strated that cell clones were decreased with increase of the concentration of procyanidins. PI staining a- nalysis showed that cell cycle was a

Size: px

Start display at page:

Download "strated that cell clones were decreased with increase of the concentration of procyanidins. PI staining a- nalysis showed that cell cycle was a"

窃施
7 years ago
Views:

1 32 2 Vol. 32 No Journal of Jinan University Medicine Edition Apr HepG HepG2 MTT WST-8 HepG2 PI Annexin V-FITC /PI Western blotting HepG2 - P < HepG2 48 h IC g /L HepG2 HepG2 G g /L 81% GFP - LC3 HepG2 LC3 LC3- II HepG2 R A Grape seed proanthocyanidins induce apoptosis and autophagic cell death in human hepatoma HepG2 cells WANG Wei 1 CHEN Jing-hong 1 WANG Xin-ning 2 LI Qiang 1 CHEN Qing 1 JIANG Jian-wei 1 1. Department of Biochemistry Medical College Jinan University Guangzhou China 2. Department of Oncology the First Affiliated Hospital of Guangdong Pharmaceutical University Guangzhou China Abstract Aim To investigate the inhibiory effect of procyanidins on human hepatoma HepG2 cell lines and the possible mechanism. Methods Improved MTT assay WST-8 method and cell colony formation inhibitory assay were used to investigate the inhibition effect of procyanidins on human hepatoma HepG2 cell lines. Changes in cell cycle DNA ploidy and apoptotic cell percentage were calculated by flow cytometry. Laser confocal microscopy and Western blotting assay were used to determine cell autophagy. Results The results of WST-8 method shows that procyanidins with different concentrations inhibited human hepatoma HepG2 cells in a dose-dependent manner. The IC 50 for procyanidins against human hepatoma HepG2 cells for 48 hours was g /L. Cell colone formation inhibition assay demon B A Tel E - mail @ qq. com

2 strated that cell clones were decreased with increase of the concentration of procyanidins. PI staining a- nalysis showed that cell cycle was arrested in G 1 phase. DNA ploidy analysis showed that the percentage of hypodiploid cells was accumulated in a dose-dependent manner. Annexin V-FITC / PI staining analysis also showed a dose-dependent effect on late apoptotic and necrotic cells. By using laser scanning confocal microscopy it was observed that LC3 aggregates significantly the green fluorescence of control group was dispersed. Western-blotting assay showed that the LC3-II protein expression levels increased gradually with increasing of procyanidins. Conclusion Procyanidins inhibits the cell survival of human hepatoma HepG2 cells in a dose-dependent manner and arrestes cells at G 1 phase. Procyanidins inhibits cell growth by inducing apoptotic and autophagic death. Key words procyanidins PC human hepatoma HepG2 cell line apoptosis autophagy proanthocyanidin procyanidin PC RPMI /ml μl μl g /L % CO 2 48 h 10 μl CCK-8 1 h A450 HepG2 % = 1 - A /A 100% 95% CCK-8 Cell Count Kit- 8 AnnexinV- 4 PI FITC PI Sigma GAPDH LC3 HepG2 Cell Signaling Technology Opti-MEM /ml 6 1 ml 12 h Gibco Lipofectamine TM 2000 Invitrogen ECL Pierce HepG2 6 min mol /L PBS 37 5% CO % % PBS % HepG2 10% 3 HepG2 15% RPMI HepG2 GFP-LC g /L 37 5% CO 2 7 d PBS 2 V V = min Giemsa 30 min 5min g /L 48 h r /min RNA MTT WST g /L 30 min 300

3 2 HepG2 183 DNA Annexin V-FITC /PI 2. 1 HepG MTT WST /ml 6 HepG % CO 2 48 h r /min 6 min PBS 2 10 μl Annexin V-FITC 5 μl propidine iodide PI 15 min μl ± ± ± ± ± h PBS r /min 10 min PBS 2 ρ PBS 15 min / g L r /min 15 min BCA % SDS PAGE PVDF % 2 h ECL HepG2 5min X 2 P < HepG2 48 h IC g /L ρ HepG2 x 珔 ± s / g L -1 A 450 /% 6 GFP-LC ± ± μl Opti-MEM ± ± Lipofectamine TM μg ± ± P < μl 30 min Opti-MEM HepG CO h HepG2 10% RPMI G d GFP-LC3 1 HepG / ml 2. 3 PI 10% RPMI1640 PI GFP-LC3 HepG2 6 HepG2 G g /L S G 2 /M 2 2 HepG2 /% G 0 /G 1 S G 2 /M 8 SPSS g /L ± x 珋 ± s 3 P < 0. 05

4 HepG2 2 HepG2

5 2 HepG HepG Annexin V-FITC /PI UR HepG2 48 h Annexin V-FITC /PI LL LR LU 4 4 HepG2

6 LC g /L GFP-LC3 HepG2 5 GFP-LC Western blotting LC3 SW480 S / G 2 /M Faria g /L MCF-7 II LC3 MDA-MB468 Dox MCF-7 6 G 1 MDA-MB468 G 1 8 B16 S 9 HepG2 DNA g /L. 6 Weston blotting HepG2 LC3 PC-3 MCF SMMC PI MCF-7 Aa g /L 3 - U-87 5 Annexin V-FITC /PI HepG2 Lovo HT29 SW480 Lovo HT29 G 1 G2 G 1 S G 2 /M HepG2 G 1 Hela

7 2 HepG GORETTAA L A ROMANCZYKB L J RODRIGUEZA C A et al. Cytotoxic effects of digalloyl dimer procyanidins in human cancer cell lines J. Nutritional Biochemistry LC3 Atg8 LC3 Pro- 5 ZHANG F J YANG J Y MOU Y H et al. Inhibition of LC3 LC3-I U-87 human glioblastoma cell proliferation and formyl LC3-I peptide receptor function by oligomer procyanidins F2 Atg7 Atg 3 isolated from grape seeds J. Chemico Biological Interactions LC3-II II 6 MALDONADO-CELIS M E SOUAD B FRANCINE G AVO et al. Apple procyanidins activate apoptotic signaling pathway in human colon adenocarcinoma cells by a lipid raft independent mechanism J. Biochemi Biophysical 14 Res Com FARIA A CALHAU C DEFREITAS V et al. Procyanidins as antioxidants and tumor cell growth modulators GFP-LC3 HepG2 GFP-LC3 J. J Agric Food Chem LC3 8. GFP-LC3 B16 J Western blotting GAPDH LC3-II 9. HepG2 J Hela livin Caspase-3 J. HepG PC-3 J SUN J LIU R H. Apple phytochemical extracts inhibit proliferation of estrogen-dependent and estrogen-independent human breast cancer cells through cell cycle modulation J. J Agric Food Chem SMMC J. J DPPH J J. 14 TANIDA I MINEMATSU-IKEGUCHI N UENO T et al. Lysosomal turnover but not a cellular level of endogenous LC3 is a marker for autophagy J. Autophagy 嫄

马丽媛, 等. 油茶皂苷体外诱导人白血病 Jurkat 细胞凋亡及其可能机制 1073 力争将茶油作为食用油的主流品种 [5] 茶麸系油茶果实榨除茶油后的废弃残渣, 药用名为茶子饼,

马丽媛, 等. 油茶皂苷体外诱导人白血病 Jurkat 细胞凋亡及其可能机制 1073 力争将茶油作为食用油的主流品种 [5] 茶麸系油茶果实榨除茶油后的废弃残渣, 药用名为茶子饼, 1072 基础研究 Basic Research www.tumorsci.org 肿瘤 2011 年 12 月第 31 卷第 12 期 TUMOR Vol. 31, December, 2011 DOI: 10.3781/j.issn.1000-7431.2011.12. 004 Copyright 2011 by TUMOR 油茶皂苷体外诱导人白血病 Jurkat