Marcel Benadiba

Gamma-linolenic acid inhibits both tumour cell cycle progression and angiogenesis in the orthotopic C6 glioma model through changes in VEGF, Flt1, ERK1/2, MMP2, cyclin D1, pRb, p53 and p27 protein expression.

BackgroundGamma-linolenic acid is a known inhibitor of tumour cell proliferation and migration in both in vitro and in vivo conditions. The aim of the present study was to determine the mechanisms by which gamma-linolenic acid (GLA) osmotic pump infusion alters glioma cell proliferation, and whether it affects cell cycle control and angiogenesis in the C6 glioma in vivo.MethodsEstablished C6 rat gliomas were treated for 14 days with 5 mM GLA in CSF or CSF alone. Tumour size was estimated, microvessel density (MVD) counted and protein and mRNA expression measured by immunohistochemistry, western blotting and RT-PCR.ResultsGLA caused a significant decrease in tumour size (75 ± 8.8%) and reduced MVD by 44 ± 5.4%. These changes were associated with reduced expression of vascular endothelial growth factor (VEGF) (71 ± 16%) and the VEGF receptor Flt1 (57 ± 5.8%) but not Flk1. Expression of ERK1/2 was also reduced by 27 ± 7.7% and 31 ± 8.7% respectively. mRNA expression of matrix metalloproteinase-2 (MMP2) was reduced by 35 ± 6.8% and zymography showed MMP2 proteolytic activity was reduced by 32 ± 8.5%. GLA altered the expression of several proteins involved in cell cycle control. pRb protein expression was decreased (62 ± 18%) while E2F1 remained unchanged. Cyclin D1 protein expression was increased by 42 ± 12% in the presence of GLA. The cyclin dependent kinase inhibitors p21 and p27 responded differently to GLA, p27 expression was increased (27 ± 7.3%) while p21 remained unchanged. The expression of p53 was increased (44 ± 16%) by GLA. Finally, the BrdU incorporation studies found a significant inhibition (32 ± 11%) of BrdU incorporation into the tumour in vivo.ConclusionOverall the findings reported in the present study lend further support to the potential of GLA as an inhibitor of glioma cell proliferation in vivo and show it has direct effects upon cell cycle control and angiogenesis. These effects involve changes in protein expression of VEGF, Flt1, ERK1, ERK2, MMP2, Cyclin D1, pRb, p53 and p27. Combination therapy using drugs with other, complementary targets and GLA could lead to gains in treatment efficacy in this notoriously difficult to treat tumour.

Inhibition of C6 rat glioma proliferation by [Ru2Cl(Ibp)4] depends on changes in p21, p27, Bax/Bcl2 ratio and mitochondrial membrane potential

The ruthenium compound [Ru(2)Cl(Ibp)(4)] (or RuIbp) has been reported to cause significantly greater inhibition of C6 glioma cell proliferation than the parent HIbp. The present study determined the effects of 0-72h exposure to RuIbp upon C6 cell cycle distribution, mitochondrial membrane potential, reactive species generation and mRNA and protein expression of E2F1, cyclin D1, c-myc, pRb, p21, p27, p53, Ku70, Ku80, Bax, Bcl2, cyclooxygenase 1 and 2 (COX1 and COX2). The most significant changes in mRNA and protein expression were seen for the cyclin-dependent kinase inhibitors p21 and p27 which were both increased (p<0.05). The marked decrease in mitochondrial membrane potential (p<0.01) and modest increase in apoptosis was accompanied by a decrease in anti-apoptotic Bcl2 expression and an increase in pro-apoptotic Bax expression (p<0.05). Interestingly, COX1 expression was increased in response to a significant loss of prostaglandin E(2) production (p<0.001), most likely due to the intracellular action of Ibp. Future studies will investigate the efficacy of this novel ruthenium-ibuprofen complex in human glioma cell lines in vitro and both rat and human glioma cells growing under orthotopic conditions in vivo.

Gamma-linolenic acid alters Ku80, E2F1, and bax expression and induces micronucleus formation in C6 glioma cells in vitro.

Gamma‐linolenic acid (GLA) is an inhibitor of tumor cell proliferation in both in vitro and in vivo conditions. The aim of this study was to investigate the effects of 150 μM GLA on the expression of E2F1, cyclin D1, bax, bcl2, Ku70, and Ku80 in C6 rat glioma cells. The Ku proteins were chosen as previous studies have shown that loss or reduction in their expression causes increased DNA damage and micronucleus formation in the presence of radiation. The fact that GLA exposure is known to enhance the efficacy of radiation treatment raised the question whether the Ku proteins could be involved in this effect as seen for other molecules such as roscovitine and flavopiridol. GLA altered the mRNA expression of E2F1, cyclin D1, and bax, but no changes were found for bcl2, Ku70, and Ku80. Alterations in protein expression were observed for bax, Ku80, and E2F1. The 45% decrease in E2F1 expression was proportional to decreased cell proliferation (44%). Morphological analysis found a 25% decrease in mitotic activity in the GLA‐treated cells, which was accompanied by a 49% decrease in S‐phase by FACS analysis. A 39% increase in the number of micronuclei detected by Hoechst fluorescence points to GLAs effects on cell division even at concentrations that do not produce significant increases in apoptosis. Most important was the finding that Ku80 expression, a critical protein involved in DNA repair as a heterodimer with Ku70, was decreased by 71%. It is probable that reduced Ku80 is responsible for the increase in micronucleus formation in GLA‐treated cells in a similar manner to that found in Ku80 null cells exposed to radiation. The decreased expression of Ku80 and E2F1 could make cells more susceptible to radiotherapy and chemotherapy.

The novel ruthenium-gamma-linolenic complex [Ru(2)(aGLA)(4)Cl] inhibits C6 rat glioma cell proliferation and induces changes in mitochondrial membrane potential, increased reactive oxygen species generation and apoptosis in vitro.

The present study reports the synthesis of a novel compound with the formula [Ru2(aGLA)4Cl] according to elemental analyses data, referred to as Ru2GLA. The electronic spectra of Ru2GLA is typical of a mixed valent diruthenium(II,III) carboxylate. Ru2GLA was synthesized with the aim of combining and possibly improving the anti‐tumour properties of the two active components ruthenium and γ‐linolenic acid (GLA). The properties of Ru2GLA were tested in C6 rat glioma cells by analysing cell number, viability, lipid droplet formation, apoptosis, cell cycle distribution, mitochondrial membrane potential and reactive oxygen species. Ru2GLA inhibited cell proliferation in a time and concentration dependent manner. Nile Red staining suggested that Ru2GLA enters the cells and ICP‐AES elemental analysis found an increase in ruthenium from <0.02 to 425 mg/Kg in treated cells. The sub‐G1 apoptotic cell population was increased by Ru2GLA (22 ± 5.2%) when analysed by FACS and this was confirmed by Hoechst staining of nuclei. Mitochondrial membrane potential was decreased in the presence of Ru2GLA (44 ± 2.3%). In contrast, the cells which maintained a high mitochondrial membrane potential had an increase (18 ± 1.5%) in reactive oxygen species generation. Both decreased mitochondrial membrane potential and increased reactive oxygen species generation may be involved in triggering apoptosis in Ru2GLA exposed cells. The EC50 for Ru2GLA decreased with increasing time of exposure from 285 µM at 24 h, 211 µM at 48 h to 81 µM at 72 h. In conclusion, Ru2GLA is a novel drug with antiproliferative properties in C6 glioma cells and is a potential candidate for novel therapies in gliomas. Copyright

(11)C- and (18)F-Labeled Radioligands for P-Glycoprotein Imaging by Positron Emission Tomography.

P‐Glycoprotein (P‐gp) is an efflux transporter widely expressed at the human blood–brain barrier. It is involved in xenobiotics efflux and in onset and progression of neurodegenerative disorders. For these reasons, there is great interest in the assessment of P‐gp expression and function by noninvasive techniques such as positron emission tomography (PET). Three radiolabeled aryloxazole derivatives: 2‐[2‐(2‐methyl‐(11C)‐5‐methoxyphenyl)oxazol‐4‐ylmethyl]‐6,7‐dimethoxy‐1,2,3,4‐tetrahydroisoquinoline ([11C]‐5); 2‐[2‐(2‐fluoromethyl‐(18F)‐5‐methoxyphenyl)oxazol‐4‐ylmethyl]‐6,7‐dimethoxy‐1,2,3,4‐tetra‐hydroisoquinoline ([18F]‐6); and 2‐[2‐(2‐fluoroethyl‐(18F)‐5‐methoxyphenyl)oxazol‐4‐ylmethyl]‐6,7‐dimethoxy‐1,2,3,4‐tetrahydroisoquinoline ([18F]‐7), were tested in several in vitro biological assays to assess the effect of the aryl substituent in terms of potency and mechanism of action toward P‐gp. Methyl derivative [11C]‐5 is a potent P‐gp substrate, whereas the corresponding fluoroethyl derivative [18F]‐7 is a P‐gp inhibitor. Fluoromethyl compound [18F]‐6 is classified as a non‐transported P‐gp substrate, because its efflux increases after cyclosporine A modulation. These studies revealed a promising substrate and inhibitor, [11C]‐5 and [18F]‐7, respectively, for in vivo imaging of P‐gp by using PET.

New Perspectives in Nuclear Neurology for the Evaluation of Parkinson's Disease

The pathophysiology of Parkinsons disease (PD) has not yet been completely elucidated. However, during the past few years, significant progress has been made in understanding the intra- and extracellular mechanisms by which proteins such as alpha-synuclein and neuroinflammatory molecules may display impaired function and/or expression in PD. Recent developments in imaging techniques based on positron emission tomography (PET) and single photon emission computed tomography (SPECT) now allow the non-invasive tracking of such molecular targets of known relevance to PD in vivo. This article summarizes recent PET and SPECT studies of new radiopharmaceuticals and discusses their potential role and perspectives for use in the fields of new drug development and early diagnosis for PD, as well to aid in differential diagnosis and monitoring of the progression of PD.

PET Imaging of ABC Transporters in the BBB

Neuroscience has never been so close to early diagnosis of complex neurological disorders like Alzheimer’s and Parkinson’s disease. As a consequence, new treatments can now be designed and pharmacotherapy be prescribed with better promise of a favorable clinical outcome, especially for resistant neurodegenerative diseases. Protein expression and function (particularly of membrane-bound receptors or enzymes) can be imaged and quantified in a noninvasive manner with positron emission tomography (PET). This technique can also be applied to the ABC transporter family of proteins, among which P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) have been major topics of study. [11C]Verapamil, for example, is a widely used radiotracer to assess P-gp functionality in the blood–brain barrier (BBB) using PET. However, significant limitations of this tracer have led to the search for new radiopharmaceuticals for exploring BBB transporter function. This search aims to identify an optimal tracer to specifically assess P-gp function and expression at the BBB. Translating in vitro results to the in vivo situation may be difficult. Thus, studies in different animal species and strains are of great value. Transporter imaging may help clinicians with a more reliable early diagnosis of neurodegenerative syndromes such as Alzheimer’s and Parkinson’s disease with greater accuracy, preferably in a presymptomatic stage of the disease and before pharmacoresistance has developed.