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.

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.

The Neuro-Protective Effects of the TSPO Ligands CB86 and CB204 on 6-OHDA-Induced PC12 Cell Death as an In Vitro Model for Parkinson's Disease.

Parkinson's disease (PD) is a progressive neurodegenerative disorder which is characterized by the degeneration of dopaminergic neurons in substantia nigra (SN). Oxidative stress or reactive oxygen species (ROS) generation was suggested to play a role in this specific type of neurodegeneration. Therapeutic options which can target and counteract ROS generation may be of benefit. TSPO ligands are known to counteract with neuro-inflammation, ROS generation, apoptosis, and necrosis. In the current study, we investigated an in vitro cellular PD model by the assessment of 6-hydroxydopamine (6-OHDA, 80 µM)-induced PC12 neurotoxicity. Simultaneously to the exposure of the cells to 6-OHDA, we added the TSPO ligands CB86 and CB204 (25 µM each) and assessed the impact on several markers of cell death. The two ligands normalized significantly (57% and 52% respectively, from 44%; whereas the control was 68%) cell proliferation at different time points from 0-24 h. Additionally, we evaluated the effect of these two TSPO ligands on necrosis using propidium iodide (PI) staining and found that the ligands inhibited significantly the 6-OHDA-induced necrosis. As compared to control, the red count was increased up to 57-fold whereas CB86 and CB204 inhibited to 2.7-fold and 3.2-fold respectively. Necrosis was also analyzed by LDH assay which showed significant effect. Both assays demonstrated similar potent anti-necrotic effect of the two TSPO ligands. Reactive oxygen species (ROS) generation induced by 6-OHDA was also inhibited by the two TSPO ligand up to 1.3 and 1.5-fold respectively, as compared to 6-OHDA group. CB86 and CB204 inhibited also normalized the cell viability up to 1.8-fold after the exposure to 6-OHDA, as assessed by XTT assay. The two TSPO ligands also inhibited apoptosis significantly (1.3-fold for both) as assessed by apopxin green staining. In summary, it appears that the two TSPO ligands CB86 and CB204 can suppress cell death of PC12 induced by 6-OHDA. The results may be relevant to the use of these two TSPO ligands as therapeutic option neurodegenerative diseases like PD.

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.

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.

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.

Sunitinib-Containing Carborane Pharmacophore with the Ability to Inhibit Tyrosine Kinases Receptors FLT3, KIT and PDGFR-ß, Exhibits Powerful In Vivo Anti-Glioblastoma Activity.

Malignant gliomas are the most common malignant and aggressive primary brain tumors in adults, the prognosis being-especially for glioblastomas-extremely poor. There are no effective treatments yet. However, tyrosine kinase receptor (TKR) inhibitors and boron neutron capture therapy (BNCT), together, have been proposed as future therapeutic strategies. In this sense in our ongoing project of developing new anti-glioblastoma drugs, we identified a sunitinib-carborane hybrid agent, 1, with both in vitro selective cytotoxicity and excellent BNCT-behavior. Consequently, we studied the ability of compound 1 to inhibit TKRs, its promotion of cellular death processes, and its effects on the cell cycle. Moreover, we analyzed some relevant drug-like properties of 1, i.e., mutagenicity and ability to cross the blood-brain barrier. These results encouraged us to perform an in vivo anti-glioblastoma proof of concept assay. It turned out to be a selective FLT3, KIT, and PDGFR-ß inhibitor and increased the apoptotic glioma-cell numbers and arrested sub-G1-phase cell cycle. Its in vivo activity in immunosuppressed mice bearing U87 MG human glioblastoma evidenced excellent anti-tumor behavior.

The Efficacy of the Novel TSPO Ligands 2-Cl-MGV-1 and 2,4-Di-Cl-MGV-1 Compared to the Classical TSPO Ligand PK 11195 to Counteract the Release of Chemokines from LPS-Stimulated BV-2 Microglial Cells.

The impact of ligands of the 18 kDa translocator protein (TSPO) on the release of chemokines is not vastly investigated. In the present study, we assessed the effect of our novel TSPO ligands 2-Cl-MGV-1 and 2,4-Di-Cl-MGV-1 compared to the classical TSPO ligand PK 11195 on chemokine release in LPS-stimulated BV-2 microglial cells. As per the effect of 2-Cl-MGV-1, CCL2, CCL3, and CCL5 were inhibited by 90%, CCL8 by 97%, and IL-2 by 77% (p < 0.05 for all). 2,4-Di-Cl-MGV-1 inhibited CCL2 release by 92%, CCL3 by 91%, CCL5 by 90%, CCL8 by 89%, and IL-2 by 80% (p < 0.05 for all). PK 11195 exhibited weaker inhibitory effects: CCL2 by 22%, CCL3 by 83%, CCL5 by 34%, CCL8 by 41%, and the cytokine IL-2 by 14% (p < 0.05 for all). Thus, it appears that the novel TSPO ligands are potent suppressors of LPS-stimulated BV-2 microglial cells, and their inhibitory effect is larger than that of PK 11195. Such immunomodulatory effects on microglial cells may be relevant to the treatment of neurodegenerative and neuroinflammatory diseases.

Efficaciousness of Low Affinity Compared to High Affinity TSPO Ligands in the Inhibition of Hypoxic Mitochondrial Cellular Damage Induced by Cobalt Chloride in Human Lung H1299 Cells.

The 18 kDa translocator protein (TSPO) plays an important role in apoptotic cell death, including apoptosis induced by the hypoxia mimicking agent cobalt chloride (CoCl2). In this study, the protective effects of a high (CB86; Ki= 1.6 nM) and a low (CB204; Ki= 117.7 nM) affinity TSPO ligands were investigated in H1299 lung cancer cell line exposed to CoCl2. The lung cell line H1299 was chosen in the present study since they express TSPO and able to undergo programmed cell death. The examined cell death markers included: ATP synthase reversal, reactive oxygen species (ROS) generation, mitochondrial membrane potential (Δψm) depolarization, cellular toxicity, and cellular viability. Pretreatment of the cells with the low affinity ligand CB204 at a concentration of 100 µM suppressed significantly (p < 0.05 for all) CoCl2-induced cellular cytotoxicity (100%), ATP synthase reversal (67%), ROS generation (82%), Δψm depolarization (100%), reduction in cellular density (97%), and also increased cell viability (85%). Furthermore, the low affinity TSPO ligand CB204, was harmless when given by itself at 100 µM. In contrast, the high affinity ligand (CB86) was significantly effective only in the prevention of CoCl2-induced ROS generation (39%, p < 0.001), and showed significant cytotoxic effects when given alone at 100 µM, as reflected in alterations in ADP/ATP ratio, oxidative stress, mitochondrial membrane potential depolarization and cell death. It appears that similar to previous studies on brain-derived cells, the relatively low affinity for the TSPO target enhances the potency of TSPO ligands in the protection from hypoxic cell death. Moreover, the high affinity TSPO ligand CB86, but not the low affinity ligand CB204, was lethal to the lung cells at high concentration (100 µM). The low affinity TSPO ligand CB204 may be a candidate for the treatment of pulmonary diseases related to hypoxia, such as pulmonary ischemia and chronic obstructive pulmonary disease COPD.

18-kDa Translocator Protein Ligands Protect H9C2 Cardiomyocytes from Cigarette Smoke-induced Cell Death: In Vitro Study.

BACKGROUND: Cigarette smoke (CS) can induce cellular damage via alterations in 18 kDa translocator protein (TSPO)-related functions, leading to cardiovascular diseases. The current study focused on the possible protective effect of TSPO ligands against CS-induced damage to cardiac cells. MATERIALS AND METHODS: H9C2 Cardiomyocyte cell line of rat origin was pre-treated with TSPO ligands. Cell death, TSPO binding, and TSPO protein expression levels were assessed following 30-min CS exposure with/without TSPO ligands. RESULTS: CS exposure of H9C2 cells significantly incensed cell death (by 26%, p<0.001). Pre-treatment with TSPO ligands at two concentrations prevented cell death. Neither CS nor ligands affected TSPO protein expression in H9C2 cells. CS led to increased cell death and reduced TSPO binding. CONCLUSION: Reduced TSPO binding may have a role in CS-induced cell death, and TSPO ligand MGV-1 can prevent suppression of TSPO binding and corresponding cell death. These results may be relevant to treatment of cardiovascular diseases associated with CS.

The protective effect of the TSPO ligands 2,4-Di-Cl-MGV-1, CB86, and CB204 against LPS-induced M1 pro-inflammatory activation of microglia.

We have shown previously, that the 18 â€‹kDa translocator protein (TSPO) synthetic ligands quinazoline derivatives (2-Cl-MGV-1 and MGV-1) can inhibit activation of in BV-2 microglial cells. In the present study we assessed the impact of novel TSPO ligands on lipopolysaccharide (LPS)-induced microglial activation as expressed by release of pro-inflammatory molecules, including cytokines [interleukin-6 (IL-6), IL-1ß, interferon- γ (IFN-γ)] nitric oxide (NO), CD8, and cyclo-oxygenase-2 (COX-2). The TSPO ligands 2,4-Di-Cl-MGV-1, CB86, and CB204 counteracted with the LPS-induced microglial activation. Exposure to LPS along with the TSPO ligand 2,4-Di-Cl-MGV-1 (25 â€‹µM) reduced significantly the release of NO by 24-, IL-6 by 14-, IL-ß by 14-, IFN- γ by 6-, and TNF-α by 29-folds, respectively. In contrast to the anti-neuroinflammatory effect of the TSPO ligands, the effect of diclofenac sodium (DS; 25 â€‹µM) did not reach statistical significance. No alterations in IL-10 and IL-13 were detected (M2 anti-inflammatory pathway) during the inhibition of M1 pro-inflammatory pathway.

Effects of Cigarette Smoke on TSPO-related Mitochondrial Processes.

The 18 kDa translocator protein (TSPO) is an initiator of the mitochondrial apoptosis cascade. Cigarette smoke (CS) exposure provokes alterations in TSPO expression as well as upregulation of its related functions such as mitochondrial membrane potential (ΔψM) and reactive oxygen species generation, which are associated with cell death. In the current study, H1299 lung cancer cell line exposed to CS for various time periods (30 mins, 60 mins and 120 mins) and TSPO expression and cell death processes were studied. CS exposure for 30 mins resulted in a non-significant increase in TSPO expression by 24% (p > 0.05 vs. control). CS exposure for 60 mins and 120 mins resulted in a significant increase by 43% (p < 0.05 vs. control) and by 47% (p < 0.01 vs. control), respectively. Furthermore, TSPO-related mitochondrial functions were upregulated at the 120 mins time point following CS exposure. TSPO expression is upregulated by CS, suggesting that TSPO plays a role in cell death processes induced by CS exposure. Alterations in TSPO-related cell death processes suggest that TSPO may be involved in the tissue damage caused by CS.

Inhibitory Effects of the Two Novel TSPO Ligands 2-Cl-MGV-1 and MGV-1 on LPS-induced Microglial Activation.

The 18 kDa translocator protein (TSPO) ligands 2-Cl-MGV-1 and MGV-1 can attenuate cell death of astrocyte-like cells (U118MG) and induce differentiation of neuronal progenitor cells (PC-12). Lipopolysaccharide (LPS) is a bacterial membrane endotoxin that activates cellular inflammatory pathways by releasing pro-inflammatory molecules, including cytokines and chemokines. The aim of the present study was to assess the immuno-modulatory effect of TSPO ligands in activated microglial cells. We demonstrated that the TSPO ligands 2-Cl-MGV-1 and MGV-1 can prevent LPS-induced activation of microglia (BV-2 cell line). Co-treatment of LPS (100 ng/mL) with these TSPO ligands (final concentration- 25 µM) reduces significantly the LPS-induced release of interleukin-6 (IL-6) from 16.9-fold to 2.5-fold, IL-ß from 8.3-fold to 1.6-fold, interferon-γ from 16.0-fold to 2.2-fold, and tumor necrosis factor-α from 16.4-fold to 1.8-fold. This anti-inflammatory activity seems to be achieved by inhibition of NF-κB p65 activation. Assessment of initiation of ROS generation and cell metabolism shows significant protective effects of these two novel TSPO ligands. The IL-10 and IL-13 levels were not affected by any of the TSPO ligands. Thus, it appears that the ligands suppress the LPS-induced activation of some inflammatory responses of microglia. Such immunomodulatory effects may be relevant to the pharmacotherapy of neuro-inflammatory diseases.

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.

Regulation of Mitochondrial, Cellular, and Organismal Functions by TSPO.

In 1999, the enigma of the 18kDa mitochondrial translocator protein (TSPO), also known as the peripheral-type benzodiazepine receptor, was the seeming disparity of the many functions attributed to TSPO, ranging from the potential of TSPO acting as a housekeeping gene at molecular biological levels to adaptations to stress, and even involvement in higher emotional and cognitive functioning, such as anxiety and depression. In the years since then, knowledge regarding the many functions modulated by TSPO has expanded, and understanding has deepened. In addition, new functions could be firmly associated with TSPO, such as regulation of programmed cell death and modulation of gene expression. Interestingly, control by the mitochondrial TSPO over both of these life and death functions appears to include Ca++ homeostasis, generation of reactive oxygen species (ROS), and ATP production. Other mitochondrial functions under TSPO control are considered to be steroidogenesis and tetrapyrrole metabolism. As TSPO effects on gene expression and on programmed cell death can be related to the wide range of functions that can be associated with TSPO, several of these five elements of Ca++, ROS, ATP, steroids, and tetrapyrroles may indeed form the basis of TSPO's capability to operate as a multifunctional housekeeping gene to maintain homeostasis of the cell and of the whole multicellular organism.

In vitro Effects of the Specific Mitochondrial TSPO Ligand Ro5 4864 in Cultured Human Osteoblasts.

The 18 kDa mitochondrial translocator protein (TSPO) ligands (10 µM), e. g., protoporphyrin IX, PK 11195 and FGIN-1-27, have different effects on metabolism and protein expression in human osteoblasts. In this study, we investigated the archetypical TSPO specific ligand Ro5-4864 (10 µM) effect in primary osteoblasts in culture aiming to further understand the TSPO role in these mature metabolically active cells.We found that following exposure to Ro5-4864, cellular [18F]-FDG incorporation and ATP content were reduced by 48% (p<0.001) and 44% (p<0.001), respectively. The mitochondrial membrane potential (ΔΨm) increased by 50% (p<0.01), mRNA synthesis of TSPO and voltage dependent anion channel (VDAC1) decreased both by 70%, the TSPO and VDAC1 protein expression decreased by 80% and 68%, respectively (p<0.001). Ro5 4864 caused a decrease in the proportion of cells in the G1 phase (by 20%, p<0.05), shifting the cell cycle to the S and G2/M phases. Furthermore, 63% decrease in hexokinase 2 protein expression (p<0.001) was found. However, we found no significant effects on hexokinase 2 mRNA expression (by RT-PCR). We also did not see significant changes in mitochondrial mass (MitoTracker Green assay), apoptosis rate (TUNEL assay), overall cell death (LDH assay), cellular proliferation (BrdU assay), cell maturation (cellular alkaline phosphatase assay), and the number of cells in the culture.Therefore, an overall effect of Ro5-4864 exhorts is via pathways related to the mitochondrial activity, which is only partly like PK 11195, but not to the other TSPO ligands.

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.