Anthracyclines are chemotherapeutic agents employed in the treatment of a wide range of cancer such as breast cancer, leukemia, lymphoma, lung cancer, multiple myeloma, and sarcoma. The clinical use of anthracyclines, in particular doxorubicin, is well-known to be limited by the cumulative dose-related toxicity, the reduction in left ventricular ejection fraction (LVEF), and the irreversible cardiotoxicity [1
]. In particular, cardiotoxicity usually occurs one year after the completion of chemotherapy. Moreover, a rare form of acute cardiotoxicity, characterized by pericarditis and arrhythmias, has been described [2
]. The heart is a target organ of oxidative stress-related injuries because of its very high energetic metabolic demand and lower levels of antioxidant agents and enzymes [3
]. The chronic cardiotoxicity of doxorubicin induces cardiomyocyte death through free radical generation caused by mitochondrial Nicotinamide adenine dinucleotide (NADH) dehydrogenase-catalyzed reduction of anthracycline and by interference with iron metabolism catalyzing the Fenton reaction [4
Many studies have demonstrated that healthy lifestyle and diet can help to prevent the insurgence of chronic pathologies such as cardiovascular and neurodegenerative diseases, diabetes, and cancer [5
]. Nowadays, high attention has been addressed to natural components in fruits and vegetables with potential antioxidant, anti-inflammatory, anticarcinogenic and antimutagenic properties [6
]. In particular, some scientific evidence described the potential health effects that dietary polyphenols are able to exercise on biological systems through antioxidant, cardioprotective, anti-inflammatory and anti-proliferative activities [10
]. The consumption of dietary supplements and nutraceuticals in cancer therapy is mainly aimed at chemoprevention to reduce the drug resistance, to the identification of synergistic effects with anticancer drugs in order to decrease drug concentrations and, consequently, the side effects associated with current cancer treatments. In fact, chemotherapy protocols including surgery, pharmacotherapy, radiotherapy, and biologically based therapies, have unintended side effects, which compromise both health and well-being of patients [11
]. In this regard, dietary polyphenols have been widely demonstrated to be able to not only reduce oxidative and inflammatory stress typical of the onset and development of cancer, but also to counteract the side effects associated with drug therapy [12
Different species of plants are able to protect against cancer including Vitis vinifera
and Citrus sinensis
]. Red grape juice (Vitis vinifera
L. cv. Aglianico N) demonstrated an appreciable direct radical-scavenging activity, able to contrast the doxorubicin-induced oxidative stress in cardiac-derived myocytes, decreasing ROS levels and depressing caspase-3 activity [15
]. It has been also reported the antioxidant and anti-inflammatory potential of Citrus sinensis
. The methanolic extract was able to reduce macrophages pro-inflammatory mediators and different cytokines. Furthermore, Citrus sinensis
polyphenols have been shown to have antioxidant effect by reducing reactive oxygen species (ROS) and increasing cytoprotective enzymes expression [16
]. In this regard, in the present study, we evaluated the ability of Vitis vinifera
and Citrus sinensis
smoothies, and relative mixes, to inhibit and counteract the doxorubicin-induced oxidative stress in embryonic rat heart-derived cells (H9c2). Moreover, the antiproliferative activity of the smoothies in human breast adenocarcinoma cell line (MCF-7) exposed to the anthracycline was also evaluated.
Citrus fruits and red grape juice are considered worldwide as a healthy food due to the high content of bioactive compounds and they are increasingly used in pharmaceutical and nutraceuticals fields for their antioxidant, anti-inflammatory, anti-proliferative and potential anticancer activities [23
]. In this regard, the use of nutraceutical formulations based on phytochemical extracts both in chemoprevention and as adjuvants in support of pharmacological therapies, is increasingly being proposed [25
Although anthracyclines effectiveness is still a gold standard in the treatment of various cancer, the clinical use of doxorubicin is limited by severe cardiotoxicity [26
]. This cytotoxic side effect occurs through multiple mechanisms, which mainly involve doxorubicin accumulation in myocardial mitochondrial membrane and subsequent reactive oxygen species production. In addition, doxorubicin quinone-containing structure is prone to ROS release by redox-cycling reaction that, associated to a reduction in endogenous antioxidants, increase oxidative stress, ultimately leading to myocyte apoptosis [27
]. Among several strategies to limit doxorubicin adverse effects, natural polyphenolic compounds are notable as a cardioprotective agent because of the low toxicity and good antioxidant properties through radical-scavenging activity, carbonyl reductase-inhibitory effects, as well as, modulation of endogenous antioxidant enzymes [28
]. Apart from their antioxidant effects, also apoptosis modulation is involved in the protective properties of the flavonoids against doxorubicin toxicity [32
In this work, we evaluated the use of different mixed ratio of commercial nutraceutical smoothies, obtained from Citrus sinensis and Vitis vinifera fruits, in chemotherapeutics protocols to alleviate anthracyclines adverse effects, but also enhance drugs selectivity, lowering their dosage, without interfere with pharmacological effect on cancer cells.
flavonoids have been shown to reduce cardiovascular disease prominently due to their antioxidant effects [33
]. In this regard, hesperidin, abundant flavanone glycoside present in citrus
fruits, has been demonstrated to modulate the inflammatory responses and antioxidant status following acute myocardial infarction and could be considered as a promising candidate to alleviate cardiotoxic effect of doxorubicin [34
]. In addition, hesperitin, the aglycone of hesperidin, has a cardioprotective effect increasing GHS levels and preventing oxidative DNA damage in rat models [35
]. Furthermore, has been reported that Pummelo (Citrus maxima
) phytocomplex exert protection in cardiomyocytes by reduction of intracellular oxidative stress, maintaining GSH availability, and enhancing GST enzyme activity and expression in rat cardiac cells [36
Some scientific evidence has attributed to grape proanthocyandins the contribution to iron and calcium levels normalizations and apoptotic signaling pathways regulation in human cardiomyocytes cells subjected to doxorubicin effects [37
]. However, grape is also a rich source of anthocyanins that possess a good antioxidant activity. Indeed, anthocyanins have gained increasing interest for their protective effect on doxorubicin-induced cardiotoxicity due to the superoxide anion scavenging activity in rat cardiomyocytes [39
]. Furthermore, has been recently found that dietary supplementation of bilberry extract protects against doxorubicin-induced cardiotoxicity in rats [40
LC-MS analyses were used to assess the stability of the polyphenolic profile in the smoothies. In particular, in the hydro-alcoholic extract of orange smoothie, 22 polyphenols and 4 limonoids glycosides were identified. In accordance with our previous results [8
], the most abundant compounds were the flavanone narirutin and hesperidin. However, narirutin content was higher in smoothies than fresh juice. The increase may be due to the stage of maturation of the fruits used [42
]. Furthermore, the anthocyanin profile of grape remained almost unchanged, and malvidin 3-O
-glucoside was the main representative anthocyanin. It can be concluded that employment of innovative sterilization and packaging techniques developed and employed by the producer, are able to preserve the organoleptic, nutritional and functional properties of relative fruits, and has no influence on the quantitative and qualitative polyphenolic composition.
After analytical characterization, the investigated matrices were tested for their ability to reduce the doxorubicin-induced cardiotoxicity in embryonic cardiomyocyte cell lines (H9c2) and for their synergistic role with the anticancer effect of the drug on human breast cancer cell lines (MCF7).
Initial antiproliferative screening, showed that both the single smoothies and all the possible mix did not influence cellular proliferation, at the concentrations used in this study. The evaluation of antiproliferative activity indicated that both orange and red grape protected cardiomyocytes from doxorubicin, even if this effect was slightly higher for orange. On the other hand, red grape was more active in maintaining the doxorubicin antiproliferative effect on MCF-7 cells but previous experiments indicated also a concentration-related antiproliferative effect of red grape alone on cardiomyocytes (>25 µg/mL; data not shown). In this regard, the employment of mixed phytochemical combination is an attractive option. Thus, we decided to reduce red grape concentration and to add orange in different combinations to identify a mixture able to reduce doxorubicin-induced cardiotoxicity, without altering its therapeutic effect on cancer cells. Moreover, the wide differences in polyphenolic structures among the two matrices could be responsible for different mechanisms of action, integrating the radical scavenging activity with the stimulation of the endogenous antioxidant response [43
Interestingly, among the tested mixture, the MIX 1:1 was able to protect cardiomyocytes from doxorubicin-induced antiproliferative effect without reducing the effect on MCF-7 cells. Moreover, while red grape alone did not affect doxorubicin activity on MCF-7 cells only at the lower concentration (1 µg/mL), this activity for the MIX 1:1 was observed at the two lowest concentrations (5 and 1 µg/mL) maintaining the same protective effects on cardiomyocytes (Figure 2
). This might be due to an optimal balance of antioxidant agent concentration in the MIX 1:1 in comparison with the single smoothies.
To assess the cardioprotective effects of the smoothie mixtures, H9c2 and MCF-7 cells were exposed to different concentrations of orange and red grape mixtures in combination with doxorubicin. Selected concentrations were chosen based on previous investigations, to avoid the pro-oxidant effect of the polyphenols at high doses [18
]. MIX 1:1 provided an appreciable radical-scavenging activity, significantly reducing ROS release in H9c2 cells at all the tested concentrations, while only at the highest dose in MCF-7 cells. ROS release is one of the main mechanisms triggered by chemotherapy agents to induce cellular death and, on the other hand, able to mediate chemotherapy-associated toxicity in healthy cells/tissue [45
]. Therefore, the ability of the MIX 1:1 to differently act on these two cells type points out its potential to reduce the myocardial injury induced by anthracycline, without interfering with its anticancer effect.
Interestingly, we observed that following doxorubicin treatment, H9c2 cells present significantly high ROS levels compared to MCF-7 cells. This may be due to endogenous deficiency in cardiomyocytes of antioxidants and cytoprotective enzymes as HO-1 and NQO1 [3
]. These metabolizing enzymes, play a key role both in reducing ROS generation and in the cellular detoxification of highly reactive molecules, generated by the interaction of ROS with biological macromolecules. HO-1 is the primary rate-limiting enzyme in Heme catabolism [46
]. NQO1 catalyzes the two-electron reduction of electrophilic quinone compounds, thus limiting the formation of semi-quinone radicals through one electron reduction and the subsequent generation of ROS [47
]. Several polyphenols present in citrus and grapefruits have been reported to induce HO-1 expression in several cells [32
]. Our experiments showed that the MIX 1:1 induced a significant increase in their expression, particularly in cardiomyocytes. Therefore, the coordinated action of radical scavenger and metabolizing enzymes ensure effective detoxification of ROS and contribute to the protective effects against the doxorubicin cardiotoxicity.
As stated before, doxorubicin mediated apoptosis plays an important role in its cardiotoxicity, by activating both intrinsic and extrinsic pathways. Interestingly, seems that apoptosis process is linked to intracellular H2
formation and p53 protein activation in a different manner between healthy and cancer cells [50
]. In this regard, it has been reported that Citrus aurantifolia
ethanolic extract increase apoptosis induction on MCF-7 cells synergistically with doxorubicin, through regulation of protein p53 and Bcl-2 expression [51
]. In addition, grape extract, besides its ability to suppress apoptotic process in cardiomyocytes [38
], exert a pro-apoptotic function on MCF-7 by a mechanism that involves a transient increase of gap junction intracellular communication [52
]. Therefore, the combination of orange and grapes phytochemicals can further strengthen both their cardioprotective potential and the synergistic activity with doxorubicin. In accordance with these considerations, we found that MIX 1:1 reduced apoptosis more in cardiomyocytes than in MCF-7 cells.