肽类抗生素放线菌素D（Act D）被临床应用于抑制恶性肿瘤的生长。Act D结合DNA的转录起始复合物，以防止RNA的延伸。Act D会导致DNA损伤、生长抑制和细胞死亡。髓细胞白血病（Mcl-1）是一种抗凋亡的Bcl-2家族成员蛋白，本研究探究了Act D引起的Mcl-1下调的效应和分子机制。通过细胞实验方法，利用人腺癌A549细胞检查Act D的细胞毒性信号通路，特别是在凋亡机制方面。检查了特定阻断剂对凋亡因子的可能作用。我们发现，Act D会造成细胞生长抑制和凋亡。基于丙碘留流式细胞分析和免疫染色的结果确认了细胞凋亡。Act D处理导致DNA损伤，之后出现p53非依赖性的细胞死亡。Western blotting显示Mcl-1表达显著降低，线粒体跨膜电位下降和caspase-9/caspase-3级联激活。蛋白酶体抑制剂MG132逆转了Act D引起的Mcl-1下调。然而，利用药物抑制糖原合成酶激酶-3、p53表达、内质网应激、自噬和小泡酸化等调节Mcl-1的信号通路不能挽救这些效果。值得注意的是，Cullin-Ring E3连接酶部分介导了Mcl-1的下调。给予转化生长因子-β可促进间充质细胞分化，但Act D仍然会导致Mcl-1降低并引起细胞凋亡。所有这些数据表明，Act D有可能通过促进Mcl-1非规范性下调而具有促凋亡作用。版权所有 ©2023 Elsevier Inc.。保留所有权利。

Actinomycin (Act) D, a polypeptide antibiotic, is used clinically to inhibit the growth of malignant tumors. Act D binds to DNA at the transcription initiation complex to prevent the elongation of RNA. Act D causes DNA damage, growth inhibition, and cell death. Myeloid cell leukemia (Mcl-1) is an anti-apoptotic Bcl-2 family member protein, and the present study explored the effects and molecular mechanism of Act D-induced Mcl-1 downregulation.Human adenocarcinoma A549 cells were used to check the cytotoxic signaling pathways of Act D, particularly in apoptotic mechanism, in a cell-based study approach. Specific blockers targeting the apoptotic factors were examined for their possible roles.We found that Act D caused cell growth inhibition and apoptosis. Propidium iodide-based flow cytometric analysis and immunostaining confirmed cell apoptosis. Treatment with Act D caused DNA damage, followed by p53-independent cell death. Western blotting showed a significant decrease in Mcl-1 expression, mitochondrial transmembrane potential loss, and caspase-9/caspase-3 cascade activation. The proteasome inhibitor MG132 reversed Act D-induced Mcl-1 downregulation. However, pharmacological inhibition of glycogen synthase kinase-3, p53 expression, ER stress, autophagy, and vesicle acidification, which are Mcl-1-regulating signaling pathways, did not rescue these effects. Notably, Cullin-Ring E3 ligase partially mediated Mcl-1 downregulation. Administration of transforming growth factor-β induced mesenchymal cell differentiation, but Act D still decreased Mcl-1 and caused cell apoptosis.All of these data show a potential pro-apoptotic effect for Act D by facilitating Mcl-1 uncanonical downregulation.Copyright © 2023 Elsevier Inc. All rights reserved.