Gianpiero Pescarmona

Mode of antimalarial effect of methylene blue and some of its analogues on Plasmodium falciparum in culture and their inhibition of P. vinckei petteri and P. yoelii nigeriensis in vivo

The antimalarial action of methylene blue (MB) was first noted by Paul Ehrlich in the late 19th century. Although it has only sporadically been adopted as a serviceable drug, the resolution of its antimalarial action seems warranted, as it is currently used for the treatment of various methemoglobinemias. In this work we have used MB, and its analogues Azures A (AZA), B (AZB), C (AZC), and thionin (TH), as well as the oxazine Celestine blue (CB) and azine Phenosaphranin (PS). All MB analogues inhibit the growth of various strains of Plasmodium falciparum in culture with IC50s in the 2 x 10(-9)-1 x 10(-7) M range, with the rank order MB approximately AZA > AZB > AZC > TH > PS > CB. The IC50s for a mammalian cell line were in the 3 x 10(-6)-4 x 10(-5) M range, and the rank order was TH approximately AZB > AZA approximately PS > AZC approximately CB > MB. As MB could affect cell growth through the oxidation of NADPH, we tested the action of the various compounds on the hexose-monophosphate shunt activity. Appreciable activation of the shunt was observed at 1 x 10(-5) M in both cell types, thus accounting for inhibition of growth of mammalian cells but not of parasites. All compounds were found to complex with heme in a rank order similar to their antimalarial effect. It is therefore suggested that MB and its congeners act by preventing the polymerization of heme, which is produced during the digestion of host cell cytosol in the parasite food vacuole, into hemozoin. In this respect, these compounds seem to act similarly to the 4-aminoquinoline antimalarials. All compounds effectively suppressed the growth of P. vinckei petteri in vivo with IC50 in the 1.2-5.2 mg/kg range, and MB and AZB suppressed P. yoelii nigeriensis in the 9-11 mg/kg range (i.e. at doses similar to those of chloroquine). The potential toxicity of these compounds may restrict their clinical use, but their impressive antimalarial activities suggest that the phenothiazine structure could serve as a lead compound for further drug development.

Free cytoplasmic Ca++ at rest and after cholinergic stimulus is increased in cultured muscle cells from Duchenne muscular dystrophy patients

We used a fluorescent dye, quin 2, to measure intracellular free calcium ([Ca+ +]i) in cultured skeletal muscle cells and skin fibroblasts from five Duchenne muscular dystrophy (DMD) patients and from five controls. We observed an enhanced [Ca+ +]i level, at rest and after acetylcholine (ACh) stimulation, in DMD muscle cells, but we did not detect any difference between DMD and normal skin fibroblasts. The abnormally higher [Ca+ +]i transient induced by ACh suggests that it plays a critical role in muscle degeneration. The skin fibroblast results suggest that there is no generalized membrane defect.

Glioblastoma cancer stem cells: heterogeneity, microenvironment and related therapeutic strategies

Glioblastoma Multiforme (GBM) is an incurable malignancy. GBM patients have a short life expectancy despite aggressive therapeutic approaches based on surgical resection followed by adjuvant radiotherapy and concomitant chemotherapy. Glioblastoma growth is characterized by a high motility of tumour cells, their resistance to both chemo/radio‐therapy, apoptosis inhibition leading to failure of conventional therapy. Cancer Stem Cells (CSCs), identified in GBM as well as in many other cancer types, express the membrane antigen prominin‐1 (namely CD133). These cells and normal Neural Stem Cells (NSC) share surface markers and properties, i.e. are able to self‐renew and differentiate into multiple cell types. Stem cell self‐renewal depends on microenvironmental cues, including Extracellular Matrix (ECM) composition and cell types. Therefore, the role of microenvironment needs to be evaluated to clarify its importance in tumour initiation and progression through CSCs. The specific microenvironment of CSCs was found to mimic in part the vascular niche of normal stem cells. The targeting of GMB CSCs may represent a powerful treatment approach. Lastly, in GBM patients cancer‐initiating cells contribute to the profound immune suppression that in turn correlated with CSCs STAT3 (CD133 + ). Further studies of microenvironment are needed to better understand the origin of GMB/GBM CSCs and its immunosuppressive properties. Copyright

Mitochondrial Localization of Vitamin D Receptor in Human Platelets and Differentiated Megakaryocytes

Background Like other steroid hormones, vitamin D elicits both transcriptional events and rapid non genomic effects. Vitamin D receptor (VDR) localization and mechanisms of VDR-triggered non genomic responses are still controversial. Although anticoagulant effects of vitamin D have been reported and VDR signalling has been characterized in monocytes and vascular cells, nothing is known about VDR expression and functions in human platelets, anucleated fragments of megakaryocytes which are known targets of other steroids. Methodology/Principal Findings In this study we characterized the expression and cellular localization of VDR in human platelets and in a megakaryocyte lineage. Human platelets and their TPA-differentiated precursors expressed a classical 50 kDa VDR protein, which increased with megakaryocytes maturation. By biochemical fractionation studies we demonstrated the presence of the receptor in the soluble and mitochondrial compartment of human platelets, and the observation was confirmed by immunoelectron microscopy analysis. Similar localization was found in mature megakaryocytes, where besides its classical nuclear localization the receptor was evident as soluble and mitochondria resident protein. Conclusions The results reported here suggest that megakaryocytopoiesis and platelet activation, which are calcium-dependent events, might be modulated by a mitochondrial non genomic activity of VDR. These data open challenging future studies on VDR physiological role in platelets and more generally in mitochondria.

Acetylcholine and Dopamine Promote the Production of Platelet Activating Factor in Immature Cells of Chick Embryonic Retina

Abstract: We have previously shown that platelet‐activating factor (PAF), a naturally occurring lipid mediator of cell‐to‐cell communication, was produced by 3‐day‐old chick retina stimulated with acetylcholine (ACh) and dopamine (DA), but not with other neurotransmitters. ACh and DA stimulated PAF synthesis via a dithiothreitol (DTT)‐insensitive choline‐phosphotransferase, without affecting the acetyltransferase pathway, which was stimulated only by the calcium ionophore A23187. Therefore, we attempted to study the effects of neurotransmitters on PAF production and on the activities of the DTT‐insensitive cholinephosphotransferase and acetyltransferase in the developing chick embryo retina up to hatching. Our results show that PAF was produced already at 8 days of development, when retinal cells are still rather immature and ganglion and Mueller cells are the only differentiated cells. The stimulation of PAF production occurred with ACh and not with other neurotransmitters. In older stages, DA also stimulated PAF production, as already described in the chick after hatching. DTT‐insensitive cholinephosphotransferase and acetyltransferase activities were present in 8‐day‐old embryos, the earliest stage analyzed. Both enzymatic activities increased with age; DTT‐insensitive cholinephosphotransferase increased rapidly from day 12 up to day 18, whereas acetyltransferase activity increased linearly up to the time of hatching. To promote PAF production, ACh and DA activate DTT‐insensitive cholinephosphotransferase, but not acetyltransferase. The developmental sequence of PAF stimulation suggests that PAF production occurs through stimulation of ACh and D2‐dopamine receptors, which are mostly present in ganglion and amacrine cells. Synaptic maturation enhances the effects of ACh and DA. Our data taken together suggest that PAF may play a physiological role after the time of hatching, since light stimulation induces neurotransmitter release which can eventually lead to PAF production.

Activation of Nuclear Factor-κB Pathway by Simvastatin and RhoA Silencing Increases Doxorubicin Cytotoxicity in Human Colon Cancer HT29 Cells

Doxorubicin efficacy in cancer therapy is hampered by the dose-dependent side effects, which may be overcome by reducing the drugs dose and increasing its efficacy. In the present work, we suggest that the activation of the nuclear factor-κB (NF-κB) pathway and of nitric-oxide (NO) synthase increases the doxorubicin efficacy in human colon cancer HT29 cells. To induce NF-κB, we took into account the effect of doxorubicin itself and of the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor simvastatin; as NF-κB inhibitors, we chose the sesquiterpene lactones parthenolide and artemisinin. Simvastatin increased the NF-κB activity and NO synthesis, elicited the tyrosine nitration of the multidrug resistance-related protein 3, and enhanced the doxorubicin intracellular accumulation and cytotoxicity. Simvastatin potentiated the effect of doxorubicin on the NF-κB pathway and the inducible NO synthase expression. The effects of simvastatin were due to the inhibition of the small G-protein RhoA and of its effector Rho kinase. Parthenolide and artemisinin prevented all of the statin effects by inducing RhoA/Rho kinase activation. On the other hand, they did not reduce the NF-κB translocation and doxorubicin intracellular content when RhoA was silenced by small interfering RNA (siRNA). It is interesting that RhoA siRNA was sufficient to increase NF-κB translocation, NO synthase activity, doxorubicin accumulation, and cytotoxicity also in non-stimulated cells. Our results suggest that artemisinin, a widely used antimalarial drug, may impair the response to doxorubicin in colon cancer cells; on the contrary, simvastatin and RhoA siRNA may represent future therapeutic approaches to improve doxorubicin efficacy, reducing the risk of doxorubicin-dependent adverse effects.

Role of Na+/H+ exchange in thrombin- and arachidonic acid-induced Ca2+ influx in platelets

Platelet activation is accompanied by an increase of cytosolic free Ca2+ concentration, [Ca2+]i, (due to both extracellular Ca2+ influx and Ca2+ movements from the dense tubular system) and an Na+ influx associated with H+ extrusion. The latter event is attributable to the activation of Na+/H+ exchange, which requires Na+ in the extracellular medium and is inhibited by amiloride and its analogs. The present study was carried out to determine whether a link exists between Ca2+ transients (measured by the quin2 method and the 45CaCl2 technique) and Na+/H+ exchange activation (studied with the pH-sensitive intracellular probe, 6-carboxyfluorescein) during platelet stimulation. Washed human platelets, stimulated with thrombin and arachidonic acid, showed: (1) a large and rapid [Ca2+]i rise, mostly due to a Ca2+ influx through the plasma membrane; (2) a marked intracellular alkalinization. Both phenomena were markedly inhibited in the absence of extracellular Na+ or in the presence of an amiloride analog (EIPA). Monensin, a cation exchanger which elicits Na+ influx and alkalinization, and NH4Cl, which induces alkalinization only, were able to evoke an increase in [Ca2+]i, mostly as an influx from the extracellular medium. Our results suggest that Ca2+ influx induced by thrombin and arachidonic acid in human platelets is strictly dependent on Na+/H+-exchange activation.

Protein kinase C and cyclic AMP modulate thrombin-induced platelet-activating factor synthesis in human endothelial cells

Stimulation of human endothelial cells (EC) by thrombin elicits a rapid increase of intracellular free Ca2+ [(Ca2+]i), platelet-activating factor (PAF) production and 1-O-alkyl-2-lyso-sn-glycero-3- phosphocholine (lyso-PAF): acetyl-CoA acetyltransferase (EC 2.3.1.67) activity. The treatment of EC with thrombin leads to a 90% decrease in the cytosolic protein kinase C (PKC) activity; this dramatic decline is accompanied by an increase of the enzymatic activity in the particulate fraction. The role of PKC in thrombin-mediated PAF synthesis has been assessed: (1) by the blockade of PKC activity with partially selective inhibitors (palmitoyl-carnitine, sphingosine and H-7); (2) by chronic exposure of EC to phorbol 12-myristate 13-acetate (PMA), which results in down-regulation of PKC. In both cases, a strong inhibition of thrombin-induced PAF production is observed, suggesting obligatory requirement of PKC activity for PAF synthesis. It is suggested that PKC regulates EC phospholipase A2 (PLA2) activity as thrombin-induced arachidonic acid (AA) release is 90% inhibited in PKC-depleted cells. Brief exposure of EC to PMA strongly inhibits thrombin-induced [Ca2+]i rise, acetyltransferase activation and PAF production, suggesting that, in addition to the positive forward action, PKC provides a negative feedback control over membrane signalling pathways involved in the thrombin effect on EC. Forskolin and iloprost, two agents that increase the level of cellular cAMP in EC, are very effective in inhibiting thrombin-evoked cytosolic Ca2+ rise, acetyltransferase activation and PAF production; this suggests that endogenously generated prostacyclin (PGI2) may modulate the synthesis of PAF in human endothelial cells.

RhoA Silencing Reverts the Resistance to Doxorubicin in Human Colon Cancer Cells

The efficacy of doxorubicin in the treatment of cancer is limited by its side effects and by the onset of drug resistance. Reverting such resistance could allow the decrease of the dose necessary to eradicate the tumor, thus diminishing the toxicity of the drug. We transfected doxorubicin-sensitive (HT29) and doxorubicin-resistant (HT29-dx) human colon cancer cells with RhoA small interfering RNA. The subsequent decrease of RhoA protein was associated with the increased sensitivity to doxorubicin in HT29 cells and the complete reversion of doxorubicin resistance in HT29-dx cells. RhoA silencing increased the activation of the nuclear factor-κB pathway, inducing the transcription and the activity of nitric oxide synthase. This led to the tyrosine nitration of the multidrug resistance protein 3 transporter (MRP3) and contributed to a reduced doxorubicin efflux. Moreover, RhoA silencing decreased the ATPase activity of P-glycoprotein (Pgp) in HT29 and HT29-dx cells as a consequence of the reduced expression of Pgp. RhoA silencing, by acting as an upstream controller of both MRP3 nitration and Pgp expression, was effective to revert the toxicity and accumulation of doxorubicin in both HT29 and HT29-dx cells. Therefore, we suggest that inactivating RhoA has potential clinical applications and might in the future become part of a gene therapy protocol. (Mol Cancer Res 2008;6(10):1607–20)

Modulation of doxorubicin resistance by the glucose-6-phosphate dehydrogenase activity.

How anti-neoplastic agents induce MDR (multidrug resistance) in cancer cells and the role of GSH (glutathione) in the activation of pumps such as the MRPs (MDR-associated proteins) are still open questions. In the present paper we illustrate that a doxorubicin-resistant human colon cancer cell line (HT29-DX), exhibiting decreased doxorubicin accumulation, increased intracellular GSH content, and increased MRP1 and MRP2 expression in comparison with doxorubicin-sensitive HT29 cells, shows increased activity of the PPP (pentose phosphate pathway) and of G6PD (glucose-6-phosphate dehydrogenase). We observed the onset of MDR in HT29 cells overexpressing G6PD which was accompanied by an increase in GSH. The G6PD inhibitors DHEA (dehydroepiandrosterone) and 6-AN (6-aminonicotinamide) reversed the increase of G6PD and GSH and inhibited MDR both in HT29-DX cells and in HT29 cells overexpressing G6PD. In our opinion, these results suggest that the activation of the PPP and an increased activity of G6PD are necessary to some MDR cells to keep the GSH content high, which is in turn necessary to extrude anticancer drugs out of the cell. We think that our data provide a new further mechanism for GSH increase and its effects on MDR acquisition.