| objective1.to explore the effect of ATL-1 on non-small cell lung cancer cells proliferation though decreasing PDK1 protein expression,and to uncover its molecular mechanism2.to explore the effect of ATL-1 and erlotinib on non-small cell lung cancer cells proliferation,though decreasing EZH2 protein expression,and to uncover its molecular mechanismMethodsCell viability and cell cycle distribution were measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)and flow cytometry assays,respectively.Western blot analysis was performed to examine the phosphorylation and protein expression of extracellular signaling-regulated kinase 1/2(ERK1/2),signal transducer and activator of transcription 3(Stat3),3-phosphoinositide dependent protein kinase-1(PDK1),transcription factor SPland enhancer of zeste homolog 2(EZH2).QRT-PCR was used to examine the mRNA levels of PDK1 gene.Exogenously expressions of SP1,Stat3,PDK1,EZH2and HOTAIR were carried out by transient transfection assays.PDK1 promoter activity was measured by Secrete-Pair Dual Luminescence Assay Kit.RNA-binding proteinimmunoprecipitation assays were used for the analysis of HOTAIR interactionwith EZH2.A nude mice xenograft model was used to confirm the findings in vitro.Results1.We showed that ATL-1 inhibited human lung cancer cell growth and induced cell cycle arrest.Furthermore,we found that ATL-1 stimulated phosphorylation of ERK1/2,inhibited phosphorylation and protein expressions of Stat3 and SP1;the latter were abrogated in the presence of MEK/ERK inhibitor PD98059.Moreover,ATL-1 reduced the protein,mRNA expression and promoter activity of PDK1.Intriguingly,exogenously expressed Stat3 and SP1 overcame ATL-1-inhibited SP1 and Stat3,and PDK1 protein expressions,respectively.Moreover,overexpression of PDK1 resisted the ATL-1-inhibited lung cancer cell growth.In consistent with the results in vitro,ATL-1 inhibited tumor growth,protein expressions of Stat3,SP1 and PDK1,and induced phosphorylation of ERK1/2 in vivo.2.We showed that combination of ATL-1 and EGFR-TKI erlotinib further inhibitedgrowth and induced cell arrest of human lung cancer cells asdetermined by both MTT andflow cytometry assays.ATL-1 inhibited protein expression of EZH2,which was reversed incells with overexpressed PDK1.Interestingly,we observed that ATL-1 inhibited theexpression levels of HOTAIR.While silencing of HOTAIR inhibited expression of PDK1 andEZH2,overexpressed HOTAIR resisted the ATL-1-reduced PDK1 and EZH2 protein3expressions.In addition,ATL-1 reduced HOTAIR RNA bind to EZH2 protein.Moreover,wefound that exogenously expressed EZH2 antagonized the effect of ATL-1 on cell growthinhibition.In consistent with the results in vitro,there was additive effect or potential synergy in combination of ATL-1 and erlotinib inthis process.Conclusion1.In summary,ourresults show that ATL-1 inhibits lung cancer cell growth through activation of ERK1/2,followed by suppressing SP1 protein expression.ATL-1 also reduces phosphorylation and protein levels of Stat3.These are mutual regulation between Stat3 and SP1 proteins affected by ATL-1.This ultimately suppresses PDK1 gene expression.This study reveals a novel mechanism by which ATL-1 inhibits growth of lung cancer cells.Thus,targeting PDKI pinpoints a potential in the lung cancer treatment.2.We provide the first evidence showing that ATL-1 inhibits lung cancer cellgrowth through inhibition not only PDK1 but also IncRN,A HOTAIR,this results in reduction ofone downstream effectors EZH2 expression.The novel interplay between HOTAIR and EZH2,and reciprocal repression of PDK1 and HOTAIR coordinate to the overall effects of ATL-1.More importantly,there is an enhancement of combination of ATL-1 and EGFR-TKI erlotinibin this process.Thus,targeting PDK1-and HOTAIR-mediated downstream molecule EZH2 bycombination of ATL-1 and erlotinib potentially facilitates the development of an additionalnovel strategy for combating lung cancer. |
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