Cannabinoid derivatives exert a potent anti-myeloma activity both in vitro and in vivo

12 p.-6 fig.-1 tab.Although hematopoietic and immune system show high levels of the cannabinoid receptor CB2, the potential effect of cannabinoids on hematologic malignancies has been poorly determined. Here we have investigated their anti-tumor effect in multiple myeloma (MM). We demonstrate that cannabinoids induce a selective apoptosis in MM cell lines and in primary plasma cells of MM patients, while sparing normal cells from healthy donors, including hematopoietic stem cells. This effect was mediated by caspase activation, mainly caspase-2, and was partially prevented by a pan-caspase inhibitor. Their pro-apoptotic effect was correlated with an increased expression of Bax and Bak, a decrease of Bcl-xL and Mcl-1, a biphasic response of Akt/PKB and an increase in the levels of ceramide in MM cells. Inhibition of ceramide synthesis partially prevented apoptosis, indicating that these sphingolipids play a key role in the pro-apoptotic effect of cannabinoids in MM cells. Remarkably, blockage of the CB2 receptor also inhibited cannabinoid-induced apoptosis. Cannabinoid derivative WIN-55 enhanced the anti-myeloma activity of dexamethasone and melphalan overcoming resistance to melphalan in vitro. Finally, administration of cannabinoid WIN-55 to plasmacytoma-bearing mice significantly suppressed tumor growth in vivo. Together, our data suggest that cannabinoids may be considered as potential therapeutic agents in the treatment of MM.Grant sponsor: Junta de Andalucía; Grant numbers: PI-0355–2013 and AC-0062–2013; Grant sponsor: Instituto de Salud Carlos III;Grant numbers: PI14/02074 and CP12/03273.Peer reviewe

Genetically Modeled Mice with Mutations in Mitochondrial Metabolic Enzymes for the Study of Cancer

Mitochondrial dysfunction has long been implicated in progression of cancer. As a paradigm, the “Warburg effect,” which by means of a switch toward anaerobic metabolism enables cancer cells to proliferate in oxygen limiting conditions, is well established. Besides this metabolic transformation of tumors, it has been discovered that mutations in genes encoding mitochondrial proteins are the etiological factors in different types of cancer. This confers to mitochondrial dysfunction a causative role, rather than resultant, in tumor genesis beyond its role in tumor progression and development. Mitochondrial proteins encoded by tumor-suppressor genes are part of the succinate-dehydrogenase, the fumarate-hydratase, and the mitochondrial isocitrate-dehydrogenase enzymes, all of them participating in the Krebs cycle. The spectrum of tumors associated with mutations in these genes is becoming larger and varies between each enzyme. Several mechanisms of tumorigenesis have been proposed for the different enzymatic defects, most of them based on studies using cellular and animal models. Regarding the molecular pathways implicated in the oncogenic transformation, one of the first accepted theories was based on the constitutive expression of the hypoxia-inducible factor 1α (Hif1α) at normal oxygen tension, a theory referred to as “pseudo-hypoxic drive.” This mechanism has been linked to the three types of mutations, thus suggesting a central role in cancer. However, other alternative molecular processes, such as oxidative stress or altered chromatin remodeling, have been also proposed to play an onco-pathogenic role. In the recent years, the role of oncometabolites, a new concept emerged from biochemical studies upon these tumors, has acquired relevance as responsible for tumor formation. Nevertheless, the actual contribution of each of these mechanisms has not been definitively established. In this review, we summarize the results obtained from mouse strains genetically modified in the three different enzymes.Peer reviewedPeer Reviewe

Absolute requirement of GDNF for adult catecholaminergic neuron survival

33 páginas, 7 figuras, 1 tabla.GDNF is a potent neurotrophic factor that protects catecholaminergic neurons from toxic damage and induces fiber outgrowth. However, the actual role of endogenous GDNF in the normal adult brain is unknown, even though GDNF-based therapies are considered promising for neurodegenerative disorders. We have generated a conditional GDNF-null mouse to suppress GDNF expression in adulthood, hence avoiding the developmental compensatory modifications masking its true physiologic action. After Gdnf ablation, mice showed a progressive hypokinesia and a selective decrease of brain tyrosine hydroxylase (Th) mRNA, accompanied by pronounced catecholaminergic cell death, affecting most notably the locus coeruleus, which practically disappears; the substantia nigra; and the ventral tegmental area. These data unequivocally demonstrate that GDNF is indispensable for adult catecholaminergic neuron survival and also show that, under physiologic conditions, downregulation of a single trophic factor can produce massive neuronal death.Support was obtained from the Juan March Foundation, the Marcelino Botín Foundation, the Spanish Ministry of Science and Education, the Spanish Ministry of Health (TERCEL), and the Andalusian Government. CIBERNED is funded by the “Instituto de Salud Carlos III”.Peer reviewe

Differential impairment of catecholaminergic cell maturation and survival by genetic mitochondrial complex II dysfunction

The SDHD gene (subunit D of succinate dehydrogenase) has been shown to be involved in the generation of paragangliomas and pheochromocytomas. Loss of heterozygosity of the normal allele is necessary for tumor transformation of the affected cells. As complete SdhD deletion is lethal, we have generated mouse models carrying a >floxed> SdhD allele and either an inducible (SDHD-ESR strain) or a catecholaminergic tissue-specific (TH-SDHD strain) CRE recombinase. Ablation of both SdhD alleles in adult SDHD-ESR mice did not result in generation of paragangliomas or pheochromocytomas. In contrast, carotid bodies from these animals showed smaller volume than controls. In accord with these observations, the TH-SDHD mice had decreased cell numbers in the adrenal medulla, carotid body, and superior cervical ganglion. They also manifested inhibited postnatal maturation of mesencephalic dopaminergic neurons and progressive cell loss during the first year of life. These alterations were particularly intense in the substantia nigra, the most affected neuronal population in Parkinson's disease. Unexpectedly, TH + neurons in the locus coeruleus and group A13, also lacking the SdhD gene, were unaltered. These data indicate that complete loss of SdhD is not sufficient to induce tumorigenesis in mice. They suggest that substantia nigra neurons are more susceptible to mitochondrial damage than other catecholaminergic cells, particularly during a critical postnatal maturation period. © 2012, American Society for Microbiology.Support was obtained from the Marcelino Botín Foundation, the Spanish Ministries of Science and Health (TERCEL), and the Andalusian Government.Peer Reviewe

Efecto antileucémico de derivados cannabinoides: un nuevo grupo terapéutico en LMA

2 p.-3 fig.Los cannabinoides, componentes activos del cannabis, se emplean como tratamiento paliativo en pacientes con cáncer en virtud de su efecto antiemético y analgésico. Además, hay evidencias de que estos compuestos inhiben el crecimiento de algunas células tumorales en animales de laboratorio. Estos compuestos actúan uniéndose a receptores específicos. Los principales receptores son el CB1 (mayoritario en SNC) y CB2 (en tejido hematopoyético). El objetivo de este estudio fue confirmar el efecto terapéutico de derivados cannabinoides sintéticos específicos del receptor CB2 en LMA.Peer reviewe

Animal models for neurodegenerative diseases

38 páginas, 8 figuras, 1 tabla. Fecha de prioridad: 07/12/2007. IPC: A01K-067/00; A61K-049/00; C07K-014/00; C12N-015/00. Solicitante: Universidad de Sevilla.The invention provides a non-human animal model of Parkinson's disease based on animals which carry a modified version of the GDNF gene which comprises recombinase target sites flanking an essential part of said gene and a transgene which encodes for a recombinase specific for the target sites in the GDNF gene and which can be activated at will by administering a given compound to the animal. Activation of the recombinase in adult animals leads to a progressive catecholaminergic neuronal death. The invention also provides the use of said animals for the study of the pathogenesis of Parkinson's disease and for the identification of GDNF targets (which preserve the neurons from degeneration) as well as GDNF agonists and activators.Peer reviewe

Effect of pomalidomide on T cell polarization is mediated through epigenetic modifcations

Resumen del póster presentado al 22nd Congress of the European Hematology Association, celebrado en Madrid (España) del 22 al 25 de junio de 2017.-- Alvarez Laderas, Isabel et al.[Background]: There is conflicting evidence regarding the potential use of IMIDs and particularly pomalidomide after allogeneic stem cell transplantation (allo-HSCT). It has been well described that IMIDs polarize naïve T cells towards a Th1 phenotype increasing IFN-γ cytokine production via the augmentation of T-bet transcription factor. This effect might increase the risk of GvHD after allo-HSCT. Nevertheless, a recent trial has reported a potential benefit on the use of pomalidomide as GVHD treatment. [Aims]: In the current study, we have analyzed the effect of pomalidomide in the polarization of CD45RA+ cells and the epigenetic mechanisms that might be involved in this effect. [Methods]: Isolated CD45RA+ T cells from healthy donor’s Buffy Coats werestimulated with anti-CD3 plus anti-CD28 in the presence of several cytokines to polarize towards Th1 (IL-12, INF-γand anti-IL4) or Th2 (IL-4, IL-2, anti-IFN-γ and anti-IL-12) for 5 days. Pomalidomide at two different doses (10 and 100 nM) were added into the culture and the effect on T cells polarization was analyzed by flow cytometry after staining with anti-CD25, anti-IFNγ, anti-CD4 and anti-IL2 for Th1 cell polarization and anti-CD25, anti-IL10, anti-CD3 and anti-IL4 for Th2 cell polarization. In addition, the release of cytokines (IL-2, IL-4, IL-6, IL-10, TNF-α and IFN-γ) in cell culture supernants were measured with BD Human Th1/Th2 Cytokine CBA kit (BDBiosciences) and T-bet and GATA-3 expression were analyzed by Western Blot. Chromatin immunoprecipitation (ChIP) assays were performed to assess the trimethylation of H3K4 (associated with gene activation) and the trimethylation of H3K27 (associated with gene repression) in the TBET and GATA-3 gene promoters. [Results]: Pomalidomide increased the expression of INF-γ and IL-2 as determined by flow cytometry in Th1 cell culture conditions. By contrast, in the presence of Th2 promoting cytokines, we observed an increase for both IL-10 and IL-4 upon adding pomalidomide to the culture. In addition, the exposure to pomalidomide increased the levels of TNF-α, INF-γ and IL-2 in the Th1 polarizing culture while, under Th2 promoting conditions, an increased concentration of IL-4 and IL-2 in supernatant was observed after exposure to pomalidomide. Furthermore, exposure to pomalidomide led to an increased expression of T-bet as assessed by western-blot in naïve CD45RA+ cells activated with anti-CD3 plus anti-CD28 and supplemented with IL-12, INF-γ and anti-IL4. By contrast, in Th2 polarization conditions, pomalidomide increased GATA-3 expression.We next studied whether or not the effect of pomalidomide in T cell polarization might be mediated by epigenetic mechanisms: in the presence of Th1 promoting conditions there was a significant increase of the activation marker H3K4me3 at the TBET promoter and a significant decrease in H3K27me3 upon exposure to the drug while, under Th2 promoting conditions, a significant increase in H3K4me3 at the promoter of GATA-3 gene was observed among T cells exposed to pomalidomide. [Conclusion]: Pomalidomide favours both Th1 and Th2 cell differentiation of CD45RA+ cells depending on the cytokines present in the medium. Treatment of naïve T cells with pomalidomide induces epigenetic modifications during T cell polarization which might favour the process of differentiation of the naïve T cells.Peer Reviewe

Human acute myeloid leukemia cells express Neurokinin-1 receptor, which is involved in the antileukemic effect of Neurokinin-1 receptor antagonists

The substance P/neurokinin-1 receptor system has been implicated in tumor cell proliferation. Neurokinin-1 receptor has been identified in different solid tumors but not frequently in hematopoietic malignant cells. We investigated the presence of the Neurokinin-1 receptor in acute myeloid leukemia cell lines (KG-1 and HL-60), demonstrating that acute myeloid leukemia cell lines overexpress the truncated Neurokinin-1 receptor isoform compared with lymphocytes from healthy donors. Using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) method, we demonstrated that substance P induced cell proliferation in both acute myeloid leukemia cell lines. We also observed that four different Neurokinin-1 receptor antagonists (L-733,060, L-732,138, CP 96–345 and aprepitant) elicited inhibition of acute myeloid leukemia cell growth lines in a concentration-dependent manner, while growth inhibition was only marginal in lymphocytes; the specific antitumor action of Neurokinin-1 receptor antagonists occurs via the Neurokinin-1 receptor, and leukemia cell death is due to apoptosis. Finally, administration of high doses of daily intraperitoneal fosaprepitant to NOD scid gamma mice previously xenografted with the HL60 cell line increased the median survival from 4 days (control group) to 7 days (treated group) (p = 0.059). Taken together, these findings suggest that Neurokinin-1 receptor antagonists suppress leukemic cell growth and may be considered to be potential antitumor drugs for the treatment of human acute myeloid leukemia

Sdhd ablation promotes thyroid tumorigenesis by inducing a stem-like phenotype.

Mutations in genes encoding enzymes in the tricarboxylic acid cycle (TCA, also known as the Krebs cycle) have been implicated as causative genetic lesions in a number of human cancers, including renal cell cancers, glioblastomas and pheochromocytomas. In recent studies, missense mutations in the succinate dehydrogenase (SDH) complex have also been proposed to cause differentiated thyroid cancer. In order to gain mechanistic insight into this process, we generated mice lacking the SDH subunit D (Sdhd) in the thyroid. We report that these mice develop enlarged thyroid glands with follicle hypercellularity and increased proliferation. In vitro, human thyroid cell lines with knockdown of SDHD exhibit an enhanced migratory capability, despite no change in proliferative capacity. Interestingly, these cells acquire stem-like features which are also observed in the mouse tumors. The stem-like characteristics are reversed by α-ketoglutarate, suggesting that SDH-associated tumorigenesis results from dedifferentiation driven by an imbalance in cellular metabolites of the TCA cycle. The results of this study reveal a metabolic vulnerability for potential future treatment of SDH-associated neoplasia