背景:替莫唑胺(TMZ)耐药性是多形性胶质母细胞瘤(GBM)治疗面临的主要障碍。Muscone,麝香(Moschus)的主要活性药理成分之一,可以穿过血脑屏障(BBB),并且正在作为抗肿瘤药物进行研究。然而,Muscone治疗GBM的研究很少,其可能的机制尚不清楚。
目的:本研究旨在评估麝香酮对TMZ抗性GBM细胞的影响和潜在的分子机制。
方法:使用GEO2R筛选TMZ抗性GBM细胞和TMZ敏感GBM细胞之间的差异表达基因(DEGs)。通过逐步提高TMZ浓度,建立了相对稳定的抗TMZ人GBM细胞系。通过CCK-8测定和MGMT和TOP2A表达的蛋白质印迹分析评估U251-TR细胞的耐药性特征。细胞活力,细胞增殖,细胞迁移能力,和药物协同作用通过CCK-8检测,集落形成试验,伤口愈合试验,和药物相互作用关系测试,分别。Anoikis通过钙黄绿素-AM/EthD-1染色定量,MTT测定,和流式细胞术。细胞周期停滞的测量,凋亡,线粒体膜电位(MMP),和活性氧(ROS)使用细胞周期染色进行,膜联蛋白V-FITC/PI标签,JC-1测定,和ROS测定,分别。DNA损伤通过TUNEL测定来测量,碱性彗星试验,和γ-H2AX病灶测定。GEPIA用于研究失巢凋亡标记(FAK)/耐药基因与EGFR/整合素β1信号通路中关键蛋白之间的联系。分子对接用于预测麝香酮的可能靶标。使用免疫荧光显示EGFR和FAK的细胞内共定位和表达。使用慢病毒转导构建稳定过表达EGFR的U251-TR细胞系,以评估EGFR相关信号传导在失巢凋亡抗性中的参与。WesternBlot用于检测迁移相关蛋白的表达,细胞周期蛋白,与之相关的蛋白质,DNA损伤/修复相关蛋白,和相关的途径蛋白。
结果:DEGs分析在TMZ耐药GBM细胞中鉴定出97个化疗耐药基因和3779个上调基因。随后的实验验证了在连续低剂量TMZ诱导的U251-TR细胞中TMZ抗性和DNA修复相关基因(TOP2A和MGMT)的高表达。Muscone对U251-TR细胞迁移和增殖表现出剂量依赖性抑制,其与TMZ共同给药显示出增强治疗效果的潜力。通过下调FAK,麝香酮降低了锚定非依赖性U251-TR细胞的抗肛门凋亡性。它还通过上调p21并下调CDK1,CDK2和CyclinE1而导致G2/M期细胞周期停滞。Muscone诱导的失巢凋亡伴随线粒体膜电位崩溃,ROS生产,BAX/Bcl-2比值增加,以及细胞色素c(Cytc)水平升高,切割的caspase-9和切割的caspase-3。这些发现表明,麝香酮可能通过ROS的产生触发线粒体依赖性失巢凋亡。此外,显著的DNA损伤,DNA双链断裂(DSB),γ-H2AX灶的形成,和TOP2A表达的减少也与麝香酮诱导的失巢凋亡有关。EGFR在U251-TR细胞中的过表达促进整合素β1,FAK的表达,β-连环蛋白,TOP2A,而麝香酮抑制EGFR的表达水平,整合素β1,β-连环蛋白,FAK,TOP2AMuscone可能会影响关键DNA修复酶的表达,TOP2A,通过抑制EGFR/整合素β1/FAK途径。
结论:我们首先证明了麝香酮通过EGFR/整合素β1/FAK途径抑制TOP2A表达,从而恢复TMZ抗性GBM细胞的失巢凋亡敏感性。这些数据表明,麝香酮可能是增强GBM治疗的有前途的联合治疗剂,特别是在TOP2A表达升高的TMZ抗性GBM的情况下。
BACKGROUND: Temozolomide (TMZ) resistance is the main obstacle faced by glioblastoma multiforme (GBM) treatment. Muscone, one of the primary active pharmacological ingredients of Shexiang (Moschus), can cross the blood-brain barrier (BBB) and is being investigated as an antineoplastic medication. However, muscone treatment for GBM has received little research, and its possible mechanisms are still unclear.
OBJECTIVE: This study aims to evaluate the effect and the potential molecular mechanism of muscone on TMZ-resistant GBM cells.
METHODS: The differentially expressed genes (DEGs) between TMZ-resistant GBM cells and TMZ-sensitive GBM cells were screened using GEO2R. By progressively raising the TMZ concentration, a relatively stable TMZ-resistant human GBM cell line was established. The drug-resistance traits of U251-TR cells were assessed via the CCK-8 assay and Western Blot analysis of MGMT and TOP2A expression. Cell viability, cell proliferation, cell migration ability, and drug synergism were detected by the CCK-8 assay, colony formation assay, wound healing assay, and drug interaction relationship test, respectively. Anoikis was quantified by Calcein-AM/EthD-1 staining, MTT assay, and flow cytometry. Measurements of cell cycle arrest, apoptosis, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) were performed using cell cycle staining, Annexin V-FITC/PI labeling, JC-1 assay, and ROS assay, respectively. DNA damage was measured by TUNEL assay, alkaline comet assay, and γ-H2AX foci assay. GEPIA was used to investigate the link between the anoikis marker (FAK)/drug resistance gene and critical proteins in the EGFR/Integrin β1 signaling pathway. Molecular docking was used to anticipate the probable targets of muscone. The intracellular co-localization and expression of EGFR and FAK were shown using immunofluorescence. The U251-TR cell line stably overexpressing EGFR was constructed using lentiviral transduction to assess the involvement of EGFR-related signaling in anoikis resistance. Western Blot was employed to detect the expression of migration-related proteins, cyclins, anoikis-related proteins, DNA damage/repair-related proteins, and associated pathway proteins.
RESULTS: DEGs analysis identified 97 deregulated chemotherapy-resistant genes and 3779 upregulated genes in TMZ-resistant GBM cells. Subsequent experiments verified TMZ resistance and the hyper-expression of DNA repair-related genes (TOP2A and MGMT) in continuously low-dose TMZ-induced U251-TR cells. Muscone exhibited dose-dependent inhibition of U251-TR cell migration and proliferation, and its co-administration with TMZ showed the potential for enhanced therapeutic efficacy. By downregulating FAK, muscone reduced anoikis resistance in anchorage-independent U251-TR cells. It also caused cell cycle arrest in the G2/M phase by upregulating p21 and downregulating CDK1, CDK2, and Cyclin E1. Muscone-induced anoikis was accompanied by mitochondrial membrane potential collapse, ROS production, an increase in the BAX/Bcl-2 ratio, as well as elevated levels of Cytochrome c (Cyt c), cleaved caspase-9, and cleaved caspase-3. These findings indicated that muscone might trigger mitochondrial-dependent anoikis via ROS generation. Moreover, significant DNA damage, DNA double-strand breaks (DSBs), the formation of γ-H2AX foci, and a reduction in TOP2A expression are also associated with muscone-induced anoikis. Overexpression of EGFR in U251-TR cells boosted the expression of Integrin β1, FAK, β-Catenin, and TOP2A, whereas muscone suppressed the expression levels of EGFR, Integrin β1, β-Catenin, FAK, and TOP2A. Muscone may influence the expression of the key DNA repair enzyme, TOP2A, by suppressing the EGFR/Integrin β1/FAK pathway.
CONCLUSIONS: We first demonstrated that muscone suppressed TOP2A expression through the EGFR/Integrin β1/FAK pathway, hence restoring anoikis sensitivity in TMZ-resistant GBM cells. These data suggest that muscone may be a promising co-therapeutic agent for enhancing GBM treatment, particularly in cases of TMZ-resistant GBM with elevated TOP2A expression.