677 research outputs found
Biocompatibility, endocytosis, and intracellular trafficking of mesoporous silica and polystyrene nanoparticles in ovarian cancer cells: effects of size and surface charge groups
No full textManeerat Ekkapongpisit,1 Antonino Giovia,1 Carlo Follo,1 Giuseppe Caputo,2,3 Ciro Isidoro11Laboratory of Molecular Pathology and Nanobioimaging, Department of Health Sciences, Università del Piemonte Orientale “A Avogadro”, Novara, 2Dipartimento di Chimica dell’Università di Torino, Torino, 3Cyanine Technology SpA, Torino, ItalyBackground and methods: Nanoparticles engineered to carry both a chemotherapeutic drug and a sensitive imaging probe are valid tools for early detection of cancer cells and to monitor the cytotoxic effects of anticancer treatment simultaneously. Here we report on the effect of size (10–30 nm versus 50 nm), type of material (mesoporous silica versus polystyrene), and surface charge functionalization (none, amine groups, or carboxyl groups) on biocompatibility, uptake, compartmentalization, and intracellular retention of fluorescently labeled nanoparticles in cultured human ovarian cancer cells. We also investigated the involvement of caveolae in the mechanism of uptake of nanoparticles.Results: We found that mesoporous silica nanoparticles entered via caveolae-mediated endocytosis and reached the lysosomes; however, while the 50 nm nanoparticles permanently resided within these organelles, the 10 nm nanoparticles soon relocated in the cytoplasm. Naked 10 nm mesoporous silica nanoparticles showed the highest and 50 nm carboxyl-modified mesoporous silica nanoparticles the lowest uptake rates, respectively. Polystyrene nanoparticle uptake also occurred via a caveolae-independent pathway, and was negatively affected by serum. The 30 nm carboxyl-modified polystyrene nanoparticles did not localize in lysosomes and were not toxic, while the 50 nm amine-modified polystyrene nanoparticles accumulated within lysosomes and eventually caused cell death. Ovarian cancer cells expressing caveolin-1 were more likely to endocytose these nanoparticles.Conclusion: These data highlight the importance of considering both the physicochemical characteristics (ie, material, size and surface charge on chemical groups) of nanoparticles and the biochemical composition of the cell membrane when choosing the most suitable nanotheranostics for targeting cancer cells.Keywords: nanoparticles, imaging, lysosomes, vesicular traffic, ovarian cancer, caveoli
Role of inositide signalling regulation in higher-risk MDS patients during epigenetic therapy
No full textBackground: Inositide signalling pathways are involved in cell
growth, differentiation and apoptosis and play a role in
the progression of myelodysplastic syndromes (MDS) towards
acute myeloid leukemia (AML). The combination of the DNA
methyltransferase inhibitor azacitidine (AZA) and the HDAC inhibitor
valproic acid (VPA) in patients with IPSS intermediate-2/high-risk
MDS has been demonstrated to be active and associated with a high
response rate in patients with MDS and unfavourable prognosis
(Voso MT et al, 2009).
Introduction: In the last few years, our group demonstrated not
only that phosphoinositide-phospholipase C beta1 (PI-PLCbeta1)
promoter gene is hyper-methylated in higher-risk MDS, but also that is affected by epigenetic therapy (Follo MY et al, PNAS 2009; Follo
MY et al. Leukemia 2010). Indeed, AZA, alone or in combination with
VPA was able to induce PI-PLCbeta1 demethylation and expression.
Purpose: In this study we further investigated the role of lipid
signalling pathways during epigenetic therapy, focusing on the
functional effect of AZA and VPA on PI-PLCbeta1 promoter in highrisk
MDS patients.
Materials and Methods: The study included 20 higher-risk MDS
patients (IPSS risk: intermediate-2 or high): 8 of them were treated
with AZA alone (75 mg/m2/day SC for 7 days/28 days), whereas 6 of
them received the combination of AZA with VPA (600–1500 mg/daily
orally) and the remaining 6 were treated only with best supportive
care. For each patient we analyzed the effect of epigenetic therapy
in correlation to PI-PLCbeta1 signalling, by analyzing the binding
affinity of transcription factors correlated to hematopoietic stem
cell differentiation and proliferation, as well as by quantifyng the
expression of molecules involved in the epigenetic machinery, such
as Class I HDACs.
Results: 8/20 (40%) of our MDS patients showed a favourable
hematologic response to epigenetic therapy and an increase in PIPLCbeta1
expression, as compared with the pre-treatment period,
thus confirming the involvement of this molecule in response
to demethylating agents. Moreover, MDS patients responding to
epigenetic treatment seem to involve the recruitment of specific
transcription factors on PI-PLCbeta1 promoter during the regulation
of methylation processes. Taken together, our data are consistent
with the hypothesis of a correlation between epigenetic treatment
and PI-PLCbeta1 signalling, thus hinting at a role for PI-PLCbeta1 in
monitoring the efficacy of epigenetic therapy and paving the way
for the development of innovative therapeutic strategies in MD
Effect of azacitidine on inositide-dependent signalling pathways in low-risk MDS patients
No full textBackground: Azacitidine is a DNA methyltransferase inhibitor
currently used for the treatment of higher-risk myelodysplastic
syndromes (MDS) which has also been proven an effective treatment
of lower-risk MDS (Silverman LR et al, J Clin Oncol 2002; Musto P
et al, Cancer 2010). Inositide signalling is involved in cell growth,
differentiation and apoptosis, therefore affecting cell processes and
playing a role in the pathogenesis of several malignancies.
Introduction: Our group recently demonstrated that phosphoinositide-phospholipase
C beta1 (PI-PLCbeta1) is involved in the
MDS progression to AML and is affected by epigenetic therapy
(Follo MY et al, J Cell Biochem 2010; Follo MY et al, Leukemia
2010). Namely, PI-PLCbeta1 specifically targets Cyclin D3, which
is in turn implicated in hematopoietic stem cell proliferation and
differentiation.
Purpose: In this study, we analyzed the role of PI-PLCbeta1-
dependent signalling pathways during epigenetic therapy, focusing
on the effect of azacitidine on PI-PLCbeta1 downstream target Cyclin
D3 in lower-risk MDS patients.
Materials and Methods: The analysis included 25 patients (IPSS risk:
low or intermediate-1) treated with azacitidine alone (75 mg/m2
subcutaneous daily for 5 consecutive days every 28 days, for a total
of 8 courses). For each patient we followed the effect of azacitidine
in correlation to both PI-PLCbeta1 and Cyclin D3 expression.
Results: Our results show that 9/25 (36%) of our lower-risk MDS
patients showed a hematologic response to azacitidine (Complete
Remission: 4 patients, Partial Remission: 1 patient, Hematologic
Improvements: 4 patients), as well as an increase in PI-PLCbeta1
expression, as compared with pre-treatment levels. Furthermore,
ongoing analyses are trying to disclose whether Cyclin D3 activation
is also implicated in azacitidine response, therefore affecting
hematopoietic stem cell differentiation processes.
Conclusions: Overall, our data hint at a correlation between
azacitidine therapy and PI-PLCbeta1 signalling, possibly via the
activation of Cyclin D3, even in lower-risk MDS, thus indicating
that PI-PLCbeta1 could be useful not only for evaluating the efficacy
of azacitidine but also for disclosing the molecular mechanisms
underlying this kind of treatment in lower-risk MD
Similarities and differences in the biogenesis, processing and lysosomal targeting between zebrafish and human pro-Cathepsin D: functional implications.
No full textDOPAMINE INDUCES APOPTOSIS IN APP(SWE)-OVEREXPRESSING NEURO2A CELLS FOLLOWING ENDOCYTOSIS AND PROTEOLYTIC PROCESSING OF APP
No full textEpigenetic Regulation of Lipid Signalling Pathways In Low-Risk MDS Patients During Azacitidine Treatment
No full textAzacitidine, a DNA methyltransferase inhibitor currently used for the treatment of higher-risk myelodysplastic syndromes (MDS) patients, was shown to delay the evolution into acute myeloid leukemia (AML) and prolong overall survival (Fenaux P et al, Lancet Oncol 2009). In addition, azacitidine has recently been shown to potentially be a feasible and effective treatment even for patients with lower-risk MDS (Musto P et al, Cancer 2010). Lipid signalling pathways are involved in many important biological processes, such as cell growth, differentiation and apoptosis and play a role in the progression of MDS towards AML (Follo MY et al, J Cell Biochem 2010). Moreover, we recently demonstrated that phosphoinositide-phospholipase C beta1 (PI-PLCbeta1) promoter gene is hyper-methylated in higher-risk MDS and that azacitidine treatment can induce an increase in the level of PI-PLCbeta1 splicing variants as well as a down-regulation of activated Akt (Follo MY et al, Leukemia 2008; Follo MY et al, PNAS 2009). In fact, responding patients showed an increase in PI-PLCbeta1 expression in correlation with the therapeutic response, whereas their PI-PLCbeta1 promoter methylation was reduced. Furthermore, the decrease of promoter methylation anticipated the hematologic response, since the variations in PI-PLCbeta1 gene expression were observed prior to the clinical outcome. Stemming from these data, we further investigated the role of inositide signalling pathways during the epigenetic therapy, focusing on the effect of azacitidine on lipid signal transduction pathways in lower-risk MDS patients. The study included 25 patients (IPSS risk: low or intermediate-1) treated with azacitidine (75mg/m2 subcutaneous daily for 5 consecutive days every 28 days, for a total of 8 courses). For each patient we followed the effect of azacitidine in correlation to both PI-PLCbeta1 promoter methylation and gene expression, as well as the molecular profile of key molecules involved in the regulation of methylation processes, such as histone deacetylases (HDACs), methyl-CpG binding domain proteins (MBDs), and transcription factors correlated to hematopoietic stem cell differentiation and proliferation. Our results show that 8/25 (34%) of our lower-risk MDS patients, showing hematologic improvements after azacitidine therapy, had a significant increase in PI-PLCbeta1 expression, as compared with the amount of the pre-treatment period, thus confirming the involvement of this molecule in the response to demethylating agents. As for the remaining patients, mainly showing a stable disease, we observed slight increases or almost constant levels of PI-PLCbeta1 expression. Moreover, ongoing analyses are trying to disclose whether lower-risk MDS patients responding to azacitidine show a specific molecular epigenetic profile during the regulation of methylation processes. Taken together, our data suggest a correlation between azacitidine treatment and PI-PLCbeta1 signalling even in lower-risk MDS, thus hinting at a role for PI-PLCbeta1 in the evaluation of patients likely to respond to azacitidine and paving the way for the development of innovative therapeutic strategies in lower-risk MDS patients
Effect of Erythropoietin Treatment on Lipid Signalling Pathways in Low-Risk MDS Patients.
No full textIntroduction: Phosphoinositide-phospholipase (PI-PLC) C beta1, PI-PLCgamma1 and Akt are key enzymes in nuclear signal transduction pathways, affecting both cell cycle and differentiation in normal physiology and neoplastic transformation. Our group previously showed not only that the Akt/mTOR axis is activated in patients with high-risk MDS (Follo MY et al, Cancer Res 2007), but also that there is an inverse correlation between PI-PLCbeta1 expression and Akt activation (Follo MY et al, Leukemia 2008). Moreover, we recently demonstrated that patients belonging to all of the IPSS risk groups can display a PI-PLCbeta1 mono-allelic deletion, and that this cytogenetic alteration is associated with a higher risk of evolution into Acute Myeloid Leukemia (AML) (Follo MY et al, J Clin Oncol 2009). Erythropoietin (EPO) is an effective treatment of anemia in 40-60% of low-risk MDS, often inducing a prolonged response. Interestingly, the activation of the EPO receptor has been correlated to the PI3K/Akt axis, which in turn is linked to either PI-PLCbeta1 or PI-PLCgamma1 signalling, so that EPO could affect cell proliferation and apoptosis. The aim of this study was therefore to clarify the relationship between EPO treatment and lipid signalling pathways, to investigate their role as molecular targets or predictive factors during EPO therapy. In fact, in patients who are refractory or lose response to EPO there could be a specific activation or inhibition of pathways involved in both cell cycle and differentiation.
Patients and Methods: In this study we examined the effect of EPO treatment on lipid signal transduction pathways in MDS patients. The study included 16 patients (IPSS risk: low or intermediate-1), with a favourable response to EPO in 8/16 (50%) of the cases. For each patient we had the opportunity to analyze the expression of PI-PLCbeta1, PI-PLCgamma1, p-Akt and PIP2, which is involved in both PI-PLCbeta1 and PI3K/Akt activation processes, before and during EPO treatment, in order to detect every change in both clinical and biological features. By FISH analysis, we firstly assessed the presence of PI-PLCbeta1 mono-allelic deletion. Then, we quantified PI-PLCbeta1 and PI-PLCgamma1 gene and protein expression, as well as PIP2 and the degree of Akt activation; mRNA levels were quantified by real-time PCR, whereas the protein amount was detected by both a immunocytochemical and a flow cytometric detection approach.
Results: The PI-PLCbeta1 mono-allelic deletion was found in 5/16 (31%) low-risk MDS patients: 2 of them showed a rapid evolution into AML, whilst the remaining 3 cases did not respond to EPO treatment. The molecular analyses showed a specific increase in Akt/PI-PLCgamma1 pathway for responder patients, whereas most of the patients refactory to EPO displayed a slight decrease in p-Akt levels and an activation of PI-PLCbeta1 signalling during EPO administration, so that these patients seem to counteract the lack of one PI-PLCbeta1 allele by increasing PI-PLCbeta1 gene and protein expression.
Conclusions: Our results, although obtained in a small number of cases, confirm the possible involvement of PI-PLCbeta1 pathways in the EPO signalling. Moreover, our data suggest that the presence of the PI-PLCbeta1 mono-allelic deletion is associated with a worse clinical outcome and with a lack of response to EPO treatment, even in low-risk MDS patients who apparently have a good response profile for EPO (recent diagnosis, absence of long-term transfusion dependence, low or intermediate-1 IPSS risk, serum EPO levels<500 U/L). In fact, in our series, patients with the PI-PLCbeta1 mono-allelic deletion showed an unfavourable outcome (either a rapid evolution into AML or refractoriness to EPO treatment). Moreover, our findings indicate that not only PI-PLCbeta1, but also Akt/PI-PLCgamma1 pathways are critical for cell survival and proliferation in MDS patients treated with EPO. Therefore, these signal transduction pathways could become in the future an important target for the development of innovative strategies for MDS
Nuclear phospholipase C isoenzyme imbalance leads to pathologies in brain, hematologic, neuromuscular, and fertility disorders
No full textPhosphoinositide-specific phospholipases C (PI-PLCs) are involved in signaling pathways related to critical cellular functions, such as cell cycle regulation, cell differentiation, and gene expression. Nuclear PI-PLCs have been studied as key enzymes, molecular targets, and clinical prognostic/diagnostic factors in many physiopathologic processes. Here, we summarize the main studies about nuclear PI-PLCs, specifically, the imbalance of isozymes such as PI-PLC1 and PI-PLC, in cerebral, hematologic, neuromuscular, and fertility disorders. PI-PLC1 and PI-PLC1 affect epilepsy, depression, and bipolar disorder. In the brain, PI-PLC1 is involved in endocannabinoid neuronal excitability and is a potentially novel signature gene for subtypes of high-grade glioma. An altered quality or quantity of PI-PLC contributes to sperm defects that result in infertility, and PI-PLC1 aberrant inositide signaling contributes to both hematologic and degenerative muscle diseases. Understanding the mechanisms behind PI-PLC involvement in human pathologies may help identify new strategies for personalized therapies of these conditions.—Ratti, S., M. Y. Follo, G. Ramazzotti, I. Faenza, R. Fiume, P-G. Suh, J. A. McCubrey, L. Manzoli, and L. Cocco. Nuclear phospholipase C isoenzyme imbalance leads to pathologies in brain, hematologic, neuromuscular, and fertility disorders
Protein kinase C involvement in cell cycle modulation
No full textProtein kinases C (PKCs) are a family of serine/threonine kinases which act as key regulators in cell cycle progression and differentiation. Studies of the involvement of PKCs in cell proliferation showed that their role is dependent on cell models, cell cycle phases, timing of activation and localization. Indeed, PKCs can positively and negatively act on it, regulating entry, progression and exit from the cell cycle. In particular, the targets of PKCs resulted to be some of the key proteins involved in the cell cycle including cyclins, cyclin-dependent kinases (Cdks), Cip/Kip inhibitors and lamins. Several findings described roles for PKCs in the regulation of G1/S and G2/M checkpoints. As a matter of fact, data from independent laboratories demonstrated PKC-related modulations of cyclins D, leading to effects on the G1/S transition and differentiation of different cell lines. Moreover, interesting data were published on PKC-mediated phosphorylation of lamins. In addition, PKC isoenzymes can accumulate in the nuclei, attracted by different stimuli including diacylglycerol (DAG) fluctuations during cell cycle progression, and target lamins, leading to their disassembly at mitosis. In the present paper, we briefly review how PKCs could regulate cell proliferation and differentiation affecting different molecules related to cell cycle progression
- …
