Nuclear Nox4-derived reactive oxygen species in myelodysplastic syndromes

A role for intracellular ROS production has been recently implicated in the pathogenesis and progression of a wide variety of neoplasias. ROS sources, such as NAD(P)H oxidase (Nox) complexes, are frequently activated in AML (acute myeloid leukemia) blasts and strongly contribute to their proliferation, survival, and drug resistance. Myelodysplastic syndromes (MDS) comprise a heterogeneous group of disorders characterized by ineffective hematopoiesis, with an increased propensity to develop AML. The molecular basis for MDS progression is unknown, but a key element in MDS disease progression is the genomic instability. NADPH oxidases are now recognized to have specific subcellular localizations, this targeting to specific compartments for localized ROS production. Local Nox-dependent ROS production in the nucleus may contribute to the regulation of redox-dependent cell growth, differentiation, senescence, DNA damage, and apoptosis. We observed that Nox1, 2, and 4 isoforms and p22phox and Rac1 subunits are expressed in MDS/AML cell lines and MDS samples, also in the nuclear fractions. Interestingly, Nox4 interacts with ERK and Akt1 within nuclear speckle domain, suggesting that Nox4 could be involved in regulating gene expression and splicing factor activity. These data contribute to the elucidation of the molecular mechanisms used by nuclear ROS to drive MDS evolution to AML

Specific ablation of phospholipase Cγ1 in forebrain causes manic-like behavior

It is well known that manic episodes are one of the major diagnostic symptoms in a spectrum of neuropsychiatric disorders that include schizophrenia, obsessive-compulsive disorder and bipolar disorder (BD). Despite a possible association between BD and the gene encoding phospholipase Cγ1 (PLCG1), its etiological basis remains unclear. Here, we report that mice lacking phospholipase Cγ1 (PLCγ1) in the forebrain (Plcg1f/f; CaMKII) exhibit hyperactivity, decreased anxiety-like behavior, reduced depressive-related behavior, hyperhedonia, hyperphagia, impaired learning and memory and exaggerated startle responses. Inhibitory transmission in hippocampal pyramidal neurons and striatal dopamine receptor D1-expressing neurons of Plcg1-deficient mice was significantly reduced. The decrease in inhibitory transmission is likely due to a reduced number of γ-aminobutyric acid (GABA)-ergic boutons, which may result from impaired localization and/or stabilization of postsynaptic CaMKII (Ca2+/calmodulin-dependent protein kinase II) at inhibitory synapses. Moreover, mutant mice display impaired brain-derived neurotrophic factor-tropomyosin receptor kinase B-dependent synaptic plasticity in the hippocampus, which could account for deficits of spatial memory. Lithium and valproate, the drugs presently used to treat mania associated with BD, rescued the hyperactive phenotypes of Plcg1f/f; CaMKII mice. These findings provide evidence that PLCγ1 is critical for synaptic function and plasticity and that the loss of PLCγ1 from the forebrain results in manic-like behavior

Epigenetic Regulation of Nuclear PI-PLC beta1 Signalling Pathway in Low-Risk MDS Patients During Azacitidine Treatment

Myelodysplastic syndromes (MDS) are a heterogeneous group of hematological malignancies characterized by epigenetic abnormalities and therefore treated with demethylating agents [1]. PI-PLCbeta1 has been reported to be a specific target for demethylating therapy in high-risk MDS patients, since azacitidine treatment can be associated with a PI-PLCbeta1 specific promoter demethylation and induction of both PI-PLCbeta1 gene and protein expression [1]. In the present study we investigated the role of epigenetic regulation of PI-PLCbeta1, mainly focusing on the functional role of azacitidine on the structure of the PI-PLCbeta1 promoter. We firstly examined the effect of azacitidine on PI-PLCbeta1 promoter methylation and gene expression in low-risk MDS. Moreover, we studied the expression of key molecules involved in the nuclear inositide signalling pathway, such as Cyclin D3. We also studied the correlation between the demethylating effect of azacitidine and the degree of recruitment to PI-PLCbeta1 promoter of some transcription factors implicated in hematopoietic stem cell proliferation and differentiation, as well as of the Methyl-CpG binding domain proteins (MBDs), which specifically interact with methylated DNA. Taken together, our results hint at a specific involvement of PI-PLCbeta1 in epigenetic mechanisms, and are particularly consistent with the hypothesis of a role for PI-PLCbeta1 in azacitidine- induced myeloid differentiation

Nuclear Nox4 Interaction with Prelamin A is Associated with Nuclear Redox Control of Stem Cell Aging

Mesenchymal stem cells have emerged as an important tool that can be used for tissue regeneration thanks to their easy preparation, differentiation potential and immunomodulatory activity. However, an extensive culture of stem cells in vitro prior to clinical use can lead to oxidative stress that can modulate different stem cells properties, such as self-renewal, proliferation, differentiation and senescence. The aim of this study was to investigate the aging process occurring during in vitro expansion of stem cells, obtained from amniotic fluids (AFSC) at similar gestational age. The analysis of 21 AFSC samples allowed to classify them in groups with different levels of stemness properties. In summary, the expression of pluripotency genes and the proliferation rate were inversely correlated with the content of reactive oxygen species (ROS), DNA damage signs and the onset premature aging markers, including accumulation of prelamin A, the lamin A immature form. Interestingly, a specific source of ROS, the NADPH oxidase isoform 4 (Nox4), can localize into PML nuclear bodies (PML-NB), where it associates to prelamin A. Besides, Nox4 post translational modification, involved in PML-NB localization, is linked to its degradation pathway, as it is also for prelamin A, thus possibly modulating the premature aging phenotype occurrence

Differential activation of nuclear inositide-dependent signalling pathways during erythropoiesis and myelopoiesis induced by lenalidomide and azacitidine in myelodysplastic syndromes (MDS)

Inositide-dependent signalling pathways regulated by phosphoinositide-specific phospholi- pase C (PI-PLC) beta1 have been demonstrated to play important roles in MDS pathogenesis and in cell differentiation (1). Moreover, the MDS therapy aims at inducing myeloid and/or erythroid differentiation of MDS stem cells. Indeed, azacitidine is a demethylating agent that can induce myeloid differentiation. On the other hand, lenalidomide may restore a normal erythropoiesis. The exact molecular mechanisms underlying the effect of azacitidine and lenalidomide in MDS cells are still unclear, although it is clear that these therapies regulate stem cell proliferation, differentiation and apoptosis (2). The combination of azacitidine and lenalidomide in MDS therapy is now under considera- tion, given the capability of both drugs to balance proliferation and differentiation processes (3). In this study we analyzed the molecular effect of this combination therapy on PI-PLC isoenzymes, not only studying PI-PLCbeta1, but also PI-PLCgamma1, that can be associated with erythropoiesis. We analyzed 44 patients diagnosed with high-risk MDS who were given azacitidine and lenalidomide. Given the limited number of cells, we quantified the expression of these molecules by Real-Time PCR analyses and immunocytochemical experiments. Moreover, we carried out cell cycle analyses and studied both PI-PLCbeta1 methylation status and the expression of Globin genes. In our case series, 28/44 patients were evaluable, with an overall response rate of 78.6% (22/28 cases). At a molecular level, a significant increase of PI-PLCbeta1 and/or PI-PLCgamma1 expression was associated with a favourable clinical response to the combination therapy. Responder cases also showed an increase of Beta-globin expression, hinting at a specific contri- bution of lenalidomide on erythroid activation, whilst the frequent demethylation of PI-PLCbeta1 promoter could be specifically linked to azacitidine. Taken together, our results show that the combination of azacitidine and lenalidomide can be important for activating PI-PLC isoenzymes, therefore regulating myeloid and erythroid dif- ferentiation in MDS cells

Response of high-risk MDS to azacitidine and lenalidomide is impacted by baseline and acquired mutations in a cluster of three inositide-specific genes

Specific myeloid-related and inositide-specific gene mutations can be linked to myelodysplastic syndromes (MDS) pathogenesis and therapy. Here, 44 higher-risk MDS patients were treated with azacitidine and lenalidomide and mutations analyses were performed at baseline and during the therapy. Results were then correlated to clinical outcome, overall survival (OS), leukemia-free-survival (LFS) and response to therapy. Collectively, 34/44 patients were considered evaluable for response, with an overall response rate of 76.25% (26/34 cases): 17 patients showed a durable response, 9 patients early lost response and 8 patients never responded. The most frequently mutated genes were ASXL1, TET2, RUNX1, and SRSF2. All patients early losing response, as well as cases never responding, acquired the same 3 point mutations during therapy, affecting respectively PIK3CD (D133E), AKT3 (D280G), and PLCG2 (Q548R) genes, that regulate cell proliferation and differentiation. Moreover, Kaplan–Meier analyses revealed that this mutated cluster was significantly associated with a shorter OS, LFS, and duration of response. All in all, a common mutated cluster affecting 3 inositide-specific genes is significantly associated with loss of response to azacitidine and lenalidomide therapy in higher risk MDS. Further studies are warranted to confirm these data and to further analyze the functional role of this 3-gene cluster

Nuclear Translocation of PKC is Associated with Cell Cycle Arrest and Erythroid Differentiation in Myelodysplastic Syndromes (MDSs)

PI-PLC beta 1 is involved in cell proliferation, differentiation, and myelodysplastic syndrome (MDS) pathogenesis. Moreover, the increased activity of PI-PLC beta 1 reduces the expression of PKC-alpha, which, in turn, delays the cell proliferation and is linked to erythropoiesis. Lenalidomide is currently used in low-risk patients with MDS and del(5q), where it can suppress the del(5q) clone and restore normal erythropoiesis. In this study, we analyzed the effect of lenalidomide on 16 patients with low-risk del(5q) MDS, as well as del(5q) and non-del(5q) hematopoietic cell lines, mainly focusing on erythropoiesis, cell cycle, and PI-PLC beta 1/PKC-alpha signaling. Overall, 11 patients were evaluated clinically, and 10 (90%) had favorable responses; the remaining case had a stable disease. At a molecular level, both responder patients and del(5q) cells showed a specific induction of erythropoiesis, with a reduced gamma/beta-globin ratio, an increase in glycophorin A, and a nuclear translocation of PKC-alpha. Moreover, lenalidomide could induce a selective G(0)/G(1) arrest of the cell cycle in del(5q) cells, slowing down the rate proliferation in those cells. Altogether, our results could not only better explain the role of PI-PLC beta 1/PKC-alpha signaling in erythropoiesis but also lead to a better comprehension of the lenalidomide effect on del(5q) MDS and pave the way to innovative, targeted therapies

Abilities of berberine and chemically modified berberines to interact with metformin and inhibit proliferation of pancreatic cancer cells

Pancreatic cancer is devastating cancer worldwide with few if any truly effective therapies. Pancreatic cancer has an increasing incidence and may become the second leading cause of death from cancer. Novel, more effective therapeutic approaches are needed as pancreatic cancer patients usually survive for less than a year after being diagnosed. Control of blood sugar levels by the prescription drug metformin in diseases such as diabetes mellitus has been examined in association with pancreatic cancer. While the clinical trials remain inconclusive, there is hope that certain diets and medications may affect positively the outcomes of patients with pancreatic and other cancers. Other natural compounds may share some of the effects of metformin. One "medicinal" fruit consumed by millions worldwide is berberine (BBR). Metformin and BBR both activate AMP-activated protein kinase (AMPK) which is a key mediator of glucose metabolism. Glucose metabolism has been shown to be very important in cancer and its significance is increasing. In the following studies, we have examined the effects of metformin, BBR and a panel of modified BBRs (NAX compounds) and chemotherapeutic drugs on the growth of four different human pancreatic adenocarcinoma cell lines (PDAC). Interestingly, the effects of metformin could be enhanced by BBR and certain modified BBRs. Upon restoration of WT-TP53 activity in MIA-PaCa-2 cells, an altered sensitivity to the combination of certain NAX compounds and metformin was observed compared to the parental cells which normally lack WT-TP53. Certain NAX compounds may interact with WT-TP53 and metformin treatment to alter the expression of key molecules involved in cell growth. These results suggest a therapeutic approach by combining certain pharmaceutical drugs and nutraceuticals to suppress the growth of cancer cells

Effects of the MDM-2 inhibitor Nutlin-3a on PDAC cells containing and lacking WT-TP53 on sensitivity to chemotherapy, signal transduction inhibitors and nutraceuticals

Mutations at the TP53 gene are readily detected (approximately 50-75%) in pancreatic ductal adenocarcinoma (PDAC) patients. TP53 was previously thought to be a difficult target as it is often mutated, deleted or inactivated on both chromosomes in certain cancers. In the following study, the effects of restoration of wild-type (WT) TP53 activity on the sensitivities of MIA-PaCa-2 pancreatic cancer cells to the MDM2 inhibitor nutlin-3a in combination with chemotherapy, targeted therapy, as well as, nutraceuticals were examined. Upon introduction of the WT-TP53 gene into MIA-PaCa-2 cells, which contain a TP53 gain of function (GOF) mutation, the sensitivity to the MDM2 inhibitor increased. However, effects of nutlin-3a were also observed in MIA-PaCa-2 cells lacking WT-TP53, as upon co-treatment with nutlin-3a, the sensitivity to certain inhibitors, chemotherapeutic drugs and nutraceuticals increased. Interestingly, co-treatment with nutlin-3a and certain chemotherapeutic drug such as irinotecan and oxaliplatin resulted in antagonistic effects in cells both lacking and containing WT-TP53 activity. These studies indicate the sensitizing abilities that WT-TP53 activity can have in PDAC cells which normally lack WT-TP53, as well as, the effects that the MDM2 inhibitor nutlin-3a can have in both cells containing and lacking WT-TP53 to various therapeutic agents