Curcumin, a Multifaceted Hormetic Agent, Mediates an Intricate Crosstalk between Mitochondrial Turnover, Autophagy, and Apoptosis.

Curcumin has extensive therapeutic potential because of its antioxidant, anti-inflammatory, and antiproliferative properties. Multiple preclinical studies in vitro and in vivo have proven curcumin to be effective against various cancers. These potent effects are driven by curcumin's ability to induce G2/M cell cycle arrest, induce autophagy, activate apoptosis, disrupt molecular signaling, inhibit invasion and metastasis, and increase the efficacy of current chemotherapeutics. Here, we focus on the hormetic behavior of curcumin. Frequently, low doses of natural chemical products activate an adaptive stress response, whereas high doses activate acute responses like autophagy and cell death. This phenomenon is often referred to as hormesis. Curcumin causes cell death and primarily initiates an autophagic step (mitophagy). At higher doses, cells undergo mitochondrial destabilization due to calcium release from the endoplasmic reticulum, and die. Herein, we address the complex crosstalk that involves mitochondrial biogenesis, mitochondrial destabilization accompanied by mitophagy, and cell death.

Cysteine 62 of Bax is critical for its conformational activation and its proapoptotic activity in response to H2O2-induced apoptosis.

Bax is activated and translocated onto mitochondria to mediate cytochrome c release and apoptosis. The molecular mechanisms of Bax activation during apoptosis remain a subject of debate. We addressed the question of whether reactive oxygen species could directly activate Bax for its subsequent translocation and apoptosis. Using the SW480 human colon adenocarcinoma cell line stably expressing Bax fused to GFP, we showed that H2O2 induces Bax conformational change, mitochondrial translocation, and subsequent oligomerization at mitochondria. We found that H2O2-induced Bax activation is dependent on the conserved cysteine residue 62 of Bax. Mutation of cysteine 62, but not cysteine 126, to serine or alanine abolished its activation by H2O2 but not other death stimuli, both in SW480 and Bax-deficient HCT116 cells, whereas wild type Bax sensitizes these cells to apoptosis. Cysteines of Bax could chemically react with H2O2. Mutation of Bax BH3 domain in the presence of cysteine 62 also abolished Bax proapoptotic activity. We conclude that reactive oxygen species could be a direct signal for Bax activation by reacting with cysteine residues. Our results identify a critical role of cysteine 62 in oxidative stress-induced Bax activation and subsequent apoptosis.

A novel C-terminal motif is necessary for the export of the vasopressin V1b/V3 receptor to the plasma membrane.

Little is known about endoplasmic reticulum (ER) export signals, particularly those of members of the G-protein-coupled receptor family. We investigated the structural motifs involved in membrane export of the human pituitary vasopressin V1b/V3 receptor. A series of V3 receptors carrying deletions and point mutations were expressed in AtT20 corticotroph cells. We analyzed the export of these receptors by monitoring radioligand binding and by analysis of a V3 receptor tagged with both green fluorescent protein and Myc epitopes by a novel flow cytometry-based method. This novel method allowed us to quantify total and membrane-bound receptor expression. Receptors lacking the C terminus were not expressed at the cell surface, suggesting the presence of an export motif in this domain. The distal C terminus contains two di-acidic (DXE) ER export motifs; however, mutating both these motifs had no effect on the V3 receptor export. The proximal C terminus contains a di-leucine (345)LL(346) motif surrounded by the hydrophobic residues Phe(341), Asn(342), and Leu(350). The mutation of one or more of these five residues abolished up to 100% of the receptor export. In addition, these mutants colocalized with calnexin, demonstrating that they were retained in the ER. Finally, this motif was sufficient to confer export properties on a CD8alpha glycoprotein-V3 receptor chimera. In conclusion, we have identified a novel export motif, FN(X)(2)LL(X)(3)L, in the C terminus of the V3 receptor.

Retroactive pathway involving mitochondria in electroloaded cytochrome c-induced apoptosis. Protective properties of Bcl-2 and Bcl-XL.

Cytochrome c release is thought to play an important role in the initiation of apoptosis. The nature of the control exerted by Bcl-2 and Bcl-XL on such a pathway is not precisely known. We addressed this issue by square-wave pulse electroloading of exogenous cytochrome c into Jurkat cells. Three hours after cytochrome c loading into the cells, characteristic phenotypes of apoptosis were observed. However, a significant drop in the mitochondrial membrane potential (Deltapsim) was also observed, while cytochrome c was generally considered to act downstream from the mitochondria. Related to the Deltapsim drop, there was a release of proapoptotic proteins such as AIF and Smac from the mitochondria. This release, as well as NAD(P)H and cardiolipids oxidation, are linked to previous caspase activation. Cytochrome c-linked caspase activation also led to potassium efflux out of the cell. Overexpression of Bcl-2 and Bcl-XL or N-acetyl-DEVD-aldehyde treatment not only prevented the mitochondrial membrane potential decrease, but also protected cells from the apoptosis directly induced by cytochrome c electroloading. Bcl-2 and Bcl-XL protection is based on the inhibition of the caspase-dependent retroactive pathway affecting the mitochondrial compartment.