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

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.

Targeting Glioblastoma via Selective Alteration of Mitochondrial Redox State

“…This is direct evidence that menadione does not act as a coagulant when used in combination with ascorbate in doses with a pronounced anticancer effect. M/A at concentrations up to 20/2000 µM/µM, exceeding those that can be achieved pharmacologically, did not affect the viability of normal human lymphocytes and normal human epithelial cells, which was analyzed in vitro in our previous study [46,65].…”

“…This effect is thought to be due to the redox cycling between the two molecules in the cells and the extracellular environment, which requires an excess of ascorbate and leads to the overproduction of ROS (Figure S1, see the Supporting Materials). The mechanism and the combination index are described in many articles summarized recently in [46]. M/A has attracted the attention of researchers due to the synergistic anticancer effect of the two molecules, as well as its ability to kill cancer cells without affecting the viability of normal cells [46].…”

“…The mechanism and the combination index are described in many articles summarized recently in [46]. M/A has attracted the attention of researchers due to the synergistic anticancer effect of the two molecules, as well as its ability to kill cancer cells without affecting the viability of normal cells [46]. Importantly, the strong synergistic antiproliferative and cytotoxic effect on cancer cells is inherent for the combination of ascorbate and menadione (pro-vitamin K3), but not for the combination of ascorbate and vitamin K1 or K2 [46].…”

“…M/A has attracted the attention of researchers due to the synergistic anticancer effect of the two molecules, as well as its ability to kill cancer cells without affecting the viability of normal cells [46]. Importantly, the strong synergistic antiproliferative and cytotoxic effect on cancer cells is inherent for the combination of ascorbate and menadione (pro-vitamin K3), but not for the combination of ascorbate and vitamin K1 or K2 [46]. This suggests that the prenylation of menadione and its conversion into vitamin K1 or K2 will decrease its anticancer activity in combination with ascorbate.…”

Anticancer Activity of Aqueous Extracts from Asparagus officinalis L. Byproduct on Breast Cancer Cells

“…Our results similarly showed a specific cooperative effect on HR + /HER2 − cells (MCF7), while preserving normal control fibroblasts. To further support our theory, it is of interest to cite Bakalova et al, who demonstrated that using menadione/ascorbate in combination with 13 conventional clinically used drugs for leukemia treatment did not induce cytotoxicity in cancer cells, but instead, caused irreversible metabolic changes, increasing sensitivity toward conventional therapy [23]. These observations are particularly important because they lay the foundations for future use of extracts to enhance the effects of clinically used drugs that act on breast cancer cells through the induction of oxidative stress, such as tamoxifen (used for hormonal therapy) or paclitaxel (used for chemotherapy).…”