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Inhibitors of glutamate release from breast cancer cells; new targets for cancer-induced bone-pain.

Fazzari J, Lin H, Murphy C, Ungard R, Singh G - Sci Rep (2015)

Bottom Line: Current treatment options are commonly accompanied by serious side-effects that negatively impact patient care.Positive chemical hits were based on the potency of each molecule relative to a known pharmacological inhibitor of glutamate release, sulfasalazine.Efficacy was confirmed and drug-like molecules were identified as potent inhibitors of glutamate secretion from MDA-MB-231, MCF-7 and Mat-Ly-Lu cells.

View Article: PubMed Central - PubMed

Affiliation: Michael G. DeGroote Institute for Pain Research and Care, Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario Canada.

ABSTRACT
Glutamate is an important signaling molecule in a wide variety of tissues. Aberrant glutamatergic signaling disrupts normal tissue homeostasis and induces several disruptive pathological conditions including pain. Breast cancer cells secrete high levels of glutamate and often metastasize to bone. Exogenous glutamate can disrupt normal bone turnover and may be responsible for cancer-induced bone pain (CIBP). CIBP is a significant co-morbidity that affects quality of life for many advanced-stage breast cancer patients. Current treatment options are commonly accompanied by serious side-effects that negatively impact patient care. Identifying small molecule inhibitors of glutamate release from aggressive breast cancer cells advances a novel, mechanistic approach to targeting CIBP that could advance treatment for several pathological conditions. Using high-throughput screening, we investigated the ability of approximately 30,000 compounds from the Canadian Compound Collection to reduce glutamate release from MDA-MB-231 breast cancer cells. This line is known to secrete high levels of glutamate and has been demonstrated to induce CIBP by this mechanism. Positive chemical hits were based on the potency of each molecule relative to a known pharmacological inhibitor of glutamate release, sulfasalazine. Efficacy was confirmed and drug-like molecules were identified as potent inhibitors of glutamate secretion from MDA-MB-231, MCF-7 and Mat-Ly-Lu cells.

No MeSH data available.


Related in: MedlinePlus

Cytotoxicity of SKF38393, NNDP and capsazepine in MDA-MB-231, MCF-7 and Mat-Ly-Lu cells.The cell number was quantified by crystal violet staining 48 hours post-incubation. Data are represented as the mean of n = 3 experiments ± the standard error of the mean.
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f4: Cytotoxicity of SKF38393, NNDP and capsazepine in MDA-MB-231, MCF-7 and Mat-Ly-Lu cells.The cell number was quantified by crystal violet staining 48 hours post-incubation. Data are represented as the mean of n = 3 experiments ± the standard error of the mean.

Mentions: Fluorescence was measured 10 times at 90 second intervals for each plate. The rate change of relative fluorescent units versus time was calculated and used as indicator of inhibitor potency. The results were expressed as a ratio of the rate change between a testing compound and the positive control, SSZ (Fig. 1). A ratio of 1.0 means that the test compound, at a 10 μM screening concentration, has the same potency of inhibition as 200 μM SSZ. Approximately 500 positive hits were identified from the primary screening. Among these compounds, 110 were 0.2-fold (i.e. 5 times) more potent than SSZ, 127 were less than 0.4-fold, 292 were less than 1-fold, and 320 were less than 1.1-fold. These 320 compounds were selected for secondary screening. During secondary screening, cell viability was assessed visually following treatment with the compounds selected from the primary screen. A potent cytotoxic compound would not qualify as a viable therapeutic candidate, as it most likely would have targets outside the tumour in healthy tissue when administered in vivo. Eliminating these compounds with potent cytotoxicity narrowed the range of positive hits before progressing to secondary screening. Ultimately, 7 compounds were identified as viable potent inhibitors of glutamate release with low to moderate cytotoxicity. These compounds were (R,R)-cis-Diethltetrahydro-2,8-chrysendiol, (+/−)-SKF38393 hydrochloride, N,N-dipropyldopamine hydrobromide (NNDP), capsazepine, SKF83565 hydrobromide, KM02894 and BTB01303 (Fig. 2). Among them, SKF38393, SKF83565 and NNDP are well-characterized dopamine receptor agonists, while capsazepine is a vanilloid receptor antagonist. Interestingly, these four compounds share a substituted benzazepine functional group. Substituted benzazepine derivatives (1-phenyl-1H-3-benzazepines) have been shown to have specificity for the dopamine D1 receptor51. Due to supplier availability, SKF83565 and KM02894 were not available for subsequent testing. (R,R)-cis-Diethltetrahydro-2,8-chrysendiol and BTB01303 were eliminated due to structurally predicted auto-fluorescence that may interfere with the glutamate release assay. As a result, SKF38393, NNDP and capsazepine were selected for follow-up testing to assess their cytotoxicity and inhibitory effect on glutamate release in a 96-well plate format. Ultimately, capsazepine, SKF38393 and NNDP showed a dose dependent inhibition of glutamate release (Fig. 3) and low to moderate cytotoxicity (Fig. 4).


Inhibitors of glutamate release from breast cancer cells; new targets for cancer-induced bone-pain.

Fazzari J, Lin H, Murphy C, Ungard R, Singh G - Sci Rep (2015)

Cytotoxicity of SKF38393, NNDP and capsazepine in MDA-MB-231, MCF-7 and Mat-Ly-Lu cells.The cell number was quantified by crystal violet staining 48 hours post-incubation. Data are represented as the mean of n = 3 experiments ± the standard error of the mean.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4323637&req=5

f4: Cytotoxicity of SKF38393, NNDP and capsazepine in MDA-MB-231, MCF-7 and Mat-Ly-Lu cells.The cell number was quantified by crystal violet staining 48 hours post-incubation. Data are represented as the mean of n = 3 experiments ± the standard error of the mean.
Mentions: Fluorescence was measured 10 times at 90 second intervals for each plate. The rate change of relative fluorescent units versus time was calculated and used as indicator of inhibitor potency. The results were expressed as a ratio of the rate change between a testing compound and the positive control, SSZ (Fig. 1). A ratio of 1.0 means that the test compound, at a 10 μM screening concentration, has the same potency of inhibition as 200 μM SSZ. Approximately 500 positive hits were identified from the primary screening. Among these compounds, 110 were 0.2-fold (i.e. 5 times) more potent than SSZ, 127 were less than 0.4-fold, 292 were less than 1-fold, and 320 were less than 1.1-fold. These 320 compounds were selected for secondary screening. During secondary screening, cell viability was assessed visually following treatment with the compounds selected from the primary screen. A potent cytotoxic compound would not qualify as a viable therapeutic candidate, as it most likely would have targets outside the tumour in healthy tissue when administered in vivo. Eliminating these compounds with potent cytotoxicity narrowed the range of positive hits before progressing to secondary screening. Ultimately, 7 compounds were identified as viable potent inhibitors of glutamate release with low to moderate cytotoxicity. These compounds were (R,R)-cis-Diethltetrahydro-2,8-chrysendiol, (+/−)-SKF38393 hydrochloride, N,N-dipropyldopamine hydrobromide (NNDP), capsazepine, SKF83565 hydrobromide, KM02894 and BTB01303 (Fig. 2). Among them, SKF38393, SKF83565 and NNDP are well-characterized dopamine receptor agonists, while capsazepine is a vanilloid receptor antagonist. Interestingly, these four compounds share a substituted benzazepine functional group. Substituted benzazepine derivatives (1-phenyl-1H-3-benzazepines) have been shown to have specificity for the dopamine D1 receptor51. Due to supplier availability, SKF83565 and KM02894 were not available for subsequent testing. (R,R)-cis-Diethltetrahydro-2,8-chrysendiol and BTB01303 were eliminated due to structurally predicted auto-fluorescence that may interfere with the glutamate release assay. As a result, SKF38393, NNDP and capsazepine were selected for follow-up testing to assess their cytotoxicity and inhibitory effect on glutamate release in a 96-well plate format. Ultimately, capsazepine, SKF38393 and NNDP showed a dose dependent inhibition of glutamate release (Fig. 3) and low to moderate cytotoxicity (Fig. 4).

Bottom Line: Current treatment options are commonly accompanied by serious side-effects that negatively impact patient care.Positive chemical hits were based on the potency of each molecule relative to a known pharmacological inhibitor of glutamate release, sulfasalazine.Efficacy was confirmed and drug-like molecules were identified as potent inhibitors of glutamate secretion from MDA-MB-231, MCF-7 and Mat-Ly-Lu cells.

View Article: PubMed Central - PubMed

Affiliation: Michael G. DeGroote Institute for Pain Research and Care, Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario Canada.

ABSTRACT
Glutamate is an important signaling molecule in a wide variety of tissues. Aberrant glutamatergic signaling disrupts normal tissue homeostasis and induces several disruptive pathological conditions including pain. Breast cancer cells secrete high levels of glutamate and often metastasize to bone. Exogenous glutamate can disrupt normal bone turnover and may be responsible for cancer-induced bone pain (CIBP). CIBP is a significant co-morbidity that affects quality of life for many advanced-stage breast cancer patients. Current treatment options are commonly accompanied by serious side-effects that negatively impact patient care. Identifying small molecule inhibitors of glutamate release from aggressive breast cancer cells advances a novel, mechanistic approach to targeting CIBP that could advance treatment for several pathological conditions. Using high-throughput screening, we investigated the ability of approximately 30,000 compounds from the Canadian Compound Collection to reduce glutamate release from MDA-MB-231 breast cancer cells. This line is known to secrete high levels of glutamate and has been demonstrated to induce CIBP by this mechanism. Positive chemical hits were based on the potency of each molecule relative to a known pharmacological inhibitor of glutamate release, sulfasalazine. Efficacy was confirmed and drug-like molecules were identified as potent inhibitors of glutamate secretion from MDA-MB-231, MCF-7 and Mat-Ly-Lu cells.

No MeSH data available.


Related in: MedlinePlus