## 3 Results
### Effects of \(\beta\)-Caryophyllene and CSC on Cell Migration and on Cell Migration Factors  
In order to better outline the chemoprevetitive abilities of \(\beta\)-caryophyllene towards CSC, we also assessed the cell migration in the wound healing assay (Figures 14, 15 and S4). Images were captured at each time point and processed by Gen5(tm) Microplate Reader and Imager Software (Version 3.11, BioTek, AHSI, Milan, Italy) in order to highlight the wound area to be measured. Under our experimental conditions, the treatment of MDA-MB-468 cells with \(\beta\)-caryophyllene only slightly affected the cell migration, being the wound area similar to that of the control at each time point. Conversely, CSC lowered the wound area with respect to the control, especially after 48 h exposure, thus suggesting a promotion of migration abilities. Interestingly, this effect was blocked by the co-treatment with \(\beta\)-caryophyllene, which induced an increase in the wound area, especially after 48 h exposure. Figure 14: MDA-MB-468 cells migration rate at 0 h, 4 h and 72 h (\(n=3\)) after co-treatment with \(\beta\)-caryophyllene and cigarette smoke condensate (CSC) in MDA-MB-468 cancer cells. A. Images of wound healing assay captured at each time point and processed by Gen5(tm) Microplate Reader and Imager Software (Version 3.11, BioTek, AHSI, Milan, Italy) (4\(\times\) original magnification). The quantified wound areas are displayed in black. Scale bars = 1000 \(\upmu\)m.  
Under the selected experimental conditions, we also evaluated the expression modulation of the genes involved in cell migration and metastas, including BIRC5, MMP2, TPX2 and IL-8 at each time point (i.e., zero point and after 24 h and 48 h exposure). According to the results of the RT-qPCR analysis, we found that the expression of all of the assessed genes was markedly upregulated during incubation with CSC (Figure 16). In particular, the expression of the BIRC5 gene was increased by CSC by almost three-fold after both 24 h and 48 h, while MMP2 was enhanced from at least three-fold to four-fold during incubation; similarly, the TPX2 and IL-8 genes were upregulated by 2.5-fold to 3.5-fold, respectively (Figure 16). Conversely, the presence of \(\beta\)-caryophyllene hindered the effects of CSC; indeed, the induced expression of the BIRC5 gene was halved after 24 h exposure, achieving a five-fold reduction after 48 h (Figure 16A). It is noteworthy that in this last condition, the gene expression of BIRC5 was also halved with respect to the basal level; accordingly, \(\beta\)-caryophyllene alone affected the basal level of the BIRC5 gene achieving a reduction by at least 1.5-fold after 24 h and 48 h exposure. \begin{table}
\begin{tabular}{c c c c c} \hline \hline  & **Ctrl** & \(\beta\)**-Caryophyllene** & **CSC** & \(\beta\)**-Caryophyllene + CSC** \\ \hline LC3B & - & +/ – & + & +/ – \\ \hline Beclin-1 & - & +/ – & + & +/ – \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Semiquantitative analysis of LC3B and Beclin-1 expression induced by \(\beta\)-caryophyllene and cigarette smoke condensate (CSC) in MDA-MB-468 cancer cells after 24 h exposure. Analysis has been carried out (five fields for each treatment) using a previous published grading system [42]: 0–5% = negative; 6–10% = +/ –; 11–30% = +; 31–60% = ++; >61% = +++. Figure 15: Quantification of MDA-MB-468 cells migration rate at 0 h, 4 h and 72 h (\(n=3\)) after treatment with \(\beta\)-caryophyllene and cigarette smoke condensate (CSC). 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 difference of each time point with respect to zero time (ANOVA + [PERSON]’s multiple comparison post-hoc test). *** \(p<0.001\), significant difference with respect to the control at the same time point (Student’s \(t\)-test). *** \(p<0.001\), significant difference respect to CSC at the same time point (Student’s \(t\)-test). *** \(t\)-test).