Abstract
Triple-negative breast cancers (TNBCs) are aggressive forms of breast carcinoma associated with a high rate of recidivism. In this paper, we report the production of mammospheres from three lines of TNBC cells and demonstrate that both parthenolide (PN) and its soluble analog dimethylaminoparthenolide (DMAPT) suppressed this production and induced cytotoxic effects in breast cancer stem-like cells, derived from dissociation of mammospheres. In particular, the drugs exerted a remarkable inhibitory effect on viability of stem-like cells. Such an effect was suppressed by N-acetylcysteine, suggesting a role of reactive oxygen species (ROS) generation in the cytotoxic effect. Instead z-VAD, a general inhibitor of caspase activity, was ineffective. Analysis of ROS generation, performed using fluorescent probes, showed that both the drugs stimulated in the first hours of treatment a very high production of hydrogen peroxide. This event was, at least in part, a consequence of activation of NADPH oxidases (NOXs), as it was reduced by apocynin and diphenylene iodinium, two inhibitors of NOXs. Moreover, both the drugs caused downregulation of Nrf2 (nuclear factor erythroid 2-related factor 2), which is a critical regulator of the intracellular antioxidant response. Prolonging the treatment with PN or DMAPT we observed between 12 and 24 h that the levels of both superoxide anion and hROS increased in concomitance with the downregulation of manganese superoxide dismutase and catalase. In addition, during this phase dissipation of mitochondrial membrane potential occurred together with necrosis of stem-like cells. Finally, our results suggested that the effect on ROS generation found in the first hours of treatment was, in part, responsible for the cytotoxic events observed in the successive phase. In conclusion, PN and DMAPT markedly inhibited viability of stem-like cells derived from three lines of TNBCs by inducing ROS generation, mitochondrial dysfunction and cell necrosis.
Highlights
Structure of PN exhibits the typical α-methylene-γ-lactone group, which reacts with cysteine thiol groups inducing modifications of many biological functions
PN at 5 μM was ineffective on viability of normal human mammary epithelial cells (HMECs), while at 20 μM exerted only a modest effect (−15%)
When single cells of the three lines of Triple-negative breast cancers (TNBCs) were grown for 10 days in non-adherent conditions, a small number of survival cells generated floating mammospheres, as reported by other authors.[23,24,25]
Summary
Structure of PN exhibits the typical α-methylene-γ-lactone group, which reacts with cysteine thiol groups inducing modifications of many biological functions. In prostate cancer cells,[11] ROS generation is accompanied by downregulation of antioxidant enzymes, such as manganese superoxide dismutase (MnSOD) and catalase.[12]. We showed that PN and dimethylaminoparthenolide (DMAPT), a soluble analog of PN, induced cytotoxic effects in MDA-MB231 cells,[14] the most studied line of TNBCs, by stimulating ROS generation and autophagy. DMAPT decreased tumor growth in mice bearing xenografts of MDA-MB231 cells and induced animal survival.[14] suberoylanilide hydroxamic acid, an histone deacetylase inhibitor, synergistically sensitized MDA-MB231 cells to PN effect.[15]. Many drugs, which inhibit replication of the bulk of cancer cells, are not effective for eradication of CSCs in many types of tumors.[20,21,22] new researches are needed to individuate molecules able to eliminate CSCs
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