Anti-tumoral action of cannabinoids on hepatocellular carcinoma: role of AMPK-dependent activation of autophagy

Abstract

Hepatocellular carcinoma (HCC) is the third cause of cancer-related death worldwide. When these tumors are in advanced stages, few therapeutic options are available. Therefore, it is essential to search for new treatments to fight this disease. In this study, we investigated the effects of cannabinoids--a novel family of potential anticancer agents--on the growth of HCC. We found that Δ(9)-tetrahydrocannabinol (Δ(9)-THC, the main active component of Cannabis sativa) and JWH-015 (a cannabinoid receptor 2 (CB(2)) cannabinoid receptor-selective agonist) reduced the viability of the human HCC cell lines HepG2 (human hepatocellular liver carcinoma cell line) and HuH-7 (hepatocellular carcinoma cells), an effect that relied on the stimulation of CB(2) receptor. We also found that Δ(9)-THC- and JWH-015-induced autophagy relies on tribbles homolog 3 (TRB3) upregulation, and subsequent inhibition of the serine-threonine kinase Akt/mammalian target of rapamycin C1 axis and adenosine monophosphate-activated kinase (AMPK) stimulation. Pharmacological and genetic inhibition of AMPK upstream kinases supported that calmodulin-activated kinase kinase β was responsible for cannabinoid-induced AMPK activation and autophagy. In vivo studies revealed that Δ(9)-THC and JWH-015 reduced the growth of HCC subcutaneous xenografts, an effect that was not evident when autophagy was genetically of pharmacologically inhibited in those tumors. Moreover, cannabinoids were also able to inhibit tumor growth and ascites in an orthotopic model of HCC xenograft. Our findings may contribute to the design of new therapeutic strategies for the management of HCC.

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 24 h and microtubule-associated protein 1 light chain 3α (LC3) was detected by confocal immunofluorescence. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Values on the lower right corner of each panel correspond to the number of cells with LC3 dots relative to the total number of cells (n=5; mean±standard deviation (S.D.)). (b) Immunoblot analysis of p62, LC3-I and LC3-II levels after Δ⁹-THC or JWH-015 treatment in the presence of the lysosomal protease inhibitors E64d (2.5 μg/ml) and pepstatin A (5 μg/ml; PA) for 24 h. Tubulin levels are shown as loading control. (c) HepG2 and HuH-7 cells were treated either with 8 μM Δ⁹-THC or 8 μM JWH-015 in the presence of 2.5 μg/ml E64d and 5 μg/ml PA or 1 μM 3-methyladenine (3MA) for 48 h and cell viability was analyzed by 3-[4,5-dimethylthiazolyl-2] 2,5-diphenyl-tetrazolium bromide (MTT) test. Data are the mean±S.D. of three different experiments each performed in triplicate (^**P<0.01 versus control; ^#P<0.05 and ^##P<0.01 versus cannabinoid-treated cells). (d) Effect of Δ⁹-THC or JWH-015 on the viability – as determined by the MTT test (48 h) of HepG2 cells transfected with Atg5-selective (small interfering (si)Atg5) or control (siC) siRNA. Data correspond to the mean±S.D. of three different experiments each performed in triplicate (^**P<0.01 versus control; ^##P<0.01 versus cannabinoid-treated cells). Atg5 mRNA levels (mean of the three experiments) assessed by real-time polymerase chain reactions (PCRs) are shown in the lower panel. (e) HepG2 or HuH-7 cells were incubated either with Δ⁹-THC or JWH-015 for 30 h in the presence of 1 μM 3-MA and levels of procaspase-3 and LC3 were detected by western blot. Tubulin levels are shown as loading control. The image is representative of three different experiments

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 (AMPK) through cannabinoid receptor 2 (CB₂) receptors. (a) Effect of Δ⁹-THC or JWH-015 (8 h) on the phosphorylation of eIf2α in HepG2 and HuH7 cells. Tubulin levels are shown as a loading control. The image is representative of three different experiments (b) Effect of Δ⁹-THC or JWH-015 (24 h) on tribbles homolog 3 (TRB3) mRNA levels (as determined by quantitative polymerase chain reaction (qPCR)) of HepG2 and HuH7 cells (n=4; ^**P<0.01). (c) Effect of Δ⁹-THC or JWH-015 (24 h) in the phosphorylation of AMPK, Akt, p70S6K and S6 of HepG2 and HuH7 cells. Tubulin levels are shown as a loading control. The image is representative of three different experiments. (d) Effect of Δ⁹-THC (8 μM), JWH-015 (8 μM), 1 μM SR141716A (SR1) or 2 μM SR 144528 (SR2) (24 h) on AMPK, Akt and S6 phosphorylation, as well as microtubule-associated protein 1 light chain 3 alpha (LC3) lipidation of HepG2 cells. Tubulin levels are shown as loading control. The image is representative of five different experiments. Optical density (O.D.) values (mean±standard deviation (S.D.) of the five experiments; ^*P<0.05 and ^**P<0.01 versus control) are shown under the each image

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 48 h in the presence of 0.5 μM dorsomorphin and cell viability was assayed by 3-[4,5-dimethylthiazolyl-2] 2,5-diphenyl-tetrazolium bromide (MTT). Data are the mean±standard deviation (S.D.) of three different experiments, each performed in triplicate (^**P<0.01 versus control; ^#P<0.05 and ^##P<0.01 versus cannabinoid-treated cells). (b) HepG2 and HuH-7 cells transfected either with small interfering (si)C or AMPKα-selective siRNA (siAMPK) were incubated with Δ⁹-THC or JWH-015 for 48 h and cell viability was assayed by MTT. Data correspond to the mean±S.D. of five different experiments (^**P<0.01 versus control; ^#P<0.05 and ^##P<0.01 versus cannabinoid-treated cells). AMPKα levels of a representative experiment, assessed by western blot are shown in the upper panel. (c) Effect of Δ⁹-THC or JWH-015 on AMPK, Akt and S6 phosphorylation, as well as microtubule-associated protein 1 light chain 3α (LC3) lipidation (24 h) of HepG2 and HuH-7 cells transfected with siC or siAMPK . Tubulin levels are shown as loading control. A representative western blot of three different experiments is shown

Figure 4

Δ⁹-Tetrahydrocannabinol (Δ⁹-THC) and JWH-015 activate adenosine monophosphate-activated kinase AMPK via CaMKKβ. (a) Left panel: Effect of Δ⁹-THC (8 μM) or JWH-015 (8 μM) on the viability (48 h; as determined by the 3-[4,5-dimethylthiazolyl-2] 2,5-diphenyl-tetrazolium bromide (MTT) test) of HepG2 cells transfected with small interfering (si)C or liver kinase B1 (LKB1)-selective (siLKB1) siRNA. Data correspond to the mean±standard deviation (S.D.) of four different experiments, each performed in quadruplicate (^**P<0.01 versus control). Lower panel: LKB1 mRNA levels (mean of the four experiments as determined by real-time quantitative polymerase chain reaction (PCR)) of HepG2 cells transfected with siC or siLKB1. Right panel: Effect of Δ⁹-THC or JWH-015 (24 h) on the AMPK, ACC, Akt, S6 phosphorylation and microtubule-associated protein 1 light chain 3α (LC3) lipidation of siC- and siLKB1-transfected HepG2 cells. Tubulin levels are shown as loading control. A representative western blot of four different experiments is shown. Optical density (O.D.) values (mean±S.D. of the four experiments) are shown under the each image. (b) Effect of Δ⁹-THC (8 μM) or JWH-015 (8 μM) on the viability (48 h, as determined by the MTT test) of HepG2 cells transfected with siC- or CaMKKβ-selective (siCaMKKb) siRNA. Data correspond to the mean±S.D. of four different experiments, each performed in quadruplicate (^**P<0.01 versus control; ^##P<0.01 versus cannabinoid-treated cells). Lower panel: CaMKKβ mRNA levels (mean of the four experiments as determined by real-time quantitative PCR) of HepG2 cells transfected with siC or siCaMKKb. Right panel: Effect of Δ⁹-THC or JWH-015 (24 h) on AMPK, ACC, Akt and S6 phosphorylation and LC3 lipidation of siC- and siCaMKKb-transfected HepG2 cells. Tubulin levels are shown as loading control. A representative western blot of three different experiments is shown. O.D. values (mean±S.D. of the four experiments) are shown under the each image. (c) Effect of Δ⁹-THC (8 μM) or JWH-015 (8 μM) on the viability (48 h, as determined by the MTT test) of HepG2 cells incubated in the presence or absence of the 10 μM STO609 (STO; a CaMKKα/β inhibitor). Data correspond to the mean±S.D. of four different experiments, each performed in quadruplicate (^**P<0.01 versus control; ^##P<0.01 versus cannabinoid-treated cells). Right panel: Effect of Δ⁹-THC, JWH-015 and STO (10 μM) on AMPK, ACC, Akt and S6 phosphorylation and LC3 lipidation (24 h). Tubulin levels are shown as loading control. A representative western blot of four different experiments is shown. O.D. values (mean±S.D. of the four experiments)

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 serine–threonine kinase Akt/mammalian target of rapamycin C (Akt/mTORC1) axis, and (ii) activation of adenosine monophosphate-activated kinase (AMPK) via CaMKKβ. Stimulation of autophagy by cannabinoids leads to HCC apoptosis and cell death

Figure 6

Δ⁹-Tetrahydrocannabinol (Δ⁹-THC) and JWH-015 reduce the growth of HepG2- and HuH-7 cell-derived tumor xenografts. Athymic nude mice were injected subcutaneously (s.c.) in the right flank with HepG2 cells (a and b) or HuH-7 cells (c and d). When tumors reached a 150 mm³ size, mice were daily treated during 15 days with vehicle (control), 15 mg/kg Δ⁹-THC or 1.5 mg/kg JWH-015. Tumor volumes were measured daily. (a and c) Tumor growth curve after administration of vehicle (diamonds), Δ⁹-THC (squares) or JWH-015 (triangles). Results represent the mean±standard error of mean (S.E.M.) of eight mice in each group. ^*P<0.01 versus control compared by Student's t-test. A representative image of the dissected tumors after treatment is shown. (b and d) Immunoblot analysis of adenosine monophosphate-activated kinase (AMPK), Akt and S6 phosphorylation, microtubule-associated protein 1 light chain 3α (LC3) lipidation and active-caspase-3 levels in the dissected tumors. Western blots analyses of one representative tumor for each condition are shown

Figure 7

Autophagy is required for the anti-tumoral action of Δ⁹-tetrahydrocannabinol (Δ⁹-THC) and JWH-015 on hepatocellular carcinoma (HCC) tumor xenografts. (a) Athymic nude mice were injected subcutaneously (s.c.) in the right flank with HepG2 cells. When tumors reached a 150 mm³ size, mice daily treated during 15 days with vehicle (control), 15 mg/kg Δ⁹-THC or 1.5 mg/kg JWH-015. Tumors were injected with atelocollagen complexed with control RNA or atelocollagen complexed with small interfering (si)Atg5 in days 1 and 7 of the treatment. Tumor volumes were measured daily. Tumor growth curves and final tumor volumes after administration of the treatments are shown. Results represent the mean±standard error of mean (S.E.M.) of eight mice in each group. ^**P<0.01 versus control and ^##P<0.01 versus siControl compared by Student's t-test. Expression levels of Atg5 in siC and siATG5 tumors at the end of the treatment was examined by real-time polymerase chain reaction (PCR). A representative image of the dissected tumors after the treatments is shown. (b) Athymic nude mice injected s.c. in the right flank with HepG2 cells were daily treated during 15 days with vehicle (control) (filled circles), 15 mg/kg Δ⁹-THC (filled squares), 1.5 mg/kg JWH-015 (filled triangles), vehicle plus 1 mg/kg 3-MA (open circles), 15 mg/kg Δ⁹-THC plus 1 mg/kg 3-MA (open squares) or 1.5 mg/kg JWH-015 plus 1 mg/kg 3-MA (open triangles). Tumor growth curves and final tumor volumes after administration of the treatments are shown. Results represent the mean±S.E.M. of eight mice in each group. ^**P<0.01 versus control and ^##P<0.01 versus cannabinoid-treated tumors compared by Student's t-test. A representative image of the dissected tumors after the treatments is shown

Figure 8

Anti-tumoral effect of Δ⁹-tetrahydrocannabinol (Δ⁹-THC) and JWH in an orthotopic transplantation tumor model of hepatocellular carcinoma (HCC). The orthotopic transplantation HCC model was established by intrahepatic implanting of HepG2 cells. At 1 week after injection, mice were daily treated intraperitoneally (i.p.) with vehicle (control), 15 mg/kg Δ⁹-THC or 1.5 mg/kg JWH-015 for 10 days. (a) Effect of the different treatments on liver weight and ascites development. Representative images of mice at the end of the treatment are shown. Immunoblot analysis of adenosine monophosphate-activated kinase (AMPK), Akt and S6 phosphorylation, microtubule-associated protein 1 light chain 3α (LC3) lipidation and active-caspase-3 levels in the dissected tumors. Western blots analyses of one representative tumor for each condition is shown. (b) Effect of the different treatments on α-fetoprotein levels (as determined by western Blot – left panel) and immunofluorescence (right panel) of the dissected livers are shown. A normal liver is shown for comparison