## 3 Results and Discussion
### Analysis of Apoptosis Using the Muse(r) Flow Cytometer
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 a mean \(\pm\) SD normalized to the internal reference protein (\(\beta\)-actin). Untreated negative control (UC) was set as 100% expression level. Statistical significance was based on \(t\)-test * \(p\leq 0.05\) vs. UC. The extrinsic or death receptor pathway involves the binding of signal ligands to cell surface death receptors, members of the tumor necrosis factor receptor gene (_TNFR_) superfamily, and inducing apoptosis [37]. Upon binding, the ligand to the Fas death receptor, the cytoplasmic adaptor protein FADD, exhibiting corresponding death domains, is recruited. Proaspase-8 then is recruited, forming a death-inducing signaling complex (DISC). Active caspase is then released from the DISC complex into the cytosol, and directly activates caspase-3 and -7 (effector proteins) to start the caspase cascade and initiate degradation of the cell. Active caspases can also activate the Bid protein and represent a \"cross-talk\" of the two main pathways, amplifying the apoptotic signaling from death receptors [37]. The current results revealed that juices up-regulated the expression of gene and protein involved in the extrinsic or death receptor pathway of apoptosis (Table 6; Figures 3 and 4; Supplementary Figures S3 and S4). In the DU145 cell line treated with  
Figure 4: Expression of proteins involved in proliferation and apoptosis signaling in LNCaP prostate cancer cells. LNCaP 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. 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 a mean \(\pm\) SD normalized to the internal reference protein (\(\beta\)-actin). Untreated negative control (UC) was set as 100% expression level. Statistical significance was based on t-test \({}^{*}p\leq\) 0.05 vs. UC. juices subjected to in vitro gastrointestinal digestion and absorption, an up-regulation (\(p\leq 0.05\)) expression of _FAS_ and _FADD_ genes was observed. For the LNCaP cell line, only after treatment with fresh juice of the mature vegetable was an up-regulation (\(p\leq 0.05\)) expression of _FAS_ gene observed (Table 6). Furthermore, we found that the level of the FAS receptor and adaptor protein FADD expression in DU145 significantly (\(p\leq 0.05\)) increased (exception was expression of FAS after treatment of cells with fresh juice of young shoots) (Figure 3; Supplementary Figure S3). The LNCaP cell line did not express FAS and FADD proteins. In the following binding of the TNF\(\kappa\) to TNFR1 receptor and trimerisation, the adaptor molecule TRADD is recruited, which has the ability to recruit secondary adaptors such as RIP or TRAF2 (TNF receptor-associated factor 2). These, respectively, activate the pathways related with NF-\(\kappa\beta\) and JNK/AP-1 and can stop the apoptotic signal and provide cell survival [37]. Activation of NF-\(\kappa\beta\) by extracellular stimuli (e.g., TNF cytokine) leads to rapid phosphorylation, ubiquitination, and proteolytic degradation of its inhibitor protein (I\(\kappa\beta\)), thereby resulting in the nuclear translocation of NF- \(\kappa\beta\) and initiation of the transcription of a large number of, also anti-apoptotic, genes promoting survival [38]. In this study, juices significantly (\(p\leq 0.05\)) decreased the expression of the TNFR1 receptor, adaptor protein TRADD, and secondary adaptor protein RIP in both cell lines. Fresh juice of young shoots as well as being subjected to in vitro gastrointestinal digestion and absorption, had no effect (\(p\geq 0.05\)) on the TRADD level in the DU145 cell line (Figure 3; Supplementary Figure S3).