A simple LC-MS/MS method for the simultaneous quantification of endocannabinoids in biological samples.

The arachidonic acid derivatives N-arachidonoylethanolamine (anandamide; AEA), 2-arachidonoylglycerol (2-AG), N-arachidonoyldopamine (NADA), 2-arachidonoylglycerol ether (noladin ether; 2-AGE) and O-arachidonoylethanolamine (virodhamine; VA) were identified as physiological components of the endocannabinoid (EC) system. In order to gain further profound knowledge about the different EC-induced physiological and pathophysiological effects, appropriate analytical methods are required. The method described here uses liquid chromatography in combination with positive electrospray ionization mass spectrometry (LC-MS/MS) to quantify the concentrations of the above-mentioned EC compounds in cells. Sample preparation prior to LC-MS/MS analysis was performed by means of two liquid extractions with ethyl acetate. The method has been validated according to the bioanalytical guidelines of the Food and Drug Administration (FDA). The lower limits of quantification were 0.03 ng/mL for AEA, 2 ng/mL for 2-AG, 0.03 ng/mL for NADA, 0.3 ng/mL for 2-AGE and 0.15 ng/mL for VA. Linearity was demonstrated up to 10 ng/mL (AEA, NADA, 2-AGE and VA) and 50 ng/mL (2-AG). The values for intra- and inter-day precision and accuracy were within the guideline recommended acceptance criteria for assay validation. Low matrix effects and good recovery were found for AEA, 2-AG and 2-AGE, while a higher matrix effect was observed for NADA and VA. Extraction yields were lowest for VA. The method was used for EC measurement in different cell lines and in mouse brains.

SB202190 inhibits endothelial cell apoptosis via induction of autophagy and heme oxygenase-1.

Activation of the p38 mitogen-activated protein kinase (MAPK) pathway has been implicated in various detrimental events finally leading to endothelial dysfunction. The present study therefore investigates the impact of the p38 MAPK inhibitor SB202190 on the expression of the cytoprotective enzyme heme oxygenase-1 (HO-1) as well as metabolic activity, apoptosis and autophagy of endothelial cells. Using human umbilical vein endothelial cells (HUVEC) SB202190 was found to cause a time- and concentration-dependent induction of HO-1 protein. Induction of HO-1 protein expression was mimicked by SB203580, another p38 MAPK inhibitor, but not by SB202474, an inactive structural analogue of p38 MAPK inhibitors. HO-1 induction by both SB202190 and SB203580 was also demonstrated by analysis of mRNA expression. On the functional level, SB202190 was shown to increase metabolic activity and autophagy of HUVEC along with diminishing basal apoptosis. Treatment of cells with tin protoporphyrin IX (SnPPIX), a well-characterised HO-1 enzymatic inhibitor, or HO-1 siRNA left SB202190-modulated metabolic activity and autophagy virtually unaltered but caused a significant reversal of the anti-apoptotic action of SB202190. Conversely, however, HO-1 expression by SB202190 became completely suppressed by the autophagy inhibitor bafilomycin A1. Bafilomycin A1 likewise fully reversed effects of SB202190 on metabolic activity and apoptosis, albeit significantly inducing apoptosis per se. Collectively, this work demonstrates SB202190 to confer upstream induction of autophagy followed by HO-1 induction resulting in potential protective effects against apoptosis. On the other hand, our data oppose HO-1 to contribute to SB202190-mediated increases in metabolic activity and autophagy, respectively.

Structure-Activity Relationship of Propargylamine-Based HDAC Inhibitors.

As histone deacetylases (HDACs) play an important role in the treatment of cancer, their selective inhibition has been the subject of various studies. These continuous investigations have given rise to a large collection of pan- and selective HDAC inhibitors, containing diverse US Food and Drug Administration (FDA)-approved representatives. In previous studies, a class of alkyne-based HDAC inhibitors was presented. We modified this scaffold in two previously neglected regions and compared their cytotoxicity and affinity toward HDAC1, HDAC6, and HDAC8. We were able to show that R-configured propargylamines contribute to increased selectivity for HDAC6. Docking studies on available HDAC crystal structures were carried out to rationalize the observed selectivity of the compounds. Substitution of the aromatic portion by a thiophene derivative results in high affinity and low cytotoxicity, indicating an improved drug tolerance.

A simple method for simultaneous determination of N-arachidonoylethanolamine, N-oleoylethanolamine, N-palmitoylethanolamine and 2-arachidonoylglycerol in human cells.

The endocannabinoid system has been considered as a target for pharmacological intervention. Accordingly, inhibition of fatty acid amide hydrolase (FAAH), a degrading enzyme of the endocannabinoids N-arachidonoylethanolamine (anandamide; AEA) and 2-arachidonoylglycerol (2-AG) as well as of the endocannabinoid-like substances N-oleoylethanolamine (OEA) and N-palmitoylethanolamine (PEA), can cause augmented endogenous cannabinoid tone. Using liquid chromatography coupled with positive electrospray ionisation mass spectrometry, we herein describe a method to simultaneously quantify levels of AEA, OEA, PEA and 2-AG in cultured cells. The procedure was developed according to the FDA guidelines for bioanalytical methods validation. The limits of quantification (LOQs) were 0.05 pmol for AEA, 0.09 pmol for OEA, 0.10 pmol for PEA and 0.80 pmol for 2-AG when molecular ion monitoring was used. In H460 human lung carcinoma cells, basal levels of all four analytes ranged between 2 and 17 pmol mg(-1) protein with PEA showing the lowest and OEA the highest concentrations. Endocannabinoid levels observed in mesenchymal stem cells were of the same order of magnitude when compared to those in H460 human lung carcinoma cells.

Induction but not inhibition of COX-2 confers human lung cancer cell apoptosis by celecoxib.

The antitumorigenic mechanism of the selective cyclooxygenase-2 (COX-2) inhibitor celecoxib is still a matter of debate. Among different structurally related COX-2 inhibitors, only celecoxib was found to cause apoptosis and cell death of human lung cancer cells (IC50 values of 19.96 µM [A549], 12.48 µM [H460], and 41.39 µM [H358]) that was paralleled by a time- and concentration-dependent upregulation of COX-2 and peroxisome proliferator-activated receptor γ (PPARγ) at mRNA and protein levels. Apoptotic death of celecoxib-treated cancer cells was suppressed by the PPARγ antagonist GW9662 and by siRNA targeting PPARγ and, surprisingly, also by the selective COX-2 inhibitor NS-398 and siRNA targeting COX-2. NS-398 (1 µM) was shown to suppress celecoxib-induced COX-2 activity. Among the COX-2-dependent prostaglandins (PG) induced upon celecoxib treatment, PGD2 and 15-deoxy-Δ¹²,¹4-PGJ2 were found to induce a cytosol-to-nucleus translocation of PPARγ as well as a PPARγ-dependent apoptosis. Celecoxib-elicited PPARγ translocation was inhibited by NS-398. Finally, a COX-2- and PPARγ-dependent cytotoxic action of celecoxib was proven for primary human lung tumor cells. Together, our data demonstrate a proapoptotic mechanism of celecoxib involving initial upregulation of COX-2 and PPARγ and a subsequent nuclear translocation of PPARγ by COX-2-dependent PGs.

Cannabidiol inhibits lung cancer cell invasion and metastasis via intercellular adhesion molecule-1.

Cannabinoids inhibit cancer cell invasion via increasing tissue inhibitor of matrix metalloproteinases-1 (TIMP-1). This study investigates the role of intercellular adhesion molecule-1 (ICAM-1) within this action. In the lung cancer cell lines A549, H358, and H460, cannabidiol (CBD; 0.001-3 µM) elicited concentration-dependent ICAM-1 up-regulation compared to vehicle via cannabinoid receptors, transient receptor potential vanilloid 1, and p42/44 mitogen-activated protein kinase. Up-regulation of ICAM-1 mRNA by CBD in A549 was 4-fold at 3 µM, with significant effects already evident at 0.01 µM. ICAM-1 induction became significant after 2 h, whereas significant TIMP-1 mRNA increases were observed only after 48 h. Inhibition of ICAM-1 by antibody or siRNA approaches reversed the anti-invasive and TIMP-1-upregulating action of CBD and the likewise ICAM-1-inducing cannabinoids Δ(9)-tetrahydrocannabinol and R(+)-methanandamide when compared to isotype or nonsilencing siRNA controls. ICAM-1-dependent anti-invasive cannabinoid effects were confirmed in primary tumor cells from a lung cancer patient. In athymic nude mice, CBD elicited a 2.6- and 3.0-fold increase of ICAM-1 and TIMP-1 protein in A549 xenografts, as compared to vehicle-treated animals, and an antimetastatic effect that was fully reversed by a neutralizing antibody against ICAM-1 [% metastatic lung nodules vs. isotype control (100%): 47.7% for CBD + isotype antibody and 106.6% for CBD + ICAM-1 antibody]. Overall, our data indicate that cannabinoids induce ICAM-1, thereby conferring TIMP-1 induction and subsequent decreased cancer cell invasiveness.