Peripheral-type benzodiazepine receptor overexpression and knockdown in human breast cancer cells indicate its prominent role in tumor cell proliferation.

The peripheral-type benzodiazepine receptor (PBR), an 18-kDa high affinity drug and cholesterol binding protein, is expressed at high levels in various cancers. Its expression is positively correlated with aggressive metastatic behavior in human breast cancer cells. To determine the role of PBR in tumor progression, two human mammary carcinoma cell lines were utilized: the non-aggressive MCF-7 cell line, which expresses extremely low PBR levels, and the highly aggressive MDA-MB-231 cell line, which has much higher PBR levels. We have generated stably transfected lines of the tetracycline-repressible MCF-7 cell line (MCF-7 Tet-Off) with inducible human PBR cDNA. Induction of PBR expression in MCF-7 Tet-Off cells increased PBR ligand binding and cell proliferation. Transfection of MDA-MB-231 cells with multiple siRNAs complementary to PBR (PBR-siRNAs) led to different levels of PBR mRNA knockdown. Lentiviral-mediated PBR RNA interference in MDA-MB-231 cells decreased PBR levels by 50%. Decreased PBR expression was associated with cell cycle arrest at G2 phase, decreased cell proliferation, and significant increases in the protein levels of the cyclin-dependent kinase inhibitor p21(WAF/CIP1). These changes were accompanied by p53 activation seen as increased p53 phosphorylation (Ser15). In parallel, increased proteolytic activation of caspase-3 was also observed. Taken together these results suggest that PBR protein expression is directly involved in regulating cell survival and proliferation in human breast cancer cells by influencing signaling mechanisms involved in cell cycle control and apoptosis.

Cancer-related overexpression of the peripheral-type benzodiazepine receptor and cytostatic anticancer effects of Ginkgo biloba extract (EGb 761).

The peripheral-type benzodiazepine receptor (PBR) is an 18-kDa high affinity drug- and cholesterol-binding protein that is involved in various cell functions, including cell proliferation and apoptosis. PBR was shown to be overexpressed in certain types of malignant human tumors and cancer cell lines, correlating with enhanced tumorigenicity and cell proliferation rates. The present study was conducted in order to further define the role of PBR in cancer and to extend our recent findings regarding the possible anticancer effects of the standardized Ginkgo biloba extract EGb 761. Treatment with EGb 761 decreased PBR mRNA levels and inhibited the proliferation of breast, glioma and hepatocarcinoma cell lines, further corroborating our previous contention that its mechanism of action is through the modification of PBR expression. In vivo treatment with Ginkgo biloba extract led to dose-dependent decreases in xenograft growth of both MDA-MB-231 breast cancer and U-87 glioma cell lines in nude mice, although the effects were not maintained after 50 days of treatment in the latter. The results obtained in MDA-MB-231 xenografts indicated pronounced inhibition of tumor growth, verified by MRI imaging. These results were obtained using a modified experimental protocol where the animals were treated with the extract before cell inoculation. Although an exact role for PBR in relation to the initiation and progression of various types of cancer remains to be defined, our results indicate that PBR overexpression in certain cancer cells is related to an aggressive phenotype. Since EGb 761 treatment opposes this aggressive phenotype by decreasing PBR overexpression, it could be useful in preventing or treating cancer invasiveness and metastasis.

Neuroprotective effects of green and black teas and their catechin gallate esters against beta-amyloid-induced toxicity.

Teas represent a large family of plants containing high amounts of polyphenols that may confer health benefits in various diseases. Recently, it has been hypothesized that tea consumption may also reduce the risk of age-related neurodegenerative pathologies. Considering the deleterious role of beta-amyloid (Abeta) in the aetiology of Alzheimer's disease (AD), we investigated green and black tea extracts and flavan-3-ols (present as monomers and dimers in green and black forms, respectively) against toxicity induced by Abeta-derived peptides using primary cultures of rat hippocampal cells as model. Both green and black tea extracts (5-25 microg/mL) displayed neuroprotective action against Abeta toxicity. These effects were shared by gallic acid (1-20 microm), epicatechin gallate (ECG; 1-20 microM) and epigallocatechin gallate (EGCG; 1-10 microM), the former being the most potent flavan-3-ol. In contrast, epicatechin and epigallocatechin were ineffective in the same range of concentrations. Moreover, only tea flavan-3-ol gallate esters (i.e. ECG, EGCG) and gallic acid inhibited apoptotic events induced by Abeta(25-35). Interestingly, EGCG and gallic acid inhibited Abeta aggregation and/or the formation of Abeta-derived diffusible neurotoxin ligands. Taken together, these results indicate that the catechin gallates (through the galloyl moiety) contribute to the neuroprotective effects of both green and black teas. Moreover, the protective effect of EGCG is likely to be associated, at least in part, with its inhibitory action on Abeta fibrils/oligomers formation. These data also support the hypothesis that not only green but also black teas may reduce age-related neurodegenerative diseases, such as AD.

Identification, design, synthesis, and pharmacological activity of (4-ethyl-piperazin-1-yl)-phenylmethanone derivatives with neuroprotective properties against beta-amyloid-induced toxicity.

In search of novel therapeutic approaches for Alzheimer's disease (AD), we report herein the identification, design, synthesis, and pharmacological activity of (4-ethyl-piperaz-1-yl)-phenylmethanone derivatives with neuroprotective properties against beta-amyloid-induced toxicity. (4-ethyl-piperaz-1-yl)-phenylmethanone is a common substructure shared by molecules isolated from plants of the Asteraceae genus, traditionally used as restorative of lost or declining mental functions. (4-Ethyl-piperaz-1-yl)-phenylmethanone displayed strong neuroprotective properties against Abeta1-42 and reversed Abeta1-42-induced ATP depletion on neuronal cells, suggesting a mitochondrial site of action. Abeta1-42 has been described to induce a hyperactivity of the glutamate network in neuronal cells. (4-Ethyl-piperaz-1-yl)-phenylmethanone also inhibited the neurotoxic effect that glutamate displayed on PC12 cells, suggesting that the reduction of glutamate-induced neurotoxicity may be one of the mechanisms by which this compound exerts its neuroprotective properties against the deleterious effects of the Abeta1-42. These data suggest that the identified (4-ethyl-piperaz-1-yl)-phenylmethanone chemical entity exerts neuroprotective properties and may serve as a lead compound for the development of novel therapies for AD.

Characterization of the cholesterol recognition amino acid consensus sequence of the peripheral-type benzodiazepine receptor.

We previously defined a cholesterol recognition/interaction amino acid consensus sequence [CRAC: L/V-X (1-5)-Y-X (1-5)-R/K] in the carboxyl terminus of the peripheral-type benzodiazepine receptor (PBR), a high-affinity drug and cholesterol-binding protein present in the outer mitochondrial membrane protein. This protein is involved in the regulation of cholesterol transport into the mitochondria, the rate-determining step in steroid biosynthesis. Reconstituted wild-type recombinant PBR into proteoliposomes demonstrated high-affinity 2-chlorophenyl)-N-methyl-N-(1-methyl-propyl)-3-isoquinolinecarboxamide and cholesterol binding. In the present work, we functionally and structurally characterized this CRAC motif using reconstituted recombinant PBR and nuclear magnetic resonance. Deletion of the C-terminal domain of PBR and mutation of the highly conserved among all PBR amino acid sequences Y152 of the CRAC domain resulted in loss of the ability of mutant recPBR to bind cholesterol. Nuclear magnetic resonance analysis of a PBR C-terminal peptide (144-169) containing the CRAC domain indicated a helical conformation for the L144-S159 fragment. As a result of the side-chain distribution, a groove that could fit a cholesterol molecule is delineated, on one hand, by Y152, T148, and L144, and, on the other hand, by Y153, M149, and A145. The aromatic rings of Y152 and Y153 assigned as essential residues for cholesterol binding constitute the gate of the groove. Furthermore, the side chain of R156 may cap the groove by interacting with the sterol hydroxyl group. These results provide structural and functional evidence supporting the finding that the CRAC domain in the cytosolic carboxyl-terminal domain of PBR might be responsible for the uptake and translocation of cholesterol into the mitochondria.

Ginkgo biloba extract (Egb 761) inhibits beta-amyloid production by lowering free cholesterol levels.

Ginkgo biloba extract (EGb 761) can improve cognitive function in patients with Alzheimer's disease, but the molecular mechanisms underlying this effect remain undefined. Because free cholesterol may be involved in the production of beta-amyloid precursor protein and amyloid beta-peptide, key events in the development of Alzheimer's disease, we examined EGb 761 in relation to cholesterol and amyloidogenesis. In aging rats, EGb 761 treatment lowered circulating free cholesterol and inhibited the production of brain beta-amyloid precursor protein and amyloid beta-peptide. Exposure of PC12 cells to EGb 761 decreased the processing of beta-amyloid precursor protein and abolished cholesterol-induced overproduction of this protein. Exposure of human NT2 cells to EGb 761 decreased free cholesterol influx and increased free cholesterol efflux. Our findings indicate that free circulating and intracellular cholesterol levels affect the processing of beta-amyloid precursor protein and amyloidogenesis. Our findings also provide the first demonstration that EGb 761 can influence these mechanisms.

Evidence for a mitochondrial impact of trimetazidine during cold ischemia and reperfusion.

In organ transplantation, ischemia-reperfusion injury (IRI) has been implicated in delayed graft function (DGF) as well as in short- and long-term complications. Using an autotransplant pig kidney model, changes in renal function and morphology were determined after different periods of cold ischemia in kidneys preserved in the University of Wisconsin solution (UW), high-Na(+) version of UW (HEH) or Celsior (CEL) a newly developed high-Na(+) solution, with or without trimetazidine (TMZ). Kidney function was better preserved in CEL, UW and particularly HEH in combination with TMZ, particularly after 48 and 72 h. Mitochondria integrity was improved in TMZ-preserved groups. This study indicates that TMZ is efficiently protective against IRI even after prolonged preservation and in different preservation solutions.

Inhibition of adrenal cortical steroid formation by procaine is mediated by reduction of the cAMP-induced 3-hydroxy-3-methylglutaryl-coenzyme A reductase messenger ribonucleic acid levels.

Elevated glucocorticoid levels are associated with many diseases, including age-related depression, hypertension, Alzheimer's disease, and acquired immunodeficiency syndrome. Cortisol-lowering agents could provide useful complementary therapy for these disorders. We examined the effect of procaine and procaine in a pharmaceutical formulation on adrenal cortical steroid formation. Procaine inhibited dibutyryl cyclic AMP (dbcAMP)-induced corticosteroid synthesis by murine Y1 and human H295R adrenal cells in a dose-dependent manner without affecting basal steroid formation. Treatment of rats with the procaine-based formulation reduced circulating corticosterone levels. This steroidogenesis-inhibiting activity of procaine was not observed in Leydig cells, suggesting that the effect was specific to adrenocortical cells. In search of the mechanism underlying this inhibitory effect on cAMP-induced corticosteroidogenesis, procaine was found to affect neither the cAMP-dependent protein kinase activity nor key proteins involved in cholesterol transport into mitochondria, cytochrome P450 side chain cleavage enzyme expression, and enzymatic activities associated with cholesterol metabolism to final steroid products. However, procaine reduced in a dose-dependent manner the 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA) activity and the dbcAMP-induced HMG-CoA reductase mRNA levels by affecting mRNA stability. These data suggest that the inhibitory effect of procaine on cAMP-induced corticosteroid formation is due to the reduced synthesis of cholesterol. This modulatory effect of procaine on HMG-CoA reductase mRNA expression was also seen in dbcAMP-stimulated Hepa1-6 mouse liver hepatoma cells. Taken together, these results suggest that procaine may provide a pharmacological means for the control of hormone-induced HMG-CoA reductase mRNA expression and hypercortisolemia.

Prenatal exposure of rats to Ginkgo biloba extract (EGb 761) increases neuronal survival/growth and alters gene expression in the developing fetal hippocampus.

Hippocampal neuron survival/growth and gene expression have been examined after prenatal (in utero) exposure of rats to EGb 761, a leaf extract of Ginkgo biloba. Oral administration of EGb 761 (100 or 300 mg/kg/day) to pregnant dams for 5 days increased the number of hippocampal neurons (maintained in culture) of their fetuses, indicating a neurotrophic effect of the extract. Using large-scale oligonucleotide microarrays containing over 8000 combined rat genes and expressed sequence tag clusters, it was shown that treatment of pregnant dams with EGb 761 (25, 50 or 100 mg/kg/day for 5 days) altered the expression of 187 genes in the hippocampi of male fetuses and 160 genes in those of female fetuses. Using gene-cluster analysis, these genes were grouped into 18 distinct clusters for males and 17 distinct clusters for females. Among these clusters, 35 genes shared a common expression pattern in male and female hippocampal development. Of these genes, the changes observed in insulin growth factor II, insulin growth factor binding protein 2, testosterone repressed prostate message-2, glutathione-dependent dehydroascorbate reductase, lipoprotein lipase, guanylate cyclase and DNA binding protein Brn-2 were confirmed by real-time quantitative polymerase chain reaction. These findings, which have provided the first genetic profile of the effects of EGb 761 on the developing rat hippocampus, increase our understanding of the molecular and genetic programs that are activated by the extract. These effects of EGb 761 may underlie its neuroprotective properties.

Ginkgo biloba extracts and cancer: a research area in its infancy.

Recent studies conducted with various molecular, cellular and whole animal models have revealed that leaf extracts of Ginkgo biloba may have anticancer (chemopreventive) properties that are related to their antioxidant, anti-angiogenic and gene-regulatory actions. The antioxidant and associated anti-lipoperoxidative effects of Ginkgo extracts appear to involve both their flavonoid and terpenoid constituents. The anti-angiogenic activity of the extracts may involve their antioxidant activity and their ability to inhibit both inducible and endothelial forms of nitric oxide synthase. With regard to gene expression, a Ginkgo extract and one of its terpenoid constituents, ginkgolide B, inhibited the proliferation of a highly aggressive human breast cancer cell line and xenografts of this cell line in nude mice. cDNA microarray analyses have shown that exposure of human breast cancer cells to a Ginkgo extract altered the expression of genes that are involved in the regulation of cell proliferation, cell differentiation or apoptosis, and that exposure of human bladder cancer cells to a Ginkgo extract produced an adaptive transcriptional response that augments antioxidant status and inhibits DNA damage. In humans, Ginkgo extracts inhibit the formation of radiation-induced (chromosome-damaging) clastogenic factors and ultraviolet light-induced oxidative stress - effects that may also be associated with anticancer activity. Flavonoid and terpenoid constituents of Ginkgo extracts may act in a complementary manner to inhibit several carcinogenesis-related processes, and therefore the total extracts may be required for producing optimal effects.

Oxidative stress-mediated DHEA formation in Alzheimer's disease pathology.

An alternative pathway for dehydroepiandrosterone (DHEA) synthesis has been suggested by treating rat and human brain cells with ferrous sulfate and beta-amyloid (Abeta). To determine if this pathway exists in human brain, levels of DHEA in hippocampus, hypothalamus and frontal cortex from Alzheimer's disease (AD) patients and age-matched controls were measured. DHEA is significantly higher in AD brain than control, and was highest in AD hippocampi. Cytochrome p450 17alpha-hydroxylase, responsible for peripheral DHEA synthesis, is not present in hippocampus. DHEA levels in AD cerebrospinal fluid (CSF) were significantly higher than age-matched controls. AD serum DHEA levels are lower than CSF, and not significantly different from controls. Treatment of control hippocampus, hypothalamus and serum with FeSO(4) increases DHEA, suggesting that levels of precursor are higher in control that in AD brain. This suggests that (i). an alternative precursor is present in control brain, (ii). AD brain DHEA is formed by oxidative stress metabolism of precursor, and (iii). CSF DHEA levels and serum DHEA formation in response to FeSO(4) may serve as an indicator of AD pathology.

22R-Hydroxycholesterol protects neuronal cells from beta-amyloid-induced cytotoxicity by binding to beta-amyloid peptide.

22R-hydroxycholesterol, a steroid intermediate in the pathway of pregnenolone formation from cholesterol, was found at lower levels in Alzheimer's disease (AD) hippocampus and frontal cortex tissue specimens compared to age-matched controls. beta-Amyloid (Abeta) peptide has been shown to be neurotoxic and its presence in brain has been linked to AD pathology. 22R-hydroxycholesterol was found to protect, in a dose-dependent manner, against Abeta-induced rat sympathetic nerve pheochromocytoma (PC12) and differentiated human Ntera2/D1 teratocarcinoma (NT2N) neuron cell death. Other steroids tested were either inactive or acted on rodent neurons only. The effect of 22R-hydroxycholesterol was found to be stereospecific because its enantiomer 22S-hydroxycholesterol failed to protect the neurons from Abeta-induced cell death. Moreover, the effect of 22R-hydroxycholesterol was specific for Abeta-induced cell death because it did not protect against glutamate-induced neurotoxicity. The neuroprotective effect of 22R-hydroxycholesterol was seen when using Abeta1-42 but not the Abeta25-35 peptide. To investigate the mechanism of action of 22R-hydroxycholesterol we examined the direct binding of this steroid to Abeta using a novel cholesterol-protein binding blot assay. Using this method the direct specific binding, under native conditions, of 22R-hydroxycholesterol to Abeta1-42 and Abeta17-40, but not Abeta25-35, was observed. These data suggest that 22R-hydroxycholesterol binds to Abeta and the formed 22R-hydroxycholesterol/Abeta complex is not toxic to rodent and human neurons. We propose that 22R-hydroxycholesterol offers a new means of neuroprotection against Abeta toxicity by inactivating the peptide.

Use of ginkgolide B and a ginkgolide-activated response element to control gene transcription: example of the adrenocortical peripheral-type benzodiazepine receptor.

Identification of the molecular switch controlling glucocorticoid synthesis might facilitate the development of pharmacological tools to control circulating cortisol levels. The transport of cholesterol from intracellular sources to the inner mitochondrial membrane represents the rate-determining step in the cascade of reactions leading to cortisol synthesis. A key element in this step is the peripheral-type benzodiazepine receptor (PBR). Several studies have indicated the beneficial effects of Ginkgo biloba on memory and stress control. Using pharmacological, biochemical and proteomic methods, we demonstrated that the standardized Ginkgo biloba extract EGb 761 and its isolated component ginkgolide B (GKB) inhibit PBR ligand binding and protein expression, resulting in decreased serum corticosterone levels. We further demonstrated that EGb 761- and GKB-induced inhibition of PBR protein is preceded by a decrease in mRNA-levels due to transcriptional suppression of PBR gene expression. Further studies indicated that the action of GKB is mediated by a transcription factor binding to the PBR gene promoter, thereby regulating PBR gene expression. These data indicate that EGb 761-induced inhibition of glucocorticoid production is due to specific transcriptional suppression of the adrenal PBR gene by GKB, and suggest that EGb 761 and GKB might serve as pharmacological tools to control excess glucocorticoid formation.

Common gene targets of Ginkgo biloba extract (EGb 761) in human tumor cells: relation to cell growth.

The standardized extract of Ginkgo biloba leaves (EGb 761) has been shown to inhibit aggressive human breast cancer cell proliferation both in vitro and in vivo. These results were extended to human glioma and hepatoma cells in vitro suggesting that EGb 761 may have a more widespread application for tumor growth control. To understand the mechanism by which EGb 761 acts to inhibit cell proliferation, we investigated the effects of EGb 761 on human breast cancer, glioma and hepatoma cell transcriptomes by means of various large-scale DNA array techniques. The data presented focus on genes regulated by EGb 761 that are common to the three tumor cell types and for which the data were verified by two different types of DNA microarray and/or RNA (Northern) blot analysis and real-time quantitative PCR. These results could therefore help elucidate the mechanism of cytostatic action of EGb 761 and identify genes important for tumor growth.