| Objective: Endometrial cancer is one of three major gynecologic malignancies with poor prognosis in advanced cancer or recurrence of cancer, so the treatment is a clinical problem. Hence, it is an urgent need for the effective new anti-cancer drug with small side effects and better tolerance in the clinical treatment. Arsenic trioxide(AS2O3)is a novel anti-cancer drug. It has been successfully used in clinical treatment of acute promyelocytic leukemia (APL)and certain solid tumors. The experiments in vitro and in vivo have confirmed its good effect as chemotherapy agent, but there are fresh reports about AS2O3 on the treatment of endometrial cancer all over. So the aim of this research is to explore the inhibitory effect of arsenic troixide (AS2O3)on the growth of human endometrial cancer HEC-1-A cells in vitro and in vivo and its mechanisms, to evaluate the side effects of AS2O3, then to provide the basis for the clinical application of AS2O3 on the treatment of endometrial cancer.Methods:1 Tetrazolium salt assay(MTT)was used to detect cell proliferation activity and compare the effect of AS2O3 with progesterone, medroxyprogesterone acetate (MPA) and CDDP. 2 Cell cycle progress was measured by flow cytometryHEC-1-A cells (1×106) were collected after 48 h incubation with 5μmol/L AS2O3 and were digested with protease. Cell cycle progress was measured by flow cytometry[4]. Cell cycle compartments of G0/G1-phase, G2/M-phase and S-phase of cells were determined as percentage of the total population by a computer program Cell-Quest (Becton-Dickinson). The sub- G0/G1 fraction represents apoptotic cells.3 DNA gelelectrophoresis was used to detect apoptosisHEC-1-A cells (5-10×106) were collected after 48 h incubation with 5,10μmol/L AS2O3. DNA was gently extracted by phenol/chloroform/isoamyl alcohol[5], electrophoresed on a 1.2% agarose gel. The typical apoptosis ladders of DNA were visualized under ultraviolet light.4 Human endometrial cancer transplanted tumor models were established in nude mice using HEC-1-A cells, then were divided into five groups as following: AS2O3 groups divided as low-dose group(4 mg·kg-1·d-1), medium-dose group(6 mg·kg-1·d-1), high-dose group(8 mg·kg-1·d-1), CDDP positive control group(3 mg·kg-1·d-1), and negative control group using saline. The drugs were given intraperitoneally for 14 d, then the inhibition rate of tumor volume and tumor weight were calculated. The change of body weight was measured.5 Side effect analysis: All animals were sacrificed by bleeding through the eyes under anesthesia. The blood was collected to be used blood test, serum was used for liver and kidney function. The wet weight of tumor, heart, liver, spleen, lung, kidney and uterus were measured. The tumors and organs were kept in 4% formalin and made into paraffin blocks.6 Use SPSS 11.5 of statistical software for data analysis.Results:1 In vitro studies: AS2O3, CDDP inhibited the cell growth significantly at concentration of 1～20μmol/L. It seemed AS2O3 > CDDP. AS2O3 (5μmol/L) treatment resulted in apoptosis, S and G2/M phase cell block.2 In vivo studies: The tumor volume was inhibited by 50.97%, 75.58%, 56.92%, 52.23%, and the tumor weight was inhibited by 10.15%, 29.33%, 16.67%, 14.69%, respectively, in low, medium, high-dose AS2O3 groups and CDDP group , as compared with the saline group, the difference was statistically significant respectively (P<0.05). It showed that middle-dose and high-dose of AS2O3 had a more stronger role than CDDP on inhibiting the growth of transplanted tumor.3 Observation of the general situation in nude mice: The nude mice had thoese performance before long in CDDP group after delivery: Significant reduction of activity, dispirited spirit, reduction of food intake, increase of drinking water, increasingly thinner and cachexia, so one dead in the day before killing them, but the nude mice in AS2O3 groups and the negative control group all had good state in spirit and activity, and the nude mice in AS2O3 groups had adds in eating and drinking.4 Change in body weight of nude mice: It noted that AS2O3 has little effect on body weight of nude mice before and after treatment, the average weight of nude mice in CDDP group had a significant reduction, that was (3.74±2.36)g, there were significant differences comparing with each group of AS2O3 and the negative control group (P <0.05).5 Results of routine test of blood: There was no significant impact on hemoglobin in AS2O3 and CDDP groups comparing with the negative control group(P>0.05). The numbers of leukocytes and platelets of nude mice in AS2O3 groups were lower than that of negative control group, the differences were significant (P<0.05). The number of leukocytes, red blood cells, platelets and the value of hematocrit of nude mice in CDDP group were lower than that of negative control group, the differences were significant (P<0.05). There were significant differences in the decline of the number of red blood cells and platelets in CDDP group comparing with AS2O3 groups, it showed that the impact on blood of CDDP was more obvious than that of AS2O3.6 Liver and kidney function: There were no significant differences in liver and kidney function of AS2O3 comparing with negative control group (P>0.05), it suggested that AS2O3 had no obvious effects on the liver and kidney function. The numbers of ALT, AST, TBIL and CHE in CDDP group were higher than that of negative control group and the number of AST/ALT was lower than that of negative control group, there were significant differences (P<0.05), it showed that CDDP had different levels of impacts on liver function, and comparing with AS2O3 groups, there were significant differences (P<0.05), it showed that there were more significant impacts on liver function of CDDP than that of AS2O3; The number of BUN was higher than that of negative control group, the number of GLU was lower than that of negative control group, there were significant differences (P<0.05), it showed that CDDP had different levels of impact on kidney function, and comparing with AS2O3 groups, there were significant differences (P<0.05), it noted that there were more significant effects on kidney function of CDDP than that of AS2O3.7 Comparison of wet weight of major organs and pathological observation: AS2O3 had no effect on the wet weight of the major organs, there were no significant differences from AS2O3 groups and the negative control group (P>0.05); The average wet weight of heart, liver, spleen and kidney of CDDP group were lighter than that of AS2O3 groups and the negative group, the differences were significant (P<0.05), it noted that CDDP had more significant impact on the weight of these organs than AS2O3. However, there were no obvious lesions in nude mice organs of each group under microscopy.8 The results of the expression of PCNA and of p27 protein in transplanted tumor by immunohistochemistry and observation of pathology. The expression of PCNA: the color intensity in the negative control group was strong positive, as well as negative or weakly positive in medium-dose of AS2O3. The expression of p27: the color intensity in the negative control group was negative, as well as positive partly in medium-dose of AS2O3.Conclusions:1 AS2O3 has obvious inhibitory effect on the growth of human endometrial cancer HEC-1-A cells in vitro and in vito.2 Therapeutic concentration of AS2O3 of the anti-tumor effect is not the higher the better, it should take the optimal dose, the dose of 6 mg·kg-1·d-1 is most appropriate, its inhibitory effect is superior to low-dose and high-dose of AS2O3 and CDDP.3 AS2O3 could inhibit the growth of HEC-1-A cells by inducing cell apoptosis and blocking S and G2/M phase cell. The experiment in vivo further indicates that AS2O3 could inbibit the growth of tumor and disturb the cell cycle growth, by down regulating the expression of the uclear proliferation antigen PCNA and up regulating the expression of the tumor suppressor gene p27.4 AS2O3 of therapeutic concentration has a stronger inhibitive effect than CDDP, with a lighter side effect, thus AS2O3 is a new drug for endometrial cancer treatment.
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