Histone deacetylase (HDAC) inhibitors are promising anticancer drugs but their effect on tumor treatment has been disappointing mainly due to the acquisition of HDAC inhibitor resistance. However, the mechanisms underlying such resistance remain unclear. In this study, we performed Western blot, q-PCR, and promoter assay to examine the expression of HDAC inhibitor-induced phospholipase D2 (PLD2) in MDA-MB231and MDA-MB435 breast cancer cells. Apoptosis and proliferation were analyzed by flow cytometry. In addition to invasion and migration assay, angiogenesis was further measured using in vitro tube formation and chick embryo chorioallantoic membrane model. HDAC inhibitors including suberoylanilide hydroxamic acid (SAHA), trichostatin, and apicidin, induce expression of PLD2 in a transcriptional level. SAHA upregulates expression of PLD2 via protein kinase C-ζ in breast cancer cells and increases the enzymatic activity of PLD. The combination treatment of SAHA with PLD2 inhibitor significantly enhances cell death in breast cancer cells. Phosphatidic acid, a product of PLD activity, prevented apoptosis promoted by cotreatment with SAHA and PLD2 inhibitor, suggesting that SAHA-induced PLD2 expression and subsequent activation of PLD2 might confers resistance of breast cancer cells to HDAC inhibitor. The combinational treatment of the drugs significantly suppressed invasion, migration, and angiogenesis, compared with that of either treatment. These findings provide further insight into elucidating the advantages of combination therapy with HDAC and PLD2 inhibitors over single-agent strategies for the treatment of cancer.

中文翻译：

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磷脂酶D2的抑制增加了乳腺癌中组蛋白脱乙酰基酶抑制剂诱导的细胞死亡

组蛋白脱乙酰基酶（HDAC）抑制剂是很有前途的抗癌药物，但其对肿瘤治疗的效果却令人失望，主要是由于获得了HDAC抑制剂耐药性。但是，这种抗药性的机制尚不清楚。在这项研究中，我们进行了蛋白质印迹，q-PCR和启动子测定，以检查HDAC抑制剂诱导的磷脂酶D2（PLD2）在MDA-MB231和MDA-MB435乳腺癌细胞中的表达。通过流式细胞术分析细胞凋亡和增殖。除了侵袭和迁移分析外，还使用体外管形成和鸡胚绒膜尿囊膜模型进一步测量了血管生成。HDAC抑制剂包括辛二酰氨基苯胺异羟肟酸（SAHA），曲古抑菌素和阿匹西定，可在转录水平诱导PLD2的表达。SAHA通过蛋白激酶C-ζ上调乳腺癌细胞中PLD2的表达，并提高PLD的酶促活性。SAHA与PLD2抑制剂的联合治疗显着提高了乳腺癌细胞的细胞死亡。PLD活性的产物磷脂酸可防止SAHA和PLD2抑制剂共同处理促进细胞凋亡，这表明SAHA诱导的PLD2表达和随后的PLD2激活可能赋予乳腺癌细胞对HDAC抑制剂的抗性。与任一治疗相比，药物的联合治疗显着抑制了侵袭，迁移和血管生成。这些发现为阐明HDAC和PLD2抑制剂联合治疗优于单药治疗癌症的优势提供了进一步的见解。