## 2 Results
### Abietic Acid Modulates Key- Genes Regulating Multiple Controlling Pathways  
It was essential to understand the possible molecular mechanisms regulating apoptosis that occurred after treatment of MCF-7 with both pine extract and abietic acid on the level of genetic expression in order to predict possible mechanistic changes at the early stage. Thus, it was of particular interest to investigate the expression pattern of a group of apoptosis-related genes (extrinsic and intrinsic pathways) over different time scales--4, 8, and 24 h. The treatment periods chosen for the analysis of abietic acid action were based on previous studies in the same area [40; 41]. Table 4 shows the gene expression levels of different genes of different pathways. Both pine extract and abietic acid were able to induce gene overexpression of extrinsic apoptotic driven genes, such as _Fas_, _FasL_ and _Casp8_, in all selected time scales, with maximum peaks between 4 and 8 h.  
However, abiotic acid showed markedly significant increases in gene expression levels after 24 h of _Cyto-C_, _Bax_, and _p53_, which play important roles in intrinsic pathways with lower levels of _Bcl-2_ and a higher _Bax/Bcl-2_ ratio. Furthermore, _p53_-mediated transactivation of apoptosis comes from its ability to control transcription of proapoptotic members of the _Bcl-2_ family. Additionally, _p53_ activation is able to activate other caspases, a cascade that consequently activates _Cyto-C_ and apoptosome formation [42]. _Bax_ genes and _Bcl-2_ were expressed in harmonic rhythm, wherein the _Bax_ gene was overexpressed to a maximum peak after 24 h, while the _Bcl-2_ gene was downregulated in the same ratio. Additionally, the _Bax/Bcl-2_ ratio, which acts as a rheostat and determines cell susceptibility to apoptosis [43; 44], reached its maximum (13.9) with only abietic acid treatment after 24 h.  
Finally, the _p53_ gene did not show any genetic modification after treatment with pine extract, while it was overexpressed after 24 h with abiotic acid. The _ATG5_ gene is an important interplay mediator and was shown to overexpressed with both pine extract and abietic acid after 24 h and to play a dual role in both autophagy and apoptosis. _BNIP3_ is the network mediator connecting the two types of cell death pathways; it was also \begin{table}
\begin{tabular}{c c c c c} \hline
**Apoptotic Parameters on MF-7 Cells** & **Control** & **Raptinal** & **Abietic Acid** & **Pine Extract** \\ \hline \% Viable cells (C\({}^{-}\) ) & 93.65 \(\pm\) 10.7 & 48.55 ** \(\pm\) 4.8 & 8.4 *** \(\pm\) 2.9 & 28.55 *** \(\pm\) 10.9 \\ \hline \% Early apoptotic cells (C\({}^{+}\) ) & 6.30 \(\pm\) 2.4 & 51.35 *** \(\pm\) 11.5 & 91.41 *** \(\pm\) 12.9 & 71.39 *** \(\pm\) 7.6 \\ \hline \% Late apoptotic cells (C\({}^{-}\) +) & 0.01 & 0.00 & 0.01 & 0.00 \\ \hline \% Necrotic cells (C\({}^{+}\) +) & 0.04 & 0.1 & 0.1 & 0.06 \\ \hline Apoptotic index (AI) & 0.06 \(\pm\) 0.03 & 0.51 \(\pm\) 0.4 & 1.0 \(\pm\) 0.35 & 0.71 \(\pm\) 0.11 \\ \hline \end{tabular} Data represent mean \(\pm\) SEM, \(n\) = 3. \(p\)-values for comparison with control nontreated cells are ** \(p\) < 0.001,
*** \(p\) < 0.001.  
\end{table} 
Table 3: Percentage of viable, apoptotic, late apoptotic, and necrotic MCF-7 cells using flow cytometry. MCF-7 were treated with IC\({}_{50}\) of pine extract and abietic acid and compared to both negative control (0.1% DMSO) and positive control (Raptinal). Apoptotic index, as marker of apoptosis, was calculated for Raptinal-, pine extract-, and abietic acid-treated cells. over-expressed after 8 h. This may suggest that both pine extract and abiotic acid activities were achieved within 4-8 h of MCF-7 treatment, and maximum gene modifications were observed after 24 h.  
The expression of proliferation target genes such as _VEGF, TGF-\(\beta\)1, IGF1R_ and _ATG12_, and levels of oncogenes _C-myc, TNF-\(\alpha\)_ and _NF-\(\kappa\)B_ were downregulated over all time scales and did not change significantly.