The Inhibition Of TMS(2,3′,4,5′-tetramethoxystilbene) On Human Endometrial Carcinoma Xenografts

Objective: Endometrial carcinoma is one of the most common malignancy of the female genital tract. Its incidence and morbidity increase significantly in recent years. It has been testified that the incidence of most endometrial cancer is correlated with estrogens. The risk of endometrial cancer is elevated by long-term exposure to estrogens without progesterone working. Study about estrogen metabolism and the carcinogenic effects of its metabolites has become a focus in recent years. It is reported widely that estrogen metabolites may cause DNA damage and mutation directly, then induce the occurrence of carcinoma.Cytochrome P4501B1(CYP1B1) is one member of CYP450 family, mainly expressed in extrahepatic tissues. CYP1B1 catalyzes the metabolism of endogenous and exogenous compounds, then plays an important role in extrogen local metabolism and the balance. As a tumor- associated antigen, CYP1B1 is expressed at a high frequency in malignant tissue, while at a low frequency or no expression in normal tissue. And the expression of CYP1B1 is higher in late stage endometrial carcinoma than in the tissues of early stage. Such specific high expression of CYP1B1 provides a wide prospect and theoretical basis of carcinoma diagnosis and treatment with CYP1B1 as target. High levels of CYP1B1 limit the effect of CYP1B1 inhibitor in tumor tissues, which makes the medicine kill tumor cells with most toxicity while the least toxicity to normal cells. TMS(2,3′,4,5′-tetramethoxystilbene) is found as one of the most specific CYP1B1 inhibitors at present. It can inhibit the hydroxylation of estrogens to 4-hydroxy estrogens catalyzed by CYP1B1. It has been reported that TMS can inhibit the proliferation of Ishikawa endometrial cancer cells,induce apoptosis, and arrest cell cycle.The study was designed to explore the anti-tumor effects of TMS and its mechanism in xenograft transplanted by human endometrial carcinoma cell line(Ishikawa) in nude mice. Besides, the paper intends to evaluate the side effects of TMS and provide a new way for target therapy of endometrial cancer.Methods:1 Cell culture:Refrigerated Ishikawa cells were grown in 100 ml culture flasks with 1640 culture medium including 10% fetal bovine serum(FBS), 100U/ml streptomycin and penicillin, at 37℃, 5%CO2 humidied incubator. The cells were subcultured with 0.25% steapsin, and were observed by inverted hydroxy microscope.2 Human endometrial carcinoma exnograft models were established in nude mice:Collect human endometrial Ishikawa cells in the logarithmic pase and make into cell suspension at concentration of 107/ml. Each nude mouse was injected 0.2ml(about 2×106 cells) subcutaneously in back. After two weeks, tumors appeared obviously and the volume had increased into about 100 mm3. Afterwards, nude mice were divided into two groups as following: TMS group and negative control group. Both groups were administered 0.2ml subcutaneously. Nude mice in TMS group were injected TMS at concentration 10 mg?kg-1?d-1 dissolved by olive oil, whereas, mice in the negative control group were subcutaneously injected olive oil with eaqual volume. The drugs were given subcutaneously for 14 d, then the inhibition rate of tumor volume and tumor weight were calculated. The change of body weitht was measured.3 Side effect analysis:All the animals were sacrificed by bleeding through the eyes. The blood was collected to be used for liver and kidney function. The wet weight of tumor, heart, liver, spleen, lung, kidney, uterus and ovary were measured. The tumors and organs were kept in 4% formalin and made into paraffin blocks.4 The expressions of CYP1B1, ERK, P-ERK protein were measured by immunohistochemistry in the tumor. Streptavidin/ Peroxidase(SP) method was used.5 Use SPSS 21 of statistical software for data analysis.Resutls:1 The tumor volume was inhibited by 59.70%, and the tumor weight was inhibited by 35.85%. Compared with the control group, the difference was statistically significant(P<0.05).2 Observation of the general situation in nude mice: Before the endometrial cancer exnograft models were established, all nude mice were in good state in spirit and behavior, so were the nude mice in TMS group and the control group after the treatment.3 Change in body weight of nude mice: It was noted that TMS has little effect on body weight of nude mice before and after treatment. There was no significant differences comparing with negative control group(P>0.05).4 Liver and kidney function: There was no significant difference in liver and kidney function of TMS group comparing with negative control group. The amount of ALT, AST, ALB,UREA and CREA in two groups were of no significant differences(P>0.05).5 Comparison of wet weight of major organs and pathological observation: TMS had no effect on the wet weight of the major organs, there were no significant differences between TMS groups and the negative control group(P>0.05). Meanwhile there were no obvious lesions in nude mice organs of each group under microscopy.6 The expression of CYP1B1 and ERK, p-ERK proteins in exnograft tumor by immunohistochemistry. The expression of CYP1B1 in the negative control group was strongly positive, while weakly positive in TMS group. The expression of ERK in the negative control group was strongly positive,while weakly positive in TMS group. The expression of R-ERK in the negative control group was strongly positive, as well as weakly positive in TMS groups.Conclusions:1 TMS has an obvious inhibitory effect on human endometrial carcinoma exnografts in nude mice.2 It is possible that TMS can reduce the generation of 4-OHE2 throuth its specific inhibitory effect, which furthermore reduces the incidence of endometrial carcinoma by suppressing the quick activication of ERK/MAPK signal way by phosphorylation of 4-OHE2.3 TMS has no obvious side effect in nude mice.