Faculty of Science, Department of Physiology, Neuroimmunophysiology and Chrononutrition Research Group, University of Extremadura, Badajoz, Spain.
J Pineal Res. 2013 May;54(4):442-52. doi: 10.1111/jpi.12042. Epub 2013 Feb 9.
The pro-apoptotic signalling cascades induced by tumour necrosis factor-alpha (TNF-α) have been intensively studied in multiple cellular systems. So far, it is known that TNF-α can simultaneously activate survival and apoptotic cell death responses. The balance between these signals determines the ultimate response of the cell to TNF-α. Moreover, emerging evidence suggests that melatonin may be involved in the protection of different cell types against apoptosis. Thus, the objective of this study was to evaluate the effect of melatonin on TNF-α-induced apoptosis in human leucocytes. Cells were treated with TNF-α alone or in the presence of cycloheximide (CHX), which promotes caspase-8 activation by eliminating the endogenous caspase-8 inhibitor, c-FLIP. Treatment with TNF-α/CHX led to apoptotic cell death, as ascertained by annexin V/propidium iodide (PI) staining. Likewise, in the presence of CHX, TNF-α stimulation produced cFLIP down-regulation and subsequent caspase-8 activation, thus directly triggering caspase-3 activation and causing Bid truncation and subsequent caspase-9 activation. Conversely, pre-incubation of cells with melatonin inhibited TNF-α-/CHX-evoked leucocyte apoptosis. Similarly, pretreatment of leucocytes with melatonin increased cFLIP protein levels, thereby preventing TNF-α-/CHX-mediated caspase processing. Blockade of melatonin membrane receptor MT1/MT2 or extracellular signal-regulated kinase (ERK) pathway with luzindole or PD98059, respectively, abolished the inhibitory effects of melatonin on leucocyte apoptosis evoked by TNF-α/CHX. In conclusion, the model proposed by these findings is that the MT1/MT2 receptors, which are under the positive control of melatonin, trigger an ERK-dependent signalling cascade that interferes with the anti-apoptotic protein cFLIP modulating the cell life/death balance of human leucocytes.
肿瘤坏死因子-α（TNF-α）诱导的促凋亡信号级联在多种细胞系统中得到了深入研究。到目前为止，已知 TNF-α可以同时激活细胞存活和凋亡细胞死亡反应。这些信号之间的平衡决定了细胞对 TNF-α的最终反应。此外，新出现的证据表明，褪黑素可能参与保护不同类型的细胞免受凋亡。因此，本研究的目的是评估褪黑素对人白细胞中 TNF-α诱导的凋亡的影响。细胞单独用 TNF-α处理或用环已酰亚胺（CHX）处理，CHX 通过消除内源性 caspase-8 抑制剂 c-FLIP 来促进 caspase-8 的激活。用 TNF-α/CHX 处理导致凋亡细胞死亡，如用 annexin V/碘化丙啶（PI）染色确定。同样，在 CHX 存在的情况下，TNF-α刺激导致 cFLIP 下调和随后的 caspase-8 激活，从而直接触发 caspase-3 激活并导致 Bid 截断和随后的 caspase-9 激活。相反，细胞预先用褪黑素孵育可抑制 TNF-α/CHX 诱导的白细胞凋亡。同样，褪黑素预处理增加 cFLIP 蛋白水平，从而防止 TNF-α/CHX 介导的 caspase 加工。用 luzindole 或 PD98059 分别阻断褪黑素膜受体 MT1/MT2 或细胞外信号调节激酶（ERK）通路，消除了褪黑素对 TNF-α/CHX 诱导的白细胞凋亡的抑制作用。总之，这些发现提出的模型是 MT1/MT2 受体受褪黑素的正调控，触发一个 ERK 依赖的信号级联，该级联干扰抗凋亡蛋白 cFLIP，调节人白细胞的细胞生死平衡。
