溴泰君联合加温逆转MCF-7/ADM细胞株多药耐药的实验研究
摘要
[研究目的] 肿瘤多药耐药(MDR)是肿瘤化疗失败的主要原因。药物流出泵P-糖蛋白(P-gp)过度表达是引起肿瘤多药耐药的主要原因。热化疗能提高化疗药物在耐药细胞中的作用已在一些实验中得到证实。现已发现多种中药具有逆转肿瘤耐药的作用。溴泰君(Br-Tet)是中药汉防己的有效成分粉防己碱基础上改造而成的一种新型耐药逆转剂。体内外实验证明,Br-Tet与抗肿瘤药物合用可增强耐药细胞对抗肿瘤药物的敏感性,逆转MDR。本实验目的在于观察Br-Tet联合加温是否有更强的耐药逆转作用,即二者联用是否有协同作用。
[研究方法] 以人乳腺癌细胞株MCF-7为对照,以人乳腺癌耐药细胞株MCF-7/ADM为研究对象,当阿霉素(ADM)浓度为1ug/ml时,观察在37℃,40℃,41℃,42℃条件下分别加入不同浓度的Br-Tet(包括空白对照,0.125umol/l,0.25umol/l,0.5umol/l,1.0umol/l)时,耐药细胞MCF-7/ADM的不同表现。用MTT法测定实验对象的细胞生长抑制率,用流式细胞技术测定细胞内阿霉素浓度,细胞表面P-gp表达的影响。统计学处理采用SAS8.0软件包分析。
[研究结果] 通过比较各实验条件下细胞生长抑制率,细胞内阿霉素浓度,细胞表面P-gp表达情况,结果显示:MCF-7/ADM细胞对于阿霉素的敏
[Objectives] Multidrug resistance (MDR) is the most common cause of chemotherapy failure. It is well established that the majority of tumors develop MDR through over-expression of the drug efflux pump P-glycoprotein (P-gp). Thermochemotherapy have been proved could increase sensitivity of multidurg-resistant cells to chemotherapeutic drugs .Br-Tet is a novel multidrug-resistant reversal agent, which is from a Chinese herb named tetrandrine. Experiment in vitro and vivo indicated that chemotherapeutic drugs such as ADM combined with Br-Tet could increase the sensitivity of multidrug-resistant cells to ADM by inhibiting the drug efflux pump P-gp. The purpose of this study was investigate the reversal effect of Br-Tet combined with heating and ADM on multidrug-resistant breast cancer cell line MCF-7/ADM[Methods] At different temperature 37℃, 40℃, 41℃, 42 ℃ /The multidurg resistant ,breast cancer cell line MCF-7/ADM was treated with lug/ml ADM; 0.125umol/l+lug/mlADM;0.25umol/lBr-Tet+lug/mlADM;0.5umol/lBr-Tet+ lug/mlADM;1.0umol/lBr-Tet+lug/mlADM for 1 hour, respectively. The
cytotoxicity effect was observed by using 3 - (4 , 5 - dimethylthi-azol - 2 - yl) - 2, 5 - diphenyltetrazolium bromide(MTT) assay in vitro, and intracellular drug concentration and the expression of Pgp were analysed with flow cytometry ( FCM) after treated with Br-Tet of different concentration combined with lug/ml ADM and different temperature. Statistical analysis depends on SAS8.0.[Result] Sensitivity of MCF-7/ADM to ADM was significantly lower than that of MCF-7.Br-Tet showed marked synergstic effects in MCF-7/ADM. The sensitivity of MCF-7/ADM to ADM increased after the treatment of Br-Tet combined with heating. P-gp expression was significantly decreased and intracellular concentration of ADM increased by exposure to the treatment. In the all conditions observed, lumol/1 Br-Tet combined with lug/ml ADM at 42℃ could reverse the multidrug resistance efficiently. [Conclusion] It is suggested that combined Br-Tet with heating could be used as a method to overcome multidrug resistance. Br-Tet showed marked synergstic effects with thermochemotherapy in MCF-7/ADM.
[研究方法] 以人乳腺癌细胞株MCF-7为对照,以人乳腺癌耐药细胞株MCF-7/ADM为研究对象,当阿霉素(ADM)浓度为1ug/ml时,观察在37℃,40℃,41℃,42℃条件下分别加入不同浓度的Br-Tet(包括空白对照,0.125umol/l,0.25umol/l,0.5umol/l,1.0umol/l)时,耐药细胞MCF-7/ADM的不同表现。用MTT法测定实验对象的细胞生长抑制率,用流式细胞技术测定细胞内阿霉素浓度,细胞表面P-gp表达的影响。统计学处理采用SAS8.0软件包分析。
[研究结果] 通过比较各实验条件下细胞生长抑制率,细胞内阿霉素浓度,细胞表面P-gp表达情况,结果显示:MCF-7/ADM细胞对于阿霉素的敏
[Objectives] Multidrug resistance (MDR) is the most common cause of chemotherapy failure. It is well established that the majority of tumors develop MDR through over-expression of the drug efflux pump P-glycoprotein (P-gp). Thermochemotherapy have been proved could increase sensitivity of multidurg-resistant cells to chemotherapeutic drugs .Br-Tet is a novel multidrug-resistant reversal agent, which is from a Chinese herb named tetrandrine. Experiment in vitro and vivo indicated that chemotherapeutic drugs such as ADM combined with Br-Tet could increase the sensitivity of multidrug-resistant cells to ADM by inhibiting the drug efflux pump P-gp. The purpose of this study was investigate the reversal effect of Br-Tet combined with heating and ADM on multidrug-resistant breast cancer cell line MCF-7/ADM[Methods] At different temperature 37℃, 40℃, 41℃, 42 ℃ /The multidurg resistant ,breast cancer cell line MCF-7/ADM was treated with lug/ml ADM; 0.125umol/l+lug/mlADM;0.25umol/lBr-Tet+lug/mlADM;0.5umol/lBr-Tet+ lug/mlADM;1.0umol/lBr-Tet+lug/mlADM for 1 hour, respectively. The
cytotoxicity effect was observed by using 3 - (4 , 5 - dimethylthi-azol - 2 - yl) - 2, 5 - diphenyltetrazolium bromide(MTT) assay in vitro, and intracellular drug concentration and the expression of Pgp were analysed with flow cytometry ( FCM) after treated with Br-Tet of different concentration combined with lug/ml ADM and different temperature. Statistical analysis depends on SAS8.0.[Result] Sensitivity of MCF-7/ADM to ADM was significantly lower than that of MCF-7.Br-Tet showed marked synergstic effects in MCF-7/ADM. The sensitivity of MCF-7/ADM to ADM increased after the treatment of Br-Tet combined with heating. P-gp expression was significantly decreased and intracellular concentration of ADM increased by exposure to the treatment. In the all conditions observed, lumol/1 Br-Tet combined with lug/ml ADM at 42℃ could reverse the multidrug resistance efficiently. [Conclusion] It is suggested that combined Br-Tet with heating could be used as a method to overcome multidrug resistance. Br-Tet showed marked synergstic effects with thermochemotherapy in MCF-7/ADM.
引文
1. Biedler JL, Riehm H. Cellular resistance to actionomycin D in Chinese hamster cells in vitro: Cross-resistance, radioautographic and cytogenetic studies [J]. Cancer Res, 1970, 30(4): 1174-1184.
2.杨纯正.肿瘤耐药研究进展及逆转对策.中华血液学杂志,1997;18(2):59.
3. Lehne G. P-glycoprotein as a drug target in the treatment of multidrug resistance Cancer Curr Drug Target, 2000,1: 85-99.
4. Rimet O, Mirrione A, Barra Y. Multidrug-resistant phenotype influences the differentiation of a human colon carcinoma cell line [J]. Biochem Biophys Res Commun, 1999;259(1): 43-49.
5. Ruiz Gomez MJ, Souviron Rodriguez A, Martinez Morillo M. P-glycoprotein, a membrane pump that represents a barrier to chemotherapy in cancer patients.An Med Interna,2002,19:477-485.
6. Anthony V Skach WR. Molecular mechanism of P-glycoprotein assembly into cellular membranes. Curr Protein Pept Sci, 2002,3:485-501.
7. Ruiz Gomez MJ, Souviron Rodriguez A, Martinez Morillo M. P-glycoprotein,a membrane pump that represets a barrier to chemotherapy in cancer patients. An Med Interna, 2002,19(9):477.
8. Cheng AL, Chuang SE, Fine RL, Hanahan D, Fo lkman J,Tanaka H, Taniguch i T. Inhibition of the membrane transtocation and activation of p ro tein k inase C, and potentiation of ADMorubicin2induced apoptosis of hepatocellular carcinoma cells by tamoxifen [J]. Biochem 2P harm acol, 1998;55 (4): 523-531.
9. Chou YY, Cheng AL, Hsu HC. Expression of P-glycoprotein and p53 in advanced hepatocellular carcinoma treated by single agent chemotherapy: Clinical correlation [J]. J Gastroenterol H ep atol, 1997; 12 (8): 569-575.
10. Advani R, Saba HI, Tallman MS,et al. Treatment of refractory and relapsed acute myelogenous leukemia with combination chemotherapy plus the multidrug resistance modulator PSC 833 (Valspodar).Blood, 1999,93 (3):787-795
11. Yang L Y, T rujillo JM. Bio logical characterization of multidrug resistant human co lon carcinoma sublines induced/selected by two methods[J]. Cancer R es, 1990; 50 (5):3218-3225.
12. Hettinga JV, Konings AV. Reduction of cellular cisplatin resistance by hyperthermia-a review [J]. Int J H yp ertherm ia,1997; 13 (5): 439-457.
13. Carmichael L,DeGraff WG;Gazdar AF,et al. Evaluation of a tetrazolium-based semiautomated colorimetric assay: Assessment of chemosensitivity testing. Cancer Res, 1987,47:936-942.
14.彭楠 赵彼得 主编.临床肿瘤热疗.第一版.北京:人民军医出版社 2002;63
15. Denecke J, Fideler K, Hacker-Klom U, et al. Multiple drug-resistant C6 glioma cells cross-resistant to irradiation[J]. Anticancer Res,1997,17 (6):4531-4534.
16. Robins HI, Grosen E, Katschinski DM et al. Whole body hyperthermia induction of soluble tumor necrosis factor receptors: implications for rheumatoid disease [J]. J Rheumatol, 1999,26(12):2513-2516.
17. Katschinski DM, Benndorf R, Wiedemann GJ et al. Heat shock protein antibodies in sarcoma patients undergoing 41. 8 C whole body hyperthermia [J]. J Immunother, 1999,22(1):67-70.
18. Ressel A, Weiss C, Feyerabend T. Tumor oxygenation after radiotherapy,chemotherapy, and/or hyperthermia predicts tumor free survival[J]. Int J Radiat Oncol Biol Phys, 2001,49(4):1119-1125.
19. Van der Zee J. Heating the patient: a promising approach ? [J] Ann Oncol, 2002,13(8):1173-1784.
20. Majima H, Kashiwado K, Egaswa S et al. Interaction between the kinetics of thermo to lerance and effect of cis-diamm inedichlorplatinum (11) o r bleomycin given at 37 o r 43[J]. Int J H yperthermia, 1992; 8 (2): 431-432.
21.宋燕爽,刘洪,王立梅,等.加温或化疗药对结肠癌细胞系THC-8908多药耐药基因表达的影响.中国肿瘤临床,1997,24:131-134.
2.杨纯正.肿瘤耐药研究进展及逆转对策.中华血液学杂志,1997;18(2):59.
3. Lehne G. P-glycoprotein as a drug target in the treatment of multidrug resistance Cancer Curr Drug Target, 2000,1: 85-99.
4. Rimet O, Mirrione A, Barra Y. Multidrug-resistant phenotype influences the differentiation of a human colon carcinoma cell line [J]. Biochem Biophys Res Commun, 1999;259(1): 43-49.
5. Ruiz Gomez MJ, Souviron Rodriguez A, Martinez Morillo M. P-glycoprotein, a membrane pump that represents a barrier to chemotherapy in cancer patients.An Med Interna,2002,19:477-485.
6. Anthony V Skach WR. Molecular mechanism of P-glycoprotein assembly into cellular membranes. Curr Protein Pept Sci, 2002,3:485-501.
7. Ruiz Gomez MJ, Souviron Rodriguez A, Martinez Morillo M. P-glycoprotein,a membrane pump that represets a barrier to chemotherapy in cancer patients. An Med Interna, 2002,19(9):477.
8. Cheng AL, Chuang SE, Fine RL, Hanahan D, Fo lkman J,Tanaka H, Taniguch i T. Inhibition of the membrane transtocation and activation of p ro tein k inase C, and potentiation of ADMorubicin2induced apoptosis of hepatocellular carcinoma cells by tamoxifen [J]. Biochem 2P harm acol, 1998;55 (4): 523-531.
9. Chou YY, Cheng AL, Hsu HC. Expression of P-glycoprotein and p53 in advanced hepatocellular carcinoma treated by single agent chemotherapy: Clinical correlation [J]. J Gastroenterol H ep atol, 1997; 12 (8): 569-575.
10. Advani R, Saba HI, Tallman MS,et al. Treatment of refractory and relapsed acute myelogenous leukemia with combination chemotherapy plus the multidrug resistance modulator PSC 833 (Valspodar).Blood, 1999,93 (3):787-795
11. Yang L Y, T rujillo JM. Bio logical characterization of multidrug resistant human co lon carcinoma sublines induced/selected by two methods[J]. Cancer R es, 1990; 50 (5):3218-3225.
12. Hettinga JV, Konings AV. Reduction of cellular cisplatin resistance by hyperthermia-a review [J]. Int J H yp ertherm ia,1997; 13 (5): 439-457.
13. Carmichael L,DeGraff WG;Gazdar AF,et al. Evaluation of a tetrazolium-based semiautomated colorimetric assay: Assessment of chemosensitivity testing. Cancer Res, 1987,47:936-942.
14.彭楠 赵彼得 主编.临床肿瘤热疗.第一版.北京:人民军医出版社 2002;63
15. Denecke J, Fideler K, Hacker-Klom U, et al. Multiple drug-resistant C6 glioma cells cross-resistant to irradiation[J]. Anticancer Res,1997,17 (6):4531-4534.
16. Robins HI, Grosen E, Katschinski DM et al. Whole body hyperthermia induction of soluble tumor necrosis factor receptors: implications for rheumatoid disease [J]. J Rheumatol, 1999,26(12):2513-2516.
17. Katschinski DM, Benndorf R, Wiedemann GJ et al. Heat shock protein antibodies in sarcoma patients undergoing 41. 8 C whole body hyperthermia [J]. J Immunother, 1999,22(1):67-70.
18. Ressel A, Weiss C, Feyerabend T. Tumor oxygenation after radiotherapy,chemotherapy, and/or hyperthermia predicts tumor free survival[J]. Int J Radiat Oncol Biol Phys, 2001,49(4):1119-1125.
19. Van der Zee J. Heating the patient: a promising approach ? [J] Ann Oncol, 2002,13(8):1173-1784.
20. Majima H, Kashiwado K, Egaswa S et al. Interaction between the kinetics of thermo to lerance and effect of cis-diamm inedichlorplatinum (11) o r bleomycin given at 37 o r 43[J]. Int J H yperthermia, 1992; 8 (2): 431-432.
21.宋燕爽,刘洪,王立梅,等.加温或化疗药对结肠癌细胞系THC-8908多药耐药基因表达的影响.中国肿瘤临床,1997,24:131-134.