| Ovarian cancer is one of three most gynecological malignancies, ofwhich the morbidity is in the third place in female reproductive duct. But themortality rate ranks first in gynecological tumors. Ovarian cancer was thoughtto have no symptoms until the very late stages of disease, it is easy torecurrence and metastasis.It would be difficult to cure and the prognos ispoor.Ovarian cancer is a serious threat to the majority of women’s lives andhealth. Surgery and Chemotherapy are important in treatment of ovariancancer, but resistance to chemotherapy is a significant obstacle to thetreatment of ovarian cancer. Therefore, searching new drugs and new targetsfor the therapy of ovarian cancer has become an urgent need.Genistein (4’,5,7-trihydroxyisoflavone), a weak phytoestrogen and anaturally occurring soy isoflavone, is found in legumes.The research fromhome and abroad indicates that Genistein inhibits the invasion and metastasisof cancer in many stages of neoplastic process.Genistein has multiplebiological functions such as estrogen-like effect, cell cycle regulation,induction of apoptosis and regulation of signaling pathways and so on. It hasbeen reported to inhibit the growth of many kinds of cancer.Many studiesabout Genistein have focused on breast cancer, prostate cancer, lung cancer,colorectal cancer, leukemia lymphoma melanoma and the other cancers.However the vivo studies about ovarian cancer is limited. In This experiment,we targeted at ovarian serous papillary cystadenocarcinoma in the nude mice,investigated Genistein’s inhibitory effect on HMGA2(High mobility groupprotein) and let-7a. It provides a theoretical basis for Genistein’s clinicalapplication and further development in ovarian cancer.Methods:1Cell culture and Preparation of cell suspension: Human ovarian serous papillary adenocarcinoma cell line SKOV3were maintained in RPMI-1640containing10%fetal bovine serum (FBS)，100u/L penicillin and100u/Lstreptomycin in Power Of Hydrogen7.4.The cell were subcultured in theincubator containing5%of CO2at37℃. Conventional culture SKOV3cellsuntil the cells were cultured in logarithmic growth phase, then the cell staiedclose to culture flask wall and digested the cells with0.25%trypsin, thedigestion was terminated with10%fetal bovine serum, when the cells weresufficiently dispersed, counted and resuspended in RPMI-1640containingSerum-free at a concentration of2.5×107/ml.2The establishment of the nude mouse model and the experimentalgroups:4to6-week-old BALB/C/nu/nu athymic nude mice were bred inthe SPF grade environment.1ml Syringe of the cell suspension (about5×106cells per nude in0.2ml cell suspension) was inoculated at subcutaneous of thenude mouse’s dorsal near upperlimb in sterile conditions. After7days ofinoculation, choose the successful model with transplanted tumor about3~6mm in diameter and randomly divided into six groups，group1: control groupwith6nude mice （Hydroxypropyl-β-Cyclodextrin solution）; group2:Genistein group with6nude mice (50mg/kg/day); group3:Genistein groupwith6nude mice (150mg/6kg/day);group4: Genistein group with6nudemice (450mg/kg/day);group5: Cisplatin group with6nude mice (3mg/kg/day);group6: Genistein+Cisplatin combination group with6nude mice(thedose with simple administration).3Preparation of experimental solutions: Genistein solution wereinclusion complex(containing6%Genistein,94%hydroxypropyl cyclodextrinbeta) solution which were dissolved in0.9%sodium chloride injection.Thesolution passed through a filter under aseptic conditions before it was used.It is prepared once every day.Cisplatin injection was diluted into3mg/mlconcentration by0.9%sodium chloride injection and should be used instantly.4Dosage and Administration: Genistein low-dose group（50mg/kg/day），medium-dose group（150mg/kg/day）and high-dose group（450mg/kg/day）Genistein group,intraperitoneal injection for21days; Cisplatin group （3mg/kg/day）intraperitoneal injection for7days; combination group,50mg/kg/day of Genistein intraperitoneal injection for21days and3mg/kg/day of Cisplatin intraperitoneal injection for7days; control group,intraperitoneal injection of the same volume of Hydroxypropyl-β-Cyclodextrinsolution once a day for21days.5The weight of nude mice and the xenografted tumor’s volume andweight: observed the growth, diet, activity and stool of the nude miceeveryday; from the first day of experiment, measured the weight of the nudemice and the xenografted tumor with calipers every week, the longestdiameter a and the shortest diameter b. Calculated the volume of xenograftedtumor according to the formula:V (mm3)=πab2/6.Calculated the theinhibition rate of xenografted tumor according to the formula: IR(%)=(1-theaverage tumor weight of treatment group/the average tumor weight of controlgroup)×100%. At the end of the observation the nude mice were euthanizedin a neck method and the tumor were removed and weighed and measured thevolume under aseptic conditions.6Immunohistochemisty to detection the HMGA2protein expression inthe tumor tissues: take the4%paraformaldehyde fixed pieces of xenograftedtumors (less than0.5×0.5×0.1cm3) for immunohistochemical staining, andresearch the expression of inhibitor of HMGA2.7Use RT-qPCR to detect the expression of let-7a of the xenograftedtumor tissues: Take the xenografted tumor tissues which were stored at-80℃refrigerator and without reagents fixed, using RT-qPCR technology to detectthe expression of let-7a in the xenografted tumor.8Statistical methods: The experimental data were analysed andconducted with SPSS13.0statistical software.The multiple sample meandifference was compared with the one-factor analysis of variance and factorialdesign variance analysis.To compare the level grade data after ranking theconverted multiple independent samples with variance analysis,P＜0.05fordifferences with statistical significance. Results:1The whole experimental process goes well, only the nude mice ofCisplatin group have a mental fatigue slightly and a normal diet.In the middleof the experiment, the activity is slow slightly and defecate is normal.There isno obvious toxicities in other groups.With the development of theexperimental process,the weight of experimental nude mice has increased.Theweight of nude mice in Cisplatin group is less than the control group, thedifference was statistically significant. The weight of nude mice in Genisteingroup is heavier than the control group, there was no statistically significantdifference.The weight of nude mice in Combination group was less than thecontrol group, there was no statistically significant difference.2The weight and volume of xenografted tumors in Genistein group wereless than control group, the difference was statistically significant andGenistein inhibit tumor growth in a dose dependent manner.The weight andvolume of xenografted tumors in combination group were less than cisplatingroup and low-dose Genistein group, there was no statistically significantdifference.3The expression of HMGA2in experimental group were lower than thecontrol group, the difference was statistically significant.4The expression of let-7a in Genisteine group were higher than thecontrol group, the difference was significant statistically,the expression oflet-7a was markedly coincident with Genistein dose,there was statisticallysignificant difference between two groups.The expression of let-7a incombination group was less than cisplatin group and low-dose Genisteingroup, the difference was statistically significant.Conclusions:1Genistein is a natural substance without obvious toxicities and itinhibits the growth of tumor cells in a concentration dependent manner. Thecombination of Genistein and Cisplatin can improve the antitumor effect.2The expression of HMGA2in Genistein group was lower than thecontrol group, the difference was statistically significant.The expression ofHMGA2in Cisplatin group were lower than the control group, the difference was statistically significant. It was indicated that Genistein and Cisplatin couldpromote cell apoptosis, and it probably could inhibit tumor growth byinhibiting the expression of HMGA2.3The expression of the let-7each group were higher than control group,it showed that cisplatin and Genistein can promote the expression of the let-7a.Cisplatin and Genistein both have a obvious synergy, they probably couldinhibit tumor growth by promoting the expression of the let-7a. |
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