## 3 Results and Discussion
### Analysis of Apoptosis Using the Muse(r) Flow Cytometer
\begin{table}
\begin{tabular}{c c c c c c c} \hline  & **UC** & **STS** & **ys d + a** & **ys\%** & **mv d + a** & **mv\%** \\ \hline
**live** & 87.83 \({}^{\mathrm{d}}\)\(\pm\) 2.78 & 34.25 \({}^{\mathrm{a}}\)\(\pm\) 1.55 & 30.28 \({}^{\mathrm{a}}\)\(\pm\) 2.08 & 58.65 \({}^{\mathrm{b}}\)\(\pm\) 0.35 & 62.2 \({}^{\mathrm{b}}\)\(\pm\) 9.35 & 78.73 \({}^{\mathrm{c}}\)\(\pm\) 0.57 \\
**caspase+** & 8.68 \({}^{\mathrm{a}}\)\(\pm\) 0.77 & 38.50 \({}^{\mathrm{d}}\)\(\pm\) 14.95 & 22.13 \({}^{\mathrm{b}}\)\(\pm\) 1.73 & 15.55 \({}^{\mathrm{a}}\)\(\pm\) 0.15 & 25.2 \({}^{\mathrm{c}}\)\(\pm\) 3.75 & 12.05 \({}^{\mathrm{a}}\)\(\pm\) 0.05 \\
**caspase+/dead** & 3.28 \({}^{\mathrm{a}}\)\(\pm\) 1.98 & 26.83 \({}^{\mathrm{b}}\)\(\pm\) 16.68 & 47.55 \({}^{\mathrm{c}}\)\(\pm\) 0.35 & 25.80 \({}^{\mathrm{b}}\)\(\pm\) 0.20 & 12.4 \({}^{\mathrm{a}}\)\(\pm\) 5.65 & 9.08 \({}^{\mathrm{a}}\)\(\pm\) 0.48 \\
**dead** & 0.23 \({}^{\mathrm{c}}\)\(\pm\) 0.03 & 0.43 \({}^{\mathrm{d}}\)\(\pm\) 0.18 & 0.05 \({}^{\mathrm{a}}\)\(\pm\) 0.0 & 0.0 \({}^{\mathrm{a}}\)\(\pm\) 0.0 & 0.2 \({}^{\mathrm{b}}\)\(\pm\) 0.05 & 0.1 \({}^{\mathrm{a}}\)\(\pm\) 0.1 \\
**total caspase** & 11.95 \({}^{\mathrm{a}}\)\(\pm\) 2.75 & 63.60 \({}^{\mathrm{d}}\)\(\pm\) 2.01 & 69.68 \({}^{\mathrm{d}}\)\(\pm\) 2.08 & 41.35 \({}^{\mathrm{c}}\)\(\pm\) 0.35 & 37.6\({}^{\mathrm{c}}\)\(\pm\) 9.4 & 21.18 \({}^{\mathrm{b}}\)\(\pm\) 0.48 \\ \hline \end{tabular}  
* UC, untreated control. STS, staurosporine. ys d + a, juice of young shoots subjected to in vitro gastrointestinal digestion and absorption. ys%, fresh, 5% juice of young shoots. mv d + a, juice of the mature vegetable subjected to in vitro gastrointestinal digestion and absorption. mv%, fresh, 5% juice of the mature vegetable. Results are expressed as mean \(\pm\) SD (\(n=3\)). Statistically significant differences (based on ANOVA and [PERSON]’s a posteriori test) \(p\leq 0.05\) within each row are indicated by letter code a-d.  
\end{table} 
Table 5: Cell apoptosis results of LNCaPP prostate cancer cells at treated with experimental juices. Apoptosis was analyzed using flow cytometry (Muse® Cell Analyzer) and the Muse® MultiCaspase Assay Kit. The pro-apoptotic proteins, e.g., Bax, Bid, and PUMA, bind to the outer membrane of the mitochondria to signal the release of the internal proteins. In normal cells, the Bax protein is an inactive form in the cytosol and in response to certain apoptotic, can be induced to change conformation and translocate into the mitochondria [32]. A transcription factor, p53 plays, a relevant role in the regulation of the expression of Bcl-2 family proteins [33]. The _TP53_ gene controls the transcription of many genes and regulates processes related to DNA repair, cell-cycle arrest, or apoptosis [34]. Our results for juice-treated cancer cells showed significant (\(p\leq 0.05\)) up-regulation of the _TP53_ gene and the p53 protein in both examined cell lines (Table 6, Figures 3 and 4; Supplementary Figures S3 and S4). In addition, we determined that \(BBC3\) (the p53 upregulated modulator of apoptosis, _PUMA_), \(BAD\) (Bcl-2 associated agonist of cell death), and _DIABLO_ mRNA level in the DU145 cell line was significantly (\(p\leq 0.05\)) increased after exposure to juices, especially to the juice of young shoots, both fresh and subjected to in vitro digestion and absorption (Table 6). The \(BBC3\) gene is the essential mediator of p53 dependent apoptosis. The BAD protein induced cell death, successfully competing for the binding to Bcl-xL, Bcl-2, and Bcl-w, affected the level of heterodimer of these proteins with Bax [35]. Using Western blot analysis, we found a significant increase (\(p\leq 0.05\)) in the pro-apoptotic Bcl-2 family members-Bax and PUMA proteins expression in DU145 and LNCaP prostate cancer cells treated with the experimental juices (Figures 3 and 4; Supplementary Figures S3 and S4).