The Role And Mechanism Of Integrin αⅤ In Multicellular Resistance Of Colon Adenocarcinoma

At present,available therapies cure a little more than half of cancer patients. Chemotherapy is one of the most useful methods to cure cancer but anticancer drug resistance is frequent.A successful solution to this resistance would lead to a great increase in therapeutic success as novel anticancer drugs often offer only a small improvement over older agents.Recent research using multicellular tumor spheroids has resulted in new insights in drug resistance.When grown in spheroids,cancer cells exhibit a phenomenon known as 'multicellular resistance'. Although we have already known that multicellular resistance is adhesion-dependent,until now, the mechanism of multicellular resistance is elusive.In this dissertation,we focused on integrinαV,one of cell adhesion molecules,which could be found in many kinds of cells and played a key role in cell adhesion and regulation of apoptosis.Objective:To investigate the role and mechanism of integrinαV in multicellular resistance of colon adenocarcinoma.Materials and Methods:We cultured human colon adenocarcinoma cells(HT29) as multicellular spheroids by gently convoluting them horizontally in culture dishes coated with 2% agarose.We also used LIM1863,a cell line of colon adenocarcinoma,which grows as spheroids in an automatic way.Drug sensitivity to oxaliplatin was detected by Cell counting kit-8(Dojindo, Japan) and its absorbance was detected with 450nm measurement wavelength and 630nm reference wavelength.PCR was used to clone genes of EGFP and CMV in plasmid pEGFP-N2 and genes were inserted in plasmid pSUPER.neo.Then plasmid pSUPER.neo.EGFP has been constructed.Synthesize idiosyncratic DNA which can differently RNAi integrinαV,NF kappa B, JNK1,JNK2 by the instruction of pSUPER.neo and connected them to pSUPER.neo.EGFP cut by restriction enzymes BglⅡand HindⅢ.Then cut the plasmid with restriction enzymes EcoRⅠand XhoⅠand insert the small DNA fragment which contained genes of H1 promoter, Synthesize idiosyncratic DNA,CMV promoter and EGFP into retrovirus plasmid pLXSN.Then retrovirus plasmids pLXSN.EGFP.si-IntegrinαV,pLXSN.EGFP.si-NF kappa B, pLXSN.EGFEsi-JNK1,pLXSN.EGFP.si-JNK2 had been constructed.Differently transfect retrovirus plasmids into HT29 and LIM1863 to silence integrinαV,NF kappa B,JNK1,JNK2 and then culture with G418 for a month.Then select the clone what cells express EGFP and culture them with G418,and select a clone,and repeat for five times.We calls HT29 cell lines stably express pLXSN.EGFEsi-IntegrinαV as HT29/IntegrinαV-,so do with HT29/NF kappa B-,HT29/JNK1-,HT29/JNK2-,LIM1863/IntegrinαV,LIM1863/NF kappa B-, LIM1863/JNK1-,LIM1863/JNK2-.Treat all kinds of HT29 cultured as monolayer and spheroids with oxaliplatin for one hour then use 2×SDS loading buffer to lyse cells.Detect protein concentration of samples with RC DC Protein Assay(Bio-Rad,America).Analyze the samples by Western Blot and use the cell lysate of HT29 monolayer and spheroids as control.Use NE-PER(?) Nuclear and Cytoplasmic Extraction Reagents(Pierce,America) to extract nuclear protein and detect protein concentration of samples with Bradford colourimetry(Coomassie G250).Use LightShift Chemiluminescent EMSA Kit to detect the activity of NF kappa B by chemiluminescent EMSA.Drug sensitivity of Monolayer cells and spheroids of HT29, HT29/IntegrinαV-,HT29/NF kappa B-,HT29/JNK1-,HT29/JNK2-,and spheroids of LIM1863,LIM1863/IntegrinαV-,LIM1863/NF kappa B-,LIM1863/JNK1-, LIM1863/JNK2-to oxaliplatin was detected.To confirm that retrovirus plasmid didn't influence the result,we also repeated all procedures written before with lysate of cells what transfected retrovirus plasmid.Colon carcinoma xenografts were established by subcutaneous injection of 2×10~7 cells of HT29,HT29/IntegrinαV-,HT29/NF kappa B-,HT29/JNK1-,HT29/JNK2-in flank of female,5 weeks old,BALB/c nu/nu naked mice.After injection of 48 hours,treated the mouse with oxaliplatin at 10mg/kg intravenously.Mice were put to death after 30 days and tumors were dissected and weighed.Analyze the data with one way ANOVA.Results:Firstly,we successfully grew HT29 cells as spheroids.Under inverted microscope, we observed that HT29 cells adherent to each other in 24 hours and formed spheroids after 48 hours.Under scanning electron microscope,we saw cells in HT29 spheroids adherent tightly.HE dyeing,under microscope,we could find spheroids were composed of layers of cells and necrosis and lumen of gland were found in large spheroids grew more than 1 week.Structures of HT29 spheroids were the same with that of LIM1863 except that LIM1863 hadn't necrosis.Under transmission electron microscope,few HT29 monolayer cells were found to adherent to each other and cell junction weren't found.Much cell junction,especially tight junction,was found in HT29 spheroids and LIM1863.Secondly,HT29 spheroids was multicellular resistant to oxaliplatin.Survival ratio(((?)±s)) of HT29 monolayer cells treated with oxaliplatin for 2 days at 50μg/L is 0.582±0.033,at 500μg/L is 0.0930±0.020,while that of HT29 spheroids increased to 0.865±0.047,0.429±0.040.Between survival ratio of monolayer cells and that of HT29 spheroids at the same concentration of oxaliplatin had significant difference(p＜0.05).Survival ratio of LIM1863 treated with oxaliplatin for 2 days at 50μg/L is 0.738±0.038.Thirdly, decrease expression of integrinαV can significantly increase cells sensitivity to oxaliplatin.Used by RNAi to integrinαV,expression of integrinαV in HT 29 was decreased.When integrinαV was effectively silenced,the phosphorylated-NF kappa B and activity of NF kappa B decreased while phosphorylated-JNK2 increased.Drug sensitivity to oxaliplatin of HT29 spheroids and LIM1863 were increased after silence of integrinαV.Survival ratio of HT29/IntegrinαV-spheroids treated with oxaliplatin at 500μg/L significantly decrease to 0.300±0.037(p＜0.05), while LIM1863/IntegrinαV-treated with oxaliplatin at 50μg/L significantly decrease to 0.638±0.029(p＜0.05).HT29/IntegrinαV-xenografts were more sensitive to oxaliplatin than HT29 xenografts too.Average weight of HT29/IntegrinαV-xenografts treated with oxaliplatin is 0.71±0.36(unit:g),while that of HT29 xenografts is 0.94±0.34(p＜0.05).Fourthly,decrease activity of NF kappa B can significantly increase cells sensitivity to oxaliplatin too.Used by RNAi to NF kappa B,expression of both NF kappa B and phosphorylated-NF kappa B and activity of NF kappa B in HT 29 was decreased.When NF kappa B was effectively silenced, phosphorylated-JNK2 increased.Drug sensitivity to oxaliplatin of HT29 spheroids and LIM1863 were increased after silence of NF kappa B.Survival ratio of HT29/NF kappa B-spheroids treated with oxaliplatin at 500μg/L significantly decrease to 0.346±0.038(p＜0.05),while LIM1863/NF kappa B-treated with oxaliplatin at 50μg/L significantly decrease to 0.668±0.037 (p＜0.05).Average weight of HT29/NF kappa B-xenografts treated with oxaliplatin decreased to 0.76±0.43.Fifthly,decrease activity of JNK2 can significantly decrease cells sensitivity to oxaliplatin too.Used by RNAi to JNK2,expression of both JNK2 and phosphorylated-JNK2 in HT 29 was decreased.When JNK2 was effectively silenced,drug sensitivity to oxaliplatin of HT29 spheroids and LIM1863 were decreased.Survival ratio of HT29/JNK2-spheroids treated with oxaliplatin at 500μg/L significantly decrease to 0.493±0.032(p＜0.05),while LIM1863/NF kappa B-treated with oxaliplatin at 50μg/L significantly decrease to 0.798±0.038 (p＜0.05).Average weight of HT29/NF kappa B-xenografts treated with oxaliplatin decreased to 1.10±0.46.sixth,though silence JNK1could decease expression of phosphorylated-JNK,drug sensitivity to oxaliplatin of HT29 spheroids and LIM1863 were not change significantly(p＞0.05).Conclusions:First,HT29 spheroids could simulate the small solid tumor in vivo before t vascularized.It is a good candidate for hypoxia,adhesion,cell junction and differentiation. Second,we constructed a system of plasmid-retrovirus plasmid used as RNAi media what can stably,effectively and specific to silence a gene and it itself has little influence on cells.Third, activation integrinαV in multicellular spheroids played a key role in multicellular resistance of colon adenocarcinoma.That it activated NF kappa B to inhibit JNK2 was the mechanism.Fourth, decrease expression of integrinαV or block its signal transduction could significantly reverse multicellular resistance of colon adenocarcinoma.Fifth,that silence JNK1 can decrease expression of JNK1,but couldn't effectively change drug sensitivity to oxaliplatin made us think JNK1 didn't play an important role in multicellular resistance of colon adenocarcinoma.