The anti-tumoral effects of cannabinoids have been described in different tumor systems, including pancreatic adenocarcinoma, but their mechanism of action remains unclear. We used cannabinoids specific for the CB1 (ACPA) and CB2 (GW) receptors and metabolomic analyses to unravel the potential pathways mediating cannabinoid-dependent inhibition of pancreatic cancer cell growth. Panc1 cells treated with cannabinoids show elevated AMPK activation induced by a ROS-dependent increase of AMP/ATP ratio. ROS promote nuclear translocation of GAPDH, which is further amplified by AMPK, thereby attenuating glycolysis. Furthermore, ROS determine the accumulation of NADH, suggestive of a blockage in the respiratory chain, which in turn inhibits the Krebs cycle. Concomitantly, inhibition of Akt/c-Myc pathway leads to decreased activity of both the pyruvate kinase isoform M2 (PKM2), further downregulating glycolysis, and glutamine uptake. Altogether, these alterations of pancreatic cancer cell metabolism mediated by cannabinoids result in a strong induction of autophagy and in the inhibition of cell growth.

Cannabinoid-induced autophagy depends on AMPK activation

We previously demonstrated that arachidonoyl cyclopropamide (ACPA) orGW405833 (GW), two synthetic cannabinoid ligands specific for CB1 and CB2, respectively, are able to induce ROS-mediated autophagy in pancreatic adenocarcinoma cell lines.¹⁰ To more deeply investigate the molecular mechanisms of this effect, we performed in Panc1 cells treated with ACPA or GW kinetic analyses of the Thr172 phosphorylated AMPK (p-AMPK) and of the Thr389 phosphorylated p70S6K (p-p70S6K), HIF-1α and PDHK downstream targets of mTORC1, a known autophagy inhibitor. As shown in Figure 1, after 1h of treatment with cannabinoids, the increase of AMPK phosphorylation already occurred, with a further extension of the effect up to 24h (Figure 1a). It is worthy to note that the AMPK total protein levels decreased after 12 and 24-h treatment. The ratio phospho-p70S6K/p70S6K decreased after 1-h treatment and to a larger extent at 12 and 24h (Figure 1b), although the total protein formed also decreased after treatment with cannabinoids. Instead, HIF-1α (Figure 1c) showed a reduction of the expression starting at 12-h treatment and PDHK (Figure 1d) at 12 and 24h for GW and ACPA, respectively. These results strongly suggested that AMPK could be involved in autophagy induction by cannabinoids in pancreatic adenocarcinoma cells. To further examine the role of AMPK in this effect, we treated the cells with cannabinoids in the absence or presence of compound C (CC), an AMPK inhibitor,²⁴ and we analysed AMPK phosphorylation as control (Supplementary Figure 1) and the regulation of the autophagy marker phosphoethanolaminated LC3-II (Figure 2a). CC prevented AMPK phosphorylation and autophagy induction by cannabinoids, providing a strong support to the dependence of cannabinoid-induced autophagy on AMPK activation. To rule out the nonspecific effect of CC and to characterize the upstream events of AMPK activation by cannabinoids, we performed transient transfection of Panc1 cells using a plasmid coding for the mutated γ subunit isoform 2 (γ2 of AMPK, which is unable to bind AMP). The results reported in Figures 2b and c show that overexpression of the mutant AMPK (γ2 R531G) significantly decreased both p70S6K phosphorylation inhibition and LC3-II induction by cannabinoids, whereas the wild-type AMPK (γ2wt) had no effect on those cannabinoid activities. These findings indicated that AMPK was involved in cannabinoid-induced autophagy via an AMP-dependent mechanism. To confirm the role of AMP in this event and to verify whether it could depend on ROS production, we analysed the cellular AMP/ATP ratio following GW or ACPA treatment in the absence or presence of the radical scavenger N-acetyl-_L-cysteine (NAC). As shown in Figure 2d, the level of AMP/ATP was strongly increased by cannabinoids, while it was similar to the control upon NAC pre-treatment of the cells.

Figure 1

Effect of cannabinoids on key metabolic proteins. Western blot analyses of (a) phospho-AMPK, (b) phospho-p70S6K, (c) HIF-1 alpha, and (d) pyruvate dehydrogenase kinase (PDHK) were performed after treatment of Panc1 cells for 1, 12, and 24h with …

Figure 2

Role of AMPK in cannabinoid-induced autophagy. Western blot analysis of (a) LC3-II after treatment of Panc1 cells for 12h with 200μM GW or ACPA and 20μM CC, and of (b) p-p70S6K and (c) LC3-II after treatment …

Cannabinoids inhibit the glycolytic pathway

To assess whether a restriction of the energetic metabolism by cannabinoids could be responsible for the enhancement of the cellular AMP level, we performed a targeted metabolomic analysis. We determined fold-change variations of the concentration levels of several key metabolites from the glycolitic pathway upon GW or ACPA treatment. Figure 3a shows a significant increase of glyceraldehyde 3-phosphate (G3P) and phosphoenolpyruvate (PEP) and a decrease of lactate (LACT), following a 12-h treatment with GW. Instead, ACPA treatment determined only a G3P increase (Figure 3b). To exclude the possibility that the glycolysis metabolite increase was determined by a higher glucose uptake by the cells, we measured the amount of glucose in the supernatant of treated or untreated cells, and we found that glucose incorporation did not significantly change upon cannabinoid treatment (Figure 3c).

Figure 3

Effect of cannabinoids on glycolysis. Panc1 were treated for 12h with (a) GW or (b) ACPA and the metabolites analysis was performed as reported in Materials and Methods.FDP, fructose 1,6-bisphosphate; F6P, fructose-6-phosphate; G3P, glyceraldehyde-3-phosphate; …

As the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is known to contribute to the upregulation of autophagy by translocating to the nucleus,²⁵ we analysed the cellular distribution of GAPDH after the treatment with GW or ACPA. Figure 4 shows the confocal images where GAPDH appear to be translocated into the nucleus of the cells after 12-h cannabinoid treatments. This effect was inhibited by NAC or CC, indicating its dependence on ROS and AMPK activation. These data suggested that the increased level of G3P observed in the metabolomic analysis could arise from the diminished presence of GAPDH in the cytosol of cells treated with cannabinoids.

Figure 4

Cannabinoids induce AMPK-dependent GAPDH nuclear translocation. Representative confocal images of GAPDH translocation in Panc1 cell nuclei after 12h treatment with 200μM GW or ACPA in the absence or presence of a pre-treatment …

PKM2 is an embryonic isoform of pyruvate kinase (PK) re-expressed in cancer cells, which dephosphorylates PEP to pyruvate, and determines a metabolic advantage for the cell by allowing the availability of phosphometabolites upstream of pyruvate as precursors for cellular syntheses.²⁶ To assess whether the PKM2 activity might be responsible for an increase of PEP level by GW, we analysed the activity of PKM2 on crude protein extracts from cells treated with cannabinoids for 12 or 24h. Figure 3d shows that PKM2 activity after GW treatment significantly decreased at 12h and even more at 24h, whereas it was inhibited by ACPA only after 24-h treatment. These data are consistent with the increased level of PEP observed in the metabolomic analysis after 12h of treatment with GW. Furthermore, data not shown indicate that PEP did increase after 24-h treatment with ACPA.

Taken together, these findings suggested an impairment of the glycolytic pathway by cannabinoids that correlated with the increase of the AMP/ATP ratio.

Cannabinoids inhibit the Krebs cycle

To further examine the involvement of the energetic metabolism in the induction of AMPK-mediated autophagy by cannabinoids, we analysed the critical metabolites of the Krebs cycle. As shown in Figure 5, only the levels of the α-ketoglutarate (Figures 5a and b) and, to a very large extent, those of NADH (Figure 5c) increased after cannabinoid treatments. These results suggested the occurrence of the inhibition by NADH of the α-ketoglutarate dehydrogenase, the enzyme of the Krebs cycle most sensitive to the levels of that coenzyme, and the impairment of the oxidative phosphorylation in the increased AMP/ATP ratio determined by cannabinoids. Interestingly, NAC was able to significantly reduce the increase of NADH by GW or ACPA (Figure 5c), suggesting the involvement of the oxidative stress in that effect. To verify whether the ROS-dependent NADH increase correlated with the activation of the pentose phosphate pathway and thus with the accumulation of the coenzyme NADPH, known to have a key role in the antioxidant response of the cell, we measured the amounts of the phosphogluconolactone and of the NADPH. The levels of both the compounds remained unchanged after treatment with GW or ACPA, indicating that cannabinoids were unable to activate the pentose phosphate pathway in our experimental conditions (Supplementary Figure 2).

Figure 5

Effect of cannabinoids on Krebs cycle. Panc1 were treated for 12h with 200μM (a) GW or (b) ACPA and the metabolites analysis was performed as reported in Materials and Methods.KET, alpha-ketoglutarate; MA, malate; SUCC, succinate. …

Cannabinoids inhibit the anaplerotic flux of the Krebs cycle from glutamine

Glutamine and glucose are the only two molecules catabolized in appreciable quantities in most mammalian cells in culture, to supply the cell with most of the carbon, nitrogen, free energy and reducing equivalents. To assess whether the regulation of glutamine catabolism was involved in the downregulation of the energetic metabolism, we measured the levels of glutamine and glutamate in the culture medium after the treatment of cells with cannabinoids. Figure 6a shows that glutamine incorporation was strongly reduced by both GW and ACPA, whereas glutamate release remained unchanged. The oncogene c-Myc has been described to coordinate the expression of genes involved in glutamine catabolism, including the induction of glutamine transporters.²⁷ Furthermore, it has been shown that Akt induces the upregulation of c-Myc and that Akt suppression inhibits the c-Myc expression.²⁸ We evaluated both Akt phosphorylation on serine 473, which is a marker of activation for this kinase, and c-Myc activity after GW or ACPA treatments. Figures 6b and c show that GW strongly inhibited Akt phosphorylation and c-Myc activity at 12-h treatment and even more at 24h, while ACPA determined a significant decrease of both the proteins only at 24-h treatment. Altogether, these results suggested that the inhibition of glutamine uptake by cannabinoids could depend, at least for GW, on the repression of glutamine transporters determined by the decrease of c-Myc activity.

Figure 6

Effect of cannabinoids on glutamine metabolism. (a) The analyses of glutamine uptake and glutamate release were performed after treatment of Panc1 cells for 12h with 200μM GW or ACPA. (b) Western blot analysis was performed using …

Materials

ACPA was obtained from Cayman Chemicals (Inalco, Milan, Italy); GW405833hydrochloride (1-(2,3-dichlorobenzoyl)-2-methyl-3-(2-(1-morpholine)ethyl)-5-methoxyindole) and NAC (N-acetyl--cysteine) were obtained from Sigma (Milan, Italy); Compound C (CC) was obtained from Calbiochem. Acetonitrile, formic acid, and HPLC-grade water were purchased from Sigma Aldrich (Milan, Italy). Standards (equal to or greater than 98% chemical purity) -glucose 6-phosphate, fructose 6-phosphate, -fructose 1,6 biphosphate, glyceraldehyde 3-phosphate, phosphoenolpyruvic acid, -lactic acid, α-ketoglutaric acid, -malic acid, succinic acid, ATP, NADH, NADPH, 6-phosphogluconolactic acid, -glutamic acid, glutamine, reduced glutathione, and oxidized glutathione were purchased from Sigma Aldrich.

Cell culture

Panc1 cell line was grown in RPMI 1640 supplemented with 2mM glutamine, 10% FBS, and 50μg/ml gentamicin sulphate (BioWhittaker, Lonza, Bergamo, Italy), and incubated at 37°C with 5% CO₂.

Immunoblot analysis

Cells (1.2 × 10⁶ cells/dish 10cm) were treated as reported in the figures and then lysated with RIPA buffer for total protein extract. Protein concentration was measured with the Bradford protein assay reagent (Pierce, Rockford, IL, USA) using bovine serum albumin as a standard. Forty μg of protein extracts was electrophoresed through SDS–polyacrylamide gel and electroblotted onto polyvinylidene difluoride membranes (Millipore, Milan, Italy). Membranes were then incubated for 1h at room temperature with blocking solution (5% low-fat milk in 100mM Tris pH 7.5, 0.9% NaCl, 0.1% Tween 20) and probed overnight at 4°C with the primary monoclonal antibodies (1:1000 in blocking solution). Light chain protein-II (LC-3II), AMPK, phospho-AMPK Thr172, p70S6K, phopsho-p70S6K Thr389, Akt, phospho-Akt Ser473, and PDHK were obtained from Cell Signaling, Euroclone, Milan, Italy, HIF-1 alpha antibody from Novus Biologicals, Milan, Italy, and α-tubulin antibody from Oncogene, Cambridge, MA, USA. Horseradish peroxidase-conjugated secondary antibodies IgG (1:8000 in blocking solution; Upstate Biotechnology, DBA Italia, Milan, Italy) were used to detect specific proteins. Immunodetection was carried out using chemiluminescent substrates (Amersham Pharmacia Biotech, Euroclone, Milan, Italy) and recorded using HyperfilmECL (Amersham Pharmacia Biotech). The bands for total and phosphorylated proteins were scanned as digital peaks and the areas of the peaks were calculated in arbitrary units using the public domain NIH Image software (http://rsb.info.nih.gov/nih-image/) and then normalized with α-tubulin expression. Quantifications were obtained as fold induction relative to controls and, for phosphorylated proteins, quantifications represent the ratio phosphorylated/total protein.

Metabolite extraction

Metabolomic analyses after treatment for 12h with 200μM GW or ACPA and 20mM NAC were performed as previously reported.⁴¹ Cells were prepared following the protocol by Sana et al.,⁴² with minor modifications as previously reported.⁴¹ The sample was resuspended by adding 0.15ml of ice-cold ultra-pure water (18MΩ) to lyse cells. The tubes were plunged into dry ice or a circulating bath at −25°C for 0.5min and then into a water bath at 37°C for 0.5min. To each tube was added 0.6ml of −20°C methanol and then 0.45ml of −20°C chloroform. The tubes were mixed every 5min for 30min. Subsequently, 0.15ml of ice-cold pH-adjusted ultra-pure water was added to each tube and these were centrifuged at 1000 × g for 1min at 4°C, before being transferred to −20°C for 2–8h. After thawing, liquid phases were recovered and an equivalent volume of acetonitrile was added to precipitate any residual protein. The tubes were then transferred to a refrigerator (4°C) for 20min, centrifuged at 10000 × g for 10min at 4°C and the collected supernatants were dried to obtain visible pellets. Finally, the dried samples were re-suspended in 1ml of water, 5% formic acid and transferred to glass autosampler vials for LC/MS analysis.

Rapid-resolution reverse-phase HPLC

An Ultimate 3000 Rapid Resolution HPLC system (LC Packings, DIONEX, Sunnyvale, CA, USA) was used to perform metabolite separation. A Dionex Acclaim RSLC 120 C18 column 2.1mm × 150mm × 2.2μm was used to separate the extracted metabolites. A 0–95% linear gradient of solvent A (0.1% formic acid in water) to B (0.1% formic acid in acetonitrile) was employed over 15min followed by a solvent B hold of 2min, returning to 100% A in 2min and a 6-min post-time solvent A hold.

ESI mass spectrometry

Metabolites were directly eluted into a High Capacity ion Trap HCTplus (Bruker-Daltonik, Bremen, Germany). Mass spectra for metabolite extracted samples were acquired in positive ion mode. ESI capillary voltage was set at 3000V (+) ion mode. The liquid nebulizer was set to 30psig and the nitrogen drying gas was set to a flow rate of 9l/min. Dry gas temperature was maintained at 300°C. Data were stored in centroid mode. Internal reference ions were used to continuously maintain mass accuracy. Data were acquired at a rate of 5 spectra/s with a stored mass range of m/z 50–1500. Data were collected using Bruker Esquire Control (v. 5.3 – build 11) data acquisition software. In MRM analysis, m/z values of interest were isolated, fragmented and monitored (either the parental or the fragment ions) throughout the whole RT range. Validation of HPLC on-line MS-eluted metabolites was performed by comparing transitions fingerprint, upon fragmentation and matching against the standard metabolites through direct infusion with a syringe pump (infusion rate 4μl/min). Standard curve calibration was performed either on precursor or on fragment ion signals. Double analyses were performed on both parental and product ion species and results were adopted for quantitation. Transitions were monitored to validate each detected metabolite.

Metabolite data elaboration

LC/MS data files were processed by Bruker DataAnalysis 4.0 (build 234) software, Milan, Italy. Files from each run were either analysed as ‘.d’ files or exported as ‘.mzXML’ files, to be further elaborated for spectra alignment, peak picking and quantitation with InSilicos Viewer 1.5.4 (Insilicos LLC; Seattle, WA, USA).

Quantitative analyses of standard compounds were performed on MRM data. Each standard metabolite was run in triplicate, at incremental dilution until LOD and LOQ were reached. The limit of detection for each compound was calculated as the minimum amount injected that gave a detector response higher than three times the signal-to-noise ratio (S/N). Linearity of the observed quantities, slope, intercept and linear correlation values were all calculated via Microsoft Excel (Microsoft, Redmond, WA, USA).

Analysis of metabolite uptake and release

Cells (1.2 × 10⁶/dish 10cm) were treated for 12h with 200μM GW or ACPA. The cell culture medium was collected and analysed with Bioprofile Flex (Nova Biomedical, Waltham, MA, USA).

Immunofluorescence analysis

Cells (1.6 × 10⁴) were grown on coverslips and treated for 12h with 200μM GW or ACPA and 20μM CC or 20mM NAC. Cells were incubated with rabbit GAPDH antibody (1:100) at RT for 90min and then incubated with Alexa Fluor 488 anti-rabbit IgG antibody (1:500) at RT for 60min. To assess nuclear morphology, cells were incubated with HOECHST for 2min at RT. Fluorescence was visualized using excitation/emission wavelengths of 488/520nm (green) and 350/460nm (blue) for GAPDH and HOECHST, respectively. Cells were examined using TCS-SP5 Leica confocal microscope, Milan, Italy, at × 40 and × 63 magnification.

PKM2 activity assay

One μg of total protein extract obtained from Panc1 cells treated for 12 and 24h with 200μM GW or ACPA was used to analyse PKM2 activity as previously reported.⁴³

c-Myc activity assay

Cells (4 × 10⁵ cells/dish 6cm) were treated for 1, 12 or 24h with 200μM GW or ACPA and then were lysed with nuclear extract kit (Active Motif, Vinci Biochem, Florence, Italy) for nuclear and cytosolic protein extracts. Five μg of the nuclear extract was used to measured c-Myc activity through ELISA assay (Active Motif, TransAM, c-Myc).

Transfection experiments

Cells (2.5 × 10⁵ cells/dish 6cm) were transfected with the pcDNA5/FRT expression vector containing the human AMPK gamma-2 subunit wt or R531G mutated using TransIT-LT1 transfection reagent (Mirus Bologna, Italy) according to the manufacturer’s directions. Cells were incubated for 72h and then treated with 200μM GW or ACPA for 12h, to evaluate the role of AMP production on AMPK induction. Transfection efficiency was assessed by cytofluorimetric analysis and was ~56%. The expression vectors for the AMPK wt and mutant R531G γ2 subunit were kindly provided by Dr. Hawley (University of Dundee, Scotland, UK).

Statistical analysis

ANOVA (post hoc Bonferroni) and graphical presentations were performed by GraphPad Prism 5. P-values of *P<0.05, **P<0.01, or ***P<0.001 are indicated in the figures.

This work was supported by Associazione Italiana Ricerca Cancro (AIRC), Milan, Italy; Fondazione CariPaRo, Padova, Italy; Progetti di Ricerca di Interesse Nazionale (PRIN, MIUR), Rome, Italy. We thank Prof. Michela Giuliano and Prof. Maurizio Bifulco for their critical reading of the manuscript.

LC-MS

liquid chromatography–mass spectrometry

AMPK

AMP-activated protein kinase

ACPA

arachidonoyl cyclopropylamide

GW

GW405833 hydrochloride

NAC

N-acetyl--cysteine

CC

Compound C

GAPDH

glyceraldehyde 3-phosphate dehydrogenase

PDHK

pyruvate dehydrogenase kinase

ROS

reactive oxygen species

PKM2

pyruvate kinase M2

CaMKKβ

Ca²⁺/calmodulin-dependent protein kinase

F6P

fructose-6-phosphate

G6P

glucose-6-phosphate

FDP

fructose 1,6-bisphosphate

G3P

glyceraldehyde-3-phosphate

PEP

phosphoenolpyruvate

LACT

lactate

KET

alpha-ketoglutarate

SUCC

succinate

MA

malate

PPP

pentose phosphate pathway

PGL

phosphogluconolactone

Supplementary Information accompanies this paper on Cell Death and Disease website (http://www.nature.com/cddis)

Edited by G Melino

Supplementary Figure 1

Supplementary Figure 2

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