PO.MCB05.02 · 分子与细胞生物学
2-羟基黄烷酮增强PARP抑制剂在BRCA野生型乳腺癌细胞系中的活性
Potentiation ofPARP inhibitor activity in BRCA -wildtype breast cancer cell lines by 2-hydroxyflavanone
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：2-羟基黄烷酮（2HF）是一种柑橘来源的化合物，已在体外证明对多种恶性肿瘤具有抗癌活性。其存在可抑制RLIP——一种由RALBP1基因编码的蛋白，其功能为ATP依赖性外排泵，转运谷胱甘肽-亲电试剂偶联物、氧化及烷基化中间体，并介导表皮生长因子受体的内吞。因此，RLIP通过调节生长以及借助谷胱甘肽介导的异生物质外排产生化疗耐药能力来促进肿瘤发生。2HF降低RLIP表达可减少p53缺失小鼠模型中的自发性癌变，表明其在DNA修复缺陷型肿瘤中的应用价值。BRCA1、BRCA2和PALB2是参与同源重组（HR）的关键基因，其突变可被聚（ADP-核糖）聚合酶通路（PARP）抑制剂靶向。通过本研究，我们评估了PARP抑制剂AZD2461单独及与2HF联合对MDA-MB-231和MCF-7乳腺癌细胞系细胞活力的影响。
方法：在MDA-MB-231（三阴性）和MCF-7（ER+）乳腺癌细胞系暴露于2HF和LNA后，使用聚合酶链反应的定量循环评估BRCA1、BRCA2和PALB2基因的mRNA表达。测量单独或联合使用2HF或AZD2461处理的细胞在72小时时的LDH释放，并与对照进行比较。使用存活细胞产生的还原型WST-8的吸光度测量细胞毒性。
结果：2HF耗竭RLIP将HR基因BRCA1、BRCA2和PALB2的mRNA表达降低至未处理对照水平的2%-20%。PARP抑制剂随着2HF剂量（10、20和40 μM）递增而诱导LDH释放，在40 μM 2HF时细胞毒作用最强（p<0.001）。在PARP抑制剂AZD2461中加入2HF增加了MCF-7和MDA-MB-231细胞的细胞裂解，但仅在MCF-7中降低了细胞活力，而在MDA-MB-231中未降低。
讨论：PARP抑制剂在具有HRD突变的肿瘤中非常有效，但在涉及非HRD通路突变的受试者中其应用受限。用2HF抑制RALBP1可降低参与HR通路基因的表达，从而激发出类似于HRD突变细胞的转录组环境。因此，2HF可能使BRCA野生型ER+乳腺癌以及可能的三阴性乳腺癌对PARP抑制剂敏感。这有望在可能受益于PARP抑制剂、但处于当前适应证（如BRCA或HRD缺陷突变）之外的患者中具有临床应用价值。通过这项工作，我们期望启发更多关于2HF与PARP抑制剂临床探索的研究。
查看英文原文 English abstract
Background: 2-hydroxyflavanone (2HF) is citrus derived compound which has demonstrated in vitro anticancer activity against multiple malignancies. Its presence inhibits RLIP, a protein encoded by RALBP1 gene which functions as an ATP dependent efflux pump for glutathione-electrophile conjugates, oxidative and alkylating intermediates and, mediates epidermal growth factor receptor endocytosis. Thus RLIP promotes oncogenesis with growth modulation and ability to resist chemotherapy with glutathione mediated xenobiotic efflux. Reduced RLIP expression by 2HF reduces spontaneous carcinogenesis in p53 null mouse models, indicating its utility in tumors with DNA repair-deficiency. BRCA1, BRCA2, and PALB2 are the pivotal genes involved in homologous recombination (HR) mutation which can be targeted by poly(ADP-ribose) polymerase pathway (PARP) inhibitors. Through this study we assessed the effects of PARP inhibitor AZD2461 alone and in combination with 2HF on cell viability of MDA-MB-231 and MCF-7 breast cancer cell lines.
Methods: Expression of mRNA of BRCA1, BRCA2 and PALB2 genes was assessed in MDA-MB-231 (triple negative) and MCF-7 (ER+) breast cancer cell lines after exposure to 2HF and LNA by using quantification cycle in Polymerase chain reaction. LDH release at 72 hours was measured and compared with controls in cells treated with 2HF or AZD2461 alone or in combination. Cytotoxicity was measured using absorbance of reduced WST-8 produced by the viable cells.
Results: RLIP depletion by 2HF reduced the expression of mRNA of HR genes BRCA1, BRCA2, and PALB2 to 2% - 20% of the level of untreated controls. PARP inhibitor induced LDH release with incremental dose of 2HF at 10, 20 and 40 µM, with greatest cytotoxic effects at 40 µM 2HF (p < 0.001). Addition of 2HF to PARP inhibitor AZD2461 increased cell lysis in MCF-7 and MDA-MB-231 cells, however only diminished cell viability in MCF-7 but not in MDA-MB-231.
Discussion: PARP inhibitors are very effective in tumors with HRD mutation, however their utility is limited in subjects with mutations involving non HRD pathways. RALBP1 inhibition with 2HF decreases the expression of gene involved in HR pathway thus stimulating a transcriptomic environment that is similar to HRD mutated cells. 2HF could thus sensitize BRCA-wildtype ER+ and possibly triple negative breast cancers to PARP inhibitors. This potentially has clinical application among patients who may benefit from PARP inhibitors outside of current indications such as BRCA or HRD deficient mutations. Through this work, we look forward to inspire more work on clinical exploration of 2HF with PARP inhibitors.
利益披露 Disclosure
H. Khosla, None.