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.

中文翻译：

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氧化还原活性治疗佐剂的癌性线粒体的选择性靶向和肿瘤生长的抑制。

氧化还原活性物质及其组合，例如醌/抗坏血酸，尤其是甲萘醌/抗坏血酸（M / A；也称为Apatone®），以其杀死癌细胞的异常能力而又不影响正常细胞的活力而引起关注。以及两个分子的协同抗癌作用。到目前为止，M / A介导的抗癌作用的主要机制尚未与线粒体相关。我们研究的目的是阐明这种“组合药物”是否特别影响癌细胞中的线粒体功能。对癌细胞（Jurkat，Colon26和MCF7）和正常细胞（正常淋巴细胞，FHC和MCF10A）进行了研究，分别用不同浓度的甲萘醌，抗坏血酸和/或其组合（2 / 200、3 / 300， 5/500，10/1000，和20/2000  μM / μ M / A的M）。M / A对癌细胞的生长表现出高度特异性和协同抑制作用，但在药理学上可达到的浓度下对正常细胞的活力没有不利影响。在经M / A处理的癌细胞中，细胞抑制/细胞毒性作用伴随着（i）线粒体超氧化物的极高产生（是对照水平的15倍），（ii）线粒体膜电位的显着降低，（ iii）ATP，琥珀酸，NADH和NAD的稳态水平降低⁺（iv）程序性细胞死亡配体1（PD-L1）（主要的免疫检查点之一）的表达降低。这些作用是剂量依赖性的。双香豆酚对NQO1的抑制作用增加了线粒体超氧化物歧化并使癌细胞对M / A敏感。在正常细胞中，M / A诱导线粒体超氧化物的相对较低且剂量依赖性的增加和轻度的氧化应激，这似乎具有良好的耐受性。这些数据表明，M / A的所有抗癌作用均来自与癌细胞线粒体紧密相关的特定机制。在低/可耐受的剂量M / A的（1 / 100-3 / 300  μ M / μM）通过口服和肠胃外给药可在癌症中获得，M / A使癌细胞对常规抗癌药物敏感，表现出协同或累加的细胞毒性，并伴随令人印象深刻的凋亡诱导。研究了M / A与13种抗癌药的组合（ABT-737，barasertib，博来霉素，BEZ-235，硼替佐米，顺铂，依维莫司，洛莫斯汀，lonafarnib，MG-132，MLN-2238，palbociclib和PI-103）。低/可耐受剂量的M / A不会在癌细胞中诱导不可逆的细胞毒性，但会引起不可逆的代谢变化，包括：（i）琥珀酸盐和NADH减少，（ii）线粒体膜去极化，以及（iii）过量生产超氧化物仅存在于癌细胞的线粒体中。此外，M / A抑制了黑色素瘤小鼠口服后体内肿瘤的生长，并且该药物下调了黑色素瘤细胞中的PD-L1。实验数据表明，通过增加癌细胞对常规抗癌疗法以及免疫系统的敏感性，同时保留正常细胞，M / A具有有益的抗癌作用的巨大潜力。我们假设M / A介导的抗癌作用是由这两种物质的氧化还原循环触发的，特别是在功能异常的线粒体中。M / A还可能对免疫系统产生有益作用，使癌细胞“可见”，更容易受到天然免疫反应的影响。实验数据表明，通过增加癌细胞对常规抗癌疗法以及免疫系统的敏感性，同时保留正常细胞，M / A具有有益的抗癌作用的巨大潜力。我们假设M / A介导的抗癌作用是由这两种物质的氧化还原循环触发的，特别是在功能异常的线粒体中。M / A还可能对免疫系统产生有益作用，使癌细胞“可见”，更容易受到天然免疫反应的影响。实验数据表明，通过增加癌细胞对常规抗癌疗法以及免疫系统的敏感性，同时保留正常细胞，M / A具有有益的抗癌作用的巨大潜力。我们假设M / A介导的抗癌作用是由这两种物质的氧化还原循环触发的，特别是在功能异常的线粒体中。M / A还可能对免疫系统产生有益作用，使癌细胞“可见”，更容易受到天然免疫反应的影响。