| Introduction:Colorectal cancer(CRC)is the third leading cause of cancer death worldwide,and the incidence is still rising due to environmental deterioration,aged tendency of population,Western diet.The low screening rate results in most of Chinese patient were in terminal when they are diagnosed.The survival rate of CRC is less than 30%when there is metastasis.Explore the mechanism involved in onset,invasion and metastasis of CRC is important to specific therapeutic agents developing.Cancer formation is widely attributed to dysfunction of many gene and cellular pathways,and cross talk of them.One of these,the Notch pathway,involves direct cell-cell signaling and is highly conserved across evolution.Its proper function is imperative to normal cell development,differentiation,proliferation and apoptosis.Abnormal Notch signaling along with genetic mutations in Notch signaling associated factors,impact the tumorigenicity and proliferation of cells in a variety of cancers.In particular,disturbances in the Notch pathway have been highly noted in CRC.In humans,there are five ligands and four receptors for the Notch pathway.It has been reported that Notch signaling is dysregulated in CRC and Notch signaling is maintained throughout tumorigenesis.Enhanced levels of Notch-1 have been correlated with progression,tumor grade,and metastasis which could be related to inhibition of apoptosis promoted by Notch-1 expression.In normal intestinal tissue growth,WNT,Notch,Hedgehog,and BMP serve as primary signaling pathways,and their interaction with each other is crucial for normal cell growth,differentiation,and homeostasis.CSCs use aberrant and dysregulated cross-talks of these pathways for self-renewal,differentiation,and proliferation in tumorigenesis.In mice with defective Tcf4(loss of WNT signaling),activation of Notch signaling does not lead to cell proliferation,indicating that intestinal cell proliferation is dependent on WNT signaling?Conversely,inhibiting Notch signaling in mice with APC loss(active WNT signaling)leads to differentiation of adenoma proliferating cells?These results suggest that active Notch crosstalk with WNT is important for tumorigenesis and cell proliferation?WNT signaling can also influence Notch pathways through either modulation of Notch ligands?Notch may also exert influence on WNT signaling through various signaling intermediaries,including Notchregulated ankyrin repeat protein(Nrarp),atonal homolog 1(ATOH 1),and glycogen synthase kinase 3?(GSK3P),that influence either activation or inhibition of crosstalk between these two pathways in CRC.Wnt signaling pathway is a highly conserved pathway,which plays an important role in many biological processes.If it is abnormal,it will lead to many kinds of tumor and metabolic diseases.Activation of the Wnt signal at the bottom of the intestinal crypt is of great significance in maintaining the number and tissue balance of stem cells,but there is an abnormal activation in the tumor,especially in colorectal cancer.Various factors caused by promoter activation of this pathway in the cytoplasm of ?-catenin concentration,free beta excess ?-catenin into nucleus initiated overexpression of downstream target genes,resulting in enhanced cell proliferation,apoptosis initiate or promote the occurrence of colorectal cancer.As previously stated,there is interaction between the Wnt signaling pathway and the Notch signaling pathway to promote the occurrence of colorectal cancer.Therefore,this paper intends to prove that Wnt signal pathway blocker rDKK-1 and Notch signal pathway blocker on colorectal cancer cell line HCT-116 cells express the existence of inhibitory effect by inhibiting one pathway detection under the condition of another pathway change,to clarify the existence of interaction.And further through the analysis of signal pathway blocking agents on the cell cycle,proliferation,invasion effect to clarify the mechanism of signal pathway blockers,clinical application prospects.In the third part of the study,the transplantation tumor animal experiment was carried out to verify the results of the in vitro experiment.The inhibitory effect of the signal pathway inhibitor on the expression of Notch and Wnt in HCT-116 cellsObjectve:Analysis of signal pathway blocker on colorectal cancer cell line HCT-116 expression inhibition effect,and by blocking one pathway after another to detect the expression of mRNA signaling pathway,protein,understanding the interaction between the two pathways.Methods:HCT-116 cells were divided into control group(DMSO),Wnt signal blocking group(rDKK-1 treatment),Notch group(LY374973)signal blocking drug treatment after 48 hours,QRT-PCR was used to detect the expression of Notch signaling pathway related genes Notchl,Notch2,Hes1,DLL4,mRNA and Wnt signaling pathway related gene expression ?-catenin,Pra-1,beta c-myc mRNA.Western blot was used to detecte proteins mentioned above.Results:LY374973 can lead to Notch signaling pathway related genes Hesl and DLL4 expression down(P<0.05),had no effect on the expression of Notch2 and Notchl(P>0.05).rDKK-1 reduced Wnt pathway related gene expression of ?-catenin and c-myc(P<0.05),and had no effect on the expression of Pra-1(P>0.05).In addition,r-DKKl in addition to effect the expression of the two genes affect their own pathway,but also caused the Notch signaling pathway Hes1 and DLL4 expression down(P<0.05).Notch pathway inhibitor LY374973 had no effect on the expression of Wnt pathway related genes(P>0.05).Co-treatment group can cause the detection of 7 gene expression down.Blotting Western results support the above QRT-PCR test results.Conclusions:Signaling pathway blockers can reduce the expression of Notch and Wnt pathway related genes.When Wnt pathway was blocked can reduce the expression of Notch pathway related gene Hes1 and DLL4,while blocking the Notch signaling pathway has no significant effect on the expression of Wnt signaling pathway gene.Effects of Notch signaling pathway and Wnt signaling pathway on cell invasion,migration,cell cycle and proliferation of colon cancer cell lineObjectve:To investigate the effects of Notch signaling pathway and Wnt signal blocking on invasion,migration,cell cycle and proliferation of colorectal cancer cell line and its possible mechanism.Methods:Cell culture and drug treatments were the same as the first part,using MTT method to study the effect on cell proliferation inhibitor,PI staining and flow cytometry to detect the cell cycle,the invasive ability of Transwell in vitro evaluation of cells,cell scratch experiment evaluation after treatment of cell migration,cell immunofluorescence detection of beta-catenin protein and Notchl protein expression and locationResults:Experiment 1:cell proliferation:after 24 hours of r-DKK-1 treatment group(0.836±0.017),LY374973 group(0.829±0.032),compared with the control group DMSO(1.2166±0.022)was significantly decreased(P<0.01),while the two signal athways(rDKK-1+LY374973)block after more obvious inhibition(0.444±0.055).After 48 hours,the two drug treated cells were still in a state of inhibition compared to the control group.2.cell cycle:48 hours of rDKK-1 or LY374973 alone,G0/G1 period,S period,the proportion of cells did not change significantly(P<0.05),and the combined effect(rDKK-1+LY374973)after the proportion of cells in G0/G1 phase increased significantly(from 33.507±2.561%to 61.723±0.766%),with significant differences(P<0.05).There were no differences between the three treatments and the control group during the period of S.In the G2/M phase,the proportion of the three treated cells was significantly decreased(P<0.05).3.Transwell invasion experiment:DMSO group was(76.667±7.5055)/HP,rDKK-1 group was(56.667±5.033)/HP,the difference was statistically significant(P<0.05).Group LY374973(47.667+4.163)/HP,the difference was statistically significant(P<0.05),rDKK-1+LY374973 group(31.333 ± 2.082)/HP,the difference was statistically significant(P<0.05),but there is no significant difference between rDKK-1 and LY374973(P>0.05).3:cell scratch experiment:rDKK-1 group 24 hours after wound healing rate was(37.309±4.170%),LY374973 group(38.872 ±0.937%),group rDKK-1+LY374973 scratch repair rate was(23.455±0.738),the difference was statistically significant(P<0.05).4:cell immunofluorescence:rDkk-1 can significantly reduce the expression of ?-catenin protein,while LY3 74973 has no effect on its expression.LY374973 and rDkk-1 did not affect the expression of Notch1 protein.Conclusions:Signal pathway blocker can reduce the proliferation,invasion and migration of colon cancer cell line HCT-116Animal transplanted tumor modelObjectve:The experimental results of cell line and the effect of the signal pathway blocker on the growth of transplanted tumor were further verified by subcutaneous transplantation tumor model in nude mice.Methods:The establishment of subcutaneous tumor model in nude mice,successful tumor were randomly divided into the control group(DMSO injection);the rDKK-1(5mg/kg,administered once a week,a total of 3 drug treatment group);the LY374973(4mg/kg,administered once a week,a total of 3 drug treatment group);4.rDKK-1+LY374973 treatment group.Take out the tumor tissue,using the same Western,blot QRT-PCR in the first part of the nude mice transplanted tumor in the first part of the expression of gene protein,and compared to the cell experiment.The expression of different proteins in tumor tissues was observed by HE staining and immunohistochemical staining.Results:The treatment group was treated with drug therapy for three times,and the growth of LY474937 and rDKK-1 were significantly different(P<0.05)after 12 days.And as the treatment time goes on,it gradually became clear.Tumor detection results suggest that:compared with the control group,Wnt signal pathway inhibitor rDKK-1 did not significantly affect the Wnt pathway 3 gene expression changes of Notch signal pathway inhibitor LY374973 also did not affect the Wnt pathway gene expression.-catenin and c-myc expression was down regulated in two drug combinations.Notch signaling pathway blocker LY374973 could down regulate the expression of Notch2 and Hesl genes in Notch pathway,and had no effect on the expression of Notch1 and DLL4.Effect of rDKK-1 on the expression of Notch pathway related genes in the same LY3 74973.The two drug combination treatment group could down regulate the expression of Notch2,DLL4 and Hesl genes.Blot Western detection supports QRT-PCR detection results.Conclusions:The signal pathway blocker has obvious inhibitory effect on the growth of xenografted tumor in nude mice,two kinds of drugs more obvious inhibition,tumor mRNA and protein expression and in vitro cell line experiments similar but not identical... |
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