Intramitochondrial Ascorbic Acid Enhances the Formation of Mitochondrial Superoxide Induced by Peroxynitrite via a Ca2+-Independent Mechanism

Guidarelli, Andrea; Cerioni, Liana; Fiorani, Mara; Cantoni, Orazio

doi:10.3390/ijms18081686

Cited by 5 publications

(6 citation statements)

References 50 publications

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“…Both mechanisms are based on the assumption of overproduction of hydrogen peroxide due to one-electron menadione/ascorbate redox cycling [ 23 – 25 , 28 , 30 , 57 , 79 ]. All these events are reported at high concentrations of M/A (>5/500 μ M/ μ M), and conclusions are based on indirect evidence such as (i) effects of catalase, metal chelators, antioxidants, and end-products of oxidative stress in M/A-treated cells [ 23 – 25 , 28 , 57 , 65 ] or (iii) production of superoxide and/or hydrogen peroxide in cells treated with menadione or ascorbate alone [ 79 – 84 ]. Based on our knowledge, there are no data about direct detection of production and/or degradation of superoxide and/or hydrogen peroxide in M/A-treated cells.…”

Section: Resultsmentioning

confidence: 99%

Selective Targeting of Cancerous Mitochondria and Suppression of Tumor Growth Using Redox-Active Treatment Adjuvant

Bakalova

Semkova

Ivanova

et al. 2020

Oxidative Medicine and Cellular Longevity

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Redox-active substances and their combinations, such as of quinone/ascorbate and in particular menadione/ascorbate (M/A; also named Apatone®), attract attention with their unusual ability to kill cancer cells without affecting the viability of normal cells as well as with the synergistic anticancer effect of both molecules. So far, the primary mechanism of M/A-mediated anticancer effects has not been linked to the mitochondria. The aim of our study was to clarify whether this “combination drug” affects mitochondrial functionality specifically in cancer cells. Studies were conducted on cancer cells (Jurkat, Colon26, and MCF7) and normal cells (normal lymphocytes, FHC, and MCF10A), treated with different concentrations of menadione, ascorbate, and/or their combination (2/200, 3/300, 5/500, 10/1000, and 20/2000 μM/μM of M/A). M/A exhibited highly specific and synergistic suppression on cancer cell growth but without adversely affecting the viability of normal cells at pharmacologically attainable concentrations. In M/A-treated cancer cells, the cytostatic/cytotoxic effect is accompanied by (i) extremely high production of mitochondrial superoxide (up to 15-fold over the control level), (ii) a significant decrease of mitochondrial membrane potential, (iii) a decrease of the steady-state levels of ATP, succinate, NADH, and NAD+, and (iv) a decreased expression of programed cell death ligand 1 (PD-L1)—one of the major immune checkpoints. These effects were dose dependent. The inhibition of NQO1 by dicoumarol increased mitochondrial superoxide and sensitized cancer cells to M/A. In normal cells, M/A induced relatively low and dose-independent increase of mitochondrial superoxide and mild oxidative stress, which seems to be well tolerated. These data suggest that all anticancer effects of M/A result from a specific mechanism, tightly connected to the mitochondria of cancer cells. At low/tolerable doses of M/A (1/100-3/300 μM/μM) attainable in cancer by oral and parenteral administration, M/A sensitized cancer cells to conventional anticancer drugs, exhibiting synergistic or additive cytotoxicity accompanied by impressive induction of apoptosis. Combinations of M/A with 13 anticancer drugs were investigated (ABT-737, barasertib, bleomycin, BEZ-235, bortezomib, cisplatin, everolimus, lomustine, lonafarnib, MG-132, MLN-2238, palbociclib, and PI-103). Low/tolerable doses of M/A did not induce irreversible cytotoxicity in cancer cells but did cause irreversible metabolic changes, including: (i) a decrease of succinate and NADH, (ii) depolarization of the mitochondrial membrane, and (iii) overproduction of superoxide in the mitochondria of cancer cells only. In addition, M/A suppressed tumor growth in vivo after oral administration in mice with melanoma and the drug downregulated PD-L1 in melanoma cells. Experimental data suggest a great potential for beneficial anticancer effects of M/A through increasing the sensitivity of cancer cells to conventional anticancer therapy, as well as to the immune system, while sparing normal cells. We hypothesize that M/A-mediated anticancer effects are triggered by redox cycling of both substances, specifically within dysfunctional mitochondria. M/A may also have a beneficial effect on the immune system, making cancer cells “visible” and more vulnerable to the native immune response.

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Section: Resultsmentioning

confidence: 99%

Selective Targeting of Cancerous Mitochondria and Suppression of Tumor Growth Using Redox-Active Treatment Adjuvant

Bakalova

Semkova

Ivanova

et al. 2020

Oxidative Medicine and Cellular Longevity

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“…Importantly, Vitamin C is highly concentrated within mitochondria [7–11]. For example, when U937 cells (a human leukaemia cell line) were incubated for only 15 minutes in media containing 3 μM Vitamin C, it was very efficiently transported to the mitochondria, reaching a level of 5 mM – being concentrated ~1,700-fold [8]. Mitochondrial transport of Vitamin C is accomplished by the sodium-coupled Vitamin C transporter 2 (SVCT2) [7–10], also known as SLC23A2, although other novel mitochondrial transporters have been suggested [11].…”

Section: Discussionmentioning

confidence: 99%

Doxycycline, Azithromycin and Vitamin C (DAV): A potent combination therapy for targeting mitochondria and eradicating cancer stem cells (CSCs)

Fiorillo

Tóth

Sotgia

et al. 2019

Aging

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Here, we devised a new strategy for eradicating cancer stem cells (CSCs), via a “synthetic-metabolic” approach, involving two FDA-approved antibiotics and a dietary vitamin supplement. This approach was designed to induce a “rho-zero-like” phenotype in cancer cells. This strategy effectively results in the synergistic eradication of CSCs, using vanishingly small quantities of two antibiotics. The 2 metabolic targets are i) the large mitochondrial ribosome and ii) the small mitochondrial ribosome. Azithromycin inhibits the large mitochondrial ribosome as an off-target side-effect. In addition, Doxycycline inhibits the small mitochondrial ribosome as an off-target side-effect. Vitamin C acts as a mild pro-oxidant, which can produce free radicals and, as a consequence, induces mitochondrial biogenesis. Remarkably, treatment with a combination of Doxycycline (1 μM), Azithromycin (1 μM) plus Vitamin C (250 μM) very potently inhibited CSC propagation by >90%, using the MCF7 ER(+) breast cancer cell line as a model system. The strong inhibitory effects of this DAV triple combination therapy on mitochondrial oxygen consumption and ATP production were directly validated using metabolic flux analysis. Therefore, the induction of mitochondrial biogenesis due to mild oxidative stress, coupled with inhibition of mitochondrial protein translation, may be a new promising therapeutic anti-cancer strategy. Consistent with these assertions, Vitamin C is known to be highly concentrated within mitochondria, by a specific transporter, namely SVCT2, in a sodium-coupled manner. Also, the concentrations of antibiotics used here represent sub-antimicrobial levels of Doxycycline and Azithromycin, thereby avoiding the potential problems associated with antibiotic resistance. Finally, we also discuss possible implications for improving health-span and life-span, as Azithromycin is an anti-aging drug that behaves as a senolytic, which selectively kills and removes senescent fibroblasts.

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“…As an example, we found that preexposure of U937 cells to AA enhances their susceptibility to the deleterious effects mediated by various hydroperoxides, in particular peroxynitrite [ 11 , 46 ]. We also found that the mitochondrial fraction of AA is specifically linked to the enhanced cyto-genotoxicity induced by peroxynitrite [ 30 , 46 ].…”

Section: Resultsmentioning

confidence: 99%

“…We also employed a different strategy based on our previous findings indicating that preexposure of undifferentiated U937 cells to AA enhances their susceptibility to the deleterious effects mediated by peroxynitrite [ 11 , 46 ]. These enhancing effects are based on the notion that mitochondrial AA increases the mitochondrial formation of superoxide mediated by peroxynitrite, via a Ca 2+ -independent mechanism associated with inhibition of complex III [ 30 , 46 ]. As expected, preexposure to 10 μ M AA significantly increased mitochondrial superoxide formation and its downstream deleterious effects, induced by peroxynitrite in the undifferentiated cells.…”

Section: Discussionmentioning

confidence: 99%

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Differentiation of Promonocytic U937 Cells to Monocytes Is Associated with Reduced Mitochondrial Transport of Ascorbic Acid

Scotti

Fiorani

Guidarelli

et al. 2018

Oxidative Medicine and Cellular Longevity

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Growth of promonocytic U937 cells in the presence of DMSO promotes their differentiation to monocytes. After 4 days of culture in differentiating medium, these cells ceased to proliferate, displayed downregulated ryanodine receptor expression, and responded to specific stimuli with enhanced NADPH-oxidase-derived superoxide formation or cytosolic phospholipase A2-dependent arachidonic acid release. We found that the 4-day differentiation process is also associated with downregulated SVCT2 mRNA expression, in the absence of apparent changes in SVCT2 protein expression and transport rate of ascorbic acid (AA). Interestingly, under the same conditions, these cells accumulated lower amounts of the vitamin in their mitochondria, with an ensuing reduced response to external stimuli sensitive to the mitochondrial fraction of AA. Further analyses demonstrated an unexpected increase in mitochondrial SVCT2 protein expression, however, associated with reduced SVCT2-dependent AA uptake in isolated mitochondria. A decrease in the transporter Vmax, with no change in affinity, was found to account for this response. Differentiation of promonocytic cells to monocytes is therefore characterized by decreased SVCT2 mRNA expression that, even prior to the onset of SVCT2 protein downregulation or apparent changes in plasma membrane transport activity, impacts on the mitochondrial accumulation of the vitamin through a decreased Vmax of the transporter.

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Intramitochondrial Ascorbic Acid Enhances the Formation of Mitochondrial Superoxide Induced by Peroxynitrite via a Ca2+-Independent Mechanism

Cited by 5 publications

References 50 publications

Selective Targeting of Cancerous Mitochondria and Suppression of Tumor Growth Using Redox-Active Treatment Adjuvant

Selective Targeting of Cancerous Mitochondria and Suppression of Tumor Growth Using Redox-Active Treatment Adjuvant

Doxycycline, Azithromycin and Vitamin C (DAV): A potent combination therapy for targeting mitochondria and eradicating cancer stem cells (CSCs)

Differentiation of Promonocytic U937 Cells to Monocytes Is Associated with Reduced Mitochondrial Transport of Ascorbic Acid

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