Effect Of TMS (2,3’,4,5’-tetramethoxystilbene) To Human Endometrial Carcinoma Cells

Objective: Endometrial cancer is a malignant neoplasm which isoriginated from endometrial epithelium, and it is also one of the most commongynecologic malignancies of the female urogenital tract, harm to women heath.It accounts about7%female cancer and20%~30％gynaecological genitalcancer. In recent years, the incidence and mortality rates of endometrial canceris on the rise. As a kind of sex hormone dependent carcinoma, it is consideredas a risk of developing endometrial cancer that long-term estrogen exposurewithout progesterone against. Traditional therapy are surgery, radiotherapy,chemotherapy and hormone therapy. However, the anti-cancer medicine hasharm to propagating cells at the same time of killing the tumor cells andinfluences the effect of the chemotherapy, during the process. So thechemotherapy agent becomes one of the focuses, which can kill the tumorcells most but has the least toxicity to normal cells. In recent years, moleculetarget therapy is gradually becoming the focus of researchers’, which is also anew way to treat endometrial cancer.Cytochrome P4501B1(CYP1B1) is an enzyme that is expressed highly inmany tumors and related to hormone, and it could metabolize the estrogen intocarcinogen and activates the procarcinogen-aromatic hydrocarbons. Previousresearch has shown that CYP1B1is highly expressed in endometrial cancertissues, while in the normal tissues, while its level is lower in the normaltissues, indicating that CYP1B1may play an important role in the occurrenceof endometrial cancer. TMS (2,3′,4,5′-tetramethoxystilbene) is found as one ofthe CYP1B1special inhibitors in recent years, and it can inhibit the reactionthat CYP1B1catalyzes the hydroxylation of estrogens to4-hydroxyestrogens.This study was designed to investigate the effect of TMS on the proliferation and apoptosis of human endometrial carcinoma cell line Ishikawaand to explore the internal mechanism, then to provide a new way for targettherapy of endometrial cancer.Methods:1Cell culture: Refrigerated Ishikawa cells were grown in100ml cultureflasks with DMEM including10%fetal bovine serum (FBS),100U/mlstreptomycin and penicillin, at37℃,5%CO2humidied incubator. The cellswere subcultured with0.25%steapsin， and were observed by invertedmicroscope.2The expression of CYP1B1protein was measured byimmunocytochemistry in Ishikawa cells: Streptavidin/Peroxidase (SP) methodwas used.3MTT method was used to measure cell proliferation after Ishikawa cellswere treated with TMS, and got the cell inhibitory rate curve of each group. Atthe same time, the morphology of the cells was observed by invertedmicroscope.4Flow cytometry was used to investigate the ratio of cell apoptosis and cellcycle after Ishikawa cells were treated with TMS. Ishikawa cells weresuspended in100ml culture flasks at a density of2.5×105/ml. After the cellssticked to walls, each group was added with various concentrations（0,10,20,40,80μmol/L）of TMS. In control group, the same amount of culture mediumwas added. After48h, the cells were harvested with steapsin (0.25%) and fixedby cold ethanol (70%).The cells were stained with propidium iodide(PI) later,then were measured by flow cytometry (Beckman Coulter Company,America).5The expression of Bcl-2, Bax and Survivin were detected by flowcytometry after Ishikawa cells were treated with TMS. After Ishikawa cellswere treated with different concentration（0,10,20,40,80μmol/L）of TMS for48h, the cells were harvested with steapsin (0.25%) and fixed by cold ethanol(70%). Each group was added with Bcl-2, Bax and Survivin antibodie（s1:50）, and then was added with matched FITC-IgG antibody（1:50）, then weremeasured by flow cytometry.6Statistical analysis was performed using SPSS13.0software package,andthe results were described as x±s. Comparison of many groups wasperformed by the One-Way ANOVA, and multiple comparisons betweengroups used SNK. With uneven variance, the comparison of many groups wasperformed by the non-parametric test, and multiple comparisons betweengroups used Kruskal-Wallis H. P<0.05was considered significant.Results:1CYP1B1protein was expressed in human endometrial carcinoma Ishikawacells, with brown granules in cell plasma or cell nucleus.2After the Ishikawa cells were treated with different concentrations（0,10,20,40,80μmol/L）of TMS for24h,48h and72h, the proliferation of Ishikawacells was significantly inhibited by TMS in a dose and time dependent manner,and the inhibition rate of various concentrations of TMS showed significantdifference(P<0.05).3Ishikawa cell morphology was observed by inverted microscope. Growthbehavior of Ishikawa cells in control group is good. The cells extended aspolygon. As time went on, the cell number increased gradually in alogarithmic growth and the cells contacted tightly. When the Ishikawa cellswere treated with different concentration（10,20,40,80μmol/L）of TMS for48h, degeneration and necrosis of Ishikawa cells were found under invertedmicroscope. The cells were suspended, and the shape became from polygoninto round. The vacuole was seen in cytoplasm and intercellular spaceenlarged. The cell-substance concentrated and became round, and the typicalmorphologic changes of cell apoptosis appeared, such as cell nucleusconcentration, nuclear fragmentation and so on, but apoptotic body wasn’tseen. Along with the concentration of TMS increasing, the apoptotic cellincreased and the living cells, sticked to the wall, decreased.4Effects of TMS on cell apoptosis and cell cycle were measured. AfterIshikawa cells were treated with various concentrations of TMS（10,20,40, 80μmol/L）for48h, FCM showed that the ratio of cell apoptosis and cell cyclewere changed. The ratio of cell apoptosis detected by FCM were（2.34±0.69）%，（6.09±0.91）%，（12.00±0.90）%，（16.63±0.55）%，（26.04±1.02）%, so the ratio of cell apoptosis was increased with theconcentration. So TMS could promote the apoptosis of Ishikawa cells in adose-dependent way. Cell cycle phase analysis revealed that the proportion ofIshikawa cells in G0/G1phase decreased, while the proportion of Ishikawacells in G2/M phase increased in a concentration-dependent manner, andshowed significant differences (P<0.05). Thus TMS apparently bloked theIshikawa cells at G2/M phase.5Effects of TMS on the expression of Bcl-2, Bax and Survivin weremeasured. After Ishikawa cells were treated with different concentrations ofTMS（0,10,20,40,80μmol/L） for48h, FCM showed that the expression ofBcl-2and Survivin was down-regulated and the expression of Bax wasup-regulated in a concentration-dependent manner, and showed significantdifferences (P<0.05).Conclusion:1The expression of CYP1B1protein is found in human endometrialcarcinoma Ishikawa cells, so that TMS has an effect on Ishikawa cells mainlythrough CYP1B1protein.2TMS significantly inhibits the proliferation of human endometrialcarcinoma Ishikawa cells in vitro in a dose and time dependent manner. Withcertain concentration of TMS and certain time, the cell-substance concentrated,and the typical morphologic changes of cell apoptosis such as cell nucleusconcentrated, nuclear fragmentation and so on, but apoptotic body isn’t seen.3TMS induces apoptosis of human endometrial carcinoma Ishikawa cells invitro, and it blocks the Ishikawa cells at G2/M phase. At the same time, theratio of cell apoptosis was increased with the TMS concentration. Themechanism might be through down-regulation of Bcl-2and Survivin, and theup-regulation of Bax.4TMS might become a new target therapy drug for endometrial carcinoma.