The Role Of ILK In Multicelluar Drug Resistance Of Colon Adenocarcinoma

Background and Objective:Previous researches have identified drug resistance is the main reason for the poor chemotherapy.Recent researches using multicellular tumor spheroids have resulted in new insights in drug resistance.Although we have known that multicellular drug resistance is adhesion-dependent,until now,the mechanism of multicellular resistance remains elusive.ILK,as the key link gene located between cells and extracellular matrix(ECM) is the key point in vivo signal transduction pathway.Study have identified that ILK has been involved in the process of proliferation,adhesion, migration,apoptosis and invasion of various tumor cells,and even in tumor drug resistance.This study sought to culture human colon adenocarcinoma cells(HT29) as multicellular spheroids.We further examined whether inhibition of ILK by gene silencing using mammalian expression vectors expressing small interfering RNA(siRNA) might lead to inhibition of ILK expression,primary tumor growth,apoptosis and multicellular drug resistance in HT29 cells.Methods:We constructed HT29 multicellular spheroids using cultured human colon adenocarcinoma cells(HT29) by liquid overlay technique.RNAi vector that can express siRNA targeting ILK or siRNA that does not match any known human coding mRNA was designed,constructed,and lipofected into HT29 cells line.HT29 cells carrying ILK siRNA Vector(HT29/siILK) or Control siRNA Vector(HT29/Control) were selected with 1000μg/mL G418-sulfate.Stably transfected HT29/siILK and HT29/Control cells were obtained and routinely maintained in selection media containing 400μg/mL of G418-sulfate to avoid overgrowth of nontransfected cells.Expressions of ILK mRNA and protein were examined by RT-PCR,Western blot in HT29,HT29/siILK and HT29/Control cells respectively.We detected the cell proliferation and drug sensitivity to Oxaliplatin in HT29,HT29/siILK and HT29/siILK cells with MTT and Cell Counting,and analyzed the apoptosis with Tunel.Results:First,we successfully cultured HT29 multicelluar spheroids.Under inverted microscope,we observed these HT29 cells adherent and aggregated to each other tightly.Second,HT29 spheroids was multicellular resistant to Oxaliplatin.Survival ratio of HT29 monolayer cells treated with Oxaliplatin at 50μg/ml,100μg/m were 0.565±0.014,0.425±0.032,while that of HT29 spheroids increased to 0.797±0.004,0.715±0.017. Third,Through Western blot,we inentified that ILK expression in HT29 multicellular spheroids is obviously high compared with the express in HT29 cells.Four,RNAi vector that can express siRNA targeting ILK or siRNA that does not match any known human coding mRNA was designed,constructed,and lipofected into HT29 cells line.By RT-PCR test,the expression of ILK mRNA in HT29/siILK cells(0.16±0.11) was significantly lower than in HT29/Control cells(0.53±0.10).That ILK protein expression in HT29/siILK was lower than HT29 cells and HT29/Control cells(P＜0.05) obviously still can be indicated by Western blot.Five,We can observed through MTT that proliferation of HT29/siILK cells was less active than HT29 cells and drug sensitivity to Oxaliplatin of HT29 multicellular spheroids decreased signitificantly.After transfection,the survival ratio of HT29/siILK treated with Oxaliplatin at 100μg/ml was 0.525±0.054 with no significant difference with HT29,suggesting ILK plays an important role in Multicelluar Drug Resistance of Colon Adenocarcinoma.Six,the analysis of cells apoptosis by Tunel showed HT29/siILK's apoptosis radio inceased rapidly and there was significant difference(P＜0.05) with HT29 cells,while comparing HT29/C ontrol cells with HT29 cells,there was no significant difference(P＞0.05).Conclusions:First,we constructed HT29 multicellular shperoids,which can simulate the solid colon tumor in vivo.Second,we constructed specific RNAi plasmid vector designed for ILK gene to stably, effectively silence ILK gene expression.Third,the inhibition of ILK gene expression could inhibit proliferation of HT29 cells and increase the HT29 cells apoptosis ratio treated with Oxaliplatin.Fourth,the inhibition of ILK could increase the HT29 multicellular spheroids sensitivity to drugs,suggesting it play a significant role in multicellular resistance of colon adenocarcinoma.