Resibufogenin Suppresses Colorectal Cancer Growth And Metastasis Through RIP3-mediated Necroptosis

Background:In 2018,the population of colorectal cancer(CRC)in the United States was taken as the research object.The results showed that the number of new cases and deaths in the disease is expected to be 140250 and 50630,respectively,accounting for 8.1%and 8.3%of the total number of cancer cases and deaths.Since 2010,cancer has become the leading cause of death in China.In 2015,the number of CRC cases and deaths reached 376300 and 191000 respectively,ranking the fifth in the number of new cancer cases and deaths.Toad puff,a traditional Chinese medicine,has been recorded in the theory of medicinal properties in Tang Dynasty for the treatment of carbuncle,sores and other diseases.Resibufogenin is an effective active component of toad venom.In this study,resibufogenin is used as the main research material.The results show that it has the effect of preventing and treating colorectal cancer,and clarify the regulatory mechanism of resibufogenin on programmed cell necrosis,which provides a potential target for clinical treatment of colorectal cancer.Methods:The ectopic tumor model of colon cancer SW480 in nude mice and the liver metastasis model of ectopic tumor injected into the spleen of mice were used to observe the effect of resibufogenin on cell necrosis,cell growth and liver metastasis.HCT116 and SW480 cells were stained by PI,FCM and TEM in vitro.At the same time,N-acetyl-L-cysteine(L-NAC)and caspase inhibitor were used to observe the cell viability.Receptor interacting protein 3(RIP3)knockout of mouse embryonic fibroblasts(MEF)can save cell death.The activities of RIP3 mediated energy metabolizing enzymes were observed in wild-type and RIP3 knockout mouse embryonic fibroblasts.At the same time,the expression of EMT related proteins in mice was detected by in vitro cell Transwell,scratch testing and Western blot.Results:1.Resibufogenin inhibited the growth of colon cancer in vitro and in vivo.Compared with the model group,the tumor volume was measured after treatment with resibufogenin.In HCT116 and SW480 cell lines,MTT and plate cloning experiments showed that resibufogenin significantly reduced the survival rate and proliferation ability of cells,and was significantly related to the intervention time and dose of resibufogenin.2.The colon cancer cells were used as experimental materials,which were induced by resibufogenin.Swelling and disintegration of lysosomes,mitochondria and endoplasmic reticulum were observed under electron microscope.PI staining was positive with bright red fluorescent spots.The results of Annexin v/7AAD double staining showed that the necrosis rate of HCT116 cells induced by resibufogenin was significantly higher than that of the control group(P<0.05).The activity of lactate dehydrogenase(LDH)increased significantly,accompanied by the activity of reactive oxygen species(ROS).After pretreatment with antioxidant L-NAC for 5 hours,the survival rate of the treated group was significantly higher than that of the control group(P<0.05).High concentration of resibufogenin can induce inflammatory oxidative stress and eventually lead to cell death.3.Resibufogenin induced RIP3 dependent death by activating glycogen phosphorylase(PYGL),glutamine synthetase(GLUL),glutamate dehydrogenase 1(GLUD1).Western blotting,quantitative PCR and immunofluorescence staining showed that RIP3 increased significantly under the effect of resibufogenin.Using wild-type and RIP3-/-mouse embryonic fibroblasts(MEFs)to further study the role of RIP3 in resibufogenin induced cell death.After the concentration of 10 μm and 20 μm of resibufogenin for 24 hours,the survival rate of RIP3-/-cells was higher than that of wild-type cells(P<0.05).The expression of PYGL,GLUL and GLUD1 in wild-type cells was significantly higher than that in RIP3-/-cells,especially 24 hours after 10 μm treatment(P<0.05).These results indicate that RIP3 is an essential element in the cell necrosis induced by resibufogenin.4.Resibufogenin inhibited liver metastasis of colorectal cancer in mice.By fluorescence imaging analysis,resibufogenin significantly reduced liver metastasis.HE staining and immunofluorescence further study confirmed that resibufogenin inhibited liver cell metastasis in mice with primary colorectal cancer.Conclusion:Resibufogenin inhibited the growth and metastasis of colorectal cancer through stimulating RIP3-mediated necrosis.