| Background and objectivesGalectin-4(Gal-4)has two similar carbohydrate cognition domains(CRD),it is a tandem complex galectin which is expressed intracelluar and extracelluar.Gal-4 participates in cell proliferation,differentiation,apoptosis and adhesion.At present,Gal-4 expression has been detected in many cancers,such as colorectal cancer,hepatocellular carcinoma,breast cancer,gastric cancer,etc.Currently,there are few research on the progression of Gal-4 in cancer,and it has been only reported that Gal-4 can promote the proliferation and migration of tumor cells in colorectal cancer.In lung adenocarcinoma,high level of Gal-4 expression has been only reported to be an independent predictor of metastasis.The previous study of our group firstly showed that Gal-4 played an opposite role in the invasion and migration of two non-small cell lung cancer(NSCLC)cells,A549 and H1299 cells.The underlying mechanism still remains unclear.In the present study,the effects of Gal-4 on the adhesion,invasion and migration in NSCLC cells were confirmed by adding another two types of NSCLC cells,H460 and Calu-1 cells,and subsequently elucidating the underlying mechanism of the opposite effects.The results will support Gal-4 to be an independent predictor of different NSCLC metastasis and clinical therapy target for NSCLC treatment.Methods and ResultsMethods:1.Effects of intracellular Gal-4 on the adhesion,invasion and migration of H460 and Calu-1 cellsIn the present study,NSCLC H460 cells with high expression of Gal-4 and Calu-1 cells with low expression of Gal-4 were chose to investigate the effects of Gal-4 on the adhesion,invasion and migration of different NSCLCs.We interfered the expression of Gal-4 in H460 and Calu-1 cells by siRNA technique.The expression of Gal-4 in Calu-1 cells was increased by plasmid transfection technique,and the expression of Gal-4 was detected by Western blot method.After interference or overexpression of intracellular Gal-4,the effects of Gal-4 on adhesion,invasion and metastasis of H460 cells and Calu-1 cells were examined by wound scratch assay,adhesion assay and transwell invasion assay.2.Regulation mechanism of intracellular Gal-4 on the adhesion,invasion and migration in H460 and Calu-1 cellsFirstly,adhesion molecules N-cadherin,CD44 and E-cadherin,which were involved in the regulation of invasion,metastasis and adhesion of H460 and Calu-1 cells,were detected by adhesion assay,Transwell invasion assay and migration assay.Changes of N-cadherin,CD44 and E-cadherin protein expression in cells after interference with Gal-4 were detected by Western blot method.Then we study the influences between Gal-4 and the expression of transcriptional factors,NF-?B,STAT3 and ?-catenin to reveal the regulation of Gal-4 on adhesion molecules in NSCLC cells.The upstream and downstream relationship of the two transcriptional regulators NF-?B,p-STAT3 was determined by introducing NF-?B inhibitor PDTC and p-STAT3 inhibitor HO-3867 to cell culture system.After interfering with or overexpressing Gal-4 in cells,the NF-?B inhibitor PDTC and the?-catenin inhibitor ICG-001 were added to determine whether the involvement of Gal-4 in the invasion,metastasis and adhesion of NSCLC cells was related to the regulation on transcriptional factors NF-?B and ?-catenin.3.Correlation between the opposite regulation of intracellular Gal-4 on the invasion and metastasis of NSCLCs and the expression of intracellular E-cadherinWestern blot and PCR were used to detect the protein and gene expression levels of E-cadherin in A549,H1299,Calu-1 and H460 cells.After interfering with Gal-4 in cells,E-cadherin/?-catenin complex was extracted by immunoprecipitation,and the expression of ?-catenin was detected by Western blot.4.Detecting the correlation between the opposite regulation of Gal-4 on the invasion and metastasis of NSCLCs cells and the expression level of intracellular tumor suppressor gene p53Western blot and PCR were used to detect the protein and gene expression levels of p53 in A549,H1299,Calu-1 and H460 cells.The effects of p53 inhibitor pifithrin-?on the migration of different NSCLC cells(A549,H1299,Calu-1 and H460 cells)were assessed using wound scratch assay.The effect of p53 on related adhesion molecules was determined by detecting the expression of adhesion molecules by Western blot method.After interfering or overexpressing intracellular Gal-4,the role of p53 on the migration of NSCLC cells were determined by wound scratch assy using p53 inhibitor pifithrin-?.5.Correlation between the opposite regulation of intracellular Gal-4 on the invasion and metastasis of NSCLCs and the expression of intracellular proto-oncogene RasThe effect of Ras inhibitor salarisib on the migration of NSCLC cells(A549,H1299,Calu-1 and H460 cells)was examined by wound scratch assay.To further explore the role of Ras in the regulation of Gal-4 on the metastasis of NSCLC cells,the Ras inhibitor salarisib was used after interfering or overexpressing intracellular Gal-4 to validate the role of Ras in the regulation of Gal-4 on the invasion and metastasis of NSCLC cells using wound scratch assay,adhesion assay and Transwell invasion assay.6.Effects of intracellular Gal-4 on lung metastasis of A549 cells or H1299 cells in nude miceThe opposite effects of Gal-4 on the adhesion,invasion and migration of A549 and H1299 cells was observed in vivo lung metastasis model by injection of A549-Luc cells and H1299 cells which interfered with siGal-4 into the tail vein of immunodeficient nude mice.The mice were photographed every 10 days by using the bioluminescence system to observe the lung metastasis of A549-luc cells.The lung metastasis of H1299 cells was determined by observing the lung nodules after dissecting nude mice(the days of dissecting mice was decided by observing tumor formation in a pre-experiment).Results:1.Intracellular Gal-4 inhibits the adhesion,invasion and migration of H460 cells,promotes the adhesion,invasion and migration of Calu-1 cellsWestern blot analysis showed that interference with siGal-4 could decrease the expression of Gal-4 in H460 cells and Calu-1 cells,while transfection with Gal-4 plasmid could increase the expression of Gal-4 in Calu-1 cells.In wound scratch assay,adhesion assay and transwell invasion assay,by interference with Gal-4,the adhesion,invasion and migration of H460 cells was increased,while the adhesion,invasion and migration of Calu-1 cells were decreased.After overexpression of Gal-4 in Calu-1 cells,the adhesion,invasion and migration of Calu-1 cells were higher than those of the control group.Results showed that intracellular Gal-4 inhibited the invasion and metastasis of H460 cells and promoted the invasion and metastasis of Calu-1 cells.2.Intracellular Gal-4 involves the invasion and metastasis of H460 and Calu-1 cells by regulating the expression of adhesion molecules,N-cadherin and CD44After interfering with N-cadherin and CD44 in H460 and Calu-1 cells by siRNA technique,the adhesion and invasion ability were decreased in both cells,indicating that the adhesion molecules,N-Cadherin and CD44,promotes the invasion and adhesion process of H460 and Calu-1 cells.At the same time,by interfering withGal-4 of H460 and Calu-1,both N-cadherin and CD44 expression were up-regulated in H460 cells,while both N-cadherin and CD44 expression was down-regulated in Calu-1 cells.This indicates that intracellular Gal-4 regulates the expression of adhesion molecules,N-cadherin and CD44,to regulate invasion and adhesionof H460 and Calu-1 cells.3.Intracellular Gal-4 regulates the expression of transcription factor p-STAT3/NF-?B to regulate the expression of downstream adhesion molecules After interfering with siGal-4 in H460 and Calu-1 cells,p-STAT3 and NF-?B were up-regulated in H460 cells,while downregulated in Calu-1 cells.After addition of NF-?B inhibitor PDTC and p-STAT3 inhibitor HO-3867,NF-?B was down-regulated after adding p-STAT3 inhibitor HO-3867,however,the expression of p-STAT3 has no significant change after adding p-STAT3 inhibitor HO-3867,indicating that p-STAT3 was located upstream of NF-?B.After adding NF-?B inhibitor PDTC,NF-?B promotes the migration of H460 andCalu-1 cells.Moreover,NF-?B could regulate the expression of downstream adhesion molecules N-cadherin,E-cadherin and CD44,and MMP-2.After interfering Gal-4 in both cells,NF-?B inhibitor PDTC decreased the invasion,migration of both cells and the adhesion function to vascular endothelial cells.The results of Western blot showed that Gal-4 regulates the expression of downstream adhesion molecules E-cadherin,N-cadherin and CD44,and MMP-2 by regulating the transcription factor p-STAT3/NF-?B.4.Intracellular Gal-4 regulates the expression of transcription factor ?-catenin to regulate the expression of downstream adhesion molecules After interference with siGal-4 in H460 and Calu-1 cells,?-catenin was up-regulated in H460 cells,while down-regulated in Calu-1 cells.After adding?-catenin inhibitor ICG-001,the migration of H460 and Calu-1 cells was inhibited.The Western blot results indicated that ?-catenin could upregulate downstream adhesion molecules(N-cadherin and CD44)and MMP-2 expression.After interfering with siGal-4 of both cells,the ?-catenin inhibitor ICG-001 decreased the invasion,adhesion and migration of both cells.Western blot analysis showed that Gal-4 regulated downstream adhesion molecules E-cadherin,N-cadherin and CD44,and MMP-2 by interacting with transcription factor ?-catenin.5.Intracellular Gal-4 promotes the formation of E-cadherin/?-catenin complex in A549 and H460 cellsThe results of PCR and Western blot showed that E-cadherin was highly expressed in A549 and H460 cells,but not expressed in H1299 cells,and lower expressed inCalu-1 cells.After interfering with siGal-4 in these four cells,the intracellularE-cadherin/?-catenin complex was isolated by immunoprecipitation assay.Western blot results showed that Gal-4 up-regulated the expression of E-cadherin/?-catenin complex in A549 and H460 cells.However,no significant E-cadherin expression wasdetected in H1299 and Calu-1 cells.This resutls suggested that Gal-4 inhibited the invasion and metastasis of A549 and H460 cells by promoting the formation E-cadherin/?-catenin complex.6.p53 inhibits the migration of A549 and H460 cells by down-regulating the expression of N-cadherin,CD44 and MMP-2 PCR and Western blot showed that p53 was highly expressed in both A549 andH460 cells,but almost not expressed in H1299 cells,and little expressed in Calu-1 cells.The mobility of A549 and H460 cells was increased after the addition of p53 inhibitor pifithrin-?,indicating that p53 inhibited the migration of A549 and H460 cells.Western blot results showed that the addition of the p53 inhibitor pifithrin-?increased the expression of N-cadherin,CD44 and MMP-2 in A549 and H460 cells.The results indicated that p53 couldinhibit the migration of A549 and H460 cells by down-regulating the protein expression of adhesion molecules N-cadherin and CD44.7.Intracellular Gal-4 does not directly regulate the expression of p53 to inhibit the migration of A549 and H460 cellsAfter the addition of the p53 inhibitor pifithrin-? to Gal-4 interfering cells,no significant change was observed on the migration of A549 and H460 cells,indicating that Gal-4 does not affect the cell migration of NSCLC through direct interaction with p53.8.Ras promotes migration of four NSCLC cellsThe migration of A549,H1299,Calu-1 and H460 cells was decreased by the Ras inhibitor salirasib in a concentration dependent manner.This indicates that Ras promotes the migration of these four types NSCLC cells9.Intracellular Gal-4 promotes migration,adhesion and invasion of H1299 and Calu-1 cells,inhibits migration,adhesion and invasion of A549 and H460 cells by regulating Ras expressionAfter interfering with siGal-4 in A549,H1299,Calu-1 and H460 cells,the Ras inhibitor salarisib was added to cells to reverse the promotion of siGal-4 on the adhesion,invasion and migration of A549 and H460 cells,the inhibition of siGal-4 on the adhesion,invasion and migration of H1299 and Calu-1 cells.Moreover,the Ras inhibitor salirasib also reversed the promotion of overexpression of Gal-4 on the adhesion,invasion and migration of Calu-1 cells.The above results indicated that intracellular Gal-4 promoted the migration,adhesion and invasion of H1299 and Calu-1 cells,and inhibited the migration,adhesion and invasion of A549 and H460 cells by regulating Ras expression in these NSCLC cells.10.Interference with Gal-4 expression increases lung metastasis of A549 cells in nude mice,and reduces lung metastasis of H1299 cells in nude miceIn vivo experiments,the luminescence intensity of the A549-Luc cells after interference with siGal-4 in lungs was significantly increased compared with control cells without interference with siGal-4.The number of lung metastasis nodules of A549 cells in siGal-4 A549 cell groupwas higher than that in the control group after mice were dissected.However,the number of lung nodules of H1299 cells in siGal-4 H1299 cell group was lower than that in the control group.The results indicated that the interference of Gal-4 expression in A549 cells could increase the lung metastasis of A549 cells in nude mice,but the interference of Gal-4 expression in H1299 cells could reduce the lung metastasis of H1299 cells in nude mice.Conclusion1.Intracellular Gal-4 inhibits the migration,adhesion and invasion of H460 cells,and promotes the migration,adhesion and invasion of Calu-1 cells.Intracellular Gal-4 regulates the expression of adhesion molecules N-cadherin and CD44 to regulate the metastasis of H460 and Calu-1 cells.In H460 cells,intracellular Gal-4 inhibits transcription factors p-STAT3/NF-?B and ?-catenin to decrease the expression of adhesion molecules.In Calu-1 cells,intracellular Gal-4 promotes transcription factors p-STAT3/NF-?B and(3-catenmn to increase the expression of adhesion molecules.Among the transcription factors,Gal-4 shows more obvious regulation on ?-catenin.2.Gal-4 promotes the formationof E-cadherin/?-catenin complex in both A549 and H460 cells,and the level of ?-catenin which interacts with Gal-4 is reduced due to the increased level of complex formation.Therefore,Gal-4 in A549 and H460 cells showed inhibition on the metastasis of NSCLC cells.Whereas in the other two cells,there was no E-cadherin/?-catenin complex formation due to the absence of E-cadherin expression,so Gal-4 directly promotes the translocation of ?-catenin into the cellular nucleus,thereby mediating the promotion of invasion and metastasis of H1299 and Calu-1 cells.3.There was p53 expression in A549 and H460 cells,but no p53 expression in H1299 and Calu-1 cells.p53 inhibits the migration of A549 and H460 cells by down-regulating the expression of N-cadherin,CD44 and MMP-2.However,we found that the regulation of Gal-4 onthe metastasis of NSCLC cells did not directly interaction with p53.4.In NSCLC,Ras promotes the migration of four NSCLC cells.Intracellular Gal-4 regulates Ras expression to promote the migration of H1299 and Calu cells and to inhibit the migration of A549 and H460 cells.In vivo,Gal-4 inhibits the lung metastasis of A549 cells and promotes the lung metastasis of H1299 cells,which is consistent with the results of in vitro cell experiments. |
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