The elevated levels of intracellular antioxidant such as

The elevated levels of intracellular antioxidant such as reduced glutathione (GSH) are normally oberserved is various tumor LY2157299 which confers the chemoresistant to chemotherapy [59]. Therefore, the induction of oxidative stress via elevation of intracellular ROS and dimunition of intracellular GSH levels are thought to perturb cellular redox homeostasis leading to dissipation of Δψm, irreversible oxidative modifications of lipids, proteins, or DNA and eventually, cellular apoptosis [66], [67]. Furthermore, various reports have demonstrated that enhanced production of intracellular ROS can promote apoptosis through the perturbation of mitrohondria redox leading to oxidative stress and the release of apoptogenic factors [68], [69]. In this regard, we hypothesized that citral promoted mitochondrial-mediated apoptosis partly by shifting cellular redox homeostasis towards prooxidant state by elevating the intracellular ROS and decreasing the total GSH level in HCT116 and HT29 cells. To support this, the data revealed that treatment with citral stimulated the production of intracellular ROS level while attenuating the total GSH level in both HCT116 and HT29 cells (Fig. 4). Interestingly, the pretreatment with NAC (2mM, 30min) significantly inhibited the elevation of intracellular ROS by citral but sustained the high levels of total GSH that resulted in the maintained level of HCT116 and HT29 cell viability. Thus, the current data confirmed that the ability of citral to induce mitochondrial-mediated apoptosis is at least, partially due to the induction of oxidative sress-induced apoptosis via increased production intracellular ROS production and diminution of total GSH levels that disrupted the cellular redox balance and mitochondrial integrity in HCT116 and HT29 cells. Our current findings are similar to another report that demonstarted Melissa officinalis essential oil (EO) and its major component, citral, reduced cell viability and promoted apoptosis via elevation of ROS. Moreover, they reported the presence of antioxidant mitigated citral-induced cell death and following elevation of ROS, citral downmodulated the activity and inhibited the expression of multidrug resistance associated protein 1 (MRP1) [70]. The p53 is a tumor suppressor protein that plays imperative roles in regulating cellular response to DNA damage and oncogenic stress as its loss of function through mutation or deletion often results in tumorigenesis malignancies. The activation of p53 through its phosphorylation is important in inducing cell cycle arrestment and concomitant apoptosis in various cancer models. For example, the activation of p53 serves as a checkpoint during the G1/S-phase of the cell cycle that inhibits cell proliferation and promotes apoptosis by suppressing the expression of cyclin E and cyclin A/cdk2 following DNA damage [71], [72]. Additionally, p53 has been reported to promote apoptosis through activation of the mitochondria-mediated apoptosis pathway by augmenting the proapoptotic and inhibiting the expression of antiapoptotic Bcl-2 family [62], [73]. The current data further support these findings where citral treatment (200μM) induced the phosphorylation of p53 as early as 6 and 12h in HCT116 and HT29 cells (Fig. 6). Moreover, the expression of p53 was more prominent when the cells were incubated with citral for 24h. These strong activation of p53 could be corelated with the concomitant increased expression of Bax and Bcl-2 proteins in both HCT116 and HT29 cells. p53 activation has been reported to influence the Bax gene transcription directly and in a non-transcription manner, p53 can directly promote depolarization of Δψm by forming complexes with the proapoptotic Bcl-2 family proteins [74]. For instance, the activated p53 can interact with Bcl-2 protein which then promoted the proapoptotic activity of Bax in elevating MPTP [75], [76]. Additionally, increased production of intracellular ROS is one of the signals that triggers the p53-induced apoptosis [77], [78]. The current findings are in line with another report that demonstrated the inhibition of glutathione synthesis by L-buthionine sulfoxamine promoted the anticancer activity of citral in p53-expressing ECC-1 and OVCAR-3 cells while the addition of NAC significantly attenuated activation of p53 and apoptosis in the ECC-1 and OVCAR-3 cells [34]. They demonstrated that citral activated the p53 (via phosphroylation of ser-15) which led to apoptosis by increasing Bax, PUMA and NOXA expression, decreasing Bcl-2 expression, promoting cell cycle arrestment at G1/S phase and activation of caspase-3. In contrast, when the cells were treated with pifithrin-α, p53 activation was inhibited leading to mitigation of apoptosis.Thus, the current data urther suggest that citral promoted mitochondrial-mediated apoptosis with the association between p53 and ROS HCT116 and HT29 cells.