32 2 Vol. 32 No. 2 2011 4 Journal of Jinan University Medicine Edition Apr. 2011 HepG2 1 1 2 1 1 1 1. 510632 2. 510080 HepG2 MTT WST-8 HepG2 PI Annexin V-FITC /PI Western blotting HepG2 - P < 0. 01 HepG2 48 h IC 50 1. 304 10-1 g /L HepG2 HepG2 G 1 1. 6 10-1 g /L 81% GFP - LC3 HepG2 LC3 LC3- II HepG2 R285. 5 A 1000-9965 2011 02-0181 - 07 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 510632 China 2. Department of Oncology the First Affiliated Hospital of Guangdong Pharmaceutical University Guangzhou 510080 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 1. 304 10-1 g /L. Cell colone formation inhibition assay demon- 2011-01 - 10 2009B080701051 A2009348 1987 - Tel 020-85220306 E - mail 329255726@ qq. com
182 32 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-1640 5. 0 10 4 /ml 96 100 μl 5 000 1961 150 μl 5 0 10-3 4. 0 10-2 8. 0 10-2 1. 2 10-1 1. 6 10-1 2. 0 10-1 g /L 1 37 5% 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 1. 0 10 5 /ml 6 1 ml 12 h Gibco Lipofectamine TM 2000 Invitrogen ECL Pierce 1. 2 1 HepG2 6 min 0. 01 mol /L PBS 37 5% CO 2 2 70% 4 3 1 70% PBS 3 0. 25% HepG2 10% 3 HepG2 15% RPMI- 1 1640 12 300 5 1. 1 0 10-3 4. 0 10-2 8. 0 10-2 1. 2 HepG2 GFP-LC3 10-1 1. 6 10-1 2. 0 10-1 g /L 37 5% CO 2 7 d PBS 2 V V = 3 1 10 min Giemsa 30 min 5min 0 10-3 4. 0 10-2 8. 0 10-2 1. 2 10-1 1. 6 10-1 g /L 48 h 1 000 r /min RNA 5. 0 2 MTT WST-8 10-2 g /L 30 min 300
2 HepG2 183 DNA 10 000 5 Annexin V-FITC /PI 2. 1 HepG2 1. 0 MTT WST-8 10 5 /ml 6 HepG2 0 10-3 4. 0 10-2 8. 0 10-2 1. 2 10-1 1. 6 10-1 37 5% CO 2 48 h 1 000 r /min 6 min PBS 2 10 μl Annexin V-FITC 5 μl propidine iodide PI 15 min 1 200 μl 0 0. 913 ± 0. 038 0 4. 0 10-2 0. 788 ± 0. 020 0. 136 ± 0. 022 1 10 000 8. 0 10-2 0. 616 ± 0. 034 0. 325 ± 0. 037 1 36 h PBS 2 2 7 1 000 r /min 10 min PBS 2 ρ PBS 15 min / g L - 1 12 000 r /min 15 min BCA 0 46. 8 40. 2 13. 0 12% 4. 0 10-2 52. 0 35. 7 12. 3 - SDS - 8. 0 10 PAGE - 2 65. 9 23. 7 10. 4 1. 2 10-1 75. 8 19. 9 4. 3 PVDF 1. 6 10-1 78. 3 19. 0 2. 7 5% 2 h ECL HepG2 5min X 2 P < 0. 01 1 HepG2 48 h IC 50 1. 304 10-1 g /L ρ HepG2 x 珔 ± s / g L -1 A 450 /% 6 GFP-LC3 1. 2 10-1 0. 475 ± 0. 016 0. 479 ± 0. 018 1 50 μl Opti-MEM 1. 6 10-1 0. 406 ± 0. 030 0. 556 ± 0. 033 1 Lipofectamine TM 2. 0 10 2000 1 μg - 1 0. 320 ± 0. 024 0. 650 ± 0. 026 1 1 P < 0. 01 2. 5 μl 30 min Opti-MEM HepG2 2. 2 CO 2 37 6 h HepG2 10% RPMI-1640 12 G418 10 d GFP-LC3 1 HepG2 1. 5 10 5 / ml 2. 3 PI 10% RPMI1640 PI GFP-LC3 HepG2 6 HepG2 G 1 0 10-3 1. 5 10-1 g /L S G 2 /M 2 2 HepG2 /% G 0 /G 1 S G 2 /M 8 SPSS 13. 0 1. 6 10-1 g /L ± x 珋 ± s 3 P < 0. 05
184 32 1 HepG2 2 HepG2
2 HepG2 185 3 HepG2 2. 4 Annexin V-FITC /PI UR HepG2 48 h Annexin V-FITC /PI LL LR LU 4 4 HepG2
186 32 2. 5 5 LC3 1. 5 10-1 g /L GFP-LC3 HepG2 5 GFP-LC3 2. 6 Western blotting LC3 SW480 S / G 2 /M 0 10-3 4. 0 10-2 8. 0 6 Faria 7 10-2 1. 2 10-1 1. 6 10-1 g /L MCF-7 II LC3 MDA-MB468 Dox MCF-7 6 G 1 MDA-MB468 G 1 8 B16 S 9 HepG2 DNA 1 2 3 4 4. 0 10-2 8. 0 10-2 1. 2 10-1 1. 6 10-1 g /L. 6 Weston blotting HepG2 LC3 PC-3 MCF-7 3 10 - SMMC-7721 12 2 PI MCF-7 Aa427 1. 6 10-1 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
2 HepG2 187 11661-11667. 13 4 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 2008 19 12 797-808. 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 2009 179 3 419-429. 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 2009 388 2 372-376. 7 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 2006 22 54 6 2392-2397. LC3 8. GFP-LC3 B16 J. 2007 29 Western blotting 12 1828-1830. GAPDH LC3-II 9. HepG2 J. 2008 24 6 891-893. 10. Hela livin Caspase-3 J. HepG2 2007 17 19 2349-2353. 11. PC-3 J. 2009 28 1 3 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 2008 56 24 47-49. 12. SMMC-7721 1. J. J. 2004 2007 35 1 37-40. 7 761-764. 13. 2. DPPH J. 2008 39 1 37-40. J. 14 TANIDA I MINEMATSU-IKEGUCHI N UENO T et 2006 25 2 102-106. al. Lysosomal turnover but not a cellular level of endogenous LC3 is a marker for autophagy J. Autophagy 2005 1 2 84-91. 嫄