Study On Biological Characterization And Mechanisms Of Acquired Resistance To Irinotecan In Human Colon Cancer

BackgroundIrinotecan(CPT-11)is mainly used in colorectal cancer(CRC)diagnosed patients with metastases,with recorded relapse or progression after application of standard 5FU-based therapy.However,the response rate to these regimens is only in the range of about 30-55%and the 5-year survival rate is less than 10%.Resistance to chemotherapy is a major limitation to the outcome in CRC.Twist is a basic helix-loop-helix transcription factor,which is one of the master regulators of EMT process,including Twistl and Twist2.Different studies have also reported that high expression of Twistl is closely associated with more aggressive behaviors of cancers,including epithelium-mesenchymal transition(EMT),cell proliferation and differentiation,multidrug resistance(MDR),tumor invasion and metastasis,cancer stem cells(CSC).In addition,Our previous studies have indicated that Twistl is overexpressed in colon cancer tissues,its positive expression is related to histological grade,TNM stage,recurrence and poor overall survival and is a significant independent prognostic indicator in CRC patients.And it plays an important role in multidrug resistance and invasion and metastasis of colon cancer cells.However,the underlying mechanisms of irinotecan resistance induced by Twistl are still ambiguous in CRC.Object1.To establish irinotecan-resistance cell subline and study the malignant biological characteristics;2.To explore the mechanisms of irinotecan resistance induced by Twistl in CRC.MethodPart 1:The establishment,evaluation and malignant biological characteristics of irinotecan-resistance cell of human colon cancerIrinotecan-resistant LoVo cells were established by gradual adaptation of the original cell lines to increasing irinotecan concentrations,named LoVo/CPT-11R cells.After treated by different concentration of irinotecan,the cell viability of LoVo/CPT-11R cells was detectedd by CCK-8.The morphological changes of LoVo/CPT-11R cells visualized via light microscopy.The cell viability to various anticancer drugs used to treat CRC,including 5-FU,DDP and Cur,were detected using the CCK-8 assay;the expression of ABC transporter genes(ABCB1,ABCC1 and ABCG2)on the mRNA level were detected by qRT-PCR assay and the protein level of ABCB1(P-gp)by Western blot in LoVo cells and LoVo/CPT-11R cells.The staining localizations of EMT markers(E-cadherin,Vimentin)were determined by Immunofluorescence assay,the expression of EMT(E-cadherin,ZO-1,Vimentin,N-cadherin),transcriptuon factors(Twistl,Slug,Snail,Zeb1)and CSC markers(CD44,CD 133)were detected on defferent levels by qRT-PCR and Western blot in LoVo cells and LoVo/CPT-11R cells.Part 2:The mechanisms induced by Twistl of multidrug resistance to irinotecan and malignant biological behaviors in colon cancerThe overexpressed Twistl LoVo cells were bult by transfection with overexpressed Twistl lentiviruses,and the control cells were transfected by empty vector lentiviruses,respectly named LoVo/Twistl cells and LoVo/Vector cells.After treatment with different concentrations of irinotecan,the cell viability was detected by CCK-8 in LoVo/Twistl cells and LoVo/Vector cells.The expression of EMT(E-cadherin,ZO-1,Vimentin,N-cadherin),transcriptuon factors(Twistl,Slug,Snail,Zebl)and CSC markers(CD44,CD133)on defferent levels by qRT-PCR and Western blot in LoVo/Twistl cells and LoVo/Vector cells.To assess the migratory and invasive properties using Transwell assays and detecct the expression of MMP2,MMP9 using Western blot in LoVo/Twistl cells.Downregulation of Twistl by transfected respectively with NC siRNA and Twistl-targeted siRNA in LoVo cells and LoVo/CPT-11R cells,then cells were treated with different concentrations of irinotecan,every group cell viabilities were detected by CCK-8 assays.Moreover,the expression of EMT and CSC-like markers were detected respectively by RT-PCR and Immunofluorescence assays.ResultPart 1:1.The LoVo/CPT-11R cells were able to grow steadily in the complete culture containing 70 ?g/mL irinotecan.The drug resistance index was 5.79.The celluar morphology was significantly changed,and the long spindle fibrous cells increased and the diffusion was irregular.2.The cell viabilities of LoVo/CPT-11R cells were significantly increased at different concentrations of CPT-11,5-FU,DDP and Cur treatment.The expression of ABCB1(P-gp)was up-regulated in both mRNA and protein level.3.The staining of E-cadherin was located mainly in cell membrane of LoVo cells,but E-cadherin was located mainly in cytoplasm of LoVo/CPT-11R cells.The Vimentin protein was more distinctly stained in cytoskeletal localization in LoVo/CPT-11R cells.The E-cadherin and ZO1 were downregulated in LoVo/CPT-11R cells.In contrast,the Vimentin and N-cadherin were upregulated.In addition,the expression of Twistl was higher in LoVo/CPT-11R cells than LoVo cells.However,the changes of expression of Snail,Slug and Zebl were not statistically significant.Furtherly,the expression of CD44 and CD 133 were increased in mRNA and protein levels in LoVo/CPT-11R cells compared to LoVo cells.It was confirmed that the LoVo/CPT-11R cells possessed EMT and CSC-like phenotypes.Part 2:1.The cell viabilities of LoVo/Twistl cells were distinctly increased compared with the LoVo/Vector cells.The expression of CD44 was up-regulated and E-cadherin was down-regulated.2.The overexpression of Twistl enhanced the migratory and invasive potentials of LoVo cells.The expression of MMP2 protein was up-regulated.3.Inhibition of Twistl expression increased sensitivity to irinotecan of LoVo cells,and reversed resistance to irinotecan of LoVo/CPT-11R cells.4.Inhibition of Twistl expression down-regulate the expression of ABCB1,Vimentin and CD44.ConclusionPart 1:1.The irinotecan-resistant colon cancer performs multidrug resistance to anticancer drugs,associating to increased expression of ABCB1;2.The irinotecan-resistant colon cancer shows EMT,CSC-like phenotypic change,and Twistl was up-regulated;Part 2:1.EMT and CSC-like phenotype induced by Twistl contribute to resistance to irinotecan in colon cancer.2.Twistl mediates the up-regulation of ABCB1 and MMP2 to promote the resistance of irinotecan and enhance the ability of invasion and migration in colon cancer.