## 2 Results  
### Abietic Acid Induces Growth Inhibition in MCF-7 Sensitive Cell Line  
In the current study, MTT assay was used to assess the antiproliferative activity of both pine extract and its pure abietic acid active compound against three different cancer cell lines. These were also compared to normal human fibroblast cell lines. Two positive controls of documented antiproliferative activities were used for comparison (Raptinal [21] and Doxorubicin [22]). The IC\({}_{50}\) was calculated according to three parameters and the logistic dose-response sigmoidal curve using GraphPad Prism 4.0 (Figure S1). Table 1 shows positive controls, pine extract, and abietic acid with variable antiproliferative activities against the different cancer cell lines (HCT-116, HepG-2 and MCF-7). In HepG-2 hepatocellular carcinoma cells, Raptinal and Doxorubicin showed antiproliferative activity with IC\({}_{50}\) = 0.74 and 0.8 \(\upmu\)M, respectively, which was similar to the documentedvalues in the literature [23]. Pine extract showed superior antiproliferative activity with IC\({}_{50}\) = 0.2\(\upmu\)g/mL. The selectivity index (SI) is defined as the ratio of the toxic concentration of a sample against its therapeutic concentration [24]. The ideal drug for continuation in drug screening should have a relatively high toxic concentration in order to cause toxicity at very low doses for therapeutic effect. Candidate drug should have generally SI value > 1 and fall into one of the following criteria: SI value = 1-1.99 is of low selectivity (general toxicity), SI value = 2-2.99 is of moderate selectivity and SI value >3 is of high selectivity [25; 26; 27; 28; 29; 30]. Therefore, evaluation of SI values for any herbal drug and/or isolated compound is crucial for determining whether further work can be done. Pine extract's SI value was 2, indicating less cytotoxic effects than Raptinal (SI = 7). Abietic acid did not show any significant antiproliferative effects on HepG-2 cells. The same scenario was observed in HCT-116 human colon cancer cells, where pine extract showed superior antiproliferative activity, with IC\({}_{50}\)= 0.25 \(\upmu\)g/mL, and minimal cytotoxic effects, with SI = 1.7, as compared to the nonsignificant effects of abietic acid. MCF-7 was the most sensitive cell line, with minimum cytotoxicity for both pine extract and its pure isolated abietic acid (IC\({}_{50}\) = 0.11 \(\upmu\)g/mL, SI = 3.8 and 0.06 \(\upmu\)M, SI > 1, respectively (Table 1). SI > 1 for abietic acid indicated that it was the safest, since very high concentrations, i.e., above 100 \(\upmu\)M, were required for cytotoxic effects on WI-38, as compared to IC\({}_{50}\) on MCF-7 cells. The IC\({}_{50}\) value for abietic acid was not applicable on WI-38, HepG-2, or HCT-116, as we tested several x-fold concentrations and dilutions of abietic acid on both extremities without 50% cellular viability inhibition. Therefore, there was no need to perform further experiments with them on these cell lines; there was almost no chance for them to be used as therapeutic drugs on these cell lines. Any dose above 100 \(\upmu\)M or 0.5 mg/mL is considered within cytotoxic margins, as documented by many studies [31; 32; 33; 34; 35] and, consequently, calculation of SI value was not applicable. Abietic Acid Induces MCF-7 Cells Apoptosis with Cell Cycle Arrests at Both G0-G1 and G2/M Phases and SubG0-G1 Sub-Population  
The SubG0-G1 subpopulation is an important fraction of the nondividing cells that tend to enter cell cycle division. Hence, compounds that are able to induce cell cycle accumulation in this subpopulation have a powerful apoptotic effect and inhibit scell growth at early stage [36; 37; 38]. Since abiotic acid showed potent antiproliferative activity against MCF-7 cells, the next step was to investigate its apoptotic and cell cycle effects in comparison to pine extract on MCF-7 cell cycle. MCF-7 cells were incubated with IC\({}_{50}\) of abietic acid and pine extract for 24 h and flow cytometry assay was undertaken.