## 3 Discussion
massoniana_ bark hydroalcoholic extract on a human breast cancer cell line was demonstrated at a higher IC\({}_{50}\) value than 32.5 \(\mathrm{\SIUnitSymbolMicro}\)/mL [25]. Compared to our results, the IC\({}_{50}\) values of _P. eldarica_ bark essential oil and hexane, aqueous, and methanolic extracts were 17.9, 18.0, 25.1, and 209 \(\mathrm{\SIUnitSymbolMicro}\)/mL, respectively. _P. eldarica_ essential oil and extracts have been shown to prevent the growth of several cancer cell lines, with the essential oil selectively inhibiting the growth of human breast cancer MCF-7 and HeLa cells, presenting IC\({}_{50}\) values of 0.032 and 0.038 \(\mathrm{\SIUnitSymbolMicro}\)/mL, respectively [24]. Herein, we observed that the pollen hexane extract, bark essential oil, and methanolic needle extract of _P. eldarica_ significantly suppressed the migration of lung cancer cells at 24 and 48 h. The migration rates of experimental groups were calculated, with the methanolic needle extract revealing the lowest rate (1.87 \(\mathrm{\SIUnitSymbolMicro}\)/h in 48 h). Recent studies have shown that pine extracts can inhibit lung cancer cell migration by reducing the expression of matrix metalloproteinases [29; 30]. Matrix metalloproteinases are proteolytic enzymes that are secreted from tumor cells, involved in tumor metastasis by degrading the extracellular matrix. The result of the colony formation assay showed that colony numbers in treated groups decreased significantly compared to the control group (_p_ < 0.01). Some studies suggest that natural products can inhibit colony formation by the change in tumor suppressor genes expression [31]. We explored the anticancer mechanisms of _P. eldarica_ extracts and essential oils on A549 lung cancer cells. Apoptosis is a crucial pathway for reducing mutated or growing cancer cells [32]. Reportedly, some natural products have been shown to inhibit the growth of cancer cell lines by inducing apoptosis [33; 34]. To evaluate the effects of _P. eldarica_ extracts and essential oils on apoptosis, several experiments, including DAPI staining, Annexin V/PI staining, and cell cycle analysis, was performed. Morphological changes during apoptosis were observed by DAPI staining under a fluorescence microscope. This occurs by nucleic acid denaturation in the cytoplasm. DAPI binds to DNA and forms a complex that emits bluish-white fluorescence by ultraviolet light exciting, so small amounts of DNA can be visualized. Condensed and fragmented chromatin, as specific properties of apoptotic cells, were detected in all extract- and essential oil-treated groups, with a large number of apoptotic cells observed in the methanolic needle extract-treated group containing 0.3 \(\mathrm{\SIUnitSymbolMicro}\)/mL of extract; these findings were in agreement with our results obtained following the Annexin V/PI test. In the Annexin V/PI assay, the apoptotic cell population of A549 cells was increased from 0.5% in the control group to 66.6%, 56.5%, and 29.7% in the methanolic needle extract-, bark essential oil-, and pollen hexane extract-treated groups, respectively. The methanolic needle extract-treated cells showed the highest population of apoptotic cells when compared with the other two groups. Thus, we conclude that this group showed suitable efficacy as a potential therapeutic option. In the cell cycle analysis, A549 cells treated with the methanolic needle extract (0.3 \(\mathrm{\SIUnitSymbolMicro}\)/mL) and pollen hexane extract (31.7 \(\mathrm{\SIUnitSymbolMicro}\)/mL) revealed cell accumulation in the sub-G1 phase, and the percentage of cells in the S phase was reduced in the methanolic needle extract-treated group. [PERSON] et al. have investigated the cytotoxic effects of _P. massoniana_ extract on HeLa cells, demonstrating that cell cycle arrest occurs in the sub-G1 phase [35]; this was in agreement with our results. Sub-G1 cells were non-apoptotic and viable.