Withaferin A induces apoptosis in human melanoma cells through generation of reactive oxygen species and down-regulation of Bcl-2.

A high resistance and heterogeneous response to conventional anti-cancer chemotherapies characterize malignant cutaneous melanoma, the most aggressive and deadly form of skin cancer. Withaferin A (WFA), a withanolide derived from the medicinal plant Withania somnifera, has been reported for its anti-tumorigenic activity against various cancer cells. For the first time, we examined the death-inducing potential of WFA against a panel of four different human melanoma cells and investigated the cellular mechanisms involved. WFA induces apoptotic cell death with various IC50 ranging from 1.8 to 6.1 µM. The susceptibility of cells toward WFA-induced apoptosis correlated with low Bcl-2/Bax and Bcl-2/Bim ratios. In all cell lines, the apoptotic process triggered by WFA involves the mitochondrial pathway and was associated with Bcl-2 down regulation, Bax mitochondrial translocation, cytochrome c release into the cytosol, transmembrane potential (ΔΨm) dissipation, caspase 9 and caspase 3 activation and DNA fragmentation. WFA cytotoxicity requires early reactive oxygen species (ROS) production and glutathione depletion, the inhibition of ROS increase by the antioxidant N-acetylcysteine resulting in complete suppression of mitochondrial and nuclear events. Altogether, these results support the therapeutic potential of WFA against human melanoma.

The adenine nucleotide translocase 2, a mitochondrial target for anticancer biotherapy.

Apoptosis or programmed cell death is one of the most important signaling pathways, which controls the cell fate and is frequently impaired in cancer cells. The major consequences of apoptosis inhibition are the accumulation of mutated cells and their enhanced resistance to chemotherapeutic agents. More generally, intrinsic or acquired apoptosis resistance may favor tumor growth and dissemination of mutated cells, and this resistance can be responsible of treatment failure. Mitochondria are central organelles in the signaling pathway of apoptosis and have been proposed as favorite candidates for anticancer biotherapy because they accommodate potential biological targets. Indeed, although cancer cells are highly glycolytic and become energetically independent of oxidative phosphorylation. Mitochondrial proteins involved in the so-called mitochondrial membrane permeabilization (MMP), such as the adenine nucleotide translocase (ANT) can be instrumental to elicit cancer cell death. Thus, multiple pharmacological and molecular studies revealed ANT could be a promising therapeutic target for the following reasons: (i) ANT is a bi-functional protein, it mediates the vital exchange of cytosolic ADP and mitochondrial ATP and participates to MMP via its capacity to become a lethal pore in the mitochondrial inner membrane; (ii) both ANT functions are under the control of the (anti)-oncogenes from the Bax/Bcl-2 family, (iii) several chemotherapeutic agents directly modulate the pore-forming activity of ANT and (iv) ANT2 isoform, which is anti-apoptotic, can be overexpressed in human cancers and its invalidation sensitize cells to apoptosis. In this review, we will introduce the knowledge of the role of ANT in MMP, illustrate the modulation of ANT by several strategies and propose the possibility to target preferentially the ANT2 isoform for induction of cancer cell apoptosis.

The fourth isoform of the adenine nucleotide translocator inhibits mitochondrial apoptosis in cancer cells.

The adenine nucleotide translocator (ANT) is a mitochondrial bi-functional protein, which catalyzes the exchange of ADP and ATP between cytosol and mitochondria and participates in many models of mitochondrial apoptosis. The human adenine nucleotide translocator sub-family is composed of four isoforms, namely ANT1-4, encoded by four nuclear genes, whose expression is highly regulated. Previous studies have revealed that ANT1 and 3 induce mitochondrial apoptosis, whereas ANT2 is anti-apoptotic. However, the role of the recently identified isoform ANT4 in the apoptotic pathway has not yet been elucidated. Here, we investigated the effects of stable heterologous expression of the ANT4 on proliferation, mitochondrial respiration and cell death in human cancer cells, using ANT3 as a control of pro-apoptotic isoform. As expected, ANT3 enhanced mitochondria-mediated apoptosis in response to lonidamine, a mitochondriotoxic chemotherapeutic drug, and staurosporine, a protein kinase inhibitor. Our results also indicate that the pro-apoptotic effect of ANT3 was accompanied by decreased rate of cell proliferation, alteration in the mitochondrial network topology, and decreased reactive oxygen species production. Of note, we demonstrate for the first time that ANT4 enhanced cell growth without impacting mitochondrial network or respiration. Moreover, ANT4 differentially regulated the intracellular levels of hydrogen peroxide without affecting superoxide anion levels. Finally, stable ANT4 overexpression protected cancer cells from lonidamine and staurosporine apoptosis in a manner independent of Bcl-2 expression. These data highlight a hitherto undefined cytoprotective activity of ANT4, and provide a novel dichotomy in the human ANT isoform sub-family with ANT1 and 3 isoforms functioning as pro-apoptotic while ANT2 and 4 isoforms render cells resistant to death inducing stimuli.

A partial form of recessive STAT1 deficiency in humans.

Complete STAT1 deficiency is an autosomal recessive primary immunodeficiency caused by null mutations that abolish STAT1-dependent cellular responses to both IFN-alpha/beta and IFN-gamma. Affected children suffer from lethal intracellular bacterial and viral diseases. Here we report a recessive form of partial STAT1 deficiency, characterized by impaired but not abolished IFN-alpha/beta and IFN-gamma signaling. Two affected siblings suffered from severe but curable intracellular bacterial and viral diseases. Both were homozygous for a missense STAT1 mutation: g.C2086T (P696S). This STAT1 allele impaired the splicing of STAT1 mRNA, probably by disrupting an exonic splice enhancer. The misspliced forms were not translated into a mature protein. The allele was hypofunctional, because residual full-length mRNA production resulted in low but detectable levels of normally functional STAT1 proteins. The P696S amino acid substitution was not detrimental. The patients' cells, therefore, displayed impaired but not abolished responses to both IFN-alpha and IFN-gamma. We also show that recessive STAT1 deficiencies impaired the IL-27 and IFN-lambda1 signaling pathways, possibly contributing to the predisposition to bacterial and viral infections, respectively. Partial recessive STAT1 deficiency is what we believe to be a novel primary immunodeficiency, resulting in impairment of the response to at least 4 cytokines (IFN-alpha/beta, IFN-gamma, IFN-lambda1, and IL-27). It should be considered in patients with unexplained, severe, but curable intracellular bacterial and viral infections.