Wasilewski Andrzej, Krajewska Urszula, Owczarek Katarzyna, Lewandowska Urszula, Fichna Jakub
Department of Biochemistry, Faculty of Medicine, Medical University of Lodz, Łódź, Poland.
Department of Pharmaceutical Biochemistry and Molecular Diagnostics, Faculty of Pharmacy, Medical University of Lodz, Łódź, Poland.
Acta Biochim Pol. 2017;64(3):519-525. doi: 10.18388/abp.2017_1520. Epub 2017 Aug 30.
Earlier reports suggest that the endocannabinoids may play a role of endogenous tumor growth modulators. In this study, we investigated whether inhibition of the enzymes involved in the synthesis and degradation of endocannabinoids may reduce colorectal cancer cell invasion and migration. The human colon adenocarcinoma Colo-205 cells were incubated with PF-3845, JZL-184 and RHC-80267 (fatty acid amide hydrolase (FAAH), mono- (MAGL) and diacylglycerol lipase (DAGL) inhibitors, respectively) for 48 h. The MTT colorimetric assay was performed to quantify cell viability. Next, Colo-205 cells were incubated with PF-3845 alone or with PF-3845 together with selected antagonists: AM 251, AM 630, SB 366791, RN 1734 and G-15 (CB, CB, TRPV1, TRPV4 and GPR30 antagonists, respectively). Western blot assay was applied to identify the changes in CB and CB receptor expression. Migration and invasion assays were employed to characterize the effect of PF-3845 on colorectal cancer cell invasion. We found that of all the inhibitors used, the FAAH inhibitor PF-3845 reduced the Colo-205 cell line viability the most effectively (IC0=52.55 μM). We also showed that the effect of decreased cell viability was enhanced when Colo-205 cells were incubated with PF-3845 and RN-1734, a TRPV4 antagonist (IC0=30.54 μM). Western blot assay revealed significantly decreased CB receptor expression levels, while CB expression was increased in response to PF-3845 when compared to control. Furthermore, PF-3845 inhibited migration and invasion of Colo-205 cell line. These results suggest that pharmacological inhibition of FAAH and consequent enhancement of the endocannabinoid levels may reduce the colorectal cancer growth and progression.
早期报告表明，内源性大麻素可能发挥内源性肿瘤生长调节剂的作用。在本研究中，我们调查了抑制参与内源性大麻素合成和降解的酶是否可以减少结肠癌细胞的侵袭和迁移。将人结肠腺癌Colo-205细胞分别与PF-3845、JZL-184和RHC-80267（分别为脂肪酸酰胺水解酶（FAAH）、单酰甘油脂肪酶（MAGL）和二酰基甘油脂肪酶（DAGL）抑制剂）孵育48小时。进行MTT比色法以量化细胞活力。接下来，将Colo-205细胞单独与PF-3845孵育，或与PF-3845以及选定的拮抗剂一起孵育：AM 251、AM 630、SB 366791、RN 1734和G-15（分别为CB、CB、TRPV1、TRPV4和GPR30拮抗剂）。应用蛋白质印迹分析来鉴定CB和CB受体表达的变化。采用迁移和侵袭试验来表征PF-3845对结肠癌细胞侵袭的影响。我们发现，在所使用的所有抑制剂中，FAAH抑制剂PF-3845最有效地降低了Colo-205细胞系的活力（IC0=52.55μM）。我们还表明，当Colo-205细胞与TRPV4拮抗剂RN-1734（IC0=30.54μM）一起与PF-3845孵育时，细胞活力降低的效果增强。蛋白质印迹分析显示CB受体表达水平显著降低，而与对照相比，PF-3845处理后CB表达增加。此外，PF-3845抑制了Colo-205细胞系的迁移和侵袭。这些结果表明，对FAAH的药理学抑制以及随之而来的内源性大麻素水平的提高可能会降低结肠癌的生长和进展。
