骨肉瘤（OS）的特点是生长迅速、肺转移频繁。东革阿里 (Eurycoma longifolia Jack) 是一种主要产于东南亚国家的开花植物，常用于传统草药。其根提取物主要用于对抗癌症、疟疾、寄生虫等疾病。其根提取物中的活性化合物广酰马酮（EUR）已被证明可以抑制肺癌和肝癌的增殖。我们的研究旨在探讨EUR对OS生长和转移的抑制作用及其潜在分子机制。体外实验：蛋白质印迹(WB) 筛选了 41 种抑制 GRP78 表达的化合物，并评估了 GRP78、PARP、cleaved-PARP、MMP2 和 MMP9 的蛋白水平。使用CCK-8、EdU、集落形成测定评估细胞增殖，并通过流式细胞术评估细胞凋亡。进行 Transwell、伤口愈合和管形成测定，分别确定 EUR 对肿瘤侵袭、迁移和血管生成的影响。定量实时聚合酶链 (qRT-PCR) 和双荧光素酶活性测定检测了 EUR 和毒胡萝卜素处理后的 GRP78 mRNA 稳定性和转录水平。 RNA-Seq 鉴定了 EUR 抑制的信号通路。体内实验：通过 H 评估 EUR 对小鼠的影响

Osteosarcoma (OS) is characterized by rapid growth and frequent pulmonary metastasis. Eurycoma longifolia Jack, a flowering plant primarily found in Southeast Asian countries, is commonly used in traditional herbal medicine. Its root extract is mainly used for against cancer, malaria, parasites and other conditions. The active compound in its root extract, eurycomanone (EUR), has been proven to inhibit lung and liver cancer proliferation.Our research aimed to investigate the inhibitory effect and underlying molecular mechanism of EUR on OS growth and metastasis.In vitro experiments: western blotting (WB) screened 41 compounds that inhibited GRP78 expression and evaluated the protein levels of GRP78, PARP, cleaved-PARP, MMP2, and MMP9. Cell proliferation was evaluated using CCK-8, EdU, colony formation assay, and cell apoptosis was assessed by flow cytometry. Transwell, wound healing, and tube formation assays were performed to determine the effect of EUR on tumor invasion, migration, and angiogenesis, respectively. Quantitative real-time polymerase chain (qRT-PCR) and dual-luciferase activity assays detected GRP78 mRNA stability and transcription levels post-EUR and thapsigargin treatment. RNA-Seq identified signaling pathways inhibited by EUR. In vivo experiments: effects of EUR in mice were evaluated by H&E staining to detect lung metastasis and potential toxic effects in tissues. Immunohistochemical (IHC) staining detected the expression of Ki-67, CD31, and cleaved caspase-3 in tumors.GRP78 is highly expressed in OS and correlated with poor prognosis. In vitro, eurycomanone (EUR) significantly downregulated GRP78 expression, inhibited cell proliferation, migration, invasion, tube formation, and induced apoptosis. Moreover, it enhanced trichostatin A (TSA) sensitivity and exhibited inhibitory effects on other cancer types. Mechanistically, EUR decreased GRP78 mRNA stability and transcription. In vivo, EUR inhibited proliferation and invasion in tibial and PDX models.Our study demonstrated that EUR inhibits the growth and metastasis of OS by reducing GRP78 mRNA stability and inhibiting its transcription, which offers a novel approach for clinical treatment of OS.Copyright © 2024. Published by Elsevier B.V.