ESD for Laterally Spreading Tumors in Colon: Experience for a Tertiary Unit

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G A A b st ra ct s However the relations between chemoresistance, cytoskeleton and EMT are inevitable. In the present study, we investigated the effect of chemoresistance on cell motility, cytoskeleton, and EMT. METHODS: HT-29, a human colon cancer cell, was exposed to increasing doses of 5FU or oxaliplatin for 6 months to achieve resistance at clinically relevant doses. cDNA microarray analysis was performed for parent and resistant cells. Rates of proliferation and sensitivity to drugs were assessed using WST-1 Assay. Cell movement was analyzed by Timelapse imaging; the images were captured under an Axiovert 200M microscope (CarlZeiss) for eight hours at intervals of 15 min. For cytoskeleton study, morphologic and molecular changes investigated by immunofluorescence staining and PCR analyses. RESULTS; HT29 cells were finally resistant to 5FU (2μg/mL) and oxaliplatin (2μmol/L). cDNA microarray studies showed alteration of cytoskeletal gene expression (actin related genes including caldesmon were upregulated) and EMT-related genes (TGF-beta1, ZEB2, and Vimentin were upregulated and E-cadherin was downregulated) in both 5FU resistant cells and oxaliplatin resistant cells. The both resistant cells exhibited a decrease in cellular proliferation and EMTlike morphologic changes; spindle-cell shape, loss of polarity, intercellular separation, and pseudopodia formation. Time-lapse imaging showed random and accelerated cell motility. Immunofluorescence staining revealed that E-cadherin expression was decreased and caldesmon expression was enhanced. Caldesmon is reported to act as a potent repressor of cancer cell invasion, however in both resistant cells of this study, overexpressed caldesmon was not associated with F-actin and stress fiber was not developed, which was not typical EMT phenotype. Depletion of caldesmon with siRNA restored the chemoresistant cells to parental cell-like adherent epithelial shape. CONCLUSIONS: These results indicate that chemoresistant CRC developed altered cytoskeleton, accelerated cell motility and atypical EMT phenotype, and that caldesmon is involved in chemoresistance-related phenotypical changes, suggesting that the cytoskeleton related genes are potent molecular targets to chemoresistance and tumor invasion/metastasis.