## 3 Results
### Volatile Chemical Profile of Cigarette Smoke Condensate (CSC)
Conversely, when CSC was assessed in the presence of \(\beta\)-caryophyllene, the G1 phase was markedly reduced (about a three-fold lowering) with respect to CSC, the G2/M phase almost quadrupled with a slight increase in the S phase. Moreover, comparing cell cycle distribution in the co-treatment of \(\beta\)-caryophyllene and CSC with respect to the control, the G1 phase results at least halved, while the G2/M phase almost doubled, with a slight lowering of the S phase. These findings suggest that \(\beta\)-caryophyllene induces a G2/M phase arrest in MDA-MB-468 cells which can lower their progression and also activate checkpoints, thus promoting cell death: this hypothesis agrees with the previous evidence in cholangiocarcinoma cells [32]. Figure 9: Levels of \(\gamma\)H2 AX induced by cigarette smoke condensate (CSC) and \(\beta\)-caryophyllene in MDA-MB-468 cancer cells after 24 h exposure. (**A**) Representative images of cells stained by Alexa Fluor® 647 anti-gamma H2A.X (phospho S139) antibody and Hoechst 33,258 dye. (**B**) Data are displayed as mean \(\pm\) SE of at least three independent experiments with at least two technical replicates (\(n=6\)). *** \(p<0.001\), significant lowering of cell viability with respect to the control (ANOVA + [PERSON]’s multiple comparison post-hoc test). Scale bars = 20 \(\mu\)m.  
Effects of \(\beta\)-Caryophyllene and Cigarette Smoke Condensate (CSC) on Apoptosis and Autophagic Cell Death  
In order to clarify the cell death mechanisms involved in the increased cytotoxicity induced by the co-treatment of \(\beta\)-caryophyllene and CSC, the apoptosis rate was determined by both immunofluorescence and flow cytometric analysis (Figure 11). In particular, imaging was executed by staining cells with a Annexin-V-FITC probe and apoptosis was revealed as a green fluorescence around the cells. For the flow cytometric analysis, we exploited a previously published method [32], which allows for the detection of both apoptotic and viable cells, using Annexin-V-Cy3 and carboxyfluorescein diacetate (CFDA). The live and apoptotic cells were selected by their typical cell size and granularity, measured through the forward light scatter (FSC) and side scatter (SSC). Under our experimental condition, immunofluorescence analysis highlighted the presence of apoptosis (green fluorescence around the cells) in the cells treated with both CSC and CSC plus \(\beta\)-caryophyllene, without signs in the other treatments (Figure 11A). Flow cytometric analysis confirmed the imaging results and highlighted an increased apoptosis rate by about 50% with CSC and by about 40% with the co-treatment of CSC and \(\beta\)-caryophyllene; moreover, a slight 10% lowering of viable cells was induced by both \(\beta\)-caryophyllene and CSC, achieving 25% in combination (Figure 11B). As expected, the positive control doxorubicin lowered the viable cell rate by about 30% and increased apoptosis by at least 40% (Figure 11B). An early apoptosis activation was also found after 3 h exposure at imaging analysis; in these conditions, the effect of the co-treatment of \(\beta\)-caryophyllene and CSC appeared slightly  
Figure 10: Modulation of cell cycle phases induced by \(\beta\)-caryophyllene and cigarette smoke condensate (CSC) in MDA-MB-468 cancer cells after 24 h exposure. (**A**) Histograms showing the percentages of cells stained by Hoechst 33,258 in different cell cycle phases under the scheduled treatments. (**B**) Data are displayed as mean \(\pm\) SE of at least three independent experiments with at least three technical replicates (\(n=9\)). \({}^{n}p<0.05\) and \({}^{n}p<0.001\), significant lowering of cell viability with respect to the control (ANOVA + [PERSON]’s multiple comparison post-hoc test). \({}^{o}00p<0.001\), significant difference with respect to CSC (Student’s \(t\)-test). higher than that induced by CSC (Figure S3), suggesting that apoptosis could be quickly induced, as a consequence of the marked oxidative and genotoxic damage and of the activation of cell cycle checkpoints.