Δ9-THC and JWH-015 inhibit the growth of the human HCC lines HepG2 and HuH-7 via autophagy stimulation

It has been recently shown that cannabinoids induce human glioma cell death via autophagy stimulation in vitro and in vivo.13, 23 We therefore examined whether Δ9-THC and JWH-015 activate a similar mechanism in HCC cells. Upon autophagy stimulation, the autophagy protein LC3 (microtubule-associated protein 1 light chain 3α) becomes conjugated to phosphatidylethanolamine (PE), which targets this protein to the membrane of the autophagosomes. The lipidated autophagosome-associated form of LC3 (LC3-II) can be monitored by immunofluorescence (autophagic cells exhibit a characteristic pattern of LC3 puncta) or western blot (LC3-II has higher electrophoretic mobility than non-lipidated LC3). Immunofluorescence analysis revealed that LC3 exhibited a punctuated distribution, consistent with its translocation to the autophagosome, in cells that had been treated with Δ9-THC or JWH-015 (Figure 1a). Likewise, incubation of HepG2 and HuH-7 cells with Δ9-THC or JWH-015 increased the levels of the lipidated form of LC3 (Figure 1b). Furthermore, pharmacological inhibition with E64d and pepstatin A (PA) of lysosomal proteases (the enzymes responsible for the degradation of the autophagosome content after fusion with the lysosome) enhanced the accumulation of LC3-II (as well as of the autophagosome cargo p62) in cells that had been treated with THC or JWH-015, thus supporting the fact that cannabinoid treatment leads to dynamic autophagy in HCC cells (Figure 1b).

Figure 1

Δ9-Tetrahydrocannabinol (Δ9-THC) and JWH-015 treatment induces autophagy in hepatocellular carcinoma (HCC) cells. (a) HepG2 and HuH-7 cells were treated with Δ9-THC or JWH-015 for 24h and microtubule-associated protein …

Next, we investigated whether autophagy was directly involved in the mechanism of cannabinoid-induced cell death. As shown in Figure 1c, cell death was inhibited when autophagy was pharmacologically blocked at a very early stage (by incubation with 3-methyladenine (3-MA), an inhibitor of Vps34, a class III phosphatidyl inositol-3-kinase that has a crucial role in autophagy initiation24) or at a final stage (by incubation with E64d and PA). Likewise, knock down of Atg5 (an essential autophagy gene that is part of one of the two protein conjugation systems required for autophagosome elongation25, 26) impaired THC- or JWH-015-induced cell death (Figure 1d). Taken together, these observations strongly support that autophagy is required for cannabinoid-induced HCC cell death.

Many lines of evidence indicate that there is a cross-talk between autophagy and apoptosis.27 To investigate whether cannabinoid-induced autophagy was involved in apoptosis induction, HepG2 and HuH-7 cells were incubated with either Δ9-THC or JWH-015 in the presence of the 3-MA, and levels of procaspase-3 were detected by immunoblot. As shown in Figure 1e, pre-incubation with 3-MA prevented the cleavage of procaspase-3, suggesting that autophagy induction by cannabinoids was previous to and necessary for apoptosis.

AMPK activation and TRB3 upregulation are involved in Δ9-THC- and JWH-015-induced autophagy and apoptosis of HCC cells

The mechanisms of autophagy stimulation by cannabinoids in glioma and other types of cancer cells relies on the stimulation of an ER stress-related pathway, which leads to the upregulation of the pseudokinase tribbles homolog 3 (TRB3). This latter protein interacts with serine–threonine kinase Akt (Akt) and promotes the inhibition of the mammalian target of rapamycin C1 (mTORC1) complex, which leads to autophagy stimulation.23 As shown in Figure 2, THC and JWH-015 increased the phosphorylation of the α-subunit of the eukaryotic translation initiation factor 2 (eIF2α, a hallmark of the ER stress response; Figure 2a), increased TRB3 levels (Figure 2b) and decreased the phosphorylation of Akt, p70S6 kinase (a well-established substrate of mTORC1) and the ribosomal protein S6 (a target of p70S6 kinase) in HepG2 and HuH-7 cells (Figure 2c). Furthermore, selective knockdown of TRB3 abrogated cannabinoid-induced inhibition of the Akt/mTOR pathway, autophagy and cell death (Supplementary Figure 2), thus supporting the fact that the mechanism by which cannabinoids promotes glioma cell death also operates in HCC cells.

Figure 2

Δ9-Tetrahydrocannabinol (Δ9-THC) and JWH-015 upregulate tribbles homolog 3 (TRB3), inhibit the serine–threonine kinase Akt/mammalian target of rapamycin C1 (Akt/mTORC1) pathway and activate adenosine monophosphate-activated kinase …

Of note, we observed that treatment of HepG2 and HuH-7 cells with THC or JWH-015 increased the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK), an important intracellular nutrient status sensor that has been proposed to have a critical role in the regulation of autophagy as induced by hypoxia or nutrient deprivation (Figure 2b). In addition, pharmacological blockade of CB2 receptors with SR2 abrogated the effect of Δ9-THC and JWH-015 on Akt, S6 and AMPK phosphorylation, as well as autophagy (Figure 2d and Supplementary Figure 1C). We therefore asked whether AMPK may also have a role in the regulation of the antiproliferative effect evoked by cannabinoids in HCC cells. In line with this notion, when AMPK was pharmacologically blocked with dorsomorphin (Figure 3a) or genetically inhibited with small interfering RNA (siRNA) (Figure 3b), HepG2 and HuH-7 cells were more resistant to cannabinoid-induced cell death. Likewise, AMPK knockdown prevented LC3 lipidation (Figure 3c and Supplementary Figure 2C). These observations support the fact that activation of AMPK is necessary for the stimulation of autophagy-mediated cell death by cannabinoids in HCC cells.

Figure 3

Δ9-Tetrahydrocannabinol (Δ9-THC) and JWH-015 induce autophagy via adenosine monophosphate-activated kinase (AMPK). (a) HepG2 and HuH-7 cells were incubated with Δ9-THC or JWH-015 for 48h in the presence of 0.5 …

AMPK and TRB3 regulate cannabinoid-induced autophagy of HCC cells through different mechanisms

AMPK has been shown to inhibit mTORC1.28 Unlike Akt, AMPK activates tuberous sclerosis complex 2 (TSC2), a GTPase-activating protein responsible for the blockade of mTORC1.28 Therefore, we next studied whether this was the mechanism by which AMPK stimulated autophagy in our system. As shown in Figure 3c and Supplementary Figure 2C, AMPK silencing abrogated the effect of Δ9-THC and JWH-015 in the phosphorylation of AMPK and its downstream target acetyl-CoA carboxylase (ACC), as well as on autophagy, but did not modify the effect of cannabinoid treatment on the phosphorylation of Akt, TSC2 (data not shown) or the mTORC1-related substrates 4EBP1 and S6. By contrast, TRB3 knockdown did not affect AMPK or ACC phosphorylation, but did inhibit the cannabinoid-induced decrease in Akt, TSC2, 4EBP1 and S6 phosphorylation and LC3 lipidation. Likewise, pharmacological blockade of ceramide biosynthesis by using ISP-1 (a pharmacological inhibitor of serine palmitoyltransferase, one of the upstream events that trigger TRB3 upregulation in response to cannabinoid treatment29) abrogated the effect of Δ9-THC on Akt, TSC2, 4EBP1 and S6 phosphorylation, as well as LC3 lipidation, but did not affect AMPK phosphorylation. These observations support that the cannabinoid-evoked stimulation of autophagy on HCC cells relies on two different mechanisms: (i) inhibition of the Akt/mTORC1 axis via TRB3 upregulation and (ii) stimulation of AMPK. (A scheme is shown in Figure 5.)

Figure 5

Schematic of the proposed mechanism of cannabinoid-induced hepatocellular carcinoma (HCC) cell death. Cannabinoid treatment stimulates autophagy via two different mechanism: (i) upregulation of tribbles homolog 3 (TRB3) and subsequent inhibition of the …

Activation of AMPK by cannabinoids relies on CAMKK

We next investigated the mechanism by which cannabinoids activate AMPK. Among the different kinases proposed to act as AMPKKs, the human tumor suppressor liver kinase B1 (LKB1) and the calmodulin-activated kinase kinase (CaMKK) are now widely accepted as the most relevant ones.30 Inhibition of LKB1 expression with siRNA did not have any significant effect on the viability of cannabinoid-treated HepG2 (Figure 4a) or HuH7 (Supplementary Figure 3) cells. Likewise, the effect of cannabinoid treatment on AMPK activation, Akt/mTORC1 pathway inhibition and autophagy (LC3 lipidation) was not affected by LKB1 silencing (Figure 4a), supporting the fact that LKB1 is not involved in cannabinoid-induced AMPK activation and autophagy in HCC cells. By contrast, selective knockdown of CaMKKβ or incubation with the CaMKK pharmacological inhibitor STO609 prevented the cannabinoid-evoked decrease in HCC cell viability (Figures 4b and c and Supplementary Figure 3), increase in AMPK and ACC phosphorylation (Figures 4b and c) and autophagy (Figures 4b and c), indicating that AMPK activation by cannabinoids in HCC cells relies on CaMKKβ. Of note, genetic or pharmacological blockade of CaMKKβ did not modify the inhibition of the Akt/mTORC1 pathway evoked by these agents, which again supports the fact that mTORC1 inhibition by cannabinoids in HCC occurs independently of AMPK activation (Figures 4b and c).

Figure 4

Δ9-Tetrahydrocannabinol (Δ9-THC) and JWH-015 activate adenosine monophosphate-activated kinase AMPK via CaMKKβ. (a) Left panel: Effect of Δ9-THC (8μM) or JWH-015 (8μM) on the viability …

Cell cultures

Human HCC HepG2 cells (ATCC, HB-8065) (Rockville, MD, USA) were cultured according to suppliers. The human hepatoma cell line HuH-7 was kindly supply by Dr. Lisardo Boscá (IKnstituto de Investigaciones Biomédicas Alberto Sols, Madrid, Spain). Cells were routinely growth in DMEM/10% FBS supplemented with 1% non-essential amino acids and 100IU/ml penicillin G sodium, 100μg/ml streptomycin sulfate, 0.25μg/ml amphotericin B (Invitrogen, Paisley, UK). One day before the experiments, the medium was changed to 0.5% FBS medium. Experiments were carried out when cell monolayers were 80% confluent.

RT-PCR analysis

Total RNA was isolated from cells by Trizol Reagent from Gibco (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s protocol. One microgram total RNA was retrotranscribed to cDNA with the M-MLV Reverse transcriptase kit (Life Technologies, Carlsbad, CA, USA). Two microliters of RT reaction were then PCR amplified with specific primers for CB1: sense primer, 5′-TATATTCTCTGGAAGGCTCACAGCC-3′ antisense primer, 5′-GAGCATA CTGCAGAATGCAAACACC-3′(for amplification of a 270-bp product for human CB1); and CB2: sense primer, 5′-TTTCCCACTGATCCCCAATG-3′ antisense primer, 5′-GAGCATACTGCAGAATGCAAACACC-3′ (for amplification of a 333-bp product for human CB2). PCR products were analyzed by electrophoresis on ethidium bromide-stained 2% agarose gels and DNA was detected by exposure under UV light.

Western blot

After different treatments according to the experiments cells were lysed in ice-cold lysis buffer (50mM Tris (pH 7.4), 0.8M NaCl, 5mM MgCl2, 0.1% Triton X-100, 1mM PMSF, 10μg/ml soybean trypsin inhibitor, 1μg/ml aprotinin and 5μg/ml leupeptin), and cleared by microcentrifugation. Equivalent protein amounts of each sample were separated on SDS-PAGE gels and blotted to PVDF transfer membrane. After blocking with 5% skim dried milk, immunoblot analysis was performed followed by enhanced chemo luminescence detection.

Cell viability assay

Cells in logarithmic phase were cultured at a density of 5000 cells per cm2 in a 12-well plate. The cells were exposed to various concentrations of Δ9-THC and JWH-015 for indicated times. The 3-[4,5-dimethylthiazolyl-2] 2,5-diphenyl-tetrazolium bromide (MTT) cell viability assay was used to evaluate the effects of cannabinoids on cell growth and to determine the IC50.

Confocal microscopy

After 48h in culture, the cells were fixed in 4% paraformaldehyde in PBS and incubated with 0.1% Triton X-100 for permeabilization. Immunolabeling with the anti-LC3 polyclonal antibody was performed by incubation at room temperature for 1h. Secondary labeling was performed with Alexa Flour 594, conjugated to anti-rabbit IgG and Alexa Flour 488 (Invitrogen). Imaging was with a Leica TCS SP5 laser-scanning confocal microscope with LAS-AF imaging software, using a 63 × oil objective.

siRNA transfections

Cells were seeded at 2 × 105 cells/35mm well the day before transfection. Cells were then transfected in 1ml OPTIMEN containing 4μg Lipofectamine 2000 (Invitrogen), with 100nM siRNA duplexes or control scrambled RNA according to the manufacturer’s protocols. At 24h after transfection, the medium was removed and replaced for DMEM containing 10% fetal bovine serum. Cells were then treated with cannabinoids for 48h and used for MTT cell viability assays.

For each transfection, the following sequences were used: Atg 5 sense sequence, 5′-GUGAGAUAUGGUUUGAAUAdTdT-3′ α1 subunit AMPK first sense sequence, 5′-CCCAUAUUAUUUGCGUGUAdTdT-3′ second sense, sequence 5′-GAATCCTGTGACAAGCACAdTdT-3′ sense sequences of the Dharmacon Smart Pool, 5′-CCAUACCCUUGAUGAAUUAUU-3′ 5′-GCCCAGAGGUAGAUAUAUGUU-3′ 5′-GAGGAUCCAUCAUAUAGUUUU-3′ and 5′-ACAAUUGGAUUAUGAAUGGUU-3′. TRB3 sense sequence 5′-GUGCGAAGCCGCCACCGUAdTdT-3′ LKB1 sense sequence, 5′-GUACUUCUGUCAGCUGAUUdTdT-3′ CaMKKβ sense sequence, 5′-GCUCCUAUGGUGUCGUCAAdTdT-3′ (Sigma).

Real-time quantitative PCR

cDNA was obtained from cells using Transcriptor (Roche Applied Science, Mannheim, Germany). Real-time quantitative (qPCR) assays were performed using the FastStart Universal Probe Master mix with Rox (Roche Applied Science), and probes were obtained from the Universal ProbeLibrary Set (Roche Applied Science); Atg 5 sense primer, 5′-GACGCTGGTAACTGACAAAGTGA-3′ Atg 5 antisense primer, 5′-TAGGAGATCTCCAAGGGTATGCA-3′ TRB3 sense primer, 5′-GCCACTGCCTCCCGTTCTTG-3′ TRB3 antisense primer, 5′-GCTGCCTTGCCCGAGTATGA-3′ LKB1 sense primer, 5′-GGCATGCAGGAAATGCTGGACAGC-3′ LKB1 antisense primer, 5′-GTGTCCAGGCCGTTGGCAATCTCG-3′ CaMKKβ sense primer, 5′-TCGAGTACTTGCACTGCCAGAAGATC-3′ CaMKKβ antisense primer, 5′-GGGGTTCTTGTCCAGCATACGGGT-3′ 18S sense primer, 5′-GCTCTAGAATTACCACAGTTATCCAA-3′ 18S antisense primer, 5′-AAATCAGTTATGGTTCCTTTGGTC-3′. Amplifications were run in a 7900 HT-Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Each value was adjusted by using 18S RNA levels as a reference.

Animal care and handling

Athymic nude (nu/nu) 5-week-old male mice were obtained from Harlan Iberica Laboratory (Barcelona, Spain) and maintained under specific pathogen-free conditions with the approval of the Institutional Animal Care and Use Committee of Alcala University. All animal studies were conducted in accordance with the Spanish institutional regulation for the housing, care and use of experimental animals and met the European Community directives regulating animal research. Recommendations made by the United Kingdom coordinating Committee on Cancer Research (UKCCCR) have been kept carefully.

In vivo studies

To study the in vivo antitumor activity of cannabinoids, hepatocarcinoma tumors were induced in athymic mice by subcutaneal injection or by liver implantation. Mice were injected subcutaneously in the right flank with 10 × 106 HepG2 or HuH-7 cells in 0.1ml of PBS+0.5% BSA. At 2 weeks after transplantation, tumors had grown to an average volume of 150mm3. Mice were then divided into different experimental groups of eight animals each, which received the following treatments as subcutaneous injections according to the experiment: saline (control); 15mg/kg b.w. Δ9-THC; 1.5mg/kg b.w. JWH-015; and 1mg/kg b.w. 3-MA. The injection was repeated every day and treatment was continued for 15 days. Tumor volumes were monitored every day using calliper measurements and were calculated by the formula: (4π/3) × (w/2)2 × (l/2). The body weight of the animals was recorded daily. For in vivo Atg5 knockdown, xenograft tumors were induced as indicated and 1nmol specific Atg5 atelocollagen-complexed siRNA or control siRNA was injected peritumorally on days 1 and 7 of the treatment. Mice were treated daily for 15 days with saline, 15mg/kg b.w. Δ9-THC or 1.5mg/kg b.w. JWH-015.

When tumor cells were implanted in the liver, the treatments were initiated 1 week after cells injection and administered intraperitoneally. Eight animals were used in each experimental group. The study was performed for 10 days to minimize the trauma of the host animals according to UKCCCR recommendations. At the end of the treatment, the animals were killed and xenografted tumors and livers weighted and frozen.

This work was supported by Ministerio de Ciencia e Innovación (Grant SAF2008-03220 to ID, PS09/01401 to GV and SAF2006/00918 to MG), Comunidad de Madrid (Grants CAM/UAH CCG08-UAH/BIO-3914 and CAM S-SAL-0261-2006), Comunidad Castilla-LaMancha (Grant PII1/09-0165-0822) and Santander-Complutense (Grant PR34/07-15856 to GV). NO-H and DV received fellowships from the University of Alcalá. MS was recipient of a fellowship from MEC and of a formation contract from Comunidad de Madrid. We thank Sonia Hernández, Mar Lorente and Sofía Torres for their technical advice as well as other members of our laboratories for their continuous support.