Evidences display that oxidative stress induces a variety of cardiomyocyte deaths, consisting of apoptosis, necrosis and necroptosis [26–28]. In this context, ample researches have shown that the therapeutic agents generating ROS cause cardiotoxicity [29]. Among different types of therapeutics, certain classes of chemotherapeutic agents induce more common and frequent cardiotoxic effects. On the other hand, heart dysfunction along with the promotion of cardiac hypertrophy and the loss of contractile occur due to apoptosis of cardiomyocyte, which in turn develops cardiovascular diseases [30, 31]. The anti-breast cancer activities of two novel synthesized compounds (A and B) have been previously studied [18]. Our findings revealed that these two compounds act through inducing apoptosis. Keeping in mind that apoptosis plays an important role in cardiotoxicity and in consequence heart dysfunction and knowing that these two compounds likely resemble to COX-2 inhibitors with tri-aryl structures exerting their effects via apoptosis induction [19], herein, we attempted to verify that these compounds are nontoxic to cardiac cells. Hence, we performed the in vitro experiments on H9c2 cells. This is the first study to find out the effectiveness of compounds A and B to preserve the cardiomyocyte viability.
In the current study, for an exposure time of 24 h, compounds A and B concentration-dependently decreased H9c2 cell viability. For longer period of exposure (48 hours), these compounds also caused a decrease in cell viability implying a time –dependent cell cytotoxicity. Furthermore, our findings exhibited that the higher dose of compounds A and B (50 µM) can trigger the apoptotic process in H9c2 cells upon 24 h of treatment in which early apoptosis to a large extent was involved in the cell death. Interestingly, a concentration of 10 µM by which a remarkable anticancer activity had been seen for the compounds against breast cancer cells [18], could activate no apoptotic process at 24 and even 48 hours on cardiac cells.
A large number of studies have elucidated that intracellular ROS generation is closely associated with cell apoptosis [32]. Consistent with the apoptosis data, our results from determining the intracellular ROS level revealed no shift in the fluorescent intensity in H9c2 cells after cell exposure to 10 µM of both compounds for 24 h. Surprisingly, when the cells were subjected to 50 µM of compounds A and B for 24 h, the level of ROS remained unchanged in H9c2 cells which can be explained regarding the time that the ROS level was determined in the treated cells. Indeed, we assumed that following an initial increase in ROS level, it reaches the original level after 24 h. Thus, a large population of the cells was in the early of apoptosis at 24 h, while no significant difference in ROS concentration was detected in the treated cells compared with the control. Moreover, after incubation of the cells with 10 µM of compounds A and B for 48 h, no apoptosis process was induced, whereas a small level of ROS was produced suggesting the triggering of oxidative stress which was not strong enough to induce cell apoptosis. Our data support previous findings and indicated that ROS contributes to the regulation of apoptosis but it is generated in two steps; early and late stages in which time of ROS determination plays a key role [33].
Deregulation of the different cell death pathways causes pathological outcomes for oxidative stress-associated diseases including ischemia-reperfusion injury that has to be considered as adverse effects of drugs [34]. The generated ROS can inaugurate and enhance important mitochondrial alterations by depolarizing Δψm. In other words, ROS mainly causes depolarization and bulging of the mitochondria and then augments apoptotic mechanism through mitochondrial involvement [35, 36]. Thus, as a consequence, cytochrome c that is a key mediator in the mitochondrial pathway of cell death is released from mitochondria [37]. At last, upon activation of caspase-3, cell apoptosis is induced [38]. Our results demonstrated that 50 µM of compounds A and B disturbed mitochondrial membrane potential ψm of H9c2 at 24 h, whereas no mitochondrial function impairment was observed following treatment of H9c2 with 10 µM of both compounds for 24 h confirming our apoptosis results. Interestingly, it was shown that excessive ROS can damage mitochondria as well as open its permeability transition pore (PTP), and thereby induces mitochondrial permeability transition. The mitochondrial depolarization and outer membrane rupture due to these alterations result in cell apoptosis or death [39, 40]. These findings corroborate our obtained results and indicate that low dose of both compounds can induce ROS production after 48 h of treatment; however, this level of ROS was unable to induce apoptosis. Interestingly, high dose of these compounds after 24 h damaged mitochondrial potential and induced apoptosis, although it did not affected ROS generation suggesting an undetectable level of ROS due to the inappropriate time of measurement in the cells exposed to a high concentration of the compounds.