三氧化二砷对A549细胞凋亡及survivin、cyclin D1基因表达的影响
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摘要
目的:了解三氧化二砷对肺腺癌A549细胞株细胞增殖、细胞周期,细胞凋亡以及细胞survivin、cylin D1基因表达的影响。
方法:分别以不同浓度的三氧化二砷作用于A549细胞,作用不同时间后对细胞增殖活性、细胞周期、细胞凋亡及细胞survivin、cyclin D1基因表达的变化进行检测。
结果:稍高浓度(＞2μmol/L)的三氧化二砷可抑制A549细胞增殖,并呈剂量和时间依赖性。逆转录聚合酶链式反应(RT-PCR)检测survivin、cyclin D1基因转录水平和细胞爬片免疫组化检测survivin、cyclin D1基因蛋白表达的相对强度,发现三氧化二砷作用组survivin和cyclin D1基因的表达与对照组相比明显减低(P＜0.05)。三氧化二砷抑制A549细胞由G1期进入S期
结论:三氧化二砷可有效抑制A549细胞增殖,诱导A549细胞凋亡,抑制细胞G1进入S期,并与三氧化二砷剂量和作用时间正相关,这一过程可能与三氧化二砷抑制survivin、cyclin D1表达有关。
Objective : To investigat the effect of arsenic trioxide on cell proliferation,cell cycle, cell apoptosis and survivin,cyclin D1 gene expression in lung adenocarcinoma A549 cells. Methods: After A549 were treated with arsenic trioxide in different doses and with different time, MTT was used to detect the proliferation of A549 cells. The Annexin V-FITC /PI double staining analysis by flow cytometry was used to evaluate apoptosis rate. Cell cycle was elaluated by PI staining FCM anlysis. Survivin ,cyclin D1 gene expression was detected by RT-PCR and immunohistochemesitry. Results: Arsenic trioxide could inhibit cell proliferation, induce cell apoptosis and stop cell cycle at G1/S transition in A549 cells. The apoptotic rate was both dose depedent and time dependent. Survivin and cyclin D1 gene expression was down regulated in arsenic trioxide group compared with the control. Conclusion: Arsenic trioxide can effectively inhibit cell proliferatioin and induce apoptosis in lung adenocarcinoma A549 cells,this may result from its actioin of reduce survivin and cyclin D1 gene expression. Arsenic trioxide stop cell cycle at G1 phase in lung adenocarcinoma A549 cells, this may result from its actioin of reduce cyclin D1 gene expression.
方法:分别以不同浓度的三氧化二砷作用于A549细胞,作用不同时间后对细胞增殖活性、细胞周期、细胞凋亡及细胞survivin、cyclin D1基因表达的变化进行检测。
结果:稍高浓度(＞2μmol/L)的三氧化二砷可抑制A549细胞增殖,并呈剂量和时间依赖性。逆转录聚合酶链式反应(RT-PCR)检测survivin、cyclin D1基因转录水平和细胞爬片免疫组化检测survivin、cyclin D1基因蛋白表达的相对强度,发现三氧化二砷作用组survivin和cyclin D1基因的表达与对照组相比明显减低(P＜0.05)。三氧化二砷抑制A549细胞由G1期进入S期
结论:三氧化二砷可有效抑制A549细胞增殖,诱导A549细胞凋亡,抑制细胞G1进入S期,并与三氧化二砷剂量和作用时间正相关,这一过程可能与三氧化二砷抑制survivin、cyclin D1表达有关。
Objective : To investigat the effect of arsenic trioxide on cell proliferation,cell cycle, cell apoptosis and survivin,cyclin D1 gene expression in lung adenocarcinoma A549 cells. Methods: After A549 were treated with arsenic trioxide in different doses and with different time, MTT was used to detect the proliferation of A549 cells. The Annexin V-FITC /PI double staining analysis by flow cytometry was used to evaluate apoptosis rate. Cell cycle was elaluated by PI staining FCM anlysis. Survivin ,cyclin D1 gene expression was detected by RT-PCR and immunohistochemesitry. Results: Arsenic trioxide could inhibit cell proliferation, induce cell apoptosis and stop cell cycle at G1/S transition in A549 cells. The apoptotic rate was both dose depedent and time dependent. Survivin and cyclin D1 gene expression was down regulated in arsenic trioxide group compared with the control. Conclusion: Arsenic trioxide can effectively inhibit cell proliferatioin and induce apoptosis in lung adenocarcinoma A549 cells,this may result from its actioin of reduce survivin and cyclin D1 gene expression. Arsenic trioxide stop cell cycle at G1 phase in lung adenocarcinoma A549 cells, this may result from its actioin of reduce cyclin D1 gene expression.
引文
[1]Parkin DM,Bray F,Ferlay J,et al.Global cancer statistics,2002[J].CA Cancer J Clin,2005,55(2):74-108.
[2]Chantalat L,Skoufias DA,Kleman J P,et al.Crystal structure of human survivin reveals a bow tie-shaped dimer with two unusual α-helicals extensions[J].Mol Cell,2000,6(1):183-189
[3]Jin M,Inoue S,Umemura T,et al.Cyclin D1,p16 and retinoblastoma gene product expression as a predictor for prognosis in non-small cell lung cancer at stages Ⅰ and Ⅱ[J].Lung Cancer 2001;34:207-218.
[4]曹琦,韦国桢,郭红荣.三氧化二砷诱导恶性肿瘤细胞凋亡机制的研究进展[J].现代肿瘤医学,2007,15(5):731-734.
[5]Escuin D,Rosell R.,The anti-apoptosis survivin gene and its role in human cancer:an overview.[J]Clin Lung Cancer.1999;1(2):138-143.
[6]Altieri DC,Survivin,versatile modulation of cell division and apoptosis in cancer.[J].Oncogene.2003;22(53):8581-8589.
[7]Gautschi O,Hugli B,Ziegler A et al,Cyclin D1(CCND1)A870G gene polymorphism modulates smoking-induced lung cancer risk and response to platinum-based chemotherapy in non-small cell lung cancer(NSCLC)patients.[J].Lung Cancer.2006;51(3):303-311
[8]Liang RY,Liao ZS,Jiang SP,et al,Expression of cyclin D1 and vascular endothelial growth factor(VEGF)in non-small cell lung carcinoma and their association with the prognosis.[J].Ai Zheng. 2003 ;22(1):86-90
[9]Giodini ,kallio MJ, Wall NR,et al. Regulation of microtuble stability and mitotic progression by survivin. [J].Cancer Res,2002;62:2462-2467
[10]Miller WH, Jr., Schipper HM, Lee JS, Singer J, Waxman S. Mechanisms of action of arsenic trioxide [J]. Cancer Res 2002;62:3893-3903.
[11]Synergistic induction of apoptosis by sulindac and arsenic trioxide in human lung cancer A549 cells via reactive oxygen species-dependent down-regulation of survivin. [J].Biochem Pharmacol. 2006 Nov 15;72(10):1228-1236
[12]Ning S, Knox SJ, Optimization of combination therapy of arsenic trioxide and fractionated radiotherapy for malignant glioma. [J].Int J Radiat Oncol Biol Phys. 2006;65(2):493-498
[13]Zaffaroni N, Pennati M, Daidone MG. Survivin as a target for new anticancer interventions. [J].J Cell Mol Med. 2005;9(2):360-372.
[1]Chen GQ,Zhu J,Shi XG,et al.In vitro studies on cellular and molecular mechanisms of arsenic trioxide(ATO)in the treatment of acute promyelocytic leukemia:ATO induces NB4 cell apoptosis with downregulation of Bcl-2 expression and modulation of PML-RAR alpha/PML proteins[J]Blood,1996,88(3):1052-1061.
[2]Hannun YA.Apoptosis and the dilemma of cancer therapy[J].Blood,1997,89(6):1845-1853.
[3]Twomey C,McCarthy JV.Pathways of apoptosis and importance in development[J]J Cell Mol Med,2005,9(2):345-359.
[4]马晓冬,乔东访,田雪梅,等.三氧化二砷诱导线粒体通透性转变孔道开放的机制研究[J].癌症.2006,5(1):17-21.
[5]石玉枝,柳英兰,霍建民等.三氧化二砷诱导小细胞肺癌凋亡及p53、bcl-2基因表达的研究[J].中华结核和呼吸杂志,2002,11,25(11):665-666.
[6]Szegezdi E,Cahill S,Meyer M,et al.TRAIL sensitisation by arsenic trioxide is caspase-8 dependent and involves modulation of death receptor components and Akt[J].British Journal of Cancer,2006,94:398 406.
[7]Shen ZY,Shen NY,Chen MH,et al.,Reactive oxygen species and antioxidants in apoptosis of esophageal cancer cells induced by ATO[J].Int J Mol Med,2003,11:479-484.
[8]Shin S,Sung BJ,Cho YS,et al.An anti-apoptotic protein human Survivin is a direct inhibitor of caspase-3 and -7[J].Bio Chemistry,2001,40(4):1117-1123.
[9]Maeda H,Hori S,Ohizumi H,et al.Effective treatment of advanced solid tumors by the combination of arsenic trioxide and L-buthionine sulfoximine[J].eell Death Differ,2004,11:737 746.
[10]Kajiguchi T,Yamamoto K,Iida S,et al.Sustained activation of c-jun-N-terminal kinase plays a critical role in arsenic trioxide-induced cell apoptosis in multiple myeloma cell lines[J].Cancer Sci,2006,97(6):540 545.
[11]Giafis N,Katsoulidis E,SassanoA,et al.Role of the p38mitogen-activated protein kinase pathway in the generation of arsenic trioxide-dependent cellular responses[J].Cancer Res,2006,66(13):6763-6771.
[12]Kannan-Thulasiraman P,Katsoulidis E,Tallman MS,et al,Activation of the Mitogen-and Stress-activated Kinase 1 by Arsenic Trioxide[J].J Biol Chem,2006,281(32):22446-22452.
[13]Ye J,Li A,Liu Q,et al.Inhibition of mitogen-activated protein kinase kinase enhances apoptosis induced by arsenic trioxide in human breast cancer MCF-7 cells[J].Clin Exp Pharmacol Physiol,2005,32(12):1042-1048.
[14]郭振兴,金洁,ATO对KM3细胞端粒酶及其逆转录酶作用的研究[J].中国实验血液学杂志,2004,12(3):346-349.
[15]Ai Z,Lu W,Qin X,Arsenic trioxide induces gallbladder carcinoma cell apoptosis via downregulation of Bcl-2 [J] .Biochem Biophys Res Commun, 2006 , 348 (3): 1075-1081. Epub 2006 Aug 7.
[16] Zhang Y, Shen WL. Bel-2 antisense oligodeoxynucleotide increases the sensitivity of leukemic cells to arsenic trioxide[J].Cell Bio Int, 2003, 27: 953 958.
[17] Lemarie A, Morzadec C, Merino D, et al. Arsenic trioxide induces apoptosis of human monocytes during macrophagic differentiation through nuclear factor-kappaB-related survival pathway down-regulation [J].J Pharmacol Exp Ther,2006 ,316(1): 304-314.
[18] Wei LH, Lai KP, Chen CA,et al. Asenic trioxide prevents radiation-enhanced tumor invasiveness and inhibits matrix metalloproteinase-9 through downregulation of nuclear factor B[J] Oncogene, 2005,24:390-398.
[19]Michael K, Nuclear factor-κB in cancer development and progression[J]. Nature, 2006, 441 (25): 431-436.
[20]Thomas FF, Christoph PH, Lisa S, et al. PI3K/Akt and apoptosis: size matters [J]. Oncogene, 2003,22:8983 8998.
[21]Au WY, Kumana CR, Lam CW, et al. Solid tumors subsequent to arsenic trioxide treatment for acute promyelocytic leukemia [J]. Leuk Res. 2006, May 23; [Epub ahead of print].
[22] Terek MC, Karabulut B, Selvi N, et al. Arsenic trioxide loaded,microemulsion-enhanced cytotoxicity on MDAH 2774ovarian carcinoma cell line[J].Int J Gynecol Cancer.2006,Mar-Apr,16(2):532-537.
[2]Chantalat L,Skoufias DA,Kleman J P,et al.Crystal structure of human survivin reveals a bow tie-shaped dimer with two unusual α-helicals extensions[J].Mol Cell,2000,6(1):183-189
[3]Jin M,Inoue S,Umemura T,et al.Cyclin D1,p16 and retinoblastoma gene product expression as a predictor for prognosis in non-small cell lung cancer at stages Ⅰ and Ⅱ[J].Lung Cancer 2001;34:207-218.
[4]曹琦,韦国桢,郭红荣.三氧化二砷诱导恶性肿瘤细胞凋亡机制的研究进展[J].现代肿瘤医学,2007,15(5):731-734.
[5]Escuin D,Rosell R.,The anti-apoptosis survivin gene and its role in human cancer:an overview.[J]Clin Lung Cancer.1999;1(2):138-143.
[6]Altieri DC,Survivin,versatile modulation of cell division and apoptosis in cancer.[J].Oncogene.2003;22(53):8581-8589.
[7]Gautschi O,Hugli B,Ziegler A et al,Cyclin D1(CCND1)A870G gene polymorphism modulates smoking-induced lung cancer risk and response to platinum-based chemotherapy in non-small cell lung cancer(NSCLC)patients.[J].Lung Cancer.2006;51(3):303-311
[8]Liang RY,Liao ZS,Jiang SP,et al,Expression of cyclin D1 and vascular endothelial growth factor(VEGF)in non-small cell lung carcinoma and their association with the prognosis.[J].Ai Zheng. 2003 ;22(1):86-90
[9]Giodini ,kallio MJ, Wall NR,et al. Regulation of microtuble stability and mitotic progression by survivin. [J].Cancer Res,2002;62:2462-2467
[10]Miller WH, Jr., Schipper HM, Lee JS, Singer J, Waxman S. Mechanisms of action of arsenic trioxide [J]. Cancer Res 2002;62:3893-3903.
[11]Synergistic induction of apoptosis by sulindac and arsenic trioxide in human lung cancer A549 cells via reactive oxygen species-dependent down-regulation of survivin. [J].Biochem Pharmacol. 2006 Nov 15;72(10):1228-1236
[12]Ning S, Knox SJ, Optimization of combination therapy of arsenic trioxide and fractionated radiotherapy for malignant glioma. [J].Int J Radiat Oncol Biol Phys. 2006;65(2):493-498
[13]Zaffaroni N, Pennati M, Daidone MG. Survivin as a target for new anticancer interventions. [J].J Cell Mol Med. 2005;9(2):360-372.
[1]Chen GQ,Zhu J,Shi XG,et al.In vitro studies on cellular and molecular mechanisms of arsenic trioxide(ATO)in the treatment of acute promyelocytic leukemia:ATO induces NB4 cell apoptosis with downregulation of Bcl-2 expression and modulation of PML-RAR alpha/PML proteins[J]Blood,1996,88(3):1052-1061.
[2]Hannun YA.Apoptosis and the dilemma of cancer therapy[J].Blood,1997,89(6):1845-1853.
[3]Twomey C,McCarthy JV.Pathways of apoptosis and importance in development[J]J Cell Mol Med,2005,9(2):345-359.
[4]马晓冬,乔东访,田雪梅,等.三氧化二砷诱导线粒体通透性转变孔道开放的机制研究[J].癌症.2006,5(1):17-21.
[5]石玉枝,柳英兰,霍建民等.三氧化二砷诱导小细胞肺癌凋亡及p53、bcl-2基因表达的研究[J].中华结核和呼吸杂志,2002,11,25(11):665-666.
[6]Szegezdi E,Cahill S,Meyer M,et al.TRAIL sensitisation by arsenic trioxide is caspase-8 dependent and involves modulation of death receptor components and Akt[J].British Journal of Cancer,2006,94:398 406.
[7]Shen ZY,Shen NY,Chen MH,et al.,Reactive oxygen species and antioxidants in apoptosis of esophageal cancer cells induced by ATO[J].Int J Mol Med,2003,11:479-484.
[8]Shin S,Sung BJ,Cho YS,et al.An anti-apoptotic protein human Survivin is a direct inhibitor of caspase-3 and -7[J].Bio Chemistry,2001,40(4):1117-1123.
[9]Maeda H,Hori S,Ohizumi H,et al.Effective treatment of advanced solid tumors by the combination of arsenic trioxide and L-buthionine sulfoximine[J].eell Death Differ,2004,11:737 746.
[10]Kajiguchi T,Yamamoto K,Iida S,et al.Sustained activation of c-jun-N-terminal kinase plays a critical role in arsenic trioxide-induced cell apoptosis in multiple myeloma cell lines[J].Cancer Sci,2006,97(6):540 545.
[11]Giafis N,Katsoulidis E,SassanoA,et al.Role of the p38mitogen-activated protein kinase pathway in the generation of arsenic trioxide-dependent cellular responses[J].Cancer Res,2006,66(13):6763-6771.
[12]Kannan-Thulasiraman P,Katsoulidis E,Tallman MS,et al,Activation of the Mitogen-and Stress-activated Kinase 1 by Arsenic Trioxide[J].J Biol Chem,2006,281(32):22446-22452.
[13]Ye J,Li A,Liu Q,et al.Inhibition of mitogen-activated protein kinase kinase enhances apoptosis induced by arsenic trioxide in human breast cancer MCF-7 cells[J].Clin Exp Pharmacol Physiol,2005,32(12):1042-1048.
[14]郭振兴,金洁,ATO对KM3细胞端粒酶及其逆转录酶作用的研究[J].中国实验血液学杂志,2004,12(3):346-349.
[15]Ai Z,Lu W,Qin X,Arsenic trioxide induces gallbladder carcinoma cell apoptosis via downregulation of Bcl-2 [J] .Biochem Biophys Res Commun, 2006 , 348 (3): 1075-1081. Epub 2006 Aug 7.
[16] Zhang Y, Shen WL. Bel-2 antisense oligodeoxynucleotide increases the sensitivity of leukemic cells to arsenic trioxide[J].Cell Bio Int, 2003, 27: 953 958.
[17] Lemarie A, Morzadec C, Merino D, et al. Arsenic trioxide induces apoptosis of human monocytes during macrophagic differentiation through nuclear factor-kappaB-related survival pathway down-regulation [J].J Pharmacol Exp Ther,2006 ,316(1): 304-314.
[18] Wei LH, Lai KP, Chen CA,et al. Asenic trioxide prevents radiation-enhanced tumor invasiveness and inhibits matrix metalloproteinase-9 through downregulation of nuclear factor B[J] Oncogene, 2005,24:390-398.
[19]Michael K, Nuclear factor-κB in cancer development and progression[J]. Nature, 2006, 441 (25): 431-436.
[20]Thomas FF, Christoph PH, Lisa S, et al. PI3K/Akt and apoptosis: size matters [J]. Oncogene, 2003,22:8983 8998.
[21]Au WY, Kumana CR, Lam CW, et al. Solid tumors subsequent to arsenic trioxide treatment for acute promyelocytic leukemia [J]. Leuk Res. 2006, May 23; [Epub ahead of print].
[22] Terek MC, Karabulut B, Selvi N, et al. Arsenic trioxide loaded,microemulsion-enhanced cytotoxicity on MDAH 2774ovarian carcinoma cell line[J].Int J Gynecol Cancer.2006,Mar-Apr,16(2):532-537.
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