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Antitumorigenic mechanisms of the monoacylglycerol lipase inhibitor JZL184

Subject Area Pharmacology
Pharmacy
Term from 2016 to 2022
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 323010209
 
Final Report Year 2023

Final Report Abstract

Cannabinoids have been shown to exert antitumor effects in numerous preclinical in vivo and in vitro models. This includes anti-invasive, antimetastatic and antiangiogenic effects in addition to the wellstudied proapoptotic and antiproliferative properties. Besides the administration of corresponding receptor agonists, activation of cannabinoid receptors can also be achieved by increasing cannabinoid receptor-activating and antitumor endocannabinoids (EC) in the respective pathological foci. The latter strategy can be achieved by inhibiting the EC-degrading enzymes fatty acid amide hydrolase (FAAH; most important enzyme for the degradation of the EC anandamide) or monoacylglycerol lipase (MAGL). MAGL, an enzyme of the serine hydrolase superfamily, catalyzes the hydrolysis of monoacylglycerols to free fatty acids and glycerol and is the most important degradation enzyme for the EC 2-arachidonoylglycerol (2-AG). The mechanism of anticancer action of MAGL inhibitors is either via inhibition of degradation of 2-AG or via reduced concentrations of protumorigenic free fatty acids resulting from inhibition of degradation of other monoacylglycerides, or via both processes. The studies of this project focused on the mechanisms of potential anti-invasive/antimetastatic as well as antiangiogenic effect of MAGL inhibition in human lung cancer cells (A549, H358). An initial study addressed the effects of MAGL inhibition on lung cancer cell invasion and metastasis. Here, significantly increased 2-AG levels were detected in A549 cells treated with the MAGL inhibitor JZL184. JZL184 suppressed A549 cell metastasis in athymic nude mice in a dose- and CB1 receptordependent manner and showed an equally CB1-dependent anti-invasive effect in vitro. In contrast, MAGL inhibition-associated reduction of free fatty acids as a cause of the anti-invasive effect could be excluded by add-back experiments with palmitic acid. Both JZL184 and the MAGL substrate 2-AG 7 resulted in increased formation of tissue inhibitor of metalloproteinase-1 (TIMP-1), with suppression of TIMP-1 by siRNA significantly attenuating the anti-invasive effect of both compounds, suggesting a role for TIMP-1 as a mediator of the anti-invasive effect. Similarly, MAGL inhibitors JW651 and MJN110 or transfection with MAGL-siRNA caused a reduction in cellular invasion rates associated with TIMP-1 upregulation. A CB1- and TIMP-1-dependent anti-invasive effect was also confirmed for JZL184 in H358 cells. In another study focusing on antiangiogenic effects, conditioned medium (CM) from A549 and H358 cells treated with increasing concentrations of JZL184 or 2-AG caused a concentration- and CB1 receptor-dependent reduction in human umbilical vein endothelial cell (HUVEC) migration and tube formation compared with CM from vehicle-treated cancer cells. Experiments with JW651 and MJN110 showed the same results. Again, on the basis of siRNA experiments, TIMP-1 proved to mediate the antiangiogenic effect of JZL184 and 2-AG. On the other hand, angiogenic properties were not significantly altered by exposure to CM from JZL184- or 2-AG-treated noncancerous bronchial epithelial cells (BEAS-2B). Moreover, JZL184 induced a dose-dependent regression of A549 tumor xenografts in athymic nude mice, which was associated with a decreased number of CD31-positive cells and an upregulation of TIMP-1-positive cells in xenograft tissues. Thus, TIMP-1 formed via MAGL inhibition appears to mediate also antiangiogenic effects in addition to the previously demonstrated anti-invasive effect. Because hypoxia is an important factor in tumor biology and response to cancer therapy, a third study addressed the antiangiogenic effect of MAGL inhibition under hypoxic conditions. Here, it was shown that treatment of HUVECs with CM of A549 or H358 cells cultured for 48 h under hypoxic conditions was associated with a substantial increase in migration and tube formation, which was inhibited when the cancer cells were incubated with JZL184. Thereby, a significant increase in 2-AG levels by JZL184 was also detected under hypoxia. Analysis of a panel of proangiogenic factors revealed inhibition of hypoxia-induced vascular endothelial growth factor (VEGF) expression by JZL184. The described effects of JZL184 proceeded with partial involvement of the cannabinoid receptors CB1 and CB2. The functional importance of VEGF for angiogenesis in the selected system was supported by findings showing that CM of hypoxic cancer cells treated with JZL184 or spiked with a neutralizing VEGF antibody elicited significant inhibition of VEGF receptor 2 (VEGFR2) phosphorylation in HUVECs. On the other hand, JZL184 did not exert any direct effect on VEGFR2 activation induced by recombinant VEGF, so there seems to be no downstream effect on already released VEGF. Collectively, this work revealed a novel mechanism of anti-angiogenic action of JZL184 under conditions of hypoxic tumor-endothelial communication.

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