## 4 Discussion
Moreover, a reduced level of caspase-2 was noticed upon Fas stimulation [31] and we also presented that treatment of CRC cells only with anti-Fas Ab did not exert a prominent effect on the caspase-2 level. In the same samples we found significantly elevated CD133+ CSCs count. At the same time, simultaneous stimulation of CRC cells with ASA and anti-Fas Absignificantly (2-5 times) increased caspase-2 level in both studied cell lines with parallel lowering of CSCs proportion. Based on previous observations that caspase-2 is not involved in the anti-apoptotic and pre-cancerous functions of Fas signaling [31], we hypothesized that this protein might antagonize the Fas pathway by being the natural inhibitor driving the elimination of CRC cells. Additionally, CSCs seem to be specific targets of such stimulation. Moreover, caspase-2 was described as an agent that triggers DICE, a necrotic form of mitotic catastrophe characterized by cell swelling, ROS production causing DNA damage and mitochondrial outer membrane permeabilization [31; 36]. DICE was suggested to be the last resort allowing the specific elimination of cells lacking Fas and/or FasL. We included into our study DCs to assess if lysates prepared from cancer cells treated with both active compounds would influence their activity. The analysis of DCs' phenotype seems to confirm that pretreatment of cancer cells before their engagement into in vitro modification of DCs can be beneficial for the final effect. We found that the lysates obtained from HCT116 colorectal cancer cells treated with our active compounds led to significantly enhanced expression of CD80 and CD83 markers on DCs surface, commonly associated with activation status of these cells. HT29-derived lysates exerted a less prominent effect on DCs what is probably associated with diverse cancer progression status of both CRC cell lines (HCT116-TNM3, HT29-TNM2). Nonetheless, this issue is open for further investigation since many different aspects of DC features and functions should be taken into consideration. Moreover, many previous results proved the influence of caspases in cancer milieu on the activity of immune cells, including DCs. Additionally, it has recently been suggested that mutations in caspase-3 might increase tumor recurrence risk after T cell-based cancer immunotherapy [37]. Previously, it was found that the number of mature CD11c+ MHCII+ DCs was significantly lower in caspase 3 gene knockout mice in comparison to wild type. The Authors suggested that caspase 3 may be involved in the regulation of maturation and anti-cancerous activity of DCs [38]. Moreover, it was demonstrated that DC and cytokine-induced killer cells significantly enhanced the apoptosis ratio of cancer stem cells of human hepatocellular carcinoma by, among others, increasing caspase-3 protein expression [39]. As we previously reported, anti-Fas stimulation has rather pro-cancerous effect since we found increased number of CD133+ and CD29+ CSCs, an increased sphere sizes, decreased apoptosis rate and most of these differences were significant comparing to untreated control cells [20] and the effect of anti-Fas treatment depended on the cell line used. The increased level of caspase-2 confirmed the association of Fas signaling with DICE (a necrotic form of mitotic catastrophe) which is believed to be characteristic for CSC population. Mentioned above pro-tumorigenic activity may be ceased by ASA, what was confirmed in the presented study by the increased apoptosis mediated by elevated caspase-3. Additionally, we noticed a decrease of CD133+, CD44+ and CD29+ CSCs in the total population of cancer cell lines. The increased number of CD44+ CD29+ cells among both CD133- and CD133+ populations (data not shown) is suggested to be associated with increased adhesive properties of remaining cells and was responsible for maintaining colonospheres' integrity.