title: Honokiol activates AMP-activated protein kinase in breast cancer cells via an LKB1-dependent pathway and inhibits breast carcinogenesis.
abstract: BACKGROUND
  Honokiol, a small-molecule polyphenol isolated from magnolia species, is widely known for its therapeutic potential as an antiinflammatory, antithrombosis, and antioxidant agent, and more recently, for its protective function in the pathogenesis of carcinogenesis. In the present study, we sought to examine the effectiveness of honokiol in inhibiting migration and invasion of breast cancer cells and to elucidate the underlying molecular mechanisms.
  
  
  METHODS
  Clonogenicity and three-dimensional colony-formation assays were used to examine breast cancer cell growth with honokiol treatment. The effect of honokiol on invasion and migration of breast cancer cells was evaluated by using Matrigel invasion, scratch-migration, spheroid-migration, and electric cell-substrate impedance sensing (ECIS)-based migration assays. Western blot and immunofluorescence analysis were used to examine activation of the liver kinase B1 (LKB1)-AMP-activated protein kinase (AMPK) axis. Isogenic LKB1-knockdown breast cancer cell line pairs were developed. Functional importance of AMPK activation and LKB1 overexpression in the biologic effects of honokiol was examined by using AMPK-null and AMPK-wild type (WT) immortalized mouse embryonic fibroblasts (MEFs) and isogenic LKB1-knockdown cell line pairs. Finally, mouse xenografts, immunohistochemical and Western blot analysis of tumors were used.
  
  
  RESULTS
  Analysis of the underlying molecular mechanisms revealed that honokiol treatment increases AMP-activated protein kinase (AMPK) phosphorylation and activity, as evidenced by increased phosphorylation of the downstream target of AMPK, acetyl-coenzyme A carboxylase (ACC) and inhibition of phosphorylation of p70S6kinase (pS6K) and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1). By using AMPK-null and AMPK-WT (MEFs), we found that AMPK is required for honokiol-mediated modulation of pACC-pS6K. Intriguingly, we discovered that honokiol treatment increased the expression and cytoplasmic translocation of tumor-suppressor LKB1 in breast cancer cells. LKB1 knockdown inhibited honokiol-mediated activation of AMPK and, more important, inhibition of migration and invasion of breast cancer cells. Furthermore, honokiol treatment resulted in inhibition of breast tumorigenesis in vivo. Analysis of tumors showed significant increases in the levels of cytoplasmic LKB1 and phospho-AMPK in honokiol-treated tumors.
  
  
  CONCLUSIONS
  Taken together, these data provide the first in vitro and in vivo evidence of the integral role of the LKB1-AMPK axis in honokiol-mediated inhibition of the invasion and migration of breast cancer cells. In conclusion, honokiol treatment could potentially be a rational therapeutic strategy for breast carcinoma.
authors.affiliation: Department of Oncology, Johns Hopkins University School of Medicine and the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore MD 21231, USA.
authors.forename: Arumugam
authors.initials: A
authors.lastname: Nagalingam
authors.forename: Jack L
authors.initials: JL
authors.lastname: Arbiser
authors.forename: Michael Y
authors.initials: MY
authors.lastname: Bonner
authors.forename: Neeraj K
authors.initials: NK
authors.lastname: Saxena
authors.forename: Dipali
authors.initials: D
authors.lastname: Sharma
mesh_terms: D000091162:AMP-Activated Protein Kinase Kinases; D055372:AMP-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D001713:Biphenyl Compounds / Q000494:pharmacology* / Q000627:therapeutic use; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000473:pathology; D018797:Cell Cycle Proteins; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D002471:Cell Transformation, Neoplastic / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D015870:Gene Expression; D006801:Humans; D017705:Lignans / Q000494:pharmacology* / Q000627:therapeutic use; D051379:Mice; D008819:Mice, Nude; D009361:Neoplasm Invasiveness; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D019893:Ribosomal Protein S6 Kinases / Q000378:metabolism; D018874:Spheroids, Cellular / Q000187:drug effects / Q000502:physiology; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism; D047368:Tumor Burden / Q000187:drug effects; D023041:Xenograft Model Antitumor Assays; D000498:Allyl Compounds; D010636:Phenols
chemical_list: D000091162:AMP-Activated Protein Kinase Kinases; D055372:AMP-Activated Protein Kinases; D048868:Adaptor Proteins, Signal Transducing; D000970:Antineoplastic Agents; D001713:Biphenyl Compounds; D018797:Cell Cycle Proteins; D017705:Lignans; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D019893:Ribosomal Protein S6 Kinases; D058570:TOR Serine-Threonine Kinases; C087000:EIF4EBP1 protein, human; C005499:honokiol; D000498:Allyl Compounds; D010636:Phenols; C546842:MTOR protein, human; C494918:AKT1 protein, human; C109790:STK11 protein, human