## 3 Results
### Volatile Chemical Profile of Cigarette Smoke Condensate (CSC)
\begin{table}
\begin{tabular}{l c} \hline \hline  & **IC50 [\(\upmu\)g/mL] (CL)** \\ \cline{2-3}
**Tested Sample** & **MDA-MB-468** \\ \hline CSC & 134.8 (119.6–152.7) *** \\ \(\upbeta\)-Caryophyllene & 18.6 (15.9–21.7) *** \\ Doxorubicin & 4.7 (3.8–6.7) \\ \hline \hline \end{tabular}
\end{table} 
Table 3: IC50 values of \(\upbeta\)-caryophyllene, cigarette smoke condensate (CSC) and the positive control doxorubicin in triple-negative breast cancer MDA-MB-468 cells. Data represent the mean \(\pm\) SE of at least three independent experiments with at least three technical replicates (\(n\) = 9). Figure 5: Cytotoxic effects of \(\beta\)-caryophyllene and cigarette smoke condensate (CSC) in triple-negative breast cancer MDA-MB-468 cells under pre-, co- and post-treatment protocols, measured as cell viability (A, C, E) and LDH release (B, D, F). 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). *** \(p<0.001\), significant difference the respect to CSC (Student’s \(t\)-test). In order to clarify the phenomenon and to better understand the processes responsible for CSC-induced chemoresistance and for the protective effects of \(\beta\)-caryophyllene, different cell parameters, including the redox state, genomic instability, autophagic and apoptotic rates, and cell migration were studied under the selected experimental conditions, as explained in the following sections. Effects of \(\beta\)-Caryophyllene and Cigarette Smoke Condensate (CSC) on Intracellular Oxidative Stress  
The redox state of triple-negative breast cancer MDA-MB-468 cells subjected to a 24 h co-treatment with \(\beta\)-caryophyllene and CSC was determined in terms of levels of reactive oxygen species (ROS) and reduced glutathione (GSH). The results highlighted the ability of both \(\beta\)-caryophyllene and CSC to upregulate the intracellular oxidative stress, as shown by the increased ROS levels induced by both the tested samples and their combination (Figure 7). At imaging analysis, the oxidative stress was highlighted by the green DCF-fluorescence into the cells, as displayed in Figure 7A. Quantification of the DCF-fluorescence showed that the ROS levels were enhanced by about 30% by \(\beta\)-caryophyllene, and by at least 70% by CSC, while more than doubled by their co-treatment (Figure 7B). The increased oxidative stress can be responsible for the activation of cell-death signaling, thus explaining the higher cytotoxicity highlighted by the co-treatment with \(\beta\)-caryophyllene and CSC with respect to the samples alone in all of the assessed protocols of exposure. According to the marked oxidative stress, CSC induced a three-fold reduction of the ratio between the GSH levels and GSSG levels, despite a slight lowering (about 1.09 fold) found with \(\beta\)-caryophyllene; conversely, the co-treatment of \(\beta\)-caryophyllene and CSC almost restored the GSH/GSSG ratio, inducing about a 2.7-fold increase (Figure 8). These results suggest that the treatment with \(\beta\)-caryophyllene only slightly affected the glutathione levels, despite a marked depletion (about a four-fold lowering with respect to the control) induced by CSC. However, considering that the GSSG amount was similar to that of the control, GSH depletion by CSC could be due to other mechanism than ROS species neutralization, such as conjugation of the toxicants contained in the smoke mixture. Figure 6: Effect of the co-treatment of \(\beta\)-caryophyllene and cigarette smoke condensate (CSC) in triple-negative breast cancer MDA-MB-468 cells after 24 h exposure and subsequent 72 h cell recovery. Recovery abilities were expressed as a recovery index, which represents the ratio between the cell number at each time point of recovery and that achieved after 24 h treatment. Cell number was determined by trypan blue exclusion assay at each time point.