title: Equol inhibits proliferation of human gastric carcinoma cells via modulating Akt pathway.
abstract: OBJECTIVE
  To investigate the anti-tumor effects of equol in gastric cancer cells and the underlying molecular mechanisms.
  
  
  METHODS
  MGC-803 cells were employed for in vitro experiments in this study. Cells were treated with control (vehicle, 0.1% DMSO) or equol under specified dose titration or time courses. Cell viability was examined by MTS assay, and the levels of Ki67 were determined by qPCR and immunofluorescent assay. Changes in cell cycle distribution and apoptosis rate were detected by flow cytometry. The mRNA expression of cyclin E1 and P21(WAF1) was determined by qPCR. The protein levels of cell cycle regulators, PARP and Caspase-3 cleavage, and the phosphorylation of Akt were examined by Western blot. In addition, to characterize the role of elevated Akt activation in the anti-tumor effect exerted by equol, Ly294002, a PI3K/AKT pathway inhibitor, was used to pretreat MGC-803 cells.
  
  
  RESULTS
  Equol (5, 10, 20, 40, or 80 μmol/L) inhibited viability of MGC-803 cells in a dose- and time-dependent manner after treatment for 24, 36, or 48 h (P < 0.05 for all). Equol also decreased the mRNA (P < 0.05 for 12 and 24 h treatment) and protein levels of Ki67. Equol treatment significantly induced G0/G1 cell cycle arrest (P < 0.05), with the percentages of G0/G1 cells of 32.23% ± 3.62%, 36.31% ± 0.24%, 45.58% ± 2.29%, and 65.10% ± 2.04% for equol (0, 10, 20, or 30 μmol/L) treatment, respectively, accompanied by a significant decrease of CDK2/4 (P < 0.05 for 24 and 48 h treatment) and Cyclin D1/Cyclin E1 (P < 0.05), and an increased level of P21(WAF1) (P < 0.05). A marked increase of apoptosis was observed, with the percentages of apoptotic cells of 5.01% ± 0.91%, 14.57% ± 0.99%, 37.40% ± 0.58%, and 38.46% ± 2.01% for equol (0, 5, 10, or 20 μmol/L) treatment, respectively, accompanied by increased levels of cleaved PARP and caspase-3. In addition, we found that equol treatment increased P-Akt (Ser473 and Thr308) at 12 and 24 h compared to vehicle-treated control; longer treatment for 48 h decreased P-Akt (Ser473 and Thr308). P-Akt at Thr450, however, was decreased by equol treatment at all time points examined (P < 0.05 for all). Moreover, Akt inhibition by Ly294002 could not prevent but led to enhanced G0/G1 arrest and apoptosis.
  
  
  CONCLUSIONS
  Equol inhibits MGC-803 cells proliferation by induction of G0/G1 arrest and apoptosis. Its anti-cancer effects are likely mediated by dephosphorylation of Akt at Thr450.
authors.affiliation: Zhi-Ping Yang, Fang Huang, Jie Chen, Xiao-Nan Wu, Public Health College, Fujian Medical University, Fuzhou 350108, Fujian Province, China.
authors.forename: Zhi-Ping
authors.initials: ZP
authors.lastname: Yang
authors.affiliation: Zhi-Ping Yang, Fang Huang, Jie Chen, Xiao-Nan Wu, Public Health College, Fujian Medical University, Fuzhou 350108, Fujian Province, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Zhi-Ping Yang, Fang Huang, Jie Chen, Xiao-Nan Wu, Public Health College, Fujian Medical University, Fuzhou 350108, Fujian Province, China.
authors.forename: Fang
authors.initials: F
authors.lastname: Huang
authors.affiliation: Zhi-Ping Yang, Fang Huang, Jie Chen, Xiao-Nan Wu, Public Health College, Fujian Medical University, Fuzhou 350108, Fujian Province, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Chen
authors.affiliation: Zhi-Ping Yang, Fang Huang, Jie Chen, Xiao-Nan Wu, Public Health College, Fujian Medical University, Fuzhou 350108, Fujian Province, China.
authors.forename: Ya-Hong
authors.initials: YH
authors.lastname: Yao
authors.affiliation: Zhi-Ping Yang, Fang Huang, Jie Chen, Xiao-Nan Wu, Public Health College, Fujian Medical University, Fuzhou 350108, Fujian Province, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Li
authors.affiliation: Zhi-Ping Yang, Fang Huang, Jie Chen, Xiao-Nan Wu, Public Health College, Fujian Medical University, Fuzhou 350108, Fujian Province, China.
authors.forename: Xiao-Nan
authors.initials: XN
authors.lastname: Wu
mesh_terms: D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D002277:Carcinoma / Q000188:drug therapy* / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D053148:Caspase 3 / Q000378:metabolism; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D019927:Cyclin E / Q000235:genetics / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D060754:Equol / Q000494:pharmacology*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D019394:Ki-67 Antigen / Q000235:genetics / Q000378:metabolism; D015513:Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013274:Stomach Neoplasms / Q000188:drug therapy* / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D013997:Time Factors
chemical_list: D000972:Antineoplastic Agents, Phytogenic; C475820:CCNE1 protein, human; C493956:CDKN1A protein, human; D019927:Cyclin E; D050759:Cyclin-Dependent Kinase Inhibitor p21; D019394:Ki-67 Antigen; D015513:Oncogene Proteins; D047428:Protein Kinase Inhibitors; D060754:Equol; D011065:Poly(ADP-ribose) Polymerases; D051057:Proto-Oncogene Proteins c-akt; C505407:CASP3 protein, human; D053148:Caspase 3
keywords: Apoptosis; Chemoprevention; Equol; G0/G1 arrest; Gastric cancer