## 3 Biological Activities
### Antiproliferative Effect against Cancer Cells

Table 6: _Cont._pathway with direct involvement of the mitochondria and the extrinsic pathway mediated through death receptors. Caspases are a family of proteins responsible for activating and executing apoptosis cell death. Activation of intrinsic apoptosis involves the activation of caspase-9 and caspase-3, while the extrinsic pathway promotes the activation of caspase-8 and caspase-3. Caspase-3 is responsible for diverse biochemical changes in the cell, such as DNA fragmentation and the degradation of cytoskeleton proteins. Diverse stimuli such as ROS can trigger intrinsic pathway activation by modulating Bcl-2 proteins. The translocation of phosphatidylserine from the inner membrane to the outer membrane is another biochemical change in cells that die by apoptosis. This change can be detected by flow cytometry using annexin V-FITC/PI [97]. In HCT116 and HT29 cancer cells, citral induces mitochondrial-mediated apoptosis via activation of p53 and increasing the intracellular levels of ROS. Flow cytometry analysis using annexin V-FITC/PI demonstrated that citral induces apoptosis cell death in a dose- and time-dependent manner. In addition, the capacity of citral to increase the intracellular level of ROS and decrease the GHS levels was detected by flow cytometry. By Western blot analysis, it was observed that citral induces the activation of p53, _Bax_, and caspase-3 proteins, while the expression of _Blc-2_ and _Bcl-xL_ decreased after the treatment with citral [9]. [PERSON] et al. [91] investigated the apoptotic effect of citral in AGS cells. The Hoechst and PI stainings showed that citral induces chromatin condensation, while the ability of citral to cause DNA degradation in this cell line was observed by electrophoresis. The annexin V-FITC/PI analysis demonstrated that citral is a strongly apoptosis-inductive agent in AGS cells. After the treatment with citral, a high accumulation of AGS cells in early and late apoptosis phases was observed. In addition, the transcriptome analysis showed the capacity of citral to modify the expression of some genes associated with MAPK, NF-\(\kappa\)B, p53, and PI3K-Akt signaling pathways that control the cells' survival, apoptosis, and cell cycle. On the other hand, in Caco-2 and HT-29 cells, citral induces cycle arrest in G2/M [9]. In metastatic B16F10 murine cancer cells, the apoptotic induction capacity of citral was demonstrated by annexin V/PI staining and TUNEL assay, while the effect of citral to induce the formation of autophagic vacuoles was observed with the use of mono Jansyl cadaverine staining (MDC). Through the analysis of GSH and malondialdehydehehyde levels, as well as the evaluation of the cytotoxic effect of citral by 3(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazololium bromide (MTT) in the presence of specific scavengers of ROS (histidine, trolox, tempol), the capacity of citral to induce oxidative stress in B16F10 was demonstrated. The above was consistent with the ability of citral to cause DNA damage, which was observed through the comet assay. Citral interferes with the activation of some signaling pathways in B16F10; after the treatment, citral decreases the expression of ERK1/2, PI3K, and AKT and increases the expression of p53. Likewise, citral inhibits the nuclear translocation of NF-\(\kappa\)B in B16F10 cells [9]. In the same way, [PERSON] et al. reported that the level of caspase-3 activated was higher in M624 with respect to HaCaT cells, which is associated with the greatest cytotoxic/apoptotic effect of citral on M624 in comparison to HaCaT cells [9]. Citral interferes with lipid metabolism, inhibits colony formation, and induces apoptosis in PC-3 cells. Using Oil Red O staining (OSO), it was observed that this compound inhibits the survival of the cells and induces the expulsion of lipid droplets.