Selective lysine-specific demethylase 1 inhibitor, NCL1, could cause testicular toxicity via the regulation of apoptosis.
Selective lysine-specific demethylase 1 inhibitor, NCL1, could cause testicular toxicity via the regulation of apoptosis.
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作者：
Nozaki S;Naiki T;Naiki-Ito A;Iwatsuki S;Takeda T;Etani T;Nagai T;Iida K;Kato H;Suzuki T;Takahashi S;Umemoto Y;Yasui T
最近的研究表明，表观遗传改变，例如涉及赖氨酸特异性去甲基酶 1 (LSD1) 的改变，会导致致癌激活，并将此类改变作为治疗靶点。然而，目前尚缺乏评估 LSD1 抑制剂对男性生育能力影响的研究。我们分析了新型选择性 LSD1 抑制剂 N-[(1S)-3-[3-(反式-2-氨基环丙基)苯氧基]-1-(苄基氨基甲酰基)丙基]苯甲酰胺 (NCL1) 在睾丸中的潜在毒性。对人类睾丸样本进行免疫组织化学分析。六周龄雄性 C57BL/6J 小鼠腹膜内注射二甲亚砜媒介物 (n = 15)，或每两周注射 1.0 (n = 15) 或 3.0 (n = 15) mg/kg NCL1。五周后，利用 RNA 序列数据和定量逆转录酶 (qRT)-PCR，通过独创性途径分析 (IPA) 分析睾丸样本的毒性和基因表达；对血液样本中的荷尔蒙损害进行了分析。通过细胞活力、染色质免疫沉淀、流式细胞术和蛋白质印迹分析 NCL1 处理的 GC-1、TM3 和 TM4 细胞系。 LSD1主要在人类支持细胞和生殖细胞中表达，LSD1水平以减数分裂依赖性进行性显着降低；生殖细胞在正常精子发生和早/晚成熟停滞中表现出相似的表达模式。组织学检查显示，与对照组相比，3.0 mg/kg NCL1 治疗的小鼠异常生精小管水平显着增加，细胞脱离、脱落、空泡化、嗜酸性粒细胞变化和 TUNEL 阳性细胞增加。 IPA 和 qRT-PCR 显示 NCL1 治疗下调了 LSD1 活性。 NCL1 还降低了血清总睾酮水平。小鼠睾丸样本的蛋白质印迹显示，NCL1 诱导裂解的半胱天冬酶 3、7 和 8 以及连接蛋白 43 蛋白显着升高。 NCL1 治疗以剂量依赖性方式显着降低 GC-1 细胞活力，但不降低 TM3 和 TM4 细胞活力。在流式细胞术分析中，NCL1 诱导 GC-1 细胞凋亡。针对 LSD1 的高剂量 NCL1 治疗导致精子发生功能障碍并诱导 caspase 依赖性细胞凋亡。这表明LSD1抑制剂可能通过调节细胞凋亡而引起睾丸毒性。
Recent studies have shown that epigenetic alterations, such as those involving lysine‐specific demethylase 1 (LSD1), lead to oncogenic activation and highlight such alterations as therapeutic targets. However, studies evaluating the effect of LSD1 inhibitors on male fertility are lacking. We analyzed the potential toxicity of a new selective LSD1 inhibitor, N‐[(1S)‐3‐[3‐(trans‐2‐aminocyclopropyl)phenoxy]‐1‐(benzylcarbamoyl)propyl] benzamide (NCL1), in testes. Human testicular samples were immunohistochemically analyzed. Six‐week‐old male C57BL/6J mice were injected intraperitoneally with dimethyl sulfoxide vehicle (n = 15), or 1.0 (n = 15) or 3.0 (n = 15) mg/kg NCL1 biweekly. After five weeks, toxicity and gene expression were analyzed in testicular samples by ingenuity pathway analysis (IPA) using RNA sequence data and quantitative reverse transcriptase (qRT)–PCR; hormonal damage was analyzed in blood samples. NCL1 treated GC‐1, TM3, and TM4 cell lines were analyzed by cell viability, chromatin immunoprecipitation, flow cytometry, and Western blot assays. LSD1 was mainly expressed in human Sertoli and germ cells, with LSD1 levels significantly decreased in a progressive meiosis‐dependent manner; germ cells showed similar expression patterns in normal spermatogenesis and early/late maturation arrest. Histological examination revealed significantly increased levels of abnormal seminiferous tubules in 3.0 mg/kg NCL1–treated mice compared to control, with increased cellular detachment, sloughing, vacuolization, eosinophilic changes, and TUNEL‐positive cells. IPA and qRT–PCR revealed NCL1 treatment down‐regulated LSD1 activity. NCL1 also reduced total serum testosterone levels. Western blots of mouse testicular samples revealed NCL1 induced a marked elevation in cleaved caspases 3, 7, and 8, and connexin 43 proteins. NCL1 treatment significantly reduced GC‐1, but not TM3 and TM4, cell viability in a dose‐dependent manner. In flow cytometry analysis, NCL1 induced apoptosis in GC‐1 cells. High‐dose NCL1 treatment targeting LSD1 caused dysfunctional spermatogenesis and induced caspase‐dependent apoptosis. This suggests the LSD1 inhibitor may cause testicular toxicity via the regulation of apoptosis.
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