Department of Neurosurgery, The Third People's Hospital of Dalian, Non‑Directly Affiliated Hospital of Dalian Medical University, Dalian, Liaoning 116033, P.R. China.
Department of Neurology, Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310009, P.R. China.
Int J Mol Med. 2021 May;47(5). doi: 10.3892/ijmm.2021.4901. Epub 2021 Mar 2.
Glioblastoma (GBM) is the most common aggressive brain tumor and is associated with an extremely poor prognosis, as the current standard of care treatments have limited efficacy. Natural compounds have attracted increasing attention as potential anticancer drugs. Alantolactone (ATL) is a natural small molecule inhibitor, that has antitumor properties. In the present study, U87MG and U251 cells were treated ATL and changes in actin/G‑actin/F‑actin/cofilin pathway were detected in whole cells, in the cytoplasm and mitochondria by western blot analysis. Immunofluorescence and immunoprecipitation analysis identified changes in the expression levels of target proteins and interactions, respectively. A LIMK enzyme inhibitor was also applied to assess the effects of ATL on the migration and invasion of GBM cells. Flow cytometry was used to detect the levels of apoptosis of GBM cells. The expression of matrix metalloproteinase (MMP)‑2/MMP‑9, caspase‑3/caspase‑9/poly(ADP‑ribose) polymerase (PARP)/cytochrome , were determined by western blot analysis to assess the effects of targeting LIMK. The findings were verified by characterizing changes in the expression of cofilin/LIMK in xenograft tumors in immunodeficient mice. It was found that ATL activated cofilin through the targeted inhibition of LIMK enzyme activity and it thus upregulated the ratio of G/F actin, and inhibited GBM cell migration and invasion. Conversely, the activation of cofilin and G‑actin could be co‑transferred to the mitochondria to initiate the mitochondrial‑cytochrome pathway to induce apoptosis. On the whole, the findings of the present study further illustrate the molecular mechanisms through which ATL inhibits the metastatic phenotype of GBM cells and induces apoptosis. Given previous findings, it can be deduced that ATL can function through multiple pathways and has multiple targets in GBM models, highlighting its potential for use in clinical applications.
胶质母细胞瘤（GBM）是最常见的侵袭性脑肿瘤，预后极差，因为目前的治疗标准疗效有限。天然化合物作为潜在的抗癌药物越来越受到关注。冬凌草甲素（ATL）是一种天然的小分子抑制剂，具有抗肿瘤特性。在本研究中，通过 Western blot 分析检测了 U87MG 和 U251 细胞中 ATL 处理后整个细胞、细胞质和线粒体中肌动蛋白/肌动蛋白/F-肌动蛋白/丝切蛋白途径的变化。免疫荧光和免疫沉淀分析分别鉴定了靶蛋白表达水平和相互作用的变化。还应用 LIMK 酶抑制剂来评估 ATL 对 GBM 细胞迁移和侵袭的影响。流式细胞术用于检测 GBM 细胞的凋亡水平。通过 Western blot 分析确定基质金属蛋白酶 2/9（MMP-2/9）、半胱天冬酶 3/9/多聚（ADP-核糖）聚合酶（PARP）/细胞色素的表达，以评估靶向 LIMK 的效果。通过表征免疫缺陷小鼠异种移植肿瘤中丝切蛋白/ LIMK 的表达变化来验证结果。结果发现，ATL 通过靶向抑制 LIMK 酶活性激活丝切蛋白，从而上调 G/F 肌动蛋白的比值，抑制 GBM 细胞的迁移和侵袭。相反，丝切蛋白和 G 肌动蛋白的激活可以共转移到线粒体，启动线粒体细胞色素途径诱导细胞凋亡。总的来说，本研究的结果进一步说明了 ATL 抑制 GBM 细胞转移表型和诱导细胞凋亡的分子机制。鉴于先前的研究结果，可以推断 ATL 在 GBM 模型中可以通过多种途径发挥作用，具有多个靶点，突出了其在临床应用中的潜力。
