title: Cryptotanshinone activates AMPK-TSC2 axis leading to inhibition of mTORC1 signaling in cancer cells.
abstract: Cryptotanshinone (CPT), a fat-soluble phenanthraquinone from Salvia miltiorrhiza Bunge, has been demonstrated to inhibit phosphorylation of p70 S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1), a couple of direct downstream effectors of the mammalian target of rapamycin complex 1 (mTORC1), resulting in cancer cell arrested in G0 phase and subsequent inhibition of proliferation. However, its concrete molecular mechanism about how CPT inhibits mTORC1 signaling pathway is unclear.
  
  
  
  one solution was used to check cell viability and western blotting for determining expression of the indicated proteins. Molecular docking was performed to assess the binding of CPT with mTOR. The co-immunoprecipitation assay was to analyze whether CPT could disrupt the mTORC1 and TSC1/TSC2 complex. Recombinant adenoviral dominant-negative AMPKα was used to downregulate expression of AMPKα and lentiviral AMPK and TSC2 to silence the AMPK and TSC2 in Rh30 cells.
  
  
  
  Primarily, Rh30 cells expressing rapamycin-resistant mutant mTOR are also sensitive to CPT, while the molecular docking result for CPT binding to mTOR is negative, suggesting that CPT inhibition of mTORC1 is different from rapamycin. Then the related proteins of PTEN-PI3K pathway was proved not to be affected, but the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) was activated by a concentration- and time- dependent manner, meaning that it may be associated with AMPK. Further results indicated that compound C, inhibitor of AMPK, could clearly reversed CPT inhibitory effect on Rh30 cells, and dominant-negative AMPK in cancer cells conferred resistance to CPT inhibition of 4E-BP1 and phosphorylation of S6K1, as well as sh-AMPK. Furthermore, compared with AMPK-positive MEF cells, AMPK-negative MEF cells are less sensitive to CPT by the findings that 4E-BP1 and phosphorylation of S6K1 express comparatively more. Additionally, phosphorylation of tuberous sclerosis complex 2 (TSC2) was activated under the treatment of CPT, and down-expression of TSC2 by shRNA slightly recovered expression of 4E-BP1 and phosphorylation of S6K1, while co-immunoprecipitation of TSC2 did not alter expression of TSC1 by CPT.
  
  
  
  CPT inhibiting mTORC1 pathway was mostly due to activation of AMPK-TSC2 axis rather than specific binding to mTORC1. CPT is a potent anticancer agent targeting AMPK.
authors.affiliation: School of Pharmacy, Nanjing University of Chinese Medicine, NO.138, Xianlin Avenue, Nanjing, Jiangsu Province, 210023, China. chenwxcn@hotmail.com.|Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing, Jiangsu Province, 210023, China. chenwxcn@hotmail.com.
authors.forename: Wenxing
authors.initials: W
authors.lastname: Chen
authors.affiliation: School of Pharmacy, Nanjing University of Chinese Medicine, NO.138, Xianlin Avenue, Nanjing, Jiangsu Province, 210023, China.
authors.forename: Yanhong
authors.initials: Y
authors.lastname: Pan
authors.affiliation: School of Pharmacy, Nanjing University of Chinese Medicine, NO.138, Xianlin Avenue, Nanjing, Jiangsu Province, 210023, China.
authors.forename: Siliang
authors.initials: S
authors.lastname: Wang
authors.affiliation: School of Pharmacy, Nanjing University of Chinese Medicine, NO.138, Xianlin Avenue, Nanjing, Jiangsu Province, 210023, China.
authors.forename: Yuping
authors.initials: Y
authors.lastname: Liu
authors.affiliation: College of Chemistry and Chemical Engineering, Hainan Normal University, Haikou, 571158, China.
authors.forename: Guangying
authors.initials: G
authors.lastname: Chen
authors.affiliation: School of Pharmacy, Nanjing University of Chinese Medicine, NO.138, Xianlin Avenue, Nanjing, Jiangsu Province, 210023, China.
authors.forename: Liang
authors.initials: L
authors.lastname: Zhou
authors.affiliation: School of Pharmacy, Nanjing University of Chinese Medicine, NO.138, Xianlin Avenue, Nanjing, Jiangsu Province, 210023, China.
authors.forename: Wenting
authors.initials: W
authors.lastname: Ni
authors.affiliation: School of Pharmacy, Nanjing University of Chinese Medicine, NO.138, Xianlin Avenue, Nanjing, Jiangsu Province, 210023, China.
authors.forename: Aiyun
authors.initials: A
authors.lastname: Wang
authors.affiliation: School of Pharmacy, Nanjing University of Chinese Medicine, NO.138, Xianlin Avenue, Nanjing, Jiangsu Province, 210023, China. luyingreen@126.com.|Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing, Jiangsu Province, 210023, China. luyingreen@126.com.
authors.forename: Yin
authors.initials: Y
authors.lastname: Lu
mesh_terms: D000263:Adenylate Kinase / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D015536:Down-Regulation; D004354:Drug Screening Assays, Antitumor; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D061986:MCF-7 Cells; D000076222:Mechanistic Target of Rapamycin Complex 1 / Q000378:metabolism*; D009369:Neoplasms / Q000188:drug therapy / Q000378:metabolism*; D010616:Phenanthrenes / Q000494:pharmacology*; D010766:Phosphorylation; D015398:Signal Transduction / Q000187:drug effects; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins / Q000378:metabolism*
chemical_list: D010616:Phenanthrenes; C000624653:TSC2 protein, human; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; C037886:cryptotanshinone; D000076222:Mechanistic Target of Rapamycin Complex 1; D000263:Adenylate Kinase
keywords: AMPK; Cryptotanshinone; TSC2; mTORC1