Lagler Charlotte, El-Mesery Mohamed, Kübler Alexander Christian, Müller-Richter Urs Dietmar Achim, Stühmer Thorsten, Nickel Joachim, Müller Thomas Dieter, Wajant Harald, Seher Axel
Department of Oral and Maxillofacial Plastic Surgery, University Hospital Wuerzburg, Wuerzburg, Germany.
Department of Biochemistry, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt.
PLoS One. 2017 Oct 13;12(10):e0185720. doi: 10.1371/journal.pone.0185720. eCollection 2017.
Multiple myeloma (MM), a malignancy of the bone marrow, is characterized by a pathological increase in antibody-producing plasma cells and an increase in immunoglobulins (plasmacytosis). In recent years, bone morphogenetic proteins (BMPs) have been reported to be activators of apoptotic cell death in neoplastic B cells in MM. Here, we use bone morphogenetic protein 2 (BMP2) to show that the "apoptotic" effect of BMPs on human neoplastic B cells is dominated by anti-proliferative activities and cell cycle arrest and is apoptosis-independent. The anti-proliferative effect of BMP2 was analysed in the human cell lines KMS12-BM and L363 using WST-1 and a Coulter counter and was confirmed using CytoTox assays with established inhibitors of programmed cell death (zVAD-fmk and necrostatin-1). Furthermore, apoptotic activity was compared in both cell lines employing western blot analysis for caspase 3 and 8 in cells treated with BMP2 and FasL. Additionally, expression profiles of marker genes of different cell death pathways were analysed in both cell lines after stimulation with BMP2 for 48h using an RT-PCR-based array. In our experiments we observed that there was rather no reduction in absolute cell number, but cells stopped proliferating following treatment with BMP2 instead. The time frame (48-72 h) after BMP2 treatment at which a reduction in cell number is detectable is too long to indicate a directly BMP2-triggered apoptosis. Moreover, in comparison to robust apoptosis induced by the approved apoptotic factor FasL, BMP2 only marginally induced cell death. Consistently, neither the known inhibitor of apoptotic cell death zVAD-fmk nor the necroptosis inhibitor necrostatin-1 was able to rescue myeloma cell growth in the presence of BMP2.
多发性骨髓瘤(MM)是一种骨髓恶性肿瘤,其特征是产生抗体的浆细胞病理性增加以及免疫球蛋白增加(浆细胞增多)。近年来,有报道称骨形态发生蛋白(BMPs)是MM肿瘤性B细胞凋亡性细胞死亡的激活剂。在此,我们使用骨形态发生蛋白2(BMP2)来表明BMPs对人肿瘤性B细胞的“凋亡”作用主要由抗增殖活性和细胞周期停滞主导,且与凋亡无关。使用WST-1和库尔特计数器在人细胞系KMS12-BM和L363中分析了BMP2的抗增殖作用,并使用既定的程序性细胞死亡抑制剂(zVAD-fmk和坏死素-1)通过细胞毒性试验进行了证实。此外,采用蛋白质印迹分析对用BMP2和FasL处理的细胞中的半胱天冬酶3和8进行检测,比较了两种细胞系中的凋亡活性。另外,使用基于RT-PCR的芯片分析了用BMP2刺激48小时后两种细胞系中不同细胞死亡途径的标记基因的表达谱。在我们的实验中,我们观察到绝对细胞数量并没有减少,而是在用BMP2处理后细胞停止了增殖。BMP2处理后可检测到细胞数量减少的时间范围(48 - 72小时)过长,无法表明是由BMP2直接引发的凋亡。此外,与经批准的凋亡因子FasL诱导的强烈凋亡相比,BMP2仅轻微诱导细胞死亡。一致地,在存在BMP2的情况下,已知的凋亡细胞死亡抑制剂zVAD-fmk和坏死性凋亡抑制剂坏死素-1均无法挽救骨髓瘤细胞的生长。