Visnagin—a new protectant against doxorubicin cardiotoxicity? Inhibition of mitochondrial malate dehydrogenase 2 (MDH2) and beyond

Doxorubicin (DOX) is a broad-spectrum and potent anthracycline antibiotic that has been widely used since 1960s as a chemotherapeutic agent to treat a variety of human cancers (1). Despite its superior anti-cancer efficacy, the clinical use of DOX is often limited by dose-dependent cardiotoxicity, which may lead to irreversible dilated cardiomyopathy and congestive heart failure (2,3). Currently predominant theories for explaining DOX cardiotoxicity include the DOX-induced increase of oxidative stress in cardiomyocytes (4), alteration of mitochondrial energetics (5,6), and direct effect on DNA. Particularly, anthracyclines promote the formation of reactive oxygen species (ROS) through redox cycling of their aglycones and anthracycline-iron complexes (7). Other suggested contributing factors to DOX cardiotoxicity comprise platelet-activating factor, prostaglandins, histamine, calcium, and C-13 hydroxy anthracycline metabolites, etc. (8,9). DOX is metabolized to doxorubicinol, which is implicated for its cardiotoxicity, possibly by causing perturbation of the iron homeostasis (4).

Mitochondria are abundant in cardiomyocytes and may take up to 35% of the cell volume. Since cardiac cells rely upon mitochondria-generated ATP to sustain dynamic contractile function, any interference with structural or functional integrity of mitochondria is likely to cause selective toxicity to the heart (5). Evidently, mitochondria have been considered as the primary targets for DOX cardiotoxicity (5,6). Accumulation of DOX aglycones in the inner membrane of mitochondria can interfere with electron carriers of the respiratory chain and cause the release of cytochrome c (10). DOX also disturb calcium homeostasis in cardiac mitochondria (11). DOX cardiotoxicity is associated with excessive ROS generation in mitochondria (12,13).

Due to the complex multi-factorial cellular and molecular drivers underlying DOX cardiotoxicity, the optimal therapeutic approaches for protection against DOX cardiotoxicity have not yet been identified, despite over 40 years of extensive research. Notably Herman et al. in 1972 first introduced bisdioxopiperazine compound as a cardioprotective agent against DOX cardiotoxicity (14). The subsequent research in this area led to identification of dexrazoxane, the only drug currently approved by the United States Food and Drug Administration (FDA) for reducing DOX cardiotoxicity (15). Dexrazoxane acts by displacing iron from anthracycline-iron complexes or by chelating free cellular iron and in turn preventing the site-specific iron-catalyzed ROS overproduction (7). However, critical reassessment of the so-called “ROS and iron” hypothesis indicated that numerous exogenous antioxidants failed to alleviate DOX cardiotoxicity in clinical trials (7). Several chelators that are stronger and more selective for iron than dexrazoxane did not protect against DOX cardiotoxicity (16,17). Another concern about dexrazoxane is its adverse effects of worsening myelosuppression and interfering with the anti-cancer efficacy of DOX, resulting in increased risk of secondary malignancy in pediatric patients with Hodgkin’s lymphoma (18). Therefore, there is an ongoing and urgent need to search for a better and safer cardioprotectant against DOX toxicity.

In an article published on December 10, 2014 in Science Translational Medicine, entitled “Visnagin protects against doxorubicin-induced cardiomyopathy through modulation of mitochondrial malate dehydrogenase” (19), Liu and colleagues from Massachusetts General Hospital and Harvard Medical School elegantly demonstrated protective effects of visnagin against DOX cardiotoxicity. These investigators first established a zebrafish model of DOX-induced cardiomyopathy (indicated by cardiac myocyte apoptosis and contractility decline) and screened ~3,000 compounds using this model. They found that visnagin and diphenylurea rescue the DOX-induced cardiac dysfunction and/or circulatory defects in zebrafish and mice. Importantly, visnagin did not reduce the chemotherapeutic efficacy of DOX in several cultured tumor cell lines, zebrafish, and mouse xenograft models (19).

First, in terms of methodological advances, Liu et al. (19) and a few other groups (20,21) have utilized zebrafish as an effective model for high-throughput screening a number of synthetic or natural chemical formula that possess protective effects against DOX cardiotoxicity. These simple and low-cost zebrafish models of tractable heart failure represent a good alternative approach differed from the much more expensive mammalian models for large scale and non-biased drug discovery process targeting the complex heart failure pathologies, e.g., DOX cardiomyopathy in adult zebrafish and aristolochic acid-caused cardiac defects in zebrafish embryos (19-21). It is plausible to predict that this kind of approaches may lead to additional discoveries of novel drug candidates for prevention and treatment of DOX cardiotoxicity.

One of the exciting findings from the work of Liu et al. is the identification of mitochondrial malate dehydrogenase (MDH2) as a novel target for reducing DOX cardiotoxicity (19). By means of affinity chromatography, these researchers revealed that visnagin binds to MDH2, a key enzyme in the tricarboxylic acid cycle. They further demonstrated that treatment with other known structurally diverse inhibitors of MDH2 (i.e., mebendazole, thyroxine, iodine, malate) also prevented DOX cardiotoxicity, suggesting that modulation of MDH2 activity is responsible for cardioprotective effects of visnagin. MDH are active enzymes in glyoxysomes, mitochondria, peroxisomes, chloroplasts and the cytosol (22) that interact with the malate-aspartate shuttle (MAS), which constitutes the primary metabolic pathway for transfer of reducing equivalents from the cytosol into the mitochondria for oxidation. The MAS enzymatic reactions include the coupled cytosolic and mitochondrial transamination of aspartate and glutamate and inter-conversion of malate and oxaloacetate by MDH. The enzymes of MAS co-localize with enzymes of the tricarboxylic acid cycle at the inner mitochondrial membrane. MAS also shares intermediates with the tricarboxylic acid cycle (oxaloacetate, malate, α-ketoglutarate) and the electron transport chain (glutamate, malate) ensuring a tight co-ordination between shuttle activity and mitochondrial respiration (23). The flux of MAS is tightly linked to the flux of the tricarboxylic acid cycle and the electron transport chain and MAS is also an important regulator of cytosolic and mitochondrial calcium homeostasis. It was reported that inhibition of MAS during ischemia and early reperfusion led to down-regulation of mitochondrial respiration during lethal ischemia and in turn afforded cardioprotective effects similar to ischemic preconditioning (23). Hence, it is conceivable that visnagin induces cardioprotection against DOX cardiotoxicity—a type of non-ischemic tissue injury, likewise via inhibition of MDH2 and in turn the MAS.

Remarkably, an inhibitor of MDH2 such as visnagin may also be beneficial against resistance to chemotherapy in cancer cells. A recent study showed that MDH2 confers docetaxel resistance in prostate cancer via regulations of JNK signaling and oxidative metabolism (24). It was shown that MDH2 is overexpressed in clinical prostate cancer specimens. Patients with MDH2 overexpression had a significantly shorter period of relapse-free survival after chemotherapy. Whereas MDH2 expression was elevated in prostate cancer cell lines compared to benign prostate epithelial cells, stable knockdown of MDH2 via shRNA in the cancer cells decreased cell proliferation and increased docetaxel sensitivity. More recently, Lo et al. reported that MDH2 plays a role in the development of DOX-resistant uterine cancer (25). Therefore, the potential benefits in both cardioprotective and anti-cancer fronts would make visnagin more desirable as an adjunct agent for cancer chemotherapy.

Nevertheless, it remains relatively uncertain if MDH2 inhibition is the only mechanism underlying the visnagin-induced cardioprotection. Previous studies from Duarte and colleagues demonstrated a significant vasodilatory effect of visnagin that can lead to a systemic hypotensive response to in vivo administration of this compound (26,27). Interestingly, our previous studies showed that several chemical or natural compounds with vasodilatory property can also afford protective effects against DOX cardiotoxicity in mice. One of the cardioprotective modalities is dietary supplementation of inorganic nitrate, which improves post-DOX ventricular contractile function, cell survival, and mitochondrial respiratory chain function (28,29). We also revealed cardioprotective effects of phosphodiesterase 5 (PDE5) inhibitors—sildenafil (30) and tadalafil (31), two well-known vasodilators, against DOX cardiotoxicity. Intriguingly, visnagin is also reported to be a weak pan-inhibitor of PDE (32). The exact contribution of the vasoactive effects of visnagin during its in vivo administration to the drug-induced cardioprotection remains to be evaluated.

In addition, as a possible mechanistic perspective, it will be interesting to find out whether or not vasnagin and other MDH2 inhibitors affect topoisomerase 2 (Top2)—a key regulator of DNA replication, transcription, and recombination. Recent studies from Dr. Yeh’s group at the MD Anderson Cancer Center in Houston, Texas demonstrated that DOX inhibits both Top2α (found in the rapidly proliferating tumor cells) and Top2β (found in the less actively dividing cells, such as cardiomyocytes) and inhibition of Top2β plays a key role in mediating DOX cardiotoxicity (33,34). These investigators also showed that Top2β deletion protected mice from cardiotoxic effects of DOX. Therefore, based on the demonstrated important role played by Top2β in development of DOX cardiotoxicity (35), Top2β should be considered as a potential target for cardioprotective therapy, including the use of visnagin.

After all, can we translate the cardioprotective effects of visnagin observed in zebrafish and rodents (19) into an effective cardioprotectant in helping cancer patients receiving DOX chemotherapy? The ultimate answer depends upon a series of rigorous pre-clinical validation in larger mammalian species as well as clinical trials in cancer patients for attesting the therapeutic usefulness of visnagin against DOX cardiotoxicity in humans. In terms of translational value, it is noteworthy that visnagin is a natural product extracted from the plant Ammi Visnaga, which grows wild in the Eastern Mediterranean countries. This compound was traditionally prescribed by Arabic folk physicians as a diuretic and antispasmodic in cases of ureteral stones and it was also studied in animal models for ameliorating angina pectoris (36). The potential utilities of such botanical derivatives as reported by Liu et al. on visnagin (19) and by our laboratory on inorganic nitrate (beetroot juice) (29,37-39) deserve further studies and validations, in order to develop them into potentially effective and affordable therapies for protecting thousands of cancer patients undergoing DOX chemotherapy against its devastating cardiotoxicity and eventually to improve the clinical outcome and quality of life of the cancer survivors.