The current findings proved that citral treatment
The current findings proved that citral treatment induced mitochondrial-mediated apoptotic cell death in both HCT116 and HT29 through the p53 and ROS. As such, citral promoted apoptosis as evident by the externalization of PS and dissipation of Δψm at 24h of treatment in HCT116 and HT29 cells. These observations further increased when the Spautin-1 were treated for 48h as shown by the higher annexin V positive cells and further diminution of Δψm. Moreover, the induction of mitochondrial-mediated apoptosis was further evident by the shift balance between anti- and pro-apoptotic protein members resulting in elevated ratio of Bax/Bcl-2 and Bax/Bcl-xL ratios and the activation of caspase-3 in both HCT116 and HT29 cells. These observations were preceded by the elevation of intracellular ROS (4h) and the phosphorylation of p53 protein that further indicated that citral treatment perturbed cellular redox and mitochondria homeostasis. Furthermore, since one of p53 downstream targets is Baxprotein,therefore, citral treatment that induced p53 activation also increased Bax expression and decreased Bcl-2 expression which ultimately created a cellular environment that favored apoptosis. Therefore, it is noteworthy that treatment with citral induced p53- and ROS-induced mitochondrial mediated apoptosis through the modulation of Bcl-2 family members that ultimately activated caspase-3. To the best of our knowledge, this is the first report on the apoptosis inducing effects of citral on colorectal cancer, HCT116 and HT29 cells and via the activation of p53 and ROS. Nonetheless, the current data is still limited to in vitro evidences and therefore further mechanistic and in vivo studies are still greatly desired to underscore the translational value of citral to be developed as chemotherapeutic agent for colorectal cancer.
Conclusions Based on experimental data, it can be concluded that citral for the first time was demonstrated to inhibit the growth and induce apoptosis in HCT116 and HT29 cells. Citral treatment induced apoptosis via p53 activation and increased intracellular ROS level with concomitant upregulation of Bax and downregulation of Bcl-2 and Bcl-xL that led to increase in the Bax/Bcl-2 and Bax/Bcl-xL ratios. Consequently, these events in turn induced depolarization of Δψm and mitochondrial release of apoptogenic factors that ultimately led to the activation of caspase-3. These findings merit further mechanistic and in vivo studies of citral as potential anticancer agent for colorectal cancer.
Conflicts of interest
Introduction Saponins, mainly produced by plants, are widely used in medicine for their multiple biological and pharmacological activities including immunomodulatory, anti-inflammatory or anti-cancer (Lorent et al., 2014a). Cytotoxic and hemolytic activities for most saponins (e.g. digitonin (Korchowiec et al., 2015; Sudji et al., 2015; Frenkel et al., 2014), α-tomatine (Keukens et al., 1996), α-hederin (Lorent et al., 2016; Lorent et al., 2014b), seem to be ascribed to their ability to interact with membrane lipids, especially cholesterol (de Groot and Muller-Goymann, 2016). For example, digitonin induces membrane permeability in the presence of cholesterol by removing cholesterol from the membrane and forming complexes with the latter (Sudji et al., 2015). Cholesterol plays a crucial role in the stability, dynamics and organization of the plasma membrane, serving as a spacer between hydrocarbon chains of phospho- and sphingo-lipids (Ikonen, 2008). It is distributed heterogeneously in the plasma membrane (Carquin et al., 2016), notably enabling the formation of “lipid rafts” (Simons and Sampaio, 2011; Lingwood and Simons, 2010; Simons and Ikonen, 1997), transient ordered nanometric domains enriched in cholesterol and sphingolipids. Rafts are proposed to serve as platforms capable of promoting various cellular signaling including pro- and anti-apoptotic pathways such as the Fas death receptor and the phosphatidylinositol-3 kinase (PI3K)/Akt cell survival pathways, respectively (George and Wu, 2012; Mollinedo and Gajate, 2015).