Study On The Enhancement Of Tumor Cell Killing Of Cyclophosphamide By Ginkgo Biloba Extract

Background:Breast cancer is a common malignant tumor in the world which seriously endangers women's health.According to the Journal of the American Cancer Society in 2018,the incidence of breast cancer among women cancer is 24.2%worldwide,and the new incidence is about 2 million,which is the highest incidence of cancer in women.According to the National Cancer Center of China,the number of new breast cancer cases in China was 27.49 per 10,000 cases in 2014,the incidence is also on the top,and the prevalence of breast cancer is increasing year by year,and the age of onset is tending to be younger.Nowadays,with the development of medical technology,the treatment ofbreast cancer has entered the era of comprehensive treatment,the treatment methods include surgery,chemotherapy,radiotherapy,endocrine therapy,molecular targeted therapy and biological immunotherapy.Surgery is an essential treatment for breast cancer.doctor will according to the pathological stage of breast cancer,hormone sensitivity,genes,etc,and then give different adjuvant treatment options.But no matter which protocol is used,most patients will be exposed to chemotherapy,so chemotherapy plays a very important role in the treatment of breast cancer.As a commonly used chemotherapeutic drug,cyclophosphamide(CTX)is inactive in vitro.After entering the human body,it undergoes biotransformation through the liver.The metabolite phosphoramide mustard damages tumor cell DNA,blocks cell cycle,inhibits tumor cell proliferation and promotes tumor apoptosis,but the large dose use of CTX in clinical long-term treatments can cause strong side effects.CTX metabolism in the body produces a large amount of reactive oxygen species(ROS),which interferes with the redox balance in the body and increase the adverse reactions of various organs in the body.Therefore,if a preparation capable of effectively scavenging free radicals and assisting in killing tumor cells product can be found,the toxic side effects caused by CTX can be effectively alleviated,and the killing effect of CTX on tumor cells can be enhanced.Ginkgo biloba extract(GBE)is an active ingredient extracted from Ginkgo biloba leaves,it's main ingredient is flavonoids,has a good function of scavenging free radicals and antioxidants,and has the effect of killing tumor cells.Therefore,this experiment established a mouse EMT6 breast cancer model,alone and in combination with CTX and GBE,to evaluate the effect of GBE on enhancement the effect of CTX on killing tumor cells and explore its possible mechanism.Objective:To study the effect of Ginkgo biloba extract on the enhancement of killing of tumor cells by cyclophosphamide and its possible mechanism.Method:1.Cell culture:Mouse breast cancer EMT6 cells were cultured.2.Animal treatment:60 healthy female ICR mice were randomly divided into five groups,including Control,PBS,CTX,GBE,GBE+CTX group.Except the Control group,the other four groups were subcutaneously inoculated with 1×10~6EMT6 cells on the lateral side of the left hind limb of the mouse for modeling.The dose of CTX was 2 mg/kg,intraperitoneal injection every other day,The dose of GBE was 3 mg/kg,intraperitoneal injection every day,no treatment in the Control group,and the same amount of PBS was injected daily in the PBS group,body weight was weighed daily.3.Evaluation of the effect of GBE and CTX on killing tumor cells:The latency of tumors in each group of mice was recorded and calculated,the tumor volume was calculated,measured tumor weight after peeling and calculated tumor inhibition rate.4.Enzymology test:The serum and tumor tissue malondialdehyde(MDA)content and superoxide dismutase(SOD)activity of each group were detected.5.Detection of NF-?B pathway-related proteins in mouse tumor tissue:Western bIot method was used to detect the expression levels of PI3K,Akt,IKB-?,P-I?B-?,NF-?B and P-NF-?B proteins.6.Detection of apoptosis-related proteins in mouse tumor cells:Western bIot method was used to detect the expression levels of Bax,Bcl-2,Caspase-8,FADD,Cyto-C and P53 proteins.Result:1.Evaluation of the effect of killing tumor cells:The tumor weight of GBE+CTX,CTX and GBE groups was significantly lower than that of PBS group.The tumor weight of GBE+CTX group was significantly lower than that of CTX and GBE groups,the difference was statistically significant(P<0.05).The tumor volume of GBE+CTX,CTX and GBE group was significantly lower than that of PBS group.The tumor volume of GBE+CTX group was significantly lower than that of CTX and GBE group,the difference was statistically significant(P<0.05).The tumor latency of GBE+CTX group was higher than that of PBS,GBE and CTX groups.The tumor latency of CTX and GBE group was higher than that of PBS group,the difference was statistically significant(P<0.05).The tumor inhibition rate of GBE+CTX group was higher than that of CTX and GBE group,and the difference was statistically significant(P<0.05).2.The changes of MDA content and SOD activity in serum and tumor tissue of mice:The serum MDA content of PBS group was significantly higher than that of Control group.The serum MDA content of GBE+CTX group was significantly lower than that of PBS,GBE and CTX groups.The serum SOD activity in the PBS group was significantly lower than that in the Control group.The serum SOD activity in the GBE+CTX group was significantly higher than that in the PBS,GBE and CTX groups,and the difference was statistically significant(P<0.05).The MDA content of tumor tissue in GBE+CTX,group were significantly lower than that in PBS,GBE and CTX group,the difference was statistically significant(P<0.05).The SOD activity in tumor tissue of GBE+CTX group were significantly higher than that in the PBS,GBE and CTX groups,and the difference was statistically significant(P<0.05).3.Expression of NF-?B pathway-related proteins in mouse tumor tissue:The expression levels of NF-?B,P-NF-?B and P-NF-?B/NF-?B in GBE+CTX group were significantly lower than those in PBS,CTX and GBE groups.The expression levels of P-NF-?B/NF-?B protein in CTX and GBE groups were significantly lower than those in PBS group.The differences were statistically significant(P<0.05).The expression levels of I?B-?,P-I?B-?and P-I?B-?/I?B-?in GBE+CTX group were significantly lower than those in PBS,CTX and GBE groups.The expression levels of P-I?B-?/I?B-?protein in CTX and GBE groups were significantly lower than those in PBS group.The differences were statistically significant(P<0.05).The expression levels of PI3K,Akt in GBE+CTX group were significantly lower than those in PBS,CTX and GBE groups.The expression levels of PI3K,Akt protein in CTX and GBE groups were significantly lower than those in PBS group.The differences were statistically significant(P<0.05).4.Expression of apoptosis-related proteins in mouse tumor tissue:The expression levels of Caspase8,FADD and Cyto-C protein in GBE+CTX group were significantly higher than those in PBS,CTX and GBE groups.The expression levels of Caspase8,FADD and Cyto-C protein in CTX and GBE group were significantly higher than those in PBS group,and the difference was statistically significant(P<0.05).The expression of Bax protein in GBE+CTX group was significantly higher than that in PBS,CTX and GBE groups.The expression of Bax protein in CTX and GBE group was significantly higher than that in PBS group.The difference was statistically significant.(P<0.05).The expression of Bcl-2 protein in GBE+CTX group was significantly lower than that in PBS,CTX and GBE groups.The expression of Bcl-2 protein in CTX and GBE group was significantly lower than that in PBS group.The difference was statistically significant.(P<0.05).The ratio of Bax/Bcl-2 in GBE+CTX group was significantly higher than that in PBS,CTX and GBE groups.The ratio of Bax/Bcl-2 in CTX and GBE group was significantly higher than that in PBS group.The difference was statistically significant.(P<0.05).The expression of P53 protein in GBE+CTX group was significantly higher than that in PBS,CTX and GBE groups.The expression of P53 protein in CTX and GBE group was significantly higher than that in PBS group.The difference was statistically significant.(P<0.05).Conclusion:1.The combination of GBE and CTX inhibited the growth of mouse breast cancer cells significantly better than CTX alone.2.The combination of GBE and CTX can improve the antioxidant capacity of mice.3.GBE can enhance the effect of CTX killing tumor cells by regulating the expression of apoptosis-related proteins in mouse breast cancer tissue.4.GBE can enhance the effect of CTX killing tumor cells by regulating the expression of NF-?B pathway-related proteins in mouse breast cancer tissue.