br Materials and methods br Results br Discussion The

Materials and methods
Results
Discussion The current treatment modalities for colorectal cancer are hampered by various issues such as high acquired resistance, long-term complication with potential of cancer recurrence, severe side-effects and poor therapeutic index [50], [51], [52]. Complementary and alternatives medicines through the utilization of bioactive compounds from traditional medicinal plants that possess the ability to specifically target malfunctioned genes and signaling pathways in cancers has gained great attention in developing safer and effective immunostimulants and anticancer agents [53], [54].In support of these, we have demonstrated the cytotoxic and apoptosis inducing effects of citral against colorectal cancer, HCT116 and HT29 which is associated with p53 and ROS-induced mitochondrial mediated apoptosis. A higher dose of citral (50, 100 and 200μM) were selected based on IC50 values in both HCT116 and HT29 BKT140 (Table 1) in order to achieve the desirable apoptosis-inducing effect. The MTT cell viability data illustrated that citral suppressed the growth of HCT116 and HT29 cells dose- and time-dependent manner. Although the cytotoxic and reduction of cell viability activities of citral as compared to paclitaxel that exhibited IC50 in lower range of doses, the treatment of citral did not exhibit cytotoxicity (72h) in normal colon cells, CCD841-CoN (Fig. 1). To elucidate the apoptosis inducing effects of citral in HCT116 and HT29 cells, the citral-treated cells were subjected to a series of apoptosis assay. Apoptosis hold the cardinal role in the normal physiological and homeostasis processes as well as defense mechanisms against various insults that is systematically orchestrated which will result in the demise of the older or damaged cell [39], [55]. Generally, apoptosis is characterized by several biochemical and the induction of apoptosis can be characterized by several archetypal biochemical and morphological hallmarks which includes nuclear condensation (pyknosis), nuclear fragmentation (karyorrhexis), membrane blebbing, externalization of PS and dissipation of Δψm[56], [57]. For instance, during early apoptosis, the plasma membrane losses its asymmetry due to the externalization of PS from the inner to the outer membrane [39], [58]. Thus, the detection of externalized PS by Annexin V would aides in the detection of apoptotic cell death. The current data showed that citral treatment (50, 100 and 200μM) dose- and time-dependently increased of the combined early and late apoptotic cells (Annexin V-positive cells) which confirmed that citral induced apoptotic cell death in HCT116 and HT29 cells (Fig. 2). Furthermore, it is noteworthy that at every treatment dose and exposure time point, citral preferably promoted apoptotic cell death in both HCT116 and HT29 cells as the necrotic cell population was small (below 10%). Apoptosis can be mediated via extrinsic (death receptor) or intrinsic (mitochondrial mediated) pathways which ultimately promote the activation of caspases [59]. To detect the induction of intrinsic apoptotic cell death, the citral-treated cells were analyzed by flow cytometric analysis of Δψm. During early intrinsic apoptosis, the pro-apoptotic protein can elevate the permeability of outer mitochondrial membrane causing dissipation of Δψm that leads to the formation of mitochondrial pores and consequential mitochondrial release of apoptogenic factors [59], [60]. Treatment with citral (50, 100 and 200μM) was shown to promote the dissipation of Δψm in both HCT116 and HT29 cells in dose- and time-dependent manner (Fig. 3A and B). This observation was found to be similar to paclitaxel treatment (10nM) and thus, provided the evidences that citral induced mitochondrial-mediated apoptosis in both HCT116 and HT29 cells. The disruption of mitochondria membrane integrity is known to be regulated by the interplay between pro- and anti-apoptotic Bcl-2 family members [61], [62]. The proapoptotic proteins such as Bax and Bid, act as promoter of apoptosis whereas the anti-apoptotic proteins, such as Bcl-2 and Bcl-xL function as inhibitors of apoptosis. Following apoptotic stimuli, proapoptotic proteins such as Bax and Bak oligomers can translocate themselves onto the mitochondrial outer membrane and promoted the elevation of the mitochondria permeability transition pores (MPTP) leading to the leakage of apoptogenic factors such as cytochrome c, apoptosis inducing factor (AIF) and second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI (Smac/DIABLO) into cytosol to form apoptosome complex with apoptotic protease-activating factor-1 (Apaf-1) [62], [63]. Contrary to this, the antiapoptotic protein can suppress the apoptotic cascade by binding onto the proapoptotic proteins that inhibited the elevation of MPTP and mitochondrial release of apoptogenic factors [64]. The current results demonstrated that citral treatment upregulated the Bax protein expression in both HCT116 and HT29 cells and downregulated the Bcl-2 andBcl-xL protein expression (Fig. 5). These observations further confirmed that citral promoted mitochondrial-mediated apoptosis by shifting the balance from anti- to pro-apoptotic proteins leading to a cellular environment that favored apoptosis. Furthermore, this was further exemplified by the increase in both Bax/Bcl-2 and Bax/Bcl-xL ratios in HCT116 and HT29 as shown in (Fig. 5F). Following these events, citral treatment was hypothesized to induce apoptotic cell death through the activation of caspases. Caspases-3 is a member of the cysteine proteases family that acts as one of the major executioner caspases that proteolytically cleave various cellular proteins leading to loss of cellular structure and functions [65]. Further examination demonstrated that citral treatment induced the activation of caspase-3 in both HCT116 and HT29 cells as shown by the increased expression of cleaved caspase-3 (Fig. 5A–B and G). Hence, it is noteworthy that citral treatment promoted mitochondrial-mediated apoptosis in HCT116 and HT29 cells by increasing the Bax/Bcl-2 and Bax/Bcl-xL ratios and activation of caspase-3.