BACKGROUND 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. MATERIALS AND METHODS 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. RESULTS 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 MDR1 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 MDR1 expression. Moreover, PAD4 activity was required for activating proapoptotic gene transcripts. CONCLUSION 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.

中文翻译：

---

肽精氨酸脱亚胺酶 4 过表达通过 GSK3β/p53 激活使 MCF-7/ADR 乳腺癌细胞对阿霉素重新敏感。

背景技术阿霉素(ADR)广泛用于乳腺癌的临床化疗。但由于获得多药耐药性 (MDR)，其疗效受到极大限制。因此，获得对 ADR 的耐药性仍然是乳腺癌患者化疗失败的主要原因。据报道，肽精氨酸脱亚胺酶 IV (PAD4) 靶向非组蛋白进行瓜氨酸化，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 的发展尚不清楚。材料和方法 我们通过实时 PCR 和蛋白质印迹分析检测了 PAD 家族成员的表达，包括 ADR 抗性 MCF-7 细胞中的 PAD4 与亲本对照细胞相比。拯救 MCF-7/ADR 细胞中的 PAD4 表达，以评估 PAD4 是否可以恢复 MCF-7/ADR 细胞对细胞计数试剂盒 8、流式细胞术、TUNEL、细胞核和细胞质提取物制剂的 ADR 处理的敏感性，以及免疫荧光染色分析。结果 ADR 抗性细胞中 PAD2 和 PAD4 均显着降低。然而，只有 PAD4 过表达可以增加 MCF-7/ADR 细胞对 ADR 治疗的敏感性并降低 MDR1 基因表达。PAD4 在 MCF-7/ADR 细胞中的过表达通过诱导细胞凋亡来抑制细胞增殖。在 ADR 处理下，PAD4 的过表达促进了糖原合酶激酶 3β 和 p53 的核积累，这进一步激活了促凋亡基因的表达并下调了 MDR1 的表达。而且，PAD4 活性是激活促凋亡基因转录本所必需的。结论 我们证明了 PAD4 在逆转 MCF-7/ADR 细胞中的 ADR 耐药性中以前未被重视的作用，并有助于将 PAD4 确立为乳腺癌 MDR 预后和化疗靶点的候选生物标志物。