml,10μg/ml,20μg/ml,40μg/ml; Combined groups were half the IC₅₀ of IND and half the IC₅₀ of Gen. 1.In vitro growth inhibitory effects of IND and Gen on BGC-823 were assessed by MTT performed according to the previously described protocol with a slight modification.After cultured for several generations, the cells were digested and transferred into 96-well culture plates, 5×10³ cells in each well. After cultured for 24h, changed the liquid of RPMI-1640 medium. Added different concentration drugs into the wells and each concentration drug had 4 same wells. The cells were incubated for 24h and 48h. Subsequently, 20μl of MTT (5g/L)was added to each well, followed by incubation for 4h at 37℃. Formazan crystals were dissolved in DMSO.Absorbance was determined with an enzyme-linked immunosorbent assay reader at 492nm.The cell inhibition was examined using MTT assays. Each assay was performed 5 times. The controls received medium and DMSO.The result were expressed as mean ±SE. The MTT method was used to obtain the IC₅₀ at the time of 48h of the drugs on BGC-823 cells. The IC₅₀ of each drug and half the IC₅₀ of IND added half the IC₅₀ of Gen was used to treat BGC-823 cells for 48h in order to obtain the inhibition rate. 2.For detection of the apoptosis affected by the drugs, the cells were harvested and stained by Acridine Orange and Propidium Iodide Fluorescence to determine the morpological alteration of apoptotic cells and the apoptotic rate. 3.The cells DNA were extracted using saturation phenol/chloroform and stained by Ethidium Bromide to evaluate DNA strands breaks using agarose gel electrophoresis and observed with UV lamp. 4.In preparation of flow cytometry(FCM), BGC-823 cells were centrifuged 48h after transfection. The cells were washed with PBS, and fixed by 70% cold ethanol. Propidium Iodide stained the cells, then cell cycle alteration and apoptotic rate ofthe cells were determinned by flow cytometry. Apoptotic cells appeared in the cell cycle distribution as cells with DNA contents less than G1 cells, and the percentage of apoptotic cella was calculated. Results: It was revealed by MTT assay that indomethacin and genistein could inhibit the growth of gastric cancer cells in a dose-and time-dependent manner. At the time of 48h IC₅₀ of Genistein was 16.61μg/ml, while indomethacin was 67.92μg/ml. After exposure for 48h to these drugs, gastric cancer cells BGC-823 presented some morphologic features of apoptosis, including cell shrinkage, nuclear condensation, DNA fragmentation, and formation of apoptotic bodies. The effect of growth inhibition was more obvious in cotreatment group with IND and Gen than respective drug group. The apoptotic rates of the cells observed through PI and AO staining were 2.27±0.28%, 18.00±0.41%, 18.53±0.19% and 33.80±0.38% for the control group, indomethacin and Genistein, respectively. The cellular DNA strand breskages were found obviously both in indomethacin group and genistein group by agarose gel electrophoresis. It was found that the cells induced by genistein or indomethacin in a dose-dependent and non-linear manner and increased the proportion of cells in the G₂/M while reduced the proportion of cells in the S phase and G₀/G₁ phase. The apoptotic rates of cells measured by using flow cytometry were 1.1%, 24.8%, 22.6% and 32.8% each for the control group, IND group, Gen group, individually, and combined group.