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Specific binding of benzodiazepines to human breast cancer cell lines
A Beinlich1, R Strohmeier, M Kaufmann
1Department of Obstetrics and Gynecology, Johann-Wolfgan-Goethe University, Frankfurt, Germany.
Abstract:
Binding of [3H]Ro5-4864, a peripheral benzodiazepine receptor (PBR) agonist, to BT-20 human, estrogen- (ER) and progesterone- (PR) receptor negative breast cancer cells was characterized. It was found to be specific, dose-dependent and saturable with a single population of binding sites. Dissociation constant (K(D)) was 8.5 nM, maximal binding capacity (Bmax) 339 fM/10(6) cells. Ro5-4864 (IC50 17.3 nM) and PK 11195 (IC50 12.3 nM) were able to compete with [3H]Ro5-4864 for binding, indicating specificity of interaction with PBR. Diazepam was able to displace [3H]Ro5-4864 from binding only at high concentrations (>1 microM), while ODN did not compete for PBR binding. Thymidine-uptake assay showed a biphasic response of cell proliferation. While low concentrations (100 nM) of Ro5-4864, PK 11195 and diazepam increased cell growth by 10 to 20%, higher concentrations (10-100 microM) significantly inhibited cell proliferation. PK 11195, a potent PBR ligand, was able to attenuate growth of BT-20 cells stimulated by 100 nM Ro5-4864 and to reverse growth reduction caused by 1 and 10 microM Ro5-4864, but not by 50 microM and 100 microM. This indicates that the antimitotic activity of higher concentrations of Ro5-4864 is independent of PBR binding. It is suggested, that PBR are involved in growth regulation of certain human breast cancer cell lines, possibly by supplying proliferating cells with energy, as their endogenous ligand is a polypeptide transporting Acyl-CoA.
Insights
Peripheral benzodiazepine receptors (PBR) are involved in regulating the growth of certain human breast cancer cells. Ro5-4864 and PK 11195 show biphasic effects on cell proliferation, with higher concentrations inhibiting growth independently of PBR binding.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- Peripheral benzodiazepine receptors (PBR) are implicated in various cellular functions.
- Estrogen receptor (ER)- and progesterone receptor (PR)-negative breast cancer cells present unique therapeutic challenges.
- Understanding the role of PBR in cancer cell proliferation is crucial for developing novel treatment strategies.
Purpose of the Study:
- To characterize the binding of [3H]Ro5-4864, a PBR agonist, to BT-20 human breast cancer cells.
- To investigate the effect of PBR ligands on the proliferation of these cancer cells.
- To elucidate the role of PBR in the growth regulation of ER/PR-negative breast cancer.
Main Methods:
- Radioligand binding assays using [3H]Ro5-4864 to determine binding kinetics (K(D), Bmax).
- Competition binding assays with Ro5-4864, PK 11195, diazepam, and ODN to assess specificity.
- Thymidine-uptake assays to evaluate the effect of ligands on cell proliferation.
Main Results:
- Specific, dose-dependent, and saturable binding of [3H]Ro5-4864 to BT-20 cells with K(D) of 8.5 nM and Bmax of 339 fM/10(6) cells.
- Ro5-4864 and PK 11195 competed for PBR binding, confirming specificity.
- Low concentrations of Ro5-4864, PK 11195, and diazepam enhanced cell growth, while higher concentrations inhibited it, with antimitotic activity at high doses being PBR-independent.
Conclusions:
- PBR are present and functional in ER/PR-negative human breast cancer cells.
- PBR ligands exhibit a biphasic effect on cancer cell proliferation, suggesting complex regulatory mechanisms.
- The antimitotic effects of high-concentration Ro5-4864 are not solely mediated by PBR binding, indicating additional cellular targets or pathways.