Nrf2/ARE信号在化疗所致血液毒性/骨髓抑制个体化预测及预防中的作用研究

Hematotoxicity (or myelosuppression) is the most common toxicity and dose-limiting response of chemotherapies. Currently there has no reliable approaches to predict the incidence and severity of chemotherapy-induced myelosuppression; and the clinical management of chemotherapy-induced myelosuppression relies on colony stimulating factors (CSFs) treatment after myelosuppression has been diagnosed. Thus chemotherapy-induced myelosuppression has significantly limited the efficacy of chemotherapeutic regimens, lead to deterioration of survivor's life quality and increasing of medical expenses. The nuclear factor-erythroid 2 p45-related factor 2 (Nrf2), which controls the basal and inducible expression of many antioxidant/detoxifying genes driven by antioxidant response element (ARE), is a pivotal coordinator of cellular defensive machinery, which protects cells against chemical stresses such as oxidative species and chemotherapeutic drugs. Our preliminary studies indicate that the mRNA level of Nrf2 and related genes in the peripheral blood nucleated cells of tumor patients displayed significant inter-individual variations, which significantly reversely correlates with the incidence and severity of leucopenia and neutropenia. In the present study, we will further investigate the correlation of the expression and activity of Nrf2/ARE signaling and its target genes to chemotherapy-induced myelosuppression as well as other clinical indications and outcomes, to discover biomarkers that can personalized predict the incidence and severity of chemotherapy-induced myelosuppression in advance, and to explore the possible molecular events underlying the inter-individual variations. Furthermore, we will study the role of Nrf2 in chemotherapy-induced myelosuppression and the function of chemoprotectants such as amifostine employing Nrf2 knockout mice and cell models, to define the possibility of Nrf2 as a drug target of chemoprotectants agaisnt chemotherapy-induced myelosuppression.

血液毒性/骨髓抑制是化疗最常见的毒性反应和剂量限制效应，临床上还没有有效的预测和预防方法，只能靠经验和集落刺激因子等发生后干预段，极大的影响了化疗效果和患者生存质量，并导致医疗费用显著上升。转录因子Nrf2控制着抗氧化响应元件驱动的抗氧化和药物代谢酶表达，是细胞化学防御系统的枢纽。前期研究发现肿瘤患者外周血有核细胞中Nrf2表达水平存在显著个体差异，且与化疗后骨髓抑制程度呈显著负相关。本研究将进一步明确外周血细胞中Nrf2/ARE信号通路及其调控的基因表达及活性的个体差异与不同化疗方案下骨髓抑制发生情况及其它临床数据的相关性，发现可用于个体化预测化疗所致骨髓抑制发生情况的生物标志物和合适的检测方法，初步探讨其个体差异产生的分子机制；进而利用细胞和Nrf2基因敲除小鼠等模型研究Nrf2/ARE信号及其激动剂对化疗所致血液毒性/骨髓抑制的保护作用，探讨Nrf2作为化疗保护剂作用靶标的可能。

正常组织的毒性和癌细胞的耐药性是化疗失败的两大主要原因。化疗毒副作用包括血液毒性、消化道毒性、肝肾毒性、耳毒性、心脏毒性和外周神经损伤等，其中血液毒性是化疗最常见的毒性反应和剂量限制效应。Nrf2是细胞防御系统的核心调控分子，在化疗耐药性中的作用吸引了大量的研究者注意，但其在化疗毒性中的作用却极少有人涉足。我们认为，Nrf2是潜在的个体化预测化疗毒性的生物标志物以及化疗保护药物的作用靶标，并围绕上述科学假设展开了从细胞、动物到临床的系统研究，作出了以下重要发现：.1. Nrf2表达水平是决定环磷酰胺、顺铂、紫杉醇等临床常用药物血液毒性的重要因素，也可以作为化疗毒性保护药物的靶标，同时外周血单个核细胞的Nrf2表达水平与临床上紫杉醇、吉西他滨所导致的骨髓抑制毒性显著负相关；其分子机制与Nrf2调控的抗氧化和代谢解毒基因表达相关。该发现基本实现了本项目的研究目标，回答了本项目的关键科学问题。.2. 敲除Nrf2显著加剧小鼠中伊立替康所致脂肪性肝炎/肠毒性和奥沙利铂所致外周神经毒性，其中伊立替康所致脂肪性肝炎在野生小鼠中几乎观察不到；而激活Nrf2则可以显著减轻上述毒性，通过选择适当的激动剂（sulforaphane和isoliquiritigenin）可以达到仅激活正常组织（血液、骨髓、肝、肠、外周神经）中Nrf2而不影响肿瘤中Nrf2活性，从而选择性保护正常组织而不降低化疗疗效。.3. 利用体外培养的原代野生型和Nrf2敲除骨髓细胞、肝细胞和胚胎成纤维细胞对阿霉素类、铂类、生物碱类、拓扑异构酶抑制剂类、抗肿瘤抗生素类、烷化剂类、TKI类、抗代谢类、蒽环类、紫杉醇等25种药物的毒性进行了筛选，发现其中约1/3药物毒性与Nrf2相关。还利用CRISPR/Cas9技术构建了Nrf2敲除的结肠癌、肝癌和乳腺癌细胞系，对上述药物的耐药性也进行了系统筛选，发现超过2/3的药物耐药性都与Nrf2相关。.此外，我们从大量的天然产物中筛选发现了若干种Nrf2激动剂，探索了运动对不同组织中Nrf2的激活作用，并研究了其对化疗毒性的保护作用，同时也研究了Nrf2在其它毒性中的作用。