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Effects of cannabinoids and related fatty acids upon the viability of P19 embryonal carcinoma cells

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Abstract

Compounds acting on the cannabinoid (CB) receptors are involved in the control of cell fate, and there is an emerging consensus that CBs have anticancer effects. However, the CB-mediated effects are contradictory since some studies suggest stimulatory effects on cancer cell proliferation, and CBs have been shown to stimulate both proliferation and differentiation of other mitotic cells such as stem and progenitor cells. In this study, the concentration-dependent effects of synthetic and endogenous CBs on the viability of mouse P19 embryonal carcinoma (EC) cells have been examined by using fluorescence assays of cell membrane integrity, cell proliferation, oxidative stress, and detection of apoptosis and necrosis. All compounds examined produced a concentration-dependent decrease in cell viability in the micromolar range, with the potent CB receptor agonist HU 210 and the enantiomer HU 211 (with no CB receptor activity) being the most potent compounds examined with apparent IC50 values of 1 and 0.6 μM, respectively. The endogenous CB anandamide showed similar potency and efficacy as structurally related polyunsaturated fatty acids with no reported activity at the CB receptors. The rapid (within hours) decrease in cell viability induced by the examined CBs suggests cytocidal rather than antiproliferative effects and is dependent on the plating cell population density with the highest toxicity around 100 cells/mm2. The CB-induced cytotoxicity, which appears to involve CB receptors and the sphingomyelin–ceramide pathway, is a mixture of both apoptosis and necrosis that can be blocked by the antioxidants α-tocopherol and N-acetylcysteine. In conclusion, both synthetic and endogenous CBs produce seemingly unspecific cytotoxic effects in the P19 EC cells.

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Authors and Affiliations

  1. Department of Pharmacology and Clinical Neuroscience, Umeå University, 901 87, Umeå, Sweden

    Sofia B. Gustafsson, Anders Wallenius, Hanna Zackrisson, Dina Popova, Linus Plym Forshell & Stig O. P. Jacobsson

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  1. Sofia B. Gustafsson
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Correspondence to Stig O. P. Jacobsson.

Electronic supplementary material

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204_2013_1051_MOESM1_ESM.pdf

Online Resource Fig. 1 HU 210 does not alter cell proliferation in P19 cells. P19 cells treated with either 0.5 % DMSO, 0.1 μM HU 210 or 10 μM HU 210 were harvested by trypsinization after 72 h, centrifuged and washed in PBS. Following a second centrifugation step, cell pellets were dissolved in 0.5 ml Vindelöv solution and incubated at 4 °C until analyzed by fluorescence activated cell sorting using a Guava flow cytometer (Millipore, Billerica, USA). HU 210, at a concentration of 0.1 μM did not affect the cell proliferation compared to untreated or solvent controls (DMSO), whereas 10 μM HU 210 induced a significant cell death (Sub/G1). Cell cycle profiles are representatives of triplicate treatments (PDF 51 kb)

204_2013_1051_MOESM2_ESM.tiff

Online Resource Fig. 2 Relationship between the cytotoxicity produced by 5 h incubation with (a) HU 210, (b) AEA and the plating density of P19 EC cells. Data are the means of five separate experiments. Linear regression analysis revealed significant deviation from zero (P = 0.0034 and P < 0.0001 for 1 µM and 10 µM HU 210, respectively, and P < 0.0001 for 10 µM and 30 µM AEA). Dashed lines indicate the 95 % confidence intervals for the regression line. (TIFF 103 kb)

204_2013_1051_MOESM3_ESM.pdf

Online Resource Fig. 3 Time-dependent effects of (a) Triton X-100, (b) 10 µM staurosporine, (c) 3 µM HU 210, and (d) 30 µM AEA on YO-PRO-1 (open circles) and propidium iodide (filled circles) fluorescence in P19 EC cells. Data are mean ± SEM of eight separate experiments. (PDF 37 kb)

204_2013_1051_MOESM4_ESM.pdf

Online Resource Fig. 4 Effects of the nitric oxide synthase inhibitor L-NAME upon the cytotoxicity in P19 EC cells induced by 5 h exposure to indicated concentration of AEA or HU 210. Data (mean ± SEM of four independent experiments) are expressed as percentage calcein fluorescence of untreated controls. Statistical treatment of data was undertaken using one-way ANOVA with Dunnett’s post hoc multiple comparison test: *P < 0.05 when the effect of the treatment is compared with the corresponding AEA or HU210 control. (PDF 49 kb)

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Gustafsson, S.B., Wallenius, A., Zackrisson, H. et al. Effects of cannabinoids and related fatty acids upon the viability of P19 embryonal carcinoma cells. Arch Toxicol 87, 1939–1951 (2013). https://doi.org/10.1007/s00204-013-1051-3

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