In summary, we found that Dox (0.5–50 μM) dose-dependently reduced H9c2 cell viability. The effects were similar as those caused by a common oxidative stress inducer, H2O2. However, unlike H2O2, there was no difference in Dox cytotoxicity when Dox was added to a fresh medium or 1-day-old medium. Thereafter, we showed that mitochondrial targeted antioxidants, SKQ1or MitoQ, but not the common antioxidant, vitamin C, significantly mitigated Dox-induced cell damage when given as co-treatment or pretreatment. Interestingly, pretreatment of MitoQ provided significantly higher efficacy of cell protection when compared to MitoQ co- treatment or SKQ1 pretreatment. Lastly, we demonstrated that Dox dose-dependently increased intracellular ROS and mitochondrial SO. By contrast, co-treatment or pretreatment of SKQ1 or MitoQ significantly reduced Dox-induced intracellular ROS and mitochondrial SO. However, pretreatment shower higher reduction only at higher doses than co-treatment.
Dox is a powerful chemotherapeutic drug and widely used in solid cancer treatment [19]. Dox usage is limited due to induction of irreversible cardiotoxicity [1, 20]. This damage is characterized by cardiac cell apoptosis or necrosis which can lead to cardiomyopathy [21]. Our study showed that Dox (0.5–50 μM) dose-dependently decreased H9c2 myoblast cell viability after incubation for 24 h. The cell viability was evaluated by measuring intracellular dehydrogenase activity of viable cells and confirmed by calcein staining. Our results are consistent with a study by Zhang et al. showing that Dox induced cell damage in a dose- and time-dependent manner. Dox (5 μM) caused about 30% reduction in H9c2 cell viability after incubation for 24 h [20], which is similar to our data. By contrast, Dallons et al. showed Dox (1–50 μM) had no effect on cell viability after incubation for 4 h, whereas Dox (1 μM) showed 60% reduction in cell viability after 24 h treatment when cell viability was measured by crystal violet assay [21]. The difference of Dox induced cell damage is possibly due to the sensitivity of methodology used to evaluate cell viability. Moreover, Dallons seeded cells at 3 × 104 cells/cm2 and experiments started after cell seeding for 48 h. By contrast, we seeded cells at 6 × 104 cells/cm2 and experiments started after cell seeding for 24 h. Researchers have found that cell numbers can influence cell damage effects induced by oxidizer [22].
In this study, we also showed that cell damage induced by H2O2 could be significantly impacted by fresh medium when compared to 1-day-old medium. The difference may be due to antioxidant effects provided by fetal bovine serum and α-keto acid in fresh medium [23]. However, we did not see any significant difference in cell viability by Dox between fresh medium and 1-day-old medium. Therefore, we could add Dox into the fresh medium after washing out MitQ or SKQ1 after both drugs were given as pretreatment.
Oxidative stress is a major mechanism mediating Dox-induced cardiac damage. It has been found that Dox, a cationic compound, can accumulate in mitochondria 100 times more than in the cytosol due to larger mitochondrial membrane potential (e.g., − 160 to -180 mV). Moreover, Dox interacts with cardiolipin forming electrostatic bonds and disrupting the mitochondrial electron transport chain, particularly complex I and II [24]. In consequence, the dysfunctional electron transport chain facilitates SO generation and overwhelms the mitochondrial antioxidant capacity in the heart. Meanwhile, Dox itself undergoes redox cycling and directly reduces to a semiquinone species through interaction with complex I. This reduced semiquinone version of Dox can oxidize oxygen and form SO. Additionally, Dox can regulate mitochondrial NADPH oxidase increasing ROS production [25]. In this study, we found that Dox increased intracellular ROS in a dose and time-dependent manner. Intracellular ROS measured by DCF fluorescence intensity started to increase after Dox incubation for 10 min and it continued to rise until 24 h. Moreover, we specifically measured mitochondrial SO levels by MitoSOX after Dox incubation for 24 h. We found that the dose-response of Dox-induced mitochondrial SO increase was very similar as the dose-response of Dox-induced intracellular ROS increase at 24 h. The data is consistent with Kuznetsov et al. showing Dox increased intracellular ROS evaluated by measuring DCF fluorescence intensity paralleled with mitochondrial redox state and membrane potential [26]. Asensio-Lopez et al. also showed that Dox dose and time-dependently increased intracellular ROS. Moreover, by utilizing MitoTracker green to locate mitochondria, they found that Dox started to appear in mitochondria at 15 min after administration. It was accompanied with increased ROS fluorescence at mitochondria as well. Both intensities increased as a function of time [25]. In consequence, increased ROS levels in mitochondria lead to membrane potential collapse, ATP production decrease, lipid peroxidation, cytochrome C release and mitochondrial DNA damage [1, 20]. A large amount studies have confirmed that Dox induces apoptosis in cardiomyocytes by increasing cytochrome C and caspase 3 and 9 activity [1, 20, 21].
Because oxidative stress is a major and early event after giving Dox, there are numerous researchers investigating if antioxidants can protect the heart against Dox-induced cardiotoxicity. Most preclinical studies found that common antioxidants, such as vitamin C, can protect the heart against Dox. Viswanatha Swamy et al. showed that vitamin C (20 mg/kg orally) when given 15 days before or 15 days after Dox significantly reduced heart cell damage and oxidative stress by enhancing antioxidant enzymes (e.g., superoxide dismutase, catalase) in rats [27]. Similarly, Akolkar et al. demonstrated better cardiac structure and function in rats when vitamin C (50 mg/kg, orally) was given a week before and continued till 2 weeks after Dox injection. Moreover, they illustrated vitamin C significantly mitigated Dox-induced nitrosative stress, proapoptotic proteins (e.g., caspase 3), inflammatory cytokines (e.g., interleukin 6), and the relevant signaling proteins (e.g., p53, nuclear factor kappa light chain enhancer of activated B cells, autophagy) in rats and isolated cardiomyocytes [5, 8, 28]. However, it is still a lack of sufficient clinical research evidence to prove the effectiveness of vitamin C in the attenuation of cardiotoxicity caused by Dox [29, 30].
In this study, we expected that vitamin C would mitigate Dox-induced cell damage. Moreover, it could serve as an effective control to compare mitochondrial-targeted antioxidants’ effects. We first found that vitamin C (1–2000 μM) alone only slightly increased cell viability after incubation for 24 h. However, when vitamin C (1–2000 μM) was applied concurrently with Dox, vitamin C failed to show any protective effects. Moreover, higher doses of vitamin C (1000 μM and 2000 μM) showed slightly lower cell viability than Dox alone. So far, vitamin C has not shown any protection against Dox-induced cardiotoxicity in clinical studies [29, 30]. This may be because vitamin C is not able to reach sources of ROS (e.g. mitochondria) quickly enough to scavenge ROS or its potential pro-oxidant property. Vitamin C is hydrophilic and it gets into the cell or cellular organelles by Na-dependent vitamin C transporter or glucose transporter. AKolkar et al. showed that Dox downregulated the expression of both transporter proteins in the cardiomyocytes [8]. Moreover, the distributions of vitamin C in different intracellular compartments vary in different tissues. For example, vitamin C concentration is five times less in the mitochondria of mouse skeletal muscle than that in the liver [31]. Furthermore, studies indicate that vitamin C can become pro-oxidant at higher concentration (e.g., > 1000 μM), higher intracellular transition metal ions, or dysfunctional mitochondria [32,33,34]. In addition, a higher Dox concentration (e.g., 40 μM) was used in this study than other studies (e.g., 10 μM). All the above factors may contribute to why vitamin C showed no cell protection against Dox in this study.
Due to Dox mainly accumulating in the mitochondria to induce oxidative stress, mitochondrial-targeted antioxidants would be more efficient to mitigate Dox-induced cell damage. MitoQ and SKQ1 are two well-studied mitochondrial-targeted antioxidants [35]. They are ubiquinone and plastoquinone, respectively, conjugated to a TPP, a lipophilic cation. The conjugated drugs accumulate several hundred times more in the mitochondria than cytosol due to larger membrane potential within the cell [10, 36]. Inside mitochondria, MitoQ and SKQ1 switch between their reduced and oxidized form via the electron transport chain, with the reduced form able to scavenge ROS [15, 37]. However, MitoQ and SKQ1 can be pro-oxidants at higher doses [15]. We found that MitoQ (0.05–5 μM) and SKQ1 (0.05–10 μM) alone slightly increased cell viability when compared to non-treated control. By contrast, a higher dose of MitoQ (10 μM) slightly reduced H9c2 cell viability by 5%. Mendez et al. also showed that MitoQ (10 μM) is cytotoxic to platelets [38].
It is well-known that heart preconditioning by transient ischemia/reperfusion episodes allows the heart develop resilience to endure a harsher insult, such as prolonged ischemia/reperfusion injury [39]. Currently, this strategy has been tried to mitigate Dox-induced cardiotoxicity. Maulik et al. showed that simulated preconditioning by hypoxia/reoxygenation attenuated Dox-induced cell damage in primary adult cardiac myocytes. However, a ROS scavenger, N-acetyl cysteine, failed to show any protection when given concurrently with Dox [16]. Similarly, Galan-Arriola et al. found that remote ischemia preconditioning before intracoronary injection of Dox preserved significantly better left ventricular ejection fraction, mitochondrial morphology, and DNA copies in pigs’ heart [17]. Instead of using ischemic preconditioning, we pretreated cells with mitochondrial targeted antioxidants, MitoQ or SKQ1, 24 h prior to Dox in this study. We also compared the effects of pretreatment to those of co-treatment. We found both co-treatment and pretreatment mitigated Dox-induced cell damage in H9c2 myoblast cells. However, the dose-responses were different between co-treatment and pretreatment. Co-treatment showed significant protection at intermediate doses of MitoQ (0.5–1 μM) and SKQ1 (1 μM); whereas higher doses showed reduced protection. By contrast, when given as pretreatment, higher doses of MitoQ (1–10 μM) and SKQ1 (5 μM) were required to protect the cells against Dox. Moreover, the efficacy of protection by MitoQ pretreatment was significantly better than its co-treatment. We further found that the protection may be related to the dose-dependent reduction in intracellular ROS and mitochondrial SO. It is noticeable that both drug’s antioxidant effects shared the similar shift of dose-dependent response between co-treatment and pretreatment. To our knowledge, the present study showed that pretreatment of mitochondrial-targeted antioxidants has higher efficacy against Dox-induced cell damage than co-treatment for the first time. The difference possibly is due to the following reasons: 1. Accumulation of MitoQ or SKQ1 in mitochondria depends on mitochondrial membrane potential [10]. When the drugs were co-administered with Dox, Dox dissipated the mitochondrial membrane potential which possibly reduced the drug accumulation in mitochondria; 2. It has been reported that higher doses of MitoQ and SKQ1 can be prooxidants. In particular, MitoQ shows higher pro-oxidant property at lower doses than SKQ1 in vitro studies [13]. Moreover, Huang et al. found that MitoQ (10 μM) reduced mitochondrial membrane potential in pancreatic acinar cells [40]. We found that higher doses of SKQ1 or MitoQ (e.g. 5 and 10 μM) when given as co-treatment showed less cell protection or no protection at all. 3. MitoQ/SKQ1 and Dox exert effects via the electron transport chain and cardiolipin, which may lead to interference when given at the same time. For example, Dox needs to be converted to semiquinone causing ROS increase by complex I. By contrast, MitoQ relies on complex I and II to recycle between the reduced and oxidized forms [41]. Similarly, SKQ1 recharges itself between reduced and oxidized forms via complex II and complex III [37]. Furthermore, Dox and SKQ1 showed higher affinity for mitochondrial cardiolipin than MitoQ. However, Dox binding disrupts cardiolipin whereas SKQ1or MitoQ protects cardiolipin from oxidation to preserve mitochondria’s normal function [24, 42]. By contrast, when MitoQ or SKQ1 was applied before Dox, drug accumulation into mitochondria and the antioxidant capacity was unlikely influenced. All the above presumptions warrant further investigation to support that pretreatment would be a better strategy when giving mitochondrial-targeted antioxidants.
Additionally, we demonstrated that MitoQ exhibited significantly higher efficacy against Dox than SKQ1 when both were given as pretreatment. The cellular protection may be partially related to its higher reduction on intracellular ROS and mitochondrial SO than SKQ1 as our data suggested. Plenty of studies support the concept that MitoQ can mitigate Dox-induced cardiotoxicity by reducing oxidative stress [1, 13]. By contrast, SKQ1 has not been widely studied in Dox-induced cardiotoxicity. However, it has been used in eye drops as a defense against oxidative stress due to dry eye syndrome [43]. SKQ1 has been also studied to promote survival of kidney epithelial cells and significantly improved survival of rats subjected to ischemia/reperfusion injury by reducing oxidative stress [44]. In vitro studies suggest that SKQ1 has antioxidant effects at lower concentrations than MitoQ [13]. Moreover, the reduced form of SKQ1 has a four-fold higher decrease in peroxyl radicals than the reduced form of MitoQ [11]. In addition to their antioxidant ability, Hu et al. recently indicated that MitoQ pretreatment activated Nrf2 signaling to enhance antioxidant capacity and to protect mitochondrial DNA in an intestinal ischemia/reperfusion model [18]. The role of Nrf2 signaling in cardioprotection provided by pretreatment of MitoQ or SKQ1 needs to be further elucidated.
We acknowledge that this study was performed on a rat H9c2 cardiomyoblast cell line instead of primary cultured cardiomyocytes. However, Kuznetsov et al. compared H9c2 cells’ mitochondrial biogenesis, function and response to hypoxia/reoxygenation to primary cardiomyocytes’. They suggested that H9c2 cells were very similar to primary heart cells regarding the energy metabolism and mitochondrial properties [45]. Therefore, we would like to further validate the effects of pretreatment of mitochondrial-targeted antioxidants in a Dox-induced cardiotoxicity animal model. Moreover, we did not evaluate whether Dox’s anti-cancer effects would be compromised by using mitochondrial-targeted antioxidants as co-treatment or pretreatment. Rao et al. demonstrated that MitoQ exerted 30 times more cytotoxicity to breast cancer cell lines than to healthy mammary epithelial cells [46]. Moreover, they also found that MitoQ not only increased Dox’s anti-cancer effects but also mitigated Dox-induced cardiotoxicity [47]. Similarly, SKQ1 also showed to attenuate cell growth in fibrosarcoma and rhabdomyosarcoma tumor cell lines and related animal models [48]. However, given the possibility of competition when mitochondrial-targeted antioxidants are co-treated with Dox, it will be very intriguing to find out if administration of MitoQ or SKQ1 as pretreatment prior to Dox would provide better anti-cancer effects than co-treatment. It will also shine some light on how cancer patients can safely take antioxidants and not interfere with anti-cancer treatments’ benefits.
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