High-intensity Exercise Inhibits Growth Of Mouse Colon Cancer Cells Through Multiple Mechanisms

Background and objective:Colorectal cancer(CRC)is one of the most common malignant gastrointestinal cancers in the world.Its incidence and mortality rate are increasing year by year and pose a serious threat and influence to the health and life of people.CRC is a heterogeneous disease that occurs mainly in the colon and rectum,and its specific etiology is currently unknown.Studies have shown that CRC is a complex disease caused by a combination of factors such as the individual's lifestyle,family inheritance,and living environment.Due to the rapid economic development and adaptability of Western lifestyles,the risk of exposure to certain environment and lifestyle have increased,unhealthy dietary choices and consumption of red meat,lack of physical activity,and obesity have greatly increased the incidence rate of CRC.Screening for precancerous lesions and colorectal cancer screening can effectively reduce the incidence and mortality of CRC.In 2011,the American Cancer Institute and the World Cancer Research Fund released a new report.There is convincing evidence that physical activity can significantly reduce the risk of colon cancer,suggesting that moderately increased physical activity in colon cancer patients may contribute to their rehabilitation.And there are several meta-analyses showing that physical activity can significantly reduce the risk of colon cancer.Exercise can improve health status,which may be related to the regulation of immune system function.By shifting the overall immune response to a Th1 response pattern,moderate intensity exercise can reduce the risk of infection.Epidemiological data show that exercise can prevent and reduce the occurrence and development of certain tumors,especially breast and colon cancer.It also includes reducing the risk of endometrial cancer,advanced prostate cancer,and pancreatic cancer,Epidemiological data show that exercise can prevent and reduce the occurrence and development of certain tumors,especially breast and colon cancer.It also includes reducing the risk of endometrial cancer,advanced prostate cancer,and pancreatic cancer.It can also reduce the risk of other chronic diseases such as heart disease,diabetes,osteoporosis,and hypertension.Exercise may improve the body's immune surveillance,immune defense and immune response functions by promoting the activation of the body's immune cells,enhancing the digestion and absorption of the gastrointestinal system,accelerating the elimination of unfavorable metabolites and alleviating oxidative stress.It plays an important supporting role in the prevention and treatment of tumors.Animal experimental studies have also demonstrated that exercise training can slow down the growth of experimental tumors,tending to believe that enhanced immune function may be the main factor in resisting tumor growth,but its detailed mechanism of action has not yet been elucidated.Moreover,the opposite result is also reported.We believe that this may be related to the intensity of exercise,the reactivity of the body's immune system,and the histological characteristics and site of growth of the tumor.Therefore,this study focused on comparing the effect of different exercise intensity on the growth and metabolism of colon cancer cells in mice and the relationship with the cycle regulatory proteins.Observe the relationship between the composition of Th17,Th22 and Treg of CD4~+T cell subsets and growth of colon cancer after exercise intervention.Methods:(1)Colon cancer CT26 cells were subcutaneously inoculated into BALB/c mice to establish a colon cancer mouse model.(2)The tumor-bearing mice were randomly divided into high-intensity exercise group,low-intensity exercise group and sit-in group.Tumor-bearing mice are trained on treadmills for different exercise intensity.The high-intensity exercise group was running at a speed of 14m/min,exercised 60 minutes a day,performed 5 times a week for 5 weeks,and the low-intensity exercise group was running at a speed of 14 m/min,exercised 30minutes a day,performed 5 times a week for 5 weeks,and the sit-in group of mice remained in the cages in the treadmill room during exercise to expose them to similar noise and external pressure that might be associated with treadmill running.Weigh each group of mice daily,measure the tumor volume and weigh dead tumors,observe the survival time of mice.(3)HE staining and TUNEL staining were used to determine the effect of different exercise intensity on the tumor pathological morphology of CT26 mice.Flow cytometry further confirmed the apoptotic state of tumor cells.(4)The expression levels of cell cycle-related genes or proteins p21,p27,p53,p73,Rb and apoptosis-related genes or protein Bcl-xL were detected by qPCR or/and Western blotting to investigate whether cell cycle-associated proteins are associated with exercise-inhibiting CT26 cell growth.(5)Flow cytometry was used to detect the early,middle and late activation of T cells and the changes of Th22,Th17and Treg subpopulations in tumor bearing mice with different exercise intensity,and the expression levels of IL-4,IL-6,IL-12,TNF-?,IFN-?,IL-1?and TGF-?were detected by ELISA.(6)22 metabolic markers related to metabolic function were analyzed by automatic biochemical analyzer to determine the effect of high-intensity exercise on metabolism of colon cancer mice.Results:High-intensity exercise can inhibit the growth of CT26 in mice,increase the survival rate of tumor-bearing mice,and it is associated with an increase in the proportion of apoptotic cells in tumor tissues of mice with severe necrosis of tumor tissue.The expressions of cyclin inhibitor proteins p53 and p21 in the tumor tissues of high-intensity exercise mice were significantly higher than those in the sit-in group and the low-intensity exercise group,while the expression of anti-apoptosis-related protein Bcl-xL was significantly lower than that of the sit-in group.Compared with the control group,the percentage of activation of T cells in mice increased significantly with exercise intensity,especially early and late T cell activation was significantly higher in the high-intensity exercise group than in the low-intensity exercise group.High-intensity exercise can significantly down-regulate the level of Treg cells and up-regulate the level of Th22 cells in CT26 mice,while there is no significant change in the level of Th17 cells.High-intensity exercise increased the expression of IL-12,TNF-?,IFN-?and IL-6 in the serum of tumor-bearing mice,while the expression levels of IL-1?,IL-4 and TGF-?decreased.The composition ratio of CD4~+T cell subsets and changes in functional cytokines indicate that high-intensity exercise significantly enhances the function of T cell immune responses.Interestingly,the expression levels of AST,LDH,CKMB,?-HBDH,UREA,and ADA increased in tumor-bearing mice compared to normal mice,whereas the expression levels of ALP and GSP decreased,Suggesting high-intensity exercise can reverse AST,CKMB,GSP and UREA to normal levels.Conclusion:High-intensity exercise may inhibit mouse colon cancer cell growth through multiple pathways:Activate P53/P21/P73/Rb signaling to block cell proliferation andinhibit Bcl-xL to promote cell apoptosis;Activate T cells,downregulate Treg cells,upregulate Th22 cells,promote the expression of IL-12,TNF-?,IFN-?,and IL-6,thereby enhancing the effect of effector T cells on tumor cells;can partially reverse the disorder of metabolic function caused by tumors.