The Inhibition Effect Of IP6+Inositol On Epithelial Ovarian Cancer Xenograft In Nude Mice

Background: Ovarian malignancy mortality,threatening women's health and life,is the first in gynecologic malignant tumor,especially the epithelial ovarian carcinoma.In recent years,lacking of early prevention screening,diagnosis and timely methods,and the lasting of unresolved chemotherapy drug resistance,the five-year survival rate is still hovering around 20% 25% which is based on ideal cytoreductive surgery,although the chemotherapy has made important progress.Therefore,to find new effective anti-cancer agents has become one of the important subject of ovarian cancer therapy research.IP6?Inositol hexaphosphate?exist in cereal bran and beans in the form of Inositol phosphate calcium and magnesium salts.With its strong antioxidant and anti-cancer effect,has it become a hotspot in anticancer drugs in recent years.And in vivo and in vitro experimental study of various types of malignant tumors,such as colon cancer,lung cancer,breast cancer,prostate cancer and other types of cancer,the results show that IP6 have effective anticancer effect.IP6 and inositol can act in synergy,which means combining them together can strengthen each other's role.At present,the main mechanism of IP6 involve the immunologic cytotoxicity on tumor cells,promoting the apoptosis of cancer cells,cytotoxicity,anti mutation and DNA repair,inhibition of telomerase and antioxidant protection ect.EMT?epithelial-to-mesenchymal transition?has been confirmed a crucial step of tumor invasion and metastasis.MicroRNA-200?miRNA-200?family can induce the epithelial cell differentiation and EMT.N-cadherin and E-cadherin as members of Cadherin family are considered to be the marker protein of tumor invasion and metastasis.Studies show that N-cadherin promotes tumor progression,on the contrary,E-cadherin act as invasion inhibition factor.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 IP6+Inositol and low dose IP6+Inositol puls cisplatin,by observing the transplanted tumors volume,weight,inhibitory rate and miRNA-200 a and N-cadherin protein expression,discuss IP6+Inositol inhibition on epithelial ovarian cancer xenograft in nude mice,as to provide theoretical basis for clinical application of IP6+Inositol.Methods:1 Cell culture : Human ovarian carcinoma SKOV3 cells are cultured in RPMI-1640 medium.Put culture bottles in temperature of 37?,containing 5%CO₂.2 Establish nude mouse transplantation tumor model and the experimental groups: BALB/c Nude nude mice,female,4⁶ weeks-old,16¹8g,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 5 days,select the kind of transplantation tumor model,whose tumor diameter are from 3⁵mm.And randomly divided into six groups:The control group,IP6+Inositol low dose group,IP6+Inositol high dose group,Cisplatin group,Cisplatin puls IP6+Inositol 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 IP6+Inositol respectively according to the content of IP6+Inositol 300mg/kg and 600mg/kg,receive Gavage,once a day,for 28 days.The control group is the,receive 0.9% sodium chloride by Gavage,once a day,for 28 days.Intraperitoneal injection of cisplatin group according to 3mg/kg,once a day,stay 7 days,after 28 days to be put to death.IP6+Inositol 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.In the end,after stopping the drug sacrifice the nude mice,weigh tumor tissue and get the volume according to V?mm³?=?ab²/6.the longest diameter?a?and shortest path?b?of transplanted tumor.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 mi RNA-200 a in the transplanted tumor tissue by Real Time-PCR technology.7 Detect the expression of N-cadherin protein in transplanted tumor tissue by immunohistochemical staining.8 Statistical methods: Use SPSS13.0 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)In aspects of mental state,diet,activity,the mice of the cisplatin group are less than the combination's.Comparing both of different doses IP6+Inositol groups and the control group,there was no difference;and one of other groups was better than the cisplatin and the combination groups.2)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 IP6+Inositol >the low dose group.2 Expression of miRNA-200 a in transplanted tumor tissue: all groups had expression of miR200 a.The expression in the different dose group of IP6+Inositol and the combination group was higher than others.With increase of IP6+Inositol dose,expression were enhanced.except the cisplatin group compared with the control group,and the low dose with the combination group,differences of comparison both of the rest had no statistically significance.3 Expression of N-cadherin protein in transplanted tumor tissue: there were N-cadherin expression in transplanted tumor tissue of all groups.The expression in the control,the cisplatin,the combination and IP6+Inositol low dose group was high.In others' the expression was low,and with increase dose of IP6+Inositol,the expression was weaken.Among the cisplatin,the combination,the low dose and the control,compared with each other,there was no statistically significant difference.Among the two different dose groups of IP6+Inositol and the control,except the high dose compared with the control,difference of the two other groups had no statistically significant.Conclusions:1 IP6+Inositol 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 IP6+Inositol and cisplatin can inhibit tumor growth,and with the increase of IP6+Inositol,the inhibition also enhanced;combination therapy has stronger inhibitory effect than alone.And the there was statistically significant difference.3 IP6+Inositol can increase expression of miRNA200 a,and it showed dose dependent.With increase dose of genistein,the expression of N-cadherin was weaken.