| Objective:Ovarian cancer is one of the three common gynecological malignancies.Epithelial ovarian cancer(EOC)is the most common form of ovarian cancer accounting for 90% of it.Because of the ovaries located in the bottom of the pelvic cavity and most early lesions are not easy to be found,so once developed symptoms,they have reached advanced.In recent years,although the method that primary cytoreductive surgery combined with chemotherapy have made great progress,the 5-year survival rate of EOC is still stuck at 20%-30%.It's mortality is the highest and prognosis is the worst during the gynecologic malignancies.Currently,platinum based chemotherapy is the main adjuvant therapy after surgery,but a series of adverse reactions caused by chemotherapy drugs which influence patients' psychology and physiology,to some extent,affect the prognosis.Consequently,it's crucial to look for low toxicity and high efficiency methods.Recently,inositol hexaphosphate(IP6)which found in the dietary fiber have important inhibiting effect on tumors during the related studies.Dr.Abulkalam M.Shamsuddin from University of Maryland and researchers from American Institute for Cancer Research(AICR)investigated IP6 could prevent and treat many diseases.It can inhibit the growth of cancer cells and shrink the tumor volume;suppress free radicals and protect cells against free radical damage;prevent kidney stones;reduce blood lipid concentration;protect the myocardial cells avoiding heart disease;prevent atherosclerosis,etc.A large number of literature reports,IP6 can prevent cancers by regulating cell signal transduction pathways,or promoting or inhibiting the expression of certain genes and proteins.But IP6 should be used along with inositol to play it's best effectiveness,as IP6+inositol can react to IP3 in the body.There are many studies and reports about animal experiments suggested that IP6+inositol can prevent cancers effectively,such as intestine cancer,colon cancer,liver cancer,prostate cancer,breast cancer and so on.Will it be effective on the ovarian cancer? We don't know,because it hasn't been reported at home and abroad.This experiment aims to explore the inhibition mechanism of IP6 on the ovarian cancer by observing and analyzing IP6+inositol inhibit the epithelial ovarian cancer xenograft in nude mice and affect the expression of apoptosis related protein Bax and miRNA-21 and as well added to cisplatin.Methods:1 Cell culture: The human epithelial ovarian cancer cell lines SKOV3 were cultured as usual to establish the xenograft in nude mice.2 The establishing of the xenograft in nude mice and experiment groups: Balb/c/ Nude mice,4~6 weeks-old,16~18g,female,were fed in the environment of SPF,while SKOV3 were regular training in a sterile environment.Digesting the SKOV3 cells when they were in logarithmic phase,and making into single cell suspension,which concentration was about 2.5× 10 7 / ml,injecting to subcutaneous inoculation in nude mice in a sterile environment,each inoculation only 0.2 ml(about 5 × 10 6 / only).Seven days later,we selected the nude mice transplanted tumor diameter in 3 to 5 mm to the groups.They were randomly divided into 4 groups,six mice respectively,control group,IP6 + inositol group,cisplatin group and IP6 + inositol in combination with cisplatin group.3 The preparation of experimental drugs: All drugs in this experiment were configured to the required concentration with 0.9% sodium chloride injection,and using them right after they were ready.4 Dosing method: The IP6+inositol group were given the content of IP6+inositol 300mg/kg by gavage every day,for 28 days;the cisplatin group were given the content of cisplatin 3mg/kg by intraperitoneal injection once a day,for 7 days;the control group were given the same volume of 0.9% sodium chloride injection every day,intraperitoneal injection the same volume of 0.9% sodium chloride injection every day;the combination group gavage 300mg/kg/d IP6+inositol,and 3mg/kg/d cisplatin intraperitoneal injection.5 Weight of nude mice & weight and volume of xenograft: We observed the state of the mice every day since begining of this experiment,such as mental status,diet,urine and excrement and so on.We measured the weight of the mice and the longest diameter of transplanted tumor(a)and the shortest(b)before using drugs and every week after using drugs respectively,then calculated the volume of the xenografted tumor according to V(mm3)=?ab2/6 and the tumor inhibition rate according to IR(%)=(1-the average tumor volume of the treatment group / the average tumor volume of the control group)×100%.We terminated of observation in drug withdrawal 24 hours and then killed the mice by cervical dislocation,wrung subcutaneous tumor tissue and weighted.6 Immunohistochemical staining to detect the expression of Bax protein in the transplantation tumor tissue: We took the transplanted tumor tissue which was fixed by the 4% paraformaldehyde(less than 0.5cm × 0.5cm × 0.5 cm)to stain through immunohistochemical,and studied the expression of apoptosis proteins,Bax.7 RT-PCR to detect the expression of mi RNA-21 in the transplantation tumor tissue: We took part of xenograft in-80 ?refrigerator without any reagent treatment to determinate the expression of miRNA-21 with RT-PCR.8 Statistical methods: We processed the datum with SPSS13.0 statistical software.Variety comparison between the mean was used the single factor analysis of variance,and the hierarchy data was used K-W rank-sum test.The experimental results were expressed in (?)ąS,P < 0.05 for the difference was statistically significant.Results:1 The experimental process smoothly,tumor cell growth in good condition during the cell cultured process,established nude mice model successfully.The status of the nude mice during the experiment good to bad sort: control group,IP6 + inositol group,combination group and cisplatin group.At the begin of the experiment,the weight of nude mice of the cisplatin group loss.With the experimental progress,the weight of each group of nude mice has increased than earlier,cisplatin group than control group in slow growth,difference was statistically significant;the other two groups closely growed with the control group,there was no statistically significant difference.The volume of the xenograft grew than earlier as well,and the volume of each treatment group was less than the control group of the same time,and the combination group's was less than the two single treatment groups',the differences were statistically significant(P < 0.05).The weight of xenograft of each treatment group was less than the control group at the 29 day,and the combination group's was less than the two single treatment groups',the differences were statistically significant(P < 0.05).2 The expression of the Bax in all three treatment groups was higher than the control group's,the difference was statistically significant(P < 0.05).Although the Bax of the combination group was significantly higher than the IP6+inositol group(P < 0.05),not significantly higher than the cisplatin group(P?0.05).3 The expression of miRNA-21 in IP6+inositol group and combination group was lower than the control group's,the difference was statistically significant(P < 0.05).But the cisplatin group's was closely to control group's.Conclusions:1 The volume and weight of transplantation tumor of IP6+inositol group were less than the control group's,and the combination group's was less than cisplatin group,and the differences had statistical significance.It shows that IP6+inositol has the anti-tumor effect on epithelial ovarian cancer.And they have synergistic effect.2 The influences of general status on and weight of nude mice of IP6+inositol group and combination group were less than cisplatin groups'.It shows that IP6 has the characteristics of low toxic side effects,and the combination can reduce chemotherapy side effects.3 The expression of Bax of the treatment groups was higher than the control group's and the combination group was higher than the IP6+inositol group,the differences were statistically significant.Suggesting that the IP6 plays anti-tumor effect maybe through up-regulating the expression of Bax probably.4 The expression of miRNA-21 of the IP6+inositol and combination groups was lower than the control group's,the difference was statistically significant.Suggesting that the IP6 plays anti-tumor effect through down-regulating the expression of miRNA-21 probably. |
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