The Monoacylglycerol Lipase Inhibitor JZL184 Inhibits Lung Cancer Cell Invasion and Metastasis via the CB₁ Cannabinoid Receptor

Cannabinoid compounds have been shown to exert antitumor effects via multiple mechanisms in a large number of preclinical in vivo and in vitro models. In this context, it was shown that cannabinoids, in addition to their well-known proapoptotic and antiproliferative properties, also exert potent anti-invasive and antimetastatic effects (for review see ref. 1). Activation of cannabinoid receptors can be achieved by administering agonists or by increasing cannabinoid receptor-activating endocannabinoids in the respective pathologic foci (for review see ref. 2). This latter strategy has likewise attracted substantial interest for cancer therapy based on findings showing elevated endocannabinoid levels in certain cancers (3–5). In view of the fact that the endocannabinoids produced after the onset of cancer could counteract neoplasia in a site-specific manner, drugs that selectively inhibit endocannabinoid degradation could be expected to effectively support this form of endogenous tumor defense (for review, see refs. 1, 2). The first of these strategies is the inhibition of the enzyme fatty acid amide hydrolase (FAAH), a member of the serine hydrolase family identified as the major catabolic enzyme of the endocannabinoid anandamide (N-arachidonoylethanolamine, AEA; ref. 6). The second strategy to combat tumor promotion by increasing endocannabinoids is based on the inhibition of monoacylglycerol lipase (MAGL), an enzyme of the serine hydrolase superfamily that catalyzes the hydrolysis of monoacylglycerols to free fatty acids and glycerol. MAGL was originally described as contributing to lipolysis in fatty tissue (7) and has since been identified as the most important degrading enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG; ref. 8).

A valuable tool to study the contribution of MAGL in influencing the fate of tumor cells has been developed with the potent and selective MAGL inhibitor JZL184 (9), which irreversibly inhibits MAGL by carbamoylating the serine nucleophile of the enzyme (10). In the pioneering work published by Nomura and colleagues (11), it was shown that both JZL184 and MAGL knockdown by small hairpin (sh) RNA inhibit the increased pathogenicity (tumor cell invasion, migration, tumor growth in vivo) caused by the overexpression of MAGL in nonaggressive cancer cells. A follow-up study of the same group finally revealed that the inhibition of the aggressiveness of prostate cancer cells achieved by JZL184 or MAGL shRNA was partially reversed by single administration of fatty acids or a CB₁ receptor antagonist and completely when administered in combination (12). Therefore, both an increase of 2-AG and a decrease of protumorigenic free fatty acids seem to be involved in the corresponding MAGL inhibitory effects, at least in prostate carcinoma cells. Meanwhile, an anti-invasive effect of JZL184 has also been demonstrated for colorectal (13), hepatocellular (14), prostate (15), and breast cancer cells (15), although the underlying molecular mechanisms have not been addressed. In the latter study, JZL184 was further shown to impair bone metastasis of osteotropic prostate and breast cancer cells in mice and to inhibit metastasis of osteosarcoma cells (15). Finally, another study revealed MAGL knockdown to be associated with reduced lymph node metastasis in MAGL-overexpressing nasopharyngeal carcinoma cells (16). However, the mechanisms of antimetastatic properties have not yet been sufficiently investigated, also with regard to the cannabinoid receptors involved in these processes. Small molecules should be the focus of such studies considering that therapeutic interventions in the treatment of aggressive tumors require pharmacologic tools and that different lipidomic profiles can be induced by JZL184 and MAGL shRNA (11).

For these reasons, the present study investigated the effect of MAGL inhibition using JZL184 on metastasis and invasion of human lung cancer cells. For the first time, we provide evidence that JZL184 inhibits cancer cell metastasis via a CB₁ receptor–dependent process. In the case of the likewise CB₁ receptor–dependent anti-invasive effect, an increased expression of the tissue inhibitor of metalloproteinase-1 (TIMP-1) was demonstrated as the underlying mechanism.

JZL184, 2-AG, AM-251, AM-630, palmitic acid, and pure standards for LC-MS analysis of AEA, 2-AG, OEA, PEA, and AEA-d8 were from Cayman Chemical. Dimethyl sulfoxide (DMSO), ethylenediaminetetraacetic acid (EDTA), glycerol, glycine, sodium chloride (NaCl), Tris hydrocloride (Tris-HCl), and Tris ultrapure were purchased from AppliChem. 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) was bought from Ferak. JW651, MJN110, capsazepine, aprotinine, bovine serum albumin (BSA), HCl, hydrogen peroxide (H₂O₂), luminol, orthovanadate, para-coumaric acid, para-formaldehyde, phenylmethylsulfonyl fluoride (PMSF), poly-D-lysine hydrobromide, and Triton X-100 were from Sigma-Aldrich. Leupeptin was purchased from Biomol. Crystal violet and acrylamide (Rotiphorese Gel 30) were obtained from Carl Roth GmbH. Dulbecco's modified eagle medium (DMEM) with 4.5 g/L glucose and with UltraGlutamine I was from Lonza. Penicillin–streptomycin was purchased from Invitrogen. Phosphate-buffered saline (PBS) and fetal calf serum (FCS) were bought from PAN Biotech. Milk powder was obtained from Bio-Rad Laboratories GmbH. Lipofectamine RNAiMAX Transfection Reagent and Opti-MEM I Reduced Serum Medium were from Thermo Fisher Scientific Inc.

A549 human lung carcinoma cells were purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ cat. No. ACC-107, RRID:CVCL_0023). Species confirmation as human was carried out by the supplier using isoelectric focusing of malate dehydrogenase, nucleosid phosphorylase, and fingerprint. Multiplex PCR of minisatellite markers revealed a unique DNA profile. NCl-H460 (ATCC cat. No. HTB-177, RRID:CVCL_0459 assigned as H460 cells) and NCl-H358 (ATCC cat. No. CRL-5807, RRID:CVCL_1559 assigned as H358 cells) were obtained from ATCC-LGC. Cell line confirmations of H460 and H358 were carried out by the supplier using cytogenetic analyses. All cell lines were frozen in large stocks at early passages and were used within 6 months following resuscitation. A549, H358, and H460 lung carcinoma cells were cultured in DMEM containing 10% heat-inactivated FCS, 100 U/mL penicillin, and 100 μg/mL streptomycin. The cells were cultured in a humidified incubator at 37°C and 5% CO₂. All incubations were carried out in serum-free DMEM. Test substances (2-AG, JZL184, JW651, MJN110, AM-251, AM-630, capsazepine) were dissolved in DMSO, or ethanol (palmitic acid), after which the stock solutions were diluted with PBS. The final solvent concentrations per substance used in the cell incubates were 0.1% (v/v) DMSO or 0.1% (v/v) ethanol. Even when combining different test substances, the final concentration of DMSO in media was never higher than 0.3% (v/v) DMSO. The vehicle control contained the respective concentrations of DMSO and ethanol. In all experiments, the incubation media of the vehicle and substance-treated cells contained the same amount of solvent. 2-AG was supplied by the manufacturer as a mixture of 2-AG and 1-AG (9:1). Therefore, all molar concentrations in the in vitro experiments refer to nine parts 2-AG and one part 1-AG.

Cell invasiveness was quantified by a modified Boyden chamber assay using Falcon cell culture inserts (8-μm pore size; Corning Inc.). In this assay, the cells seeded onto the inserts must pass a reconstituted basement membrane (Matrigel) by proteolytic degradation of a matrix gel layer and subsequently migrate through a polyethylene terephthalate membrane with 8-μm pores to a chemoattractant in the 24-well companion plate (lower compartment). In brief, the upper sides of the inserts were coated with 28.4-μg Matrigel (Corning Matrigel Basement Matrix, Corning Inc.) per insert. Cells were placed at a density of 5 × 10⁵ cells in a volume of 500-μL serum-free DMEM per insert and treated with test substances or vehicles for the indicated times. DMEM with 10% FCS served as a chemoattractant in the 24-well companion plate. After incubation in a humidified incubator at 37°C and 5% CO₂ for the indicated times, the noninvaded cells remaining on the upper side of the inserts were removed with a cotton swab. Invaded cells adhering to the lower side of the insert were quantified by using the colorimetric WST-1 assay (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1.3-benzene disulfonate; Sigma-Aldrich). For calculation of migration, experiments were performed with uncoated inserts. Invasion was expressed as an invasion index, where readings from WST-1 assays (absorbance at 450 nm with a reference filter at 690 nm) of approaches in which cells invaded through Matrigel-coated Boyden chambers were divided by readings from cells that migrated through uncoated control inserts with the same treatment [(invasion/migration) × 100%]. To visualize the basic effect of JZL184 on invasion and migration, cells after having been invaded or migrated were fixed in ethanol and stained with 0.1% (w/v) crystal violet. Images of cells were then taken under a phase contrast microscope at 100× magnification. The treatment protocol for invasion and migration assays following MAGL small interfering (si) RNA transfections is described in the respective Method section (“siRNA transfections”).

To exclude the possibility that nonspecific cytotoxicity was involved in the anti-invasive effects of MAGL inhibitors and 2-AG, cell viability was quantified in each case under similar conditions. To arrange comparable conditions with the invasion assays, cells were seeded on 48-well plates at 5 × 10⁵ cells per well in a volume of 500-μL DMEM per well. Thereafter, cells were immediately treated with test substance or vehicle for another 72 hours followed by evaluation of viability using the WST-1 test. The treatment protocol for cytotoxicity assays following MAGL siRNA transfections is described in the respective Method section (“siRNA transfections”).

In addition, WST-1 tests were also performed at lower cell densities (Supplementary Fig. S4). For this purpose, A549 cells were seeded at a density of 5 × 10³ cells per well of a 96-well plate in DMEM at 10% (v/v) FCS and allowed to adhere for 6 hours. The cells were then cultivated under serum-free conditions for 12 hours before being treated with vehicle or the specified concentrations of JZL184 or 2-AG in serum-free DMEM or DMEM with 10% (v/v) FCS for 48 hours.

To examine the modulation of migration with a second method, scratch wound tests were also carried out. For this purpose, cells with a density of 1 × 10⁵ cells per well were seeded in DMEM with 10% (v/v) FCS on 24-well plates. After 2 days, the confluent monolayers of cancer cells were scratched with a sterilized pipette tip (from a 10-μL pipette) to create a “wound” devoid of cells. The cells were then washed with PBS and incubated with JZL184 or vehicle in serum-free DMEM. Images of the wound region were taken after the specified incubation times (0, 6, 24, or 48 hours) under a phase contrast microscope at 100× magnification. For each individual migration course examined, the mechanical coordinate scale on the microscope was always relocated to the same position, the coordinates of which were determined at time 0, respectively. In order to record the wound area digitally, the wound edges were traced with the public domain software ImageJ (RRID:SCR_003070). For this procedure, the captured images were converted to gray scales, the edges found (Process → Find Edges), and the image was blurred 40 times (Process → Smooth) before the sharp changes in intensity were highlighted (Process → Find Edges). As a result, the areas with cells were white, whereas the areas without cells remained black. A MinError threshold was then applied (Figure → Adjust → Auto Threshold: MinError) to automatically detect the wound area. The wound areas were then quantified (Analyze → Analyze Particles [size: 100,000–infinite]). Wound closure (%) was calculated as follows: [(wound area immediately after scratching – wound area after the respective incubation)/wound area immediately after scratching] × 100%.

The influence of test substances on the proliferation of A549 cells was determined with the bromodeoxyuridine (BrdU) Cell Proliferation ELISA Kit (colorimetric; Abcam). In short, 5 × 10³ A549 cells were seeded on 96-well plates in DMEM with 10% FCS (v/v) and allowed to adhere for 6 hours. The cells were then washed with PBS and subsequently kept under serum-free conditions for 12 hours followed by 48-hour incubation with different concentrations of JZL184 or 2-AG in serum-free DMEM or DMEM with 10% (v/v) FCS. The BrdU reagent was added 24 hours before analysis. At the end of incubation, the supernatant was removed and the assay was performed according to the manufacturer's instructions.

To demonstrate the colony-forming properties of A549 cells, Petri dishes with a diameter of 10 cm were coated with a poly-D-lysine hydrobromide solution (0.05 mg/mL). 1 × 10³ A549 cells were seeded in DMEM with 10% (v/v) FCS per dish. After 48 hours the first treatment with vehicle or JZL184 in DMEM with 10% (v/v) FCS was performed. The treatment was repeated after 7 days and continued for another 7 days. Then the cells were fixed in ethanol, stained with 0.1% (w/v) crystal violet, and photographed. Colonies were counted as total number per Petri dish.

The quantitative analysis of β-actin (internal standard), TIMP-1, and MAGL mRNA levels was performed using real-time reverse transcriptase polymerase chain reaction (RT-PCR) as described previously (17). In brief, the cells were seeded on 24-well plates with a density of 1 × 10⁵ cells per well and cultivated in DMEM with 10% FCS for 24 hours. The cells were then washed and, in the case of TIMP-1 analyses, incubated with vehicle or the specified concentrations of JZL184 or 2-AG for 6 hours. For MAGL mRNA analyses, A549 cells were seeded on 24-well plates at a density of 1 × 10⁵ cells per well and incubated with a final concentration of 10 nmol/L MAGL siRNA or nonsilencing siRNA as negative control according to the manufacturer's instructions in the chapter “Reverse Transfection.” After 24 hours, the cells were washed and incubated with serum-free DMEM for another 24 hours. Subsequently, cells were lysed and subjected to RNA isolation using the RNeasy Mini Kit (Qiagen GmbH). Total RNA was used to perform RT-PCR with the Applied Biosystems TaqMan RNA-to-CT 1-Step Kit and the Applied Biosystems TaqMan Gene Expression Assays (Thermo Fisher Scientific Inc.) according to the manufacturer's instructions.TIMP-1 and MAGL mRNA levels were normalized to β-actin.

For the Western blot analysis of TIMP-1 and β-actin in cell lysates, 2 × 10⁵ cells per well were seeded on 6-well plates. After 24 hours, the cells were incubated with test substances or vehicles in serum-free DMEM. After a 72-hour incubation period, cells were used for analysis of TIMP-1 and β-actin from cell lysates. The treatment protocol for monitoring the expression of TIMP-1, MAGL, and β-actin after TIMP-1 or MAGL siRNA transfection is described in the following Method section (“siRNA transfections”).

For analysis of the cell lysates, the cells were washed, harvested, and lysed in solubilization buffer (50 mmol/L HEPES, pH 7.4; 150 mmol/L NaCl; 1 mmol/L EDTA; 1% (v/v) Triton X-100; 10% (v/v) glycerol; 1 mmol/L PMSF; 1 μg/mL leupeptin; 0.5 mmol/L orthovanadate; and 10 μg/mL aprotinin). Lysis was performed on ice for at least 30 minutes and frequent mixing on the vortex mixer, followed by centrifugation at 20,817 × g for 5 minutes. Supernatants were used for Western blot analysis.

In the case of Western blot analyses of membrane fractions (Fig. 2E), the latter were prepared from the harvested cells using the ProteoExtract Native Membrane Protein Extraction Kit (Merck Millipore; cat. No. 444810) according to the manufacturer's instructions under “Extraction of membrane proteins from adherent tissue culture cells.” The membrane fractions were then concentrated with the Amicon Ultra-0.5 centrifugal filter unit (Merck Millipore; cat. No. UFC501024) with a cutoff of 10 kDa according to the manufacturer's instructions.

Both the total protein in cell culture lysates and the proteins of membrane fractions were measured with the bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific Inc.). Equal amounts of denatured proteins were then separated on a 10% sodium dodecyl sulfate polyacrylamide gel. After transfer to nitrocellulose and blocking of the membranes with 5% milk powder or BSA, the blots were incubated overnight at 4°C with specific antibodies against TIMP-1 (Millipore; cat. No. MAB3300, RRID:AB_2204544), MAGL (Abgent, supplied by Biomol, Hamburg; Abgent; cat. No. AP16876a, RRID:AB_11135343), CB₁ receptor (Cell Signaling Technology; cat. No. 93815, RRID:AB_2756361), CB₂ receptor (Cayman Chemical; cat. No. 101550, RRID:AB_10079370), TRPV1 (Novus Biological Abingdon; cat. No. NBP1-97417, RRID:AB_11137892), or β-actin (Sigma-Aldrich; cat. No. A5316, RRID:AB_476743). The membranes were then exposed to horseradish peroxidase–conjugated Fab-specific anti-mouse IgG (in the case of TIMP-1 and β-actin; Cell Signaling Technology; cat. No. 7076, RRID:AB_330924) or an anti-rabbit IgG (in the case of MAGL, CB₁, CB₂, and TRPV1; Cell Signaling Technology; cat. No. 7074, RRID:AB_2099233) for 1 hour at room temperature.

Antibody binding was visualized by a chemiluminescent solution (100 mmol/L Tris-HCl pH 8.5, 1.25 mmol/L luminol, 200 mmol/L p-coumaric acid, 0.09% [v/v] H₂O₂, 0.0072% [v/v] DMSO). The densitometric analysis of the band intensities was achieved by optical scanning and quantification with the Quantity One 1-D analysis software (Bio-Rad Laboratories GmbH; RRID:SCR_014280). A precolored SDS-PAGE standard (Broad Range; Bio-Rad) was used to identify the band sizes. To determine a uniform protein loading in Western blots of cell lysates, the membranes were probed with an antibody raised against β-actin. The densitometric values of TIMP-1 and MAGL obtained from the analyses of the cell lysates were normalized to those of β-actin. For the evaluation of changes in protein expression, the vehicle controls were defined as 100%.

For reasons of comparison, the TIMP-1 protein levels in cell culture supernatants were determined in one experiment. Thereby, equal volumes of cell culture supernatants per group were used for Western blot analyses.

The cells were transfected with siRNA targeting TIMP-1 (Qiagen, cat. No. 1027418, SI00745318), MAGL (Thermo Fisher Scientific Inc., siRNA ID# 43371), or with a nonsilencing negative control RNA (Eurogentec; cat. No. SR-CL000-005) using the Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific Inc.) according to the manufacturer's instructions under slightly modified conditions. SiRNA-Lipofectamine RNAiMAX complexes were prepared in Opti-MEM I Reduced Serum Medium (Thermo Fisher Scientific Inc.). The manufacturer protocols define transfections with siRNA complexes of already adherent cells as “forward” transfections and of cells seeded in medium with siRNA complexes within the wells as “reverse” transfections. For siRNA experiments targeting TIMP-1 in A549 and H358 cells, transfections were performed with a final siRNA concentration of 2 nmol/L TIMP-1 siRNA or nonsilencing siRNA as negative control according to the manufacturer's protocol under “Reverse Transfection.” Due to the less effective MAGL knockdown, a protocol was established for these experiments that included several consecutive transfections. Accordingly, A549 cells were seeded at a density of 1 × 10⁵ cells per well of a 6-well plate and transfected with a final siRNA concentration of 10 nmol/L MAGL siRNA or nonsilencing siRNA as negative control according to the manufacturer's instructions under “Reverse Transfection” with slight modifications. After 24 hours, the media were rinsed and replaced by new media containing the transfection reagent and the same siRNA concentrations with 10% FCS according to the manufacturer's instructions under “Forward Transfection.” The latter transfection procedure was repeated once. Following another 24 hours, the cells were transfected again in serum-free medium. After this last transfection step (i.e., after a total of four transfections), the treatment protocols differed according to the respective posttransfection procedure. For the Western blot analyses, the incubation of the A549 cells was continued for another 48 hours before the subsequent lysis of the cells. In the case of viability, invasion, and migration assays, A549 cells were incubated for a further 24 hours before being washed, trypsinated, and centrifuged at 1,000 × g. The cells were than seeded at a density of 5 × 10⁵ cells in a volume of 500 μL per well of a 48-well plate (viability assay) or per cell culture insert (migration, invasion) and incubated for a further 24 hours before the measurements of invasion, migration, or viability by the WST-1 test were performed.

A549 cells were seeded on Petri dishes with a diameter of 10 cm at a density of 2 × 10⁶ cells per dish and cultivated at 37°C in DMEM supplemented with 10% FCS, 100 U/mL penicillin, and 100 μg/mL streptomycin. At 24 hours after seeding, cells were washed once with PBS. Cells were then treated in serum-free DMEM with vehicle or the appropriate concentration of JZL184 for 6 hours before harvesting. For each individual sample (vehicle or JZL184), six Petri dishes were pooled. Cell harvesting was performed by scraping the cells. Cell pellets obtained after centrifugation at 2,000 rpm for 10 minutes at 4°C were immediately frozen in liquid nitrogen and stored at −80°C before analysis. To determine the endocannabinoids, the cell pellets were added to 1 mL 20 mmol/L Tris-HCl buffer (pH 6.8), mixed with 20 ng/mL AEA-d8, further resuspended and lysed three times with a Sonopols U-tip Sonifier with a 15 × 5-second pulse at 75% power followed by a 60-second pause. The lysates were transferred to ice-cold, screw-capped glass tubes. Parallel to the standard solutions the samples were extracted and analyzed as described recently (18). In brief, the extracted samples (30–60 μL) were analyzed on a Waters HPLC 2695 Separation Module with a Multospher 120 C18 column 125 × 2 mm, 5-μm particle size (CS-Chromatography Service GmbH) coupled with a guard column (20 × 2 mm, 5-μm particle size). Endocannabinoids and endocannabinoid-like substances were separated with mobile phase A (water with 0.2% formic acid) and mobile phase B (acetonitrile/2-propanol [60:40, v/v] with 0.2% formic acid) at a flow rate of 0.15 mL/minute. The elution scheme was as follows: linear increase of mobile phase B from 65% to 80% in 10 minutes, isocratic at 80% of phase B in 3 minutes and linear to 100% of phase B in the following 6 minutes. Finally, the system was rebalanced at 35% phase A for 4 minutes. The HPLC column effluent was introduced into a Micromass Quatro Micro API mass spectrometer and analyzed by electrospray ionization in positive mode and single ion monitoring (SIM) mode: m/z 300.8 for PEA, m/z 326.8 for OEA, m/z 348.8 for AEA, m/z 379.8 for 2-AG, and m/z 356.8 for the internal standard (AEA-d8). The mass spectrometer and source parameters were set as follows: capillary voltage 3.5 kV; cone voltage 20 and 24 V for AEA/AEA-d8/2-AG or PEA/OEA; source temperature 120 °C; desolvation temperature 350°C; desolvation gas flow rate 700 L/hour. The dwell and delay times were 0.05 and 0.1 seconds, respectively. All instrument parameters for the monitored analytes were set by injecting standard solutions in a concentration of 100 ng/mL at 10 μL/minute flow rate using a syringe pump. The data were acquired using MassLynx software version 4.1 (Micromass Ltd.; RRID:SCR_014271). After quantification, the signals obtained for each analyte were normalized to the amount of internal standard observed in the corresponding sample. No influence of additives was observed when establishing the calibration curves for each of the standards used. The values shown correspond to the measured concentrations of the analytes normalized to the cellular protein concentrations. For protein measurement, 10 μL of each lysate was used for protein determination with the BCA protein assay kit (Thermo Fisher Scientific Inc.).

Immunodeficient female athymic nude mice (NMRI-nu/nu) received injections of A549 cells (1 × 10⁶ cells in 100-μL PBS per 10 g body weight) through the lateral tail vein. Mice were treated intraperitoneally with the respective test substance or vehicle for the first time 24 hours later. The test substances or their vehicles were then administered every 72 hours for 28 days. AM-251 was administered 30 minutes prior to injection of JZL184 to ensure CB₁ receptor blockage prior to injection of JZL184. The mice were sacrificed 1 day after the last administration and the entire lung was examined for metastases. To contrast the pulmonary nodules, the lungs were fixed in Bouin's fluid (saturated picric acid, formaldehyde, glacial acetic acid, 15:5:1 [v/v/v]) and the metastatic nodules were examined under a stereomicroscope. Lung specimens were fixed in 4% (v/v) formalin and embedded in paraffin for histopathologic examination. To better visualize the metastatic nodules, the paraffin sections were stained with hematoxylin and eosin. The experiments were conducted in accordance with German legislation and EU Directive 2010/63/EU and were approved by the local authorities. The mice were provided by the Animal Core Facility of the Rostock University Medical Center and were kept under specified pathogen-free conditions.

Comparisons between two groups were carried out using a Student unpaired t test. Comparisons between more than two groups were performed by one-way ANOVA with Bonferroni or Dunnett post hoc test. All statistical analyses were conducted with GraphPad Prism 7.0 (GraphPad Software, Inc.; RRID:SCR_002798).

To demonstrate the efficiency and selectivity of JZL184 with respect to the turnover of endocannabinoids and endocannabinoid-like substances by A549 cells, a previously established LC-MS/MS method (18) was used to simultaneously quantify the endocannabinoids AEA and 2-AG and the endocannabinoid-like substances oleoylethanolamide (OEA) and palmitoylethanolamide (PEA). The analysis of lysates of cells treated with JZL184 showed significantly increased intracellular concentrations of 2-AG in the presence of JZL184 (Fig. 1A). No increases were observed in FAAH substrates OEA and PEA. In the same samples, the major FAAH substrate AEA was below the limit of quantification.

To evaluate the influence of MAGL inhibition on experimental metastasis, athymic nude mice were intravenously injected with A549 lung cancer cells followed by the administration of JZL184 over a period of 4 weeks. As shown in Fig. 1B, Supplementary Figs. S1A and S2A, JZL184 caused a dose-dependent antimetastatic effect. Accordingly, in animals treated with JZL184 at a dose of ≥ 8 mg/kg every 72 hours, the number of metastatic nodules in the lung was significantly reduced.

In addition, a significant inhibition of the antimetastatic effect of JZL184 was observed when mice were cotreated with the CB₁ receptor antagonist AM-251, indicating the important role of CB₁ in this response (Fig. 1C; Supplementary Figs. S1B and S2B). Interestingly, the number of metastatic nodes was slightly but significantly reduced in the group treated with AM-251 alone, suggesting that AM-251 has an antimetastatic effect per se.

To determine the mechanism underlying the antimetastatic effect of JZL184, the in vitro effect of the MAGL inhibitor JZL184 on the invasion of A549 cancer cells was investigated next. As previously described, 2-AG, the primary MAGL substrate, reduces both the invasiveness and metastasis of these cells (19). JZL184 was also shown to have a concentration-dependent, anti-invasive effect (Fig. 2A, black bars; Supplementary Fig. S3A, top). The reduced invasion by treatment with JZL184 was not associated with a decrease in migration through membranes not coated with Matrigel (Fig. 2A, gray bars; Supplementary Fig. S3A, bottom). The absence of an antimigratory effect of JZL184 was further confirmed in scratch wound experiments (Fig. 2C; Supplementary Fig. S3B). It is noteworthy that none of the JZL184 concentrations tested elicited a cytotoxic response to A549 (Fig. 2A, white bars) when tested under conditions comparable with the Matrigel invasion test (5 × 10⁵ cells per 500 μL per well of a 48-well plate). This allowed the exclusion of a nonspecific toxicity-related phenomenon under the high cell density conditions prevailing in the invasion assay. In a time-course experiment, after a 24-hour incubation period with JZL184, a significant suppression of the invasiveness of A549 cells was observed, which increased in intensity after 48 and 72 hours (Fig. 2B).

Regardless of the lack of cytotoxic effect of JZL184 under conditions of high cell density (Fig. 2A, white bars), further control experiments were conducted to investigate whether JZL184 and 2-AG, in addition to the anti-invasive effect shown, might have an effect on the proliferation and viability of A549 cells under conditions of lower cell density. The corresponding parameters were investigated in the BrdU and WST-1 assay, respectively. For this purpose, A549 cells (5 × 10³ cells per well of a 96-well plate) were treated with JZL184 or 2-AG in serum-free medium or DMEM with 10% FCS (v/v) in concentrations between 0.01 and 10 μmol/L. In all experiments, DMEM with 10% FCS (v/v) profoundly induced proliferation (Supplementary Fig. S4A and S4C) and led to increased cell viability (Supplementary Fig. S4B and S4D) after an incubation period of 48 hours compared with serum-free medium. According to Supplementary Fig. S4A and S4B, neither proliferation nor viability was significantly reduced by JZL184 under both serum and serum-free conditions compared with the respective vehicle-treated A549 cells. At a final concentration of 10 μmol/L JZL184, there was only a 7% or 13% reduction in viability of cells incubated in serum-free or serum-containing medium, respectively, versus corresponding vehicle control (Supplementary Fig. S4B). For 2-AG, a significant decrease in BrdU incorporation under serum-free and serum-containing conditions (Supplementary Fig. S4C) as well as in viability under serum-free conditions could only be demonstrated at a concentration of 10 μmol/L (Supplementary Fig. S4D). In the case of JZL184, a comparable lack of inhibitory effect on the proliferative potential of A549 cells could also be confirmed using colony-forming assays (Supplementary Fig. S5).

In a previous work of this group, the expression of the cannabinoid receptors CB₁ and CB₂ as well as the transient receptor potential vanilloid 1 (TRPV1) was proven in the lung cancer cell lines A549, H460, and H358 (20). To investigate whether the anti-invasive effect caused by MAGL inhibition is due to the activation of these receptors, the influence of antagonists to CB₁ (AM-251), CB₂ (AM-630), and TRPV1 (capsazepine) on the invasiveness of A549 cells was tested. All antagonists were used at a concentration of 1 μmol/L, which has been shown to be efficient in detecting CB₁-, CB₂-, and TRPV1-dependent events (19–23). In accordance with the data obtained in the metastasis model, the anti-invasive effect of JZL184 was also completely reversed by the CB₁ antagonist AM-251 (Fig. 2D, black bars). In contrast, the CB₂ antagonist AM-630 and the TRPV1 antagonist capsazepine were virtually inactive in this respect (Fig. 2D, black bars). Remarkably, none of the antagonists used showed a significant effect on the invasiveness of A549 cells per se (Fig. 2D, gray bars). In view of the fact that CB₁ agonism can be accompanied by a downregulation and consequently desensitization of the CB₁ receptor (24–27), further analyses addressed the influence of JZL184 on the membrane protein levels of CB₁ (Fig. 2E, white bars), but also of CB₂ (Fig. 2E, gray bars) and TRPV1 (Fig. 2E, black bars). For none of the receptors investigated a reduction of the protein concentration could be proven. In the case of the CB₁ receptor, there was a moderate but nonsignificant increase in the corresponding membrane receptor levels by 1 μmol/L JZL184 (Fig. 2E, white bars).

To exclude a reduction of free fatty acids by MAGL inhibition as a further underlying pathway of the anti-invasive effect of JZL184, additional invasion assays with palmitic acid were performed. Here the free fatty acids potentially reduced by JZL184 should be normalized in the sense of an add-back. However, in contrast to another study performed with prostate cancer cells (12), the addition of palmitic acid could not reverse the anti-invasive effect caused by MAGL inhibition and per se had no significant effect on invasiveness (Fig. 2F).

In previous studies, upregulation of the endogenous matrix metalloproteinase (MMP) inhibitor TIMP-1 has been associated with the anti-invasive effect of several cannabinoids as well as the FAAH inhibitors URB597 and AA-5HT (19, 20, 22, 23). Therefore, in a next step, the influence of JZL184 on TIMP-1 expression was tested. Western blot analysis showed a significant TIMP-1 upregulation in response to JZL184 (Fig. 3A) and 2-AG (Fig. 3B). Interestingly, TIMP-1 upregulation was less pronounced when cells were treated with 10 μmol/L 2-AG. It is worth mentioning that an increase of TIMP-1 was also observed in cell culture media following a 72-hour treatment with vehicle and JZL184 or 2-AG. Here TIMP-1 was increased to 193% ± 7% and 223% ± 12% compared with vehicle (100% ± 4%, mean values ± SEM of n = 4 experiments) in response to treatments with 1 μmol/L JZL184 or 2-AG each. In the lysates of the identical experimental approaches, the corresponding induction of TIMP-1 was 230% ± 16% (JZL184) and 166% ± 37% (2-AG) compared with vehicle (100% ± 5%), suggesting a comparable TIMP-1 induction in medium and lysate at least in this experiment. In accordance with the protein data, TIMP-1 mRNA levels were elevated after a 6-hour exposure to JZL184 and 2-AG [123% ± 12% (JZL184; 1 μmol/L) and 139% ± 3% (2-AG; 1 μmol/L) vs. vehicle (100% ± 6%), mean ± SEM of n = 4 experiments].

To investigate a causal relationship between TIMP-1 induction and the anti-invasive effect of MAGL inhibition, a specific siRNA targeting TIMP-1 was tested for its influence on the reduced invasiveness and increased TIMP-1 expression caused by JZL184. As shown in Fig. 3C, the anti-invasive effect of JZL184 was almost completely eliminated when cells were transfected with TIMP-1 siRNA, whereas there was no significant alteration by the nonsilencing siRNA. A similar effect was observed with the administration of 2-AG and TIMP-1 siRNA, with a less pronounced reversal of the anti-invasive effect of 2-AG due to the knockdown of TIMP-1 (Fig. 3D). Analysis of TIMP-1 levels confirmed a complete inhibition of JZL184- or 2-AG–induced TIMP-1 expression in cells transfected with TIMP-1 siRNA (Fig. 3C and D).

To illustrate the signaling pathway derived from the data shown so far, the relationship between the individual parameters is shown schematically in Fig. 3E.

To further exclude possible nonspecific effects of JZL184, invasion tests with two additional MAGL inhibitors, JW651 and MJN110, were performed. Both MAGL inhibitors caused a reduced invasion of A549 cells (Fig. 4A and B, left panel each) as well as TIMP-1 upregulation (Fig. 4A and B, right panel each), whereas migration through membranes not coated with Matrigel and viability tested under comparable conditions remained virtually unchanged (Fig. 4A and B, left panel each).

In addition, the effect of MAGL knockdown on the invasiveness of A549 cells was investigated using a specific siRNA targeting MAGL. MAGL siRNA led to a significantly reduced expression of MAGL mRNA and protein (Fig. 4C). As expected from the results described above, the MAGL knockdown was associated with an increase in TIMP-1 protein expression (Fig. 4C) and a profound decrease in invasion (Fig. 4D). Although the MAGL siRNA-induced inhibition of migration through uncoated Boyden chambers and of viability reached statistical significance (Fig. 4D), the corresponding effects were relatively small compared with the anti-invasive effect.

Finally, a selective anti-invasive effect could be confirmed for JZL184 even when using two additional human lung cancer cell lines, H460 and H358 (Fig. 5A, black bars; Supplementary Fig. S6A, top).

Similar to the A549 cells, no inhibition of viability and migration through uncoated Boyden chambers was observed for H460 and H358 cells when treated with JZL184 under conditions comparable with the Matrigel invasion test (Fig. 5A, open and gray bars; Supplementary Fig. S6A, bottom). Furthermore, induction of TIMP-1 after treatment with JZL184 was also shown in H460 and H358 cells (Fig. 5B). Despite a lower wound-healing rate compared with the vehicle control, a significant antimigrative effect of JZL184 could also be excluded in the scratch wound assay for H460 cells (Fig. 5C; Supplementary Fig. S6B, left) and H358 cells (Fig. 5D; Supplementary Fig. S6B, right).

Confirmatory studies conducted on H358 also addressed the role of CB₁ receptor as well as TIMP-1 in the anti-invasive effect of JZL184.

In accordance with the data obtained on A549 cells, it was shown that the anti-invasive effect of JZL184 was at least partially abolished by the CB₁ antagonist AM-251, but was not significantly altered in the presence of the CB₂ antagonist AM-630 (without coincubation with AM-251) or the TRPV1 antagonist capsazepine (Fig. 5E, black bars). Also in H358 cells, none of the antagonists showed a significant increase in basal invasiveness of A549 cells per se (Fig. 5E, gray bars). Similarly, in accordance with the data collected in A549 cells, palmitic acid did not lead to a reversal of the anti-invasive effect of JZL184 (Fig. 5F), thereby ruling out a reduction of free fatty acids as another underlying pathway of the anti-invasive effect of JZL184.

A possible causal relationship between TIMP-1–inducing and anti-invasive effect of JZL184 was again investigated using TIMP-1 siRNA. The anti-invasive effect of JZL184 was found to be completely abolished in H358 cells transfected with TIMP-1 siRNA, whereas treatment of cells transfected with a nonsilencing siRNA control did not significantly alter the anti-invasive effect (Fig. 6A). A similar pattern was observed in 2-AG–treated cells transfected with TIMP-1 siRNA compared with nonsilencing siRNA (Fig. 6B). As previously shown for A549 cells, analysis of TIMP-1 levels confirmed complete inhibition of JZL184- or 2-AG–induced TIMP-1 expression in H358 cells transfected with TIMP-1 siRNA (Fig. 6A and B).

As expected from the results of the experiments with A549 cells, a nonspecific effect of JZL184 could be excluded by tests with the MAGL inhibitors JW651 and MJN110. Both compounds proved to be invasion inhibitors without affecting migration through uncoated Boyden chambers (Fig. 6C and D, left panel each). Here, too, the anti-invasive effect of the respective test substance was accompanied by a significant TIMP-1 upregulation (Fig. 6C and D, right panel each).

With the present data we provide for the first time evidence for a CB₁ receptor–dependent antimetastatic effect of the MAGL inhibitor JZL184 and a likewise CB₁-dependent molecular mechanism of the anti-invasive effect of JZL184, which lies in an increased synthesis of the MMP inhibitor TIMP-1 in lung cancer cells (Fig. 3E).

There are several pieces of evidence supporting this notion. First, both the antimetastatic and anti-invasive effects of JZL184 were inhibited by the CB₁ antagonist AM-251. In this context, a CB₁ receptor downregulation previously shown after chronic administration of JZL184 (26) could not be confirmed for our set-up by analysis of A549 cell membranes for the cannabinoid receptor content. Furthermore, a MAGL inhibition-associated reduction of free fatty acids as cause of the anti-invasive effect of JZL184 in both A549 and H358 cells could be excluded by add-back experiments with palmitic acid. Therefore, the latter mechanism shown on prostate carcinoma cells (12) does not seem to play a role at least for lung cancer cells studied. With regard to the significant inhibition of the anti-invasive effect of JZL184 by AM-251 in A549 and H358 cells, our data indeed suggest a pivotal role of the CB₁ agonist 2-AG, which under these circumstances is prevented from degrading, as mediator of the anti-invasive effect of JZL184. This view is secondly corroborated by the facts that JZL184 induced a selective upregulation of the MAGL substrate 2-AG in A549 cells and that the anti-invasive effect of JZL184 was mimicked by exogenous 2-AG in both A549 and H358 cells. Third, in each of the two cell lines, both JZL184 and 2-AG elicited an increased formation of TIMP-1, whose knockdown resulted in a significant reduction of the anti-invasive effect of both substances. Fourth, off-target effects of JZL184 could be ruled out by demonstrating an anti-invasive and TIMP-1-upregulating action of further MAGL inhibitors (JW651, MJN110) and MAGL siRNA. In the overview of the findings, mechanistic investigations carried out on two cell lines (A549, H358) and the principle proof of the JZL184-mediated anti-invasive and TIMP-1–inducing effect in a third cell line (H460) exclude a cell line–specific effect.

Although antimetastatic effects of pharmacologic (15) or genetic MAGL inhibition (16) have already been described, this is the first study that shows corresponding properties with simultaneous confirmation of an underlying CB₁ receptor–dependent effect. Regarding the comparable experimental conditions, the antimetastatic effect of JZL184 proved here was similar to that of the two FAAH inhibitors URB597 and AA-5HT (19) and the phytocannabinoid cannabidiol (20, 23). In one of these earlier reports (19), 2-AG also produced an antimetastatic effect. Interestingly, in our hands the CB₁ antagonist AM-251 itself inhibited metastasis of cancer cells. This result is consistent with previous studies indicating the CB₁ antagonist rimonabant to exhibit multiple anticarcinogenic effects, such as reducing the growth of breast and colon cancer cells and increasing the apoptosis of leukemia cells (28–31). In accordance with our results, Bifulco and colleagues showed that rimonabant counteracts the anticancer effect of 2-methyl-2′-F-AEA while exerting significant antitumor effects on thyroid cancer cells in vitro and in vivo (32). These results suggest a central role of endocannabinoid tone in controlling cancer progression, with further studies needed to investigate under which circumstances both the blocking and activation of CB₁ receptors lead to anticarcinogenic effects.

The results of the present study suggest that the invasion of lung cancer cells through Matrigel-coated inserts is selectively inhibited by JZL184, whereas their migration potential through uncoated Boyden chambers is not susceptible to treatment with JZL184. Thereby, the absence of a significant antimigratory effect of JZL184 was confirmed for three different lung cancer cell lines in scratch wound assays. Accordingly, MAGL inhibition by JZL184 seems to block the step of overcoming tissue boundaries rather than the actual process of cell migration. A specific anti-invasive effect has also been demonstrated for JW651 and MJN110 in A549 and H358 cells, two drugs with selective inhibitory effect on MAGL (33, 34) and is consistent with our previous studies on cannabinoids (19, 22, 23) as well as with the work of other groups that have shown an anti-invasive while lacking an antimigratory effect for various substances or transfections in cancer cells (35–37). On the other hand, the apparently more persistent effect of the MAGL siRNA on MAGL expression and activity was accompanied by a slight but significant inhibition of migration, which was, however, quantitatively below the corresponding anti-invasive effect.

Other authors reported antimigratory effects of JZL184 at final concentrations of 1 μmol/L (11, 12) or 10 μmol/L (15), which could be explained by cell type–specific properties and differently chosen experimental conditions. In contrast to the data presented here, no effect of JZL184 on the invasion of A549 cells could be demonstrated in another study (38). In the latter work, however, the invasion was analyzed only after a 24-hour incubation with 1 μmol/L JZL184. According to our results, the anti-invasive effect of JZL184 increases continuously over 72 hours and is significantly detectable after 24 hours already. Because the time course was determined at a higher concentration of 10 μmol/L JZL184, there is not necessarily a contradiction to the findings presented by Tang and colleagues (38).

In the case of 2-AG, previous work has shown that the inhibition of endogenous 2-AG synthesis with the diacylglycerol lipase inhibitor RHC-80267 leads to an inhibition of invasion in various prostate cancer cells (39). Interestingly, in the same study an anti-invasive effect was found for noladin ether, a nonhydrolyzable 2-AG analogue, but not for exogenous 2-AG itself, which was explained by the rapid metabolism of 2-AG. The results with MAGL siRNA are further consistent with studies in which MAGL knockdown reduced the invasiveness (11–16, 38) and migration (11, 12, 15, 16, 38) of various cancer cell lines.

The anti-invasive effect of JZL184 may be due to a number of mechanisms such as increased MMP-9 expression recently described for JZL184 in neuronal cells (40). In the present study, mechanistic investigations focused on the role of TIMP-1 as a possible mediator of the anti-invasive effect of JZL184. Physiologically, TIMP-1 inhibits collagen-degrading enzymes such as MMP-2 and MMP-9, which play an important role in promoting cancer metastasis (for review see ref. 41). As a matter of fact, several studies have linked the overexpression of TIMP-1 to a reduction in tumor growth, invasiveness of cancer cells and metastasis (for review see ref. 42). However, there are also contrary findings on the influence of TIMP-1 on the invasiveness of tumor cells (43).

In the present investigation, a profound intra- and extracellular TIMP-1 upregulation was found as a response to JZL184 and 2-AG treatment. Combined treatment with TIMP-1 siRNA significantly suppressed the anti-invasive effect of JZL184 and 2-AG in A549 and H358 cells, suggesting a causal relationship between inhibition of MAGL, induction of TIMP-1, and inhibition of invasion. Remarkably, the anti-invasive effect of both substances was only partially reversed in A549 cells, suggesting that mechanisms other than the upregulation of TIMP-1 may also be involved in this response. In the case of 2-AG, the rapid conversion of 2-AG into 1-AG (44) and the increased metabolism by enzymes other than MAGL (e.g., by arachidonate 15-lipoxygenase or cyclooxygenases) could be a reason for this finding (45, 46). In accordance with the data shown here, previous work of our group has shown that TIMP-1 mediates the anti-invasive effect of other cannabinoids, including cannabidiol (20, 23), Δ⁹-tetrahydrocannabinol (22), AEA (19), and the FAAH inhibitors URB597 and AA5-HT (19). Interestingly, in a former study of our group, only a low induction of TIMP-1 was observed in A549 cells stimulated with 2-AG (19). This difference might be due to a different experimental setting. In contrast to the TIMP-1 analyses of cell lysates in the present study, the Western blots on TIMP-1 in the previous investigation were performed with cell culture media from the upper Boyden chambers (19), with a correspondingly higher cell density (5 × 10⁵ cells per insert) than in the present study (2 × 10⁵ cells per well of a 6-well plate). The effect of cannabinoids in such an experimental set-up may therefore be less pronounced due to an “inoculum effect,” which causes a reduced availability of drug molecules at their binding sites, as described earlier (23, 47, 48).

Because the survival of tumor cells also depends on cell density (23), the viability of JZL184 was additionally tested in A549 cells under conditions of lower cell density compared with the viability assays performed in the context of the invasion tests. Even in the experiments carried out with a lower cell density, no significant effect of JZL184 on the viability and proliferation of A549 cells could be demonstrated, whereas MAGL siRNA already led to a slight but still significant decrease in viability under conditions of high cell density. Indeed, divergent data have been published on the influence of JZL184 on cancer cell viability. In line with our results, Nomura and colleagues (11) showed that the inhibition of breast cancer cell viability registered in the presence of MAGL shRNA could not be confirmed by JZL184 (1 μmol/L) under serum-starved conditions. To explain this phenomenon, the authors suggested that long-term inhibition of MAGL may be necessary to achieve the desired effect. Furthermore, a 72-hour incubation of neuroblastoma cells with JZL184 (10 μmol/L) exhibited no effect on viability (49). Similarly, a one-week incubation of PC-3 prostate cancer cells with JZL184 (1 μmol/L) did not lead to a decrease but rather to an increase in cell density (50). In a parallel approach of the latter study, significant inhibition of epidermal growth factor–induced cell proliferation by JZL184 was demonstrated (50). On the other hand, a significant reduction in viability could be demonstrated for various endometrial carcinomas (51) and, contrary to the study already cited (50), for PC-3 prostate cancer cells treated with JZL184 at 1 μmol/L (12). Finally, Ye and colleagues (13) and Zhang and colleagues (14) apparently used suprapharmacological concentrations of JZL184 (0.5 μg/μL) to detect inhibition of the viability of colorectal cancer and hepatocellular carcinoma cells, respectively. In agreement, survival rates of the colon adenocarcinoma cell line Colo-205 clearly demonstrate that a toxic effect of JZL184 at 48 hours incubation only becomes evident at a concentration of 1,000 μmol/L but not at concentrations up to 100 μmol/L (52). In the case of exogenous 2-AG, the antiproliferative or viability-reducing effect reported in the literature for this endocannabinoid in other cancer cell types (21, 53, 54) could only be demonstrated in our study at a 2-AG concentration of 10 μmol/L. Thereby, the viability was only reduced when administered in our hands under serum-free conditions. In summary, differences in the experimental set-up including cell density and serum addition as well as cell type–specific reactions may explain the divergent findings.

Taken together, our data show that inhibition of MAGL induces a CB₁-dependent anti-invasive and antimetastatic effect on human lung cancer cells by inhibiting 2-AG degradation. TIMP-1 upregulation was identified as a mediator of the anti-invasive effect. Provided that these findings are verified at the clinical level, MAGL inhibitors could, in addition to alleviating chemotherapy-related side effects, offer an additional benefit for cancer patients as “antimetastatic agents” in combination with the established chemotherapy regimen.

B. Hinz reports grants from Deutsche Forschungsgemeinschaft (grant HI 813/9-1 to B. Hinz) during the conduct of the study. J.L. Prüser reports grants from Deutsche Forschungsgemeinschaft (grant HI 813/9-1 to B. Hinz) during the conduct of the study. R. Ramer reports grants from Deutsche Forschungsgemeinschaft (grant HI 813/9-1 to B. Hinz) during the conduct of the study. F. Wittig reports grants from Deutsche Forschungsgemeinschaft (grant HI 813/9-1 to B. Hinz) during the conduct of the study. I. Ivanov reports grants from Deutsche Forschungsgemeinschaft (grant HI 813/9-1 to B. Hinz) during the conduct of the study. J. Merkord reports grants from Deutsche Forschungsgemeinschaft (grant HI 813/9-1 to B. Hinz) during the conduct of the study.

J.L. Prüser: Data curation, software, formal analysis, validation, investigation, methodology, writing–original draft. R. Ramer: Conceptualization, data curation, software, formal analysis, validation, investigation, and methodology. F. Wittig: Data curation, software, formal analysis, validation, investigation, and methodology. I. Ivanov: Data curation, software, formal analysis, validation, investigation, and methodology. J. Merkord: Data curation, formal analysis, validation, investigation, methodology. B. Hinz: Conceptualization, supervision, funding acquisition, investigation, writing-original draft, project administration, writing–review and editing.

This work was supported by a grant of the Deutsche Forschungsgemeinschaft: HI 813/9–1 (to B. Hinz).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.