胰腺导管腺癌（PDAC）是一种高度侵袭性的癌症类型，迫切需要有效的治疗策略。穿心莲内酯是穿心莲中富含的一种劳丹烷二萜化合物，对多种癌症类型具有抗癌作用，但其抗癌活性和针对 PDAC 的机制在很大程度上尚未得到表征。本研究探索穿心莲内酯针对 PDAC 的新药物靶点和潜在分子机制。使用 MTT、克隆形成实验和 Transwell 迁移实验测量 PDAC 细胞、PANC-1 和 MIA PaCa-2 细胞的恶性表型。 PDAC异种移植动物模型用于评估体内肿瘤生长。使用蛋白质印迹、免疫荧光和免疫组织化学来测量蛋白表达。分析 TCGA 数据库以评估启动子甲基化状态、基因表达及其与患者生存率的关系。 RT-qPCR用于检测mRNA表达。报告基因检测用于检测信号转导途径。通过亚硫酸氢钠处理和甲基化特异性 PCR (MSP) 测定启动子 DNA 甲基化。通过基因过表达来测量参与药物作用的特定基因的生物学功能和作用。穿心莲内酯治疗抑制了PDAC细胞的增殖和迁移，并损害了体内肿瘤的生长。此外，穿心莲内酯诱导 PDAC 细胞中锌指蛋白 382 (ZNF382) 的 mRNA 和蛋白表达。 ZNF382 的过度表达可抑制恶性表型和癌症相关信号通路（AP-1、NF-κB 和 β-catenin）以及癌基因（ZEB-1、STAT-3、STAT-5 和 HIF-1α）。 ZNF382 的过表达延迟了 PANC-1 细胞体内的生长。胰腺癌患者肿瘤组织中ZNF382 mRNA和蛋白表达量低于癌旁正常组织。 TCGA 数据库分析发现，ZNF382 启动子在原发性胰腺肿瘤中高度甲基化，这与其低表达相关。此外，穿心莲内酯抑制 DNA 甲基转移酶 3 β (DNMT3B) 的表达，并增加 PDAC 细胞中 ZNF382 启动子的去甲基化。 DNMT3B 的过度表达减弱了穿心莲内酯抑制的 PDAC 细胞的增殖和迁移。我们的研究结果表明，ZNF382 在胰腺癌中充当抑癌基因，穿心莲内酯恢复 ZNF382 表达以抑制胰腺癌，为胰腺癌提供了新的分子靶点和有前景的治疗方法。治疗胰腺癌。版权所有 © 2024 Elsevier GmbH。版权所有。

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer type that urgently requires effective therapeutic strategies. Andrographolide, a labdane diterpenoid compound abundant in Andrographis paniculata, has anticancer effects against various cancer types, but its anticancer activity and mechanism against PDAC remain largely uncharacterized.This study explores novel drug target(s) and underlying molecular mechanism of andrographolide against PDAC.The malignant phenotypes of PDAC cells, PANC-1 and MIA PaCa-2 cells, were measured using MTT, clonogenic assays, and Transwell migration assays. A PDAC xenograft animal model was used to evaluate tumor growth in vivo. Western blot, immunofluorescence and immunohistochemistry were used for measuring protein expression. The TCGA database was analyzed to evaluate promoter methylation status, gene expression, and their relationship with patient survival rates. RT-qPCR was used for detecting mRNA expression. Reporter assays were used for detecting signal transduction pathways. Promoter DNA methylation was determined by sodium bisulfite treatment and methylation-specific PCR (MSP). The biological function and role of specific genes involved in drug effects were measured through gene overexpression.Andrographolide treatment suppressed the proliferation and migration of PDAC cells and impaired tumor growth in vivo. Furthermore, andrographolide induced the mRNA and protein expression of zinc finger protein 382 (ZNF382) in PDAC cells. Overexpression of ZNF382 inhibited malignant phenotypes and cancer-associated signaling pathways (AP-1, NF-κB and β-catenin) and oncogenes (ZEB-1, STAT-3, STAT-5, and HIF-1α). Overexpression of ZNF382 delayed growth of PANC-1 cells in vivo. ZNF382 mRNA and protein expression was lower in tumor tissues than in adjacent normal tissues of pancreatic cancer patients. Analysis of the TCGA database found the ZNF382 promoter is hypermethylated in primary pancreatic tumors which correlates with its low expression. Furthermore, andrographolide inhibited the expression of DNA methyltransferase 3 beta (DNMT3B) and increased the demethylation of the ZNF382 promoter in PDAC cells. Overexpression of DNMT3B attenuated the andrographolide-suppressed proliferation and migration of PDAC cells.Our finding revealed that ZNF382 acts as a tumor suppressor gene in pancreatic cancer and andrographolide restores ZNF382 expression to suppress pancreatic cancer, providing a novel molecular target and a promising therapeutic approach for treating pancreatic cancer.Copyright © 2024 Elsevier GmbH. All rights reserved.