## 3 Results
### Volatile Chemical Profile of Cigarette Smoke Condensate (CSC)
Under our experimental conditions, we also evaluated the rate of autophagic cell death, which is known to be involved in chemoresistance, especially for triple-negative breast cancer. To this end, the autophagosome formation and expression of autophagic factors were measured (Figures 12 and 13). In particular, the autophagosome levels were significantly increased by CSC and the positive control sorafenib with respect to the control, while reduced by the presence of \(\beta\)-caryophyllene with respect to CSC; \(\beta\)-caryophyllene induced a slight but not significant increase in autophagomes (Figure 12A). Quantification analysis showed that CSC induced a 50% increase in autophagy, which was reduced by at least 25% by \(\beta\)-caryophyllene. The effect of the co-treatment of \(\beta\)-caryophyllene and CSC was similar to that of the positive control sorafenib (Figure 12B). According to these results, we also evaluated at immunofluorescence analysis the expression of the autophagic factors Beclin 1 (or BECN1) and LC3 II, which play a crucial role in autophagy regulation and autophagosome formation [36]. The presence of both factors in the cells was highlighted by a red and green fluorescence, respectively. Under our experimental conditions, we found that CSC markedly increased the expression of both Beclin 1 and LC3, which are two factors involved in autophagome formation. Conversely, their levels were lowered by the co-treatment with \(\beta\)-caryophyllene (Figure 13A,B; Table 4). Altogether, these results suggest that inhibiting autophagy induced by CSC can represent a chemoprevention mechanism by which \(\beta\)-caryophyllene counteracts the chemoresistance induced by this toxicant to promote cell survival and progression. Moreover, it does not interfere with apoptosis but the process could be speeded up by \(\beta\)-caryophyllene, probably as a consequence of a cell cycle arrest and checkpoint activation. Figure 11: Apoptosis rate induced by \(\beta\)-caryophyllene, cigarette smoke condensate (CSC) and the positive control doxorubicin in MDA-MB-468 cancer cells after 24 h exposure. **(A)** Representative images of cells stained by Annexin V and Hoechst 33,258 dye. **(B)** 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). \({}^{\circ\circ}\)\(p<0.01\), significant difference with respect to CSC (Student’s \(t\)-test). Scale bars = 20 μm. Figure 12: Autophagic cell death induced by \(\beta\)-caryophyllene, cigarette smoke condensate (CSC) and the positive control sorafenib in MDA-MB-468 cancer cells after 24 h exposure. (**A**) Representative images of cells stained by autophagosome detection reagent. (**B**) Bar graphs of fluorescence intensity as determined by quantitative analysis using autophagosome detection reagent. 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). \(\circ\circ\circ\)\(p<0.001\), significant difference with respect to CSC (Student’s \(t\)-test). Scale bars = 10 \(\upmu\)m.  
Figure 13: Immunofluorescentence analysis of Beclin-1 and LC3 II expression induced by \(\beta\)-caryophyllene and cigarette smoke condensate (CSC) in MDA-MB-468 cancer cells after 24 h exposure. (**A**) Representative images of cells stained by a primary anti-Beclin 1 antibody, nucleic acid DAPI dye and secondary red fluorescent antibody. (**B**) Representative images of cells stained by a primary anti-LC3 II antibody, nucleic acid DAPI dye and secondary green fluorescent antibody. Data are displayed as mean \(\pm\) SE of at least three independent experiments with at least three technical replicates (\(n=9\)). Scale bars = 20 \(\upmu\)m.