MiR-29 family dysregulation occurs in various cancers including breast cancers. We investigated miR-29b-1 functional role in human triple negative breast cancer (TNBC) the most aggressive breast cancer subtype. We found that miR-29b-1-5p was downregulated in human TNBC tissues and cell lines. To assess whether miR-29b-1-5p correlated with TNBC regenerative potential, we evaluated cancer stem cell enrichment in our TNBC cell lines, and found that only MDA-MB-231 and BT-20 produced primary, secondary and tertiary mammospheres, which were progressively enriched in OCT4, NANOG and SOX2 stemness genes. MiR-29b-1-5p expression inversely correlated with mammosphere stemness potential, and miR-29b-1 ectopic overexpression decreased TNBC cell growth, self-renewal, migration, invasiveness and paclitaxel resistance repressing WNT/βcatenin and AKT signaling pathways and stemness regulators. We identified SPINDLIN1 (SPIN1) among predicted miR-29b-1-5p targets. Consistently, SPIN1 was overexpressed in most TNBC tissues and cell lines and negatively correlated with miR-29b-1-5p. Target site inhibition showed that SPIN1 seems to be directly controlled by miR-29b-1-5p. Silencing SPIN1 mirrored the effects triggered by miR-29b-1 overexpression, whereas SPIN1 rescue by SPIN1miScript protector, determined the reversal of the molecular effects produced by the mimic-miR-29b-1-5p. Overall, we show that miR-29b-1 deregulation impacts on multiple oncogenic features of TNBC cells and their renewal potential, acting, at least partly, through SPIN1, and suggest that both these factors should be evaluated as new possible therapeutic targets against TNBC.

Triple-negative breast cancers (TNBCs) are clinically aggressive forms associated with a poor prognosis. We evaluated the cytotoxic effect exerted on triple-negative MDA-MB231 breast cancer cells both by parthenolide and its soluble analogue dimethylamino parthenolide (DMAPT) and explored the underlying molecular mechanism. The drugs induced a dose- and time-dependent decrement in cell viability, which was not prevented by the caspase inhibitor z-VAD-fmk. In particular in the first hours of treatment (1–3 h), parthenolide and DMAPT strongly stimulated reactive oxygen species (ROS) generation. The drugs induced production of superoxide anion by activating NADPH oxidase. ROS generation caused depletion of thiol groups and glutathione, activation of c-Jun N-terminal kinase (JNK) and downregulation of nuclear factor kB (NF-kB). During this first phase, parthenolide and DMAPT also stimulated autophagic process, as suggested by the enhanced expression of beclin-1, the conversion of microtubule-associated protein light chain 3-I (LC3-I) to LC3-II and the increase in the number of cells positive to monodansylcadaverine. Finally, the drugs increased RIP-1 expression. This effect was accompanied by a decrement of pro-caspase 8, while its cleaved form was not detected and the expression of c-FLIPS markedly increased. Prolonging the treatment (5–20 h) ROS generation favoured dissipation of mitochondrial membrane potential and the appearance of necrotic events, as suggested by the increased number of cells positive to propidium iodide staining. The administration of DMAPT in nude mice bearing xenografts of MDA-MB231 cells resulted in a significant inhibition of tumour growth, an increment of animal survival and a marked reduction of the lung area invaded by metastasis. Immunohistochemistry data revealed that treatment with DMAPT reduced the levels of NF-kB, metalloproteinase-2 and -9 and vascular endothelial growth factor, while induced upregulation of phosphorylated JNK. Taken together, our data suggest a possible use of parthenolide for the treatment of TNBCs.

Extracellular vesicles (EVs), including exosomes, are membranous particles released by cells into the extracellular space. They are involved in cell differentiation, tissue homeostasis, and organ remodelling in virtually all tissues, including the central nervous system (CNS). They are secreted by a range of cell types and via blood reaching other cells whose functioning they can modify because they transport and deliver active molecules, such as proteins of various types and functions, lipids, DNA, and miRNAs. Since they are relatively easy to isolate, exosomes can be characterized, and their composition elucidated and manipulated by bioengineering techniques. Consequently, exosomes appear as promising theranostics elements, applicable to accurately diagnosing pathological conditions, and assessing prognosis and response to treatment in a variety of disorders. Likewise, the characteristics and manageability of exosomes make them potential candidates for delivering selected molecules, e.g., therapeutic drugs, to specific target tissues. All these possible applications are pertinent to research in neurophysiology, as well as to the study of neurological disorders, including CNS tumors, and autoimmune and neurodegenerative diseases. In this brief review, we discuss what is known about the role and potential future applications of exosomes in the nervous system and its diseases, focusing on cell–cell communication in physiology and pathology.

The proteasome inhibitor bortezomib is an efficacious apoptotic agent in many tumor cells. This paper shows that bortezomib induced apoptosis in human hepatoma HepG2 cells associated with many modifications in the expression of survival or death factors. Although bortezomib increased the level of the protective factors HSP70 and HSP27, the effects of the drug that favour cell death were predominant. These events include accumulation of c-Jun, phospho-c-Jun and p53; increase in FasL level with activation of caspase-8; changes related to members of Bcl-2 family with increase in the level of pro-apoptotic members and decrease in that of anti-apoptotic ones; dissipation of mitochondrial potential with cytochrome c release and activation of caspase-3. In contrast, Chang liver cells exhibited a very low susceptibility to bortezomib-induced apoptosis, which was accompanied by modest modifications in the expression of apoptotic factors. In HepG2 cells bortezomib markedly increased AP-1 activity and the expression of its transcriptional targets such as c-Jun, FasL, BimEL, which are involved in apoptosis. Moreover, AP-1 induced its own production by increasing c-Jun content in the composition of the same AP-1 complex. In addition, bortezomib caused activation of JNK1, which in turn increased the level of phospho-c-Jun as well as stimulated the activation of caspase-3 and t-Bid, two fundamental apoptotic factors. Interestingly, siRNA silencing of c-Jun or JNK1 reduced HepG2 cell susceptibility to apoptosis and prevented the increase in AP-1 activity. Both JNK-1 and AP-1 thus exerted a crucial role in bortezomib-induced apoptosis. Differently, in Chang liver cells the different composition of AP-1 complex as well as the failure of JNK activation seemed to be responsible for the low susceptibility to apoptosis. Given the high susceptibility of hepatoma cells to bortezomib, our results suggest the potential application of this compound in clinical trials for liver cancers.

Histone deacetylase (HDAC) inhibitors represent a promising group of anticancer agents. This paper shows that the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) stimulated at 5-10 microM apoptosis in human hepatoma HepG2 and Huh6 cells, but was ineffective in primary human hepatocytes (PHH). In HepG2 cells SAHA induced the extrinsic apoptotic pathway, increasing the expression of both FasL and FasL receptor and causing the activation of caspase-8. Moreover, SAHA enhanced the level of Bim proteins, stimulated alternative splicing of the Bcl-X transcript with the expression of the proapoptotic Bcl-Xs isoform, induced degradation of Bid into the apoptotic factor t-Bid and dephosphorylation and inactivation of the anti-apoptotic factor Akt. Consequently, SAHA caused loss of mitochondrial transmembrane potential, release of cytochrome c from mitochondria, activation of caspase-3 and degradation of PARP. Interestingly, a combination of suboptimal doses of SAHA (1 microM) and bortezomib (5-10 nM), a potent inhibitor of 26S proteasome, synergistically induced apoptosis in both HepG2 and Huh6 cells, but was ineffective in PHH. Combined treatment increased with synergistic effects the expression levels of c-Jun, phospho-c-Jun and FasL and the production of Bcl-Xs. These effects were accompanied by activation of Bid, caspase-8 and 3. In conclusion, SAHA stimulated apoptosis in hepatoma cells and exerted a synergistic apoptotic effect when combined with bortezomib. In contrast, these treatments were quite ineffective in inducing apoptosis in PHH. Thus, our results suggest the potential application of the SAHA/bortezomib combination in clinical trials for liver cancer.

This article shows that HepG2, Hep3B, and SK-Hep1 cells, three lines of human hepatocellular carcinoma (HCC) cells, are resistant to apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Parthenolide, a sesquiterpene lactone found in European feverfew, has been shown to exert both anti-inflammatory and anti-cancer activities. This article demonstrates that co-treatment with parthenolide and TRAIL-induced apoptosis with synergistic interactions in the three lines of HCC cells. In order to explain these effects we ascertained that parthenolide increased either at protein or mRNA level the total content of death receptors TRAIL-R1 and -R2 as well as their surface expression. These effects were found in the three cell lines in the case of TRAIL-R2, while for TRAIL-R1 they were observed in HepG2 and SK-Hep1 cells, but not in Hep3B cells. We suggest that the effects of parthenolide on death receptors depend on the decrease in the level of phosphorylated and active forms of STAT proteins, an event which could be a consequence of the inhibitory effect exerted by parthenolide on the activation of JAK proteins. In agreement with this hypothesis treatment with STAT3 siRNA increased in HCC cells the effect of parthenolide on the expression of death receptors. Sensitization by parthenolide to TRAIL stimulated in the three cell lines the extrinsic mechanism of apoptosis with the activation of both caspases 8 and 3, whereas mitochondria were not involved in the process. Our results suggest that co-treatment with parthenolide and TRAIL could represent a new important therapeutic strategy for hepatic tumors.

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.

Osteosarcoma (OS) is the most common type of bone cancer, with a peak incidence in the early childhood. Emerging evidence suggests that treatments targeting cancer stem cells (CSCs) within a tumor can halt cancer and improve patient survival. MicroRNAs (miRNAs) have been implicated in the maintenance of the CSC phenotype, thus, identification of CSC-related miRNAs would provide information for a better understanding of CSCs. Downregulation of miRNA-29 family members (miR-29a/b/c; miR-29s) was observed in human OS, however, little is known about the functions of miR-29s in human OS CSCs. Previously, during the characterization of 3AB-OS cells, a CSC line selected from human OS MG63 cells, we showed a potent downregulation of miR-29b. In this study, after stable transfection of 3AB-OS cells with miR-29b-1, we investigated the role of miR-29b-1 in regulating cell proliferation, sarcosphere-forming ability, clonogenic growth, chemosensitivity, migration and invasive ability of 3AB-OS cells, in vitro. We found that, miR-29b-1 overexpression consistently reduced both, 3AB-OS CSCs growth in two- and three-dimensional culture systems and their sarcosphere- and colony-forming ability. In addition, while miR-29b-1 overexpression sensitized 3AB-OS cells to chemotherapeutic drug-induced apoptosis, it did not influence their migratory and invasive capacities, thus suggesting a context-depending role of miR-29b-1. Using publicly available databases, we proceeded to identify potential miR-29b target genes, known to play a role in the above reported functions. Among these targets we analyzed CD133, N-Myc, CCND2, E2F1 and E2F2, Bcl-2 and IAP-2. We also analyzed the most important stemness markers as Oct3/4, Sox2 and Nanog. Real-time RT-PCR and western-blot analyses showed that miR-29b-1 negatively regulated the expression of these markers. Overall, the results show that miR-29b-1 suppresses stemness properties of 3AB-OS CSCs and suggest that developing miR-29b-1 as a novel therapeutic agent might offer benefits for OS treatment.