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Importance of oxygen microenvironment in verifying the therapeutic potential of cannabinoids
Rationale: Human colonic epithelial cell lines used for inflammation and cancer models are cultured at ambient oxygen concentrations (atmosO2) with non-physiological nutrients, without consideration of their normal physiological setting (physO2). Cancer cell lines (and some primary) will adapt to non-physiological oxygen levels and glucose-rich medium, but this distorts our interpretation of their responses. Lower oxygen supply, normal within the gut, will provoke adapted mitochondrial respiration, which will determine the transcriptional programmes important for our analyses of their responses. Colonic tumours arise from low oxygen environments and gut research needs to be directed towards targets that can be validated within physiological metabolic environments. Compounds with therapeutic potential should be more accurately validated in a relevant ex vivo setting. Tissue oxygen in the healthy human colon ranges from 2-6% in the lumen to 10-12% at the basement membrane. Cells cultured under pathophysiological hypoxic conditions (<2% O2) have been compared with atmosO2 but how well this knowledge compares to the physiological environment is unknown. The aim of this study was to compare the impact of cannabinoids on proliferation of a human colonic epithelial cell line cultured in either physO2 or atmosO2.
Methods: The Caco2 colonic epithelial cell line was cultured and passaged routinely in humidity- and temperature- (37°C) controlled environments: atmosO2 (5% CO2, 20% O2) and physO2 (5% CO2, 6% O2). After 6 weeks adaption in physO2, cycling cells (105 cells/well, 24-well plates) were treated daily with a range of cannabinoids: phytocannabinoid cannabidiol (CBD) (0.01μM – 25μM), CB65 (CB2 receptor agonist), ACEA (CB1 receptor agonist), CB13 (dual CB1/CB2 agonist), AM251 (CB1 antagonist/inverse agonist), AM630 (CB2 antagonist/inverse agonist) for 72h in medium (MEM) with serum (1% or 5%) and supplements, concurrent with cells in atmosO2. Viable cell numbers (CyQUANT(1)) and metabolic activity (XTT(2)) was measured and expressed as mean % of vehicle (DMSO) control ± S.E.M., n = 4. Data were analysed by one-way ANOVA and Dunnett’s post hoc test.
Results: Cell numbers and metabolic activity dose-response curves revealed a left shift in physO2 versus atmosO2, significantly for CBD: (EC50 = 4.1 ± 1.8μM (5% serum) and 1.25 ± 0.9μM (1% serum) vs 18.6 ± 2.1μM (5%) and 7.5 ± 1.9μM (1%), respectively, p≤0.05. The increased sensitivity to CBD was reflected by the fold-reduction in the dose required for the maximal response: 2.68 ± 0.7 (5%) and 3.36 ± 0.4 (1%), p≤0.05.
Conclusions: Differential cannabinoid-mediated anticancer effects under physO2 conditions versus atmosO2 implies that ex vivo cell models used for drug target validation would provide more accurate and reliable information if the physiological microenvironment was taken into account.
(1) Jones et al., J Immunol Methods 2001, 254(1-2), 85-98. |
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