## 3 Discussion
The activation of NF-\(\kappa\)B signaling, induced by _TNF-\(\alpha\)_, has also been shown to be a key element in resistance of apoptosis-based tumor mechanisms. Thus, it is an attractive target for modulation by abiotic acid in therapy in order to facilitate the enhancement of _TNF-\(\alpha\)_-mediated apoptosis for anticancer treatment in MCF-7 cells [20; 69]. These results could support the potency of pine extract and abiotic acid in any combined adjuvant remedy with anticancer therapies due to the inhibition of _TNF-\(\alpha\)_-induced cancer cell invasion and angiogenesis. Abietic acid also showed modulating effects on some kinases' expression, such that their activity played a key role in controlling different carcinogenesis and malignant transformation pathways through ATP regulation and enhancement of anticancer sensitivity and cellular responses [70]. Moreover, dysregulation of kinases is considered an oncogenic prognostic marker for apoptotic resistance and spread of cancer cells [70]. For example, higher levels of _PKC-\(\alpha\)_ genes in MCF-7 have been associated with higher proliferation rates and transformation of epithellioid morphological appearances into tumors in nude mice [71]. MCF-7-inducing higher _PKC-\(\alpha\)_ genes exhibited significant reductions in estrogen receptor expression and decreases in estrogen-dependent gene expression [71]. These findings suggested that the inhibition of the _PKC_ pathway could modulate the progression of breast cancer to a more aggressive neoplastic process, which was observed in our study, as well. Abietic acid and pine extract were able to inhibit the growth of _PKC-\(\alpha\)_. Protein Kinase AMP-Activated Catalytic Subunit Alpha 1 (_PRKAA1_) is another regulating factor in carcinogenesis [72]. Cancer growth and metastasis is a result of imbalances between energy-producing systems and consumption of the energy for growth. AMP-activated protein kinase (_AMPK_) has been shown to regulate this process through regulation of AMP and ADP levels. [72]. In carcinogenesis, AMPK signaling is inhibited and cells produce energy for growth and motility, opposing the actions of insulin and growth factors. Increasing AMPK activity could prevent the proliferation and metastasis of tumor cells [72]. In addition, AMPK suppresses aromatase, which is responsible for production of estrogen and breast cancer growth [72]. Thus, inhibition of AMPK is a good target in anticancer therapy. Our observed data coincided with previously noted studies and could provide additional approaches for the use of abiotic acid in the prevention of breast cancer neoplasia, growth, and metastasis. Cyclin-dependent kinase 4 (_CDK-4_) was shown, in MCF-7, to lead into uncontrolled cell division, downstream of many mitogenic signaling pathways. This has implications for resistance [73]. Many CDK-4 inhibitors were recently discovered and shown to be effective inhibitors of MCF-7 poor prognosis and resistance [74]. Both pine extract and abietic acid were able to suppress expression levels of the _CDK-4_ gene after 24 h.  
It was essential, in this study, to investigate the role of the activation of all the previously mentioned pro- and apoptotic genes in activating the final executioner protein of apoptosis on the mitochondrial outer membrane (MOM). MOM plays a key role in intrinsic apoptosis, exaggerated by permeabilization and cytochrome c release to the cytoplasm [75]. Cytochrome-C release is activated by several proapoptotic stimuli, such as _Bax_ and _Bcl-2_ gene levels and, once activated, it triggers the activation of a class of protease enzymes called caspases that further activate the formation of apoptosome. Thus, investigating protein levels of cytochrome-C is of substantial importance to confirm the activation of pro-apoptotic (_Bax/Bcl-2_) and apoptotic (_Casp-3_ and _casp-8_) genes and apoptosome formation. It has been suggested that the apoptotic effects of abietic acid and pine extract are mediated through intrinsic apoptosis.