Abstract
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.
Highlights
The scientific community and the pharmaceutical industry have invested enormous efforts and resources in the discovery and development of drugs with the goal of targeting cancerous mitochondria
The studies published in the literature suggest two mechanisms of anticancer effects of menadione/ascorbate: (i) extracellular generation of hydrogen peroxide due to ascorbate/menadione one-electron redox cycling and subsequent induction of oxidative stress, accompanied by activation of poly-[ADP ribose] polymerase 1 (PARP1), inhibition of glycolysis, depletion of NAD+ and ATP, and subsequent cell death [4, 24,25,26, 110, 111] and (ii) intracellular (“cytosolic”) generation of hydrogen peroxide due to ascorbate/menadione redox cycling and severe oxidative and replicative stress due to induction of Fenton’s reactions [28, 30, 79]
Both mechanisms are nonspecific and cannot explain (i) why M/A attacks cancer cells but not normal cells and (ii) why the anticancer effects of M/A in vivo are demonstrated at significantly lower plasma concentrations than those inducing cancer cell death in vitro [1, 3]
Summary
The scientific community and the pharmaceutical industry have invested enormous efforts and resources in the discovery and development of drugs with the goal of targeting cancerous mitochondria. This goal dates from the middle of the last century, when Warburg’s studies suggested that the mitochondria are dysfunctional in cancer. This is considered a hallmark of carcinogenesis and used as a molecular platform for the development of “targeted” anticancer therapy. The nature of mitochondrial dysfunction in cancer has not yet been discovered. There are serious debates and doubts about whether they are selective for cancerous mitochondria or they attack the mitochondria of normal cells too
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