## 3 Results and Discussion
### p53 and Caspases Activity  
The process of apoptosis is primarily accomplished by the activation of caspases (caspase-3 and caspase-9) and the expression of p53 protein, which specifically take down a number of intracellular components into pieces without harming or inflaming the neighboring normal cells [35; 57]. The p53 is a well-known tumor suppressor protein that demonstrates a crucial role in cell cycle regulation and DNA repair. When DNA damage occurs, p53 is activated and induces either cell cycle arrest or apoptosis depending on the severity of the damage. Caspase-3 and caspase-9 are also the key players in the apoptotic pathway, which is initiated by various signals, including oxidative stress, DNA damage or cytokine withdrawal. Once activated, these caspases cleave specific cellular components and cause the typical morphological changes in the apoptotic cells. Thus, aiming the p53 protein and caspases (caspase-3 and caspase-9) was explored as a potential therapeutic approach for the treatment of cancers. In the current study, GEF pure, F1 and F3 have shown significantly (\(p\) < 0.05) higher levels of p53 expression than the untreated cells (control), as represented in Figure 10a. This demonstrated that F3 > F1 > GEF pure effectively induced apoptosis in A549 cells, as evidenced by the fact that p53 expression was 5.9-, 4.6- and 3.04-fold higher in the treated groups with F3, F1 and GEF pure, respectively, than that of control group. The results of the current investigation were consistent with the earlier study, which demonstrated that p53 increased GEF-mediated growth inhibition and apoptosis in NSCLC [35; 57]. Figure 10: Effects of F3, F1 and GEF pure on the overexpression of p53 level (**a**); caspase-3 level (**b**) and caspase-9 level (**c**) levels in A549 cells. The concentration of GEF for all these experiments was kept at 3.125 \(\upmu\)M. The data were the mean and standard deviations of three measurements (mean \(\pm\) SD, \(n\) = 3). The * \(p\) < 0.05 indicates the significantly improved activities of F3 on A549 cells as compared to F1, GEF pure and untreated cells (control). The # \(p\) < 0.05 signifies the enhanced activities of F1 on A549 cells as compared to GEF pure and untreated cells (control). The $ \(p\) < 0.05 suggests the increased activities of GEF pure on A549 cells as compared to untreated cells (control). Additionally, F3-, F1- and GEF-pure-treated A549 cells had significantly higher expressions of caspase-3 (Figure 10b) and caspase-9 (Figure 10c) than the untreated cells (control), which serves as another piece of evidence of the powerful anti-apoptotic effects of GEF formulations. This was also observed in macrophages during GEF-induced apoptosis, which was mediated by caspase-3 [58]. The caspase-3 expression increased around 5.38-, 3.78- and 3.01-fold in comparison to untreated (control) cells, whereas the caspase-9 expression increased approximately 5.35-, 3.76- and 2.47-fold in F3-, F1- and GEF-pure-treated cells. These results can be supported by a prior demonstration in which si-MIR31 HG lncRNAs were transfected into PC9-R cells, leading to noticeably higher levels of caspase-3 and caspase-9 [59]. The higher level of caspase-3, caspase-9 and p53 expressions in the GEF SDS-treated cells compared to the GEF-pure-treated cells is due to the high aqueous solubility of SDs, which could result in increased drug concentration surrounding the treated cells. The higher drug concentration in F3 might enhance the anticancer activity against the NSCLC (A549) because of reduced p-glycoprotein (Pgp) efflux activity of TPGS [60].