Molecular Mechanisms Of Human 67kDa Laminin Receptor (67LR) In Cell Adhesion Mediated Multidrug Resistance On Colon Cancer Cell Line SW480

The major cause of chemotherapeutic failure for colon cancer is multidrug resistance (MDR). It is highlighted that a novel mechanism of MDR, named cell adhesion mediated drug resistence (CAM-DR), induced by adhering to the extracellular matrix (ECM) around cancer cells. It is reported that lots of colon cancer malignant phenotypes are correlated with laminin-human 67kDa laminin receptor(LN-67LR) ligation. The aim of our studies is exploration the effect on drug resistance caused by the LN-67LR ligation and characterization of the functions of 67LR, acting as a cell adhesion molecule (CAM), and its role in MDR will help to understand the development and modulation of colon cancer MDR phenotype.Objective The purposes of present works are to study the chemotherapeutic drug sensitivity, exploit the mechanism of drug resistance in colon cancer cells adhering to the ECM substrate, characterize the role and illuminate the molecular mechanisms of 67LRP interacting with its ligand laminin in colon cancer CAM-DR. MethodsPart I: 1. By addhesion assay, the ability of colon cancer cell line SW480 to adhere to LN and BSA was detected. 2. SW480 cells were pre-adhered to LN and BSA, then MTT assay was used to determine the sensitivity of those cells to chemotherapeutic drugs. 3. Adriamycin accumulation and retention in pre-adhered cells were analyzed using flow cytometry. 4. Annexin V/PI staining was used to detect the chemotherapeutic drug-induced apoptosis of the pre-adhered cells.Part II: After construction of 67LR psilencer vector and transfection into SW480 cells, the transfected SW480 cell subline which express down-regulated 67LR will be established.Part III: 1. By addhesion assay, the ability of transfected cell sublines SW480-si67LR to adhere to LN and BSA was detected. 2. SW480-si67LR was pre-adhered to LN and BSA, MTT assay was used to determine the sensitivity of those cells to chemotherapeutic drugs. 3. Annexin V/PI staining was used to detect the chemotherapeutic drug-induced apoptosis of SW480-si67LR which was pre-adhered to LN and BSA. 4. By Western blotting, the expression of pFAK, tFAK, Bcl-2 and Bax in SW480-si67LR which was pre-adhered to LN and BSA were detecteded.ResultsPart I: 1. Cell adhesion assay revealed a significant increase of adhesive cellular number in SW480 cell adhesion to LN, compared with the cells adhesion to BSA as control (p<0.05). 2. MTT assay revealed that the IC50 of chemotherapeutics toward SW480 cells adhesion to LN were higher than that of adhering to BSA, which represented significant decrease of drug sensitivity in SW480 cells adhesion to LN. Flow cytometry analysis has shown a decrease apoptosis index induced by chemotherapeutics and a decrease accumulation and retention of ADR (p<0.05).Part II: Western blot revealed that transfected SW480 cell of down-regulated 67LR have been established. Part III: 1. Cell adhesion assay revealed a significant decrease of adhesion cellular number in 67LR downregulated cell sublines SW480-si67LR adhesion to LN, compared with control (p<0.05). 2. After adhesion to LN, the expression of pFAK is significantly decreased than that of in control cells (p<0.05). 3. After adhesion to BSA, there was no expression of pFAK in SW480-si67LR and control cells. 4. There was no significant difference of tFAK expression in SW480-si67LR and control cells after adhesion to LN or BSA (p<0.05). 5. MTT assay revealed a lower IC50 of chemotherapeutics toward SW480-si67LR cell subline adhesion to different matrix substrates, which represented significant increase of drug sensitivity in SW480-si67LR. Flow cytometry analysis has shown a increase apoptosis index induced by chemotherapeutics in SW480-si67LR adhesion to different matrix substrate. Western blot analysis of lysates derived from SW480-si67LR adhesion to different matrix substrate revealed a decrease in expression of Bcl-2, a increase in expression of Bax (p<0.05).Conclusion: 1. Adhesion to ECM substrates may promote MDR phenotype of colon cancer cells by two means: i. 67LR plays a role in development of colon cancer MDR which decreases the accumulation of chemotherapeutic drugs; ii. 67LR confers partial resistance of colon cancer cells to apoptosis mediated by chemotherapeutic drugs. 2. 67LR is intimately associated with the adhesion ability of colon cancer cell. 3. The interaction of 67LR and its ligand LN which helped the adherence of colon cells initiated the FAK phosphoration. The downstream molecule Bcl-2 was then upregulated and led to the resistance of apoptosis of adherent colon cancer cells.