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: 8552580
• 负责人: Michael Gottesman
• 金额: $ 90.87万
• 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/8552580
• 关键词: ABCB1 gene; ABCC1 gene; ABCG2 gene; ATP-Binding Cassette Transporters; Affect; Amino Acids; Anthracyclines; Biological Assay; Biological Factors; Blood - brain barrier anatomy; Brain; Cancer Cell Growth; Cancer cell line; Cell Culture Techniques; Cell Line; Cell model; Cells; Chemicals; Clinical; Collaborations; Cystinosis; Cytotoxic agent; Detection; Development; Doxorubicin; Drug Efflux; Drug resistance; Exposure to; Failure; Family; Family suidae; Functional RNA; Genes; Genetic Polymorphism; Goals; HIV Protease Inhibitors; Haplotypes; Human; In Vitro; Kaposi Sarcoma; LLC-PK1 Cells; Ligands; Loperamide; Lysosomes; Malignant Neoplasms; Messenger RNA; MicroRNAs; Molecular; Molecular Conformation; Multi-Drug Resistance; Multidrug Resistance Gene; National Institute of Mental Health; Natural Product Drug; New Agents; Normal tissue morphology; P-Glycoprotein; Paclitaxel; Patients; Pattern; Pharmaceutical Preparations; Phenotype; Pluripotent Stem Cells; Positron-Emission Tomography; Property; Protease Inhibitor; Proteins; Pump; Reactive Oxygen Species; Regulation; Resistance; S Phase; Sampling; Signal Transduction; Solubility; Specificity; Structure; System; Tariquidar; Techniques; Technology; Time; Vinca Alkaloids; Work; Xenograft procedure; analog; base; cancer cell; chemotherapeutic agent; chemotherapy; density; established cell line; genetic analysis; high throughput screening; improved; in vivo; inhibitor/antagonist; killings; member; monolayer; neoplastic cell; novel; novel strategies; overexpression; protein folding; resistance mechanism; thiosemicarbazide; 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. 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 ABCB5. To explore the possibility that other members of the ABC family of transporters may be involved in drug resistance in cancer, we have developed real-time PCR for detection of most of the 48 known ABC transporters; these techniques have been used to correlate expression of novel ABC transporters in cancer cell lines of known drug resistance. Expression of approximately 30 ABC transporters has been shown to correlate in the NCI-60 cell lines with resistance to specific cytotoxic drugs. Furthermore, this analysis has revealed that some drugs are more toxic to P-gp-expressing cells than to non-expressors, suggesting a novel approach to treatment of MDR cancers. Several different chemical classes with this property, including thiosemicarbazides, have been identified. One compound, NSC73306, has been studied in detail and shown to kill P-gp-expressing cells with high specificity by blocking them in S phase. Treatment with NSC73306 and related drugs also results in increased turnover of ABCB1 mRNA. Cells that survive NSC73306 treatment do not express P-gp and are sensitive to chemotherapy with natural product drugs such as anthracyclines, paclitaxel and Vinca alkaloids. A quantitative structure activity analysis of NSC73306 analogs and a further correlation analysis in the NCI-60 cell lines has yielded many additional compounds with improved ability to kill selectively P-gp-expressing cells, but also with improved solubility properties. In addition, the compound tiopronin, which is a sulfhydryl donor used clinically to treat cystinosis, and some of its derivatives have been shown to be powerful selective agents for killing MDR cells. In this case, expression of P-gp is not required for multidrug-resistant cells to be sensitive to tiopronin, which appears to kill through Reactive Oxygen Species. Technology enabling a high-throughput screen for new agents that are substrates, inhibitors or specifically kill P-gp-expressing cells has been developed and preliminary screens reveal new chemical entities that are P-gp substrates of kill P-gp-expressing cells. In cell lines derived from patients with Kaposis sarcoma, exposure to HIV protease inhibitors results in overexpression of ABCB1, for which the protease inhibitors are important substrates.Studies on the normal function of P-gp suggest that it is involved in normal uptake and distribution of many drugs. C11-desmethoxy-loperamide has been developed in collaboration with Robert Innis in NIMH to PET image distribution of this specific P-gp substrate in cancers and in the brain, with and without treatment with potent inhibitors of P-gp such as tariquidar. PET ligands that are weak bases are trapped in lysosomes, amplifying the uptake signal in the brain, in some normal tissues, and in cancers. We have shown that among three most prominent transporters at the blood-brain barrier (ABCB1, ABCC1, ABCG2), this compound is specific for ABCB1 (P-gp). A synonymous polymorphism of P-gp (C3435T, no amino acid change) in the setting of a specific P-gp haplotype can affect efficiency of P-gp pumping by altering the rhythm of protein folding and changing substrate and inhibitor interactions with P-gp. This haplotype appears to change mRNA folding, and cause a major translational delay which results in altered conformation of P-gp. Stable transfectants of porcine LLC-PK1 cells with the haplotype form of P-gp show altered drug resistance and inhibitor sensitivity compared to wild-type P-gp transfectants. We have created a highly sensitive, quantitative assay for ABC transporter mRNAs in human cancer samples using TaqMan Low Density Arrays. In initial studies of several cancers whose cell lines are represented in the NCI-60 cell lines we have found that patterns of expression of ABC transporter genes in clinical cancers differ substantially from those of the established cell lines, irrespective of the manner in which these cells are cultured (monolayer, 3D culture or xenografts). This result indicates that current in vitro cancer cell models may not be adequate to study drug resistance in cancer and efforts are underway to develop novel ex vivo cancer cell growth conditions that maintain in vivo patterns of expression of MDR genes. Studies on regulation of expression of ABCG2 during development indicate that it is regulated during development post-transcriptionally by a microRNA (miRNA) that targets the 3 prime non-coding region of ABCG2 mRNA. ABCG2 mRNA levels are high in pluripotent stem cells, but are not expressed as protein until the cells begin to differentiate.

由于特定蛋白质表达的内在或后天变化，癌细胞对化疗产生耐药性。 我们研究了对天然产物化疗药物（如阿霉素、长春花生物碱和紫杉醇）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（MDR1 或 ABCB1 基因的产物）或 ABC 转运蛋白家族的其他成员（包括 ABCB5）的表达所致。为了探索 ABC 转运蛋白家族的其他成员可能参与癌症耐药性的可能性，我们开发了实时 PCR 来检测 48 种已知的 ABC 转运蛋白中的大多数；这些技术已被用来关联已知耐药性的癌细胞系中新型 ABC 转运蛋白的表达。 NCI-60 细胞系中约 30 种 ABC 转运蛋白的表达与对特定细胞毒性药物的耐药性相关。此外，该分析表明，某些药物对 P-gp 表达细胞的毒性比对非表达细胞的毒性更大，这提出了一种治疗 MDR 癌症的新方法。 已经鉴定出具有这种特性的几种不同的化学类别，包括氨基硫脲。 一种化合物 NSC73306 已被详细研究，并显示可通过在 S 期阻断表达 P-gp 的细胞来高度特异性地杀死它们。 NSC73306 和相关药物治疗还会导致 ABCB1 mRNA 周转增加。经过 NSC73306 治疗后存活的细胞不表达 P-gp，并且对天然产物药物（例如蒽环类药物、紫杉醇和长春花生物碱）的化疗敏感。 NSC73306 类似物的定量结构活性分析和 NCI-60 细胞系中的进一步相关性分析产生了许多其他化合物，这些化合物具有改进的选择性杀死表达 P-gp 的细胞的能力，而且还具有改进的溶解度特性。此外，化合物硫普罗宁（一种临床上用于治疗胱氨酸病的巯基供体）及其一些衍生物已被证明是杀死MDR细胞的强大选择性药物。在这种情况下，多药耐药细胞不需要表达 P-gp 即可对硫普罗宁敏感，硫普罗宁似乎通过活性氧来杀死细胞。已经开发出能够高通量筛选作为底物、抑制剂或特异性杀死 P-gp 表达细胞的新药剂的技术，并且初步筛选揭示了作为杀死 P-gp 表达细胞的 P-gp 底物的新化学实体。在来自卡波西肉瘤患者的细胞系中，暴露于 HIV 蛋白酶抑制剂会导致 ABCB1 过度表达，而蛋白酶抑制剂是 ABCB1 的重要底物。对 P-gp 正常功能的研究表明，它参与许多药物的正常摄取和分布。 C11-去甲氧基洛哌丁胺是与 NIMH 的 Robert Innis 合作开发的，用于在接受或不接受 Tariquidar 等强效 P-gp 抑制剂治疗的情况下，对癌症和大脑中这种特定 P-gp 底物的 PET 图像分布进行研究。弱碱基 PET 配体被捕获在溶酶体中，放大大脑、某些正常组织和癌症中的摄取信号。我们已经证明，在血脑屏障的三种最重要的转运蛋白（ABCB1、ABCC1、ABCG2）中，该化合物对 ABCB1 (P-gp) 具有特异性。在特定 P-gp 单倍型的情况下，P-gp 的同义多态性（C3435T，无氨基酸变化）可以通过改变蛋白质折叠的节奏以及改变底物和抑制剂与 P-gp 的相互作用来影响 P-gp 泵的效率。 这种单倍型似乎会改变 mRNA 折叠，并导致主要的翻译延迟，从而导致 P-gp 构象改变。与野生型 P-gp 转染子相比，具有 P-gp 单倍型形式的猪 LLC-PK1 细胞的稳定转染子显示出改变的耐药性和抑制剂敏感性。我们使用 TaqMan 低密度阵列创建了一种对人类癌症样本中 ABC 转运蛋白 mRNA 进行高灵敏度定量检测的方法。在对以 NCI-60 细胞系为代表的几种癌症的初步研究中，我们发现临床癌症中 ABC 转运蛋白基因的表达模式与已建立的细胞系的表达模式有很大不同，无论这些细胞的培养方式如何（单层、3D 培养或异种移植）。这一结果表明，当前的体外癌细胞模型可能不足以研究癌症的耐药性，并且正在努力开发新的体外癌细胞生长条件，以维持 MDR 基因的体内表达模式。对发育过程中 ABCG2 表达调节的研究表明，它在发育过程中受到针对 ABCG2 mRNA 3 Prime 非编码区的 microRNA (miRNA) 的转录后调节。 ABCG2 mRNA 水平在多能干细胞中很高，但直到细胞开始分化时才表达为蛋白质。