Inhibition Of Tight Junction Protein CLDN6on Malignant Phenotypes Of Colon Cancer Cell SW1116

The tight junction(TJ) is composed of four kinds of proteins which are occludin,Claudins(CLDNs), junctional adhesion molecules(JAMs) and zonulaoccludens(ZO).Among the total, CLDNs, as the major part of TJs which were first found by furuse etal in1998, are the most important backbone proteins. CLDNs play determinativeroles in cell adhesion, maintaining cell polarity and signal transduction, andparticipate in the regulation of cell proliferation, differentiation and metastasis. Weconsider that CLDNs in tumor tissues and cells express in a tissue specific manner.Previous studies identified that the down-regulation or loss of CLDN proteins areimportant reasons for the abnormal structure and function of TJ, however, recentstudies have revealed that abnormal changes in the expression levels, whether it behigher or lower, can lead to structural and functional disruption of TJs, thus promotingtumorigenesis and progression. For example, some CLDN proteins appear highexpression levels in tissues and cells[3].Found in gastric cancer cell AGS CLDN6,higher CLDN7and CLDN9expression can enhance the proliferation, invasion andmigration abilityof the cancer cells[4].While some others appear the opposite[2]. Foundlow CLDN7expression in colorectal cancer. Down regulating CLDN7expression notonly associates with lymph node metastasis, but also transforms colorectal adenomasinto colorectal cancer[7].CLDNs protein expression in the colon is not the same, butthe result will be the damage of normal structure, resulting in abnormalmoleculardistributionbetweenthecells, and disappearancein a series of cell polarity[6].Despite the variety of changes in CLDNs individually, together they can ultimatelygive rise to the disruption of normal TJs, and bring about a series of structural andfunctional changes, such as abnormal molecules distribution between cells and theloss of cell polarity.CLDN6gene is a member of CLDNs family which has27known genes. Localizedin chromosome16p13.3, it encodes219amino acids guiding the synthesis of a proteinof23kDa, which includes4transmembrane regions. The two extracellular loops of CLDN6can serve as potential therapeutic targets, and the carboxy-terminal tail isrelated to signal transduction. CLDN6plays a major role in maintaining thefunctionality and integrity of the TJs, and its carboxy-terminal tail contains aPDZ-domain-binding motif that allows CLDN6to interact with cytoskeletal proteinsand signal transduction proteins, taking part in cellular response against externalsignals and internal signal transduction.Delightful progress has already been achieved in the study of the vital function ofCLDN6in epithelium originated tumor. Over expression of CLDN6evidently inducescell apoptosis, significantly inhibits cell proliferation, migration and invasion; CLDN6can inhibits the malignant phenotypes of lung adenocarcinoma cell H1299. In thepreliminary work, we identified CLDN6gene, which appears high levels ofexpression in normal colon epithelium NCM460, but low expression levels in coloncancer cell SW1116. To explore the effect of CLDN6on the malignant phenotype ofcolon tumor cells, we gained SW1116sublines with high levels of CLDN6bytransfection with a pIRES2-EGFP-CLDN6expression vector. Cell clones with stableexpression of CLDN6are selected using G418Filtration. Detect the effect of CLDN6on cell proliferation, invasion and metastasis ability,apoptosis, tumorigenic ability.Methods:1.RT-PCR and Western Blot evaluate the expression of CLDN6in human colonepithelial cell line NCM460and human colon tumor cell line SW1116;2.Transfect SW1116with pIRES2-EGFP-CLDN6expression vector bylipidosome. Screen and identify SW1116cell clones with stable CLDN6overexpression by G418Filtration;3.RT-PCR, Western Blot and cytoimmunity stain evaluate CLDN6transfectionefficiency and CLDN6expression orientation;4.CCK-8and two-dimentional clone forming essay evaluate cell proliferativeability;5.Wound Healing Essay and Transwell Migration Essay evaluate cell migrationand metastatic ability;6.DAPI and DNA Ladder experimental Essay evaluates cell apoptotic;7.Nude Mouse Transplantation Tumor Experiment evaluates tumorigenic abilityin nude mouse,Immunohistochemical method to detect nude mice into tumor tissue inthe expression and localization of CLDN6. Results:1.CLDN6had a high expression in normal colon epithelial cell line NCM460,and low expression in colon tumor cell line SW1116;2.Screened and identified three clones over-expressing CLDN6stably;3.RT-PCR and Western Blot showed that stable expression clones had higherlevels of CLDN6expression than empty vector group; The cytoimmunity stain resultsshowed that CLDN6localized on the cell membranes and nuclear membranes instable expression clones and revealed absent in vector groups;4.CCK8showed the over expression groups had a lower proliferation speed thanthe vector groups; Two-dimensional clone forming assay revealed that compared tothe vector group cells, over expression group cells had fewer clone formation,andtheir proliferative ability were significantly lower; Wound healing assay found thatcell migration ability was inhibited in over expression cells; Transwell assay showedthat invasive ability was inhibited in over expression cells; DAPI and DNA Ladderexperimental stain proved more cell apoptosis in over expression groups than vectorgroup; Nude Mouse Transplantation Tumor Experiment showed that tumors in overexpression groups were smaller both in size and number, and they showed lowertumorigenic ability; Immunohistochemistry of the nude mouse tumor tissue showedthat CLDN6localized on the cell membrane and cytoplasm.Conclusions:CLDN6significantly inhibits colon tumor cell SW1116proliferation, invasionand migration, induces cell apoptosis, and inhibits SW1116tumor forming ability innude mouse.