微量元素铜对人口腔癌细胞的影响
摘要
背景:恶性肿瘤严重威胁着人类的生命与健康,临床上常用的治疗方法是手术,放射治疗,化学药物治疗或这几种方法的综合治疗。手术与放疗对肿瘤远处转移的癌细胞作用甚微,化疗虽可以杀伤肿瘤细胞,但受其耐药性所限,常导致肿瘤复发。铜是真核细胞中必须的金属元素,与肿瘤密切相关,研究发现很多类型的肿瘤患者的血清中含有异常高水平的铜,而且在化疗过程中同样伴随着铜水平的变化;近年来的研究又发现铜与化疗药物顺铂(DDP)关系密切,铜的吸收载体CTR1,运输载体ATP7B 也介导DDP 的运输,提示在化疗中引入铜后可能影响化疗疗效,但肿瘤发生过程中铜在体内分布和代谢的异常对原发肿瘤患者化疗疗效的影响未见报道。
目的:本实验通过药物筛选,检测细胞凋亡等技术手段从细胞水平研究铜对DDP 介导的口腔癌细胞化疗反应的影响及其可能机理,为肿瘤临床化疗提供新的科学依据和启示,开辟新的治疗方案。此外,本实验还利用绿色荧光蛋白(GFP)作报告基因,积极开创一种新型的快速高效的研究药物处理后的细胞生存能力的方法。
方法:以口腔恶性肿瘤化疗药物顺铂(DDP)为例,采用Sigma公司生产的醋酸铜,以不同组织来源的口腔肿瘤细胞株作为研究对象(包括Tca8113 细胞,来源于人舌部;Acc-2,Acc-3 细胞,来源于人涎腺)。采用MTT 法观察铜对肿瘤细胞的影响,利用流式细胞术分析铜对DDP 介导的细胞凋亡和细胞周期的影响;以GFP 为代表,采用
Background: Malignant tumors seriously hurt human health. Surgery, radiotherapy, chemotherapy or combination with these several methods is ordinary therapy in clinic. Surgery and radiotherapy have little effect on metastatic cells. Because of drug resistance, chemotherapy often cause tumor recur. As an essential metal element of eukaryotic organisms, copper has close realation with tumors. It has been identified that many tumor patients have high serum copper and abnormality of copper metabolism and distribution in the prosess of tumor chemotherapy. It is also found that copper has close realation with DDP: the influx transporter of copper CTR1 and the enflux transporter of copper ATP7B also transport DDP. While The realationship between fluctucation of copper concentration in blood and chemotherapeutic responses remains unclear in patients with primary or acquired drug resistant tumor.
Object: The aim of our study was to determine the influence of copper on cytotoxicity of tumor cells at the cell lever by a series of tests and search a new method to solve drug resistance of DDP. Our study also tried to build a new method to analyze the influence of drugs on cells continuously fast and accurately useing a modern reporter gene (GFP).
Method: DDP was adopted to study the influence of copper on cytotoxicity of oral tumor cells (Tca8113 cell comes from human tongue, Acc-2 and Acc-3 cell come from human salivary) in the experiment. With
MTT assay, copper acetate was used to investigate the cytotoxicity of copper on different origin of oral tumor cell lines including. The flow cytometric analysis was used to detect the influence of copper on apoptosis of tumor cells and cell cycle. Through liposome transfection to transfect the combinant plasmid (HSP/GFP) into Tca8113 and Acc-3 cells and used G418 to select clone stably expressing GFP genes.
Resulte: MTT showed that in presence of 20～400μM copper acetate, the proliferation inhibition of three tumor cells was significantly enhanced. In presence of 25μM copper acetate combined with various doses of DDP, the result showed that copper acetate enhanced the proliferation inhibition of Tca8113 cell and Acc-2 cell than that exposed to DDP alone; as to Acc-3 cell, copper acetate enhanced the proliferation inhibition of cell exposed to less than 25μM DDP and decreases exposed to more than 25μM DDPcompared with rqual dose of DDP.In presence of 25μM copper acetate combined with various doses of DDP, the IC50 of Tca8113 cell was 2.72±0.38 μM/L, it is much lower than that treated with DDP alone which was 10.84±1.02 μM/L. While for Acc-2 and Acc-3 cells, the IC50 of them exposed to 25μM copper acetate combined with various doses of DDP were only a little lower than that treated with DDP alone, which changed from 5.2±1.3 μM/L and 5.12±0.38 μM/L to 6.88±1.02 μM/L and 7.37±0.71 μM/L. The flow cytometric analysis was used to detect the influence of copper on apoptosis of tumor cells and cell cycle. The result showed that 25μM copper acetate increased the apoptosis of cells exposed to 7.5μM DDP, while decreased the apoptosis under 25μM DDP slightly; it was also showed that 25μM copper acetate slowed down tumor cell to move to S phase combined with 7.5μM DDP while increased tumor cell to move to S phase combined with 25μM DDP. HSP/GFP can be transfected into tumor cells. The transfected cells were observed green fluorescent after 42oC heat shocked for 3h and the transfected efficiency and fluorescent were more enhanced overnight.
The green fluorescent could maintain 4 days. The clone stably expressing GFP could be obtained by G418 selection for 40 days.
Conclusion: Copper enhanced the proliferation inhibition of low dose DDP influence tumor cells; low dose of copper increased apoptosis of tumor cells exposed to DDP and change cell cycle of tumor cells exposed to DDP. Using G418 to select the transfected cells could obtain a new cell line that can contribute to analyze the influence of drugs on cells continuously fast and accurately.
目的:本实验通过药物筛选,检测细胞凋亡等技术手段从细胞水平研究铜对DDP 介导的口腔癌细胞化疗反应的影响及其可能机理,为肿瘤临床化疗提供新的科学依据和启示,开辟新的治疗方案。此外,本实验还利用绿色荧光蛋白(GFP)作报告基因,积极开创一种新型的快速高效的研究药物处理后的细胞生存能力的方法。
方法:以口腔恶性肿瘤化疗药物顺铂(DDP)为例,采用Sigma公司生产的醋酸铜,以不同组织来源的口腔肿瘤细胞株作为研究对象(包括Tca8113 细胞,来源于人舌部;Acc-2,Acc-3 细胞,来源于人涎腺)。采用MTT 法观察铜对肿瘤细胞的影响,利用流式细胞术分析铜对DDP 介导的细胞凋亡和细胞周期的影响;以GFP 为代表,采用
Background: Malignant tumors seriously hurt human health. Surgery, radiotherapy, chemotherapy or combination with these several methods is ordinary therapy in clinic. Surgery and radiotherapy have little effect on metastatic cells. Because of drug resistance, chemotherapy often cause tumor recur. As an essential metal element of eukaryotic organisms, copper has close realation with tumors. It has been identified that many tumor patients have high serum copper and abnormality of copper metabolism and distribution in the prosess of tumor chemotherapy. It is also found that copper has close realation with DDP: the influx transporter of copper CTR1 and the enflux transporter of copper ATP7B also transport DDP. While The realationship between fluctucation of copper concentration in blood and chemotherapeutic responses remains unclear in patients with primary or acquired drug resistant tumor.
Object: The aim of our study was to determine the influence of copper on cytotoxicity of tumor cells at the cell lever by a series of tests and search a new method to solve drug resistance of DDP. Our study also tried to build a new method to analyze the influence of drugs on cells continuously fast and accurately useing a modern reporter gene (GFP).
Method: DDP was adopted to study the influence of copper on cytotoxicity of oral tumor cells (Tca8113 cell comes from human tongue, Acc-2 and Acc-3 cell come from human salivary) in the experiment. With
MTT assay, copper acetate was used to investigate the cytotoxicity of copper on different origin of oral tumor cell lines including. The flow cytometric analysis was used to detect the influence of copper on apoptosis of tumor cells and cell cycle. Through liposome transfection to transfect the combinant plasmid (HSP/GFP) into Tca8113 and Acc-3 cells and used G418 to select clone stably expressing GFP genes.
Resulte: MTT showed that in presence of 20～400μM copper acetate, the proliferation inhibition of three tumor cells was significantly enhanced. In presence of 25μM copper acetate combined with various doses of DDP, the result showed that copper acetate enhanced the proliferation inhibition of Tca8113 cell and Acc-2 cell than that exposed to DDP alone; as to Acc-3 cell, copper acetate enhanced the proliferation inhibition of cell exposed to less than 25μM DDP and decreases exposed to more than 25μM DDPcompared with rqual dose of DDP.In presence of 25μM copper acetate combined with various doses of DDP, the IC50 of Tca8113 cell was 2.72±0.38 μM/L, it is much lower than that treated with DDP alone which was 10.84±1.02 μM/L. While for Acc-2 and Acc-3 cells, the IC50 of them exposed to 25μM copper acetate combined with various doses of DDP were only a little lower than that treated with DDP alone, which changed from 5.2±1.3 μM/L and 5.12±0.38 μM/L to 6.88±1.02 μM/L and 7.37±0.71 μM/L. The flow cytometric analysis was used to detect the influence of copper on apoptosis of tumor cells and cell cycle. The result showed that 25μM copper acetate increased the apoptosis of cells exposed to 7.5μM DDP, while decreased the apoptosis under 25μM DDP slightly; it was also showed that 25μM copper acetate slowed down tumor cell to move to S phase combined with 7.5μM DDP while increased tumor cell to move to S phase combined with 25μM DDP. HSP/GFP can be transfected into tumor cells. The transfected cells were observed green fluorescent after 42oC heat shocked for 3h and the transfected efficiency and fluorescent were more enhanced overnight.
The green fluorescent could maintain 4 days. The clone stably expressing GFP could be obtained by G418 selection for 40 days.
Conclusion: Copper enhanced the proliferation inhibition of low dose DDP influence tumor cells; low dose of copper increased apoptosis of tumor cells exposed to DDP and change cell cycle of tumor cells exposed to DDP. Using G418 to select the transfected cells could obtain a new cell line that can contribute to analyze the influence of drugs on cells continuously fast and accurately.
引文
1. Rosenberg b, Vancamp L and Krigas T. Inhibition of cell division in Escherichia coli by electrolysis products from a palatinum electrode. Nature, 1965, 205: 698-699
2. Loehrer PJ and Einhorn LH. Drugs five years later cisplatin. Anc Internal Med, 1984,100:704-713
3. Mese H, Sasaki A, Alcalde RE, et al. Chemotherapy, 1998,44:414-420
4. Nakagawa M, Nomura Y, Kohno K, et al. Reduction of drug accumulation in cisplatin-resistant variants of human prostatic cancer PC-3 cell line. J Urol, 1993,150:1970-1973
5. Fujli R,Mutoh M,Sumizawa T et al. Adenosine triphoshate-dependent transport of leukotriants C4 by membrane vesicles prepared from cisplatin-resistant human epidermoid carcinoma tumor cells. J Natl Cancer Inst, 1994,86:1781-1784
6. Tew KD. Glutathione-associated enzymes in anticancer drug resistance. Cancer Res, 1994,54:4313-4320
7. Kelly SL, Basu A, Teicher BA, et al. Overexpression of metallothionein confers resistance to anticancer drugs. Science, 1988,241:1813-1815
8. Yokomizo A, Ono M, Nanri H, et al. Cellular levers of thioredoxin associated with drug sensitivity to cisplatin,mitomycin C,doxorubicin and etoposide. Cancer Res, 1995,55:4293-4296
9. Cohen SM, Zamble DB and Lippard SJ. Tends Biochem Sci, 1995, 20: 435-439
10. Chu G and Chang E. Cisplatin-resistance cells express increased levels of a factor that recognizes damaged DNA. Proc Natl Acad Sci.USA, 1990,87: 3323-3328
11. Morton KO. Role of trace elements in cancer. Cancer Res, 1975,35:3475-3470
12. Cole SP. Rapid chemosensitivity testing of human lung tumor cells using the MTT assay. Cancer Chemother Pharmacol, 1986,17(3):259-263
13. Rossi L, Marchese E, Lombardo MF, et al. Increased susceptibility of copper-deficient neuroblastoma cells to oxidative stress-mediated apoptosis. Free Radical Biology & Medicine, 2001 May 15; 30(10):1177-1187
14. Darzynkiewicz Z, Bruno S, Del Bino G, et al. Features of apoptotic cells measured by flow cytometry. Cytometry, 1992,13(8):795-808
15. Gately DP and Howell SB. Cellular accumulation of anticancer agent cisplatin: a review. Br J Cancer, 1993, Jun; 67(6): 1171-1176.
16. Nicholson DL, Purser SM, Maier RH. Differential cytotoxicity of trace metals in cisplatin-sensitive and -resistant human ovarian cancer cells. Biometals, 1998, Sep; 11(3): 259-263
17. Komatsu M, Sumizawa T, Mutoh M, et al. Copper-transporting P-type adenosine triphosphatase ATP7B) is associated with cisplatin risistance. Cancer Res, 2000 Mar 1; 60(5)1312-1316
18. Nakayama K, Miyazaki K, Kanzaki A, et al. Expression and cisplatinsensitivity of copper-transporting P-type adenosine triphosphatase (ATP7B) in human solid carcinoma cell lines. Oncol Rep, 2001 Nov-Dec,8(6) 1285-1287
19. Ishida S, Lee J, Thicle DJ, et al. Uptake of the anticancer drug cisplatin mediated by the copper transporter Ctr1 in yeast and mammals. Proc Natl Acad Sci U S A, 2002 Oct 29; 99(22): 14298-14302
20. Samali A and Cotter TG. Heat shock proteins increase resistant to apoptosis. Exp Cell Res, 1996,223:163-170
21. Em Greagh, D Sheehan and TG Cotter. Heat shock proteins modulators of apoptosis in tumor cells. Leukemia, 2000,14:1161-1173
2. Loehrer PJ and Einhorn LH. Drugs five years later cisplatin. Anc Internal Med, 1984,100:704-713
3. Mese H, Sasaki A, Alcalde RE, et al. Chemotherapy, 1998,44:414-420
4. Nakagawa M, Nomura Y, Kohno K, et al. Reduction of drug accumulation in cisplatin-resistant variants of human prostatic cancer PC-3 cell line. J Urol, 1993,150:1970-1973
5. Fujli R,Mutoh M,Sumizawa T et al. Adenosine triphoshate-dependent transport of leukotriants C4 by membrane vesicles prepared from cisplatin-resistant human epidermoid carcinoma tumor cells. J Natl Cancer Inst, 1994,86:1781-1784
6. Tew KD. Glutathione-associated enzymes in anticancer drug resistance. Cancer Res, 1994,54:4313-4320
7. Kelly SL, Basu A, Teicher BA, et al. Overexpression of metallothionein confers resistance to anticancer drugs. Science, 1988,241:1813-1815
8. Yokomizo A, Ono M, Nanri H, et al. Cellular levers of thioredoxin associated with drug sensitivity to cisplatin,mitomycin C,doxorubicin and etoposide. Cancer Res, 1995,55:4293-4296
9. Cohen SM, Zamble DB and Lippard SJ. Tends Biochem Sci, 1995, 20: 435-439
10. Chu G and Chang E. Cisplatin-resistance cells express increased levels of a factor that recognizes damaged DNA. Proc Natl Acad Sci.USA, 1990,87: 3323-3328
11. Morton KO. Role of trace elements in cancer. Cancer Res, 1975,35:3475-3470
12. Cole SP. Rapid chemosensitivity testing of human lung tumor cells using the MTT assay. Cancer Chemother Pharmacol, 1986,17(3):259-263
13. Rossi L, Marchese E, Lombardo MF, et al. Increased susceptibility of copper-deficient neuroblastoma cells to oxidative stress-mediated apoptosis. Free Radical Biology & Medicine, 2001 May 15; 30(10):1177-1187
14. Darzynkiewicz Z, Bruno S, Del Bino G, et al. Features of apoptotic cells measured by flow cytometry. Cytometry, 1992,13(8):795-808
15. Gately DP and Howell SB. Cellular accumulation of anticancer agent cisplatin: a review. Br J Cancer, 1993, Jun; 67(6): 1171-1176.
16. Nicholson DL, Purser SM, Maier RH. Differential cytotoxicity of trace metals in cisplatin-sensitive and -resistant human ovarian cancer cells. Biometals, 1998, Sep; 11(3): 259-263
17. Komatsu M, Sumizawa T, Mutoh M, et al. Copper-transporting P-type adenosine triphosphatase ATP7B) is associated with cisplatin risistance. Cancer Res, 2000 Mar 1; 60(5)1312-1316
18. Nakayama K, Miyazaki K, Kanzaki A, et al. Expression and cisplatinsensitivity of copper-transporting P-type adenosine triphosphatase (ATP7B) in human solid carcinoma cell lines. Oncol Rep, 2001 Nov-Dec,8(6) 1285-1287
19. Ishida S, Lee J, Thicle DJ, et al. Uptake of the anticancer drug cisplatin mediated by the copper transporter Ctr1 in yeast and mammals. Proc Natl Acad Sci U S A, 2002 Oct 29; 99(22): 14298-14302
20. Samali A and Cotter TG. Heat shock proteins increase resistant to apoptosis. Exp Cell Res, 1996,223:163-170
21. Em Greagh, D Sheehan and TG Cotter. Heat shock proteins modulators of apoptosis in tumor cells. Leukemia, 2000,14:1161-1173