## 4 Discussion
It is known that low GSH levels activate autophagy as an adaptive stress response [80], which has been reported to be a key feature of MDA-MB-468 cells [81]. In this respect, we found that CSC raised both autophagy levels, as revealed by the levels of autophagosomes, and apoptotic cell death with respect to the untreated cells. The increased autophagy triggering was also confirmed by the upregulation of the autophagic factors Beclin 1 and LC3 II. In the presence of \(\beta\)-caryophyllene, CSC-induced autophagy was partly lowered, while apoptotic cell death was retained; accordingly, the expression of Beclin 1 and LC3 II was reduced by the sesquiterpene with respect to CSC. Autophagy is a catabolic process, activated by cells for recycling cytoplasmic constituents in autolysosomes under stress conditions, such as nutrient deficiency, oxidative stress, hypoxia and drug treatment; it is usually considered as a cell-protective mechanism; however, several pieces of evidence highlighted that excessive autophagy can also lead to cell death, thus suggesting that it represents a programmed and regulated process of self-elimination [82]. The process starts when the damaged intracellular components are encompassed by a double-membrane vesicle that leads to the autophagosome formation, which in turn is fused with lysosome to form an autolysosome; this last is responsible for intracellular material degradation and recycling products, which will be exploited by cells for metabolic purposes [83]. The entirety of the process includes five phases (i.e., initiation, elongation, maturation, fusion and degradation) which are regulated by key autophagy-related factors [83]. Among them, Beclin 1 is essential in the initiation phase, since it leads to the nucleation of autophagic vesicles, while LC3 (microtubule-associated protein 1 light chain 3) is involved in autophagosome elongation and maturation [83]. The last factor is available in a cytosolic LC3-I form which is converted in a LC3-phosphatidylethanolamine conjugate (LC3-II) recruited during autophagosome formation [83]. In cancer cells, autophagy has been reported to play a dual role, initially acting as a tumor suppressor process, and as promoting tumor progression during the advanced stages; an intricate link between autophagy and oxidative stress has also been reported [84]. Moreover, autophagy is strictly interconnected with apoptosis and different cross-talk between them have been highlighted depending on the type of cells [85]. Indeed, they can act as partners, leading to cooperative or synergistic effects, or with opposite trends, when autophagy blocks apoptotic cell death, thus promoting cell survival; at last, apoptosis may induce cell death both directly and through autophagy suppression [85]. Our results highlight that both autophagic and apoptotic cell death can contribute to the cytotoxic effects of CSC in the MDA-MB-468 cells. This could suggest a cooperating role of these processes to induce cell death, however, the results obtained with the co-treatment of \(\beta\)-caryophyllene and CSC highlighted that cytotoxicity increased when autophagy was partly lowered by the sesquiterpene, and the apoptotic rate was retained. In this context, our hypothesis is that apoptosis and autophagy are activated as opposite processes, leading apoptosis to cell death and autophagy to a buffering survival response. In support, autophagy-regulating mechanisms other than apoptosis, such as GSH depletion and genome instability, could be involved. In the presence of \(\beta\)-caryophyllene, the autophagic rise induced by CSC in the MDA-MB-468 cells was partly hindered although apoptosis was retained, leading to the activation of other undefined cell death processes. According to our results, an upregulation of the autophagic cascade was postulated to be a mechanism by which CSC induces mitochondrial dysfunction in granulosa cells [86].