title: AG4, a compound isolated from Radix Ardisiae Gigantifoliae, induces apoptosis in human nasopharyngeal cancer CNE cells through intrinsic and extrinsic apoptosis pathways.
abstract: 3β-O-{α-L-Pyran rhamnose-(1→3)-[β-D-xylopyranose-(1→2)]-β-D-glucopyranose-(1→4)-[β-D-lucopyranose-(1→2)]-α-L-pyran arabinose}-cyclamiretin A (AG4) is a saponin component obtained from the Giantleaf Ardisia Rhizome (Rhizoma Ardisiae Gigantifoliae). The present study aimed to investigate the antitumor potential of AG4 and its possible mechanisms in human nasopharyngeal carcinoma cells (CNE). We exposed tumor cells to AG4 to investigate which cell line was the most sensitive to AG4. Cell viability was assessed using the MTT reduction assay, and the effects of AG4 on apoptosis, reactive oxygen species (ROS) content, mitochondrial membrane potential (MMP), and cell cycle were detected using a flow cytometer; the glutathione, superoxide dismutase and malondialdehyde activities were measured using colorimetric methods. The relative expressions of Bax, Bad, Bid, Bcl-2, and Fas mRNA were calculated using the (Equation is included in full-text article.)comparative method by real-time PCR studies and protein was detected by western blotting. AG4 markedly inhibited the growth of CNE cells by decreasing cell proliferation, inducing apoptosis, and blocking the cell cycle in the S phase. The release of caspase-3, caspase-8, and caspase-9 was stimulated by AG4 in CNE, and the decreased proliferation induced by AG4 was blocked by the inhibitor of pan caspase (Z-VAD-FMK). Moreover, the MMP was decreased in AG4-treated cells, and AG4-induced cell apoptosis was accompanied by a rapid and lasting increase in ROS, which was abolished by N-acetyl-L-cysteine (NAC); glutathione, superoxide dismutase, and malondialdehyde were regulated by AG4. AG4 inhibited Bcl-2 mRNA and protein expression and stimulated Bax, Bad, Bid, Fas mRNA, and protein expression in CNE cultures, suggesting an effect at the transcriptional and protein level. In addition, both the FasL inhibitor (AF-016) and the Bcl-2 family inhibitor (GX15-070) could prevent the cell apoptosis induced by AG4. The findings suggested that AG4-induced apoptosis in CNE cells involved a death receptor pathway and a Bcl-2 family-mediated mitochondrial signaling pathway by decreasing the MMPs in an ROS-dependent manner and regulating genes and proteins relative to apoptosis; also, regulation of cell cycles may also play a role in the antitumor mechanism of AG4.
authors.affiliation: Departments of aClinical Pharmacology bPharmaceutical Care, Chinese PLA General Hospital, Beijing cCollege of Pharmacy, Tianjin University of Traditional Chinese Medicine, Tianjin dCollege of Pharmacy, Bengbu Medical College, Bengbu, China.
authors.forename: Xian-Zhe
authors.initials: XZ
authors.lastname: Dong
authors.forename: Ting-Ting
authors.initials: TT
authors.lastname: Xie
authors.forename: Xiao-Jiang
authors.initials: XJ
authors.lastname: Zhou
authors.forename: Li-Hua
authors.initials: LH
authors.lastname: Mu
authors.forename: Xiao-Li
authors.initials: XL
authors.lastname: Zheng
authors.forename: Dai-Hong
authors.initials: DH
authors.lastname: Guo
authors.forename: Ping
authors.initials: P
authors.lastname: Liu
authors.forename: Xiao-Yue
authors.initials: XY
authors.lastname: Ge
mesh_terms: D000972:Antineoplastic Agents, Phytogenic / Q000302:isolation & purification / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D031644:Ardisia / Q000737:chemistry*; D002277:Carcinoma; D053148:Caspase 3 / Q000378:metabolism; D053181:Caspase 8 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D004354:Drug Screening Assays, Antitumor; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D000077274:Nasopharyngeal Carcinoma; D009303:Nasopharyngeal Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D017382:Reactive Oxygen Species / Q000378:metabolism; D012503:Saponins / Q000302:isolation & purification / Q000494:pharmacology*
chemical_list: C000602456:3-O-(pyran rhamnose-(1-3)-(xylopyranose-(1-2))-glucopyranose-(1-4)-(lucopyranose-(1-2))-pyran arabinose)cyclamiretin A; D000972:Antineoplastic Agents, Phytogenic; D017382:Reactive Oxygen Species; D012503:Saponins; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9