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The sesquiterpene lactone Parthenolide (PN) exerted a cytotoxic effect on MDA-MB231 cells, a triple-negative breast cancer (TNBC) cell line, but its effectiveness was scarce when employed at low doses. This represents an obstacle for a therapeutic utilization of PN. In order to overcome this difficulty we associated to PN the suberoylanilide hydroxamic acid (SAHA), an histone deacetylase inhibitor. Our results show that SAHA synergistically sensitized MDA-MB231 cells to the cytotoxic effect of PN. It is noteworthy that treatment with PN alone stimulated the survival pathway Akt/mTOR and the consequent nuclear translocation of Nrf2, while treatment with SAHA alone induced autophagic activity. However, when the cells were treated with SAHA/PN combination, SAHA suppressed PN effect on Akt/mTOR/Nrf2 pathway, while PN reduced the prosurvival autophagic activity of SAHA. In addition SAHA/PN combination induced GSH depletion, fall in m, release of cytochrome c, activation of caspase 3 and apoptosis. Finally we demonstrated that combined treatment maintained both hyperacetylation of histones H3 and H4 induced by SAHA and down-regulation of DNMT1 expression induced by PN. Inhibition of the DNA-binding activity of NF-kB, which is determined by PN, was also observed after combined treatment. In conclusion, combination of PN to SAHA inhibits the cytoprotective responses induced by the single compounds, but does not alter the mechanisms leading to the cytotoxic effects. Taken together our results suggest that this combination could be a candidate for TNBC therapy. J. Cell. Physiol. 230: 1276-1289, 2015. (c) 2014 Wiley Periodicals, Inc., A Wiley Company

Il carcinoma mammario triplo negativo è una forma di carcinoma mammario particolarmente aggressiva, a prognosi spesso infausta. Tale forma tumorale è resistente alle terapie tradizionalmente impiegate per il trattamento del carcinoma mammario, pertanto risulta strettamente necessario individuare una valida strategia terapeutica. In questa tesi viene valutato l’effetto del Partenolide (PN), un composto di origine naturale, sulle cellule di carcinoma mammario umano triplo negativo MDA-MB231. I risultati ottenuti evidenziano che il PN induce morte delle cellule MDA-MB231 mediante un meccanismo caspasi-indipendente, infatti l’inibitore generale delle caspasi z-VAD non è in grado di prevenire gli effetti citotossici del composto. Durante le prime ore di trattamento (1-3 h) il PN induce l’attivazione dell’enzima NADPH ossidasi con conseguente produzione di specie reattive dell’ossigeno (ROS). I ROS mediano diversi effetti indotti dal PN quali la deplezione dei gruppi tiolici e del GSH, l’attivazione di JNK e l’inibizione del fattore di trascrizione NF-kB. Durante questa fase il PN determina anche la comparsa di segni di autofagia, evidenziati dalla positività alla monodansilcadaverina e dall’incremento di noti marker autofagici quali LC3II e beclina-1. Prolungando il trattamento (16 h) lo stress ossidativo indotto dal PN determina una caduta del potenziale di membrana mitocondriale e la comparsa di segni di necrosi, evidenziata da un’elevata percentuale di cellule positive allo ioduro di propidio. In conclusione i dati ottenuti suggeriscono che il PN possa essere un ottimo candidato per il trattamento del carcinoma mammario triplo negativo.

We showed that the sesquiterpene lactone Parthenolide (PN) exerts strong cytotoxic effects on triple negative breast cancer MDA-MB231 cells. Our recent results suggest that PN exerts in these cells a cytoprotective effect, which is due to the activation of mTOR pathway. To inhibit this protective response we employ the HDAC inhibitor SAHA, which is known to prevent AKT/mTOR pathway. We show that PN activates Akt, mTOR, p70S6kinase and NRF2 while SAHA abolishes these effects. Further cell pretreatment with SAHA synergistically sensitizes the cells to the cytotoxic effect of PN. Moreover SAHA alone activates the autophagic process. The addition of PN to SAHA reduces this effect and induces apoptosis. SAHA/PN combination also inhibits DNMT1 and produces hyperacetylation of histones. Epigenetic changes caused by these effects are responsible for the increased expression of oncosuppressor gene products, such as p21 and p27 and for the decrease of Bcl2 and p65 levels. In conclusion SAHA suppresses protective response exerted by PN; PN inhibits SAHA effect on autophagy and finally SAHA/PN combination induces epigenetic modifications with changes in gene expression.

Parthenolide, a sesquiterpene lactone found in European feverfew, is used in traditional medicine for its anti-inflammatory activity. In addition, parthenolide has been considered as a novel and effective anti-tumor agent because it induces cytotoxic effects in several tumor cell lines. Our studies demonstrated that parthenolide exerted strong cytotoxic effects in osteosarcoma MG63 and melanoma SK-Mel28 cells in culture. Staining with Hoechst 33342 revealed in most cells after brief periods of treatments (3-5h) chromatin condensation and fragmentation, while only few cells were PI-positive. Prolonging the treatment (5-14h) PI-positive cells strongly augmented, denouncing the increase of necrotic effects. All these effects were prevented by NAC, while caspase inhibitors were ineffective, thus suggesting a caspase-independent cell death. The study of the mechanism of action provided evidence that treatment with parthenolide rapidly stimulated (1-2 h) ROS generation, in particular by inducing activation of extracellular signal-regulated kinase1/2 and NADPH oxidase. This event caused depletion of thiol groups and glutathione, NF-κB inhibition, JNK activation and cell detachment from the matrix. ROS generation together with mitochondrial accumulation of Ca2+ favoured dissipation of Δψm, which appeared primarily determined by the opening of the permeability transition pore (PTP), since Δψm loss was partially prevented by cyclosporin A, an inhibitor of PTP opening. Recently, we focused our attention on MDA-MB231 cells, a very aggressive and poorly differentiated breast cancer cell line, which is negative for estrogen receptor alpha. Preliminary results suggested that parthenolide induced cell death in these cells with a mechanism similar to that demonstrated in osteosarcoma and melanoma cells. Interestingly, we demonstrated that in MDA-MB231 cells the effect of parthenolide was potentiated by the addition of z-VAD-fmk, a general inhibitor of caspases. Studies are in progress to elucidate the mechanism of this interaction which could suggest new strategies for the treatment of ER-α negative breast cancer.

Parthenolide, the major bioactive sesquiterpene lactone present in Feverfew (Tanacetum parthenium), has recently attracted considerable attention because of its complex pharmacological action involving anti-microbial, anti-inflammatory and anti-cancer effects. However, the mechanism of its cytotoxic effect on tumor cells still remains scarcely defined today. The aim of this study was to analyse the mechanism of parthenolide action on two lines of cancer cells, the human osteosarcoma MG63 and the melanoma SK-MEL-28 cells, on which parthenolide exerted its action inducing similar effects. Staining with Hoechst 33342 showed that parthenolide induced in the first phase of treatment (0-5 h) in most of cells of both the lines condensation of chromatin while only few cells were PI-positive. Moreover, cells assumed a rounded shape, detached from substrate and showed a reduction of their volume. In the second phase of treatment (5-15 h) a progressive increase in the percentages of PI-positive cells was observed, suggesting that extensive damage of cellular plasma membranes occurred only after long periods of treatment. All these events were not counteracted by z-VAD-fmk and other caspase inhibitors, but were dependent on oxidative stress. In fact the study of mechanism of action of partenolide revealed that all cytotoxic effects were prevented by NAC and after a short period of time (1-2 h) ROS production occurred by inducing activation of extracellular signal-regulated kinase1/2 (ERK1/2) and NADPH oxidase. This event caused depletion of thiol groups and glutathione, NF-kB inhibition, JNK activation, cell detachment from the matrix and cellular shrinkage. The increase of ROS generation together with the mitochondrial accumulation of Ca2+ also favoured dissipation of ∆ψm, which seemed primarily determined by PTP opening, since ∆ψm loss was partially prevented by the inhibitor cyclosporin A. In addition, immunofluorescence analysis revealed that at this stage AIF translocated from mitochondria to the nucleus and co-localized with areas of condensed chromatin. Prolonging the treatment (5-15h) ATP content declined while PI-positive cells strongly augmented, denouncing the increase of necrotic effects. All these effects were prevented by NAC, while caspase inhibitors were ineffective. We suggest that AIF exerts a crucial role in parthenolide action. In accordance, down-regulation of AIF markedly inhibited parthenolide effect on the production of cells with apoptotic or necrotic signs. Taken together our results demonstrate that parthenolide causes in the two cell lines a caspase-independent cell death, which is mediated by AIF.