The novel COVID-19 pandemic is affecting the world’s population differently: mostly in the presence of conditions such as aging, diabetes and hypertension the virus triggers a lethal cytokine storm and patients die from acute respiratory distress syndrome, whereas in many cases the disease has a mild or even asymptomatic progression. A common denominator in all conditions associated with COVID-19 appears to be the impaired redox homeostasis responsible for reactive oxygen species (ROS) accumulation; therefore, levels of glutathione (GSH), the key anti-oxidant guardian in all tissues, could be critical in extinguishing the exacerbated inflammation that triggers organ failure in COVID-19. The present review provides a biochemical investigation of the mechanisms leading to deadly inflammation in severe COVID-19, counterbalanced by GSH. The pathways competing for GSH are described to illustrate the events concurring to cause a depletion of endogenous GSH stocks. Drawing on evidence from literature that demonstrates the reduced levels of GSH in the main conditions clinically associated with severe disease, we highlight the relevance of restoring GSH levels in the attempt to protect the most vulnerable subjects from severe symptoms of COVID-19. Finally, we discuss the current data about the feasibility of increasing GSH levels, which could be used to prevent and subdue the disease.
Multidrug resistance (MDR) is a phenomenon by which cancer cells evade the cytotoxic effects of chemotherapeutic agents. It may occur through different mechanisms, but it often correlates with the overexpression of integral membrane transporters, such as P-glycoprotein (Pgp) and MDR-associated proteins (MRPs), with resulting decrease of drug accumulation and cellular death. Doxorubicin is a substrate of Pgp; it has been suggested that its ability to induce synthesis of nitric oxide (NO) could explain, at least in part, its cytotoxic effects. Culturing the human epithelial colon cell line HT29 in the presence of doxorubicin, we obtained a doxorubicin-resistant (HT29-dx) cell population: these cells accumulated less intracellular doxorubicin, were less sensitive to the cytotoxic effects of doxorubicin and cisplatin, overexpressed Pgp and MRP3, and exhibited a lower NO production (both under basal conditions and after doxorubicin stimulation). The resistance to doxorubicin could be reversed when HT29-dx cells were incubated with inducers of NO synthesis (cytokines mix, atorvastatin). Some NO donors increased the drug accumulation in HT29-dx cells in a guarosine-3′:5′-cyclic monophosphate–independent way; this effect was associated with a marked reduction of doxorubicin efflux rate in HT29 and HT29-dx cells, and tyrosine nitration in the MRP3 protein. Our results suggest that onset of MDR and impairment of NO synthesis are related; this finding could point to a new strategy to reverse doxorubicin resistance in human cancer.
We recently described the mitochondrial localization and import of the vitamin D receptor (VDR) in actively proliferating HaCaT cells for the first time, but its role in the organelle remains unknown. Many metabolic intermediates that support cell growth are provided by the mitochondria; consequently, the identification of proteins that regulate mitochondrial metabolic pathways is of great interest, and we sought to understand whether VDR may modulate these pathways. We genetically silenced VDR in HaCaT cells and studied the effects on cell growth, mitochondrial metabolism and biosynthetic pathways. VDR knockdown resulted in robust growth inhibition, with accumulation in the G0G1 phase of the cell cycle and decreased accumulation in the M phase. The effects of VDR silencing on proliferation were confirmed in several human cancer cell lines. Decreased VDR expression was consistently observed in two different models of cell differentiation. The impairment of silenced HaCaT cell growth was accompanied by sharp increases in the mitochondrial membrane potential, which sensitized the cells to oxidative stress. We found that transcription of the subunits II and IV of cytochrome c oxidase was significantly increased upon VDR silencing. Accordingly, treatment of HaCaT cells with vitamin D downregulated both subunits, suggesting that VDR may inhibit the respiratory chain and redirect TCA intermediates toward biosynthesis, thus contributing to the metabolic switch that is typical of cancer cells. In order to explore this hypothesis, we examined various acetyl-CoA-dependent biosynthetic pathways, such as the mevalonate pathway (measured as cholesterol biosynthesis and prenylation of small GTPases), and histone acetylation levels; all of these pathways were inhibited by VDR silencing. These data provide evidence of the role of VDR as a gatekeeper of mitochondrial respiratory chain activity and a facilitator of the diversion of acetyl-CoA from the energy-producing TCA cycle toward biosynthetic pathways that are essential for cellular proliferation.
Intraerythrocytic malaria parasites produce vast amounts of lactic acid through glycolysis. While the egress of lactate is very rapid, the mode of extrusion of H+ is not known. The possible involvement of a Na+/H+ antiport in the extrusion of protons across the plasma membrane of Plasmodium falciparum has been investigated by using the fluorescent pH probe 6-carboxyfluorescein. The resting cytosolic pH was 7.27 +/- 0.1 in ring stage parasites and 7.31 +/- 0.12 in trophozoites. Spontaneous acidification of parasite cytosol was observed in Na(+)-free medium and realkalinization occurred upon addition of Na+ to the medium in a concentration-dependent manner, with no apparent saturation. The rate of H(+)-efflux at the ring stage was higher than that at the trophozoite stage due to the larger surface/volume ratio of the young parasite stage. Na(+)-dependent H(+)-efflux was: 1) inhibited by the Na+/H+ inhibitors amiloride and 5-(N-ethyl-N-isopropyl) amiloride (EIPA), though at relatively high concentrations; 2) augmented with rising pH6 (pHi = 6.2, [Na+]o = 30 mM); and 3) decreased with increasing pHi (pHo = 7.4; [Na+]o = 30 mM). The pHi and the pHo dependencies of H(+)-efflux were almost identical at all parasite stages. Only at pHi > 7.6 efflux was totally obliterated. The target of this inhibitory effect is probably other than the antiport. Results indicate that H(+)-egress is mediated by a Na+/H+ antiport which is regulated by host and parasite pH and by the host cytosol sodium concentration. The proton transport capacity of the antiport can easily cope with all the protons of lactic acid produced by parasite's glycolysis.
Even in cells that are resistant to the differentiating effects of vitamin D, the activated vitamin D receptor (VDR) can downregulate the mitochondrial respiratory chain and sustain cell growth through enhancing the activity of biosynthetic pathways. The aim of this study was to investigate whether vitamin D is effective also in modulating mitochondria and biosynthetic metabolism of differentiating cells. We compared the effect of vitamin D on two cellular models: the primary human keratinocytes, differentiating and sensitive to the genomic action of VDR, and the human keratinocyte cell line HaCaT, characterized by a rapid growth and resistance to vitamin D. We analysed the nuclear translocation and features of VDR, the effects of vitamin D on mitochondrial transcription and the consequences on lipid biosynthetic fate. We found that the negative modulation of respiratory chain is a general mechanism of action of vitamin D, but at high doses, the HaCaT cells became resistant to mitochondrial effects by upregulating the catabolic enzyme CYP24 hydroxylase. In differentiating keratinocytes, vitamin D treatment promoted intracellular lipid deposition, likewise the inhibitor of respiratory chain stigmatellin, whereas in proliferating HaCaT, this biosynthetic pathway was not inducible by the hormone. By linking the results on respiratory chain and lipid accumulation, we conclude that vitamin D, by suppressing respiratory chain transcription in all keratinocytes, is able to support both the proliferation and the specialized metabolism of differentiating cells. Through mitochondrial control, vitamin D can have an essential role in all the metabolic phenotypes occurring in healthy and diseased skin.
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