Benzodiazepines that bind at peripheral sites inhibit cell proliferation.

Abstract

The specific binding of benzodiazepines (BZDs) has been observed in many tissues and cell types and can be pharmacologically separated into two classes. The first and most studied are the central-type receptors in brain that are thought to mediate the known clinical effects of the BZDs (1-3). The other class is the peripheral-type binding site found in a variety of tissues and cells, including kidney (4, 6), heart (5, 6), platelets (7), mast cells (8), lymphocytes (9), many cell lines (10), and brain (11,12). BZD binding to this site has no known physiological consequences. Several pharmacological effects of the BZDs have been reported (13-15); however, none has been supported by a positive correlation between the peripheral-type binding and the biological response. We now report the presence of specific, (New England Nuclear, 101.0 Ci/mmol) and unlabeled 0.5 ,uM thymidine. After 2 hr, the cells were harvested and washed over glass fiber filters by a Microharvester (Bellco Glass). The filters were dried and placed in 4 ml of Aquasol (New England Nuclear) in minivials for scintillation spectroscopy.Binding Studies. The thymnoma cells were centrifuged at 100 x g and washed once in balanced glucose salt buffer (5.0 mM KCI/120.0 mM NaCI/5.0 mM Na2HPO4/5.0 mM Tris/0.6 mM CaCl2/1.0 mM MgSO4/5.5 mM glucose, final pH = 7.4). Binding of [3H]Ro5-4864 (New England Nuclear, 73.8 Ci/mmol) to the cells was carried out in this buffer at 0°C for 40 min with the indicated concentrations of labeled ligand in a total volume of 250 ,ul.[3H]Diazepam (New England Nuclear, 87.6 Ci/mmol) binding was also carried out at 0°C but the incubation time was 15 min. Binding reaction was terminated by the addition of 3 ml of ice-cold Dulbecco's phosphate-buffered saline and rapid filtration over Whatman GF/B glass fiber filters, followed by two washes of 3 ml of ice-cold phosphate-buffered saline. The filters were air dried and placed in 4 ml of Aquasol for liquid scintillation spectroscopy. Specific binding was defined as the difference between total binding (in the absence of unlabeled ligand) and nonspecific binding (in the presence of unlabeled 10 ,M diazepam).The benzodiazepine compounds were obtained from Hoffmann-La Roche, Inc. All reagents were obtained from commercial sources. RESULTSThe specific binding of [3H]Ro5-4864, a BZD selective for peripheral-type sites (4), was saturable, whereas nonspecific binding increased linearly with increasing concentrations of the labeled ligand (Fig. 1). Scatchard analysis of the specific binding showed a single class of sites with a Kd (mean ±

Peripheral-type benzodiazepine receptor levels correlate with the ability of human breast cancer MDA-MB-231 cell line to grow in scid mice

“…Our laboratory, as well as others, has previously shown that PBR ligands regulate cell proliferation. [7][8][9][10][11][13][14][15][16][17] The in vivo behavior of the cells, however, differs from their in vitro behavior where no differences in the rates of cell proliferation between the MDA-231 PH and PL is seen. This may be due to a minimum of PBR levels required for cell proliferation.…”

“…5,6 Increased expression of PBR has been found in numerous tumor types and PBR has been shown to regulate the differentiation state and proliferation rates of several cancer cell lines. [7][8][9][10][11][12][13][14][15][16][17][18][19][20] Over the past several years our laboratory has examined the role of PBR in breast cancer using the MDA-231 cell line. While examining MDA-231 cells from various origins we observed variations in their PBR levels.…”

Differential Expressions of p53, p53R2, hRRM2 and PBR in Chronic Lymphocytic Leukemia: A Correlation with Intracellular Cholesterol

“…Recent studies demonstrated that PBR is a high affinity cholesterol binding protein [12,13]. There are studies showing abundance of PBR in tumor cells and also specifically on nuclear membrane of tumor cells [14][15][16][17][18]. The subunits of PBR act as cholesterol receptor as well as cholesterol channel.…”

Diazepam, em dose única, inibe a migração celular, a estimulação macrofágica e a atividade de TNF-α na reação inflamatória aguda induzida por LPS em camundongos

“…Receptores periféricos de BZDs (RPBs) foram identificados em tecidos endócrinos, fígado, rins, pulmão e testículos (Gavish et al, 1999; Ruff et al, 1985;Wang et al, …”

“…Os BDZs apresentam propriedades miorrelaxantes, ansiolíticas e sedativas devido à sua alta afinidade com sítios específicos de ligação no sistema nervoso central -os chamados receptores centrais de BZD (RCBs). Desta maneira, os BZDs constituem uma das classes de drogas mais freqüentemente prescritas e consumidas no Brasil, Europa e EUA (Ruiz et al, 1993).Receptores periféricos de BZDs (RPBs) foram identificados em tecidos endócrinos, fígado, rins, pulmão e testículos (Gavish et al, 1999; Ruff et al, 1985;Wang et al, …”