Effect And Mechanism Of Clostridium Butyricum Combined With Apatinib In The Treatment Of Colorectal Carcinoma

BackgroundColorectal cancer(CRC)is the fourth-leading cause of cancer-associated mortality globally.Among women,it is the second most common cancer.It is also the third most common cancer among men.Despite of the rapid development of therapies for CRC treatments,the disease still develops over time.Thus,it is particularly urgent to explore effective therapies for clinical treatments.Angiogenesis makes essential role on the CRC development and metastasis.VEGF is a signal protein that stimulates endothelial cells proliferation,migration and tube formation.Studies have shown that anti-angiogenesis has a significant improvement in the prognosis of patients with colorectal cancer.Apatinib,also called as YN968D1,a novel inhibitor of VEGFR-2 tyrosine kinase has shown remarkable effect on the solid tumors.Apatinib can be used to treat patients with metastatic gastric cancer in 2014 in China.However,the heterogeneity of CRC and the drug resistance mitigate the drug's curative effects by current research studies.Clostridium butyricum(C.butyricum)is a strictly anaerobic Gram-positive bacteria that produce butyric acid and commonly used as the probiotic to adjust the imbalance of normal intestinal flora.Moreover,it is worth noting that expression levels of C.butyricum in healthy individuals were higher than those in CRC patients.We hypothesize that combination of apatinib and C.butyricum might have synergistic antitumor effects on CRC.In order to clarify this hypothesize,mouse colon carcinoma cell line CT26.WT were cultured and xenografted into the skin of mice to get CT26.WT tumor-bearing mice,and then we treated the mice with apatinib or combination of apatinib and C.butyricum.Our results showed C.butyricum and apatinib significantly inhibited tumor growth,promoted apoptosis and decreased the expression of CD31,PCNA and Bcl-2 in model mice,meanwhile,the combination of them increased the expression of Cleaved Caspase-3.Our study will provide new reference to clinical therapy for CRC.ObjectiveOur experiment used CT26.WT mice as a model to investigate the synergistic antitumor effect of C.butyricum combined with apatinib on colorectal carcinoma in mice.Methods1.Mouse CT26.WT colon cancer cells were passaged and cultured,and then aliquoted and transplanted into 48 7-week-old,female,18-25 g,subcutaneous groin of BALB/c mice.After 3 days,each mouse was examined for tumor formation(the tumor size was measured about 5*5 mm,that is,the volume was about 60 mm3).The mice bearing the tumor were randomly divided into 4 groups,and different treatment options were started after 4 days of tumor formation.(13 days in total),including:PBS control group,C.butyricum group,apatinib group and C.butyricum combined with apatinib group.PBS control group:200?l of PBS was given to per model mouse by intragastric administration once a day.C.butyricum group:A 1000 mg/kg dose of C.butyricum(dissolved in 200?1 of PBS)was administered to the mice once a day according to the body weight of the mice.Apatinib group:model mice were intragastrically administered with a dose of 70 mg/kg of apatinib(dissolved in 200?1 of PBS)every four days(ie,1,5,9,and 13 days,4 times in total).An equal amount of PBS was administered on other days of treatment.C.butyricum combined with apatinib group:The same dose of apatinib was administered intragastrically on days 1,5,9,and 13 while the same daily dose of C.butyricum was administered.Tumor size was measured every other day,tumor volume was calculated,tumor growth changes in mice were observed,and growth curves were plotted.2.Four groups of mouse feces were collected on the 13th day,and real-time PCR was used to detect the content of C.butyricum in the intestine of mice given C.butyricum.3.At the end of drug treatment,four groups of mice(12 in each group)were dissected to obtain tumor tissues,photographed in groups,and the size changes were compared.The volume and weight of the tumors were measured,and the tumors were grouped for immunohistochemical staining(including the following indicators):CD31,PCNA,Bcl-2 and Cleaved Caspase-3,the effects of each group on the tumor growth,angiogenesis,cell proliferation and apoptosis were analyzed to observe the efficacy index.The statistical analysis tool was GraphPad Prism5 software in which statistical analysis was performed using one-way analysis of variance(ANOVA),two-way analysis of variance,and t-test.ResultsThe apatinib group alone can inhibit tumor growth in CT26.WT mice,reduce tumor angiogenesis,inhibit tumor cell proliferation,and promote tumor cell apoptosis.Tumors from mice treated with C.butyricum and apatinib group inhibited tumor growth,promoted apoptosis,decreased expression of CD31,PCNA,Bcl-2 and increased the expression of Cleaved Caspase-3 molecules compared with the apatinib group alone.The above results were statistically significant after analysis.ConclusionOur study confirmed that compared with apatinib alone in the treatment of xenograft colorectal carcinoma in mice,C.butyricum combined with apatinib treatment group can significantly inhibit tumor growth,reduce tumor angiogenesis,and promote tumor cell apoptosis,Provide reference for the future use of C.butyricum for clinical tumor treatment.