title: Neferine isolated from Nelumbo nucifera enhances anti-cancer activities in Hep3B cells: molecular mechanisms of cell cycle arrest, ER stress induced apoptosis and anti-angiogenic response.
abstract: Hepatocellular carcinoma (HCC) is one of the most aggressive malignant diseases and is highly resistant to conventional chemotherapy. Neferine, a major bisbenzylisoquinoline alkaloid derived from the embryos of Nelumbo nucifera, has been reported a few physiological activities. However, the mechanisms of anticancer effects are not well understood and its detailed activities on Hep3B cells have not been determined. Our results suggest that neferine exhibited cytotoxicity against HCC Hep3B cells, but not against HCC Sk-Hep1 and THLE-3, a normal human liver cell line. In addition, consistent with the induction of G1/S phase cell population in flow cytometry, downregulation of c-Myc, cyclin D1, D3, CDK4, E2F-1, as well as dephosphorlyation of cdc2 by western blot analysis, as evidenced by the appearance of cell cycle arrest, were observed in Hep3B cells treated with neferine. Our results demonstrated neferine induced ER stress and apoptosis, acting through multiple signaling cascades by the activation of Bim, Bid, Bax, Bak, Puma, caspases-3, -6, -7, -8 and PARP, and the protein expression levels of Bip, calnexin, PDI, calpain-2 and caspase-12 were also upregulated dramatically by neferine treatment. Overexpression of GFP-LC3B by neferine resulted in a diffuse cytosolic GFP fluorescence and the strong fluorescent spots, representing autophagosomes. The significant reduction of the migration in Hep3B cells and the capillary tube-like formation of HUVECs by neferine were also determined. These observations reveal that the therapeutic potential of neferine in treating HCC Hep3B cells, containing copies of hepatitis B virus (HBV) genomes.
authors.affiliation: Department of Microbiology, College of Natural Sciences, Pukyong National University, Namgu, Busan 608-737, Republic of Korea.
authors.forename: Jin-Soo
authors.initials: JS
authors.lastname: Yoon
authors.forename: Hwa-Mi
authors.initials: HM
authors.lastname: Kim
authors.forename: Anandam Kasin
authors.initials: AK
authors.lastname: Yadunandam
authors.forename: Nan-Hee
authors.initials: NH
authors.lastname: Kim
authors.forename: Hyun-Ah
authors.initials: HA
authors.lastname: Jung
authors.forename: Jae-Sue
authors.initials: JS
authors.lastname: Choi
authors.forename: Chi-Yeon
authors.initials: CY
authors.lastname: Kim
authors.forename: Gun-Do
authors.initials: GD
authors.lastname: Kim
mesh_terms: D020533:Angiogenesis Inhibitors / Q000302:isolation & purification / Q000494:pharmacology / Q000627:therapeutic use; D000972:Antineoplastic Agents, Phytogenic / Q000302:isolation & purification / Q000494:pharmacology* / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects; D001343:Autophagy; D044182:Benzylisoquinolines / Q000302:isolation & purification / Q000494:pharmacology* / Q000627:therapeutic use; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy / Q000473:pathology / Q000503:physiopathology; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D002460:Cell Line; D045744:Cell Line, Tumor; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects; D006515:Hepatitis B virus; D006801:Humans; D008099:Liver / Q000187:drug effects* / Q000473:pathology / Q000503:physiopathology; D008113:Liver Neoplasms / Q000188:drug therapy / Q000503:physiopathology; D031653:Nelumbo / Q000737:chemistry*; D008517:Phytotherapy; D010936:Plant Extracts / Q000737:chemistry / Q000494:pharmacology* / Q000627:therapeutic use; D012639:Seeds; D015398:Signal Transduction
chemical_list: D020533:Angiogenesis Inhibitors; D000972:Antineoplastic Agents, Phytogenic; D044182:Benzylisoquinolines; D010936:Plant Extracts; C057222:neferine
keywords: 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt; 4,6-diamidino-2-phenylindole dihydrochloride hydrate; Apoptosis; BSA; CDKs; Cell cycle arrest; DAPI; DFF; DMSO; DNA fragmentation factor; ER; ER stress; GAPDH; GFP; HBV; HCC; HUVECs; Hep3B; LC3B; MOMP; Neferine; Nelumbo nucifera; PARP; PBS; PCD; PDI; SirT6; WST-1; bovine serum albumin; cyclin-dependent kinases; dimethyl sulfoxide; endoplasmic reticulum; glyceraldehyde-3-phosphate dehydrogenase; green fluorescent protein; hepatitis B virus; hepatocellular carcinoma; human umbilical vein endothelial cells; light chain 3 B; mitochondrial outer membrane permeabilization; phosphate-buffered saline; poly(ADP-ribose) polymerase; programmed cell death; protein disulfide isomerase; sirtuin-6