Reversal effects of nomegestrol acetate on multidrug resistance in adriamycin-resistant MCF7 breast cancer cell line.
Reversal effects of nomegestrol acetate on multidrug resistance in adriamycin-resistant MCF7 breast cancer cell line.
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DOI：
10.1186/bcr303
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Chen Y
中科院分区：
文献类型：
--
作者：
Li J;Xu LZ;He KL;Guo WJ;Zheng YH;Xia P;Chen Y
化疗是乳腺癌系统治疗的重要手段。为了增强肿瘤对化学疗法的反应，注意力集中在逆转多药耐药性（MDR）的药剂和肿瘤细胞对化学药物的敏感性上。体外研究发现了数百种逆转药物，但由于其毒性，其临床应用受到限制。孕激素化合物的逆转活性已得到证实。然而，经典药物如孕酮和甲地孕酮（MG）也具有高毒性。诺美孕酮（NOM）是一种新型的孕激素衍生物，毒性很低。本文研究了NOM的逆转活性，并与维拉帕米（Verapamil，VRP）、屈洛昔芬（Droloxifene，DRO）、他莫昔芬（Tamoxifen，TAM）和MG的逆转活性进行了比较，探讨了NOM的逆转机制，即对MDR 1、谷胱甘肽S转移酶Pi（GSTπ）、MDR相关蛋白（MRP）和拓扑异构酶IIα（TopoIIα）基因表达的影响，以及对细胞内药物浓度和细胞周期的影响。本研究旨在探讨NOM对乳腺癌耐药细胞系MCF 7/ADR多药耐药的逆转作用及其作用机制。用NOM作为乙酸酯处理MCF 7/ADR细胞和MCF 7/WT（一种对ADR敏感的MCF 7乳腺癌细胞系）。采用四唑盐（3-（4，5-dimethylthiazol-2-yl）-2，5-diphenyl-2 H-tetrazolium bromide; MTT）比色法，观察不同浓度NOM对MCF 7/ADR细胞多药耐药的影响。采用逆转录聚合酶链反应（RT-PCR）免疫细胞化学方法检测5 μM NOM作用前后MDR 1、GSTπ、Topo Ⅱ α和MRP基因的表达。采用流式细胞仪（FCM）检测细胞内ADR浓度及NOM与ADR联合作用对细胞周期的影响。用Student t检验分析收集的结果。NOM可明显逆转MCF 7/ADR细胞的MDR。20、10和5 μM NOM处理后，对ADR的化疗敏感性分别增加21倍、12倍和8倍。NOM的逆转活性强于前体化合物MG，与VRP相当。5 μM NOM处理后，第2天MDR 1和GSTπ mRNA基因的表达开始下降（分别为P <0.05和P <0.01），第3天达到最低水平（均P <0.01）;但第5天表达水平又开始升高（均P <0.05）。MRP和Topo Ⅱ α表达无明显变化。P-糖蛋白（P-gp）和GSTπ的表达变化与其mRNA表达变化相似，均表现为早期下降，晚期上升。20、10和5 μM NOM作用2 h后，细胞内ADR浓度分别增加2.7倍、2.3倍和1.5倍。然而，NOM没有增加MCF 7/WT细胞中ADR的积累。流式细胞仪结果显示，NOM（20 μM）和ADR（从低到高）联合作用48 h后，随着ADR剂量的增加，MCF 7/ADR细胞逐渐阻滞于G2 M期。联合用药对ADR的抑制作用强于单一用药。MDR是恶性肿瘤细胞耐药的主要机制。为了克服MDR和增加化疗敏感性，许多逆转剂被发现，大多数孕激素化合物已被证明具有逆转作用，但我们还没有发现一种新的孕激素化合物NOM的数据。我们的研究结果表明，NOM具有很强的反转活性。逆转效果比前体化合物MG强，与VRP相当。由于NOM具有低毒性，因此具有良好的临床应用前景。采用RT-PCR和免疫细胞化学方法研究NOM对MDR相关基因的影响。结果表明，NOM能显著下调MDR 1和GSTπ的mRNA和蛋白表达水平。Topo Ⅱ α和MRP基因表达无明显变化。已知P-gp主要通过降低细胞内药物浓度来诱导肿瘤细胞中的MDR。经NOM处理后，MCF 7/ADR细胞内药物浓度显著升高。NOM和ADR联合治疗可使G2 M期阻滞。值得注意的是，NOM以时间依赖的方式引起一些MDR相关基因表达的早期降低和晚期升高。这些现象提出了一个问题，在临床上继续管理逆转剂，值得进一步研究。我们证明NOM对MCF 7/ADR细胞的多药耐药有很强的逆转作用。逆转是通过不同的途径，即下调MDR 1和GSTπ的mRNA和蛋白表达水平，增加细胞内药物浓度和将细胞阻滞在G2 M期（NOM与ADR组合）。逆转机制有待进一步研究。
Chemotherapy is important in the systematic treatment of breast cancer. To enhance the response of tumours to chemotherapy, attention has been focused on agents to reverse multidrug resistance (MDR) and on the sensitivity of tumour cells to chemical drugs. Hundreds of reversal drugs have been found in vitro, but their clinical application has been limited because of their toxicity. The reversal activity of progestogen compounds has been demonstrated. However, classical agents such as progesterone and megestrol (MG) also have high toxicity. Nomegestrol (NOM) belongs to a new derivation of progestogens and shows very low toxicity. We studied the reversal activity of NOM and compared it with that of verapamil (VRP), droloxifene (DRO), tamoxifen (TAM) and MG, and investigated the reversal mechanism, i.e. effects on the expression of the MDR1, glutathione S-transferase Pi (GSTπ), MDR-related protein (MRP) and topoisomerase IIα (TopoIIα) genes, as well as the intracellular drug concentration and the cell cycle. The aim of the study was to examine the reversal effects of NOM on MDR in MCF7/ADR, an MCF7 breast cancer cell line resistant to adriamycin (ADR), and its mechanism of action. MCF7/ADR cells and MCF7/WT, an MCF7 breast cancer cell line sensitive to ADR, were treated with NOM as the acetate ester. With an assay based on a tetrazolium dye [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; MTT], the effects of various concentrations of NOM on MDR in MCF7/ADR cells were studied. Before and after the treatment with 5 μM NOM, the expression of the MDR-related genes MDR1, GSTπ, TopoIIα and MRP were assayed with a reverse transcriptase polymerase chain reaction (RT-PCR) immunocytochemistry assay. By using flow cytometry (FCM), we observed the intracellular ADR concentration and the effects of combined treatment with NOM and ADR on the cell cycle. Results collected were analysed with Student's t test. NOM significantly reversed MDR in MCF7/ADR cells. After treatment NOM at 20, 10 and 5 μM, chemosensitivity to ADR increased 21-fold, 12-fold and 8-fold, respectively. The reversal activity of NOM was stronger than that of the precursor compound MG, and comparable to that of VRP. After treatment with 5 μM NOM, the expression of both the MDR1 and the GSTπ mRNA genes began to decline on the second day (P <0.05 and P <0.01, respectively), and reached the lowest level on the third day (both P <0.01); however, on the fifth day the expression levels began to increase again (both P <0.05). The expression of MRP and TopoIIα had no significant changes. Changes in the expression of P-glycoprotein (P-gp) and GSTπ were similar to those of their mRNA expressions, showing early declines and late increases. Two hours after treatment with 20, 10 and 5 μM NOM, the intracellular ADR concentration increased 2.7-fold, 2.3-fold and 1.5-fold respectively. However, NOM did not increase ADR accumulation in MCF7/WT cells. FCM data showed that after 48 h of combined administration of NOM (20 μM) and ADR (from low to high concentration), MCF7/ADR cells showed a gradual arrest at the G2M phase with increasing ADR dose. The arrest effect with combined drug treatment was stronger than that with the single ADR treatment. MDR is the major mechanism of drug resistance in malignant tumour cells. To overcome MDR and to increase chemosensitivity, many reversal agents have been found. Most progestogen compounds have been demonstrated to have reversal effects, but we found no data on NOM, a new progestogen compound. Our results show that NOM has strong reversal activity. The reversal effects were stronger than those of the precursor compound, MG, and were comparable to that of VRP. Because NOM has low toxicity, it might have good prospects in clinical application. Using RT-PCR and immunocytochemistry assays, we studied the effects of NOM on MDR-related genes. The results were that NOM could markedly downregulate the mRNA and protein expression levels of MDR1 and GSTπ. TopoIIα and MRP gene expression showed no significant changes. It is known that P-gp induces MDR in tumour cells mainly by decreasing the intracellular drug concentration. After treatment with NOM, the intracellular drug concentration in MCF7/ADR cells increased significantly. Combined treatment with NOM and ADR induced arrest at the G2M phase. It is worth noting that NOM caused an early decrease and a late increase in the expression of some MDR-related genes in a time-dependent manner. The phenomena raise a question for the continued administration of reversal agents in clinics that merits further study. We demonstrate that NOM has strong reversal effects on MDR in MCF7/ADR cells. The reversal is via different routes, namely downregulating the mRNA and protein expression levels of MDR1 and GSTπ, increasing intracellular drug concentration and arresting cells at the G2M phase (NOM in combination with ADR). The reversal mechanism needs further study.
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DOI：
10.1073/pnas.88.17.7654
发表时间：
1991-09-01
影响因子：
11.1
作者：
BUGG, BY;DANKS, MK;SUTTLE, DP
通讯作者：
SUTTLE, DP
影响因子：
3.6
作者：
Lee, GY;Croop, JM;Anderson, E
通讯作者：
Anderson, E
影响因子：
3
作者：
FLEMING, GF;AMATO, JM;SAFA, AR
通讯作者：
SAFA, AR
影响因子：
3.8
作者：
Claudio, JA;Emerman, JT
通讯作者：
Emerman, JT
DOI：
10.1093/jnci/84.9.711
发表时间：
1992-05-06
期刊：
JOURNAL OF THE NATIONAL CANCER INSTITUTE
影响因子：
--
作者：
HERZOG, CE;TREPEL, JB;FOJO, AT
通讯作者：
FOJO, AT