## 3 Results
### Volatile Chemical Profile of Cigarette Smoke Condensate (CSC)

Table 2: Chemical components (relative %) of cigarette smoke condensate (CSC) identified at SPME-GC/MS analysis. Figure 4: Cytotoxic effects of \(\beta\)-caryophyllene (**A**) and cigarette smoke condensate; CSC (**B**) in triple-negative breast cancer MDA-MB-468 cells after 24 h exposure. Data are displayed as mean \(\pm\) SE of at least three independent experiments with at least three technical replicates (\(n=9\)). *** \(p<0.001\), significant lowering of cell viability with respect to the control (ANOVA + [PERSON]’s multiple comparison post-hoc test). Based on the results of the cytotoxicity assay, the concentration of 10 \(\upmu\)g/mL, which induced a lower than 20% cytotoxicity, was selected for studying the chemoprevertive properties of \(\upbeta\)-caryophyllene towards CSC under different scheduled protocols of pre-, co- and post-treatment. These conditions were evaluated in line with our previous studies in which the substance was shown to modulate the injury of cigarette smoke and cigarette butt extract, acting as both a desmutagenic and a bioantimutagenic agent [33, 46]. In particular, the modulating effects of the sesquiterpene were assessed towards three nontoxic or low-toxic concentrations of CSC (i.e., 50, 75 and 100 \(\upmu\)g/mL) and the cytotoxicity was determined in terms of both the cell viability and leakage of the cytoplasmic lactate dehydrogenase (LDH) enzyme. This last assay provided information about the ability of the treatment to damage the cell membrane, causing the release of the intracellular contents, including the detected enzyme: this event is usually correlated with necrosis occurrence which in turn evokes an inflammatory response [47]. Under our experimental conditions, we found a similar behavior of the tested samples in the pre-, co- and post treatments. In particular, starting from the concentration of 75 \(\upmu\)g/mL, the cytotoxicity of CSC was associated with a progressive increase in LDH leakage, which was not significantly affected by the treatment with \(\upbeta\)-caryophyllene (Figure 5). At the concentration of 50 \(\upmu\)g/mL, CSC produced a cytotoxic effect lower than or equal to 20% with respect to the control, which was further increased by the treatment with the sesquiterpene up to a maximum of 20% in the co-treatment, without affecting LDH release (Figure 5). On the basis of these findings, the co-treatment of 10 \(\upmu\)g/mL \(\upbeta\)-caryophyllene and 50 \(\upmu\)g/mL CSC was chosen for further evaluating the recovery abilities of MDA-MB-468 cells and the modulation of chemoresistance mechanisms. The cell number was registered at the end of the treatment; in addition, the recovery abilities of triple-negative breast cancer MDA-MB-468 cells after 24 h exposure to the co-treatment were evaluated every 24 h for a total of 72 h and expressed as the recovery index (i.e., the ratio between the cell numbers at each time point of recovery and that after treatment). The results highlighted that, after a significant lowering of cell viability induced by treatments, the cell growth progressively increased, especially in the control and in CSC-treated cells (Figure S1). The recovery index of the cells subjected to CSC treatment was found markedly increased by about two- and three-fold after 48 and 72 h recovery, respectively, whereas \(\upbeta\)-caryophyllene inhibited the CSC effect, bringing the index back to the control levels (Figure 6); moreover, after 72 h recovery, a further 30% lowering with respect to the control was also highlighted. This evidence suggested that, along with an initial injury by CSC, the tumor-promoting factors can be activated by the toxicant, leading to increased cell recovery abilities and probably to chemoresistance; conversely, \(\upbeta\)-caryophyllene seemed to counteract the activation of these processes acting as a chemopreverting agent.