| Background: Ovarian malignancy mortality is the first in gynecologic malignant tumor. Epithelial ovarian cancer(ECO) account for 85%~90% of malignant ovarian tumors. About 63% of patients with ECO have widespread disease at presentation, and the five-year survival rate accounts for 27%. Based on the ideal tumor cells to destroy the loss, the important reason affecting the survival rate are ovarian cancer recurrence after chemotherapy and acquired drug resistance to chemotherapy. Therefore, to find new effective anti-cancer agents has become one of the important subject of ovarian cancer therapy research.Genistein is the strongest anti-tumor activity of natural isoflavone compounds. It is tyrosine protease inhibitors and has many biological activities, including weak estrogen-like effect, antioxidant effect and inhibiting tumor angiogenesis, and the roles of regulating the cell cycle, and so on. Some researches show that genistein has the prevention of tumor, anti-tumor effect, and can enhance sensitivity of drug-resistant tumor cells to chemotherapy drugs. And the natural medicine, genistein, receives much attention for its advantages of low toxicity and efficient. But, genistein is poorly water-soluble, and its oral absorption rate and bioavailability is poor. In most of the vivo and vitro experiments, researchers use dimethyl sulfoxide(DMSO) as solvent to dissolve, but the side effects of DMSO to cells can not be ignored. It not only affect the laboratory findings, but also limit the clinical application of drugs. In the study, we choose Hydroxypropyl-β-cyclodextrin as excipient, and the material can clathrated flavonoids. By this way, its solubility, stability and bioavailability has been improved. Lots of studies have shown that micro RNAs can regulate cell proliferation and apoptosis, and adjust the cancer related signaling pathways, play an important role in the formation and development of tumor biology function. In recent years, found epithelial-to-mesenchymal transition(EMT) is associated with invasion and metastasis of tumor cells. Mi RNAs through some related signaling pathways adjust EMT, in turn, affect the occurrence of tumor development.Objective: We treat human epithelial ovarian carcinoma in nude mice subcutaneously transplanted tumor as the research object in this study. We, giving mice different doses of genistein and low dose genistein puls cisplatin, by observing the transplanted tumors volume, inhibitory rate and E-cadherin m RNA, mi RNA-200 a and E-cadherin protein expression, discuss genistein inhibition on epithelial ovarian cancer xenograft in nude mice, as to provide theoretical basis for clinical application of genistein.Methods:1 Cell culture : Human ovarian carcinoma SKOV3 cells are cultured in RPMI-1640 medium(containing 10% fetal bovine serum). Put culture bottles in temperature of 37℃, containing 5%CO2.2 Establish nude mouse transplantation tumor model and the experimental groups: BALB/C/nu/nu nude mice, female, 4~6 weeks-old, 16~18g, were raised in the environment of SPF. Conventional sterile culture SKOV3 cells, when the cells are in logarithmic phase, prepare mixture of 2.5×107/ml single cell suspension. Each nude mouse inoculated with 0.2ml(5×10^6 cells) of single cell suspension in n subcutaneous under sterile conditions. After 7 days, select the kind of transplantation tumor model, whose tumor diameter are from 4~7mm. And randomly divided into six groups:The control group(hydroxypropyl-β-cyclodextrin), Genistein low dose group, Genistein middle dose group, Genistein high dose group, Cisplatin group, Cisplatin puls Genistein low dose group. Six nude mice are in each group.3 Preparation of experimental drugs: All drugs configured to the required concentration with 0.9% sodium chloride injection, immediate use.4 Drug delivery method: Three different doses of genistein respectively according to the content of genistein 50mg/kg、150mg/kg and 450mg/kg, intraperitoneal injection, once a day, for 21 days. The dose of hydroxypropyl-β-cyclodextrin in the control group is the same with the high dose group of genistein, intraperitoneal injection, once a day, for 21 days. Intraperitoneal injection of cisplatin group according to 3mg/kg, once a day, stay 7 days, after 21 days to be put to death. Genistein low dose puls cisplatin group: its preparation method with two drug dosing method separately.5 Weigh nude mice and measure transplanted tumors’ volume and weight: since transplanted tumors, we began to observe nude mice mental state, diet, defecate, activity and growth, everyday. Before medication and weekly after medication measure nude mice weight, the longest diameter(a) and shortest path(b) of transplanted tumor. According to V(mm3) =πab2/6, calculate the tumor volume. In the end, get and weigh tumor tissue. According to IR(%) =(1- the average tumor weight of treatment group /the average tumor weight of control group) × 100%, calculate the inhibitory rate of tumor.6 Detect the expression of E-cadherin m RNA and mi RNA-200 a in the transplanted tumor tissue by RT-q PCR technology.7 Detect the expression of E-cadherin protein in transplanted tumor tissue by immunohistochemical staining.8 Statistical methods: Use SPSS21.0 statistical software to deal with the experimental data. With P<0.05 as the standard, there is statistical significance.Results:1 Medication process was smooth.(1) During the experiment, the mice’s weight gain of the cisplatin group was not obvious. Comparing the cisplatin with the control, there was statistically significant difference. For nude mice weight growth, comparing both of three different doses of genistein groups and the control, there is no significant difference.(2) In aspects of mental state, diet, activity, the mice of the cisplatin group are less than the combination’s. Comparing both of three various doses genistein groups and the control group, there was no difference; and one of other groups was better than the cisplatin and the combination groups.(3) With the extension of time, all tumors were gradually increasing. In the end, the average tumor volume of any treatment group is smaller than the control group’s. Inhibitory rates of groups were: the combination group>the cisplatin group>the high dose group of genistein>the middle dose group>the low dose group.2 Expression of E-cadherin m RNA and mi RNA-200 a in transplanted tumor tissue: all groups had expression of E-cadherin m RNA and mi R200 a. The expression in the cisplatin and the control was lower than others. With increase of genistein dose, expression were enhanced. As for E-cadherin m RNA, comparing the control with the cisplatin, there was no statistical difference. Except comparison of the middle and the high, difference of both of three doses genistein groups and the control group was no significant. In term of mi RNA200 a expression, difference among the cisplatin, the combination, the low dose and the control was no statistical significant. Among three various dose genistein groups and the control group, except comparison the high dose with the low dose and the control, differences of comparison both of the rest had no statistically significance.3 Expression of E-cadherin protein in transplanted tumor tissue: there were E-cadherin expression in transplanted tumor tissue of all groups. The expression in the control, the cisplatin, the combination and genistein low dose group was low. In others’ the expression was high, and with increase dose of genistein, the expression was enhanced. Among the cisplatin, the combination, the low dose and the control, compared with each other, there was no statistically significant difference. Among the three various dose groups of geinstein and the control, expect the high dose and the middle dose compared with the control, difference of the two other groups had no statistically significant.Conclusions:1 Genistein while inhibiting tumor growth, will not affect the survival state of nude mice. And it may reduce the side effects of chemotherapy drugs.2 Both genistein and cisplatin can inhibit tumor growth, and with the increase of genistein, the inhibition also enhanced; combination therapy than alone has stronger inhibitory effect. But there were no significant difference, which may be related to the small sample size.3 Genistein can increase expression of E-cadherin m RNA and mi RNA200 a, and it showed dose dependent. With increase dose of genistein, the expression of E-cadherin was enhanced. |
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