## 3 Results and Discussion
### Evaluation of Cytotoxicityoxicity
#### 3.2.3 Cancer Cells Migration Is Inhibited by _A. agallocha_ and _A. malaccensis_  
Preventing cancer metastasis is a key objective of cancer treatment because it accounts for 90% of all cancer-related deaths [53]. The hallmarks of a cancer cell's ability to spread to other tissues are cell invasion and migration. The initial stage in the progression of metastatic malignancies is the invasion of malignant cells into the surrounding tissue and the vasculature [76]. To prevent and cure metastatic cancer, it would be difficult to find targets that block cancer cells from migrating and invading other tissues. A scratch migration assessment was used to inspect the impacts of A.A and A.M on the migration of A549 cells. A wound closure test was used to compare cell migration (%) before and after the treatment of A549 lung cancer cells with A.A and A.M (500 and 1000 \(\upmu\)g/mL) (Figure 8). Cells treated with A.A and A.M showed inhibited migration after 24 h. These studies propose that A.A and A.M can stop cancer cells from migrating and metastasizing. In the control group after 24 h, migratory cells had almost filled in all gaps between cell layers. Additionally, cancer cells treated with A.A. and A.M showed an enhanced ability to  
Figure 7: The effects of _A. agallocha_ and _A. malaccensis_ on (**A**) NO generation were assessed using RAW 264.7 cells activated with 1 ug/mL of LPS. (**B**) Reactive oxygen species (ROS) production within cells was contrasted with control. Data presented as \(\pm\)SEM, ***\(p\) < 0.01 vs. control cell. All medication was conducted in triplicate.  
prevent migration in contrast to the control group. The ability of A.A and A.M to inhibit growth could suggest that it has potential as an anti-lung cancer treatment.  
.2.4 By Controlling Apoptotic Gene Expression, _A. agallocha_ and _A. malaccensis_-Induced Apoptosis  
Redox signaling requires the production of ROS by mitochondria, while the redox-active transcription factor p53 inhibits cancer [77; 78]. Numerous pro- and anti-apoptotic genes involving _Bax_ and _BCL-2_ are regulated by P53. One of the apoptotic pathways, BCL-2, is stimulated in response to stressful circumstances, such as DNA damage or cytokine  
Figure 8: (**A**) ImageJ software was used to measure the scratched region’s cell-free area. The proportion of scratch cell migration seen 24 h after treatment compared to control data represents the amount of cell migration. (**B**) Untreated cells are indicated by controls. Values are revealed as mean \(\pm\) standard deviation, and statistical significance is indicated by ***\(p\) < 0.01. The scale bar indicates 10\(\times\) magnification.  
shortage [79]. This mechanism might have been triggered by mitochondrial-mediated apoptosis, as shown by the downregulation of BCL-2, the overexpression of the caspase 9/3 gene, a rise in _Bax_ level, cyto C relief into the cytoplasm, and _BCL-2_ inhibition [80]. Additionally, _A. agallocha_ and _A. malacensis_ significantly upregulated the mRNA expression of the genes _p53_, _Bax_, _Casp 9_, _and Casp 3_, and downregulated the expression of the gene _BCL-2_ when compared to the control in the RT-PCR (Figure 9). In contrast, _A. agallocha_ elevated the mRNA expression of p53 (1.44 fold), _Bax_ (1.91 fold), caspase 3 (1.92 fold) and caspase 9 (1.56 fold), whereas _BCL-2_ (0.96 fold) downregulate the expression. _A. malacensis_ showed p53, _Bax_, _BCL-2_, _Casp 3_, and _Casp 9_ gene expression by (1.56, 1.91, 0.81,1.72, 1.44) fold, respectively, compared to untreated cells. As a result, this research demonstrated that _A. agallocha_ and _A. malacensis_ were able to suppress the expression of the apoptosis gene in lung cancer cells. Therefore, to completely comprehend the biological pathways, Western blot analysis and additional studies (_in vivo_ and clinical trials) regarding molecular signaling processes are required.