Cannabinoids exert antiproliferative effects in a wide range of tumoral cells, including hepatocellular carcinoma (HCC) cells. In this study, we examined whether the PPARγ-activated pathway contributed to the antitumor effect of two cannabinoids, Δ9-tetrahydrocannabinol (THC) and JWH-015, against HepG2 and HUH-7 HCC cells. Both cannabinoids increased the activity and intracellular level of PPARγ mRNA and protein, which was abolished by the PPARγ inhibitor GW9662. Moreover, genetic ablation with small interfering RNA (siRNA), as well as pharmacological inhibition of PPARγ decreased the cannabinoid-induced cell death and apoptosis. Likewise, GW9662 totally blocked the antitumoral action of cannabinoids in xenograft-induced HCC tumors in mice. In addition, PPARγknockdown with siRNA caused accumulation of the autophagy markers LC3-II and p62, suggesting that PPARγ is necessary for the autophagy flux promoted by cannabinoids. Interestingly, downregulation of the endoplasmic reticulum stress-related protein tribbles homolog 3 (TRIB3) markedly reduced PPARγ expression and induced p62 accumulation, which was counteracted by overexpression of PPARγ in TRIB3-knocked down cells. Taken together, we demonstrate for the first time that the antiproliferative action of the cannabinoids THC and JWH-015 on HCC, in vitro and in vivo, are modulated by upregulation of PPARγ-dependent pathways.

The cannabinoids THC and JWH-015 activate PPARγ in HCC cells

To investigate the role of PPARγ in the mechanism of action of cannabinoids on HCC cells, we treated HepG2 cells with the cannabinoids THC and JWH-015, after which PPARγ expression was examined using RT-PCR and western blot. As shown in Figure 1a, there was a peak of PPARγ mRNA at 1-h treatment with both cannabinoids and a further decline at 24h. Likewise, PPARγ protein expression increased until 3h and then decreased at 8h (Figure 1b). As a further proof of PPARγ induction, we measured the PPARγ target CD36 to confirm PPARγ activation. As shown in Figure 1c, THC and JWH-015 produced an increase in CD36 mRNA levels with a peak at 6h of treatment. Lipid accumulation in liver cells is considered an indicator of PPARγ activation. Therefore, we measured neutral lipid content in HepG2 and HUH-7 cells by Oil Red O staining. Neutral lipid accumulated in both HepG2 and HUH-7 cells after THC and JWH-015 treatment. The increase in Oil Red O staining was prevented by pretreatment with the PPARγ antagonist GW9662, confirming the involvement of PPARγ in neutral lipid accumulation and PPARγ activation after cannabinoids treatment (Figure 2a). Confocal microscopy of HepG2 cells treated with THC and JWH-015 also showed a neutral lipid accumulation within the cell (Figure 2c). Therefore, these data demonstrated that cannabinoids treatment activates PPARγ in HCC cells.

Figure 1

Cannabinoid-induced PPARγ increase in HCC cells. (a) HepG2 cells were treated with Δ⁹-tetrahydrocannabinol (8μM) or JWH-015 (8μM) for different times and PPARγ mRNA levels were determined by quantitative …

Figure 2

Δ⁹-Tetrahydrocannabinol and JWH-015 increase intracellular neutral lipid content in HCC cells. (a) HepG2 cells were incubated in the presence of increasing concentrations of THC or JWH-015 for 24h, and intracellular neutral lipid content…

The activation of PPARγ by cannabinoids may be performed by direct binding to the receptor or by intracellular signalling cascades that may lead indirectly to PPARγ activation. The mechanism of action of THC on PPARγ has been extensively studied by O’Sullivan and Kendall,²⁰ but it is unknown if JWH-015 can activate PPARγ directly. To investigate whether JWH-015 joined PPARγ, we performed a binding assay using HeLa cells with a reporter luciferase gene. However, JWH-015-induced PPARγ activation was not due to an agonist activity of the compound, as JWH-015 was not able to bind to the ligand-binding domain of PPARγ in the in vitro assay (Figure 2c), suggesting that PPARγactivation must be happening indirectly through signaling pathways activated by cannabinoids.

AMPK and PPARγ are two independent pathways activated by cannabinoids

We have previously shown that THC and JWH-015 stimulate autophagy on HCC cells through two different pathways: (i) activation of adenosine monophosphate-activated kinase (AMPK) via CaMKKb, and (ii) ER stress with upregulation of tribbles homolog 3 (TRIB3) and subsequent inhibition of the serine–threonine kinase Akt/mammalian target of rapamycin C (Akt/mTORC1) axis.¹⁹ Furthermore, AMPK has been shown to participate in modulating the activity of PPARγ,²¹ thus providing a possible mechanism for cross talk between the signaling pathways activated by cannabinoids. PPARγ phosphorylation by AMPK represses both the ligand-dependent and independent trans-activating function of the receptor. Therefore, we examined whether the activities of these molecules by cannabinoids were coordinately regulated. To investigate the relationship between these two pathways, we knocked down AMPK and measured PPARγ levels after cell treatment with THC and JWH-015. Both cannabinoids, THC and JWH-015, were able to increase PPARγ levels even when AMPK was absent (Figure 3a). These findings indicate that AMPK is not necessary for PPARγ induction by cannabinoids. Moreover, PPARγ is not necessary for AMPK activation by cannabinoids, as downregulation of PPARγdid not reverse the increase in AMPK phosphorylation produced by THC or JWH-015 (Figure 3b). Therefore, these data suggest that AMPK and PPARγ are two independent pathways activated by cannabinoids in HCC.

Figure 3

TRIB3 is required for PPARγ activation. (a) HepG2 cells were transfected with control siRNA (siControl) or AMPK selective siRNA (siAMPK) and incubated with THC (8μM) or JWH-015 (8μM) for 3h, after which …

The TRIB3 protein regulates PPARγ expression in HCC cells

To investigate whether PPARγ induction was associated with the second mechanism by which cannabinoid induced cell death (ER stress/Akt/mTORC1 axis), we analysed this pathway in cells in which PPARγ was genetically blocked. However, PPARγ knockdown did not modify the inhibition of the Akt pathway and S6 phosphorylation or the increase in eIF2α phosphorylation, which we had previously reported to be modified after cannabinoids treatment (Figure 3b).

The intracellular adaptor TRIB3, a human homolog of Drosophila tribbles, has been found to interact with a variety of signalling molecules to regulate diverse cellular functions including cell proliferation, migration and morphogenesis by participating in protein complex assembly. In the liver, it functions as a negative regulator of the serine–threonine kinase Akt. We have previously shown that TRIB3 links cannabinoid-induced ceramide production and endoplasmic reticulum stress with Akt inhibition and autophagy in HCC cells.¹⁹ Recent research has shown that in 3T3-L1 adipocytes TRIB3 interacts with PPARγ to modulate its transcriptional activity.²² To investigate the role of TRIB3 in the action mechanism of cannabinoids, HCC cells were transfected with selective TRIB3 siRNA, treated with THC or JWH-015, and examined for PPARγ protein and mRNA expression. As shown in Figure 3c, when TRIB3 was knocked down, levels of PPARγ were virtually undetectable, both in control cells and in cannabinoid-treated cells, being even more effective than PPARγ silencing. This means that TRIB3 is necessary for PPARγ expression and suggests a new PPARγregulatory pathway to be explored in further research.

PPARγ contributes to cannabinoid-induced autophagy

Owing to recent data about the involvement of PPARγ in autophagy^23, 24 and as this process is necessary for the antitumoral action of cannabinoid,^19, 25 we wondered whether PPARγ receptor might also have a role in the autophagy induced by cannabinoids on HCC cells. To investigate this effect, we analyzed LC3-II levels, a hallmark of autophagy, in cannabinoid-treated cells. As shown inFigure 4, THC and JWH-015 induced an increase in LC3II, as was expected, measured by western blot (a) or confocal microscopy (b). Surprisingly, knocking down of PPARγ increased LC3II even in the control cells. Likewise, cells treated with cannabinoids showed higher LC3II levels when PPARγ was knocked down than when cells were transfected with the control siRNA. To investigate whether this increase was caused by an augmented activation of autophagy, levels of the signal adaptor protein p62, an autophagosome cargo that is eliminated by autophagy and accumulates when autophagy is deficient, were measured in control RNA-transfected cells and in PPARγ siRNA-transfected cells. As observed in Figure 4a, levels of p62 protein increased when PPARγ was silenced in control cells compared with cannabinoid-treated cells. These results indicate that in PPARγ-depleted cells, autophagy is not active, but it is blocked after autophagosome formation, and then p62 accumulates. If TRIB3 is responsible for PPARγ expression, then PPARγ overexpression in TRIB3-depleted cells should reverse p62 accumulation. Figure 4c shows that this is the case, as p62 does not accumulate in TRIB3-silenced cells in which PPARγ overexpression was induced by a mammalian expression vector, indicating that PPARγ is responsible for the autophagosome blockage. Likewise, PPARγ overexpression in TRIB3-silenced cells restored the caspase 3 cleavage (Figure 4c).

Figure 4

PPARγ participates in autophagy induction. (a) HepG2 cells were transfected with siRNA control or selective for PPARγ (siControl or siPPARγ) and treated with THC 8μM or JWH-015 8μM and PPARγ …

Autophagosome accumulation can occur for two reasons: (i) autophagosomes are not fused with lysosomes or/and (ii) lysosomes do not work correctly. As there are no data about the participation of PPARγ in the autophagosome–lysosome fusion, but there are findings showing that PPARγ regulates lysosomal proteases,^26, 27 we decided to investigate this option. Lysosomes are the key degradative compartments of the cell, because they are acidic organelles filled with hydrolases. Among the lysosomal hydrolases, proteases, especially cathepsins, have a major role. Similar to other proteases, the cathepsins are synthesized as inactive proenzymes and are activated by proteolytic removal of the N-terminal propeptide. As shown in Figure 4a, THC and JWH-015 increased the levels of cathepsines D and L (both inactive and active forms). However, PPARγ knockdown did not modify these increases, suggesting that PPARγ does not regulate cathepsin L or D in this model. Cysteine cathepsins are optimally active in a slightly acidic pH, and are mostly unstable at neutral pH. When cathepsins are outside the lysosomes or if there is no acidic pH within the lysosome, they can be relatively rapidly inactivated and proenzymes accumulate.²⁸ As SiControl and SiPPARγ-untreated or cannabinoid-treated cells had a right cleavage of these proteases and the same level of the lysosomal marker LAMP-2, we can conclude that lysosomes work correctly, suggesting that the reason for autophagosome accumulation is not lysosome failure. This is in concordance with the observation that PPARγ silencing causes an accumulation of LC3 even in control cells (Figures 4a and b). Therefore, we can conclude that in HCC cells cannabinoids induce autophagy through PPARγ activation (summarized in Figure 4d).

PPARγ participates in the antiproliferative effect evoked by cannabinoids

It has been recently described that the cannabinoid WIN induces apoptosis through PPARγ in HepG2 cells.¹⁸ Moreover, in previous studies, we demonstrated that cannabinoids induce apoptosis and autophagy in HCC cells and in xenograft tumor models. To investigate the role of PPARγ in the anti-proliferative response exerted by THC or JWH-015, HCC cells were incubated with increasing doses of both cannabinoids for 48h in the presence of the PPARγ-selective antagonist GW9669 and cell viability was measured by MTT. Pharmacological inhibition of PPARγ caused a shift of the dose–response curve to the right, thus increasing the IC50 dose in HepG2 and in HUH-7 cells (Figure 5a). Moreover, when HepG2 cells or HuH-7 cells were transfected with PPARγ siRNA, the inhibitory effect of cannabinoids on cell viability was reduced (Figure 5b). As PPARγ agonists have been evaluated as potential antitumoral agents, we decided to test the possible synergic effect between cannabinoids and the PPARγ agonist Troglitazone (TRO) in combinatorial treatment. In agreement with the results obtained by other groups,^{16, 29, 30, 31, 32, 33} we observed that TRO treatment produced a dose-dependent reduction in cell viability that reached a value of 50% when 40μM concentrations were used (Figure 5c). We therefore selected submaximal doses of Troglitazone (20μM), THC (2μM) and JWH-015 (2μM) to evaluate whether the combined administration of PPAR ligands and cannabinoids enhanced their ability to reduce cell viability. In line with this possibility, combined treatment with low doses of TRO and THC or JWH-015 reduced the viability of HepG2 cells (Figure 5d).

Figure 5

PPARγ is involved in cannabinoid-induced cell death. (a) HepG2 or HuH7 cells were incubated with different doses of THC or JWH-015 for 48h in the presence or absence of 3μM GW9662 (GW) and cell viability was assayed by …

To further investigate the role of PPARγ in cannabinoid-induced cell death, we measured the number of apoptotic Anexin-V and IP-stained cells by cytometry. Both THC and JWH-015 increased the number of apoptotic cells, which was reduced by pharmacological inhibition of PPARγ with GW9669 (Figure 6). According to this, GW9669 also reduced the cleavage of procaspase 3 induced by cannabinoids (data not shown). These observations support the fact that PPARγparticipates in cell death induced by cannabinoids.

Figure 6

Cannabinoids induce apoptosis in HepG2 and HUH-7 cells. Representative plots of Annexin V-FITC/IP staining of HepG2 cells incubated with 8μM THC or 8μM JWH-015 for 24h in the presence or absence of 3 …

PPARγ is involved in the in vivo antitumor properties of cannabinoids

As a further proof for the involvement of PPARγ in the antitumoral action of cannabinoids, we generated tumor xenografts by subcutaneous inoculation of HepG2 cells in nude mice. Mice were treated peritumorally with THC or JWH-015 alone or in combination with the PPARγ inhibitor GW9662, and tumor size was daily monitored. As shown in Figure 7, THC and JWH-015 significantly reduced the tumor growth rate. However, when tumors were treated with the cannabinoids in the presence of the PPARγ antagonist, the tumor growth was similar to those of the controls. Furthermore, we analyzed PPARγ mRNA expression of HepG2- and HuH-7-derived tumors, and we found that cannabinoid treatment increased PPARγ mRNA levels in both cases (Figure 7b). Thereby, in vivo antitumor effect of cannabinoids in HCC cells depends on PPARγ activation.

Figure 7

PPARγ is required for the in vivo antitumoral action of THC and JWH-015 on hepatocellular carcinoma tumor xenografts. (a) Athymic nude mice injected s.c. in the right flank with HepG2 cells were daily treated during 15 days with vehicle (control) …

Reagents

THC was obtained from THC Pharm GmbH (Frankfurt, Germany) and JWH-015 was purchased from Sigma (St. Louis, MO, USA). Both cannabinoids were dissolved in DMSO. The final concentration of the DMSO in the cell cultures was never >0.1%. The same quantity of DMSO was added to controls. The anti-PPARγ, anti-peiF2α, anti-pAMPK, anti-pS6, anti-pAKT-ser473 and anti-p62 polyclonal antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). The anti-LC3 polyclonal antibody was obtained from MBL International (Woburn, MA, USA) and procaspase 3, anti-cathepsin D and anti-cathepsin L were obtained from Santa Cruz Biotechnology (Bergheimer, Heidelberg, Germany). The PPARγ antagonist GW9662, the agonist Troglitazone and all other reagents were purchased from Sigma.

Cell cultures

Human HCC HepG2 cells (ATCC HB-8065, Rockville, MD, USA) and human hepatoma HuH-7 cells (kindly supplied by Dr. Lisardo Boscá, Instituto de Investigaciones Biomédicas Alberto Sols, Madrid, Spain), were maintained in DMEM medium containing 10% fetal bovine serum, 1% penicillin/streptomycin (Invitrogen, Paisley, UK) and 1 × non-essential amino acids. Cells were seeded at a density of 5000cells/cm². Sixteen hours post-seeding, medium was changed to 0.5% FBS medium and experiments were performed 24h later, when cells were 80% confluent.

Quantitative PCR

Total RNA was isolated from cells by Trizol Reagent from Gibco (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocol. One microgram total RNA was retrotranscribed to cDNA with the M-MLV Reverse transcriptase kit (Life Technologies, Carlsbad, CA, USA). Real-time quantitative PCR assays were performed using the FastStart Universal Probe Master mix with Rox (Roche Applied Science, Barcelona, Spain), and probes were obtained from the Universal Probe Library Set (Roche Applied Science); TRIB3 sense primer, 5′-GCCACTGCCTCCCGTTCTTG-3′ TRIB3 antisense primer, 5′-GCTGCCTTGCCCGAGTATGA-3′ PPARγ sense primer, 5′-GGCGAGGGCGATCTTGACAGG-3′ PPARγ antisense primer, 5′-TGCGGATGGCCACCTCTTTGC-3′. CD36 sense primer, 5′-AGTCACTGCGACATGATTAATGGT-3′ CD36 antisense primer, 5′-CTGCAATACCTGGCTTTTCTC-3′.

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 chemoluminescence detection as previously described.³⁸

DNA-binding ELISA for activated PPARγ transcription factor

Cells were treated with cannabinoids as explained in the figure legends, and scraped on washing buffer (10mM Hepes pH 7.9, 1.5mM MgCl₂, 10mM, KCl, 1mM DTT, 0.5mM PMSF, 5mM NaF, 2μg/ml leupeptin, 10μg/ml aprotinin), and then lysed in washing buffer+0.5% NP-40 for 30min at 4°C. Then, the nuclear extract was obtained by centrifugation at 15000 × g for 30min at 4°C. To determine whether the treatment activated PPARγ, 10μg nuclear extract was incubated in a 96-well plate to which oligonucleotide containing the peroxisome proliferator response element had been immobilized (Active Motif, Rixensart, Belgium). PPARs contained in nuclear extracts bind specifically to this oligonucleotide and are detected through use of an antibody directed against PPARγ. Addition of a secondary antibody conjugated to horseradish peroxidase provides a sensitive colorimetric readout that is quantified by spectrophotometry. Unspecific binding is monitored by competition with an oligonucleotide containing the consensus PPARγ response element sequence, according to the manufacturer’s protocol.

PPAR binding assay

To test the binding capacity of JWH-015 to PPARγ, HeLa cell lines expressing a chimeric protein containing the ligand-binding domain of human PPARγ were used. The ligand-binding domain of PPARγ was fused to the yeast transactivator GAL4 DNA-binding domain. HeLa cells contained a luciferase reporter gene driven by a GAL4 recognition sequence. HeLa cells were treated and binding to PPARγ was estimated according to luciferase activity (relative light units normalized against the reference compound BRL49653).

Oil red o staining

Oil Red O staining was performed after cell treatment with cannabinoids. Briefly, the cells were washed with phosphate-buffered saline twice, fixed with 10% formalin for 30min, and stained with Oil Red O for 1h. After wash with phosphate-buffered saline, the stained lipid droplets were dissolved in isopropanol and quantified at 540nm.

Confocal microscopy

After 48h in culture, the cells were fixed in 4% paraformaldehyde in phosphate-buffered saline and incubated. Immunolabelling of neutral lipid with Lipidtox (Invitrogen) was performed by incubation at room temperature for 1h. Imaging was with a Leica TCS SP5 laser-scanning confocal microscope with LAS-AF imaging software, using a × 63 oil objective.

Cell viability assay

Cells in logarithmic phase were cultured at a density of 5000cells/cm² in a 12-well plate. After treatments, as explained in the figure legends, cell viability was assayed by MTT as previously described,^19, 39 to evaluate the effects of cannabinoids on cell growth.

siRNA tranfections

Cells were then transfected in 1ml OPTIMEN containing 4μg lipofectamine 2000 (Invitrogen), with 100nM PPARγ, AMPK or TRIB3 specific siRNA duplexes (PPARγ: 5′-CCAAGUUUGAGUUUGCUGUdTdT-3′ and 5′-ACAGCAAACUCAAACUU GGdTdT-3′ AMPK: 5′-CCCAUAUUAUUUGCGUGUAdTdT-3′ and 5′-UACACGCCAAAUAAUAUGGGdTdT-3′ TRIB3: 5′-GUGCGAAGCCGCCAC CGUAdTdT-3′ and 5′-UACGGUGGCGGCUUCGCACdTdT-3′) (Sigma) or control scrambled RNA for 12h according to the manufacturer’s protocols (Invitrogen). At 24h after transfection, the medium was removed and replaced for DMEM containing 10% fetal bovine serum. At dedicated time points after transfection, cells were used for MTT cell viability assays or western blot.

PPAR gamma expression vector and transient transfections

The PPARγ expression vector pCMX-mPPARg was generously provided by Dr. Mercedes Ricote (CNIC, Madrid, Spain). HepG2 cells were transiently transfected using 4μl Lipofectamine LTX & Plus Reagent (Invitrogen) with 3μg of the expression vector in 0.2-ml OPTIMEN, according to the manufacturer’s protocols (Invitrogen). At 48h after transfection, the experiments were performed with the different treatments.

Animal study

Forty-eight (48) male athymic nude (nu/nu) mice aged 5 weeks were purchased from Harlan Iberica Laboratory (Barcelona, Spain) and housed under specific pathogen-free conditions in a 12-h light–dark cycle at 21–23°C and 40–60% humidity. All animal studies were conducted in accordance with the Spanish institutional regulation for the housing, care and use of experimental animals with the approval of the Institutional Animal Care and Use Committee of Alcala University and met the European Community directives regulating animal research. Recommendations made by the United Kingdom Co-ordinating Committee on Cancer Research (UKCCCR) have been kept carefully.

After 1 week adaptation period, mice were injected subcutaneously in the right flank with 10 × 10⁶ HepG2 cells in 0.1ml of phosphate-buffered saline+0.5% BSA to induce HCC tumors. Two weeks after transplantation, tumors had grown to an average volume of 150mm³. Then, the mice were equally divided into six groups (n=8) and daily treated with vehicle, 15mg/kg THC or 1.5mg/kg JWH-015 in the presence or absence of 1mg/kg GW9662 for 15 days. After treatment, tumors were dissected and weighed.

Statistical analysis

Data are presented as the mean±S.E. of the number of experiments indicated. Significance of differences between groups was tested by paired Student’s t-test using the SPSS statistical software (Chicago, IL, USA). Differences between groups were considered significant when P<0.05.

This work has been supported by the Spanish Ministry of Economy and Compentitivity (Grant Minneco BFU2012-31444) Comunidad de Madrid (Grant CAM S2010/BMD-2308) and University of Alcala (Grant GC2011-001). CM has a fellowship from Spanish Foreign Ministry.

Akt

serine–threonine kinase Akt

AMPK

adenosine monophosphate-activated kinase

CB1

cannabinoid receptor 1

CB2

cannabinoid receptor 2

HCC

hepatocellular carcinoma

HepG2

human hepatocellular liver carcinoma cell line

HuH-7

hepatocellular carcinoma cells

LC3

microtubule-associated protein 1 light chain 3a

THC

Δ9-tetrahydrocannabinol

mTOR

mammalian target of rapamycin

TRIB3

tribbles homolog 3

TRO

Troglitazone

Edited by Munoz-Pinedo

• Alves R. C, Alves D, Guz B, Matos C, Viana M, Harriz M, et al. Advanced hepatocellular carcinoma. Review of targeted molecular drugs. Ann Hepatol. 2011;10:21–27. [PubMed]
• Waly Raphael S, Yangde Z, Yuxiang C. Hepatocellular carcinoma: focus on different aspects of management. ISRN Oncol. 2012;2012:421673.[PMC free article] [PubMed]
• Fowler CJ, Gustafsson SB, Chung SC, Persson E, Jacobsson SO, Bergh A. Targeting the endocannabinoid system for the treatment of cancer–a practical view. Curr Top Med Chem. 2010;10:814–827. [PubMed]
• Guindon J, Hohmann AG. The endocannabinoid system and cancer: therapeutic implication. Br J Pharmacol. 2011;163:1447–1463.[PMC free article] [PubMed]
• Velasco G, Sanchez C, Guzman M. Towards the use of cannabinoids as antitumour agents. Nat Rev Cancer. 2012;12:436–444. [PubMed]
• O’Sullivan SE. Cannabinoids go nuclear: evidence for activation of peroxisome proliferator-activated receptors. Br J Pharmacol.2007;152:576–582. [PMC free article] [PubMed]
• Pertwee RG, Howlett AC, Abood ME, Alexander SP, Di Marzo V, Elphick MR, et al. International Union of Basic and Clinical Pharmacology. LXXIX. Cannabinoid receptors and their ligands: beyond CB₁ and CB₂.Pharmacol Rev. 2010;62:588–631. [PMC free article] [PubMed]
• Youssef J, Badr M. Peroxisome proliferator-activated receptors and cancer challenges and opportunities. Br J Pharmacol. 2011;164:68–82.[PMC free article] [PubMed]
• Yessoufou A, Wahli W. Multifaceted roles of peroxisome proliferator-activated receptors (PPARs) at the cellular and whole organism levels.Swiss Med Wkly. 2010;140:w13071. [PubMed]
• Zhu Y, Qi C, Korenberg JR, Chen XN, Noya D, Rao MS, et al. Structural organization of mouse peroxisome proliferator-activated receptor gamma (mPPAR gamma) gene: alternative promoter use and different splicing yield two mPPAR gamma isoforms. Proc Natl Acad Sci USA.1995;92:7921–7925. [PMC free article] [PubMed]
• Maggiora M, Oraldi M, Muzio G, Canuto RA. Involvement of PPARalpha and PPARgamma in apoptosis and proliferation of human hepatocarcinoma HepG2 cells. Cell Biochem Funct. 2010;28:571–577.[PubMed]
• Tan XW, Xia H, Xu JH, Cao JG. Induction of apoptosis in human liver carcinoma HepG2 cell line by 5-allyl-7-gen-difluoromethylenechrysin.World J Gastroenterol. 2009;15:2234–2239. [PMC free article][PubMed]
• Zhou YM, Wen YH, Kang XY, Qian HH, Yang JM, Yin ZF. Troglitazone, a peroxisome proliferator-activated receptor gamma ligand, induces growth inhibition and apoptosis of HepG2 human liver cancer cells.World J Gastroenterol. 2008;14:2168–2173. [PMC free article][PubMed]
• Shen B, Chu ES, Zhao G, Man K, Wu CW, Cheng JT, et al. PPARgamma inhibits hepatocellular carcinoma metastases in vitro and in mice. Br J Cancer. 2012;106:1486–1494. [PMC free article] [PubMed]
• Galli A, Ceni E, Mello T, Polvani S, Tarocchi M, Buccoliero F, et al. Thiazolidinediones inhibit hepatocarcinogenesis in hepatitis B virus-transgenic mice by peroxisome proliferator-activated receptor gamma-independent regulation of nucleophosmin. Hepatology. 2010;52:493–505. [PubMed]
• Yu J, Shen B, Chu ES, Teoh N, Cheung KF, Wu CW, et al. Inhibitory role of peroxisome proliferator-activated receptor gamma in hepatocarcinogenesis in mice and in vitro. Hepatology. 2010;51:2008–2019. [PubMed]
• Schmidt MV, Brune B, von Knethen A. The nuclear hormone receptor PPARgamma as a therapeutic target in major diseases. Sci World J.2010;10:2181–2197. [PubMed]
• Giuliano M, Pellerito O, Portanova P, Calvaruso G, Santulli A, De Blasio A, et al. Apoptosis induced in HepG2 cells by the synthetic cannabinoid WIN: involvement of the transcription factor PPARgamma. Biochimie.2009;91:457–465. [PubMed]
• Vara D, Salazar M, Olea-Herrero N, Guzman M, Velasco G, Diaz-Laviada I. Anti-tumoral action of cannabinoids on hepatocellular carcinoma: role of AMPK-dependent activation of autophagy. Cell Death Differ. 2011;18:1099–1111. [PMC free article] [PubMed]
• O’Sullivan SE, Kendall DA. Cannabinoid activation of peroxisome proliferator-activated receptors: potential for modulation of inflammatory disease. Immunobiology. 2010;215:611–616. [PubMed]
• Burns KA, Vanden Heuvel JP. Modulation of PPAR activity via phosphorylation. Biochim Biophys Acta. 2007;1771:952–960.[PMC free article] [PubMed]
• Takahashi Y, Ohoka N, Hayashi H, Sato R. TRB3 suppresses adipocyte differentiation by negatively regulating PPARgamma transcriptional activity. J Lipid Res. 2008;49:880–892. [PubMed]
• Zhou J, Zhang W, Liang B, Casimiro MC, Whitaker-Menezes D, Wang M, et al. PPARgamma Activation Induces Autophagy in Breast Cancer Cells. Int J Biochem Cell Biol. 2009;41:2334–2342. [PMC free article][PubMed]
• Jiang M, Fernandez S, Jerome WG, He Y, Yu X, Cai H, et al. Disruption of PPARgamma signaling results in mouse prostatic intraepithelial neoplasia involving active autophagy. Cell Death Differ. 2010;17:469–481. [PMC free article] [PubMed]
• Salazar M, Carracedo A, Salanueva IJ, Hernandez-Tiedra S, Lorente M, Egia A, et al. Cannabinoid action induces autophagy-mediated cell death through stimulation of ER stress in human glioma cells. J Clin Invest. 2009;119:1359–1372. [PMC free article] [PubMed]
• Mahmood DF, Jguirim-Souissi I, Khadija el H, Blondeau N, Diderot V, Amrani S, et al. Peroxisome proliferator-activated receptor gamma induces apoptosis and inhibits autophagy of human monocyte-derived macrophages via induction of cathepsin L: potential role in atherosclerosis. J Biol Chem. 2011;286:28858–28866. [PMC free article][PubMed]
• Nakken B, Varga T, Szatmari I, Szeles L, Gyongyosi A, Illarionov PA, et al. Peroxisome proliferator-activated receptor gamma-regulated cathepsin D is required for lipid antigen presentation by dendritic cells. J Immunol. 2011;187:240–247. [PubMed]
• Turk B, Bieth JG, Bjork I, Dolenc I, Turk D, Cimerman N, et al. Regulation of the activity of lysosomal cysteine proteinases by pH-induced inactivation and/or endogenous protein inhibitors, cystatins.Biol Chem Hoppe Seyler. 1995;376:225–230. [PubMed]
• Yu J, Qiao L, Zimmermann L, Ebert MP, Zhang H, Lin W, et al. Troglitazone inhibits tumor growth in hepatocellular carcinoma in vitroand in vivo. Hepatology. 2006;43:134–143. [PubMed]
• Yoshizawa K, Cioca DP, Kawa S, Tanaka E, Kiyosawa K. Peroxisome proliferator-activated receptor gamma ligand troglitazone induces cell cycle arrest and apoptosis of hepatocellular carcinoma cell lines. Cancer.2002;95:2243–2251. [PubMed]
• Koga H, Sakisaka S, Harada M, Takagi T, Hanada S, Taniguchi E, et al. Involvement of p21(WAF1/Cip1), p27(Kip1), and p18(INK4c) in troglitazone-induced cell-cycle arrest in human hepatoma cell lines.Hepatology. 2001;33:1087–1097. [PubMed]
• Rumi MA, Sato H, Ishihara S, Kawashima K, Hamamoto S, Kazumori H, et al. Peroxisome proliferator-activated receptor gamma ligand-induced growth inhibition of human hepatocellular carcinoma. Br J Cancer.2001;84:1640–1647. [PMC free article] [PubMed]
• Toyoda M, Takagi H, Horiguchi N, Kakizaki S, Sato K, Takayama H, et al. A ligand for peroxisome proliferator activated receptor gamma inhibits cell growth and induces apoptosis in human liver cancer cells.Gut. 2002;50:563–567. [PMC free article] [PubMed]
• Sozio MS, Lu C, Zeng Y, Liangpunsakul S, Crabb DW. Activated AMPK Inhibits PPAR{alpha} and PPAR{gamma} Transcriptional Activity in Hepatoma Cells. Am J Physiol Gastrointest Liver Physiol.2011;301:G739–G747. [PMC free article] [PubMed]
• Salazar M, Carracedo A, Salanueva IJ, Hernandez-Tiedra S, Egia A, Lorente M, et al. TRB3 links ER stress to autophagy in cannabinoid anti-tumoral action. Autophagy. 2009;5:1048–1049. [PubMed]
• Salazar M, Hernandez-Tiedra S, Torres S, Lorente M, Guzman M, Velasco G. Detecting autophagy in response to ER stress signals in cancer. Methods Enzymol. 2011;489:297–317. [PubMed]
• Wang J, Lv XW, Shi JP, Hu XS. Mechanisms involved in ceramide-induced cell cycle arrest in human hepatocarcinoma cells. World J Gastroenterol. 2007;13:1129–1134. [PubMed]
• Sanchez AM, Martinez-Botas J, Malagarie-Cazenave S, Olea N, Vara D, Lasuncion MA, et al. Induction of the endoplasmic reticulum stress protein GADD153/CHOP by capsaicin in prostate PC-3 cells: a microarray study. Biochem Biophys Res Commun. 2008;372:785–791.[PubMed]
• Malagarie-Cazenave S, Olea-Herrero N, Vara D, Diaz-Laviada I. Capsaicin, a component of red peppers, induces expression of androgen receptor via PI3K and MAPK pathways in prostate LNCaP cells. FEBS Lett. 2009;583:141–147. [PubMed]