The Influence Of Genistein On Ovarian Cancer Xenografts In Nude Mice Model

Objective:Ovarian cancer is one of the three malignancies endangeringwomen’s health,the majority of patients are at advanced stage during treatmentdue to lack early diagnosis.Based cytoreductive surgery,it is difficult tocompletely cure,ovarian cancer needs chemotherapy continuation oftreatment,it accounts for the highest mortality in the female reproductive tractdiseases.Although the application of platinum-based combinationchemotherapy works in the majority of ovarian cancer, but the long-term toxiceffects of chemotherapy, drug resistance and the recurrence rate after drugwithdrawal make chemotherapy application limited,especially in advancedovarian cancer treatment.Long-term survival in patients has almost noimprovement over the past30years，its five year survival rate is only about20﹪.To overcome these problems,looking for a new better combinationchemotherapy regimens which can both kill tumor cells and reduce thesystemic toxicity has become a major research direction.Genistein, whose chemical name is4，,5,7-trihydroxy isoflavone, is weakphytoestrogen.It is primarily found in soybeans.In vivo and in vitro studieshave shown that genistein has prominent effect in inhibiting tumor cell growthof breast, prostate and colon cancer. Epidemiological studies have also foundthat long-term intake of soy foods can reduce the incidence of breast cancer,prostate cancer,colon cancer, head and neck cancer,this is mainly due togenistein.There are multiple inhibitory effect of genistein in tumor occurrenceand development stage,its anti-tumor mechanism includs regulating estrogenreceptor,inhibitting both protein tyrosine kinase activity and angiogenesis,andenhance the activity of antioxidant enzymes,regulating the cell cycle,andinducing apoptosis,inhibitting the activity of topoisomerase.It is associatedwith the low risk of the occurrence of a variety of tumors.Genistein also can be used as an adjunct therapy to improve the sensitivity of tumor cells toradiation,to enhance the anti-tumor effect of chemotherapy drugs and toreduce tumor recurrence and metastasis.Epidemiological investigation,in vivoand in vitro experiments,anti-cancer mechanism studies have shown thatgenistein has a certain degree of tumor suppressor and anti-cancer effect,thereare conflicting results of studies of genistein,its effects requires more in vivoexperiments to verify.What’s more，we still need large sample,prospectivestudies to evaluate the anti-tumor effect of genistein,which provide strongerevidence for the prevention and treatment of ovarian cancer and othermalignancies.Undoubtedly,the soy isoflavones phytoestrogens have a widerange of potential clinical application, which needs further study.In vivostudies on the influence of Genistein on ovarian cancer are still in lack.Thisexperiment, taking human ovarian cancer xenograft as a model,studiesGenistein and paclitaxel effect of subcutaneous ovarian cancer in nudemice,providing certain theory basis for the Genistein therapy of ovariancancer.Methods:1Cell culture:Human epithelial ovarian cancer strain SKOV3is culturedaccording to conventional method,when the cells grow to logarithmicphase,they are digested with trypsin into single cells, and continue to foster insub-bottle.2Preparation of test drug：Genistein is dissolved in RPMI-1640into astock solution （30mg/ml） stored at-20℃,and diluted to the desiredconcentration(5mg/ml) during experiment.Paclitaxel injection is diluted into3mg/ml concentration by0.9%sodium chloride injection,and should be usedinstantly.3Construction of transplanted tumor model in nude mice:Totally24experimental animals use nude mice.They are female and4-5weeks old withweight of16-20g.SKOV3cells are diluted with phosphate buffer solution to aconcentration of25×106/ml,in sterile conditions are inoculated on every nudemice dorsal proximal hindlimb of subcutaneous of0.2ml.They are randomly divided into four groups(control group,Genistein group,Paclitaxelgroup,Genistein and Paclitaxel combination therapy group).4Treament methods:After7days of inoculation we administrate thenude mice.Genistein group50mg/kg/d,gavage,for4weeks.Paclitaxel group30mg/kg/w,intraperitoneal injection,for4weeks.The combination therapygroup gavage Genistein50mg/kg/d,intraperitoneal inject Paclitaxe30mg/kg/w.The control group gavage the same volume of RPMI1640everyday,intraperitoneal inject the same volume of0.9%sodium chloride injectioneveryweek.5Monitoring indicators:5.1Nude mice weight and xenograft tumor’s volume and weight:Thediet,spirit change and stool and urine are observed everyday,the nude mice’sweight and the longest diameter(a) and shortest diameter(b) of transplantedtumor are measured respectively at the beginning of the treatment and everyweekend,the tumor volume is calculated according to formula.Nude mice issacrificed24hours after drug withdrawal,stripping the tumor tissue,andmeasuring its weight,the inhibitory rate is calculated according to formula.5.2Cell morphology and apoptosis are observed under lightmicroscope.5.3RT-qPCR is used to measure miRNAlet-7expression.5.4Iummunohistochemistry is used to measure cyclinG2expression.6Statistical methods:To process experiment data with SPSS13.0statistical software.The compare of multisamplet mean use factorial designinformation variance analysis,P＜0.05for differences with statisticalsignificance.After rank transformation,level data use SNK to comparebetween two methods.Results:1The impact of four groups medication on nude mice weight,the transplanted tumor volume, weight, and tumor inhibitory rate:The nudemice body weight of four groups has no significant difference. We observe thenude mice without diarrhea,dispirited spirit,weak appetite etc.Tranplantedtumor volume of medication groups has less increase than the control group,the combination medication group with the least tumorincrease.Transplantation tumor volume of the29th day of medication group isless than control group,the difference is statistically significant.The tumorvolume of combination therapy group is less than single drug groups,but thedifference has no statistical significance.The weight of transplantation tumorof medication groups is less than that of control group,the difference isstatistically significant. The tumor weight of combination therapy group is lessthan single drug groups,but the difference has no statistical significance.Inhibitory rate of combination group is higher than that of single drug groups.2Tumor tissue morphology changes of different groups:Tumor cells with atypia are all visible in each group under lightmicroscope.Compared with the control group,the treatment group atypia haslower degree,among which the combination group’s cell atypia has the lowestdegree.Nuclear pyknosis and fragmentation is available.3Measurement of miRNAlet-7expression:The expression of let-7inmedication groups is higher than in control group,and that in combinationgroup is higher than in single drug groups. the difference is statisticallysignificant.4Measurement of cyclinG2expression: The expression of cyclinG2inmedication groups is higher than in control group,the difference is statisticallysignificant.That in the combination group is higher than in single druggroups,the difference has no statistical significance.Conclusions:1The transplanted tumor volume and weight on the29th day are lessthan that of control group,and the difference is statistically significant.Inhibitory rate of combination group is higher than that of single druggroups.All of these show Genistein has the effect of inhibiting the growth ofovarian cancer cells and the combination with Paclitaxel adds to the effect oninhibiting tumor.2Expression of miRNAlet-7in Genistein group and combination groupare higher than that of control group,the combination group’s expression is the highest,and the difference is statistically significant,showing that Genisteininhibits the proto-oncogene thus it has the effect of antitumor,and enhance therole of Paclitaxel chemotherapy.3Expression of cyclinG2in Genistein group and combination group arehigher than that of control group,and the difference is statistically significant,the combination group’s expression is the highest,showing that Genisteininhibite ovarian cancer metastasis through blocking cell cycle and inhibitingcell proliferation,and enhance the role of Paclitaxel chemotherapy.