SAHA/TRAIL combination induces detachment and anoikis of MDA-MB231 and MCF-7 breast cancer cells

SAHA, an inhibitor of histone deacetylase activity, has been shown to sensitize tumor cells to apoptosis induced by TRAIL, a member of TNF-family. In this paper we investigated the effect of SAHA/TRAIL combination in two breast cancer cell lines, the ERa positive MCF-7 and the ERa negative MDA-MB231. Treatment of MDA-MB231 and MCF-7 cells with SAHA in combination with TRAIL caused detachment of cells followed by anoikis, a form of apoptosis which occurs after cell detachment, while treatment with SAHA or TRAIL alone did not produce these effects. The effects were more evident in MDA-MB231 cells, which were chosen for ascertaining the mechanism of SAHA/TRAIL action. Our results show that SAHA decreased the level of c-FLIP, thus favouring the interaction of TRAIL with the specific death receptors DR4 and DR5 and the consequent activation of caspase-8. These effects increased when the cells were treated with SAHA/TRAIL combination. Because z-IEDT-fmk, an inhibitor of caspase-8, prevented both the cleavage of the focal adhesion-kinase FAK and cell detachment, we suggest that activation of caspase- 8 can be responsible for both the decrement of FAK and the consequent cell detachment. In addition, treatment with SAHA/TRAIL combination caused dissipation of DJm, activation of caspase-3 and decrement of both phospho-EGFR and phospho-ERK1/2, a kinase which is involved in the phosphorylation of BimEL. Therefore, co-treatment also induced decrement of phospho-BimEL and a concomitant increase in the dephosphorylated form of BimEL, which plays an important role in the induction of anoikis. Our findings suggest the potential application of SAHA in combination with TRAIL in clinical trials for breast cancer

ER+-derived breast cancer stem cells reveal a high expression of the serpin protease inhibitor PI-9.

Introduction: Breast cancers (BC) are the major cause of death in women. More than 70% of BCs express high levels of estrogen receptor-α (ERα) and are sustained for their growth by the hormone. Estrogens seem to protect BC cells from apoptosis mediated by immunosurveillance associated with cytotoxic T lymphocytes and NK cells granzyme B release. However, the production of granzyme B inhibitor PI-9 by tumor cells causes a short-circuit in immunosurveillance’s signalling. Although it has been shown the role of PI-9 in BC cells, its presence has not been investigated in tumor stem cells so far. Methods: Cell viability was evaluated by MTT, cell cycle by propidium iodide staining; mRNA and protein levels by qPCR and western blotting. Tumorspheres from ERα+BC MCF7 cells were isolated in ultra-low attachment conditions. The higher expression of stemness markers (Nanog, Oct3/4 and Sox2) was found in tertiary tumorspheres which were used in our study. Results: Low doses (10 nM-10 μM) of 17-β estradiol consistently increased the number of MCF7 cells more than tumorspheres, while higher doses (50-100 μM) reduced cell number as a consequence of G2/M cell cycle arrest. The analysis of ERα disclosed the presence of three different isoforms (66, 46 and 36 kDa) in MCF7 cells. In contrast, tumorspheres exhibited an increase in ERα36, which lacks transcriptional activity, while the level of ERα66 was undetectable. Then, we analyzed the level of PI-9, which is transcriptionally regulated by ERα66. Surprisingly, we found that tertiary tumorspheres, express higher levels of both PI-9 protein and mRNA than MCF7 cells. Conclusions: Our data provided evidence that the high level of PI-9 in ER+ tertiary tumorspheres could supply a selective advantage to BC stem cells by interfering with immune-surveillance systems. Ongoing studies aim to elucidate the relationship between the levels of different ERα isoforms and PI-9 high expression in BC-stem cells

Induction of apoptosis in human retinoblastoma cells by topoisomerase inhibitors

PURPOSE:To examine the apoptotic effect induced in human retinoblastoma Y79 cells by camptothecin, etoposide, and amsacrine, to examine the effect of these drugs on the expression of many apoptosis-related modulators, and to test the antiapoptotic effect exerted by insulin-like growth factor-I (IGF-I). METHODS:Morphologic features of apoptosis were demonstrated using acridine orange- ethidium bromide staining and electron microscopy. DNA fragmentation was determined by means of an in situ cell detection procedure (TdT-dUTP terminal nick-end labeling [TUNEL]) or by electrophoresis on agarose gels and was quantified by enzyme-linked immunosorbent assay. The expression of apoptosis-related modulators was studied by western blot analysis. The processing of latent p53 was examined by means of pulse- chase analysis. RESULTS:Camptothecin, etoposide, and amsacrine induced apoptosis in Y79 cells in a dose-dependent manner; camptothecin was the most efficacious compound. The effect, which was dependent on macromolecular synthesis, appeared after a lag of 8 hours and increased for as long as 24 hours. It was lower in cells treated with IGF-I, a potent mitogenic factor. Camptothecin and etoposide increased the p53 level after 4 hours of treatment, before the onset of apoptosis. This effect seemed to be a consequence of the conversion of latent p53 to one that is transcriptionally active. The drugs also induced an increase in p53-related proteins, such as p21, Bax, and IGF binding protein-3 (IGF-BP3), and caused a significant reduction of the Bcl-2 level. The latter effect was less evident in cells pretreated with IGF-I. CONCLUSIONS:Topoisomerase inhibitors induce apoptosis in Y79 cells. This event is accompanied by a decrease in the expression of Bcl-2, a death antagonist, and an increase in that of Bax, a death agonist. A probable consequence of these modifications is the activation of ICE-like activity with degradation of poly-(adenosine diphosphate [ADP] ribose)-polymerase. Insulin-like growth factor-I exerts an antiapoptotic action in Y79 cells, and this function is most likely reduced by the overexpression of IGF-BP3 that is induced by drug treatment

Parthenolide induces caspase-independent and AIF-mediated cell death in human osteosarcoma and melanoma cells.

The mechanism of the cytotoxic effect exerted by parthenolide on tumor cells is not clearly defined today. This article shows that parthenolide stimulates in human osteosarcoma MG63 and melanoma SK-MEL-28 cells a mechanism of cell death, which is not prevented by z-VAD-fmk and other caspase inhibitors. In particular treatment with parthenolide rapidly stimulated (1-2 h) reactive oxygen species (ROS) generation by inducing activation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and NADPH oxidase. This event caused depletion of thiol groups and glutathione, NF-κB inhibition, c-Jun N-terminal kinase (JNK) activation, cell detachment from the matrix, and cellular shrinkage. The increase of ROS generation together with the mitochondrial accumulation of Ca(2+) also favored dissipation of Δψm, which seemed primarily determined by permeability transition pore opening, since Δψm loss was partially prevented by the inhibitor cyclosporin A. Staining with Hoechst 33342 revealed in most cells, at 3-5 h of treatment, chromatin condensation, and fragmentation, while only few cells were propidium iodide (PI)-positive. In addition, at this stage apoptosis inducing factor (AIF) translocated to the nucleus and co-localized with areas of condensed chromatin. Prolonging the treatment (5-15 h) ATP content declined while PI-positive cells strongly augmented, denouncing the increase of necrotic effects. All these effects were prevented by N-acetylcysteine, 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

The Histone Deacetylase Inhibitor ITF2357 (Givinostat) Targets Oncogenic BRAF in Melanoma Cells and Promotes a Switch from Pro-Survival Autophagy to Apoptosis

Histone deacetylase inhibitors (HDACI) are epigenetic compounds that have been widely considered very promising antitumor agents. Here, we focus on the effects of the pan-HDAC inhibitor ITF2357 (Givinostat) in comparison with SAHA (Vorinostat) in melanoma cells bearing BRAF V600E oncogenic mutation. Our results indicate both ITF2357 and SAHA dose-dependently reduce the viability of BRAF-mutated SK-MEL-28 and A375 melanoma cells. The comparison of IC50 values revealed that ITF2357 was much more effective than SAHA. Interestingly, both inhibitors markedly decreased oncogenic BRAF protein expression levels, ITF2357 being the most effective compound. Moreover, the BRAF decrease induced by ITF2357 was accompanied by a decrease in the level of phospho-ERK1/2. The inhibitor of upstream MEK activity, U0126, reduced ERK1/2 phosphorylation and dramatically potentiated the antitumor effect of ITF2357, exacerbating the reduction in the BRAF level. ITF2357 stimulated an early pro-survival autophagic response, which was followed by apoptosis, as indicated by apoptotic markers evaluation and the protective effects exerted by the pan-caspase inhibitor z-VADfmk. Overall, our data indicate for the first time that ITF2357 targets oncogenic BRAF in melanoma cells and induces a switch from autophagy to classic apoptosis, thus representing a possible candidate in melanoma targeted therapy

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