恶性胶质母细胞瘤（GBM）的患者预后不良。在采用替莫唑胺（TMZ）标准化疗期间，肿瘤会获得抵抗力，因此会导致肿瘤复发。因此，解译造成TMZ抵抗的关键分子路径具有高度的治疗意义。基于质谱的蛋白质组学被用于研究GBM 蛋白组。对人类GBM组织进行阳性的卡尔旁-1或-2的免疫组化染色以定位蛋白酶的表达。采用体外细胞基于TMZ ± 卡尔旁抑制剂的测定细胞活力和初级患者衍生的GBM细胞以及已建立的GBM细胞系的存活能力。表达卡尔旁-1或卡尔旁-2的shRNA 敲低被用于研究特定亚单位的TMZ敏感性。彗星试验和ɣH2AX信号测量用于评估DNA损伤量和识别。最后，应用目标蛋白的定量实时PCR来区分转录和翻译后调控。钙依赖性卡尔旁蛋白酶，尤其是卡尔旁-2，在胶质母细胞瘤中比正常脑更丰富，并在与患者匹配的初期和复发性胶质母细胞瘤中增加。在细胞水平上，药理学卡尔旁抑制剂增加了初级胶质母细胞瘤细胞对TMZ的敏感性。在U251细胞中卡尔旁-2的遗传敲低，导致增加了卡斯蛋白-3的裂解和对新卡硝唑素的敏感性，该药物能迅速引起DNA链断裂。我们假设卡尔旁-2通过通过翻译后抑制TP53来防止强烈的DNA损伤和随后的细胞凋亡，从而使肿瘤细胞失去了对TMZ的敏感性。实际上，对U251对照组与U251卡尔旁-2敲低组进行的蛋白质组比较显示出数个与DNA损伤反应和下游通路相关的蛋白质含量的混乱，这些通路影响了TP53和NF-κB信号传导。TP53显示出了增加的蛋白质丰度，但没有转录调控。在卡尔旁-2表达存在的情况下，TMZ诱导的细胞死亡似乎有利于DNA修复和细胞存活。我们的实验表明，卡尔旁-2的表达代表了一种与通过“引导” GBM细胞对TMZ化疗进行高抵抗的蛋白质组学模式。因此，卡尔旁-2可以作为GBM结局的预后因素。©2023.作者（们）。

Glioblastoma multiforme (GBM) is characterized by an unfavorable prognosis for patients affected. During standard-of-care chemotherapy using temozolomide (TMZ), tumors acquire resistance thereby causing tumor recurrence. Thus, deciphering essential molecular pathways causing TMZ resistance are of high therapeutic relevance.Mass spectrometry based proteomics were used to study the GBM proteome. Immunohistochemistry staining of human GBM tissue for either calpain-1 or -2 was performed to locate expression of proteases. In vitro cell based assays were used to measure cell viability and survival of primary patient-derived GBM cells and established GBM cell lines after TMZ ± calpain inhibitor administration. shRNA expression knockdowns of either calpain-1 or calpain-2 were generated to study TMZ sensitivity of the specific subunits. The Comet assay and ɣH2AX signal measurements were performed in order to assess the DNA damage amount and recognition. Finally, quantitative real-time PCR of target proteins was applied to differentiate between transcriptional and post-translational regulation.Calcium-dependent calpain proteases, in particular calpain-2, are more abundant in glioblastoma compared to normal brain and increased in patient-matched initial and recurrent glioblastomas. On the cellular level, pharmacological calpain inhibition increased the sensitivities of primary glioblastoma cells towards TMZ. A genetic knockdown of calpain-2 in U251 cells led to increased caspase-3 cleavage and sensitivity to neocarzinostatin, which rapidly induces DNA strand breakage. We hypothesize that calpain-2 causes desensitization of tumor cells against TMZ by preventing strong DNA damage and subsequent apoptosis via post-translational TP53 inhibition. Indeed, proteomic comparison of U251 control vs. U251 calpain-2 knockdown cells highlights perturbed levels of numerous proteins involved in DNA damage response and downstream pathways affecting TP53 and NF-κB signaling. TP53 showed increased protein abundance, but no transcriptional regulation.TMZ-induced cell death in the presence of calpain-2 expression appears to favor DNA repair and promote cell survival. We conclude from our experiments that calpain-2 expression represents a proteomic mode that is associated with higher resistance via "priming" GBM cells to TMZ chemotherapy. Thus, calpain-2 could serve as a prognostic factor for GBM outcome.© 2023. The Author(s).