## 3 Discussion
Still, abietic acid showed more significant apoptotic effects on MCF-7 than pine extract, since it was additionally able to induce MCF-7 cell cycle arrest in G\({}_{2}\)/M phase (more so than Raptinal, a standard apoptotic agent). This observation suggested that pure isolated abietic acid could have more specific effects on cell cycles and may induce cell population shifts in different phases due to its standardization, thereby avoiding endo-interactions with other bioactive ingredients. Hence, the purity potentiates its apoptotic effects, as opposed to the complex nature of the pine extract [55; 56; 57; 58; 59; 60; 61]. Our data concurred with previously reported results showing that abietic acid showed antitumor activity through apoptosis induction and alteration of apoptosis-related proteins (Bax, caspase 3 and 9) in nasopharyngeal carcinoma (NPC) and triggered cell cycle arrest at G\({}_{2}\)/M phase [50]. Taken together, the presented results demonstrated that pine extract and pure isolated abietic acid were able to induce MCF-7 cell apoptosis with potential differences in activity. These findings were correlated at the molecular level while exploring the modulation of different key-signaling genes controlling apoptosis, resistance, carcinogenesis, vascularization, and cellular growth. Abietic acid was significantly able to induce an increase in both key intrinsic and extrinsic apoptosis genes, such as (_Fas, FasL, BNIP3, Casp8, Cyto-C, Bax_). This suggested that MCF-7 responded very well to the apoptotic genetic changes within 24 h of treatment. However, abietic acid treatment was able, after a short time (8-24 h), to render cells reliable for diminished resistance, since _Bax_ and counteracting twin _Bel-2_ family genes are good prognostic markers for cellular progression and aggressiveness and their levels were changed after treatment [62; 63]. After 8 h of _Bax_ overexpression and downregulation of _Bel-2_ genes, _Bax_ transcription and induction induced stress-activated _p53_ apoptosis on the mitochondrial membrane which, consequently, overcame the antiapoptotic effects of _Bel-2_. Thus, abietic acid might be able to induce _p53_-dependent apoptosis, after 24 h, that is attenuated in the presence of _Bax_ with diminished _Bel-2_. Some reported studies demonstrated this correlation between expression of _Bax_ and _p53_ genes [64]. Hence, the ratio of _Bax_ to _Bel-2_ gene levels could influence the fate of a cell in response to apoptosis by abietic acid. This conclusion supported the enhanced activity of abietic acid in apoptosis, as compared to pine extract. Another gene marker is _ATG5_, which is known for its potential role in controlling apoptosis/autophagy. _ATG5_ overexpression was evident after abietic acid treatment concurrently with _Fas_ and _FasL_ genes after 24 h, suggesting its previously reported role in apoptosis through interaction with _FADD_ (_Fas_-associated protein with death domain), and suggesting that this interaction mediates interferon-\(\gamma\) (IFN-\(\gamma\))-induced cell death [65]. Moreover, abietic acid was able to induce downregulation of the key gene controlling vascularity and blood supply to cancer cells such as _VEGF_ and, hence, vascular permeability, angiogenesis, proliferation, and cell resistance mediated through several cancer-causing factors [66]. Furthermore, abietic acid was shown to inhibit the expression of _c-myc_, an oncogene that belongs to a family of genes that potentially regulate transcription on the genome level. Previous studies showed that its downregulation enhanced the effects of anticancer therapies on cell cycle arrest, apoptosis, and the invasion and migration of different cancer cell in vitro [67]. This effect could be explained by regulating the HIF-1\(\alpha\)/SDF-1/CXCR4 signaling pathway [68]. Another marker deemed essential in cancer resistance is _TNF-\(\alpha\)_, which is a proinflammatory cytokine that exaggerates cellular inflammation, proliferation, and carcinogenesis in MCF-7 cells [69].