**5. A mitochondrial basis for anticancer drugs combinations: a promising approach to therapy**

The severe toxicity promoted by anticancer agents represents a substantial health care burden that may seriously affect the treatment outcome. Based on the previous sections, mitochondria take center stage within the toxicity mechanisms, and are in the first line for protection by pharmacological strategies aiming to avoid alterations that may prove deleterious both in the short and in the long term. Considering that some of these effects are irreversible or cumulative, it is desirable to prevent these events when planning the therapy of cancer patients.

As discussed in the previous section, oxidative stress has been established as one of the primary cause of mitochondrial dysfunction and toxicity induced by anticancer agents and, therefore, several antioxidants have been tested *in vitro* and *in vivo* as a prophylactic measure. In particular, naturally occurring antioxidants have been investigated as therapeutic adjuvants, as they are considered safe and well-tolerated, and may afford protection against cancer treatment-related toxicity by improving mitochondrial functions.

Alpha-lipoic acid affords protection against the neurotoxic effects promoted by cisplatin and paclitaxel through its antioxidant and mitochondrial regulatory functions [135]. The toxic effects promoted by cisplatin on rat liver mitochondria are also prevented by thiol group protecting agents [71]. Curcumin, which has anti-inflammatory and anticancerous properties, counteracts the mitochondrial lipid peroxidation and GSH levels alterations in mitochondria isolated from the brain and liver of rats treated with cisplatin, suggesting that it can abrogate the toxic effects of cisplatin on brain and liver [136]. Likewise, epicatechin prevents the renal damage and mitochondrial dysfunction promoted by cisplatin by decreasing oxidative stress; noteworthy, epicatechin does not compromise the antitumor actions of cisplatin in HeLa cells [67].

The etoposide-induced MPT is prevented by ascorbate, the primary reductant of the phenoxyl radicals generated by etoposide, and by thiol protecting agents [107].

An *in vitro* study demonstrated that Vitamin E decreases the oxidative stress induced by methotrexate in rat heart mitochondria and thereby minimizes mitochondrial dysfunction [115]. Likewise, the administration of lipoic acid decreases oxidative stress induced by methotrexate, which affects liver mitochondrial function [114].

Acetyl-L-carnitine completely blocks the effects of bortezomib on mitochondria and pain [134].

Strategies to prevent doxorubicin-induced cardiotoxicity are probably the best studied, given the significant number of patients affected and the impact on the overall success of the treatment. Many studies reported that antioxidants could afford cardioprotection against doxorubicin therapy. The broad antioxidant resveratrol markedly ameliorates the cardiac dysfunction promoted by doxorubicin, while the ROS generation is decreased, and gluta‐ thione, superoxide dismutase and catalase activities are improved [137]. Also, flavonoids, and particularly 7-monohydroxyethylrutoside, protect against the cardiac toxic effects promoted by doxorubicin both *in vitro* and *in vivo* [138]. In addition, 7-monohydroxyethylrutoside does not compromise the antitumor activity of doxorubicin in human ovarian cell lines and in the corresponding mouse xenograft models, and even inhibits the overexpression of adhesion molecules promoted by doxorubicin on vascular endothelial cells [138]. The combination of doxorubicin and vitamin E-succinate cooperates to induce apoptosis in human gastric cancer cells, by promoting doxorubicin influx and suppressing its efflux [139]. On the other hand, vitamin E also aggravates the heart damage promoted by doxorubicin in P388 tumor-bearing mice [140].

Studies in animals demonstrated that the inhibition of mitochondrial respiration and the decrease in mitochondrial calcium accumulation capacity promoted by doxorubicin are prevented by the coadministration of the beta-adrenergic receptor antagonist carvedilol [87]. The prophylactic use of carvedilol in patients receiving doxorubicin contributes to maintain left ventricle diameters constant and to preserve diastolic function [141]. Interestingly, the toxic effects promoted by doxorubicin on heart mitochondria and cardiac cell apoptosis are prevented by carvedilol, but not by atenolol, another beta-adrenergic receptor antagonist with no antioxidant action, suggesting that the antioxidant properties and not the beta-adrenergic receptor antagonism are responsible for the cardioprotective effects of carvedilol [142]. Likewise, metoprolol, which also has no antioxidative properties, failes to afford cardiopro‐ tection in lymphoma patients treated with doxorubicin [143].

Dexrazoxane, a well-studied therapeutic adjuvant for doxorubicin chemotherapy, is a free radical scavenger that was found to have cardioprotective effects by preventing the functional damage of cardiac mitochondria initiated by ROS [83, 144]. Dexrazoxane prevents or reduces cardiac injury in doxorubicin-treated children with acute lymphoblastic leukemia without affecting the antitumor activity of doxorubicin [145]. In contrast, other iron chelators have failed to afford the same degree of cardioprotection, suggesting that iron does not play a crucial role in the oxidative stress-mediated toxicity of doxorubicin [138, 146, 147].

Promising results were obtained when the potent phosphodiesterase-5 inhibitor sildenafil is combined with doxorubicin. Prophylactic treatment with sildenafil prevents cardiomyocyte apoptosis and left ventricular dysfunction in a mouse chronic model of doxorubicin-induced cardiotoxicity [148]. On the other hand, in breast cancer cells, sildenafil enhances sensitivity to doxorubicin without enhancing its toxicity in bone marrow cells or macrophages [149]. Furthermore, cotreatment with sildenafil enhances doxorubicin-induced apoptosis in prostate cancer cells and inhibits tumor growth in mice bearing prostate tumor xenografts, while attenuating left ventricular dysfunction promoted by doxorubicin [150].

Interesting results were also observed when retinoids and antiestrogens are combined. Antiestrogenic compounds inhibit the MPT-induced by retinoids in isolated liver mitochon‐ dria [127, 151, 152]. Noteworthy, the prevention of MPT by antiestrogens does not compromise the antitumor efficacy of all-*trans*-retinoic acid, as an additive/synergistic action was demon‐ strated in breast cancer [153-156] and melanoma [157] cell lines. Therefore, we propose that studies *in vivo* with combined therapies are now required to confirm that these results obtained *in vitro* will translate into more therapeutic benefits in humans while attenuate mitochondrial dysfunctions promoted by drugs used individually.
