## 4 Discussion
Interestingly, \(\beta\)-caryophyllene did not hinder the DNA damage by CSC, probably due to the failure of the DNA repair systems in the MDA-MB-468 cells, but markedly increased the G2/M cell cycle phase with respect to CSC. This evidence suggests that the cell cycle arrest induced by the sesquiterpene in the MDA-MB-468 cells can activate the checkpoints, which recognize the unsolved DNA damage induced by CSC and activate cell death, as was also found in other cancer cell models [77]. The increased cytotoxicity induced by \(\beta\)-caryophyllene in the CSC-damaged MDA-MB-468 cells can be also a consequence of the enhanced oxidative stress induced by the sesquiterpene. Oxidative stress plays an important role in cancer development and progression; indeed, despite normal cells, which usually grow under homeostatic conditions, characterized by low intracellular ROS levels, cancer cells are able to survive under altered redox balance and tolerate increased ROS levels, which also activate oncogenic signaling pathways and promote cancer cell proliferation, metastasis and chemoresistance [77]. This altered redox balance is impaired in the presence of excessive ROS levels, which overwhelmed the antioxidant systems and activated cell death [77]. Therefore, tolerable high ROS levels act as tumor-promoting factors, stimulating antioxidant cell defenses, while too high levels are tumor suppressive, since they disrupt the redox equilibrium and activate cell death. In line with this evidence, adding \(\beta\)-caryophyllene to CSC seems to trigger a tumor-suppressive oxidative stress, which is reflected in increased mitochondrial toxicity and apoptosis. The HPLC analysis also highlighted that oxidative stress by CSC was reflected in glutathione depletion. It is noteworthy that, despite a marked lowering of the reduced GSH form, the oxidized GSSG one (namely glutathione disulfide) was only slightly affected by the treatment, thus suggesting that glutathione is depleted not only for the neutralization of ROS induced by CSC, but also for other purposes. Indeed, it is known that GSH is usually exploited by cells to scavenge electrophilic and oxidant species both directly, through quenching reactive hydroxyl free radicals, and indirectly, as a co-substrate of glutathione peroxidase: this leads to the formation of the oxidized GSSG form, which in turn can be reduced to two GSH molecules [78]. Moreover, it takes part in metabolizing processes, as a substrate for conjugation metabolic reactions of electrophilic endogenous compounds and xenobiotics, mediated by glutathione-S-transferases [78]. Therefore, we hypothesize that GSH depletion induced by CSC in the MDA-MB-468 cells can be due mainly to the smoke-carcinogen conjugation, which could be activated by cells as a primary defense line to counteract injury by toxicants; however, the true compound undergoing GSH conjugation remains to be clarified. For instance, \(\alpha,\beta\)-unsaturated aldehydes (e.g., acrolein and crotonaldehyde) and \(\alpha,\beta\)-unsaturated ketone (e.g., methyl vinyl ketone), which are known smoke carcinogens, are reported to form Michael-type adducts with glutathione, thus causing intracellular GSH depletion and cell deaths [79]. Usually, GSH depletion leads to different cell death types, including necrosis and necroptosis, apoptosis and autophagy, while its upregulation has been associated with an increased chemoresistance [80]: on the basis of this evidence, the altered GSH/GSSG ratio induced by CSC in the MDA-MB-468 cells seems to represent a buffering mechanism to avoid the acute toxicity of CSC, instead of sustaining cancer resistance. Despite the CSC effects, \(\beta\)-caryophyllene alone did not affect GSH/GSSG, while it restored the basal levels lowered by CSC. In this condition, increased antioxidant capacities of MDA-MB-468 cellsshould be expected; by contrast, the combination of \(\beta\)-caryophyllene and CSC increases cell death, suggesting that the increased GSH/GSSG can also be linked to other cell processes.