WIN-induced vesiculation cooperates to the inhibition of osteosarcoma cell migration

Introduction. Animal cells release vesicles that mediate the secretion of a variety of factors in the surrounding environment affecting neighboring cells. There is increasing evidence that secreted vesicles play an important role as vehicle of intercellular communication in different biological systems and are able to influence both physiological and pathological processes. Recently, we have reported that the synthetic cannabinoid WIN55,512 is able to induce osteosarcoma MG63 cell death and negatively affect cell migration. Here, we study the effects of WIN on the induction of vesicle secretion and their possible role in WIN-dependent reduction of osteosarcoma cell migratory ability. Methods. Vesicles from MG63 cells were obtained by ultracentrifuging at 140,000g media derived from cell cultures untreated and treated for 24 h with 5 uM WIN. Purified vesicles were quantified by cytofluorimetry and by detecting acetilcholinesterase activity according to established criteria. Scratch wound healing assay was employed to monitor cell migration toward the center of a gap created in a cell monolayer. Zymographic analysis was used to evaluate metalloproteinase activities in the vesicles. Results. WIN treatment induced a significant increase (about 4-fold) in the number of vesicles released by osteosarcoma cells. Wound healing assay showed that in the presence of vesicles from WIN-treated cultures, cells only partially filled the gap with respect to those conditioned with vesicles isolated from control cells which closed the gap within about 24 h. Furthermore, zymography assay showed a reduced activity of MMP-2 and MMP-9 in the vesicles obtained from WIN-treated cells. Conclusion. Data indicate that the increase in the number of vesicles released after WIN treatment and/or their probable different composition can be responsible for the relevant inhibition of MG63 cell migration induced by the cannabinoid

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

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

Phosphatidylinositol-3-kinase activity during in vitro dendritic cell generation determines suppressive or stimulatory capacity.

Modulating PI3K at different stages of dendritic cells (DC) generation could be a novel means to balance the generation of immunosuppressive versus immunostimulatory DC. We show that PI3K inhibition during mouse DC generation in vitro results in cells that are potently immunosuppressive and characteristic of CD8alpha- CD11c+ CD11b+ DC. These DC exhibited low surface class I and class II MHC, CD40, and CD86 and did not produce TNF-alpha. In allogeneic MLR, these DC were suppressive. Although in these mixed cultures, there was no increase in the frequency of CD4+ CD25+ Foxp3+ cells, the Foxp3 content on a per cell basis was significantly increased. Sustained TLR9 signaling in the presence of PI3K inhibition during DC generation overrode the cells' suppressive phenotype

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 Anti-Cancer Effect of Mangifera indica L. Peel Extract is Associated to γH2AX-mediated Apoptosis in Colon Cancer Cells

Ethanolic extracts from Mangifera indica L. have been proved to possess anti-tumor properties in many cancer systems. However, although most effects have been demonstrated with fruit pulp extract, the underlying molecular mechanisms of mango peel are still unclear. This study was designed to explore the effects of mango peel extract (MPE) on colon cancer cell lines. MPE affected cell viability and inhibited the colony formation trend of tumor cells, while no effects were observed in human dermal fibroblasts used as a non-cancerous cell line model. These events were a consequence of the induction of apoptosis associated to reactive oxygen species (ROS) production, activation of players of the oxidative response such as JNK and ERK1/2, and the increase in Nrf2 and manganese superoxide dismutase (MnSOD). Significantly, mango peel-activated stress triggered a DNA damage response evidenced by the precocious phosphorylation of histone 2AX (γH2AX), as well as phosphorylated Ataxia telangiectasia-mutated (ATM) kinase and p53 upregulation. Mango peel extract was also characterized, and HPLC/MS (High Performance Liquid Chromatography/Mass Spectrometry) analysis unveiled the presence of some phenolic compounds that could be responsible for the anti-cancer effects. Collectively, these findings point out the importance of the genotoxic stress signaling pathway mediated by γH2AX in targeting colon tumor cells to apoptosis

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

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