A novel mechanism of CD40-induced apoptosis of carcinoma cells involving TRAF3 and JNK/AP-1 activation

Membrane-presented CD40 agonists can induce apoptosis in carcinoma, but not normal homologous epithelial cells, whereas soluble agonists are growth inhibitory but not proapoptotic unless protein synthesis is blocked. Here we demonstrate that membrane-presented CD40 ligand (CD154) (mCD40L), but not soluble agonists, triggers cell death in malignant human urothelial cells via a direct mechanism involving rapid upregulation of TNFR-associated factor (TRAF)3 protein, without concomitant upregulation of TRAF3 mRNA, followed by activation of the c-Jun N-terminal kinase (JNK)/activator protein-1 (AP-1) pathway and induction of the caspase-9/caspase-3-associated intrinsic apoptotic machinery. TRAF3 knockdown abrogated JNK/AP-1 activation and prevented CD40-mediated apoptosis, whereas restoration of CD40 expression in CD40-negative carcinoma cells restored apoptotic susceptibility via the TRAF3/AP-1-dependent mechanism. In normal human urothelial cells, mCD40L did not trigger apoptosis, but induced rapid downregulation of TRAF2 and 3, thereby paralleling the situation in B-lymphocytes. Thus, TRAF3 stabilization, JNK activation and caspase-9 induction define a novel pathway of CD40-mediated apoptosis in carcinoma cells.

Hydroxyoctadecadienoic acids regulate apoptosis in human THP-1 cells in a PPARγ-dependent manner

Macrophage apoptosis, a key process in atherogenesis, is regulated by oxidation products, including hydroxyoctadecadienoic acids (HODEs). These stable oxidation products of linoleic acid (LA) are abundant in atherosclerotic plaque and activate PPARγ and GPR132. We investigated the mechanisms through which HODEs regulate apoptosis. The effect of HODEs on THP-1 monocytes and adherent THP-1 cells were compared with other C18 fatty acids, LA and α-linolenic acid (ALA). The number of cells was reduced within 24 hours following treatment with 9-HODE (p < 0.01, 30 μM) and 13 HODE (p < 0.01, 30 μM), and the equivalent cell viability was also decreased (p < 0.001). Both 9-HODE and 13-HODE (but not LA or ALA) markedly increased caspase-3/7 activity (p < 0.001) in both monocytes and adherent THP-1 cells, with 9-HODE the more potent. In addition, 9-HODE and 13-HODE both increased Annexin-V labelling of cells (p < 0.001). There was no effect of LA, ALA, or the PPARγ agonist rosiglitazone (1μM), but the effect of HODEs was replicated with apoptosis-inducer camptothecin (10μM). Only 9-HODE increased DNA fragmentation. The pro-apoptotic effect of HODEs was blocked by the caspase inhibitor DEVD-CHO. The PPARγ antagonist T0070907 further increased apoptosis, suggestive of the PPARγ-regulated apoptotic effects induced by 9-HODE. The use of siRNA for GPR132 showed no evidence that the effect of HODEs was mediated through this receptor. 9-HODE and 13-HODE are potent—and specific—regulators of apoptosis in THP-1 cells. Their action is PPARγ-dependent and independent of GPR132. Further studies to identify the signalling pathways through which HODEs increase apoptosis in macrophages may reveal novel therapeutic targets for atherosclerosis.

Graphene quantum dots induce apoptosis, autophagy, and inflammatory response via p38 mitogen-activated protein kinase and nuclear factor-κB mediated signaling pathways in activated THP-1 macrophages.

The biomedical application of graphene quantum dots (GQDs) is a new emerging area. However, their safety data are still in scarcity to date. Particularly, the effect of GQDs on the immune system remains unknown. This study aimed to elucidate the interaction of GQDs with macrophages and the underlying mechanisms. Our results showed that GQDs slightly affected the cell viability and membrane integrity of macrophages, whereas GQDs significantly increased reactive oxygen species (ROS) generation and apoptotic and autophagic cell death with an increase in the expression level of Bax, Bad, caspase 3, caspase 9, beclin 1, and LC3-I/II and a decrease in that of Bcl-2. Furthermore, low concentrations of GQDs significantly increased the expression of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-8, whereas high concentrations of GQDs elicited opposite effects on the cytokines production. SB202190, a selective inhibitor of p38 mitogen-activated protein kinase (MAPK), abolished the cytokine-inducing effect of GQDs in macrophages. Moreover, GQDs significantly increased the phosphorylation of p38 MAPK and p65, and promoted the nuclear translocation of nuclear factor-κB (NF-κB). Taken together, these results show that GQDs induce ROS generation, apoptosis, autophagy, and inflammatory response via p38MAPK and NF-κB mediated signaling pathways in THP-1 activated macrophages.

NDRG2 promotes myoblast proliferation and caspase 3/7 activities during differentiation, and attenuates hydrogen peroxide - but not palmitate-induced toxicity

The function of the stress-responsive N-myc downstream-regulated gene 2 (NDRG2) in the control of myoblast growth, and the amino acids contributing to its function, are not well characterized. Here, we investigated the effect of increased NDRG2 levels on the proliferation, differentiation and apoptosis in skeletal muscle cells under basal and stress conditions. NDRG2 overexpression increased C2C12 myoblast proliferation and the expression of positive cell cycle regulators, cdk2, cyclin B and cyclin D, and phosphorylation of Rb, while the serine/threonine-deficient NDRG2, 3A-NDRG2, had less effect. The onset of differentiation was enhanced by NDRG2 as determined through the myogenic regulatory factor expression profiles and myocyte fusion index. However, the overall level of differentiation in myotubes was not different. While NDRG2 up-regulated caspase 3/7 activities during differentiation, no increase in apoptosis was measured by TUNEL assay or through cleavage of caspase 3 and PARP proteins. During H2O2 treatment to induce oxidative stress, NDRG2 helped protect against the loss of proliferation and ER stress as measured by GRP78 expression with 3A-NDRG2 displaying less protection. NDRG2 also attenuated apoptosis by reducing cleavage of PARP and caspase 3 and expression of pro-apoptotic Bax while enhancing the pro-survival Bcl-2 and Bcl-xL levels. In contrast, Mcl-1 was not altered, and NDRG2 did not protect against palmitate-induced lipotoxicity. Our findings show that NDRG2 overexpression increases myoblast proliferation and caspase 3/7 activities without increasing overall differentiation. Furthermore, NDRG2 attenuates H2O2-induced oxidative stress and specific serine and threonine amino acid residues appear to contribute to its function in muscle cells.