Regulation Of ASK1Signaling Pathways And Induction Of Apoptosis By CLDN6in MCF-7Breast Cancer Cells

Backgrounds:In previous work, we cloned and identified claudin-6(CLDN6) gene inCopenhagen (COP) rat mammary epithelial cells, which are almost completelyresistant to mammary cancer, and found that CLDN6may be a breast cancersuppressor gene. Further studies showed that, CLND6induced apoptosis and inhibitedproliferation, migration and invasion in MCF-7breast cancer cells. In order to clarifythe impact of CLDN6in breast cancer on biology, we used gene-chip technology toscreen the target genes and related signaling pathways in the clones, which wererelated to CLDN6on the biological function. Through the screening and analysis ofMicroarray results, we found that the expression of apoptosis signal-regulated kinase1(ASK1) significantly increased in the clone cells. Therefore, we speculated that theASK1signaling may play an important role in the process of apoptosis regulated byCLDN6in MCF-7cells.CLDNs is the most important members of tight junctions (TJ) which contain thejunctional adhesion molecules (JAMs), occludins and other associated membraneprotein. Through the cell barrier and signal transduction functions, CLDNs regulatecell proliferation, apoptosis and metastasis. Studies have shown that the mutation ofCLDNs is often associated with the occurrence and metastasis of breast cancer andother epithelial tumor cells. CLDN6is one of the members of the27Claudins proteinfamily, located on chromosome16(16p13.3), composed of220amino acids and besynthesized in an approximately23kDa protein. CLDN6has four transmembranedomains, two long extracellular loops can be used as a potential target for antibodytherapy, and two C-terminal may be related to signal transduction. So, CLDN6mayaffect the biological behavior of MCF-7cells by related signaling pathway. It wasshowed that CLDN1induced tumor apoptosis through activating the ERK1/2signaling pathway, in lung adenocarcinoma cell line NCI-of H1299, CLDN7promote apoptosis through the activation of p38protein kinase in gastric epithelial cellsMKN28.ASK1is located in chromosome6(6q22.33), composed of1,379amino acids andconstituted by an N-terminus, a kinase domain and a C-terminal regulatory region.ASK1can induce abnormal cells to undergo apoptosis, is mainly involved in thepathological process of cancer, diabetes, cardiovascular diseases andneurodegenerative diseases. By tumor necrosis factor (tumor necrosis factor-α,TNF-α), oxidative stress, calcium influx, death receptor ligand reaction andendoplasmic reticulum stress and proapoptotic factors stimulation, ASK1can beactivated and promote apoptosis. Interactions of ASK1different binding protein maydecide ASK1active and inactive status, RIP1, DAXX, TRAF2/6and TRAXX andother protein bind with ASK1can promote the activation of ASK1, ASK1and TRX1,Hsp90and14-3-3-like protein bind with ASK1can inhibit the activity of ASK1. Thephosphorylation state of serine/threonine kinase (serine/threonine kinase) as theessential component of the MAPK pathway plays an important role in the induction ofcellular cascade. Thr/Ser changes its related signal, regulates differentiation,proliferation and apoptosis of cancer cells. It was found11sites of ASK1phosphorylation, Thr-838, Ser-83, Ser-1034and Ser967regulated the kinase activityof ASK1, phosphorylation can activate Thr-838site of ASK1, while the Ser-83,Ser-1034and Ser-967phosphorylation cause inactivation of ASK1. ASK1may playan important role in the regulation of p38and JNK signaling pathway in the apoptosisprocess of tumor cell.Therefore, the main purpose of our experiment is to explore the relationshipbetween the ASK1and CLDN6expression levels in breast cancer, and then explorethe molecular mechanisms of ASK1signaling pathway in CLDN6pro-apoptoticprocess and its apoptotic function.Methods:1. The expression of CLDN6and ASK1in breast cancer MCF-7cells: RT-PCRand Western blot were used to detect the mRNA and protein expression of CLDN6and ASK1in MCF-7and the three CLDN6transfected MCF-7clone cells, andanalysis the correlation of both; 2. The expression of ASK1in breast cancer tissue: immunohistochemical methodwas used to detect the expression of ASK1in85cases of breast cancer, and analyzedits clinicopathological significance and the correlation with CLDN6;3. The activation of ASK1signaling pathway by CLDN6: Western blot assay wasused to detect the expression of ASK1phosphorylation proteins and total protein,ASK1upstream gene-RIP1protein, ASK1downstream genes-JNK and p38phosphorylation protein and total protein in clone cells;4. The appropriate conditions of TRX1role in inhibiting ASK1activation: optimalcondition of TRX1who is ASK1signaling pathways blocker inhibiting ASK1activation screened by Western blot;5. The inhibition of TRX1on ASK1signaling pathway in clone cells: TRX1treatthe clone cells, and the expression of ASK1upstream gene-RIP1protein, ASK1downstream genes-JNK and p38phosphorylation protein and total protein in clonecells was detected by Western blot;6. ASK1signaling pathway induced apoptosis in breast cancer MCF-7cells: afterTRX1treated the clone cells, Trypan Blue staining and clone formation assay wereused to detect MCF-7cell growth, TUNEL staining and DNA ladder were used todetect MCF-7cell apoptosis;7. The molecular mechanisms of CLDN6induced apoptosis by the ASK1signaling pathways in MCF-7cell: after TRX1treated the clone cells, the expressionof apoptosis-related genes Bcl-2, Bax and Caspase-3was detected by Western blot.Results:1. The expression of CLDN6and ASK1in breast cancer MCF-7cells: RT-PCRand Western blot assay showed CLDN6can significantly upregulate ASK1expressionin breast cancer cells;2. The expression of ASK1in breast cancer tissue: immunohistochemical assayresults showed that ASK1in85cases of breast cancer was mainly localized in thecytoplasm,26positive cases (30.59%), and was positively correlated with CLDN6expression;3. The activation of ASK1signaling pathway by CLDN6: Western blot assayresults showed that after CLDN6overexpression, the phosphorylation levels of ASK1 decreased, the levels of RIP1total protein, JNK and p38phosphorylation protein wereupregulated;4. The appropriate conditions of TRX1role in inhibiting ASK1activation: theoptimal condition for the ASK1signaling pathways blocker TRX1inhibiting ASK1was that10μg/ml TRX1treated clone cells48h;5. The inhibition of TRX1on ASK1signaling pathway in clone cells: after TRX1treated the clone cells, RIP1changed little, but the expression of JNK and p38phosphorylation was significantly down-regulated;6. ASK1signaling pathway induced apoptosis in breast cancer MCF-7cells:Trypan Blue staining showed that TRX1significantly increased the cell viability ofclone cells, but had no effect on cell viability of the vector cell; colony formationassay revealed that TRX1significantly enhanced cell colony formation rate of theclone cells; TUNEL staining results shown that TRX1decreased the numbers ofapoptosis cells of clone group; DNA ladder results identified that TRX1weakened theapoptosis which CLDN6induced;7. The molecular mechanisms of CLDN6induced apoptosis by the ASK1signaling pathways in MCF-7cell: the expression of Bax and Caspase-3is high andantiapoptotic gene Bcl-2is low in clone cells; but after TRX1treatment, theexpression of Bax and Caspase-3is low and Bcl-2is high.Conclusion:1. Expression of CLDN6and ASK1in breast cancer MCF-7cells and tissues werepositively correlated.2. CLDN6regulated apoptosis of MCF-7cells by activate ASK1-JNK/p38signaling pathway.3. CLDN6promoted apoptosis of MCF-7cells through the ASK1signalingpathways had relationship with Bcl-2, Bax and Caspase-3.