Large/giant congenital nevi (L/GCMN) are benign neoplasms of the melanocytic neural crest lineage covering extensive areas of skin presenting risk for melanoma. Surgical resection often leads to scarring and trauma. Histone deacetylase inhibitors (iHDACs) as topical therapeutic agents may prove beneficial as an alternative/adjunct to surgery in this disease. Here we describe the effect of in vitro treatment of iHDACs drugs on primary nevocytes isolated from L/GCMN patients. Micropthalmia transcription factor (MITF) expression in L/GCMN patients' lesions was detected by immunohistochemistry, in cultured nevocytes by immunofluorescence, immunoblot and quantitative polymerase chain reaction. Cellular senescence was detected by SA-ß galactosidase activity. Markers for melanocytic differentiation were evaluated by immunoblot analysis and extracted melanin content was estimated spectrophotometrically. Cell death was measured by lactate dehydrogenase (LDH) assay and necrosis confirmed by polymerase (PARP) cleavage and acridine orange staining of the nuclei. MITF was expressed ubiquitously in nevocytes and melanocytes in patients' lesions. In culture, iHDAC treatment suppressed MITF protein and mRNA expression resulting in a senescent-like phenotype with positive ß-galactosidase staining, progressing to necrotic cell death as evidenced by increased LDH activity, appearance of cleaved PARP and necrotic nuclei. This is the first report showing evidence of iHDACs-induced MITF suppression in congenital nevocytes in vitro leading to a morphologic change with positive ß-galactosidase staining, followed by necrotic cell death in nevocytes, indicating that iHDAC drugs could be valuable therapeutic agents for treatment of L/GCMN lesions.

中文翻译：

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组蛋白脱乙酰酶抑制剂伏立诺他 (SAHA) 抑制小眼转录因子表达并诱导大/巨大先天性黑素细胞痣的色素细胞死亡。

大/巨大先天性痣 (L/GCMN) 是黑素细胞神经嵴谱系的良性肿瘤，覆盖广泛的皮肤区域，存在患黑色素瘤的风险。手术切除常常会导致疤痕和创伤。组蛋白脱乙酰酶抑制剂（iHDAC）作为局部治疗剂可能被证明作为这种疾病手术的替代/辅助手段是有益的。在这里，我们描述了 iHDAC 药物体外治疗对从 L/GCMN 患者分离的原代神经细胞的影响。采用免疫组织化学方法检测L/GCMN患者病灶中小眼转录因子（MITF）的表达，通过免疫荧光、免疫印迹和定量聚合酶链反应检测培养的神经细胞中小眼转录因子（MITF）的表达。通过 SA-ß 半乳糖苷酶活性检测细胞衰老。通过免疫印迹分析评估黑素细胞分化的标记，并通过分光光度法估计提取的黑色素含量。通过乳酸脱氢酶（LDH）测定测量细胞死亡，并通过聚合酶（PARP）裂解和细胞核吖啶橙染色确认细胞坏死。MITF 在患者病变的色素细胞和黑素细胞中普遍表达。在培养物中，iHDAC 处理抑制 MITF 蛋白和 mRNA 表达，导致 β-半乳糖苷酶染色呈阳性的衰老样表型，并进展为坏死性细胞死亡，如 LDH 活性增加、出现裂解的 PARP 和坏死细胞核所证明。这是第一份报告，显示 iHDAC 诱导的先天性神经细胞 MITF 抑制在体外导致 β-半乳糖苷酶染色阳性的形态变化，随后神经细胞坏死，表明 iHDAC 药物可能是治疗先天性神经细胞的有价值的治疗剂。 L/GCMN 病变。