Parthenolide induces superoxide anion production by stimulating EGF receptor in MDA-MB-231 breast cancer cells.

The sesquiterpene lactone parthenolide (PN) has recently attracted considerable attention because of its anti-microbial, anti-inflammatory and anticancer effects. However, the mechanism of its cytotoxic action on tumor cells remains scarcely defined. We recently provided evidence that the effect exerted by PN in MDA-MB-231 breast cancer cells was mediated by the production of reactive oxygen species (ROS). The present study shows that PN promoted the phosphorylation of EGF receptor (phospho-EGFR) at Tyr1173, an event which was observed already at 1  h of incubation with 25  µM PN and reached a peak at 8-16  h. This effect seemed to be a consequence of ROS production, because N-acetylcysteine (NAC), a powerful ROS scavenger, prevented the increment of phospho-EGFR levels. In addition fluorescence analyses performed using dihydroethidium demonstrated that PN stimulated the production of superoxide anion already at 2-3  h of incubation and the effect further increased prolonging the time of treatment, reaching a peak at 8-16  h. Superoxide anion production was markedly hampered by apocynin, a well known NADPH oxidase (NOX) inhibitor, suggesting that the effect was dependent on NOX activity. The finding that AG1478, an EGFR kinase inhibitor, substantially blocked both EGFR phosphorylation and superoxide anion production strongly suggested that phosphorylation of EGFR can be responsible for the activation of NOX with the consequent production of superoxide anion. Therefore, EGFR phosphorylation can exert a key role in the production of superoxide anion and ROS induced by PN in MDA-MB-231 cells

The oxygen radicals involved in the toxicity induced by parthenolide in MDA-MB-231 cells

It has been shown that the sesquiterpene lactone parthenolide lowers the viability of MDA-MB-231 breast cancer cells, in correlation with oxidative stress. The present report examined the different radical species produced during parthenolide treatment and their possible role in the toxicity caused by the drug. Time course experiments showed that in the first phase of treatment (0-8 h), and in particular in the first 3 h, parthenolide induced dichlorofluorescein (DCF) signal in a large percentage of cells, while dihydroethidium (DHE) signal was not stimulated. Since the effect on DCF signal was suppressed by apocynin and diphenyleneiodonium (DPI), two inhibitors of NADPH oxidase (NOX), we suggest that parthenolide rapidly stimulated NOX activity with production of superoxide anion (O2•-), which was converted by superoxide dismutase 1 (SOD1) into hydrogen peroxide (H2O2). In the second phase of treatment (8-16 h), parthenolide increased the number of positive cells to DHE signal. Since this event was not prevented by apocynin and DPI and was associated with positivity of cells to MitoSox Red, a fluorochrome used to detect mitochondrial production of O2•-, we suggest that parthenolide induced production of O2•- at the mitochondrial level independently by NOX activity in the second phase of treatment. Finally, in this phase, most cells became positive to hydroxyphenyl fluorescein (HPF) signal, a fluorescent probe to detect highly reactive oxygen species (hROS), such as hydroxyl radical and peroxynitrite. Therefore, parthenolide between 8-16 h of treatment induced generation of O2•- and hROS, in close correlation with a marked reduction in cell viability

Parthenolide prevents resistance of MDA-MB231 cells to doxorubicin and mitoxantrone: the role of Nrf2

Triple-negative breast cancer is a group of aggressive cancers with poor prognosis owing to chemoresistance, recurrence and metastasis. New strategies are required that could reduce chemoresistance and increases the effectiveness of chemotherapy. The results presented in this paper, showing that parthenolide (PN) prevents drug resistance in MDA-MB231 cells, represent a contribution to one of these possible strategies. MDA-MB231 cells, the most studied line of TNBC cells, were submitted to selection treatment with mitoxantrone (Mitox) and doxorubicin (DOX). The presence of resistant cells was confirmed through the measurement of the resistance index. Cells submitted to this treatment exhibited a remarkable increment of NF-E2-related factor 2 (Nrf2) level, which was accompanied by upregulation of catalase, MnSOD, HSP70, Bcl-2 and P-glycoprotein. Moreover, as a consequence of overexpression of Nrf2 and correlated proteins, drug-treated cells exhibited a much lower ability than parental cells to generate ROS in response to a suitable stimulation. The addition of PN (2.0 μM) to Mitox and DOX, over the total selection time, prevented both the induction of resistance and the overexpression of Nrf2 and correlated proteins, whereas the cells showed a good ability to generate ROS in response to adequate stimulation. To demonstrate that Nrf2 exerted a crucial role in the induction of resistance, the cells were transiently transfected with a specific small interfering RNA for Nrf2. Similarly to the effects induced by PN, downregulation of Nrf2 was accompanied by reductions in the levels of catalase, MnSOD, HSP70 and Bcl-2, prevention of chemoresistance and increased ability to generate ROS under stimulation. In conclusion, our results show that PN inhibited the development of the resistance toward Mitox and DOX, and suggest that these effects were correlated with the prevention of the overexpression of Nrf2 and its target proteins, which occurred in the cells submitted to drug treatment

Synergistic effect of the HDAC inhibitor SAHA and the sesquiterpene lactone parthenolide in triple negative breast cancer cells.

Triple-negative breast cancer (TNBC) is a subtype o f breast cancer, insensitive to endocrine therapy. Chemotherapy is the main form of treatment, but is accompanied by a high rate of recidivism. The sesquiterpene lactone Parthenolide (PN) exerts a cy totoxic effect on MDA-MB231 cells, a TNBC cell line (1), but was ineffective at low doses (2-5μM). This repr esents an obstacle for a therapeutic utilization of PN. We supposed, in line with other authors (2), that PN c auses a protective response, which at low doses pre vails on the cytotoxic effect. With the aim of inhibiting this protective effect we have shown that pre-trea tment of MDA-MB231 cells with SAHA (2-5μM), an histone deace tylates inhibitor, synergistically sensitizes the c ells to the cytotoxic effect of PN, also at low doses of th is compound. SAHA/PN combination induced hyperacetylation of his tones H3 and H4 and hypomethylation of DNA. These changes cause epigenetic effects, which can be resp onsible for the increased expression of tumour suppressors p21 and p27 and decreased levels of Bcl 2 and p65, a component of NFkB. Moreover SAHA alone induced ROS generation as well as autophagy, which favours cell survival, and apoptosis. The addition of PN (8 μ M) to SAHA reduced production of ROS and autophagy, while increased the apoptotic process. Interestingly PN activates Akt, mTOR, phospho-p70S6 kinase and ULK1/2, a factor that inhibits autophagy . In addition PN caused nuclear accumulation of Nrf2 wit h stimulates antioxidant genes. SAHA prevented thes e effects. In conclusion SAHA/PN stimulated cytotoxicity throu gh many mechanisms: (i) induces epigenetic events w ith changes in gene expression, (ii) PN prevents SAHA e ffect on autophagy and (iii) SAHA suppresses the protective response exerted by PN through inactivat ion of m-TOR. Taken together our results suggest th at combination SAHA/PN can be a candidate for TNBC the rapy

Evaluation of the in vitro and in vivo antineoplastic effects of Parthenolide on MDA-MB231 breast cancer cells

Triple-negative breast cancer refers to an aggressive subtype of breast cancer in which the tumor cells lack receptors for estrogen, progesterone and the HER2 protein on their surfaces. This type of breast cancer does not respond to treatments such as hormone therapy, like tamoxifen and aromatase inhibitors, or drugs that target HER2, like Herceptin. It is important, therefore, the identification of new selective drugs for the treatment of these tumors. Parthenolide (PN), a sesquiterpene lactone extracted from the medical plant Tanacetum parthenium, exerts anticancer activity on several tumor cell lines in culture, acting through diverse molecular mechanisms. Our previous studies have shown that the PN exerts strong cytotoxic effects on MG63 osteosarcoma and SK-Mel28 melanoma cells, through a caspase-independent mechanism which is associated with production of oxidative stress. Recently, we have undertaken a study in order to investigate the antineoplastic activity of PN on MDA-MB231 cells, a triple-negative breast cancer cell line. Our results demonstrated that this compound reduced the viability of MDA-MB231 cells in a dose- and time- dependent manner. This effect was not prevented by the addition of z-VAD-fmk, a general inhibitor of caspase, thus suggesting a caspase-independent cell death. Time-course experiments provided evidence that the cytotoxic effect of PN occurs in two different phases. In the first phase of treatment (8h) cells resulted positive to monodansylcadaverine (MDC), a fluorochrome that binds to autophagic vacuoles. Prolonging the treatment (16h) MDC-positive cells lowered, and an increase of PI-positive population was found, suggesting the appearance of necrotic events. The study of the mode of PN action provided evidence that treatment with parthenolide induces ROS generation, activation of JNK and inhibition of NF-kB activity. All these effects were prevented by the addition of NAC, thus suggesting the involvement of oxidative stress. The antineoplastic activity of PN has been also assayed in vivo employing diamminoparthenolide (DMAPT), a soluble analogue of PN. Nude mice bearing breast carcinoma MDA-MB231 xenografts were treated daily with DMAPT (50 mg/Kg). It was observed that DMAPT increased survival of MDA-MB231 xenograft-bearing nude mice as well as reduced MDA-MB231 xenografts tumor growth. Moreover, immunohistochemical studies showed that DMAPT was able to decrease the expression of MMP-2, MMP-9 and VEGF, all factors involved in metastatic events. These data suggest a possible use of parthenolide for the treatment of triple negative breast cancers

Parthenolide prevents resistance of MDA-MB231 cells to doxorubicin and mitoxantrone : the role of Nrf2

Triple-negative breast cancer is a group of aggressive cancers with poor prognosis owing to chemoresistance, recurrence and metastasis. New strategies are required that could reduce chemoresistance and increases the effectiveness of chemotherapy. The results presented in this paper, showing that parthenolide (PN) prevents drug resistance in MDA-MB231 cells, represent a contribution to one of these possible strategies. MDA-MB231 cells, the most studied line of TNBC cells, were submitted to selection treatment with mitoxantrone (Mitox) and doxorubicin (DOX). The presence of resistant cells was confirmed through the measurement of the resistance index. Cells submitted to this treatment exhibited a remarkable increment of NF-E2-related factor 2 (Nrf2) level, which was accompanied by upregulation of catalase, MnSOD, HSP70, Bcl-2 and P-glycoprotein. Moreover, as a consequence of overexpression of Nrf2 and correlated proteins, drug-treated cells exhibited a much lower ability than parental cells to generate ROS in response to a suitable stimulation. The addition of PN (2.0 μM) to Mitox and DOX, over the total selection time, prevented both the induction of resistance and the overexpression of Nrf2 and correlated proteins, whereas the cells showed a good ability to generate ROS in response to adequate stimulation. To demonstrate that Nrf2 exerted a crucial role in the induction of resistance, the cells were transiently transfected with a specific small interfering RNA for Nrf2. Similarly to the effects induced by PN, downregulation of Nrf2 was accompanied by reductions in the levels of catalase, MnSOD, HSP70 and Bcl-2, prevention of chemoresistance and increased ability to generate ROS under stimulation. In conclusion, our results show that PN inhibited the development of the resistance toward Mitox and DOX, and suggest that these effects were correlated with the prevention of the overexpression of Nrf2 and its target proteins, which occurred in the cells submitted to drug treatment.peer-reviewe

A high-temporal resolution residential building occupancy model to generate high-temporal resolution heating load profiles of occupancy-integrated archetypes

A strong correlation exists between occupant behaviour and space heating energy use. In particular, the occupancy status (e.g., daytime absence) is known to have a significant influence on residential heating load profiles, as well as on cumulative heating energy consumption. In the literature, many occupancy models have been utilised to predict occupancy profiles of individual dwellings as part of the larger residential building stock. However, none of the existing models consider diversity associated with occupancy-integrated archetypes to generate high-temporal resolution heating load profiles. The current paper uses Time Use Survey (TUS) data to develop a high-temporal resolution residential building occupancy model. The key feature of the proposed model, implemented using MATLAB, is the ability to generate stochastic occupancy time-series data for national population subgroups characterised by specific occupancy profiles. It is shown that the results are capable of closely approximating data available from TUS. The developed model can be applied to improve the quality of modelled high-temporal resolution heating load profiles for generic building stock characterised by population subgroups represented by different occupancy-integrated archetypes. A case study is performed on a building stock sample located in London, UK. The developed occupancy model has been implemented in MATLAB and is available for download.European Commission Horizon 2020University College Dubli

Clustering of household occupancy profiles for archetype building models

8th International Conference on Sustainability in Energy and Buildings (SEB-16), Turin, Italy, 11-13 September 2016The continued penetration of renewable energy sources in electricity generation and the de-carbonization of the domestic space heating and hot water sectors is increasing the importance of demand side management (DSM). The development of end-use energy consumption models that can be easily integrated with electricity dispatch models is crucial for the assessment of the integration of supply and demand. The energy consumption of the domestic building stock is highly correlated with occupant behaviour, however the inclusion of occupant behaviour in energy models is challenging due to its highly variable nature. Nevertheless, in order to obtain reliable models of domestic energy consumption at high time resolution, the analysis of occupant behaviour patterns is fundamental. This paper aims to develop a new methodology to generate realistic occupancy patterns that can be representative of large numbers of households. This method is based on the clustering of household occupancy profiles using the UK 2000 Time Use Survey data as a case study. The occupancy profiles that result from this method can be used as input to residential building energy end-use models, thereby giving improved overall model performance.European Commission Horizon 202

Evaluation of advanced control strategies of electric thermal storage systems in residential building stock

This paper investigates the effect of different control strategies applied to electric thermal storage systems to provide demand response services. These results indicate how policymakers or manufacturers could target the implementation of advanced control on electric thermal storage systems and apply these to households characterised by different occupancy profiles, thereby making demand response initiatives more attractive to end users