The Inhibition Effects Of Claudin-6on Malignant Phenotypes Of Lung Adenocarcinoma Cell H1299through P38MAPK Signaling Pathway

Lung cancer has been a tumor disease of highest incidence and mortality in theworld. Lung cancer consists small cell lung cancer（SCLC） and non-small cell lungcancer（NSCLC） which includes squamous carcinoma, adenocarcinoma, large cellcarcinoma and so on. Surgry, chemotherapy, radiotherapy and molecular targetedtherapy are the main effective therapies to lung cancer. However, majority of thepatients lose the chance for surgery when the disease is diagnosed.Lung adenocarcinoma usually resists to chemotherapy and radiotherapy, some ofwhich take place distant metastasis when the tumor is less the1cm. Recently，gefitinibplays an effective role to lung adenocarcinoma as molecular targeted therapymedicine. On the other side, it remains a majority of lung adenocarcinoma that cannot been controlled by EGFR-TKI. Thus, it makes much sense to explore new ways ofmolecular targeted therapy to lung adenocarcinoma.CLDN6is a member of CLDN family which has27proteins. Previousreaserches identify many folded clones(20M24) in embryonic stem cells thoughddPCR. DNA sequencings for20M24revealed it an open reading frame with219amino acids guiding synthesis of a protein of23kDa called CLDN6which hashomology to TJ family. CLDN6localized in chromosome16p13.3which plays animportant role in maintaining TJ’ structure and function as a member of CLDN family.Previous research suggested that over expression of CLDN6could suppress theabilities of proliferation, migration and invasion in breast cancer cell MCF-7.We consider that CLDNs in lung cancer tissues and cells express specifically.Members of CLDN family in lung cancer tissues and cells show both up-regulated and down-regulated. However, whatever the situation that CLDNs express in lungcancer tissues and cells, it will inevitably give rise to the destruction of TJ’s normalstructure which leads to changes of abnormal lateral molecular diffusion betweencells, dispearance of cell polarity and so on.It is reported that some members of CLDNs influenced the biologicalbehaviours of some particular cancers by activating or inhibiting some signalingpathway. Some researches also revealed that over expression of CLDN6could inducecells apoptosis though activating ASK1/P38signaling pathway in breast cancerMCF-7. Few reports cover the effects and mechanism of CLDN6as a member of TJprotein on the biological phenotypes in lung adenocarcinoma cell line. For the firsttime，this research will identify the effects and mechanism of CLDN6on thebiological phenotype of lung adenocarcinoma cell H1299. This will contribute to notonly understand the mechanism on occurance of lung adenocarcinoma, but alsoexplore new methods to diagnosis and new medicines of molecular targetingtreatment for lung adenocarcinoma.Metheds：To explore the correlation between CLND6and some lung adenocarcinoma celllines and the effects of up-regulation of CLDN6on lung adenocarcinoma cell lineH1299, we employed the following metheds:1. We evaluated the expression of CLDN6in human bronchial epithelial cell16Hbe and lung adenocarcinoma cell lines A549, A2, and H1299.2. We designed and structured pIRES2-EGFP-CLDN6plasmid which was thentransfected into H1299cells.3. We screened and indentified H1299cells with stable over expression ofCLDN6.4. We explored the effects of CLDN6on cell growth ability.5. We explored the effects of CLDN6on cell proliferation ability.6. We explored the effects of CLDN6on cell invasive and migration ability. 7. We explored the effects of CLDN6on cell structure and function of tightjunction.8. We analysed the mechanism of effects that CLDN6worked on H1299cells.Results:1. CLDN6had a normal expression in human bronchial epithelial cell16Hbe andlow or absent expressions in lung adenocarcinoma cell lines A549, A2and H1299.2. A pIRES2-EGFP-CLDN6plasmid was structured and identified.3. Screened and identified the clones expressing CLDN6steadily by transfection:evaluated by fluorescence microscope, RT-PCR and Western blot, three clones namedNO.6,10and11with stable expression of CLDN6were confirmed. Thecytoimmunity stain results showed that CLDN6localized on the cell membranes andnuclear membranes in stable expression clones and revealed absent in vector groups.4. CCK8showed the over expression groups have a lower proliferation speedthan the vector groups. Two-dimensional clone forming assay and three-dimensionalclone forming assay revealed that comparing to the vector group cells, overexpression group cells have fewer clone formation. Flow cytometry cell cycle assaydiscovered that over expression group cells have a lower rate in S phase comparing tovector groups which suggested that over expression group have fewer cells inproliferation stage. Above all, over expression of CLDN6may decrease cellproliferation speed, two and three-dimensional clone forming abilities and change cellcycle stages.5. The effects of CLDN6on H1299cell apoptosis: DAPI stain proved more cellapoptosis in over expression groups than vector group. Hoechst33342/PI double stainshowed that the rate of early and late apoptosis cells in over expression groups werehigher than vector group. Flow cytometry cell cycle assay further proved the aboveresults. Above all, CLDN6may induce cell apoptosis.6. The effects of CLDN6on H1299cell metastatic ability: Wound healing assayfound that cell migration ability was lower in CLDN6expression cells than vector group. Transwell assay showed that invasive ability was lower in CLDN6expressioncells. Above all, CLDN6may decrease the cell migration and invasive abilities. Celladhesion assay in vitro showed there was no difference in all groups.7. Transepithelial electric resistance of monolayer culture revealed thatcomparing to the vector group cells, over expression group cells showed a higherresistance which means that over expression of CLDN6may strengthen the structureand enhance the function of tight junction between monolayer cultured cells.8. The mechanisms of CLDN6’s on H1299cell biological behaviour: Westernblot assay showed that the level of phospho-ASK1and ASK1, phospho-P38,phospho-STAT3and STAT3was higher in CLDN6expression groups than vectorgroup which meaned that over expression of CLDN6could activate P38MAPKsignaling pathway. After treating by P38inhibitor named SB203580, the expressionsof CLDN6and ASK1showed no difference and that of STAT3decreased significantlywhich suggested P38localized at downstream of CLDN6/ASK1signaling pathwayand upstream of STAT3signaling pathway. P38inhibitor could not only obviouslyinhibit the activation of P38pathway, but also partly suppress the effects of CLDN6expression on cell proliferation apoptosis, migration and invasive abilities, yet with noeffect on CLDN6expression. Above all, we believe that the effects of CLDN6on cellproliferation apoptosis, migration and invasive ability might work though P38signaling pathway.Conclusion:1. CLDN6has a normal expression in human bronchial epithelial cell16Hbe andlower expressions in lung adenocarcinoma cell lines A549, A2and H1299.2. CLDN6can inhibit proliferation, migration and invasive abilities, induce cellapoptosis in H1299cell.3. CLDN6can inhibit cell metastatic ability by strengthening the tight junction.4. Over expression of CLDN6is able to up-rgeulate P38in human lungadenocarcinoma cell line H1299. 5. The effects of CLDN6on biological phenotype of H1299are related with theactivation of P38MAPK signal pathway.