Genetic Analysis of the Multidrug Resistance Phenotype in Tumor Cells

肿瘤细胞多药耐药表型的遗传分析

• 批准号: 10925952
• 负责人: Michael Gottesman
• 金额: $ 28.4万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10925952
• 关键词: ABCB1 gene; ABCB6 gene; ABCC1 gene; ABCG2 gene; ATP-Binding Cassette Transporters; Amino Acids; Belgium; Binding; Biological Availability; Blood - brain barrier anatomy; Brain; Cause of Death; Cell Death; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Cytotoxic agent; Dimerization; Doxorubicin; Drug Efflux; Drug Interactions; Failure; Family; Fluorescence; Goals; Histone Deacetylase Inhibitor; Imidazole; Ketones; Knock-out; Laboratories; Length; Lipid Peroxidation; Malignant Neoplasms; Mediating; Molecular; Multi-Drug Resistance; Mutate; Natural Product Drug; Natural Products; Oral; Paclitaxel; Penetration; Pharmaceutical Preparations; Phenotype; Physiological; Piperazines; Process; Proteins; Renal Cell Carcinoma; Reporting; Resistance; Rhodamine; Rhodamine 123; Role; Site; System; Transcript; Transmembrane Domain; Universities; Vinca Alkaloids; Work; cancer cell; cancer drug resistance; chemotherapeutic agent; chemotherapy; erastin; genetic analysis; human tissue; inhibitor; insight; kinase inhibitor; malignant phenotype; melanoma; member; multi drug transporter; multidrug resistant cancer; neoplastic cell; overexpression; prevent; promoter; resistance mechanism; small molecule; tool; uptake; ABCB1 gene; ABCB6 gene; ABCC1 gene; ABCG2 gene; ATP-Binding Cassette Transporters; Amino Acids; Belgium; Binding; Biological Availability; Blood - brain barrier anatomy; Brain; Cause of Death; Cell Death; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Cytotoxic agent; Dimerization; Doxorubicin; Drug Efflux; Drug Interactions; Failure; Family; Fluorescence; Goals; Histone Deacetylase Inhibitor; Imidazole; Ketones; Knock-out; Laboratories; Length; Lipid Peroxidation; Malignant Neoplasms; Mediating; Molecular; Multi-Drug Resistance; Mutate; Natural Product Drug; Natural Products; Oral; Paclitaxel; Penetration; Pharmaceutical Preparations; Phenotype; Physiological; Piperazines; Process; Proteins; Renal Cell Carcinoma; Reporting; Resistance; Rhodamine; Rhodamine 123; Role; Site; System; Transcript; Transmembrane Domain; Universities; Vinca Alkaloids; Work; cancer cell; cancer drug resistance; chemotherapeutic agent; chemotherapy; erastin; genetic analysis; human tissue; inhibitor; insight; kinase inhibitor; malignant phenotype; melanoma; member; multi drug transporter; multidrug resistant cancer; neoplastic cell; overexpression; prevent; promoter; resistance mechanism; small molecule; tool; uptake

Resistance to chemotherapy occurs in cancer cells because of intrinsic or acquired changes in expression of specific proteins. We have studied resistance to natural product chemotherapeutic agents such as doxorubicin, Vinca alkaloids, and taxol and more recently, histone deacetylase inhibitors and targeted kinase inhibitors. In most cases, cells become simultaneously resistant to multiple drugs because of reductions in intracellular drug concentrations. For the natural product drugs, this cross-resistance is frequently due to expression of an energy-dependent drug efflux system (ABC transporter) known as P-glycoprotein (P-gp), the product of the MDR1 or ABCB1 gene, or to other members of the ABC transporter family, including ABCG2 and ABCB5. In collaboration with the group of Suresh Ambudkar, we have examined the basis of directional transport of compounds out of cells by P-glycoprotein. These studies have revealed a set of amino acid residues in the transmembrane regions of P-glycoprotein which can be altered to change the direction of transport of certain rhodamine compounds from out of the cell to into the cell. This process is concentration- and ATP-dependent, and gives important insight into how directionality of transport is determined in P-glycoprotein. Further studies are underway in the Ambudkar laboratory to clarify the role of specific residues and the mechanism by which the direction of transport is reversed. In collaboration with Jean-Pierre Gillet (University of Nemours, Belgium), we have further explored the role of ABCB5 in cancer drug resistance, in the malignant phenotype in melanoma (where it is frequently mutated), and as a partner as a heterodimer with other ABC B-type transporters. In pigmented cells that express ABCB5 there are both full-length and partial transcripts generated from different promoters. The partial transcripts encode a half-transporter which appears to be able to dimerize with other half transporters (notably ABCB6 and ABCB9). The localization and potential function of these heterodimeric transporters is under study. Ferroptosis is a non-apoptotic form of cell death caused by lethal lipid peroxidation. Several small molecule ferroptosis inducers (FINs) have been reported, yet little information is available regarding their interaction with the ATP-binding cassette (ABC) transporters P-glycoprotein (P-gp, ABCB1) and ABCG2. We thus sought to characterize the interactions of FINS with P-gp and ABCG2 which may provide information regarding oral bioavailability and brain penetration and predict drug-drug interactions. P-gp overexpression conferred resistance to FIN56 and the erastin derivatives imidazole ketone erastin and piperazine erastin. P-gp-mediated resistance to imidazole ketone erastin and piperazine erastin was also reversed in UO-31 renal cancer cells by CRISPR-mediated knockout of ABCB1. The FINs ML-162, GPX inhibitor 26a, and PACMA31 at 10 micromolar were able to increase intracellular rhodamine 123 fluorescence over 10-fold in P-gp-expressing MDR-19 cells. GPX inhibitor 26a was able to increase intracellular purpurin-18 fluorescence over 4-fold in ABCG2-expressing R-5 cells. We conclude that expression of P-gp may reduce the efficacy of these FINs in cancers that express the transporter and may prevent access to sanctuary sites such as the brain. The ability of some FINs to inhibit P-gp and ABCG2 suggests potential drug-drug interactions.

由于特定蛋白质表达的内在或获得的变化，癌细胞中对化疗的抗性发生在癌细胞中。我们研究了对天然产物化学治疗剂的耐药性，例如阿霉素，芬卡生物碱和紫杉醇，以及最近的组蛋白脱乙酰基酶抑制剂和靶向激酶抑制剂。在大多数情况下，由于细胞内药物浓度的降低，细胞同时对多种药物具有抗性。对于天然产物药物，这种交叉耐药通常是由于能量依赖性药物外排系统（ABC转运蛋白）称为P-糖蛋白（P-GP），MDR1或ABCB1基因的乘积，或其他ABC Transporter家族的成员，包括ABCG2和ABCB5。在与Suresh Ambudkar组合作的情况下，我们研究了P-糖蛋白从细胞中从细胞中取出的定向转运的基础。这些研究揭示了P-糖蛋白跨膜区域中的一组氨基酸残基，可以改变这些氨基酸残基，以改变某些若丹明化合物的转运方向，从细胞外到细胞。该过程依赖浓度和ATP依赖性，并且对如何在P-糖蛋白中确定转运的方向性有重要见解。在Ambudkar实验室正在进行进一步的研究，以阐明特定残基的作用以及运输方向逆转的机制。与Jean-Pierre Gillet（比利时Nemours）合作，我们进一步探讨了ABCB5在癌症耐药性中的作用，在黑色素瘤的恶性表型中（经常被突变），以及作为其他ABC B-B-type Typeters作为异二聚体的伴侣。在表达ABCB5的色素细胞中，来自不同启动子产生的全长和部分转录本。部分转录本编码一个半转运蛋白，它似乎能够与其他半转运蛋白（尤其是ABCB6和ABCB9）二聚。这些异二聚体转运蛋白的定位和潜在功能正在研究。铁凋亡是由致命脂质过氧化引起的细胞死亡的一种非凋亡形式。已经报道了几个小分子溢铁诱导蛋白（FIN），但几乎没有关于它们与ATP结合盒（ABC）转运蛋白P-糖蛋白（P-GP，ABCB1）和ABCG2相互作用的信息。因此，我们试图表征鳍与P-gp和ABCG2的相互作用，这些鳍可能提供有关口服生物利用度和脑渗透的信息，并预测药物 - 药物相互作用。 P-gp的过表达赋予了对FIN56的抵抗力和Erastin衍生物咪唑酮Erastin和哌嗪Erastin。 P-gp介导的对咪唑酮Erastin的抗性和哌嗪Erastin也通过CRISPR介导的ABCB1敲除UO-31肾脏癌细胞中的UO-31肾脏癌细胞逆转。在10微摩尔时，FINS ML-162，GPX抑制剂26A和PACMA31能够在表达P-gp的MDR-19细胞中增加细胞内若丹明123荧光。在表达ABCG2的R-5细胞中，GPX抑制剂26a能够将细胞内紫罗林-18荧光超过4倍。我们得出的结论是，P-gp的表达可能会降低表达转运蛋白的癌症中这些鳍片的功效，并可能阻止进入诸如大脑之类的避难所。某些FIN抑制P-GP和ABCG2的能力表明潜在的药物相互作用。