顺铂和阿霉素诱导U2骨肉瘤细胞周期阻滞及凋亡通路的研究
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摘要
目的:探讨化疗药顺铂和阿霉素损伤人U2骨肉瘤细胞(U2-OS)后p53、bcl-2、c-myc基因表达情况的变化及其诱导细胞周期阻滞及凋亡的情况,为骨肉瘤化疗耐药的解决提供理论依据。
方法:分别使用0.3、3.0、30μg/ml顺铂、0.06、0.6、6.0μg/ml阿霉素作用于U2-OS细胞2h后继续培养0、6、12、24、48h,采用噻唑兰比色法(MTT法)检测U2-OS细胞生长抑制率。流式细胞术、逆转录-聚合酶链反应(RT-PCR)、蛋白免疫印迹(Western Blot)检测顺铂和阿霉素损伤后继续培养6、24h后细胞细胞周期变化、凋亡率及p53、bcl-2、c-myc基因表达情况。
结果:不同浓度顺铂、阿霉素损伤U2-OS细胞后继续培养6h,流式细胞术检测各组G1期细胞分别为67.97%、79.64%、78.09%、80.21%、78.25℅、79.13℅、79.92℅,而p53基因mRNA和蛋白表达和空白对照组比较均增加,bcl-2、c-myc的表达和空白对照组比较无变化,细胞生长抑制率无明显统计学差异;继续培养24h,②、③、④、⑤、⑥、⑦组细胞生长抑制率分别为31.58%、44.12%、59.46%、23.40%、40.31%、57.56%,②、③、④组之间,⑤、⑥、⑦组之间有明显组间差异;流式细胞术检测④、⑦组细胞凋亡率分别为10.04%、14.55%,细胞周期以G2期细胞为主。②、③、④组p53、bcl-2和c-myc mRNA及蛋白表达有组间差异,⑤、⑥、⑦组p53、bcl-2和c-myc mRNA及蛋白表达有组间差异。继续培养48h,各组细胞生长抑制率增高。
结论:不同浓度的顺铂和阿霉素短时间(2h)损伤U2-OS细胞仍有细胞增殖抑制作用,这种作用与细胞DNA损伤后p53、bcl-2、c-myc基因表达变化有关;U2-OS细胞DNA损伤后6h即表现出明显的p53基因的表达增强并诱导G1期细胞周期阻滞;继续培养24h,p53、bcl-2、c-myc基因表达变化与药物浓度有关联且诱导G2细胞周期阻滞及凋亡发生。
Objective: To investigate the expression of p53﹑bcl-2 and c-myc and its effects on cell cycle arrest and apoptosis induced by cisplatin and adriamycin, the chemotherapeutics inU2-OS cells.
Methods:After the treatment of 0.3μg/ml, 3.0μg/ml, 30.0μg/ml cisplatin and 0.06μg/ml, 0.6μg/ml, 6.0μg/ml adriamycin for 2h, we keep on cultivate U2-OS cells with fresh medium in 0, 6, 12, 24, 48h. Then MTT were used to evaluate cell proliferation in vitro. The expression of wild-type p53, bcl-2,c-myc were detected by RT-PCR and Western blot, Flow Cytometry (FCM) was employed for examining the cell cycle after cultivate for 6h, 24h.
Results: After damage by different dencity of cisplatin and adriamycin, U2-OS cell were cultivated for 6h, FCM show that cell in G1 phase were incresed obviously, the ratio were 67.97%, 79.64%, 78.09%, 80.21%, 78.25℅, 79.13℅ and 79.92℅.The expression of p53 were up-regulate, bcl-2 and c-myc expression were unchangeable, the inhibite of cell grow were not manifest in statistics. After cultivate for 24h, the inhibitory rate of cell growth of group②③④⑤⑥⑦were 31.58%, 44.12%, 59.46%, 23.40%, 40.31% and 57.56%. Group②③④were different in statistics, Group⑤⑥⑦were different in statistics by contrast with each other. The ratio of apoptosis by FCM were 10.04%, 14.55%, cell cycle arrest in G2 phase. The expression of p53, bcl-2, c-myc was different obviously in statistics of group②③④, the expression of p53, bcl-2, c-myc was different obviously in statistics of group⑤⑥⑦. After cultivate for 48h, the inhibitory rate of cell growth was increased..
Conclusions:The U2-OS cells were damaged by different dencity of cisplatin and adriamycin in short time(2h), the inhibition of cell grow was also manifest. The effection were related with the diversify of the expression of p53, bcl-2, c-myc after DNA damage. The diversify of p53 induced G1 cell cycle arrest after 6h, and after 24h, the diversify of p53, bcl-2, c-myc induced G2 cell cycle arrest and apoptosis, the change was related with drug concentration.
方法:分别使用0.3、3.0、30μg/ml顺铂、0.06、0.6、6.0μg/ml阿霉素作用于U2-OS细胞2h后继续培养0、6、12、24、48h,采用噻唑兰比色法(MTT法)检测U2-OS细胞生长抑制率。流式细胞术、逆转录-聚合酶链反应(RT-PCR)、蛋白免疫印迹(Western Blot)检测顺铂和阿霉素损伤后继续培养6、24h后细胞细胞周期变化、凋亡率及p53、bcl-2、c-myc基因表达情况。
结果:不同浓度顺铂、阿霉素损伤U2-OS细胞后继续培养6h,流式细胞术检测各组G1期细胞分别为67.97%、79.64%、78.09%、80.21%、78.25℅、79.13℅、79.92℅,而p53基因mRNA和蛋白表达和空白对照组比较均增加,bcl-2、c-myc的表达和空白对照组比较无变化,细胞生长抑制率无明显统计学差异;继续培养24h,②、③、④、⑤、⑥、⑦组细胞生长抑制率分别为31.58%、44.12%、59.46%、23.40%、40.31%、57.56%,②、③、④组之间,⑤、⑥、⑦组之间有明显组间差异;流式细胞术检测④、⑦组细胞凋亡率分别为10.04%、14.55%,细胞周期以G2期细胞为主。②、③、④组p53、bcl-2和c-myc mRNA及蛋白表达有组间差异,⑤、⑥、⑦组p53、bcl-2和c-myc mRNA及蛋白表达有组间差异。继续培养48h,各组细胞生长抑制率增高。
结论:不同浓度的顺铂和阿霉素短时间(2h)损伤U2-OS细胞仍有细胞增殖抑制作用,这种作用与细胞DNA损伤后p53、bcl-2、c-myc基因表达变化有关;U2-OS细胞DNA损伤后6h即表现出明显的p53基因的表达增强并诱导G1期细胞周期阻滞;继续培养24h,p53、bcl-2、c-myc基因表达变化与药物浓度有关联且诱导G2细胞周期阻滞及凋亡发生。
Objective: To investigate the expression of p53﹑bcl-2 and c-myc and its effects on cell cycle arrest and apoptosis induced by cisplatin and adriamycin, the chemotherapeutics inU2-OS cells.
Methods:After the treatment of 0.3μg/ml, 3.0μg/ml, 30.0μg/ml cisplatin and 0.06μg/ml, 0.6μg/ml, 6.0μg/ml adriamycin for 2h, we keep on cultivate U2-OS cells with fresh medium in 0, 6, 12, 24, 48h. Then MTT were used to evaluate cell proliferation in vitro. The expression of wild-type p53, bcl-2,c-myc were detected by RT-PCR and Western blot, Flow Cytometry (FCM) was employed for examining the cell cycle after cultivate for 6h, 24h.
Results: After damage by different dencity of cisplatin and adriamycin, U2-OS cell were cultivated for 6h, FCM show that cell in G1 phase were incresed obviously, the ratio were 67.97%, 79.64%, 78.09%, 80.21%, 78.25℅, 79.13℅ and 79.92℅.The expression of p53 were up-regulate, bcl-2 and c-myc expression were unchangeable, the inhibite of cell grow were not manifest in statistics. After cultivate for 24h, the inhibitory rate of cell growth of group②③④⑤⑥⑦were 31.58%, 44.12%, 59.46%, 23.40%, 40.31% and 57.56%. Group②③④were different in statistics, Group⑤⑥⑦were different in statistics by contrast with each other. The ratio of apoptosis by FCM were 10.04%, 14.55%, cell cycle arrest in G2 phase. The expression of p53, bcl-2, c-myc was different obviously in statistics of group②③④, the expression of p53, bcl-2, c-myc was different obviously in statistics of group⑤⑥⑦. After cultivate for 48h, the inhibitory rate of cell growth was increased..
Conclusions:The U2-OS cells were damaged by different dencity of cisplatin and adriamycin in short time(2h), the inhibition of cell grow was also manifest. The effection were related with the diversify of the expression of p53, bcl-2, c-myc after DNA damage. The diversify of p53 induced G1 cell cycle arrest after 6h, and after 24h, the diversify of p53, bcl-2, c-myc induced G2 cell cycle arrest and apoptosis, the change was related with drug concentration.
引文
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[6] Kaufmann SH, Desnoyers S, Ottaviano Y,et al. Specific proteolytic cleavage of poly (ADP-ribose) polymerase: an early marker of chemotherapy-induced apoptosis. [J]Cancer Res. 1993, 53(17):3976-85.
[7] Merk O,Speit G. Detection of crosslinks with the comet assay in relationship to genotoxicity and cytotoxicity. [J]Environ Mol Mutagen.1999,33(2):167-72.
[8] Tarasov V, Jung P, Verdoodt B, et al. Differential Regulation of MicroRNAs by P53 Revealed by Massively Parallel Sequencing: miR-34a is a P53 Target that Induced Apoptosis and G1-arrest. [J]Cell cycle, 2007, 6(13):1586-93.
[9] L Gatti,R Supino,P Perego,et al. Apoptosis and growth arrest induced by platinum compounds in U2-OS cells reflect a specific DNA damage recognition associated with a different p53-mediated response. [J]Cell Death Differ.2002,9(12):1352-9.
[10] Napamatsu K, Tsuchiya F, Oquma K, et al.The Diffect of Small Interfering RNA (siRNA) against the Bcl-2 Gene on Apoptosis and Chemosensitivity in a Caninemammary Gland Tumor Cell Line. [J]Res Wet Sci, 2008, 84(1): 49-55.
[11] Mo H, Vita M, Crespin M, et al.Myc Overexpression Enhances Apoptosis Induced by Small Molecules. [J]Cell Cycle, 2006, 5(19):2191-94.
[12] Roth MJ, Tanese N, Goff SP. Purification and characterization of murine retroviral reverse transciptase expressed in Escherichia coli. [J]J Biol Chem, 1985;5(260): 9326-35.
[13] Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989;1(8): 64-97.
[14] Tang Y, Li GD. Chronic exposure to high glucose impairs bradykinin-stimulated nitric oxide production by interfering woth the phospholipase-C-implicated signaling pathway in endothelial cells: evidence for the involvement of protein kinase C. [J]Diabetologia,2004;7(47): 2093-104.
[15] Li J, Luo R, Kowluru A, et al. Novel regulation by Racl of glucose and forskolin-induced insulin secretin in INS-1 beta-cell. [J]Am J Physiol Endocrinol Metab, 2004; 3(286): E818-27.
[16] Wynne P, Newton C, Ledermann JA, et al. Enhanced Repair of DNA Interstrand Crosslinking in Ovarian Cancer Cells from Patients following Treatment with Platinum-based Chemotherapy. [J]Br J Cancer, 2007, 97(7):927-33.
[17] Tewey KM, Rowe TC, Yang L, Halligan BD and Liu LF: Adriamycin-induced DNA damage mediated by mammalian DNA topoisomerase II. [J]Science 1984; 226(4673): 466-8.
[18] Wendt J, Radetzki S, vov Haefen C, et al.Induction of P21CIP/WAF-1 and G2 Arrest by Ionizing Irradiaton Impedes Caspase-3 Mediated Apoptosis in Human Carcinoma Cells. [J]Oncogene, 2006, 25(7): 972-80.
[19] Sharma V, Hupp CD, Tepe JJ. Enhancement of Chemotherapeutic Efficacy by Small Molecule Inhibition of NF-kappaB and Chechpiunt Kinases.[J]Curr Med Chem, 2007, 14(10): 1061-74.
[20] Papai Z,Feja LN,Hanna EN et al.P53 Overexpression as an Indicator of Overall Survival and Response to Treatment in Osteosarcomas. [J]Pathol Oncol Res.1997; 3(1):15-19
[21] Song SU, Boyce FM.Cobination treatment for osteosarcoma with baculoviral vector mediat- ed gene therapy (p53) and chemotherapy (adriamycin). [J]J Exp Mol Med.2001, 33: 46-53
[22] Tsuchiya H, Mori Y, Ueda Y, et al. Sensitization and caffeine potentiation of cispliatin cytotoxity resulting from introduction of wild-type p53 gene in human osteosacoma. [J] J Aniticancer Res, 2000, 20(1A):235-42.
[23] Brajuskovic G, Orolicki SV, Cerovic S, et al. Bcl-2 and Bax protein interaction in B-lympho- cytes of peripheral blood in patients with chronic lymphocytic leukemia. [J] Vojnosanit Preql. 2005; 62(5): 357-63.
[24] Mo H, Vita M, Crespin M, et al.Myc Overexpression Enhances Apoptosis Induced by Small Molecules. [J] Cell Cycle, 2006, 5(19): 2191-94.
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1 Hayden JB, Hoang BH. Osteosarcoma: basic science and clinical implications. Orthop Clin North Am, 2006; 37(1):1-7
2 Carrle D, Bielack SS. Current strategies of chemotherapy in osteosarcoma. Int Orthop, 2006; 30(6):445-451
3 Wong RP, Tsang WP, Chau PY, et al. p53-R273H gains new function in induction of drug resistance through down-regμlation of procaspase-3. Mol Cancer Ther, 2007; 6(3):1054-1061
4 Tarasov V, Jung P, Verdoodt B, et al. Differential regμlation of microRNAs by p53 revealed by massively parallel sequencing: miR-34a is a p53 target that induces apoptosis and G1-arrest. Cell Cycle, 2007; 6(13):1586-1593
5 Papai Z, Feja CN, Hanna EN, et al. P53 overexpression as an indicator of overall survival and response to treatment in osteosarcomas. Pathol Oncol Res, 1997; 3(1):15-19
6 Zuffa E, Mancini M, Brusa G, et al. P53 oncosuppressor influences selection of genomic imbalances in response to ionizing radiations in human osteosarcoma cell line SAOS-2. Int J Radiat Biol, 2008; 84(7):591-601
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[2] Hayden JB,Hoang BH.Osteosarcoma: Basic Science and Clinical Implications. [J]Orthop Clin North Am. 2006, 37(1):1-7.
[3] Carrle D,Bielack SS. Current strategies of chemotherapy in osteosarcoma. [J]Int Orthop. 2006, 30(6):445-51.
[4] Kalifa C, Brugières L, Le Deley MC. Neoadjuvant treatment in osteosarcomas. [J]Bull Cancer.2006, 93(11):1115-20.
[5] Wong RP,Tsang WP,Chau PY,et al. p53-R273H gains new function in induction of drug resistance through down-regulation of procaspase-3. [J]Mol Cancer Ther. 2007, 6(3):1054-61.
[6] Kaufmann SH, Desnoyers S, Ottaviano Y,et al. Specific proteolytic cleavage of poly (ADP-ribose) polymerase: an early marker of chemotherapy-induced apoptosis. [J]Cancer Res. 1993, 53(17):3976-85.
[7] Merk O,Speit G. Detection of crosslinks with the comet assay in relationship to genotoxicity and cytotoxicity. [J]Environ Mol Mutagen.1999,33(2):167-72.
[8] Tarasov V, Jung P, Verdoodt B, et al. Differential Regulation of MicroRNAs by P53 Revealed by Massively Parallel Sequencing: miR-34a is a P53 Target that Induced Apoptosis and G1-arrest. [J]Cell cycle, 2007, 6(13):1586-93.
[9] L Gatti,R Supino,P Perego,et al. Apoptosis and growth arrest induced by platinum compounds in U2-OS cells reflect a specific DNA damage recognition associated with a different p53-mediated response. [J]Cell Death Differ.2002,9(12):1352-9.
[10] Napamatsu K, Tsuchiya F, Oquma K, et al.The Diffect of Small Interfering RNA (siRNA) against the Bcl-2 Gene on Apoptosis and Chemosensitivity in a Caninemammary Gland Tumor Cell Line. [J]Res Wet Sci, 2008, 84(1): 49-55.
[11] Mo H, Vita M, Crespin M, et al.Myc Overexpression Enhances Apoptosis Induced by Small Molecules. [J]Cell Cycle, 2006, 5(19):2191-94.
[12] Roth MJ, Tanese N, Goff SP. Purification and characterization of murine retroviral reverse transciptase expressed in Escherichia coli. [J]J Biol Chem, 1985;5(260): 9326-35.
[13] Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989;1(8): 64-97.
[14] Tang Y, Li GD. Chronic exposure to high glucose impairs bradykinin-stimulated nitric oxide production by interfering woth the phospholipase-C-implicated signaling pathway in endothelial cells: evidence for the involvement of protein kinase C. [J]Diabetologia,2004;7(47): 2093-104.
[15] Li J, Luo R, Kowluru A, et al. Novel regulation by Racl of glucose and forskolin-induced insulin secretin in INS-1 beta-cell. [J]Am J Physiol Endocrinol Metab, 2004; 3(286): E818-27.
[16] Wynne P, Newton C, Ledermann JA, et al. Enhanced Repair of DNA Interstrand Crosslinking in Ovarian Cancer Cells from Patients following Treatment with Platinum-based Chemotherapy. [J]Br J Cancer, 2007, 97(7):927-33.
[17] Tewey KM, Rowe TC, Yang L, Halligan BD and Liu LF: Adriamycin-induced DNA damage mediated by mammalian DNA topoisomerase II. [J]Science 1984; 226(4673): 466-8.
[18] Wendt J, Radetzki S, vov Haefen C, et al.Induction of P21CIP/WAF-1 and G2 Arrest by Ionizing Irradiaton Impedes Caspase-3 Mediated Apoptosis in Human Carcinoma Cells. [J]Oncogene, 2006, 25(7): 972-80.
[19] Sharma V, Hupp CD, Tepe JJ. Enhancement of Chemotherapeutic Efficacy by Small Molecule Inhibition of NF-kappaB and Chechpiunt Kinases.[J]Curr Med Chem, 2007, 14(10): 1061-74.
[20] Papai Z,Feja LN,Hanna EN et al.P53 Overexpression as an Indicator of Overall Survival and Response to Treatment in Osteosarcomas. [J]Pathol Oncol Res.1997; 3(1):15-19
[21] Song SU, Boyce FM.Cobination treatment for osteosarcoma with baculoviral vector mediat- ed gene therapy (p53) and chemotherapy (adriamycin). [J]J Exp Mol Med.2001, 33: 46-53
[22] Tsuchiya H, Mori Y, Ueda Y, et al. Sensitization and caffeine potentiation of cispliatin cytotoxity resulting from introduction of wild-type p53 gene in human osteosacoma. [J] J Aniticancer Res, 2000, 20(1A):235-42.
[23] Brajuskovic G, Orolicki SV, Cerovic S, et al. Bcl-2 and Bax protein interaction in B-lympho- cytes of peripheral blood in patients with chronic lymphocytic leukemia. [J] Vojnosanit Preql. 2005; 62(5): 357-63.
[24] Mo H, Vita M, Crespin M, et al.Myc Overexpression Enhances Apoptosis Induced by Small Molecules. [J] Cell Cycle, 2006, 5(19): 2191-94.
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