金属蛋白酶-解整合素ADAM8促进替莫唑胺化疗耐药性及增强人胶质母细胞瘤细胞的侵袭性。
The metalloprotease-disintegrin ADAM8 contributes to temozolomide chemoresistance and enhanced invasiveness of human glioblastoma cells.
作者信息
Dong Fangyong, Eibach Michael, Bartsch Jörg W, Dolga Amalia M, Schlomann Uwe, Conrad Catharina, Schieber Susanne, Schilling Oliver, Biniossek Martin L, Culmsee Carsten, Strik Herwig, Koller Garrit, Carl Barbara, Nimsky Christopher
机构信息
Department of Neurosurgery, Philipps-University Marburg, Marburg, Germany (F.D., M.E., J.W.B., U.S., C.Co., S.S., B.C., C.N.); Department of Neurosurgery, Tongji Hospital, Wuhan, China (F.D.); Philipps-University Marburg, Institute for Pharmacology and Clinical Pharmacy, Marburg, Germany (A.M.D., C.Cu.); Institute of Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany (O.S., M.L.B.); BIOSS Centre for Biological Signaling Studies, University of Freiburg, Freiburg, Germany (O.S.); Department of Neurology, Philipps-University Marburg, Marburg, Germany (H.S.); Biomaterials, Biomimetics and Biophotonics Research Group, King's College London Dental Institute, London, United Kingdom (G.K.).
出版信息
Neuro Oncol. 2015 Nov;17(11):1474-85. doi: 10.1093/neuonc/nov042. Epub 2015 Mar 29.
BACKGROUND
Despite multimodal treatment, glioblastoma (GBM) therapy with temozolomide (TMZ) remains inefficient due to chemoresistance. Matrix metalloproteinase (MMP) and a disintegrin and metalloprotease (ADAM), increased in GBM, could contribute to chemoresistance and TMZ-induced recurrence of glioblastoma.
METHODS
TMZ inducibility of metalloproteases was determined in GBM cell lines, primary GBM cells, and tissues from GBM and recurrent GBM. TMZ sensitivity and invasiveness of GBM cells were assessed in the presence of the metalloprotease inhibitors batimastat (BB-94) and marimastat (BB-2516). Metalloprotease-dependent effects of TMZ on mitochondria and pAkt/phosphatidylinositol-3 kinase (PI3K) and phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) pathways were analyzed by fluorescence activated cell sorting, morphometry, and immunoblotting. Invasiveness of GBM cells was determined by Matrigel invasion assays. Potential metalloprotease substrates were identified by proteomics and tested for invasion using blocking antibodies.
RESULTS
TMZ induces expression of MMP-1, -9, -14, and ADAM8 in GBM cells and in recurrent GBM tissues. BB-94, but not BB-2516 (ADAM8-sparing) increased TMZ sensitivity of TMZ-resistant and -nonresistant GBM cells with different O(6)-methylguanine-DNA methyltransferase states, suggesting that ADAM8 mediates chemoresistance, which was confirmed by ADAM8 knockdown, ADAM8 overexpression, or pharmacological inhibition of ADAM8. Levels of pAkt and pERK1/2 were increased in GBM cells and correlated with ADAM8 expression, cell survival, and invasiveness. Soluble hepatocyte growth factor (HGF) R/c-met and CD44 were identified as metalloprotease substrates in TMZ-treated GBM cells. Blocking of HGF R/c-met prevented TMZ-induced invasiveness.
CONCLUSIONS
ADAM8 causes TMZ resistance in GBM cells by enhancing pAkt/PI3K, pERK1/2, and cleavage of CD44 and HGF R/c-met. Specific ADAM8 inhibition can optimize TMZ chemotherapy of GBM in order to prevent formation of recurrent GBM in patients.
背景
尽管采用了多模式治疗，但由于化疗耐药性，替莫唑胺（TMZ）治疗胶质母细胞瘤（GBM）仍然效率低下。基质金属蛋白酶（MMP）和去整合素金属蛋白酶（ADAM）在GBM中表达增加，可能导致化疗耐药性以及TMZ诱导的胶质母细胞瘤复发。
方法
在GBM细胞系、原发性GBM细胞以及GBM和复发性GBM组织中测定金属蛋白酶的TMZ诱导性。在存在金属蛋白酶抑制剂batimastat（BB - 94）和marimastat（BB - 2516）的情况下评估GBM细胞的TMZ敏感性和侵袭性。通过荧光激活细胞分选、形态测定和免疫印迹分析TMZ对线粒体以及pAkt/磷脂酰肌醇 - 3激酶（PI3K）和磷酸化细胞外信号调节激酶1/2（pERK1/2）通路的金属蛋白酶依赖性作用。通过基质胶侵袭试验测定GBM细胞的侵袭性。通过蛋白质组学鉴定潜在的金属蛋白酶底物，并使用阻断抗体测试其侵袭情况。
结果
TMZ可诱导GBM细胞和复发性GBM组织中MMP - 1、 - 9、 - 14和ADAM8的表达。BB - 94而非BB - 2516（不抑制ADAM8）可提高具有不同O(6)-甲基鸟嘌呤 - DNA甲基转移酶状态的TMZ耐药和非耐药GBM细胞的TMZ敏感性，这表明ADAM8介导化疗耐药性，ADAM8敲低、ADAM8过表达或ADAM8的药理学抑制证实了这一点。GBM细胞中pAkt和pERK1/2水平升高，且与ADAM8表达、细胞存活和侵袭性相关。可溶性肝细胞生长因子（HGF）受体/c - met和CD44被鉴定为TMZ处理的GBM细胞中的金属蛋白酶底物。阻断HGF受体/c - met可防止TMZ诱导的侵袭性。
结论
ADAM8通过增强pAkt/PI3K、pERK1/2以及切割CD44和HGF受体/c - met导致GBM细胞产生TMZ耐药性。特异性抑制ADAM8可优化GBM的TMZ化疗，以防止患者复发性GBM的形成。
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