| Background and objectives:Among different kinds of gastrointestinal tumors,colorectal cancer which remains a high incidence is a severe threat to our healthy.Meaning while,as the development of economy and the alteration of life style and dietary structure,the incidence of colorectal cancer presented an upward tendency year after year.At present,the chief therapeutic schedule is operation,and Chemoradiotherapy is also playing an important role.However,we can't be satisfied with the low five year survival rate which is attributable to the recurrence and metastasis of colorectal cancer cells in a deep degree.Therefor,Finding a new effective method to surpress the proliferation and metastasis of colorectal cancer cells is a new research direction to cure colorectal cancer.Recently,molecular targeted therapy is paid increasing attention to on the treatment of cancer.So,to further research signal pathway associated with the proliferation and metastasis of colorectal cancer cells is beneficial to provide new theoretical basis and drug targets which is related with better treatment of colorectal cancer.cAMP as a critical second messenger is widely distributed,and plays an important role in many pathophysiological processes such as cell proliferation?differentiation?apoptosis?metastasis.The mechanism might be associated with the changes of the downstream proteins of cAMP such as Cyclin D1 and MMP9 that are correspondingly related to cell cycle?proliferation and invasiveness metastasis.In the past,cAMP signal was thought to be mediated by PKA / Rap1 signal pathway,but recent studies indicated that Epac1 / Rap1 could mediate cAMP signal as well.EPAC has two subunits: Epac1 and EPAC2.Epac1 is ubiquitously expressed and the proliferation and metastasis process of many kinds of tumor cell can be mediated by cAMP / Epac1 signal pathway.On the contrary,Epac2 mainly expresses in neure?pancreas?paranephros?pituitarium and so on,and was seldom reported to be relevant to the growth of tumor.As a result,cAMP / Epac1 signal pathway may be a key step of the growth of tumor.cAMP/Epac1 pathway has been wildly reported to be related to the proliferation and metastasis process in various kinds of tumor cells,but the role of cAMP/Epac1 pathway has not been reported in colorectal cancer cells yet.Therefor,this study aims to detect the role of cAMP/Epac1 pathway in the proliferation and metastasis process of colorectal cancer cells and the potential molecular mechanism via inhibiting the activation of cAMP/Epac1 pathway.Methods :Part one: screening out colorectal cancer cell line with high expressing level of Epac1.Human colorectal cancer Caco2?SW480?SW620 cells were chosen as abject of our study.Cells were cultivated in RPMI 1640 medium in which 10% fetal bovine serum(FBS),100 ?g/mL streptomycin and 100 U/mL penicillin were contianed,and were maintained at 37? in a humidified incubator with 5% CO2.The expressing level of Epac1 in each cell lines was measured by Western Blot,and then,colorectal cancer cell line with high expressing of Epac1 was screened out to be abject of study in the following experiment.Part two: detecting the changes of proliferation and metastasis ability of colorectal cancer cells and the potential mechanism after inhibiting cAMP/Epac1 pathway with ESI-09,a Epac-specific inhibitor.1.MTT assay was performed to measure cell proliferation of colorectal cancer.According to relevant report,5 kinds of ESI-09 concentration groups(0 umol/L,2.5 umol/L,5 umol/L,10 umol/L,20umol/L)were set,and cells were incubated with them for 12,24,48 h respectively before MTT assay was performed.2.Wound-healing assayOn the basis of MTT assay,colorectal cancer cells were treated with different concentration of ESI-09 for 6,12,24 h respectively,afterwards,wound-healing assay was carried out to measure the capacity of cell migration.3.FCM investigated cell cycleAfter treatment of ESI-09(20 umol/L)for 24 h,FCM was used to detect the cell cycle of colorectal cancer cells.4.Western Blot was employed to measure the changes of signing pathway in colorectal cancer cells.At first,we treated colorectal cancer cells with ESI-09 at the concentration of 0 umol/L,5 umol/L,10 umol/L and 20umol/L respectively for 24 h,after that,the expressing level of p-AKT,AKT,Cyclin D1 and MMP9 was measured by Western Blot.Part three: detecting the effect of Epac1 silencing on Cyclin D1 and MMP9 expressing in colorectal cancer cells.When the time Epac1-si RNA was transfected into cancer cells,Western Blot was performed to measure the expression of Cyclin D1 and MMP9.Results:1.All the three cell lines could express Epac1.The expression of Epac1 in SW620 was significantly lower than that in SW480 and Caco2(P < 0.05),while there was no significant difference between SW480 and Caco2 cells(P > 0.05).We random chose Caco2 as the following abject.2.The inhibitory effect of ESI-09 on Caco2 cells could be observed 12 h.When the concentration of ESI-09 was at 1umol/L,the inhibitory effect not significant,while there was a markedly inhibitory effect on Caco2 cells at the concentration of 2.5 ~ 20 umol/L(P<0.05),and the cell proliferative rate decreased while the concentration of ESI-09 increased.3.After treatment of ESI-09,a remarkable decrease in cell migration was observed compared with control group,especially at the concentration of 20umol/L(P < 0.05).4.Compared with control group,after treatment of ESI-09,cells in the G1-phase increased markedly,while the percentage of cells in both S-and G2/M-phase decreased(P < 0.05).5.When cells were suffered from different concentration of ESI-09,there was a significant decrease in Cyclin D1 and MMP9 expression,and the phosphorylated level of AKT decreased remarkably(P < 0.05).6.After the transfection of Epac1-si RNA,the expression of Cyclin D1 and MMP9 reduced markedly.Conclusion:1.Inhibiting the activation of cAMP/Epac1 pathway could suppress the proliferation and metastasis of colorectal cancer cells.2.cAMP/Epac1 pathway might be a new potential target for the treatment of colorectal cancer. |
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