■胶质母细胞瘤IDH-野生型(GBM)是成人中最恶性的脑肿瘤,诊断后约15个月预后不良。大多数患者在<1年内复发,这使得GBM成为危及生命的挑战。在驱动GBM侵袭性的分子机制中,由GBM内皮细胞(GECs)介导的血管生成值得考虑作为治疗转折点。在这种情况下,钙蛋白酶,一个广泛表达的钙依赖性半胱氨酸蛋白酶家族,作为一种新的治疗策略和预后组织生物标志物,成为有希望的靶标。
■为了探索这一假设,从n=10GBM活检中分离出GECs,并通过免疫荧光进行表型表征。通过qRT-PCR和Westernblot评估钙蛋白酶的表达水平,通过Pearson相关性和Kaplan-Meier生存分析估计其与患者预后的关系。通过时间和剂量依赖性增殖曲线评估钙蛋白酶靶向疗效,MTT活力测定,caspase-3/7活性,体外迁移和血管生成,以及基因和蛋白质表达水平的修饰。
■免疫荧光证实了我们的原发性GECs的内皮表型。观察到钙蛋白酶-1/2/3(CAPN)和钙蛋白酶小亚基-1/2(CAPNS1)的显着过表达,而calpastatin基因,钙蛋白酶天然抑制剂,据报道被下调了监管。CAPN1/CAPNS1与患者总生存期呈显著负相关。GEC挑战表明,钙蛋白酶-1的抑制发挥最强的促凋亡功效,所以GEC死亡率达到了80%,caspase-3/7的活性增加证实。功能测定揭示了对体外迁移和血管生成的强烈影响。基因和蛋白质表达证明了MAPK的下调,VEGF/VEGFRs,和Bcl-2,以及胱天蛋白酶和Bax家族介体的上调。
■总的来说,钙蛋白酶的差异表达及其与患者生存的相关性提示了一种新的有希望的靶途径,他们的封锁显示了精准医学策略令人鼓舞的结果。
UNASSIGNED: Glioblastoma IDH-wildtype (GBM) is the most malignant brain tumor in adults, with a poor prognosis of approximately 15 months after diagnosis. Most patients suffer from a recurrence in <1 year, and this renders GBM a life-threatening challenge. Among molecular mechanisms driving GBM aggressiveness, angiogenesis mediated by GBM endothelial cells (GECs) deserves consideration as a therapeutic turning point. In this scenario,
calpains, a family of ubiquitously expressed calcium-dependent cysteine proteases, emerged as promising targets to be investigated as a novel therapeutic strategy and prognostic tissue biomarkers.
UNASSIGNED: To explore this hypothesis, GECs were isolated from n=10 GBM biopsies and characterized phenotypically by immunofluorescence. The expression levels of
calpains were evaluated by qRT-PCR and Western blot, and their association with patients\' prognosis was estimated by Pearson correlation and Kaplan-Meier survival analysis. Calpain targeting efficacy was assessed by a time- and dose-dependent proliferation curve, MTT assay for viability, caspase-3/7 activity, migration and angiogenesis in vitro, and gene and protein expression level modification.
UNASSIGNED: Immunofluorescence confirmed the endothelial phenotype of our primary GECs. A significant overexpression was observed for calpain-1/2/3 (CAPN) and calpain-small-subunits-1/2 (CAPNS1), whereas calpastatin gene, the calpain natural inhibitor, was reported to be downregulated. A significant negative correlation was observed between CAPN1/CAPNS1 and patient overall survival. GEC challenging revealed that the inhibition of calpain-1 exerts the strongest proapoptotic efficacy, so GEC mortality reached the 80%, confirmed by the increased activity of caspase-3/7. Functional assays revealed a strong affection of in vitro migration and angiogenesis. Gene and protein expression proved a downregulation of MAPK, VEGF/VEGFRs, and Bcl-2, and an upregulation of caspases and Bax-family mediators.
UNASSIGNED: Overall, the differential expression of
calpains and their correlation with patient survival suggest a novel promising target pathway, whose blockade showed encouraging results toward precision medicine strategies.