Diterpenoid natural compound C4 (Crassin) exerts cytostatic effects on triple-negative breast cancer cells via a pathway involving reactive oxygen species

Triple-negative breast cancers (TNBC) lack expression of three common cell surface receptors, i.e., estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER2). Accordingly, TNBCs are associated with fewer treatment options and a relatively poor prognosis. Having screened a National Cancer Institute natural compound library, the purpose of this study was to investigate the bioactivity of compound C4 (Crassin) in TNBC cells. Cell viability assays were performed in two TNBC cell lines, MDA-MB-231 and 4T1, following C4 treatment in the presence or absence of the antioxidant N-acetyl-L-cysteine (NAC). Phosphorylation of Akt and ERK was assessed by Western blotting. Apoptosis, necrosis, autophagy, necroptosis, ferroptosis and cytostasis assays were performed to explain viability deficits resulting from C4 exposure. We found that the viability of the TNBC cells tested decreased in a concentration- and time-dependent fashion following C4 treatment. This decrease coincided with an unexpected increase in the expression of the cell survival effectors pAkt and pERK. In addition, we found that both the decreased cell viability and the increased pAkt/pERK levels could be rescued by the antioxidant NAC, suggesting a central role for reactive oxygen species (ROS) in the mechanism of action of C4. Necrosis, apoptosis, necroptosis and ferroptosis could be ruled out as cell death mechanisms. Instead, we found that C4 induced cytostasis downstream of ROS activation. Finally, we observed a synergistic effect between C4 and the chemotherapeutic drug doxorubicin in TNBC cells. From our in vitro data we conclude that C4 exerts cytostatic effects on triple-negative breast cancer cells via a pathway involving reactive oxygen species. Its potential value in combination with cytotoxic therapies merits deeper investigation in pre-clinical models.