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Bladder cancer cell growth and motility implicate cannabinoid 2 receptor-mediated modifications of sphingolipids metabolism

View Article: PubMed Central - PubMed

ABSTRACT

The inhibitory effects demonstrated by activation of cannabinoid receptors (CB) on cancer proliferation and migration may also play critical roles in controlling bladder cancer (BC). CB expression on human normal and BC specimens was tested by immunohistochemistry. Human BC cells RT4 and RT112 were challenged with CB agonists and assessed for proliferation, apoptosis, and motility. Cellular sphingolipids (SL) constitution and metabolism were evaluated after metabolic labelling. CB1-2 were detected in BC specimens, but only CB2 was more expressed in the tumour. Both cell lines expressed similar CB2. Exposure to CB2 agonists inhibited BC growth, down-modulated Akt, induced caspase 3-activation and modified SL metabolism. Baseline SL analysis in cell lines showed differences linked to unique migratory behaviours and cytoskeletal re-arrangements. CB2 activation changed the SL composition of more aggressive RT112 cells by reducing (p < 0.01) Gb3 ganglioside (−50 ± 3%) and sphingosine 1-phosphate (S1P, −40 ± 4%), which ended up to reduction in cell motility (−46 ± 5%) with inhibition of p-SRC. CB2-selective antagonists, gene silencing and an inhibitor of SL biosynthesis partially prevented CB2 agonist-induced effects on cell viability and motility. CB2 activation led to ceramide-mediated BC cell apoptosis independently of SL constitutive composition, which instead was modulated by CB2 agonists to reduce cell motility.

No MeSH data available.


Related in: MedlinePlus

Effect of cannabinoids on bladder cancer cell proliferation.Panel A: Dose-response cytotoxicity (MTT assay) on RT112 (black symbols) and RT4 (white symbols) cells, measured 72 hours post JWH015. Panel B: Kinetics of cytotoxicity after 20 μM JWH015 on both RT4 (white symbols) and RT112 (black symbols) cells. The effect on normal fibroblasts is also shown (grey square symbols). Panels C and D: Effect of the CB2 antagonist AM630 (0.25 μM) on CB-2 agonist-induced RT112 cell toxicity. Cells were pre-treated with AM630 for 30 min before exposure to either different concentrations of JWH015 (015, C) or JWH133 (133, D). Cell viability was assayed (MTT assay) 72 hrs post agonist challenge. Effect of antagonist alone (AM630, 0.25–10 μM range, white diamonds) is shown. *p < 0.05, ***p < 0.001 vs agonist alone, 2-ways ANOVA, Bonferroni post-test. Data are represented as mean ± sd of quintuplicate values. A single experiment is shown as representative of three independent ones with similar results. Panel E: down-modulation of CB2 mRNA (qPCR) and protein (Western blot) in RT112 cells after specific gene silencing. *p < 0.05 vs control siRNA (T test). Panel F: Effect of CB2 genetic silencing 24 and 48 hrs post JWH015 (015, 20 μM) on cytotoxicity. Data are expressed as mean ± SD of quadruplicate cell counts (Trypan Blue exclusion). ***p < 0.001 vs 015+cont siRNA (1-way ANOVA, Dunnett’s post test).
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f2: Effect of cannabinoids on bladder cancer cell proliferation.Panel A: Dose-response cytotoxicity (MTT assay) on RT112 (black symbols) and RT4 (white symbols) cells, measured 72 hours post JWH015. Panel B: Kinetics of cytotoxicity after 20 μM JWH015 on both RT4 (white symbols) and RT112 (black symbols) cells. The effect on normal fibroblasts is also shown (grey square symbols). Panels C and D: Effect of the CB2 antagonist AM630 (0.25 μM) on CB-2 agonist-induced RT112 cell toxicity. Cells were pre-treated with AM630 for 30 min before exposure to either different concentrations of JWH015 (015, C) or JWH133 (133, D). Cell viability was assayed (MTT assay) 72 hrs post agonist challenge. Effect of antagonist alone (AM630, 0.25–10 μM range, white diamonds) is shown. *p < 0.05, ***p < 0.001 vs agonist alone, 2-ways ANOVA, Bonferroni post-test. Data are represented as mean ± sd of quintuplicate values. A single experiment is shown as representative of three independent ones with similar results. Panel E: down-modulation of CB2 mRNA (qPCR) and protein (Western blot) in RT112 cells after specific gene silencing. *p < 0.05 vs control siRNA (T test). Panel F: Effect of CB2 genetic silencing 24 and 48 hrs post JWH015 (015, 20 μM) on cytotoxicity. Data are expressed as mean ± SD of quadruplicate cell counts (Trypan Blue exclusion). ***p < 0.001 vs 015+cont siRNA (1-way ANOVA, Dunnett’s post test).

Mentions: The preferential CB2 agonist JWH0151617 induced antiproliferative activity on both RT4 and RT112 cells (Fig. 2A), with a specific IC50 value around 5 μM. Cell viability reduced by 85 ± 5% and 88 ± 8% (p < 0.001) in RT4 and RT112 cells respectively, 4 days after agonist exposure (Fig. 2B). Pre-treatment with the CB2 antagonist SR144256 reduced, after 72 hrs, the JWH015-induced (20 μM) cytotoxic effect by 25.6 ± 3% (p < 0.01 vs JWH015 alone, not shown). A second, more potent CB2 antagonist (AM630) rescued significantly the RT112 cytotoxicity after both JWH015 and JWH133, a more CB2-selective agonist18 (Fig. 2C,D). No significant effects by CB2 antagonists alone (up to 10 μM) were detected. JWH015 induced similar cytotoxicity in T24, HT1376, and 5637 BC cell lines (not shown) but did not affect the viability of human primary fibroblasts (Fig. 2B). Genetic silencing of CB2 receptor in RT112 cells reduced both mRNA and protein (Fig. 2E), inhibited the agonist-induced cytotoxicity (Fig. 2F), and increased cell viability by 42.9 ± 16% after JWH015 (20 μM) challenge (MTT assay, p < 0.05 vs control siRNA).


Bladder cancer cell growth and motility implicate cannabinoid 2 receptor-mediated modifications of sphingolipids metabolism
Effect of cannabinoids on bladder cancer cell proliferation.Panel A: Dose-response cytotoxicity (MTT assay) on RT112 (black symbols) and RT4 (white symbols) cells, measured 72 hours post JWH015. Panel B: Kinetics of cytotoxicity after 20 μM JWH015 on both RT4 (white symbols) and RT112 (black symbols) cells. The effect on normal fibroblasts is also shown (grey square symbols). Panels C and D: Effect of the CB2 antagonist AM630 (0.25 μM) on CB-2 agonist-induced RT112 cell toxicity. Cells were pre-treated with AM630 for 30 min before exposure to either different concentrations of JWH015 (015, C) or JWH133 (133, D). Cell viability was assayed (MTT assay) 72 hrs post agonist challenge. Effect of antagonist alone (AM630, 0.25–10 μM range, white diamonds) is shown. *p < 0.05, ***p < 0.001 vs agonist alone, 2-ways ANOVA, Bonferroni post-test. Data are represented as mean ± sd of quintuplicate values. A single experiment is shown as representative of three independent ones with similar results. Panel E: down-modulation of CB2 mRNA (qPCR) and protein (Western blot) in RT112 cells after specific gene silencing. *p < 0.05 vs control siRNA (T test). Panel F: Effect of CB2 genetic silencing 24 and 48 hrs post JWH015 (015, 20 μM) on cytotoxicity. Data are expressed as mean ± SD of quadruplicate cell counts (Trypan Blue exclusion). ***p < 0.001 vs 015+cont siRNA (1-way ANOVA, Dunnett’s post test).
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f2: Effect of cannabinoids on bladder cancer cell proliferation.Panel A: Dose-response cytotoxicity (MTT assay) on RT112 (black symbols) and RT4 (white symbols) cells, measured 72 hours post JWH015. Panel B: Kinetics of cytotoxicity after 20 μM JWH015 on both RT4 (white symbols) and RT112 (black symbols) cells. The effect on normal fibroblasts is also shown (grey square symbols). Panels C and D: Effect of the CB2 antagonist AM630 (0.25 μM) on CB-2 agonist-induced RT112 cell toxicity. Cells were pre-treated with AM630 for 30 min before exposure to either different concentrations of JWH015 (015, C) or JWH133 (133, D). Cell viability was assayed (MTT assay) 72 hrs post agonist challenge. Effect of antagonist alone (AM630, 0.25–10 μM range, white diamonds) is shown. *p < 0.05, ***p < 0.001 vs agonist alone, 2-ways ANOVA, Bonferroni post-test. Data are represented as mean ± sd of quintuplicate values. A single experiment is shown as representative of three independent ones with similar results. Panel E: down-modulation of CB2 mRNA (qPCR) and protein (Western blot) in RT112 cells after specific gene silencing. *p < 0.05 vs control siRNA (T test). Panel F: Effect of CB2 genetic silencing 24 and 48 hrs post JWH015 (015, 20 μM) on cytotoxicity. Data are expressed as mean ± SD of quadruplicate cell counts (Trypan Blue exclusion). ***p < 0.001 vs 015+cont siRNA (1-way ANOVA, Dunnett’s post test).
Mentions: The preferential CB2 agonist JWH0151617 induced antiproliferative activity on both RT4 and RT112 cells (Fig. 2A), with a specific IC50 value around 5 μM. Cell viability reduced by 85 ± 5% and 88 ± 8% (p < 0.001) in RT4 and RT112 cells respectively, 4 days after agonist exposure (Fig. 2B). Pre-treatment with the CB2 antagonist SR144256 reduced, after 72 hrs, the JWH015-induced (20 μM) cytotoxic effect by 25.6 ± 3% (p < 0.01 vs JWH015 alone, not shown). A second, more potent CB2 antagonist (AM630) rescued significantly the RT112 cytotoxicity after both JWH015 and JWH133, a more CB2-selective agonist18 (Fig. 2C,D). No significant effects by CB2 antagonists alone (up to 10 μM) were detected. JWH015 induced similar cytotoxicity in T24, HT1376, and 5637 BC cell lines (not shown) but did not affect the viability of human primary fibroblasts (Fig. 2B). Genetic silencing of CB2 receptor in RT112 cells reduced both mRNA and protein (Fig. 2E), inhibited the agonist-induced cytotoxicity (Fig. 2F), and increased cell viability by 42.9 ± 16% after JWH015 (20 μM) challenge (MTT assay, p < 0.05 vs control siRNA).

View Article: PubMed Central - PubMed

ABSTRACT

The inhibitory effects demonstrated by activation of cannabinoid receptors (CB) on cancer proliferation and migration may also play critical roles in controlling bladder cancer (BC). CB expression on human normal and BC specimens was tested by immunohistochemistry. Human BC cells RT4 and RT112 were challenged with CB agonists and assessed for proliferation, apoptosis, and motility. Cellular sphingolipids (SL) constitution and metabolism were evaluated after metabolic labelling. CB1-2 were detected in BC specimens, but only CB2 was more expressed in the tumour. Both cell lines expressed similar CB2. Exposure to CB2 agonists inhibited BC growth, down-modulated Akt, induced caspase 3-activation and modified SL metabolism. Baseline SL analysis in cell lines showed differences linked to unique migratory behaviours and cytoskeletal re-arrangements. CB2 activation changed the SL composition of more aggressive RT112 cells by reducing (p&thinsp;&lt;&thinsp;0.01) Gb3 ganglioside (&minus;50&thinsp;&plusmn;&thinsp;3%) and sphingosine 1-phosphate (S1P, &minus;40&thinsp;&plusmn;&thinsp;4%), which ended up to reduction in cell motility (&minus;46&thinsp;&plusmn;&thinsp;5%) with inhibition of p-SRC. CB2-selective antagonists, gene silencing and an inhibitor of SL biosynthesis partially prevented CB2 agonist-induced effects on cell viability and motility. CB2 activation led to ceramide-mediated BC cell apoptosis independently of SL constitutive composition, which instead was modulated by CB2 agonists to reduce cell motility.

No MeSH data available.


Related in: MedlinePlus