title: Chalcone flavokawain B induces autophagic-cell death via reactive oxygen species-mediated signaling pathways in human gastric carcinoma and suppresses tumor growth in nude mice.
abstract: Flavokawain B (FKB), a naturally occurring chalcone in kava extracts, has been reported to possess anticancer activity. However, the effect of FKB on gastric cancer remains unclear. We examined the in vitro and in vivo anticancer activity and autophagy involvement of FKB and determined the underlying molecular mechanisms. FKB is potently cytotoxic to human gastric cancer cells (AGS/NCI-N87/KATO-III/TSGH9201) and mildly toxic towards normal (Hs738) cells and primary mouse hepatocytes. FKB-induced AGS cell death was characterized by autophagy, not apoptosis, as evidenced by increased LC3-II accumulation, GFP-LC3 puncta and acidic vesicular organelles (AVOs) formation, without resulting procaspase-3/PARP cleavage. FKB further caused p62/SQSTM1 activation, mTOR downregulation, ATG4B inhibition, and Beclin-1/Bcl-2 dysregulation. Silencing autophagy inhibitors CQ/3-MA and LC3 (shRNA) significantly reversed the FKB-induced cell death of AGS cells. FKB-triggered ROS generation and ROS inhibition by NAC pre-treatment diminished FKB-induced cell death, LC3 conversion, AVO formation, p62/SQSTM1 activation, ATG4B inhibition and Beclin-1/Bcl-2 dysregulation, which indicated ROS-mediated autophagy in AGS cells. Furthermore, FKB induces G2/M arrest and alters cell-cycle proteins through ROS-JNK signaling. Interestingly, FKB-induced autophagy is associated with the suppression of HER-2 and PI3K/AKT/mTOR signaling cascades. FKB inhibits apoptotic Bax expression, and Bax-transfected AGS cells exhibit both apoptosis and autophagy; thus, FKB-inactivated Bax results in apoptosis inhibition. In vivo data demonstrated that FKB effectively inhibited tumor growth, prolonged the survival rate, and induced autophagy in AGS-xenografted mice. Notably, silencing of LC3 attenuated FKB-induced autophagy in AGS-xenografted tumors. FKB may be a potential chemopreventive agent in the activation of ROS-mediated autophagy of gastric cancer cells.
authors.affiliation: Institute of Nutrition, College of Biopharmaceutical and Food Sciences, China Medical University, 91, Hsueh-Shih Road, Taichung, 40402, Taiwan.
authors.forename: Chia-Ting
authors.initials: CT
authors.lastname: Chang
authors.affiliation: Department of Cosmeceutics, College of Biopharmaceutical and Food Sciences, China Medical University, 91, Hsueh-Shih Road, Taichung, 40402, Taiwan. ychseu@mail.cmu.edu.tw.|Department of Health and Nutrition Biotechnology, Asia University, Taichung, 41354, Taiwan. ychseu@mail.cmu.edu.tw.
authors.forename: You-Cheng
authors.initials: YC
authors.lastname: Hseu
authors.affiliation: Department of Cosmeceutics, College of Biopharmaceutical and Food Sciences, China Medical University, 91, Hsueh-Shih Road, Taichung, 40402, Taiwan.
authors.forename: Varadharajan
authors.initials: V
authors.lastname: Thiyagarajan
authors.affiliation: Department of Medical Research, Chi-Mei Medical Center, Tainan, 710, Taiwan.
authors.forename: Kai-Yuan
authors.initials: KY
authors.lastname: Lin
authors.affiliation: Department of Life Sciences, College of Biopharmaceutical and Food Sciences, China Medical University, Taichung, 40402, Taiwan.
authors.forename: Tzong-Der
authors.initials: TD
authors.lastname: Way
authors.affiliation: Institute of Nutrition, College of Biopharmaceutical and Food Sciences, China Medical University, 91, Hsueh-Shih Road, Taichung, 40402, Taiwan.
authors.forename: Mallikarjuna
authors.initials: M
authors.lastname: Korivi
authors.affiliation: Graduate Institute of Veterinary Pathology, National Chung-Hsing University, Taichung, 402, Taiwan.
authors.forename: Jiuun-Wang
authors.initials: JW
authors.lastname: Liao
authors.affiliation: Institute of Nutrition, College of Biopharmaceutical and Food Sciences, China Medical University, 91, Hsueh-Shih Road, Taichung, 40402, Taiwan. hlyang@mail.cmu.edu.tw.
authors.forename: Hsin-Ling
authors.initials: HL
authors.lastname: Yang
mesh_terms: D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001343:Autophagy / Q000187:drug effects / Q000502:physiology; D000071183:Autophagy-Related Proteins / Q000378:metabolism; D000071186:Beclin-1 / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D003546:Cysteine Endopeptidases / Q000378:metabolism; D005260:Female; D005419:Flavonoids / Q000494:pharmacology*; D006801:Humans; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013274:Stomach Neoplasms / Q000188:drug therapy* / Q000378:metabolism* / Q000473:pathology; D023041:Xenograft Model Antitumor Assays; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D000071183:Autophagy-Related Proteins; C494826:BAX protein, human; D000071186:Beclin-1; D005419:Flavonoids; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D051028:bcl-2-Associated X Protein; C452039:flavokawain B; C507524:ATG4B protein, human; D003546:Cysteine Endopeptidases
keywords: AGS cells; ATG4B; Autophagy; Flavokawain B; ROS