## 3 Results and Discussion
### Analysis of Apoptosis Using the Muse(r) Flow Cytometer
Simultaneously, it was noted that the studied juices mediated (\(p\leq 0.05\)) down-regulation of the NF-\(\kappa\beta\) level in both prostate cancer cell lines (Figures 3 and 4; Supplementary Figures S3 and S4). Due to the fact that the mentioned proteins are involved in the activation of the nuclear factor-\(\kappa\beta\) and inhibition of NF-\(\kappa\beta\) activation is believed to suppress tumorigenesis and the progression of tumors, it can be suggested that the inhibition of prostate cancer cells proliferation and activation of programmed cell death, in this research, is related to this mechanism. Caspases are a family of protease enzymes that are formed constitutively in the cells and are present as inactive proenzymes. These molecules are activated in the early stages of apoptosis in a self-amplifying cascade [31]. As mentioned above, two major pathways of caspase cascade activation have been differentiated (activation of caspase-8 in death receptor-induced apoptosis and of caspase-9 in mitochondrial pathway). Activation of initiator caspases leads to the proteolytic activation of effector caspases-3, -6, and -7. These effector caspases are responsible for the cleaving of one of the earliest nuclear enzymes- PARP [39]. The up-regulation of caspase-3, -7, and -8 genes in the treated DU145 and LNCaP cells with juices (especially in DU145 cell line after incubation with juice of young shoots subjected to in vitro gastrointestinal digestion and absorption) was observed (Table 6). Finally, we confirmed the up-regulation (\(p\leq 0.05\)) expression of pro-apoptotic proteins: Caspase-3, -7, and -8 under juices treatment. However, the fresh juices had no measurable effect (\(p\geq 0.05\)) on caspase-8 level in the DU145 cell line (Figure 3, Supplementary Figure S3). In case of the LNCaP cell line, the fresh juice of the mature vegetable significantly decreased (\(p\leq 0.05\)) the expression of the caspase-8 level (Figure 4, Supplementary Figure S4). We also observed an increased level of PARP in both cell lines (Figures 3 and 4; Supplementary Figures S3 and S4). In the current work, we also assessed the effect of juices on selected proteins involved in the regulation of proliferation, cellular stress response, and their links to apoptosis induction. Our results showed that the juices subjected to in vitro gastrointestinal digestion and absorption led to a down-regulation (\(p\leq 0.05\)) of the _AKT1_ gene in DU145 and LNCaP cell lines (Table 6). This gene encodes the Akt1 protein, one of the members of the human Akt serine-threonine protein kinase family. One of the effector mechanisms in the cell, which plays a crucial role in signaling pathways important to cell survival and proliferation is the pathway triggered by the phosphatidylinositol 3-kinase (PI3K) and the protein kinase Akt. In simple terms, PI3K activates kinase Akt, which is involved in the activation of proteins that promote cell survival, e.g., the nuclear factor \(\kappa\beta\). The main consequence of its activation is the increased proliferation and tumor transformation and inhibition of apoptosis through its ability to phosphorylate and to inactivate several proteins, including Bad and caspase-9 [40]. In our study, we also noted that treatment with juices significantly (\(p\leq 0.05\)) decreased the level of Akt1 and p44/42 MAPK protein in prostate cancer cell lines (Figures 3 and 4; Supplementary Figures S3 and S4). p38 MAPK and SAPK/JNK MAPKs are activated by a wide range of cellular stressors. SAPK/JNK can promote the activation of both, external and mitochondrial apoptosis pathways. It has been suggested that these mechanisms can act independently or co-operate with one another to induce cell death [41]. Our results indicated a significantly (\(p\leq 0.05\)) decreased level of p38 MAPK and SAPK/JNK MAP kinases in both DU145-7 and LNCaP cell lines after treatment with juices.