title: Baicalein increases the expression and reciprocal interplay of RUNX3 and FOXO3a through crosstalk of AMPKα and MEK/ERK1/2 signaling pathways in human non-small cell lung cancer cells.
abstract: BACKGROUND
  Baicalein, a natural flavonoid obtained from the Scutellaria baicalensis root, has been reported to inhibit growth of human lung cancer. However, the detailed mechanism underlying this has not been well elucidated.
  
  
  METHODS
  Cell viability was measured using a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays. Apoptosis was detected by flow cytometry analysis and caspase 3/7 assays. The expression of RUNX3 and FOXO3a mRNA were measured by real time RT-PCR methods. Western blot analysis was performed to measure the phosphorylation and protein expression of AMP-activated protein kinase alpha (AMPKα) and extracellular signal-regulated kinase 1/2 (ERK1/2), runt-related transcription factor 3 (RUNX3) and forkhead box O3a (FOXO3a). Silencing of FOXO3a and RUNX3 were performed by small interfering RNA (siRNA) methods. Exogenous expression of FOXO3a or RUNX3 was carried out by electroporated transfection assays.
  
  
  RESULTS
  We showed that baicalein significantly inhibited growth and induced apoptosis of non-small cell lung cancer (NSCLC) cells in a time- and dose-dependent manner. Baicalein induced RUNX3 and FOXO3a protein expression, and increased phosphorylation of AMPKα and ERK1/2. Moreover, the inhibitors of AMPK and MEK/ERK1/2 reversed the effect of baicalein on RUNX3 and FOXO3a protein expression. Interestingly, while compound C had little effect on blockade of baicalein-induced phosphorylation of ERK1/2, PD98059 significantly abrogated baicalein-induced phosphorylation of AMPKα. Intriguingly, while silencing of RUNX3 abolished the effect of baicalein on expression of FOXO3a and apoptosis, silencing of FOXO3a significantly attenuated baicalein-reduced cell proliferation. On the contrary, overexpression of FOXO3a restored the effect of baicalein on cell growth inhibition in cells silencing of endogenous FOXO3a gene and enhanced the effect of baicalein on RUNX3 protein expression. Finally, exogenous expression of RUNX3 increased FOXO3a protein and strengthened baicalein-induced phosphorylation of ERK1/2.
  
  
  CONCLUSIONS
  Collectively, our results show that baicalein inhibits growth and induces apoptosis of NSCLC cells through AMPKα- and MEK/ERK1/2-mediated increase and interaction of FOXO3a and RUNX3 protein. The crosstalk between AMPKα and MEK/ERK1/2 signaling pathways, and the reciprocal interplay of FOXO3a and RUNX3 converge on the overall response of baicalein. This study reveals a novel mechanism for regulating FOXO3a and RUNX3 signaling axis in response to baicalein and suggests a new strategy for NSCLC associated targeted therapy.
authors.affiliation: Laboratory of Tumor Biology, Guangdong Provincial Hospital of Chinese Medicine, The Second Clinical Medical Collage, University of Guangzhou Traditional Chinese Medicine, Guangzhou, Guangdong Province, 510120, China. zhenfang_123@163.com.
authors.forename: Fang
authors.initials: F
authors.lastname: Zheng
authors.affiliation: Laboratory of Tumor Biology, Guangdong Provincial Hospital of Chinese Medicine, The Second Clinical Medical Collage, University of Guangzhou Traditional Chinese Medicine, Guangzhou, Guangdong Province, 510120, China. star8677@163.com.
authors.forename: Jingjing
authors.initials: J
authors.lastname: Wu
authors.affiliation: Laboratory of Tumor Biology, Guangdong Provincial Hospital of Chinese Medicine, The Second Clinical Medical Collage, University of Guangzhou Traditional Chinese Medicine, Guangzhou, Guangdong Province, 510120, China. zhaoshunyu0111@163.com.
authors.forename: Shunyu
authors.initials: S
authors.lastname: Zhao
authors.affiliation: Laboratory of Tumor Biology, Guangdong Provincial Hospital of Chinese Medicine, The Second Clinical Medical Collage, University of Guangzhou Traditional Chinese Medicine, Guangzhou, Guangdong Province, 510120, China. 18718844345@163.com.
authors.forename: Qingmei
authors.initials: Q
authors.lastname: Luo
authors.affiliation: Laboratory of Tumor Biology, Guangdong Provincial Hospital of Chinese Medicine, The Second Clinical Medical Collage, University of Guangzhou Traditional Chinese Medicine, Guangzhou, Guangdong Province, 510120, China. tangqingyanjiu@163.com.
authors.forename: Qing
authors.initials: Q
authors.lastname: Tang
authors.affiliation: Laboratory of Tumor Biology, Guangdong Provincial Hospital of Chinese Medicine, The Second Clinical Medical Collage, University of Guangzhou Traditional Chinese Medicine, Guangzhou, Guangdong Province, 510120, China. 813901541@qq.com.
authors.forename: LiJun
authors.initials: L
authors.lastname: Yang
authors.affiliation: Department of Medical Oncology, Guangdong Provincial Hospital of Chinese Medicine, The Second Clinical Medical Collage, University of Guangzhou Traditional Chinese Medicine, Guangzhou, Guangdong Province, 510120, China. liliuning97@126.com.
authors.forename: Liuning
authors.initials: L
authors.lastname: Li
authors.affiliation: Department of Medical Oncology, Guangdong Provincial Hospital of Chinese Medicine, The Second Clinical Medical Collage, University of Guangzhou Traditional Chinese Medicine, Guangzhou, Guangdong Province, 510120, China. wwanyin@126.com.
authors.forename: WanYing
authors.initials: W
authors.lastname: Wu
authors.affiliation: Laboratory of Tumor Biology, Guangdong Provincial Hospital of Chinese Medicine, The Second Clinical Medical Collage, University of Guangzhou Traditional Chinese Medicine, Guangzhou, Guangdong Province, 510120, China. swhan20010@live.com.|Higher Education Mega Center, No. 55, Neihuan West Road, Panyu District, Guangzhou, Guangdong Province, 510006, People's Republic of China. swhan20010@live.com.
authors.forename: Swei Sunny
authors.initials: SS
authors.lastname: Hann
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000378:metabolism*; D017209:Apoptosis / Q000187:drug effects; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics* / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D050677:Core Binding Factor Alpha 3 Subunit / Q000235:genetics* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D044950:Flavanones / Q000494:pharmacology*; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors / Q000235:genetics* / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics* / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000187:drug effects*
chemical_list: D050677:Core Binding Factor Alpha 3 Subunit; C499517:FOXO3 protein, human; D044950:Flavanones; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors; C006680:baicalein; D055372:AMP-Activated Protein Kinases