Mechanism of Apoptosis in Human Glioblastoma U251 Cells Induced by Diallyl Disulfi de
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摘要: 目的 研究二烯丙基二硫（diallyl disulfide, DADS）诱导人恶性胶质瘤U251细胞凋亡的作用及其分子机制。方法 光学显微镜观察DADS作用后U251细胞凋亡形态学改变；流式细胞术检测DADS对U251细胞凋亡率的影响; Western blot和免疫细胞化学分别检测凋亡相关蛋白的表达。结果 光学显微镜下，DADS处理U251细胞后，细胞呈现凋亡形态改变。MTT法显示，15、30、45、60 mg/L DADS处理U251细胞48 h后，生长抑制率分别为22.01%、38.82%、49.23%、55.27%，与未处理组比较，差异有统计学意义(P<0.05）。流式细胞术显示，15、30、45 mg/L DADS处理后，细胞凋亡率分别为（5.36±0.87）%、（28.36±3.15）% 和（44.58±3.95）%，显著高于对照组（2.14±0.45）%(P<0.05）。免疫细胞化学显示，45 mg/L DADS处理48 h后，Bcl-2表达平均吸光度值(0.34±0.03）较未处理细胞（0.75±0.06）明显下降(P<0.05)；Bax表达平均吸光度值（0.63±0.04）高于未处理组(0.26±0.03）(P<0.05)；Caspase 3表达平均吸光度值（0.41±0.05）明显高于未处理组（0.19±0.02）(P<0.05)。Western blot检测显示，15、30与45mg/L DADS处理48 h后，Bcl-2蛋白灰度值比分别为（1.21±0.18）、（0.89±0.14）与（0.41±0.09），较未处理组（2.24±0.26）显著降低(P<0.05)；Bax和Caspase 3蛋白分别为（1.12±0.19）、（1.54±0.22）与（2.08±0.27）和（0.72±0.15）、（1.39±0.21）与（2.28±0.29），明显高于未处理组（0.33±0.08）和（0.14±0.06） (P<0.05)。结论 DADS可诱导恶性胶质瘤U251细胞凋亡，机制与下调Bcl-2，上调Bax、Caspase 3有关。Abstract: Objective To investigate molecular mechanism of apoptosis induced by diallyl disulfi de (DADS) in human glioblastoma U251 cells. Methods Morphological analysis, MTT, fl ow cytometry, Western blot and immunocytochemical technique were used to observe the effects of apoptosis and its expression of related proteins in human glioblastoma U251 cells induced by DADS. Results After exposure to DADS, partial U251 cells presented characteristic morphological changes of apoptosis under the light microscopy. MTT assay showed that 15, 30, 45, 60 mg/L DADS signifi cantly inhibited proliferation of U251 cells at 48 h, its inhibition ratio were 22.01%, 38.82%, 49.23% and 55.27%, respectively, in dose-dependent manner (P<0.05). Flow cytometry analysis showed that U251 cells treated with 15, 30 and 45 mg/L DADS for 48h signifi cantly increased the percentage of apoptosis cells, its apoptosis rate were(5.36±0.87)%, (28.36±3.15)% and (44.58±3.95)%, respectively, higher than (2.14±0.45)% of untreated cells (P<0.05). Immuocytochemistry detect revealed that expression of Bcl-2 decreased from 0.75±0.06 to 0.34±0.03, and Bax and Caspase 3 expression increased from 0.26±0.03 and 0.19±0.02 to 0.63±0.04 and 0.41±0.05, respectively, in average optical value (P<0.01). Western blot showed that downregulation of Bcl-2 expression was 1.21±0.18, 0.89±0.14 and 0.41±0.09 lower than 2.24±0.26 of untreated cells, and upregulation of Bax and Caspase 3 was 1.12±0.19, 1.54±0.22 and 2.08±0.27 and 0.72±0.15, 1.39±0.21 and 2.28±0.29, higher than 0.33±0.08 and 0.14±0.06 of untreated cells, respectively, in gray scale value after U251cells treated by 15, 30 and 45 mg/LDADS (P<0.05). Conclusion DADS can induce apoptosis of U251 cells related to downregulation of Bcl-2 expression and upregulation of Bax and Caspase 3.
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Key words:
- Diallyl disulfi de /
- Glioblastoma /
- U251 cells /
- Apoptosis
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[1] Parkin DM, Bray F, Ferlay J, et al. Global cancer statistics, 20 02[J]. CA Cancer J Clin,2005,55(2):74-108. [2] Jemal A, Bray F, Center MM, et al. Global cancer statistics[J]. CA Cancer J Clin,2011,61(2):69-90. [3] Quick A, Patel D, Hadziahmetovic M, et al. Current therapeutic paradigms in glioblastoma[J]. Rev Recent Clin Trials,2010,5(1):14-27. [4] Wang XJ, Tang HL, Su Q. Research progress in the effects of garlic allyl sulfide on inhibiting nervous system tumors [J]. Zhongguo Yao Li Xue Tong Bao,2010,26(10):1274-7.[汪雪菁, 唐 海林, 苏琦. 大蒜烯丙基硫化物抗神经系统肿瘤作用研究进展 [J] 中国药理学通报,2010,26(10):1274-7. ] [5] Ling H, Wen L, Ji XX, et al. Growth inhibitory effect and Chk1- dependent signaling involved in G2/M arrest on human gastric cancer cells induced by diallyl disulfide[J]. Braz J Med Biol Res,2010,43(3):271-8. [6] Yi L, Ji XX, Lin M, et al. Diallyl disulfi de induces apoptosis in human leukemia HL-60 cells through activation of JNK mediated by reactive oxygen[J]. Pharmazie,2010,65(9):693-8. [7] Liao QJ, Su J, He J, et al. Effect of diallyl disulfi de on cell cycle arrest of human colon cancer sw480 cells[J]. Ai Zheng,2009,28(2): 13 8-41. [8] Tang HL, Song Y, Tang RJ, et al. Apoptosis of nasopharyngeal cancer cells line CNE2 induced by diallyl disulfide and its possible molecular mechanism[J]. Zhongguo Ai Zheng Za Zhi, 20 07, 17(8): 598-602. [唐海林, 宋颖, 唐荣军, 等. 二烯丙基二硫 诱导人鼻咽癌CNE2细胞凋亡及其可能的分子机制[J]. 中国癌 症杂志,2007,17(8):598-602. [9] Ferreira IC, Vaz JA, Vasconcelos MH, et al. Compounds from wild mushrooms with antitumor potential[J]. Anticancer Agents Med Chem,2010,10(5):424-36. [10] Tan H, Ling H, He J, et al. Inhibition of ERK and activation of p38 are involved in diallyl disulfi de induced apoptosis of leukemia HL-60 cells[J]. Arch Pharm Res,2008,31(6):786-93. [11] Karmakar S, Banik NL, Patel SJ, et al. Garlic compounds induced calpain and intrinsic caspase cascade for apoptosis in human malignant neuroblastoma SH-SY5Y cells[J]. Apoptosis,2007,12 (4) :671-84. [12] Lin HL, Yang JS, Yang JH, et al. The role of Ca2+ on the DADSinduced apoptosis in mouse-rat hybrid retina ganglion cells (N18) [J] Neurochem Res,2006,31(3):383-93. [13] Das A, Banik NL, Ray SK. Garlic compounds generate reactive oxygen species leading to activation of stress kinases and cysteine proteases for apoptosis in human glioblastoma T98G and U87MG cells[J]. Cancer, 2007,110(5):1083-95. [14] Ji BW, Chen QX, Tian DF, et al. Mechanism of histone deacetylase inhibitors inducing apoptosis in glioma cells[J]. Zhongguo Shen Jin Zhong Liu Za Zhi,2011,9(2): 82-7. [冀保卫, 陈谦学, 田道锋, 等. 组蛋白去乙酰化酶抑制剂诱导脑胶质瘤细 胞凋亡及其机制的研究[J]. 中国神经肿瘤杂志,2011,9(2):82-7.] [15] Yang WZ, Chen CM, Shi SS, et a1. In vitro study on increase in radiosensifivity of human gliomia cells induced by elemene[J]. Zhonghua Shi Yan Wai Ke Za Zhi, 2007, 24(4): 463-5.[杨卫忠, 陈春美, 石松生, 等. 榄香烯体外增强人脑胶质瘤U251细胞对γ 射线敏感性的研究[J]. 中华实验外科杂志, 2007, 24(4): 463-5.]
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