## 3 Discussion
Some cells frequently returned and then proceeded to mitosis (hypodiploid and hyperlipid cells), before arrested in the subsequent cell-cycle interphase. Arresting cells in sub-G1 inhibit cells entering mitosis [36]. Therefore, methanolic needle extract and pollen hexane extract caused post-mitotic arrest in proceeding cell cycles. This process was p53-dependent because when cells divided, p53 arrested the cell cycle in G1 [36]. Furthermore, we observed increased p53 protein expression levels in experimental groups when compared with those in the control group. p53 is a known tumor suppressor gene [37]. In several types of cancers, p53 is damaged owing to mutations, and p53 protein levels are decreased, leading to malignant tumors [38; 39]. The cell cycle analysis showed that the accumulation of A549 cells in the S phase was observed after 48 h treatment with the bark essential oil (17.9 \(\upmu\)g/mL). Cell cycle arrest at the S phase may result from different mechanisms of action, for example, interfering with cell cycle progression and apoptosis induction, probably by reducing Bcl-2 protein expression. Mitochondria play a critical role in apoptosis, releasing proapoptotic proteins from their membranes into the cytosol. Bcl-2 family proteins are essential mitochondrial regulators of apoptosis [40]. The primary proapoptotic member of these proteins is Bax, which can initiate the release of cytochrome c and subsequent activation of caspase-3 and caspase-9. Bcl-2 acts as an antiapoptotic protein. Bcl-2 inhibits cytochrome c translocation and inhibits caspase-3 activation and apoptosis initiation [19]. High Bax protein expression levels correlate with low Bcl-2 expression levels, which would stimulate apoptosis. The ratio of Bax/Bcl2 expression levels is a diagnostic indicator of cancer [41]. In the present study, Western blot analysis was performed to analyze changes in protein expression of Bcl-2 and Bax in the pollen hexane extract, bark essential oil, methanolic needle extract, and control groups. Based on our Western blot analysis results, the ratio of Bax/Bcl-2 expression levels, as a predictor of apoptosis, increased 6.8- and 27.2-times in the pollen hexane extract-(\(p<0.05\)) and methanolic needle extract-treated groups (\(p<0.01\)) when compared with the control, respectively. In recent studies, pine needle essential oil reportedly downregulated Bcl-2 protein expression and induced apoptosis in oral squamous cell carcinoma [42]. This was in agreement with the results of the present study, which showed that the methanolic needle extract induced apoptosis at a concentration of 0.3 \(\upmu\)g/mL by altering Bcl-2 and Bax protein expression in A549 cells more efficiently than other evaluated extracts. In normal cells, caspases exist in the proaspase form with specific molecular weights. However, upon stimulation of cells with an apoptogenic agent, caspases are activated via proteolytic cleavage. After activation, caspases can cleave other proteins, such as PARP, leading to apoptosis [43]. A previous report has reported the ability of caspases to induce apoptosis in cancer cells following treatment with natural plant-derived extracts [20]. For instance, pine cone water extract reportedly activates caspase-3 and induces apoptosis in a lung cancer cell line [20]. We observed elevated levels of cleaved caspase-3 and cleaved PARP in A549 cells treated with IC\({}_{50}\) concentrations of the pollen hexane extract, bark essential oil, and methanolic needle extract. These results suggest that activation of the caspase pathway is essential for inducing apoptosis in A549 cells following treatment with _P. eldarica_ extracts. Our results showed that _P. eldarica_ extracts mainly, methanolic needle extract, can prevent and treat cancer by various mechanisms. Cell cycle arrest, inhibition of cancer cell migration, and proliferation were some of the anticancer effects.