http / / cjbmb. bjmu. edu. cn Chinese Journal of Biochemistry and Molecular Biology COX-2 NTera-2 NTera-2 RT-PCR FasL caspase-8 caspase-3 PARP.
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1 ISSN CN / Q http / / cjbmb bjmu edu cn Chinese Journal of Biochemistry and Molecular Biology ~ 636 NTera-2 ** ** * COX-2 NTera-2 MTT NTera-2 NTera-2 Hoechest Annexin V-FITC / PI NTera-2 RT-PCR NTera-2 Fas caspase-8 FasL caspase-8 caspase-3 PARP caspase Z-VAD-FMK NTera-2 NTera-2 caspase NTera-2 NTera-2 Q789 Aspirin Induces Apoptosis in Cultured NTera-2 Human Malignant Testicular Germ Cells YI Duo ** LI Xiao-Feng ** ZHAO Xiao-Meng WANG Cheng ZHANG Xiao-Ting LIU Xi-Zhi ZHOU Chang * Department of Biochemistry and Molecular Biology Key Laboratory of Protein Chemistry and Developmental Biology Ministry of Education of China College of Life Sciences Hunan Normal University Changsha China Abstract Aspirin also known as acetylsalicylic acid has been shown to have anticancer effects with multiple activities including inhibition of proliferation induction of apoptosis and inhibition of angiogenesis The functional characteristics and the anti-tumor mechanism of aspirin are largely unknown except for the suppression of cyclo-oxygenase 2 COX-2 Aspirin administration is known to associate with lower risks of many cancer types but less reported in human testicular tumors We investigated on whether aspirin was able to induce apoptosis in NTera-2 human malignant testicular germ cells using MTT to detected cell viabilities The morphological changes formation of apoptotic bodies and cell apoptosis were assessed by fluorescence microscopy Hoechest33342 staining Annexin V-FITC / PI flow cytometry RT-PCR and Western blotting were also performed The result showed that aspirin No No No K * Tel ssipzc1995@ yahoo com cn ** Received April Accepted May Supported by National Natural Science Foundation of China No No Project of Changsha Science and Technology Plan No K * Corresponding author Tel ssipzc1995@ yahoo com cn ** Two authors contributed equally to this work
2 7 NTera inhibited the proliferation of NTera-2 cells in a dose- and time-dependent manner and Fas and caspase- 8 were up-regulated The level of FasL was down-regulated after aspirin treatment The protein levels of caspase-3 caspase-8 and poly ADP-Ribose polymerase PARP were increased Caspase inhibitor Z- VAD-FMK could attenuate the aspirin induced apoptosis We concluded that the anticancer activity of aspirin was partially due to the inhibition of cell viability and induction of apoptosis associated with caspase apoptotic pathway Key words aspirin NTera-2 cell cell apoptosis caspase 90% NTera-2 1 Hoechst Sigma PARP caspase- 20 ~ caspase-3 Fas Santa Cruz FasL Abcam Caspase Z-VAD- 50 FMK Promega 6 /10 ~ 11 /10 3 GAR GAM Jackson Immuno Research Annexin V-FITC BD Pharmingen caspase 8 80% MTT NTera-2 D h 4 h 5 mg / ml MTT 50 μl 450 μl MTT 0 5 mg / ml MTT μl DMSO 10 min nm A 1 3 Hoechest mmol / L aspirin 24 h PBS ml / 4% 30 min PBS 2 50% / 30 s 50% 2011 Rothwell 6 Lancet / 1 ml PBS 5 min 4 Hoechest min 1 ml 7-12 PBS 3 5 min Zeiss Axioskop mmol / L NTera-2 PBS 37 5% CO NTera h Annexin-Ⅴ / PI PBS μl 1 5 μl Annexin-Ⅴ 10 μl PI
3 min 400 μl Z-VAD-FMK Annexin-V PI 20 μmol / L Caspase Caspase-8 pna Western PARP μl 20 min g 4 15 min caspase NTera-2 90 μl 2 mmol / L Ac-IETD-pNA 10 μl 80 μl 10 μl 2 mmol / L Ac-DEVD-pNA 10μL caspase RT-PCR Fas Caspase-8 30 s s 30 Table 1 The primer sequences and the product lengths of semi-quantitative RT-PCR Primer Primer sequence Product / bp Fas F 5'-GCATCTGGACCCTCCTACCT-3' 210 Fas R 5'-CTTGGTGTTGCTGGTGAGTG-3' Caspase8 F 5'-GGAGGAGTTGTGTGGGGTAA-3' 205 Caspase8 R 5'-TGCATCCAAGTGTGTTCCAT-3' GAPDH F 5'-GACTGTCTCCTCCCAAATTT-3' 496 GAPDH R 5'-GCATGGACTGTGGTCATGAGT-3' 1 7 Western mmol / L Caspase-8 Caspase-8 Ac-IETD-pNA NTera-2 PBS 2 RIPA caspase-8 20 min g 20 min caspase-8 30 ~ 40 μg SDS-PAGE caspase-8 PVDF 5% Fig 4 FasL PARP caspase-3 caspase-8 β caspase h 1 h mmol / L 6 DAB 2 4 FasL caspase 3 Z-VAD-FMK NTera-2 30 min 24 h MTT NTera-2 Fig 1 10 mmol / L NTera h 24% 51% 79% 82% 2 2 NTera-2 mrna TRIZOL h mmol / L NTera-2 RNA 1 15 μg RNA DNase Promega mmol / L Table 1 RT-PCR GAPDH 20 mmol / L PCR 94 4 min s mmol / L Fig 2 Annexin V PI NTera- 2 NTera- 2 Fig 3A Annexin V PI NTera mmol / L aspirin NTera-2 24 h 8 76% 16 37% 33 67% 36 85% 47 79% 5 07% Fig 3B 2 3 caspase-8 pna 13 pna 405 nm mmol / L
4 7 NTera Fig 1 Aspirin inhibits cell viability of NTera-2 cells by a time- and dose-dependent manner NTera-2 cells were incubated with mmol / L aspirin respectively for 32 hours And cell growth was determined by the MTT assay every 8 hours ** P < 0 01 vs control group * P < 0 05 NTera-2 TRIZOL RNA RT-PCR Fas caspase-8 mrna Fig 5A Western NTera-2 FasL caspase-8 caspase-3 pro-caspase 8 pro-caspase 3 10 mmol / L PARP Fig 5B 2 5 Z-VAD-FMK Z-VAD-FMK caspase caspase Z-VAD-FMK NTera-2 30 min 24 h Fig 6A Fig 2 The morphological change of NTera-2 cell apoptosis induced by aspirin Induction of apoptosis in NTera-2 cells by treatment with mmol / L aspirin respectively for 24 hours The treated cells were stained with Hoechst and the apoptotic morphological changes in the nuclear chromatin were observed under a fluorescent microscope The apoptosis cells were indicated as arrows magnification 40 NTera-2 PARP 8 h PARP Z-VAD-FMK caspase NTera-2 NTera-2 15 mmol / L 3 NTera-2 4 h 8 h 1 Western Fig 6B 4 h PARP 8 h
5 Fig 3 Annexin V / PI analysis of apoptotic cells in aspirin-treated cells NTera-2 cells were treated for 24 hours at concentration of mmol / L aspirin or remained PBS-treated as a control A Representative data of flow cytometry assay Percentage of apoptosis was denoted on top of the dot plots and was counted by annexin V-positive / propidium iodide-negative cells B Each bar corresponded to the mean ± SD for three independent experiments Fig 4 Caspase-8 protease activity detected in aspirintreated NTera-2 cells NTera-2 cells were treated with different concentrations of aspirin Cells were harvested after 24 hours and caspase-8 protease activity was measured using a colorimetric caspase-8 activity assay kit The values were the mean the standard deviation of triplicate assays Annexin V-FITC / PI NTera-2 14 Fas Fas FasL Fas Fas Fas associated death domain FADD caspase-8 Fas / FasL DISC 15 caspase-8 caspase caspase-3 MTT NTera-2 24 h Fas caspase-8 NTera-2 mrna Fas FasL
6 7 NTera Fig 5 Aspirin modulates expression of apoptotic genes in NTera-2 cells Cells were treated with different concentrations of aspirin for 24 hours as indicated A The expression of Fas and caspase-8 in NTera-2 cells treated with aspirin were analyzed by RT-PCR GAPDH was used as control B Cells were lysed in RIPA lysis buffer FasL pro-caspase-8 pro-caspase-3 and PARP expression were assayed by Western blotting Expression was normalized with anti-β-actin One representative of three different experiments was showed Images were converted to gray scale and analyzed using Image J software FasL caspase Fas / FasL NTera-2 References caspase Caspase Urology Fas FasL FADD caspase-8 DED caspase-8 caspase NTera-2 FasL pro-caspase 8 pro-caspase 3 caspase-8 caspase-3 PARP PARP Z-VAD-FMK NTera-2 PARP Fig 6 Aspirin activates caspase-mediated apoptosis signaling pathway A NTera-2 cells were exposed to mmol / L aspirin respectively for 24 hours after treated with caspase inhibitor Z-VAD-FMK for 30 minutes The whole cell extracts were harvested and examined with anti-parp and anti-β-actin antibody by Western blotting B NTera-2 cells were incubated with 15 mmol / L aspirin in indicated times but not treated with Z- VAD-FMK Cells were harvested and the expression of PARP protein was analyzed by Western blotting β-actin was showed as an internal standard One representative of three different experiments was showed NTera-2 caspase NTera-2 1 Hara S Miyake H Nakamura I et al Increased Fas ligand expression in the tumor tissue and serum of patients with testicular germ cell tumors with seminomatous elements J 2 Horwich A Shipley J Huddart R J Lancet Ginsburg J Testicular germ-cell cancer Unanswered questions in carcinoma of the testis J Lancet Einhorn L H Curing metastatic testicular cancer J Proc Natl Acad Sci U S A Kollmannsberger C Kuzcyk M Mayer F et al Late toxicity following curative treatment of testicular cancer J Semin Surg Oncol Rothwell P M Fowkes F G Belch J F et al Effect of daily aspirin on long-term risk of death due to cancer analysis of individual patient data from randomised trials J Lancet
7 Gu Q Wang J D Xia H H et al Activation of the caspase-8 / Bid and Bax pathways in aspirin-induced apoptosis in gastric cancer J Carcinogenesis Kong S Y Kang J H Kwon Y et al Serum HER-2 concentration in patients with primary breast cancer J J Clin Pathol Merrick D T Kittelson J Winterhalder R et al Analysis of c- ErbB1 / epidermal growth factor receptor and c-erbb2 / HER-2 expression in bronchial dysplasia evaluation of potential targets for chemoprevention of lung cancer J Clin Cancer Res Pt Maxwell P H Tumor strengths and frailties Aspiring to prevent colon cancer J Nat Med Greenberg E R Baron J A Freeman D H Jr et al Reduced risk of large-bowel adenomas among aspirin users The Polyp Prevention Study Group J J Natl Cancer Inst Hossain M A Kim D H Jang J Y et al Aspirin induces apoptosis in vitro and inhibits tumor growth of human hepatocellular carcinoma cells in a nude mouse xenograft model J Int J Oncol Villa P Kaufmann S H Earnshaw W C Caspases and caspase inhibitors J Trends Biochem Sci Ashkenazi A Dixit V M Death receptors signaling and modulation J Science Chinnaiyan A M Tepper C G Seldin M F et al FADD / MORT1 is a common mediator of CD95 Fas / APO-1 and tumor necrosis factor receptor-induced apoptosis J J Biol Chem Oliver F J de la Rubia G Rolli V et al Importance of poly ADP-ribose polymerase and its cleavage in apoptosis Lesson from an uncleavable mutant J J Biol Chem Suhara T Kim H S Kirshenbaum L A et al Suppression of Akt signaling induces Fas ligand expression involvement of caspase and Jun kinase activation in Akt-mediated Fas ligand regulation J Mol Cell Biol
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213 12 33 12 TUMOR Vol. 33, December 213 www.tumorsci.org 161 Basic Research DOI: 1.3781/j.issn.1-7431.213.12. 5 Copyright 213 by TUMOR A549/DDP 1, 2 2 1 3 4 1 1 1. 3619 2. 355 3. 3611 4. 361 elemene cisplatin
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