Zhao Yi, Zhang Enfan, Lv Ning, Ma Liang, Yao Shunnan, Yan Meidi, Zi Fumin, Deng Gang, Liu Xinling, He Jingsong, Wu Wenjun, Cai Zhen, Yu Rui
Bone Marrow Transplantation Center, First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China.
Department of Pharmacy, First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China.
Cell Physiol Biochem. 2018;48(2):785-800. doi: 10.1159/000491908. Epub 2018 Jul 20.
BACKGROUND/AIMS: Patients with multiple myeloma (MM) invariably relapse with chemotherapy-resistant disease, underscoring the need for new therapeutic options that bypass these resistance mechanisms. Metformin is a widely prescribed antidiabetic drug with direct antitumor activity against various tumor cell lines. FTY720, also known as fingolimod, is an immune-modulating agent approved by the FDA as oral medication to treat the relapsing form of multiple sclerosis (MS). In recent years, FTY720 has attracted attention due to its anti-tumor activity. To explore an optimized combinational therapy, interactions between metformin and FTY720 were examined in MM cells.
MTT assays were employed to assess the viability of MM cells. An apoptotic nucleosome assay was employed to measure apoptosis. Loss of mitochondrial membrane potential (MMP, ΔΨm) and cellular levels of ROS were measured by flow cytometry. qRT-PCR was used to analyze the expression of mRNAs. Western blotting assays were applied to measure the levels of proteins involved in different signaling pathways.
Coadministration of metformin and FTY720 synergistically inhibited the proliferation of MM cells. Increased levels of apoptosis, activation of caspase-3 and cleavage of PARP were detected after cotreatment with metformin and FTY720. These events were associated with modulation of Bcl-2 proteins, loss of MMP, ER stress induction, and inhibition of the PI3K/AKT/mTOR signaling pathway. The metformin/FTY720 regimen markedly induced ROS generation; moreover, apoptosis, ER stress and inhibition of PI3K/AKT/ mTOR were attenuated by the ROS scavenger NAC.
Exposure to metformin in combination with FTY720 potently induces apoptosis in MM cells in a ROS-dependent manner, suggesting that a strategy combining these agents warrants further investigation in MM.
背景/目的：多发性骨髓瘤（MM）患者总会出现化疗耐药性疾病复发，这突出表明需要新的治疗方案来绕过这些耐药机制。二甲双胍是一种广泛使用的抗糖尿病药物，对多种肿瘤细胞系具有直接抗肿瘤活性。FTY720，也称为芬戈莫德，是一种免疫调节剂，已被美国食品药品监督管理局（FDA）批准作为口服药物用于治疗复发型多发性硬化症（MS）。近年来，FTY720因其抗肿瘤活性而受到关注。为了探索优化的联合治疗方案，研究了二甲双胍与FTY720在MM细胞中的相互作用。
采用MTT法评估MM细胞的活力。采用凋亡核小体分析法检测细胞凋亡。通过流式细胞术检测线粒体膜电位（MMP，ΔΨm）的丧失和细胞内活性氧（ROS）水平。使用qRT-PCR分析mRNA的表达。采用蛋白质印迹法检测不同信号通路中相关蛋白的水平。
二甲双胍与FTY720联合使用可协同抑制MM细胞的增殖。二甲双胍与FTY720联合处理后，检测到细胞凋亡水平升高、半胱天冬酶-3激活和聚（ADP-核糖）聚合酶（PARP）裂解。这些事件与Bcl-2蛋白的调节、MMP的丧失、内质网应激诱导以及PI3K/AKT/mTOR信号通路的抑制有关。二甲双胍/FTY720方案显著诱导ROS生成；此外，ROS清除剂NAC可减弱细胞凋亡、内质网应激以及对PI3K/AKT/mTOR的抑制作用。
二甲双胍与FTY720联合使用以ROS依赖的方式有效诱导MM细胞凋亡，这表明将这些药物联合使用的策略值得在MM中进一步研究。
