Upregulating DAB2IP expression via EGR-1 inhibition, a new approach for overcoming fractionated-irradiation-induced cross-tolerance to ionizing radiation and mitomycin C in tumor cells

Purpose: To evaluate the effect of fractionated irradiation (FI) on tumor cells’ sensitivity to ionizing radiation (IR) and antineoplastic drugs, and examine the potential of early growth response-1 (EGR-1) inhibition to sensitize tumor cells to IR. Materials and methods: PC3 and HepG2 cells were subjected 10 times to γ-rays at 2 Gy. The surviving cells were named PC3/R and HepG2/R, respectively. The cells’ sensitivity to irradiation and chemotherapeutic drugs, including cisplatin (PT), doxorubicin (DOX), mitomycin C (MMC) and 5-fluorouracil (5-FU), were identified by colony formation assay and MMT method, respectively. Quantitative real-time polymerase chain reaction (RT-qPCR) analysis was utilized to compare the difference of gene expression between radioresistant cells and parental cells. The small interfering RNA system was implemented to inhibit endogenous EGR-1 expression in radiation-resistant cells. Western blot was employed to identify the possible mechanism by which EGR-1 regulates cells’ radiosensitivity. Results: FI induced cross-resistant to IR and MMC in tumor cells. Along with the reduction of ovarian cancer-2/disabled homolog 2 (DOC-2/DAB2) interactive protein (DAB2IP) expression, EGR-1 gene was upregulated in FI-treated cells. On the other hand, downregulation of EGR-1 gene expression sensitized radioresistant cells to IR accompanied by DAB2IP overexpression and STAT3 inactivation. In addition, NF-κB inhibitor, BAY11-7082 enhanced resistant cells’ radiosensitivity and chemosensitivity. Conclusions: Conventionally FI has a higher risk of forming acquired radioresistance (ARR) in vitro. EGR-1 gene-targeted drug design could be an effective strategy to overcome DAB2IP-dysregulation-induced ARR in tumor patients.

研究目的：本研究旨在评估分次照射（fractionated irradiation, FI）对肿瘤细胞电离辐射与抗肿瘤药物敏感性的影响，并探究早期生长应答因子1（early growth response-1, EGR-1）抑制能否增强肿瘤细胞对电离辐射的敏感性。材料与方法：将PC3细胞与HepG2细胞以2 Gy剂量的γ射线连续照射10次，存活的细胞分别命名为PC3/R与HepG2/R。分别采用集落形成实验与MTT法检测细胞对电离辐射及顺铂（cisplatin, PT）、阿霉素（doxorubicin, DOX）、丝裂霉素C（mitomycin C, MMC）与5-氟尿嘧啶（5-fluorouracil, 5-FU）等化疗药物的敏感性。采用实时定量聚合酶链反应（RT-qPCR）分析比较放射抵抗细胞与亲代细胞的基因表达差异。利用小干扰RNA系统抑制放射抵抗细胞内源性EGR-1的表达。采用蛋白质免疫印迹（Western blot）实验探究EGR-1调控细胞放射敏感性的潜在分子机制。结果：分次照射可诱导肿瘤细胞对电离辐射与丝裂霉素C产生交叉耐药。经分次照射处理的细胞中，EGR-1基因表达上调，同时卵巢癌2/disabled同源物2（DOC-2/DAB2）相互作用蛋白（DAB2IP）的表达水平降低。另一方面，下调EGR-1基因表达可使放射抵抗细胞对电离辐射的敏感性增强，同时伴随DAB2IP表达上调与信号转导与转录激活因子3（signal transducer and activator of transcription 3, STAT3）信号通路失活。此外，核因子κB（NF-κB）抑制剂BAY11-7082可增强耐药细胞的放射敏感性与化学敏感性。结论：常规分次照射在体外实验中更易诱导获得性放射抵抗（acquired radioresistance, ARR）。靶向EGR-1基因的药物设计，或可成为克服肿瘤患者中由DAB2IP表达失调诱导的获得性放射抵抗的有效策略。