## 3 Results and Discussion
### CYN on Intracellular Free Calcium Concentration, [Ca\({}^{2+}\)]\({}_{\mathrm{i}}\)  
It is well known that calcium ion (Ca\({}^{2+}\)) concentration serves as an important intracellular signal for a very wide variety of cellular phenomena, including proliferation and differentiation, regulation of gene expression, stimulus-secretion coupling, and motility [23]. It is known that sustained and prolonged increases in [Ca\({}^{2+}\)]\({}_{\mathrm{i}}\), over time, are toxic to cells and are involved in triggering events that lead to cell apoptosis. [24]. The role ofGURE:S2.F1][ENDFIGURE]  
Bradykinin (BDK) is an agonist of cell membrane activity and was used as a positive control in this study. BDK induced transient increases in [Ca2+]i, likely by interacting with specific membrane receptors and activating intracellular mechanisms through the phosphoinositide-phospholipase pathway [25]. BDK was added to the cuvette at a final concentration of 100 nM. BDK elicits a rapid, transient elevation of [Ca2+]i in LoVo cells (Figure 1). The effect has two components: a first phase is a spike-like increase, followed by a slow plateau. BDK mediates both the Ca2+ release from internal pools and a small entry of Ca2+ into the cytoplasm from the extracellular space, as suggested by the plateau phase (28-30 nM) [26] (Figure 2). Figure 1: Cytotoxicity assay. Regression line for the cytotoxic effect of sesquiterpene caulerpenyne (CYN) on LoVo cells. Control (100% survival) is provided by incubation in DMSO. Time of exposure: 24 h.  
In LoVo cells that have not been pretreated with CYN, the spike-like component caused an increase in [Ca2+]i of 91.2 \(\pm\) 7.9 nM (Figure 2). In a series of experiments, spectrofluorimetric analysis was performed 24 h after incubation time in CYN in a second series of experiments, 48 h after the incubation in CYN. Different CYN incubation times were tested, and CYN was applied without preincubation (data not shown). A choice was made for 3 h of preincubation. In particular, preincubation with CYN and spectrofluorimetric analysis 24 h after CYN removal caused a decrease in the [Ca2+]i transient peak (mean \(\pm\) SEM, \(n\) = 4; Figure 3). Thus, it appears that the preincubation of cells with CYN induces a concentration-dependent decrease in the first component (spike-like) of the BDK increase in [Ca2+]i. After the transient peak, [Ca2+] dropped to a plateau level of 10-20 nM for all different CYN incubation concentrations. This plateau persisted for up to 7 min of observation. In the second series of experiments, the application of BDK and the analysis of [Ca2+]i were evaluated 48 h after preincubation in CYN. BDK alone in LoVo cells that were not pretreated with CYN (control) caused a rise in [Ca2+]i of 88.25 \(\pm\) 8.8 nM (spike-like component). Preincubation with CYN and spectrofluorimetric analysis 48 h after CYN removal caused a decrease in the [Ca2+]i transient peak (mean \(\pm\) SEM, \(n\) = 4). Again, the second component of BDK-induced calcium kinetics was a plateau level of 10-20 nM. Even in this condition, preincubation of cells with CYN induced a concentration-dependent decrease in the first component (spike-like) of the BDK-induced [Ca2+]i increase (Figure 4). A major effect is observed 24 h after preincubation in CYN (Figure 3). The effect could be related to the inhibitory action of CYN on phospholipase activity. CYN could affect the kinetics of IP3R and the calcium channel, possibly by reducing the time of the channel's open state. Figure 3: Histograms summarizing the effects of pretreatment of CYN at different concentrations (3 h of incubation) on LoVo cells. Pretreatment with CYN and spectrofluorimetric analysis 24 h after CYN removal. Data are means \(\pm\) SEM (\(n\) = 4); significance * \(p\) < 0.001 compared to control group. Different concentrations were compared between them (same letter, no statistical significance; different letters, statistical significance, \(p\) < 0.01). BDK, bradykinin; CYN, caulerpenyne; [Ca2+]i, intracellular calcium concentration.