阿司匹林诱导人胶质瘤细胞U251凋亡和自噬
摘要
目的:研究阿司匹林(Aspirin)体外抑制U251胶质瘤细胞的作用及其机制,探讨自噬在Aspirin诱导的U251细胞死亡中的作用。
方法:用不同浓度的Aspirin处理人胶质瘤细胞株U251细胞,采用MTT法观察药物对人胶质瘤细胞U251的增殖抑制作用;采用流式细胞仪检测U251细胞周期分布的变化;FITC-AnnexinⅤ/PI双标记检测U251细胞的凋亡;应用MDC荧光染色检测自噬的发生,Western Blot检测凋亡相关蛋白Bcl-2、Caspase-3、LC3、Beclin1的表达。提取U251细胞的RNA,应用Real Time PCR方法检测LC3及Beclin1的mRNA表达。台盼蓝染色和3H-TDR观察用自噬特异性抑制剂3-MA阻断自噬后Aspirin抑瘤作用的变化。
结果:Aspirin可明显抑制人胶质瘤细胞U251的增殖,且此作用呈明显的时间、剂量依赖性;Aspirin可使U251细胞阻滞在G2/M期;诱导细胞发生凋亡,下调凋亡相关蛋白Bcl-2的表达以及诱导Caspase-3激活体增加。MDC荧光染色结果显示,Aspirin给药后可诱导U251细胞发生自噬;Aspirin诱导U251细胞发生自噬时,自噬特异性蛋白MAP LC3、Beclin 1被激活,同时它们的mRNA水平与对照组相比也明显升高。用3-MA预先抑制自噬后与Aspirin单独给药组相比,Aspirin对U251细胞的抑瘤率增加。
结论:Aspirin在体外对胶质瘤细胞U251有明显的抑制作用并可能是通过线粒体凋亡途径诱导其凋亡,自噬在此过程中发挥着一定的作用,用自噬抑制剂预处理使Aspirin对U251细胞的抑瘤率增加说明自噬在早期对胶质瘤U251细胞有保护作用。
Aim: To study the mechanism and inhibitory effect of aspirin on U251 cells in vitro, and study the effects of autophagy in Aspirin-induced death of U251 cells.
Methods: The effects of aspirin on proliferation of U251 cells were assessed using the MTT assay. Cell Cycle Analysis was by flow cytometry. AnnexinⅤ-FITC Apoptosis Detection Kit was used to detect apoptosis of cells. MDC staining was used to examine autophagy induced by aspirin in U251 cells. Western blot was employed to detect expression of Bcl-2,Caspase-3,LC3,Beclin1. Total RNA was extracted from U251 cells and Real Time PCR was used to detect the mRNA levels of LC3 and Beclin1; To examine the involvement of autophagy in aspirin-induced death of U251 cells, the autophagy specific inhibitor 3-methyadenine (3-MA) was added with aspirin and the cytotoxicity of aspirin was measured by trypan blue exclusion assay and DNA synthesis assay.
Results: The growth inhibiton of U251 cells by aspirin was in a time and dose-dependent stytle. After treatment with 8 mM, cell cycle was arrested at G2/M phase. Aspirin also significantly enhanced apoptosis of U251 cells with down-regulated anti-apoptotic protein Bcl-2, and activation of Caspase-3. Autophagy was induced in U251 cells as detected by MDC staining; Western blot and Real Time PCR analysis revealed that expression of LC3 and Beclin1 increased after aspirin treatment. The blockade of autophagy with specific inhibitor 3-methyladenine(3-MA) amplified the cytotoxicity of aspirin in U251 cells.
Conclusions: Aspirin could significantly inhibit the growth of U251 cells through inducing cell apoptosis in vitro, which may depend on mitochondrial apoptotic pathway. 3-methyladenine(3-MA) can amplify the cytotoxicity of aspirin in U251 cells, this demonstrates that autophagy may paly a protective role in the early time of aspirin treatment.
方法:用不同浓度的Aspirin处理人胶质瘤细胞株U251细胞,采用MTT法观察药物对人胶质瘤细胞U251的增殖抑制作用;采用流式细胞仪检测U251细胞周期分布的变化;FITC-AnnexinⅤ/PI双标记检测U251细胞的凋亡;应用MDC荧光染色检测自噬的发生,Western Blot检测凋亡相关蛋白Bcl-2、Caspase-3、LC3、Beclin1的表达。提取U251细胞的RNA,应用Real Time PCR方法检测LC3及Beclin1的mRNA表达。台盼蓝染色和3H-TDR观察用自噬特异性抑制剂3-MA阻断自噬后Aspirin抑瘤作用的变化。
结果:Aspirin可明显抑制人胶质瘤细胞U251的增殖,且此作用呈明显的时间、剂量依赖性;Aspirin可使U251细胞阻滞在G2/M期;诱导细胞发生凋亡,下调凋亡相关蛋白Bcl-2的表达以及诱导Caspase-3激活体增加。MDC荧光染色结果显示,Aspirin给药后可诱导U251细胞发生自噬;Aspirin诱导U251细胞发生自噬时,自噬特异性蛋白MAP LC3、Beclin 1被激活,同时它们的mRNA水平与对照组相比也明显升高。用3-MA预先抑制自噬后与Aspirin单独给药组相比,Aspirin对U251细胞的抑瘤率增加。
结论:Aspirin在体外对胶质瘤细胞U251有明显的抑制作用并可能是通过线粒体凋亡途径诱导其凋亡,自噬在此过程中发挥着一定的作用,用自噬抑制剂预处理使Aspirin对U251细胞的抑瘤率增加说明自噬在早期对胶质瘤U251细胞有保护作用。
Aim: To study the mechanism and inhibitory effect of aspirin on U251 cells in vitro, and study the effects of autophagy in Aspirin-induced death of U251 cells.
Methods: The effects of aspirin on proliferation of U251 cells were assessed using the MTT assay. Cell Cycle Analysis was by flow cytometry. AnnexinⅤ-FITC Apoptosis Detection Kit was used to detect apoptosis of cells. MDC staining was used to examine autophagy induced by aspirin in U251 cells. Western blot was employed to detect expression of Bcl-2,Caspase-3,LC3,Beclin1. Total RNA was extracted from U251 cells and Real Time PCR was used to detect the mRNA levels of LC3 and Beclin1; To examine the involvement of autophagy in aspirin-induced death of U251 cells, the autophagy specific inhibitor 3-methyadenine (3-MA) was added with aspirin and the cytotoxicity of aspirin was measured by trypan blue exclusion assay and DNA synthesis assay.
Results: The growth inhibiton of U251 cells by aspirin was in a time and dose-dependent stytle. After treatment with 8 mM, cell cycle was arrested at G2/M phase. Aspirin also significantly enhanced apoptosis of U251 cells with down-regulated anti-apoptotic protein Bcl-2, and activation of Caspase-3. Autophagy was induced in U251 cells as detected by MDC staining; Western blot and Real Time PCR analysis revealed that expression of LC3 and Beclin1 increased after aspirin treatment. The blockade of autophagy with specific inhibitor 3-methyladenine(3-MA) amplified the cytotoxicity of aspirin in U251 cells.
Conclusions: Aspirin could significantly inhibit the growth of U251 cells through inducing cell apoptosis in vitro, which may depend on mitochondrial apoptotic pathway. 3-methyladenine(3-MA) can amplify the cytotoxicity of aspirin in U251 cells, this demonstrates that autophagy may paly a protective role in the early time of aspirin treatment.
引文
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1. Ming Li, Reuben L, Bernard L, et al. Aspirin induction of apoptosis in esophageal cancer: a potential for chemoprevention. Cancer Epidem Biomar. 2000; 9: 545–549.
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2. Mizushima N, Yamamoto A, Hatano M, Kobayashi Y, Kabeya Y, Suzuki K, et al. Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells. J Cell Biol. 2001; 152: 657-67.
3. Gronostajski RM, Pardee AB, Goldberg AL. The ATP dependence of the degradation of short- and long-lived proteins in growing fibroblasts. J Biol Chem.1985; 260(6): 3344-9.
4. Takeshi N, Kuninori S, YoshinoriO. Yeast autophagosome: de novo formation of a membrane structure. Trend Cell Bio. 2002; 12(5) : 231 - 5.
5. Abeliovich H, Klionsky DJ. Autophagy in Yeast: Mechanistic insights and physiological function. Microbiol Mol Biol R. 2001; 65(3): 463-479.
6. Cheong H, Yorimitsu T, Reggiori F,et al. Atg17 Regulates the Magnitude of the Autophagic Response. Mol. Biol. Cell. 2005; 16: 3438-3453.
7. Majeski AE, Dice JF. Mechanisms of chaperone-mediated autophagy. Int J Biochem Cell Biol. 2004; 36(12): 2435-44.
8. Shintani T, Klionsky DJ. Autophagy in health and disease: a double-edged sword. Science. 2004; 306(5698): 990-5.
9. Mari?o G. and López-Otín C. autophagy: molecular mechanisms, physiological functions and relevance in human pathology. Cell Mol Life Sci. 2004; 61(12): 1439-1454.
10. Knaapen MW, Davies MJ, De Bie M, et al. Apoptotic versus autophagic cell death in heart failure.2001; Cardiovasc Res. 51(2): 304-312.
11. Lockshin RA and Zakeri Z. Apoptosis, autophagy, and more. Int J Biochem Cell Biol. 2004; 36(12): 2405-2419.
12. Edinger AL and Thompson CB. Death by design: apoptosis, necrosis and autophagy. Curr Opin Cell Biol. 2004; 16(6): 663-669.
13. Longo L, Platini F, Scardino A , et al.Autophagy inhibition enhances anthocyanin-induced apoptosis in hepatocellular carcinoma. Mol Cancer Ther. 2008; 7(8):2476–85.
14. Farhana A, Ivor DB, Zsolt S, et al. Apoptotic and non-apoptotic modes of programmed cell death in MCF-7 human breast carcimona cells. Cell Biol Int. 2000; 24(4):253-260.
15. Szene B, Keri GY, Szegedi ZS, et al. Tyrphostin induces non-apoptotic programmed cell death in colon tumor cells. Cell Bio Int. 1995; 19(11): 903-911.
16. Aoki H, i TakadaY, Kondo S, et al. Evidence That Curcumin Suppresses the Growth of Malignant Gliomas in Vitro and in Vivo through Induction of Autophagy: Role of Akt and Extracellular Signal-Regulated Kinase Signaling Pathways. Mol. Pharmacol. 2007; 72: 29 - 39.
17. Meenakshi T, Kumar BV, et al. Inhibition of N-(4-hydroxyphenyl)retinamide- induced autophagy at a lower dose enhances cell death in malignant glioma cells. Carcinogenesis. 2008; 29(3): 600-609.
18. Sathornsumetee S, Reardon DA, Desjardins A, et al. Molecularly Targeted Therapy for Malignant Glioma. Cancer. 2007; 110: 13–24.
19. WANG H, LEI ZY, CHEN H, et al. Antitumor activity of PB2LY and itsmechanism in vitro. Chin Pharmacol Bull. 2008; 24(9): 1208-12.
20. Gu Q, Wang JD, Xia HH, et al. Activation of the caspase-8/Bid and Bax pathways in aspirin-induced apoptosis in gastric cancer. Carcinogenesis. 2005; 26(3): 541-546.
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