Mononetin Induces Apoptosis Of Colorectal Cancer Cells By Activating Mitochondrial Dependent MAPK Signaling Pathway

Objective:To fully explore the effect of mononetin on apoptosis and proliferation of colorectal cancer cells by regulating MAPK signaling pathway through mitochondrial dependent pathway.Materials and Methods:Select people SW1463 rectal gland cancer cells as the research object,the primary cell culture means to develop,select the third generation of monolayer rectal gland cancer cells as the research object,the following group and Control group(cells do not do processing),H-FE group(group,the high concentration of mans handle),H-EF-group I(high concentration mans,handle+ROS antagonist),H-EF-p group(high concentration mans,handle+ROS agonist)continue to develop after 24 hours,cells related projects observation:cell morphology analysis:HE dyeing observation groups of cells form;The expressions of ax,Bcl-2 and caspase-9 in each group were observed by immunohistochemical staining.Mitochondrial membrane potential analysis and ATP energy synthesis in each group;SEM was used to observe the surface morphology of cells in each group.The morphology of mitochondria in each group was observed by TEM.TUNEL detected apoptosis in each group.Cytokines:expression of interleukin(IL-6,IL-8),tumor necrosis factor and vascular endothelial factor VEGF were detected by ELISA.Determination of ROS reactive oxygen species by colorimetry;the expression of ERK,p38,JUN protein in MAPK signaling pathway and its phosphorylation concentration were detected by double luciferase method.MRNA expression of apoptosis-related proteins was detected by RT-PCR.The concentration of apoptosis-related proteins and key proteins in MAPK pathway were detected by WB.Cell proliferation in each group was detected by CCK-8.Cell invasion and migration:scratch test and Transwell test;Finally,analyze the data and summarize the analysis.Results:Mitochondrial detection:Membrane potential detection:mans,the handle after the intervention,the cell membrane potential decrease,when ROS antagonists to join,cell membrane potential increased slightly,while its agonist,after China’s entry of cell membrane potential falling,preliminary confirmed that mans handle,the intervention of the mitochondrial membrane potential and the direction of the colorectal cancer cells ROS changes has the direct correlation between the concentrations of the reactive oxygen species;The qualitative analysis trend of cell membrane potential of colorectal cancer in each group was H-EF-p<H-EF<H-EF;-i<ControlThis suggests that oxidative stress damage in the cell is gradually obvious,that the cell will continue to suffer from abnormal REDOX status of mitochondria,respiratory chain uncoupling and other phenomena,that the colorectal cancer cells will not be able to get enough energy supply,and that the possibility of cell apoptosis and necrosis is gradually increased.Membrane potential delta ΔΨm and ATP synthesis ability:mitochondrial inner membrane potential delta Ψ m analysis process,the expression of groups of cells trend for H-EF-p<H-EF<H-EF-i<Control,data comparison between groups,p<0.05,the difference is statistically significant;Furthermore,the expression trend of cells in each group was H-EF-p<H-EF<H-EF-i<Control;It is suggested that the membrane potential and ATP synthesis ability of mitochondria in colorectal cancer cells are directly correlated with the expression concentration of ROS in vivo.ROS ROS concentration expression:ROS ROS expression process,the expression trend of colorectal cancer cells in each group was Control<H-EF-i<H-EF<H-EF-p,the data of each group were compared,p<0.05,the difference was statistically significant.The expression of HSP70 showed the same trend as that of ROS.The expression trend of colorectal cancer cells in each group was Control<H-EF-i<H-EF<H-EF-p,In the S OD expression process,the expression trend of colorectal cancer cells in each group was H-EF-p<H-EF<H-EF-i<Control,In summary,it can be seen that formonolanin can fully improve the expression of mitochondrial ROS in cells and fully weaken the antioxidant capacity of cells.The key protein activity of MAPK signaling pathway was detected by the dual luciferase system:ERK1/2 signaling pathway:the protein expression activity trend of each group was Control<H-EF-i<H-EF<H-EF-p,the data of each group were compared,p<0.05,with statistically significant difference.JNK signaling pathway:the expression activity trend of proteins in each group was Control<H-EF-i<H-EF<H-EF-p,and the data of each group were compared,p<0.05,with statistically significant difference.P38 signaling pathway:the trend of protein expression activity in each group was Control<H-EF-i<H-EF<H-EF-p,and the comparison of data in each group was p<0.05,indicating a statistically significant difference.RT-PCR detection:phosphorylation of ERK1/2,JNK and p38 proteins,and the expression trend of cells in each group was Control<H-EF-i<H-EF<H-EF-p.Data in each group were compared,p<0.05,and the difference was statistically significant.Cell morphology:HE staining:the structure of the cancer cells in the control group was basically normal,and the villi of the cells were relatively positive.Often,the cell overall arrangement is relatively regular,no edema in the interstitium.After intervention with formononetin,the cells showed obvious atrophy,the mucosal layer showed atrophy,the villi of the cells gradually fell off,the cells gradually showed necrosis and edema.With the increase of ROS expression in cells,cell necrosis and apoptosis were gradually obvious.Immunohistochemical staining:during the analysis of pro-apoptotic protein Bax,the study indicated that the expression trend of cells in each group was Control<H-EF-i<H-EF<H-EF-p.Data of each group were compared,P<0.05,the difference was statistically significant.Data of each group were compared,P<0.05,the difference was statistically significant.During the analysis of the apoptotic protein Bcl-2,the study suggested that the expression trend of cells in each group was H-EF-p<H-EF<H-EF-i<Control.During the analysis of caspase-3,the expression trend of cells in each group was Control<H-EF-i<H-EF<H-EF-p,Data of each group were compared,P<0.05,the difference was statistically significant.SEM scanning electron microscope analysis:the control group had more villi on the cell surface.After the intervention of formonin,the cell surface was more wrinkled and the surface villi gradually decreased.With the increase of ROS expression,the surface roughness of colorectal cancer cells gradually increased,and the cell necrosis and apoptosis increased.TEM transmission electron microscopy showed that the mitochondria of colorectal cancer cells in the control group were normal without obvious bulges and poisoning.After intervention with formononetin,the phenomenon of mitochondrial poisoning was obvious,and apoptotic bodies could be seen in cells.With the increase of ROS expression in cells,the phenomenon of mitochondrial poisoning was gradually obvious,and the number of apoptotic bodies in colorectal cancer cells was gradually increased.The presence of intracellular apoptotic bodies was evident in colorectal cancer cells in the h-ef-p group.ELISA:the expression trend of IL-6,IL-8,TNF-α and VEGF concentration:H-EF-p<H-EF<H-EF-i<Control,P<0.05,and the difference was statistically significant.Invasion and migration of colorectal cancer cells:scratch experiment and Trans well experiment:the trend of migration speed and invasion degree of colorectal cancer cells in each group after 24 hours was H-EF-p<H-EF<H-EF-i<Control,P<0.05,and the difference was statistically significant.Proliferation and apoptosis:proliferation:the proliferation trend of colorectal cancer cells after transfection H-EF-p<H-EF<H-EF-i<Control;Apoptosis:the comparison trend of apoptosis rate of colorectal cancer cells in each group was Control<H-EF-i<H-EF<H-EF-p,and the comparison of data between the groups was p<0.05,indicating a statistically significant difference.RT-PCR detection of proliferation apoptotic protein:the expression trend of Survinvin mRNA was H-EF-p<H-EF<H-EF-i<Control,The expression trend of Bax was Control<H-EF-i<H-EF<H-EF-p,and the difference between the groups was statistically significant(p<0.05).The expression trend of Bcl-2 was H-EF-p<H-EF<H-EF-i<Control,and the difference between the groups was statistically significant(p<0.05).Western-blot analysis:Survivin protein:the expression trend of Survivin colorectal cancer cells in each group was H-EF-p<H-EF<H-EF-i<Control;Bax protein:the expression trend of colorectal cancer cells in each group was Control<H-EF-i<H-EF<H-EF-p.Bcl-2:the expression trend of colorectal cancer cells in each group was H-EF-p<H-EF<H-EF-i<Control,and the difference between the groups was statistically significant(p<0.05).Concluisons:Mononetin can decrease the proliferation trend and promote the apoptosis trend of colorectal cancer cells.As a result,mitochondrial injury of colorectal cancer cells is intensified,stress injury of cancer cells is significantly increased,membrane permeability is increased,mitochondrial membrane potential and ATP synthesis ability are decreased,which promotes the production of pro-apoptotic protein Bax,and Caspase protein kinase family,and ultimately promotes the process of apoptosis of colorectal cancer cells.Mononetin can fully promote the increased expression of ROS in the mitochondria of colorectal cancer cells,and activate the mitochondrial apoptosis pathway of colorectal cancer cells through ROS expression.ROS,as the upstream signal factor,participates in the process of colorectal cancer apoptosis.ROS induction can be used as an upstream signal to participate in the production of apoptosis mediated by mitochondrial pathway in colorectal cancer cells.The intervention of mononetin can increase the expression of key proteins of MAPK signaling pathway in colorectal cancer cells,which shows a positive trend.In conclusion,in the process of apoptosis of colorectal cancer cells,the mechanism of action of mononetin is increased expression of mononetin-ROS reactive oxygen species-activation of mitochondrial apoptosis pathway-MAPK signaling pathway-apoptosis of colorectal cancer cells.