## 3 Results and Discussion
### Analysis of Apoptosis Using the Muse(r) Flow Cytometer
A higher expression of these proteins was observed in the DU145 cell line treated with the juice of young shoots subjected to in vitro gastrointestinal digestion and absorption in comparison to that treated with juice of the mature cabbage subjected to the same processes (Figures 3 and 4; Supplementary Figures S3 and S4). The current results revealed that the juices up-regulated (\(p\leq 0.05\)) the expression of cytochrome c, Smac/Diablo, and HtrA2/Omi proteins (Figures 3 and 4; Supplementary Figures S3 and S4). However, the LNCaP cell line did not express the Smac/Diablo protein. It has been stated that the release from the mitochondria of the mentioned above proteins could be controlled by Bax and the translocation of Bax can alter the outer mitochondrial membrane's permeability, and then activate the intrinsic apoptosis pathway. Another pro-apoptotic protein released from the mitochondria during apoptosis is AIF (apoptosis-inducing factor), which translocates to the nucleus and causes DNA fragmentation and chromatin condensation [36]. Our results also showed significant (\(p\leq 0.05\)) increase in the _AIFM1_ protein coding gene mRNA expression in both cell lines (Table 6). Following the release, cytochrome c forms a complex in the cytoplasm with ATP and apoptotic protease-activating \begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline \multirow{2}{*}{
\begin{tabular}{c} **Gene** \\ **Symbol** \\ \end{tabular} } & \multicolumn{4}{c}{**DU145**} & \multicolumn{4}{c}{**LNCaP**} \\ \cline{2-9}  & **ys d + a vs.** & **ys\% vs.** & **mv d + a** & **mv\% vs.** & **ys d + a vs.** & **ys\% vs.** & **mv d + a** & **mv\% vs.** \\  & **UC** & **UC** & **vs. UC** & **UC** & **UC** & **UC** & **vs. UC** & **UC** \\ \hline _AIFM1_ & 10.888 ** & 5.813 ** & 7.402 ** & 4.843 ** & 5.971 ** & 2.399 ** & 1.474 & 3.645 ** \\ _AKT1_ & 0.105 ** & 0.860 & 0.117 ** & 1.874 & 0.221 ** & 0.281 ** & 0.209 ** & 1.006 \\ _Apaf-1_ & 6.649 ** & 1.198 & 4.854 ** & 0.952 & 1.545 * & Ns & 3.162 ** & Ns \\ _BAD_ & 2.041 * & 2.397 * & 1.676 * & 2.618 ** & 1.300 & 1.891 & 1.600 & 1.055 \\ _BBC3_ & 4.987 ** & 2.346 ** & 1.641 * & 1.422 * & Ns & Ns & Ns & Ns \\ _CASP-3_ & 10.997 ** & 1.550 & 5.606 ** & 1.429 * & 2.232 & 1.844 & 4.721 ** & 1.120 \\ _CASP-7_ & 8.893 ** & 2.511 ** & 2.498 ** & 0.754 & 3.305 ** & 2.520 * & 2.696 ** & 1.194 \\ _CASP-8_ & 5.748 ** & 0.462 ** & 1.503 ** & 0.583 & Ns & Ns & Ns & Ns \\ _DIABLO_ & 4.901 ** & 2.481 ** & 4.113 ** & 6.920 ** & Ns & Ns & Ns & Ns \\ _FADD_ & 2.242 ** & 1.500 & 2.152 * & 1.483 & 0.998 & 0.773 & 0.621 ** & 0.619 ** \\ _FAS_ & 6.617 ** & 1.124 & 4.812 ** & 3.216 * & 0.766 & 0.058 & 0.837 & 2.938 * \\ _TP53_ & 4.821 ** & 1.219 ** & 1.076 & 2.756 ** & 1.802 ** & 1.524 ** & 1.809 ** & 1.502 ** \\ \hline \hline \end{tabular}
\end{table} 
Table 6: mRNA expression of genes. factor-1 (Apaf-1). This complex will activate caspase-9 and the apoptosome is formed, which, in turn, activates caspase-3, the effector protein that initiates degradation. In the current study, we determined the up-regulation (\(p\leq 0.05\)) of the _Apaf-1_ gene, in cell lines especially after treatment with juices subjected to in vitro gastrointestinal digestion and absorption (Table 6). _Apaf-1_ encodes a cytoplasmic protein that initiates apoptosis, and accordingly, we observed the up-regulation (\(p\leq 0.05\)) of the Apaf-1 protein expression in the DU145 cell line (after treatment with juices subjected to in vitro gastrointestinal digestion and absorption increased) and in the LNCaP cell line after treatment with juices, excluding fresh mature cabbage juice (Figures 3 and 4; Supplementary Figures S3 and S4). Figure 3: Expression of proteins involved in proliferation and apoptosis signaling in DU145 prostate cancer cells. DU145 prostate cancer cells were treated for 24 h with: STS, staurosporine. ys d + a, juice of young shoots subjected to in vitro gastrointestinal digestion and absorption. ys%, fresh, 5% juice of young shoots.