Physiological oxygen levels affect intestinal cell cycle and renders epithelial cells more sensitive to cannabidiol through differential reactive oxygen species (ROS) production

Tara Macpherson1, John Westwick2, Karen Wright1. 1Lancaster University, Lancaster, UK, 2Novartis Institutes for BioMedical Research, Horsham, UK.

Background: Oxygen availability is a crucial factor in cellular microenvironments affecting a range of mechanisms from a change in hypoxia-related gene expression to metabolic shift. The majority of human colonic epithelial cell culture is carried out at an oxygen level of 21% (AtmosO2); where this is often cited as ‘normoxic’, it is seemingly artificial as many cells of the body are exposed to lower O2 levels (<14% O2). The human adenocarcinoma Caco-2 cell model is widely used as a platform to widen our knowledge of inflammatory mechanisms associated with epithelial dysfunction and, in addition to this, test the therapeutic potential of novel compounds. We have previously reported that proliferating Caco-2 cells, routinely passaged in a lower O2 environment are significantly more sensitive to cannabidiol (CBD) than AtmosO2 cells (1). It was the aim of this study to examine how O2 affects the cellular proliferation of PhysO2 Caco-2 cells and to examine possible cellular mechanisms which could account for the increased sensitivity seen in PhysO2 cells (5% O2) to CBD.

Methods: Caco-2 cells were routinely passaged in different O2 environments; AtmosO2 (21%O2, 5%CO2) and PhysO2 (5%O2, 5%CO2) at 37°C in humidity-regulated incubators. Cells were plated in 6-well plates at 105 cells/well and maintained in culture medium, complete with serum and amino acids. Every 2-3 days cells from one well were trypsinised and counted using trypan blue exclusion, for a period of 23 days. Carboxyfluorescein Diacetate Succinimidyl Ester (CellTrace™ CFSE Cell Proliferation Kit (C34554), Invitrogen) was used to dye cells and fluorescence was analysed by Flow Cytometry (BD FACS Canto™ II) at days 0-21. Changes in CB1 receptor expression was analysed by immunoblotting. CBD was used at (10-6 - 2.5x10-5M) and the induction of reactive oxygen species (ROS) production was measured using 2′,7′-Dichlorofluorescein diacetate (DCF/DA, Sigma #35845 10-4M).

Results: Haemocytometer counts (n = 3) showed that Atmos-PhysO2 cells (Cells in 5% O2 for experiment duration) could not maintain the level of proliferation seen in AtmosO2 cells resulting in a 4.97 (± 0.95 SD) fold difference by day 20 and a 7.43 (± 2.07 SD) fold difference between cell number in the environments at day 23 (p<0.05). Adapted-PhysO2 cells (in 5% O2 for 6+ weeks) showed the same growth profile seen in Atmos-PhysO2 cells. This effect was reversible. Flow Cytometry results indicate Adapted-PhysO2 cells have a longer cell cycle when compared to AtmosO2 cells. The CB1 receptor expression does not appear to be significantly different in AtmosO2 and PhysO2 cells. CBD-induced production of ROS was increased by 2.73-fold (± 0.86 SD) in the PhysO2 environment (n = 2).

Conclusions: Differences seen in Caco-2 cell proliferation and cell cycle length suggest that physiological O2 has a significant impact on cellular function. Increased sensitivity to cannabidiol may be related to changes in mitochondrial function that affect ROS production.

(1) Wright, K. (2009) http://www.pA2online.org/abstracts/Vol7Issue4abst151P.pdf