## 4 Discussion
Our experiments proved that both studied compounds (Les-4368 and Les-4370) in both studied cell lines (CACO-2 and BJ) increased ROS production in the 10-100 \(\upmu\)M concentration range. However, Les-4368 was a much stronger inducer of ROS production than Les-4370. In the case of caspase-3 activity, both studied compounds in both studied cell lines increased the caspase only at the highest (50 and 100) \(\upmu\)M concentrations. The proapoptotic role of caspase-3 in our experiments was confirmed inter alia by Hoechst 33342-based staining in which the DNA fragmentation and condensation were observed in 10 and 50 \(\upmu\)M concentrations of Les-4368  
Figure 13: Summary of the evaluation of the cytotoxic effect and some mechanisms of action of Les-4368 and Les-4370 derivatives and some structure-activity relationships (SARs). in both studied cell lines. The obtained data also suggest that in lower concentrations, the caspase-3 is not involved, or the caspase-3 activity assay is not able to detect slight changes in its activity. Interestingly, in the case of Les-4370, no significant DNA condensation or fragmentation was observed. Our results suggest that increased caspase-3 activity in the case of Les-4370 is involved in a nonapoptotic role of this enzyme. Moreover, in our study compound, Les-4368 activated caspase-3 much more strongly than Les-4360. However, in the case of CACO-2 cells treated by Les-4368 compound after 48 h, caspase-3 activity was almost unchanged. The mentioned phenomenon is most likely the result of the very strong toxicity of Les-3468 and proteolytic degradation of caspase-3 [44]. [PERSON] et al. revealed that Les-3166 increased ROS production in BJ cells after a 6 h treatment in a concentration range from 1 nM to 10 \(\upmu\)M, compared to the control [11]. Moreover, a similar trend was shown by the same team in studies on A549 cells treated with Les-3166. Treatment of cells with Les-3166 in a concentration range from 1 nM to 100 \(\upmu\)M for 6 h has also been shown to increase ROS [11]. Similarly, [PERSON] et al. showed that 6 h exposure of the SCC-15 cell line to Les-2194, Les-3377, and Les-3640 in a wide (10 nM to 100 \(\upmu\)M) range of concentrations increased ROS production, but caspase-3 activity increases only at the highest (50 and 100) \(\upmu\)M concentrations [9]. Additionally, [PERSON] et al. reported that Les-3640 and Les-6166 in concentrations of 50 and 100 \(\upmu\)M caused an increase in caspase-3 activity after 24 and 48 h of exposure in the 3T3-L1 cell line [27]. On the other hand, it is interesting that Les-236 in \(\upmu\)M concentrations decreased ROS production in the BJ, CACO-2, SCC-15, A549, and SH-SYSY cell lines [12]. However, despite the absence of ROS, increased caspase-3 activity was observed [12]. Compounds Les-2769 and Les-3266 also increased caspase-3 activity in the BJ and SCC-15 cell lines when used at the highest (50-100) \(\upmu\)M concentrations [10]. Finally, it was established that increasing the dose of another 4-thiazolione derivative Les-3288 (5-bromo-3-[2-[5-(4-methoxyphenyl)]-3-phenyl-4,5-dihydroxyrazol-1-ylyl]-4-oxo-4,5-dihydro-1,3-thiazol-5-ylidene]-2,3-dihydro-1\(H\)-indol-2-one) from 0.5 to 1.0 \(\upmu\)mL led to an increase in the amount of cleaved caspase-3 in treated U251 glioma cells [45]. It is worth mentioning that despite the classic role of caspase-3, this enzyme is also involved in the regulation of cell survival, proliferation, and tumorigenesis [44,46]. However, summarizing the data on LDH release, resazurin reduction, and ROS production, we believe that caspase-3 activity in our case is the result of apoptotic cell death. It has been well described that, despite apoptosis activation, ROSs play an important role in PPAR\(\gamma\)-related signaling pathways and cell cycle regulation [47]. It is also known that AhR is involved in the response to oxidative stress through different mechanisms [48].