title: Justicidin A-induced autophagy flux enhances apoptosis of human colorectal cancer cells via class III PI3K and Atg5 pathway.
abstract: Our previous reports showed that justicidin A (JA), a novel and pure arylnaphthalide lignan isolated from Justicia procumbens, induces apoptosis of human colorectal cancer cells and hepatocellular carcinoma cells, leading to the suppression of both tumor cell growth in NOD-SCID mice. Here, we reveal that JA induces autophagy in human colorectal cancer HT-29 cells by conversion of autophagic marker LC3-I to LC3-II. Furthermore, LC3 puncta and autophagic vesicle formation, and SQSTM1/p62 suppression were observed. Administration of autophagy inhibitor (bafilomycin A1 and chloroquine) and transfection of a tandem fluorescent-tagged LC3 (mRFP-GFP) reporter plasmid (ptfLC3) demonstrated that JA induces autophagy flux in HT-29 cells. Expression of LC3, SQSTM1, Beclin 1, and nuclear DNA double-strand breaks (representing apoptosis) were also detected in the tumor tissue of HT-29 cells transplanted into NOD-SCID mice orally administrated with JA. In addition, the expression of autophagy signaling pathway-related molecules p-PDK1, p-mTOR, p-p70S6k/p-RPS6KB2 was decreased, whereas that of class III PI3K, Beclin 1, Atg5-Atg12, and mitochondrial BNIP3 was increased in response to JA. Pre-treatment of the cells with class III PI3K inhibitor 3-methyladenine or Atg5 shRNA attenuated JA-induced LC3-II expression and LC3 puncta formation, indicating the involvement of class III PI3K and Atg5. A novel mechanism was demonstrated in the anticancer compound JA; pre-treatment with 3-methyladenine or Atg5 shRNA blocked JA-induced suppression in cell growth and colony formation, respectively, via inhibition of apoptosis. In contrast, administration of apoptosis inhibitor Z-VAD did not affect JA-induced autophagy. Our data suggest the chemotherapeutic potential of JA for treatment of human colorectal cancer.
authors.affiliation: Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
authors.forename: Shen-Jeu
authors.initials: SJ
authors.lastname: Won
authors.forename: Cheng-Hsin
authors.initials: CH
authors.lastname: Yen
authors.forename: Hsiao-Sheng
authors.initials: HS
authors.lastname: Liu
authors.forename: Shan-Ying
authors.initials: SY
authors.lastname: Wu
authors.forename: Sheng-Hui
authors.initials: SH
authors.lastname: Lan
authors.forename: Ya-Fen
authors.initials: YF
authors.lastname: Jiang-Shieh
authors.forename: Chun-Nan
authors.initials: CN
authors.lastname: Lin
authors.forename: Chun-Li
authors.initials: CL
authors.lastname: Su
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001343:Autophagy / Q000187:drug effects*; D000071187:Autophagy-Related Protein 5; D015179:Colorectal Neoplasms / Q000188:drug therapy / Q000473:pathology; D004148:Dioxolanes / Q000494:pharmacology*; D019073:HT29 Cells; D006801:Humans; D017705:Lignans / Q000494:pharmacology*; D051379:Mice; D016513:Mice, SCID; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*
chemical_list: C502778:ATG5 protein, human; C495773:Atg5 protein, mouse; D000071187:Autophagy-Related Protein 5; D004148:Dioxolanes; D017705:Lignans; D008869:Microtubule-Associated Proteins; D034741:RNA, Small Interfering; C049719:justicidins; C025946:3-methyladenine; D019869:Phosphatidylinositol 3-Kinases; D000225:Adenine