## 4 Discussion  
In the present study, the chemopreverities of the natural sesquiterpene \(\beta\)-caryophyllene towards the damage induced by cigarette smoke condensate (CSC) in triple-negative breast cancer MDA-MB-468 cells were evaluated. The obtained results highlighted the ability of the sesquiterpene to interfere with events exploited by CSC to promote cell survival and chemoresistance, including genomic instability, cell cycle progress, autophagy, apoptosis, cell migration and related pathways. In particular, \(\beta\)-caryophyllene enhanced the rate of cell death, probably promoting oxidative stress, cell cycle arrest and apoptosis, and hindered cell recovery, autophagy activation and cell migration, which were probably activated by CSC as chemoresistance mechanisms. Interestingly, a marked inhibition of the CSC-induced STAT3 activation seems to represent a key mechanism of the chemoprevertion by \(\beta\)-caryophyllene. The evidence that \(\beta\)-caryophyllene is endowed with chemopreverities agrees with previous results obtained in other in vitro models against smoke, cigarette butt extracts and other environmental pollutants [26; 46; 49; 50]. Moreover, it has been highlighted that the natural sesquiterpene protected the noncancerous cholangiocarcinomaytes towards the genotoxic damage induced by doxorubicin, despite enhancing the anticancer drug toxicity in the cholangiocarcinoma cells, thus strengthening the interest in this compound as a dual-acting chemoprevertive and chemosensitizing agent [32]. Although cigarette smoke has been reported to contain several carcinogens, among which are the polycyclic aromatic hydrocarbons (e.g., benzo[a]pyrene) and N-nitrosamines [51; 52], it is not fully understood which of them, and how, can be responsible for breast cancer exacerbation and chemoresistance development. At PME-GC/MS analysis, our CSC sample contained neophytadiene and nicotine as the most abundant identified compo  
Figure 17: Effect of \(\beta\)-caryophyllene and cigarette smoke condensate (CSC) on the expression levels of phosphorylated STAT3 at tyrosine 705 residue in MDA-MB-468 cancer cells after 24 h exposure. (**A**) Densitometric bar graph analysis (data are expressed as mean \(\pm\) standard error obtained from at least two independent experiments) (**B**) Representative Western blotting membrane, displaying phospho(Tyr705) STAT3, total STAT3 and \(\beta\) -actin, used as protein-loading controls. ***\(p<0.001\), significant lowering of cell viability with respect to the control (ANOVA + [PERSON]’s multiple comparison post-hoc test). ***\(p<0.001\), significant difference with respect to CSC (Student’s \(t\)-test). nents, along with minor components, including 7-methyl-6-tridecene, \(\alpha\)-myrcene, triacetin, (Z)-2,6,10-trimethyl-1,5,9-undecatriene, 6,11-dimethyl-2,6,10-dodecatrien-1-ol and urs-12-en-28-ol. Neophytaldiene is a tobaccoiterpene which occurs naturally in tobacco and whose concentration increases upon curing and ageing [53]; it is regarded as a possible indicator of the smoke's terpene content [54]. According to our results, it has been identified in other cigarette products (i.e., solid tobacco, cigarette smoke, cigarette papers and filters) [55; 56]. However, there is a lack in the evidence available about its potential genotoxic power; conversely, anti-inflammatory properties have been highlighted in vitro [57]. Nicotine is a characteristic alkaloid of the tobacco plant and its derived products [58], which has been reported to induce genotoxicity and genome instability in different preclinical studies [59]; however, it also suppresses DNA synthesis and the proliferation of leukemic cells [60]. Although the true mechanisms involved remain to be clarified, genetic injury by nicotine seems to be partly due to oxidative stress, which in turn could be regulated by a nicotinic receptor-dependent pathway [61; 62; 63; 64]. According to our results, high nicotine levels (55.8%) were found in CSC despite a lower relative amount (8.98%) in the CSE (cigarette smoke extract) sample.