| Objective: In gynecologic malignant tumors，the mortality rate ofovarian cancer is highest. Because of its occurrence and development has thecharacteristics of concealment, about70%of patients are in advanced whenfound (FIGO stage IIB-IV), and the five-year survival rate is less than40%.Cytoreductive surgery combined with postoperative chemotherapy hasbecome the standard scheme for ovarian cancer therapy. But the recurrence ofintravenous chemotherapy is high.Intraperitoneal chemotherapy may beextended prolong disease-free survival and overall survival, but its side effectis bigger, so its application is restricted. Therefore, to find low toxicity andeffective anti-cancer agents has become one of the important subject ofovarian cancer therapy research.There are three main kinds of flavonoids naturally present in Soybean,daidzin,genistin and glycitin. Among them, genistein is the most abundant,and most active. The chemical, called4,5,7-3hydroxy isoflavones. Studieshave shown that genistein has a variety of biological activity, such asestrogen-like effects, anti-estrogen-like effects, antioxidant and cell cycleregulation. In recent years because of its significant anti-cancer effect,it hasreceived widespread attention. However, genistein has poor solubility in water,and the oral absorption efficiency is very low. In vitro and vivo experiments,the vast majority of researchers through the use of dimethyl sulfoxide (DMSO)dissolved genistein. But the side effects of DMSO on the cells can not beignored, not only affect the experimental results, but also limit the clinicalapplication of drugs. Hydroxypropyl-β-cyclodextrin is a kind of non-toxic,small irritation material,and can be used for intravenous injection. Flavonoidsare clathrated by hydroxypropyl-β-cyclodextrin, the drug bioavailability isenhanced,and solubility and stability has been improved,too. Recent studieshave found that microRNAs is an important participant in the formation of tumor, the development of tumor is associated with abnormal microRNAsexpression.It plays the role of oncogene or tumor suppressor gene. In thisstudy,we treat human ovarian epithelial carcinoma transplantedsubcutaneously in nude mice as the research object, using the genisteinhydroxypropyl-β-cyclodextrin inclusion, to explore the inhibitory effect ofGenistein on human ovarian cancer transplanted subcutaneously in nude miceand its effect on PTEN protein and microR-21, and provide a theoretical basisfor the application of Genistein in epithelial ovarian cancer clinical treatmentand prevention.Methods:1Cell culture: Human ovarian carcinoma SKOV3cells, cultured inRPMI-containing10%fetal bovine serum medium1640, incubator fortemperature is37℃, containing5%CO2.2The establishment of the nude mouse model and the experimentalgroups: BALB/C/nu/nu nude mice,5to6-weeks-old,18-22g, female,were bredin the SPF grade environment. Conventional culture SKOV3cells until thecells reached logarithmic phase, digested with0.25%trypsin, centrifugal,preparation of2.5×107/ml single cell suspension.The cells were inoculatedin nude mice after subcutaneous limb at the back under sterile conditions.Each nude mice inoculated with0.2ml (5×106cells) of single cell suspension.7days later, transplanted tumor diameter of4~7mm in nude mice wasselected, randomly divided into six groups: The control group(hydroxypropyl-β-cyclodextrin),Cisplatin group,Genistein clathrate low dose group,Cisplatin+Genistein clathrate low dose group, Genistein clathrate middle dose group,Genistein clathrate high dose group, each group of six.3Preparation of experimental drugs: Genistein inclusion, hydroxypropyl-β-cyclodextrin, cisplatin were prepared using0.9%Sodium ChlorideInjection required concentration, immediate use.4The method of administration: Genistein inclusion complexescontaining6%genistein, the doses of genistein is50mg/kg,150mg/kg and450mg/kg, intraperitoneal injection, once a day, for three weeks; The control group according to Genistein low dose group of inclusion amounthydroxypropyl-β-cyclodextrin in abdominal cavity injection, once a day, forthree weeks; Cisplatin group at3mg/kg intraperitoneal injection, once a day,for7days, nude mice were killed after3weeks; combination group: genisteinis50mg/kg,150mg/kg and450mg/kg, intraperitoneal injection, once a day,for three weeks and Cisplatin at3mg/kg intraperitoneal injection, once a day,for7days.5The nude weight and the tansplanted tumor’s volume and weight: Weobserve in mental state, diet, stool, activity and the growth situation everyday;Before treatment, the weight of nude mice was measured,as well as thelongest diameter (a) and shortest path (b) of transplantation.After treatment,the weight of nude mice was measured weekly,as well as the longest diameter(a) and shortest path (b) of transplantation, According to the formula V (mm3)=πab2/6calculate the tumor volume.3weeks later the nude mice were killedby cervical dislocation.Obtained tumor tissue, and weighing. To calculate thetumor inhibition rate of tumor IR (%)=(1-average tumor weight of treatedgroup/mean tumor weight in control group)×100%.6Immunohistochemical staining to detect the expression of PTENprotein in transplanted tumor tissue.7Use RT-qPCR technology detecting microR-21in the transplantedtumor tissue, the expression of PTEN mMRA.8The statistical method: SPSS13.0statistical software processing of theexperimental data. Level data by rank convert multiple independent samplescomparison Student-Newman-kelus test. Factorial design and single factorvariance analysis method were used for the analysis of measurement data.With P <0.05as the standard, the difference has statistical significance.Results:1Administration process was smooth. Cisplatin group and combinationgroup nude mice appeared emaciated, mental state is poor, and with theincreased weeks of age, body weight decreased, And compared with thecontrol group, the difference had statistical significance. The other group nude mice weight all increased, but compared with the control group, no significantdifference. In the beginning of medication, no difference between the groupsof tumor volume. With the extension of time each group of tumor volumeincreased gradually, Administration for twenty-second days,the transplantedtumor average volume are smaller than in the controls. The inhibition rateswere: the combined group>cisplatin group>low dose group Genisteinclathrates; Genistein inclusion of high dose group>middle dose group>lowdose group. Cisplatin and genistein inclusion compound can inhibit the growthof tumor, but combined with cisplatin and genistein inclusion compound is notobvious synergistic effect.2PTEN protein expression in xenograft tumor tissue: The three differentdoses of genistein inclusion can increase the expression of PTEN protein,compared with the control group, the difference was statistically significant.But the cisplatin group had no obvious effect on the expression of PTENprotein. The combined group statistics showed that the two drugs had nosynergistic effect on the expression of PTEN protein. With the correspondingof PTEN protein, the expression of PTEN mRNA results also have the aboverules.3Xenograft tumor tissue miR-21expression: The three different doses ofgenistein inclusion can decrease the expression of miR-21, compared with thecontrol group, the difference was statistically significant. But the cisplatingroup had no obvious effect on the expression of miR-21. The combinedgroup statistics showed that the two drugs had no synergistic effect on theexpression of miR-21, The result showed that the cisplatin could not reducethe expression of miR-21.Conclusions:1Cisplatin and Genistein inclusion compound both have the inhibition oftumor growth, and with the dose of genistein increased inhibition alsoenhanced. Combination therapy has a little high inhibitory effect on tumorgrowth than alone therapy group, But statistical analysis had no significantdifference, May be the reason for the small sample size. 2Genistein inclusion can significantly increase the expression of PTENprotein, and showed dose dependent. But the Cisplatin had no effect on theexpression of PTEN protein, and there is no obvious synergistic effect ofcombined medication.3Genistein can inhibit the expression of miR-21. Increased theexpression of PTEN mRNA, and showed the dose dependent manner. But thecisplatin had no significant effect on the expression of miR-21and PTENmRNA, and there is no obvious synergistic effect of combined medication. |
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