Genetic Analysis of the Multidrug Resistance Phenotype in Tumor Cells

由于特定蛋白质表达的内在或获得性变化，癌细胞对化疗产生抗性。我们已经研究了对天然产物化疗剂如阿霉素、紫杉醇和紫杉醇的耐药性，最近还研究了组蛋白脱乙酰酶抑制剂和靶向激酶抑制剂的耐药性。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产生耐药性。对于天然产物药物，这种交叉耐药性通常是由于被称为P-糖蛋白（P-gp）的能量依赖性药物外排系统（ABC转运蛋白）的表达，其是MDR 1或ABCB 1基因的产物，或ABC转运蛋白家族的其他成员，包括ABCG 2和ABCB 5。与Suresh Ambudkar小组合作，我们研究了P-糖蛋白将化合物定向转运出细胞的基础。这些研究揭示了P-糖蛋白跨膜区域中的一组氨基酸残基，其可以被改变以改变某些罗丹明化合物从细胞外转运到细胞内的方向。这个过程是浓度和ATP依赖性的，并提供了重要的洞察如何在P-糖蛋白的运输方向是确定的。Ambudkar实验室正在进行进一步的研究，以澄清特定残留物的作用以及运输方向逆转的机制。在与Jean-Pierre Gillet（比利时内穆尔大学）的合作中，我们进一步探索了ABCB 5在癌症耐药性中的作用，在黑色素瘤的恶性表型中的作用（它经常突变），以及作为与其他ABC B型转运蛋白的异二聚体的伴侣。在表达ABCB 5的色素细胞中，存在由不同启动子产生的全长和部分转录物。部分转录本编码半转运蛋白，其似乎能够与另一半转运蛋白（特别是ABCB 6和ABCB 9）二聚化。这些异二聚体转运蛋白的定位和潜在功能正在研究中。铁凋亡是由致死性脂质过氧化引起的非凋亡形式的细胞死亡。已经报道了几种小分子铁凋亡诱导剂（FNs），但是关于它们与ATP结合盒（ABC）转运蛋白P-糖蛋白（P-gp，ABCB 1）和ABCG 2的相互作用的信息很少。因此，我们试图表征FINS与P-gp和ABCG 2的相互作用，这可能提供关于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过表达赋予对FIN 56和erastin衍生物咪唑酮erastin和哌嗪erastin的抗性。P-gp介导的对咪唑酮erastin和哌嗪erastin的抗性也通过CRISPR介导的ABCB 1敲除在UO-31肾癌细胞中逆转。在表达P-gp的MDR-19细胞中，10微摩尔的FIN ML-162、GPX抑制剂26 a和PACMA 31能够使细胞内罗丹明123荧光增加10倍以上。GPX抑制剂26 a能够在表达ABCG 2的R-5细胞中使细胞内红紫素-18荧光增加超过4倍。我们得出结论，P-gp的表达可能会降低这些FIN在表达转运蛋白的癌症中的疗效，并可能阻止进入避难所部位，如大脑。一些FIN抑制P-gp和ABCG 2的能力表明潜在的药物相互作用。

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.