A Potential Boron Neutron Capture Therapy Agent Selectively Suppresses High-Grade Glioma: In Vitro and in Vivo Exploration.

Glioblastoma (GBM), as the most central nervous system (CNS) intractable disease, has spoiled millions of lives due to its high mortality. Even though several efforts have been made, the existing treatments have had limited success. In this sense, we studied a lead compound, the boron-rich selective epidermal growth factor receptor (EGFR)-inhibitor hybrid 1, as a potential drug for GBM treatment. For this end, we analyzed the in vitro activity of hybrid 1 in a glioma/primary astrocytes coculture, studying cellular death types triggered by treatment with this compound and its cellular localizations. Additionally, hybrid 1 concentrated boron in glioma cells selectively and more effectively than the boron neutron capture therapy (BNCT)-clinical agent 10B-l-boronophenylalanine and thus displayed a better in vitro-BNCT effect. This encouraged us to analyze hybrid 1 in vivo. Therefore, immunosuppressed mice bearing U87 MG human GBM were treated with both 1 and 1 encapsulated in a modified liposome (recognized by brain-blood barrier peptide transporters), and we observed a potent in vivo per se antitumor activity (tumor size decrease and animal survival increase). These data demonstrate that 1 could be a promising new targeted therapy for GBM.

Liposome-based targeting of dopamine to the brain: a novel approach for the treatment of Parkinson's disease.

Delivery of drugs into the brain is poor due to the blood brain barrier (BBB). This study describes the development of a novel liposome-based brain-targeting drug delivery system. The liposomes incorporate a diacylglycerol moiety coupled through a linker to a peptide of 5 amino acids selected from amyloid precursor protein (APP), which is recognized by specific transporter(s)/receptor(s) in the BBB. This liposomal system enables the delivery of drugs across the BBB into the brain. The brain-directed liposomal system was used in a mouse model of Parkinson's disease (PD). Intra-peritoneal (IP) administration of liposomes loaded with dopamine (DA) demonstrated a good correlation between liposomal DA dose and the behavioral effects in hemiparkinsonian amphetamine-treated mice, with an optimal DA dose of 60 µg/kg. This is significantly lower dose than commonly used doses of the DA precursor levodopa (in the mg/kg range). IP injection of the APP-targeted liposomes loaded with a DA dose of 800 µg/kg, resulted in a significant increase in striatal DA within 5 min (6.9-fold, p < 0.05), in amphetamine-treated mice. The increase in striatal DA content persisted for at least 3 h after administration, which indicates a slow DA release from the delivery system. No elevation in DA content was detected in the heart or the liver. Similar increases in striatal DA were observed also in rats and mini-pigs. The liposomal delivery system enables penetration of compounds through the BBB and may be a candidate for the treatment of PD and other brain diseases.

Liposomal Carrier Conjugated to APP-Derived Peptide for Brain Cancer Treatment.

Brain tumors are hard to treat with the currently available therapy. The major obstacle in the treatment of brain tumors is the lack of therapeutic strategies capable to penetrate the blood-brain barrier (BBB). The BBB is an endothelial interface that separates the brain from the circulatory blood system and prevents the exposure of the central nervous system (CNS) to circulating toxins and potentially harmful compounds. Unfortunately, the BBB prevents also the penetration of therapeutic compounds into the brain. We present here a drug-delivery liposomal carrier, conjugated to a peptide inserted in the liposomal membrane, which is putatively recognized by BBB transporters. The peptide is a short sequence of 5 amino acids (RERMS) present in the amyloid precursor protein (APP). This APP-targeted liposomal system was designed specifically for transporting compounds with anti-cancer activity via the BBB into the brain in an effective manner. This drug-delivery liposomal carrier loaded with the anti-cancer compounds temozolomide (TMZ), curcumin, and doxorubicin crossed the BBB in an in vitro model as well as in vivo (mice model). In the in vitro model, the targeted liposomes crossed the BBB model fourfold higher than the non-targeted liposomes. Labeled targeted liposomes penetrated the brain in vivo 35% more than non-targeted liposomes. Treatment of mice that underwent intracranial injection of human U87 glioblastoma, with the targeted liposomes loaded with the three tested anti-cancer agents, delayed the tumor growth and prolonged the mice survival in a range of 45% -70%. It appears that the targeted liposomal drug-delivery system enables better therapeutic efficacy in a SCID mouse model of glioblastoma compared to the corresponding non-targeted liposomes and the free compounds.

The TSPO Ligands 2-Cl-MGV-1, MGV-1, and PK11195 Differentially Suppress the Inflammatory Response of BV-2 Microglial Cell to LPS.

The 18 kDa Translocator Protein (TSPO) is a marker for microglial activation as its expression is enhanced in activated microglia during neuroinflammation. TSPO ligands can attenuate neuroinflammation and neurotoxicity. In the present study, we examined the efficacy of new TSPO ligands designed by our laboratory, MGV-1 and 2-Cl-MGV-1, in mitigating an in vitro neuroinflammatory process compared to the classic TSPO ligand, PK 11195. We exposed BV-2 microglial cells to lipopolysaccharide (LPS) for 24 h to induce inflammatory response and added the three TSPO ligands: (1) one hour before LPS treatment (pretreatment), (2) simultaneously with LPS (cotreatment), and (3) one hour after LPS exposure (post-treatment). We evaluated the capability of TSPO ligands to reduce the levels of three glial inflammatory markers: cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and nitric oxide (NO). We compared the effects of the two novel ligands to PK 11195. Both 2-Cl-MGV-1 and MGV-1 reduced the levels of glial COX-2, iNOS, and NO in LPS-treated BV-2 cells more efficiently than PK 11195. Notably, even when added after exposure to LPS, all ligands were able to suppress the inflammatory response. Due to their pronounced anti-inflammatory activity, 2-Cl-MGV-1 and MGV-1 may serve as potential therapeutics in neuroinflammatory and neurodegenerative diseases.

2-Cl-MGV-1 Ameliorates Apoptosis in the Thalamus and Hippocampus and Cognitive Deficits After Cortical Infarct in Rats.

BACKGROUND AND PURPOSE: Focal cortical infarction causes neuronal apoptosis in the ipsilateral nonischemic thalamus and hippocampus, which is potentially associated with poststroke cognitive deficits. TSPO (translocator protein) is critical in regulating mitochondrial apoptosis pathways. We examined the effects of the novel TSPO ligand 2-(2-chlorophenyl) quinazolin-4-yl dimethylcarbamate (2-Cl-MGV-1) on poststroke cognitive deficits, neuronal mitochondrial apoptosis, and secondary damage in the ipsilateral thalamus and hippocampus after cortical infarction. METHODS: One hundred fourteen hypertensive rats underwent successful distal middle cerebral artery occlusion (n=76) or sham procedures (n=38). 2-Cl-MGV-1 or dimethyl sulfoxide as vehicle was administrated 2 hours after distal middle cerebral artery occlusion and then for 6 or 13 days (n=19 per group). Spatial learning and memory were tested using the Morris water maze. Secondary degeneration and mitochondrial apoptosis in the thalamus and hippocampus were assessed using Nissl staining, immunohistochemistry, terminal deoxynucleotidyl transferase dUTP nick end labeling, JC-1 staining, and immunoblotting 7 and 14 days after surgery. RESULTS: Infarct volumes did not significantly differ between the vehicle and 2-Cl-MGV-1 groups. There were more neurons and fewer glia in the ipsilateral thalamus and hippocampus in the vehicle groups than in the sham-operated group 7 and 14 days post-distal middle cerebral artery occlusion. 2-Cl-MGV-1 significantly ameliorated spatial cognitive impairment and decreased neuronal death and glial activation when compared with vehicle treatment (P<0.05). The collapse of mitochondrial transmembrane potential and cytoplasmic release of apoptosis-inducing factors and cytochrome c was prevented within the thalamus. Caspase cleavage and the numbers of terminal deoxynucleotidyl transferase dUTP nick end labeling+ or Nissl atrophic cells were reduced within the thalamus and hippocampus. This was accompanied by upregulation of B-cell lymphoma 2 and downregulation of Bax (P<0.05). CONCLUSIONS: 2-Cl-MGV-1 reduces neuronal apoptosis via mitochondrial-dependent pathways and attenuates secondary damage in the nonischemic thalamus and hippocampus, potentially contributing to ameliorated cognitive deficits after cortical infarction.

Classical and Novel TSPO Ligands for the Mitochondrial TSPO Can Modulate Nuclear Gene Expression: Implications for Mitochondrial Retrograde Signaling.

It is known that knockdown of the mitochondrial 18 kDa translocator protein (TSPO) as well as TSPO ligands modulate various functions, including functions related to cancer. To study the ability of TSPO to regulate gene expression regarding such functions, we applied microarray analysis of gene expression to U118MG glioblastoma cells. Within 15 min, the classical TSPO ligand PK 11195 induced changes in expression of immediate early genes and transcription factors. These changes also included gene products that are part of the canonical pathway serving to modulate general gene expression. These changes are in accord with real-time, reverse transcriptase (RT) PCR. At the time points of 15, 30, 45, and 60 min, as well as 3 and 24 h of PK 11195 exposure, the functions associated with the changes in gene expression in these glioblastoma cells covered well known TSPO functions. These functions included cell viability, proliferation, differentiation, adhesion, migration, tumorigenesis, and angiogenesis. This was corroborated microscopically for cell migration, cell accumulation, adhesion, and neuronal differentiation. Changes in gene expression at 24 h of PK 11195 exposure were related to downregulation of tumorigenesis and upregulation of programmed cell death. In the vehicle treated as well as PK 11195 exposed cell cultures, our triple labeling showed intense TSPO labeling in the mitochondria but no TSPO signal in the cell nuclei. Thus, mitochondrial TSPO appears to be part of the mitochondria-to-nucleus signaling pathway for modulation of nuclear gene expression. The novel TSPO ligand 2-Cl-MGV-1 appeared to be very specific regarding modulation of gene expression of immediate early genes and transcription factors.

Tetrapyrroles as Endogenous TSPO Ligands in Eukaryotes and Prokaryotes: Comparisons with Synthetic Ligands.

The 18 kDa translocator protein (TSPO) is highly 0conserved in eukaryotes and prokaryotes. Since its discovery in 1977, numerous studies established the TSPO's importance for life essential functions. For these studies, synthetic TSPO ligands typically are applied. Tetrapyrroles present endogenous ligands for the TSPO. Tetrapyrroles are also evolutionarily conserved and regulate multiple functions. TSPO and tetrapyrroles regulate each other. In animals TSPO-tetrapyrrole interactions range from effects on embryonic development to metabolism, programmed cell death, response to stress, injury and disease, and even to life span extension. In animals TSPOs are primarily located in mitochondria. In plants TSPOs are also present in plastids, the nuclear fraction, the endoplasmic reticulum, and Golgi stacks. This may contribute to translocation of tetrapyrrole intermediates across organelles' membranes. As in animals, plant TSPO binds heme and protoporphyrin IX. TSPO-tetrapyrrole interactions in plants appear to relate to development as well as stress conditions, including salt tolerance, abscisic acid-induced stress, reactive oxygen species homeostasis, and finally cell death regulation. In bacteria, TSPO is important for switching from aerobic to anaerobic metabolism, including the regulation of photosynthesis. As in mitochondria, in bacteria TSPO is located in the outer membrane. TSPO-tetrapyrrole interactions may be part of the establishment of the bacterial-eukaryote relationships, i.e., mitochondrial-eukaryote and plastid-plant endosymbiotic relationships.

The Effect of Cigarette Smoke on the Translocator Protein (TSPO) in Cultured Lung Cancer Cells.

Lung cancer is prevalent in cigarette smokers. The mitochondrial membrane translocator protein (TSPO), is thought to protect cells from free radical damage. We examined the effect of cigarette smoke (CS) (containing free radicals) alone and in the presence of saliva (containing redox active free iron), on survival of H1299 lung cancer cells and on their mitochondrial characteristics, and whether TSPO binding was influenced by CS and by saliva. We exposed H1299 cells to CS in the presence/absence of saliva and also characterized TSPO binding in the cells using [3H]PK 11195 as a radioligand. CS induced a significant drop in mitochondrial potential (ΔΨm), while addition of saliva did not lead to further loss of ΔΨm (42.5% vs. 39.85%). Scatchard analysis of the saturation curve of [3H]PK 11195 binding (0.2-6 nM final concentration) yielded a straight-line plot (R = 0.9). Average Bmax value was 3274 ± 787 fmol/mg of protein, and average Kd value was 9.2 ± 1.3 nM. Benzodiazepine diazepam partially prevented decrease in cell survival following exposure to CS and redox active iron containing media (saliva) while benzodiazepine clonazepam did not, indicating that this effect is TSPO-specific. Exposure of cells to CS resulted in alternation of biomolecules expressed by CLs peroxidation, reduction of TSPO binding, and depletion of the mitochondrial potential. This irreversible damage was enhanced in the presence of saliva. All these modulations may result in cellular death increase following CS exposure, enhanced in the presence of saliva.

In vitro effect of FGIN-1-27, a ligand to 18 kDa mitochondrial translocator protein, in human osteoblast-like cells.

Ligands of 18 kDa mitochondrial translocator protein (TSPO) differ in their cellular effects. We hypothesize that different TSPO ligands might exert different cellular responses. Therefore, following previous studies that showed different cellular responses to two specific TSPO ligands, PK 11195 and protoporphyrin IX, in human osteoblast-like cells in vitro, we now report the cellular response to another specific TSPO ligand, FGIN-1-27 (10(-5) M) (MW 436 kDa), in order to characterize the effects of each TSPO ligand. We found in primary culture of the human osteoblast-like cells that cell numbers were decreased by an average of 30% (p < 0.001) following exposure to 10(-5) M of FGIN-1-27 in comparison to vehicle controls. Cellular [(18)F]-FDG incorporation and ATP content were suppressed, by an average of 43% (p < 0.001) and 83% (p < 0.001), respectively. Mitochondrial mass and ΔΨm increased by an average of 26% (p < 0.01) and 425% (p < 0.0001) respectively. Lactate dehydrogenase activity was enhanced in culture media by 60% (p < 0.05), indicating overall cell death, while no increase in apoptotic levels was observed. Cellular proliferation, as determined by BrdU assay, was not affected. Synthesis of mRNA of TSPO, VDAC 1, and hexokinase 2 decreased in 0.3, 0.3 and 0.5 fold respectively, with accompanying decreases in protein expression of TSPO and Voltage Dependent Anion Channel 1 by 23% (p < 0.001) and 98% (p < 0.001), respectively, but without changes in hexokinase 2 protein expression. Thus it appears that 10(-5) M FGIN-1-27 reduces cell viability, cell metabolism, and mitochondrial function. Previously we found similar effects of PK 11195 on mitochondrial function and cell metabolism and of protoporphyrin IX on cell death in primary osteoblast-like cells.

In vitro catabolic effect of protoporphyrin IX in human osteoblast-like cells: possible role of the 18 kDa mitochondrial translocator protein.

In several pathological conditions, when conversion of Protoporphyrin (PP)IX into heme is impaired, a toxic accumulation of PPIX might occur. PPIX has been found to have affinity to the mitochondrial Translocator Protein 18 kDa. Since it is known that TSPO is abundant in human osteoblast cells, thus we assumed that PPIX can affect cellular functions via interactions with TSPO in these cells. Therefore we aimed to study the metabolic responses of human osteoblast to a high (10⁻5 M) concentration of PPIX in vitro. We found that in primary culture of human osteoblast-like cells cell numbers decreased following exposure to PPIX(10⁻5 M). Cellular [¹8F]-FDG incorporation, mitochondrial mass, ATP content were suppressed, and ΔΨm collapsed. Lactate dehydrogenase activity was enhanced in culture media, indicating overall cell death, while no increase in apoptotic levels was observed. Cellular proliferation was not affected. Protein expression of TSPO, VDAC 1, and hexokinase 2 decreased, although the synthesis of mRNA for hexokinase 2 increased. Thus, PPIX(10⁻5 M) has a cytotoxic effect on human osteoblast-like cell in vitro. Since these cells remain viable following exposure to another TSPO ligand, PK 11195 (10⁻5 M), as observed previously by us, the mode of action of PPIX on osteoblast-like cells is not identical to that of PK 11195. Accordingly pathological accumulation of PPIX may cause necrosis of osteoblasts leading to bone mass loss. We show that this phenomenon is unrelated to iron overload.

The 18 kDa translocator protein influences angiogenesis, as well as aggressiveness, adhesion, migration, and proliferation of glioblastoma cells.

BACKGROUND: It is known that the mitochondrial 18 kDa translocator protein (TSPO) is present in almost all peripheral tissues and also in glial cells in the brain. TSPO levels are typically enhanced in correlation with tumorigenesis of cancer cells including glioblastoma. Relevant for angiogenesis, TSPO is also present in almost all cells of the cardiovascular system. METHODS: We studied the effect of TSPO knockdown by siRNA on various aspects of tumor growth of U118MG glioblastoma cells in two in-vivo models: a nude mouse model with intracerebral implants of U118MG glioblastoma cells and implantation of U118MG glioblastoma cells on the chorionallantoic membrane (CAM) of chicken embryos. In vitro, we further assayed the influence of TSPO on the invasive potential of U118MG cells. RESULTS: TSPO knockdown increased tumor growth in both in-vivo models compared with the scrambled siRNA control. Angiogenesis was also increased by TSPO knockdown as determined by a CAM assay. TSPO knockdown led to a decrease in adhesion to the proteins of the extracellular matrix, including fibronectin, collagen I, collagen IV, laminin I, and fibrinogen. TSPO knockdown also led to an enhancement in the migratory capability of U118MG cells, as determined in a modified Boyden chamber. Application of the TSPO ligand 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK 11195) at a concentration of 25 µmol/l in the in-vitro models yielded results similar to those obtained on TSPO knockdown. We found no effects of PK 11195 on TSPO protein expression. Interestingly, at low nmol/l concentrations (around 1 nmol/l), PK 11195 enhanced adhesion to collagen I, suggesting a bimodal concentration effect of PK 11195. CONCLUSION: Intact TSPO appears to be able to counteract the invasive and angiogenic characteristics related to the aggressiveness of U118MG glioblastoma cells in vivo and in vitro.

Effects of 18-kDa translocator protein knockdown on gene expression of glutamate receptors, transporters, and metabolism, and on cell viability affected by glutamate.

OBJECTIVE: Previously, several important roles for glutamate have been described for the biology of primary brain tumors. For example, glutamate has been suggested to promote glioma cell proliferation by the activation of the 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid (AMPA) subtype of glutamate receptors. In the present study, we determined the potential regulatory roles of the 18-kDa translocator protein (TSPO) in the glutamatergic system in relation to cell death of brain tumor cells through knockdown of the TSPO by genetic manipulation. MATERIALS AND METHODS: With microarray analysis and validation of gene expression of particular genes using real-time PCR, we found effects because of small inhibitory RNA knockdown of the TSPO in human U118MG glioblastoma cells on gene expression of glutamate receptors, glutamate transporters, and enzymes for glutamate metabolism. We also applied antisense RNA to silence TSPO in rat C6 glioblastoma cells and assayed the effects on DNA fragmentation, indicative of apoptosis, because of glutamate exposure. RESULTS: In particular, the effects of TSPO silencing in human U118MG cells related to glutamate metabolism indicate a net effect of a reduction in glutamate levels, which may potentially protect the cells in question from cell death. The TSPO knockdown in C6 cells showed that TSPO is required for the induction of apoptosis because of glutamate exposure. CONCLUSION: These findings show that interactions between the TSPO and the glutamatergic system may play a role in tumor development of glioblastoma cells. This may also have implications for our understanding of the involvement of the TSPO in secondary brain damage and neurodegenerative diseases.

The nitric oxide donor sodium nitroprusside requires the 18 kDa Translocator Protein to induce cell death.

Various studies have shown that several lethal agents induce cell death via the mitochondrial 18 kDa Translocator Protein (TSPO). In this study we tested the possibility that nitric oxide (NO) is the signaling component inducing the TSPO to initiate cell death process. Cell viability assays included Trypan blue uptake, propidium iodide uptake, lactate dehydrogenase release, and DNA fragmentation. These assays showed that application of the specific TSPO ligand PK 11195 reduced these parameters for the lethal effects of the NO donor sodium nitroprusside (SNP) by 41, 27, 40, and 42 %, respectively. TSPO silencing by siRNA also reduced the measured lethal effects of SNP by 50 % for all of these four assays. With 2,3-bis[2-methoxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5-carboxyanilide (XTT) changes in metabolic activity were detected. PK 11195 and TSPO knockdown fully prevented the reductions in XTT signal otherwise induced by SNP. Collapse of the mitochondrial membrane potential was studied with the aid of JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine chloride). PK 11195 and TSPO knockdown reduced, respectively by 36 and 100 %, the incidence of collapse of the mitochondrial membrane potential otherwise induced by SNP. 10-N-Nonyl-Acridine Orange (NAO) was used to detect mitochondrial reactive oxygen species generation due to SNP. PK 11195 and TSPO knockdown reduced this effect of SNP by 65 and 100 %, respectively. SNP did not affect TSPO protein expression and binding characteristics, and also did not cause TSPO S-nitrosylation. However, ß-actin and various other proteins (not further defined) were S-nitrosylated. In conclusion, TSPO is required for the lethal and metabolic effects of the NO donor SNP, but TSPO itself is not S-nitrosylated.

The Effect of the Classical TSPO Ligand PK 11195 on In Vitro Cobalt Chloride Model of Hypoxia-like Condition in Lung and Brain Cell Lines.

The mitochondrial translocator protein (TSPO) is a modulator of the apoptotic pathway involving reactive oxygen species (ROS) generation, mitochondrial membrane potential (Δψm) collapse, activation of caspases, and eventually initiation of the apoptotic process. In this in vitro study, H1299 lung cells and BV-2 microglial cells were exposed to the hypoxia-like effect of CoCl2 with or without PK 11195. Exposing the H1299 cells to 0.5 mM CoCl2 for 24 h resulted in decreases in cell viability (63%, p < 0.05), elevation of cardiolipin peroxidation levels (38%, p < 0.05), mitochondrial membrane potential depolarization (13%, p < 0.001), and apoptotic cell death (117%, p < 0.05). Pretreatment with PK 11195 (25 µM) exhibited significant protective capacity on CoCl2-induced alterations in the mentioned processes. Exposure of BV-2 cells to increasing concentrations of CoCl2 (0.3, 0.5, 0.7 mM) for 4 h resulted in alterations in the same cellular processes. These alterations were obtained in a dose-dependent manner, except the changes in caspases 3 and 9. The novel ligands as well as PK 1195 attenuated the in vitro hypoxia-like effects of CoCl2. It appears that the TSPO ligand PK 11195 can prevent CoCl2-induced cellular damage in both non-neuronal and brain cell lines, and they may offer a novel approach to the treatment of hypoxia-related lung and brain diseases in some cases that fail to respond to conventional therapies.

The Effect of Natural-Based Formulation (NBF) on the Response of RAW264.7 Macrophages to LPS as an In Vitro Model of Inflammation.

Macrophages are some of the most important immune cells in the organism and are responsible for creating an inflammatory immune response in order to inhibit the passage of microscopic foreign bodies into the blood stream. Sometimes, their activation can be responsible for chronic inflammatory diseases such as asthma, tuberculosis, hepatitis, sinusitis, inflammatory bowel disease, and viral infections. Prolonged inflammation can damage the organs or may lead to death in serious conditions. In the present study, RAW264.7 macrophages were exposed to lipopolysaccharide (LPS; 20 ng/mL) and simultaneously treated with 20 µg/mL of natural-based formulation (NBF), mushroom-cannabidiol extract). Pro-inflammatory cytokines, chemokines, and other inflammatory markers were analyzed. The elevations in the presence of interleukin-6 (IL-6), cycloxygenase-2 (COX-2), C-C motif ligand-5 (CCL5), and nitrite response, following exposure to LPS, were completely inhibited by NBF administration. IL-1ß and tumor necrosis factor alpha (TNF-α) release were inhibited by 3.9-fold and 1.5-fold, respectively. No toxic effect of NBF, as assessed by lactate dehydrogenase (LDH) release, was observed. Treatment of the cells with NBF significantly increased the mRNA levels of TLR2, and TLR4, but not NF-κB. Thus, it appears that the NBF possesses anti-inflammatory and immunomodulatory effects which can attenuate the release of pro-inflammatory markers. NBF may be a candidate for the treatment of acute and chronic inflammatory diseases and deserves further investigation.

Oral cancer, cigarette smoke and mitochondrial 18kDa translocator protein (TSPO) - In vitro, in vivo, salivary analysis.

Oral cancer features high rates of mortality and morbidity, and is in dire need for new approaches. In the present study we analyzed 18 kDa translocator protein (TSPO) expression in oral (tongue) cancer tumors by immunohistochemistry. We also assayed TSPO binding in human tongue cancer cell lines and in the cellular fraction of saliva from tongue cancer patients, heavy cigarette smokers, and non-smoking healthy people as controls. Concurrently, TSPO protein levels, cell viability, mitochondrial membrane potential (Deltapsi(m)), and general protein levels were analyzed. TSPO expression could be significantly enhanced in oral cancer tumors, compared to unaffected adjacent tissue. We also found that five-year survival probability dropped from 65% in patients with TSPO negative tumors to 7% in patients with highly expressed TSPO (p<0.001). TSPO binding capacity was also pronounced in the human oral cancer cell lines SCC-25 and SCC-15 (3133+/-643 fmol/mg protein and 6956+/-549 fmol/mg protein, respectively). Binding decreased by 56% and 72%, in the SCC-25 and SCC-15 cell lines, respectively (p<0.05) following CS exposure in cell culture. In the cellular fraction of saliva of heavy smokers TSPO binding was lower than in non-smokers (by 53%, p<0.05). Also the cellular fraction of saliva exposed to CS in vitro showed decreased TSPO binding compared to unexposed saliva (by 30%, p<0.001). Interestingly, oral cancer patients also displayed significantly lower TSPO binding in the cellular fraction of saliva compared to healthy controls (by 40%, p<0.01). Our results suggest that low TSPO binding found in the cellular fraction of saliva may depend on genetic background as well as result from exposure to CS. We suggest that this may be related to a predisposition for occurrence of oral cancer.

In vitro mitochondrial effects of PK 11195, a synthetic translocator protein 18 kDa (TSPO) ligand, in human osteoblast-like cells.

The role of the TSPO in metabolism of human osteoblasts is unknown. We hypothesized that human osteoblast metabolism may be modulated by the TSPO. Therefore we evaluated the presence of TSPO in human osteoblast-like cells and the effect of its synthetic ligand PK 11195 on these cells. The presence of TSPO was determined by [(3)H]PK 11195 binding using Scatchard analysis: Bmax 7682 fmol/mg, Kd 9.24 nM. PK 11195 did not affect significantly cell proliferation, cell death, cellular viability, maturation, [(18)F]-FDG incorporation and hexokinase 2 gene expression or protein levels. PK 11195 exerted a suppressive effect on VDAC1 and caused an increase in TSPO gene expression or protein levels. In parallel there was an increase in mitochondrial mass, mitochondrial ATP content and a reduction in ΔΨm collapse. Thus, it appears that PK11195 (10(-5) M) stimulates mitochondrial activity in human osteoblast-like cells without affecting glycolytic activity and cell death.

Improvement of cerebral metabolism mediated by Ro5-4864 is associated with relief of intracranial pressure and mitochondrial protective effect in experimental brain injury.

PURPOSE: To investigate the possible impact of reduction of mitochondrial membrane permeabilization by modulation of the 18 kDa translocator protein mediated by Ro5-4864 over post-traumatic cerebral edema and metabolic crisis. METHODS: Cerebral microdialysis and intracranial pressure (ICP) monitoring were performed in Sprague-Dawley rats treated by intraperitoneal injection of either dimethylsulfoxide (vehicle) or Ro5-4864 following cortical contusion and further correlated with quantitative assessment of mitochondrial damage, water content in the injured tissue, modified neurological severity score, and lesion size. RESULTS: Ro5-4864 resulted in a profound decrease in ICP that correlated with improved cerebral metabolism characterized by significantly higher glucose and pyruvate and lower lactate concentrations in the pericontusional area in comparison with vehicle-treated animals. Reduced ICP correlated with reduced water content in the injured tissue; improved metabolism was associated with reduced mitochondrial damage evidenced by electron microscopy. Both effects were associated with a profound and significant reduction in glycerol release and lesion size, and correlated with improved neurological recovery. CONCLUSIONS: The present study shows that Ro5-4864 has a favorable effect on the fate of injured brain, presumably mediated by improvement of metabolism. It further suggests that improvement of metabolism may contribute to ICP relief.

The TSPO Ligands MGV-1 and 2-Cl-MGV-1 Differentially Inhibit the Cigarette Smoke-Induced Cytotoxicity to H1299 Lung Cancer Cells.

TSPO is involved in cigarette smoke (CS)-induced cellular toxicity, which may result in oral and pulmonary diseases and lung cancer. H1299 lung cancer cells were exposed directly to CS. The H1299 cells were pretreated with our TSPO ligands MGV-1 and 2-Cl-MGV-1 (Ki = 825 nM for both) at a concentration of 25 µM 24 h prior to CS exposure. Cell death and apoptotic markers were measured, in addition to TSPO expression levels, ATP synthase activity, generation of reactive oxygen species (ROS), depolarization of mitochondrial membrane potential (ΔΨm), cAMP and LDH levels. Pretreatment with MGV-1 and 2-Cl-MGV-1 (25 µM), 24 h prior to CS exposure, differentially attenuated the CS-induced cellular insult as well as cell death in H1299 lung cancer cells. These protective effects included prevention of ATP synthase reversal, ROS generation, depolarization of the mitochondrial membrane and elevation in LDH. The preventive efficacy of 2-Cl-MGV-1 was superior to that achieved by MGV-1. Both ligands did not prevent the elevation in cAMP. These findings may indicate a mild protective effect of these TSPO ligands in CS-related pulmonary and keratinocyte cellular pathology.