Bromopyruvate mediates autophagy and cardiolipin degradation to monolyso-cardiolipin in GL15 glioblastoma cells.

The GL15 glioblastoma cell line undergoes viability loss upon treatment with bromopyruvate. The biochemical mechanisms triggered by the antiglycolytic agent indicate the activation of an autophagic pathway. Acridine orange stains acidic intracellular vesicles already 60 min after bromopyruvate treatment, whereas autophagosomes engulfing electron dense material are well evidenced 18 h later. The autophagic process is accompanied by the expression of the early autophagosomal marker Atg5 and by LC3-II formation, a late biochemical marker associated with autophagosomes. In agreement with the autophagic route activation, the inhibitory and the activator Akt and ERK signaling pathways are depressed and enhanced, respectively. In spite of the energetic collapse suffered by bromopyruvate-treated cells, MALDI-TOF mass spectrometry lipid analysis does not evidence a decrease of the major phospholipids, in accordance with the need of phospholipids for autophagosomal membranes biogenesis. Contrarily, mitochondrial cardiolipin decreases, accompanied by monolyso-cardiolipin formation and complete cytochrome c degradation, events that could target mitochondria to autophagy. However, in our experimental conditions cytochrome c degradation seems to be independent of the autophagic process.

Differential involvement of reactive oxygen species and nucleoside transporters in cytotoxicity induced by two adenosine analogues in human prostate cancer cells.

BACKGROUND: Elevated levels of cellular oxidative stress represent a specific vulnerability of malignant cells and exposure to cytotoxic drugs is known to induce oxidative stress in cancer cells. The effects of two adenosine analogues, 2-chloroadenosine and 2-chlorodeoxyadenosine, were investigated to assess their mechanism of action in prostate cancer cells. METHODS: Androgen-independent and -sensitive (PC3 and LNCaP) prostate cancer cells and mouse primary prostate cultures were used in the study. Proliferation and cell cycle progression were analyzed in the presence of 2-chloroadenosine and 2-chlorodeoxyadenosine. Adenosine receptors and nucleoside transporters expression were determined by RT-PCR. GSH and reactive oxygen species levels were determined by DTNB and DCFH-DA, respectively. Nuclear translocation of Nrf2 was assessed by Western blotting. RESULTS: 2-Chloroadenosine marginally affected primary prostate cells viability whereas it was more potent than 2-chlorodeoxyadenosine in reducing viability and increasing apoptosis in both prostate cancer cell lines. Moreover, ROS levels and GSH content were markedly affected in PC3 whereas only ROS production was increased in LNCaP cells. The antioxidant butylated hydroxytoluene protected PC3 cells from GSH depletion and reduction in cell viability induced by 2-chloroadenosine. CONCLUSIONS: 2-Chloroadenosine, but not 2-chlorodeoxyadenosine is capable of inducing apoptosis in prostate cancer cells, an effect which may be explained at least partially by the capacity of the nucleoside analogue to modify ROS and GSH levels. These observations may offer a rationale for the use of 2-chloroadenosine to improve the clinical efficacy of GSH-dependent antitumor drugs.

Electron microscopic cytochemistry of adenylyl cyclase activity in mouse spermatozoa.

We investigated adenylyl cyclase activity of mouse spermatozoa by electron microscopic cytochemistry. Subcellular localization of enzyme activity was determined in the presence and absence of bicarbonate ions. Results confirm the existence in sperm of a bicarbonate-regulated adenylyl cyclase, which suggests microdomain signaling.

A novel role for Tm7sf2 gene in regulating TNFα expression.

We have explored the role of Tm7sf2 gene, which codifies for 3ß-hydroxysterol Δ14-reductase, an endoplasmic reticulum resident protein, in the sensitivity to endoplasmic reticulum stress and in the resulting inflammatory response. We used mouse embryonic fibroblasts, derived from Tm7sf2(+/+) and Tm7sf2(-/-) mice, to determine the in vitro effects of thapsigargin on NF-κB activation. Our results show that the Tm7sf2 gene controls the launch of the unfolded protein response and presides an anti-inflammatory loop thus its absence correlates with NF-κB activation and TNFα up-regulation. Our data also show that Tm7sf2 gene regulates liver X receptor activation and its absence inhibits LXR signalling. By expressing the hTm7sf2 gene in KO MEFs and observing a reduced NF-κB activation, we have confirmed that Tm7sf2 gene is linked to NF-κB activation. Finally we used genetically modified mice in an in vivo model of ER stress and of inflammation. Our results show a significant increase in renal TNFα expression after tunicamycin exposure and in the oedematogenic response in Tm7sf2(-/-) mice. In conclusion, we have shown that the Tm7sf2 gene, to date involved only in cholesterol biosynthesis, also controls an anti-inflammatory loop thereby confirming the existence of cross talk between metabolic pathways and inflammatory response.

Adenosine A(1) receptors contribute to mitochondria vulnerability to pro-oxidant stressors.

A(1) adenosine receptors are highly expressed in the central nervous system. Mitochondrial function is a major player in adenosine receptors-mediated effects. Here, by using mice with genetic deletion of the A(1) receptor, we addressed the existence of a relationship between mitochondria functions and adenosine A(1) receptor. Mitochondrial functions and effects of MPP(+) in primary mixed cultures are influenced by the presence of the A(1) receptor, demonstrating, for the first time, the mitochondrial localization of the adenosine A(1) receptor and suggesting a role for this receptor as a mitochondrial vulnerability factor.

Ultracytochemistry as a tool for the study of the cellular and subcellular localization of membrane-bound guanylate cyclase (GC) activity. Applicability to both receptor-activated and receptor-independent GC activity.

Membrane-bound guanylate cyclase activity was detected by ultracytochemistry at the electron microscope level in several mammalian tissues. The technique used in these studies allows the detection of active enzyme at the membrane site where it is located. In a few cases, such as normal and regenerating peripheral nerves and placenta, membrane-bound guanylate cyclase could be detected in the absence of stimulators of enzyme activity. However, in the majority of these studies membrane-bound guanylate cyclase was investigated following stimulation with natriuretic peptides, guanylin, or the Ca2+ sensor proteins, S100B and S100A1. In general, membrane-bound guanylate cyclase was localized to plasma membranes, in accordance with the functional role of this enzyme. Yet, in secretory cells the enzyme activity was localized on intracellular membranes, suggesting a role of membrane-bound guanylate cyclase in secretory processes. Finally, S100B and S100A1 were found to colocalize with membrane-bound guanylate cyclase on photoreceptor disc membranes and to stimulate enzyme activity at these sites in dark-adapted retinas in a Ca2+-dependent manner. The results of these analyses are discussed in relation to the proposed functional role(s) of this enzyme.