| Objectives:The distal metastasis is the main cause of death in patients with colon cancer,of which liver metastasis accounts for about onethird of all distant metastatic sites in colon cancer.PI3K/AKT-ERK signaling pathway is revealed to be involved in the regulation of anoikis resistance,the prerequisites to produce and mature of metastasis.Meanwhile,PI3K/AKT-ERK signaling pathway is also associated with CCL20 and C-X-C motif chemokine ligand 8(CXCL8)in combination with the induction of epithelial-mesenchymal transition(EMT)and CXCL8-induced anoikis resistance in colon cancer.Increasing studies have suggested that tyrosine receptor kinase B(TrkB)plays a central role in resisting anoikis in a variety of tumor cells,including colon cancer.Besides,TrkB is also believed to induce EMT in colon cancer cells.Thus,TrkB is considered as a powerful regulator for the survival of colon cancer cells in response to anoikis and EMT.Erlotinib is an effective,selective and orally active inhibitor of the epidermal growth factor receptor.Previous studies suggested that TrkB may be a molecular target of erlotinib and TrkB treatment in non-small-cell lung cancer.Erlotinib has also been shown to significantly inhibit ERK1/2 phosphorylation and signal transducer and activator of transcription 3(STAT3)expression in neuroblastoma,thereby inhibiting the proliferation,invasion,and metastasis of neuroblastoma.Clinical studies suggested that erlotinib combined with bevacizumab for the treatment of advanced colon cancer is a significant benefit to the survival of patients.However,the potential molecular mechanism of erlotinib in regulating cancer metastasis remains unclear thoroughly.Therefore,the study of Erlotinib inhibits the metastasis of colorectal cancer by regulating TrkB and ERK signal pathways,which may have a certain guiding significance for the treatment of patients.Methods:First,human colon cancer cell lines SW480 and Caco-2,pretreated with exogenous CXCL8 were used to evaluate the inhibitory effect of Erlotinib on tumor metastasis.1?In order to investigate the effect of erlotinib on CXCL-8-induced apoptosis in colon cancer,the colon cancer cells were pretreated with CXCL-8 for 24 h with different concentrations of erlotinib for 48 h,and the cell survival rate was determined by CCK-8 assay.2?Cell migration and invasion assay.2.1?Cell migration assay.(1)Observe the cell growth,and when the growth reaches 80-90%,the scratch wound is measured to observe the cell migration.(2)The cells were inoculated into 24-well plate at the density of 2 × 105 cells/ml and cultured for 24 hours.(3)Linear scratches were made at the tip of 10-? 1 aseptic pipette,and PBS,CXCL8 and CXCL8 Erlotinib were added into the cells.The healing of scratch wound was observed under inverted microscope(Olympus,Tokyo,Japan).2.2?Observation of invasion of colon cancer cells by transwell assay.3?To construct colon cancer cells with TrkB overexpression sequence.PDONR223 empty vector was used as control.Westernblot was used to detect the expression of apoptosis,EMT,invasion and metastasis related proteins in colorectal cancer cell lines with overexpression of TrkB.4?Westernblot was used to detect the expression of apoptosis-related proteins,EMT-related genes,TrkB,Akt/ERK phosphorylation and other related proteins.5?Apoptosis assay was performed with Annexin-V apoptosis detection kit(BDBiosciences,SanDiego,CA).The stained cells were detected by flow cytometry(BDBiosciences,SanJose,CA,USA)and analyzed by FCSExpressv2.0 software(DeNovoSoftware,LosAngeles,CA,USA).6?After adding U0126 and MK2206 which inhibit ERK and Akt respectively,the expression of EMT related protein was determined by westernblotting,and the levels of p-ERK,ERK,p-Akt and Akt in colorectal cancer cells were detected.Results:1.1?The results of flow cytometry showed that compared with SW480 cells and Caco-2 cells pretreated with CXCL8,the apoptosis rate of SW480 cells and Caco-2 cells treated with CXCL8 plus erlotinib was significantly higher than that of SW480 cells and Caco-2 cells pretreated with erlotinib(p<0.01).Western blotting analysis showed that CXCL8 treatment resulted in the activation of Caspase-3 and a significant decrease in the ratio of PARP to Bax/Bcl-2 protein,but erlotinib administration effectively restored this trend(p<0.01).These findings suggest that erlotinib can inhibit CXCL8-induced apoptosis resistance in colon cancer.1.2?The results of Transwell assay showed that erlotinib could effectively reverse the CXCL8-enhanced invasion of SW480 and Caco-2 cells(p<0.01).Similarly,the determination of cell scratch wound showed that erlotinib significantly attenuated the increase of cell migration induced by CXCL8(p<0.01).1.3?Westernblot analysis showed that the levels of E-cadherin in SW480 and Caco-2 cells pretreated with erlotinib were significantly up-regulated,while the levels of N-cadherin and vimentin in SW480 and Caco-2 cells treated with erlotinib were significantly down-regulated(p<0.05,p<0.01).1.4?CXCL8 significantly increased the level of TrkB protein in SW480 and Caco-2 cells(P<0.01),But erlotinib counteracted the regulatory effect of CXCL8(P<0.01).In addition,Western blotting showed that erlotinib significantly inhibited the upregulation of p-ERK/ERK and p-Akt/Akt induced by CXCL8.2?The colorectal cancer cells with overexpression of TrkB were successfully constructed to explore the effect of overexpression of TrkB on the progression of colon cancer.2.1?The apoptosis of colorectal cancer cells with overexpression of TrkB was detected by flow cytometry.The results showed that the apoptosis rate of SW480 cells with overexpression of TrkB was significantly lower than that of normal SW480 cells.Compared with the cells treated with erlotinib,the apoptosis rate of SW480 cells pretreated with CXCL8 overexpressed by TrkB was higher than that without erlotinib(P<0.05).2.2?Effect of erlotinib on intestinal cancer cell EMT:after treatment with erlotinib,Westernblot results showed that the level of E-cadherin in SW480 cells was up-regulated,while N-cadherin and Vimentin were down-regulated(P<0.01),but the overexpression of TrkB changed this effect(P<0.01).2.3?The results of Transwell assay showed that the invasive ability of TrkB overexpressed SW480 cells was significantly higher than that of normal SW480 cells,and the invasive ability of TrkB overexpressed SW480 cells decreased after treatment with erlotinib(P<0.01).The data of scratch test showed that the migration and invasion of colon cancer cells were significantly similar(P<0.01,p<0.05).2.4?After administration of erlotinib,the levels of TrkB,p-ERK/ERK and p-Akt/Akt in SW480 cells decreased significantly(P<0.01),but in SW480 colorectal cancer cells with overexpression of TrkB,this trend was significantly reversed(p<0.05,P<0.01).3.1?After the addition of U0126,the level of p-ERK/ERK decreased significantly(P<0.01);meanwhile,obvious changes of E-cadherin,N-cadherin,and vimentin expression were also found in CXCL8 plus U0126-treated SW480 cells compared with the cells only treated with CXCL8(P<0.01).3.2?After the addition of MK2206,the level of p-AKT/AKT was significantly inhibited(P<0.01),whereas p-ERK/ERK level,did not change conspicuously(P>0.05);besides,the expression of E-cadherin,N-cadherin,and vimentin slightly changed relative to CXCL8-treated SW480 cells(P>0.05)3.3??Anoikis rate was found to be significantly increased in both SW480 and Caco-2 cells after adding U0126(P<0.01),but the administration of MK2206 did not cause notable change(P>0.05).Similarly,a well marked decrease of cell invasion and migration capacity was also found in U0126-treated cells(P<0.01),but little change was exhibited in MK2206-treated cells(P>0.05).Conclusions:1?Erlotinib can inhibit the resistance to apoptosis,invasion and migration of CXCL8-induced colon cancer cells,as well as the occurrence of EMT.2?It is the ERK pathway but not AKT pathway was associated with erlotinib-induced metastasis resistance in colon cancer cells. |
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