Zhou Qianqian, Song Chao, Liu Xiaoqiu, Qin Hao, Miao Lixia, Zhang Xuesen
State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, China,
Key Laboratory of Pathogen Biology of Jiangsu Province, Nanjing Medical University, Nanjing, China.
Cancer Manag Res. 2019 Jan 10;11:625-636. doi: 10.2147/CMAR.S191353. eCollection 2019.
Adriamycin (ADR) is widely used in the clinical chemotherapy against breast cancer. But its efficacy is strongly limited due to the acquisition of multidrug resistance (MDR). Therefore, acquisition of the resistance to ADR is still a major cause of chemotherapy failure in breast cancer patients. Peptidylarginine deiminase IV (PAD4) is reported to target non-histone proteins for citrullination, regulate their substrate activities, and thereby play critical roles in maintaining cell phenotype in breast cancer cells. However, whether PAD4 is involved in the development of MDR in breast cancer is poorly understood.
We examined the expression of PAD family members, including PAD4 in ADR-resistant MCF-7 cells compared with the parental control cells by real-time PCR and Western blotting analyses. Rescue of PAD4 expression in MCF-7/ADR cells was performed to assess whether PAD4 could restore the sensitivity of MCF-7/ADR cells to ADR treatment with cell counting kit-8, flow cytometry, TUNEL, nuclear and cytoplasmic extract preparations, and immunofluorescence staining analyses.
Both PAD2 and PAD4 were significantly decreased in ADR-resistant cells. However, only PAD4 overexpression can increase the sensitivity of MCF-7/ADR cells to ADR treatment and decrease gene expression. Overexpression of PAD4 in MCF-7/ADR cells inhibited cell proliferation by inducing cell apoptosis. Under ADR treatment, overexpression of PAD4 promoted nuclear accumulation of glycogen synthase kinase-3β and p53, which further activated proapoptotic gene expression and downregulated expression. Moreover, PAD4 activity was required for activating proapoptotic gene transcripts.
We demonstrate the previously unappreciated role of PAD4 in reversing ADR resistance in MCF-7/ADR cells and help establish PAD4 as a candidate biomarker of prognosis and chemotherapy target for MDR in breast cancers.
阿霉素（ADR）广泛应用于乳腺癌的临床化疗。但其疗效因多药耐药（MDR）的产生而受到严重限制。因此，对ADR产生耐药性仍是乳腺癌患者化疗失败的主要原因。据报道，肽基精氨酸脱亚氨酶IV（PAD4）可使非组蛋白发生瓜氨酸化，调节其底物活性，从而在维持乳腺癌细胞的细胞表型中发挥关键作用。然而，PAD4是否参与乳腺癌MDR的发生发展尚不清楚。
通过实时PCR和蛋白质免疫印迹分析，我们检测了耐药性MCF-7细胞中包括PAD4在内的PAD家族成员的表达，并与亲本对照细胞进行比较。在MCF-7/ADR细胞中进行PAD4表达的挽救实验，通过细胞计数试剂盒-8、流式细胞术、TUNEL、细胞核和细胞质提取物制备以及免疫荧光染色分析，评估PAD4是否能恢复MCF-7/ADR细胞对ADR治疗的敏感性。
耐药细胞中PAD2和PAD4均显著降低。然而，只有PAD4过表达能增加MCF-7/ADR细胞对ADR治疗的敏感性并降低基因表达。MCF-7/ADR细胞中PAD4过表达通过诱导细胞凋亡抑制细胞增殖。在ADR处理下，PAD4过表达促进糖原合酶激酶-3β和p53的核积累，进而激活促凋亡基因表达并下调表达。此外，激活促凋亡基因转录需要PAD4活性。
我们证明了PAD4在逆转MCF-7/ADR细胞对ADR耐药性方面以前未被认识的作用，并有助于将PAD4确立为乳腺癌MDR预后的候选生物标志物和化疗靶点。
