## 3 Results
### Taheebo Induces Apoptosis by Regulating Apoptotic Gene Expression  
By eliminating damaged, old, or autoimmune cells, apoptosis plays a crucial role in the development and maintenance of multicellular organisms [59]. Anticancer mediators are vital for inducing apoptosis, which is necessary for destroying tumor cells. A substance that kills or prevents the multiplication of cancer cells by inducing apoptosis is an efficient anticancer agent [60]. Cancer cells can undergo apoptosis through intrinsic and extrinsic routes that are regulated by the caspase-dependent proteolysis of hundreds of cellular proteins, membrane blebbing, and endonucleolytic breakage of chromosomal DNA [61]. Phytochemicals that promote apoptosis via intrinsic apoptotic pathways trigger a range of intracellular stimuli. ROS produced by mitochondria are essential for redox signaling, and p53 inhibits cancer by becoming redox active. ROS cause p53 to become active, which in turn causes cancer cells to go through apoptosis [62; 63]. p53 controls the expression of the pro- and anti-apoptotic genes bax and bcl2, respectively [64]. When Bax expression is increased, it can lead to apoptosis, but when Bax expression is reduced, Bcl-2 can stop apoptosis [65]. Variation in the range of those proteins damages the mitochondrial membrane, which triggers the release of cytochrome-c and regulates the activation of caspase-9 [66]. Caspases, one of the most important types of proteases, are essential for cell death. The bax protein is an essential mitochondrial membrane component that facilitates the transport of cytochrome-c between membranes, leading to the formation of apoptotic remnants, and it activates caspase-9 and caspase-3 to produce apoptosis [67; 68; 69]. As shown by the RT-PCR results, roasted TA substantially increased the expression of the genes for p53, Bax, cyto-c, caspase 3, and caspase 9 and decreased the expression of the gene for Bcl-2, compared with both the untreated control and TA samples (Figure 7). Similarly, roasted TA downregulated the expression of bcl-2 (0.57 fold) and upregulated the expression of p53 (1.18 fold), bax (1.31 fold), cyto-c (1.28 fold), caspase 3 (1.86 fold), and caspase 9 (1.89 fold). The gene expression changes in the TA sample groups were smaller than those in the roasted TA sample groups. Thus, roasted TA can reduce the expression of anti-apoptosis  
Figure 6: Colony formation assay in A549 cells at concentrations of 250 and 500 ug/mL of TA and roasted TA The corresponding bar graph of the colony formation assay shows the number of colonies/ dish when A549 cells were treated with TA and roasted with TA. The mean \(\pm\) SD data of three replicates are shown in the bar graph. **_p_ < 0.001 shows statistically significant differences from the control groups.  
genes in lung cancer cells. To completely comprehend those biological processes, Western blot analyses and more study of the molecular mechanisms are needed.  
Figure 7: Efficacy of TA and roasted TA on mRNA expression levels of apoptosis-related genes in A549 cells. For 24 h, TA and roasted TA were administered to A549 cells at concentrations of 250 and 500 g/mL, respectively. Following total RNA extraction, qPCR was performed to assess transcript expression levels with primers targeting (**A**) p53 (**B**) BAX (**C**) Bcl-2 (**D**) Caspase 9 and (**E**) Caspase 3 (**F**) Cyto C. Each bar displays the mean \(\pm\) SE of duplicate samples from 3 independent experiments (*** \(p<\) 0.001 using the Student’s \(t\)-test compared to the non-treated control).