cbd liver cancer

December 15, 2021 By admin Off

The University Medical Center Groningen (UMCG) is to study the effect of cannabis oil on liver cancer patients who have exhausted all other treatment options. The aim of the study is to see whether cannabis oil acts as an anti-cancer agent that will shrink the liver tumours.

The first step in this study is to determine the correct dose for each patient. Patients will be given the maximum dose of cannabis oil that can be given without causing side-effects. Blood tests will be carried out and images made of the livers of those taking part after three, six and nine months to see whether the liver tumours are responding. The blood will be tested for the presence of tumour markers and to check the liver function of the participants.

The study was started after two separate reports that patients with advanced liver cancer had seen their tumours shrink after using cannabis oil. Now, two and five years after their diagnoses, the tumours have completely disappeared and the patients are cured. Although laboratory research shows that cannabis can curb the growth of liver cancer cells, a possible anti-cancer effect of cannabis oil in patients cannot be explained as no scientific research has been carried out.

Cannabis oil.

The cannabis oil being used is produced by the Transvaal apotheek, a specialist pharmacy with the certificates needed to produce drugs for research purposes. It is produced according to a fixed recipe with precise amounts of THC and CBD, two of the important substances contained in cannabis. The pharmacy only uses the cannabis flowers of the Cannabis Sativa L. variety, produced by Bedrocan. The Ministry of Health, Welfare and Sport has commissioned Bedrocan to standardize and grow the plant pharmaceutically in order to guarantee a consistent composition of the cannabis oil.

This study is being carried out with the help of patients with advanced liver cancer, for whom best supportive care is the only remaining option. As cannabis oil is broken down by the liver, all participants must have a good liver function. Patients with severe liver cirrhosis (liver damage caused by the formation of scar tissue) will not be considered for the study unless the cirrhosis is not affecting their liver function. Patients willing to join the study can be referred to the UMCG by their specialist. A total of 20 patients can take part. The study will last approximately three years.

Research design.

Patients with liver cancer who have exhausted all treatment options.

Autophagy is required for the…

Δ 9 -Tetrahydrocannabinol (Δ 9…

Schematic of the proposed mechanism…

Δ 9 -Tetrahydrocannabinol (Δ 9…

Δ 9 -Tetrahydrocannabinol (Δ 9 -THC) and JWH-015 induce autophagy via adenosine monophosphate-activated…

Figures.

Autophagy is required for the anti-tumoral action of Δ 9 -tetrahydrocannabinol (Δ 9…

Δ 9 -Tetrahydrocannabinol (Δ 9 -THC) and JWH-015 activate adenosine monophosphate-activated kinase AMPK…

Anti-tumoral effect of Δ 9…

Δ 9 -Tetrahydrocannabinol (Δ 9 -THC) and JWH-015 upregulate tribbles homolog 3 (TRB3),…

Schematic of the proposed mechanism of cannabinoid-induced hepatocellular carcinoma (HCC) cell death. Cannabinoid…

Δ 9 -Tetrahydrocannabinol (Δ 9…

Δ 9 -Tetrahydrocannabinol (Δ 9…

Δ 9 -Tetrahydrocannabinol (Δ 9 -THC) and JWH-015 reduce the growth of HepG2-…

Δ 9 -Tetrahydrocannabinol (Δ 9…

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.

Δ 9 -Tetrahydrocannabinol (Δ 9 -THC) and JWH-015 treatment induces autophagy in hepatocellular…

Anti-tumoral effect of Δ 9 -tetrahydrocannabinol (Δ 9 -THC) and JWH in an…

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 CaMKK β 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 CaMKK β . Of note, genetic or pharmacological blockade of CaMKK β 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 ).

2 Department of Biochemistry and Molecular Biology, School of Biology, Complutense University, and Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain.

Autophagy is required for the anti-tumoral action of Δ 9 -tetrahydrocannabinol (Δ 9 -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 mm 3 size, mice daily treated during 15 days with vehicle (control), 15 mg/kg Δ 9 -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 Δ 9 -THC (filled squares), 1.5 mg/kg JWH-015 (filled triangles), vehicle plus 1 mg/kg 3-MA (open circles), 15 mg/kg Δ 9 -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.

Activation of AMPK by cannabinoids relies on CAMKK.

Anti-tumoral effect of Δ 9 -tetrahydrocannabinol (Δ 9 -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 Δ 9 -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 3 α (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.

After different treatments according to the experiments cells were lysed in ice-cold lysis buffer (50 mM Tris (pH 7.4), 0.8 M NaCl, 5 mM MgCl 2 , 0.1% Triton X-100, 1 mM PMSF, 10  μ g/ml soybean trypsin inhibitor, 1  μ g/ml aprotinin and 5  μ g/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.

The authors declare no conflict of interest.

Two converging pathways have been described for AMPK regulation: one directed by LKB1, dependent on a change in cellular AMP, and another one directed by CaMKKs, dependent on changes in intracellular Ca 2+ . 34 The dramatic reduction in phospho-AMPK and phospho-ACC obtained upon silencing of CaMKK β indicates that this latter kinase rather than LKB1 is the dominant AMPKK enzyme in HCC cells in response to cannabinoids. Thus, cannabinoids induce autophagy in HCC cells, possibly by a two-pronged mechanism, one prong (similar to that operating in glioma cells) involving ER stress, TRB3 and Akt/mTORC1 inhibition, and another one reliant on AMPK stimulation via CaMKK β . A model of this mechanism of cannabinoid action in HCC cells is depicted in Figure 5 .

To further examine the role of autophagy on the anti-tumoral action of cannabinoids, another set of experiments was conducted to analyze the effect of cannabinoids on the growth of HepG2 tumor xenografts in which Atg5 expression had been knocked down in vivo . As shown in Figure 7a , Δ 9 -THC and JWH-015 failed to inhibit the growth of Atg5-silenced tumors, but not of those tumors that had been transfected with control siRNA. Furthermore, pharmacological inhibition of autophagy by using 3-MA prevented the decrease in tumor growth evoked by Δ 9 -THC and JWH-015 ( Figure 7b ). Taken together, these findings strongly support the fact that autophagy is necessary for the anti-tumoral action of cannabinoids in hepatocellular carcinoma.

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 Δ 9 -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.

Cell viability data were expressed as the mean±S.D. and evaluated by Student’s t -test. Differences were considered significant when the P -value was less than 0.05.

M Guzmán.

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′ CaMKK β sense primer, 5′-TCGAGTACTTGCACTGCCAGAAGATC-3′ CaMKK β 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.

Δ 9 -Tetrahydrocannabinol (Δ 9 -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 (CB 2 ) receptors. ( a ) Effect of Δ 9 -THC or JWH-015 (8 h) on the phosphorylation of eIf2 α in HepG2 and HuH7 cells. Tubulin levels are shown as a loading control. The image is representative of three different experiments ( b ) Effect of Δ 9 -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 Δ 9 -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 Δ 9 -THC (8  μ M), JWH-015 (8  μ M), 1  μ M SR141716A (SR1) or 2  μ M 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.

To investigate the activity of cannabinoids on HCC cells, we first analyzed the effect of Δ 9 -THC (a CB 1 /CB 2 receptor-mixed agonist that constitutes the main psychoactive ingredient of Canabis sativa ) and JWH-015 (a CB 2 receptor-selective agonist) on HepG2 (human hepatocellular liver carcinoma cell line) and HuH-7 (hepatocellular carcinoma cells) cells, two HCC lines that express CB 1 and CB 2 cannabinoid receptors (Supplementary Figure 1A). Treatment with Δ 9 -THC reduced the viability of HepG2 and HuH-7 cells, an event that was prevented by co-incubation with SR144528 (SR2, a CB 2 receptor-selective antagonist), but not with SR141716A (SR1, a CB 1 receptor-selective antagonist) (Supplementary Figure 1B). Likewise, JWH-015 decreased the viability of HCC cells, and co-incubation with SR2 abrogated this effect (Supplementary Figure 1B). These observations support that stimulation of CB 2 receptors is responsible for the decrease of cell viability triggered by cannabinoids on HCC cells.

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 Δ 9 -THC and JWH-015 activate a similar mechanism in HCC cells. Upon autophagy stimulation, the autophagy protein LC3 (microtubule-associated protein 1 light chain 3 α ) 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 Δ 9 -THC or JWH-015 ( Figure 1a ). Likewise, incubation of HepG2 and HuH-7 cells with Δ 9 -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 ).

Edited by M Piacentini.

Reagents.

To note, it has been recently described that mTOR signaling has a critical role in the pathogenesis of HCC and that mTOR inhibitors have antineoplastic activity in experimental models of HCC. 39 Moreover, decreased autophagy in HCC correlates with a more aggressive cancer cell phenotype and poor prognosis. 21, 40 Here we found that cannabinoid treatment reduces the growth of two different models of HCC subcutaneous xenografts in concert with decreased mTORC1 activation, enhanced AMPK phosphorylation and increased autophagy and apoptosis in those tumors. Moreover, knock down of the autophagic gene Atg5 as well as pharmacological inhibition of autophagy dramatically abolished the anti-tumoral activity of cannabinoids against subcutaneous HCC xenografts. Furthermore, Δ 9 -THC and JWH-015 efficiently reduced ascites development and AFP expression in an orthotopic model of HCC, which also paralleled mTORC1 inhibition, AMPK activation and autophagy stimulation in those tumors. Our data represent the first evidence for the antiproliferative action of cannabinoids in HCC cells in vivo and support that the ability of cannabinoids to inhibit mTORC1, stimulate AMPK and enhance autophagy could be therapeutically exploited for the management of HCC.

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.

Finally, we tested the anti-tumoral efficacy of cannabinoids in an orthotopic HCC model. HepG2 cells were inoculated in the liver of nude mice, and after 1 week, mice were treated intraperitoneally with vehicle (control), 15 mg/kg Δ 9 -THC or 1.5 mg/kg JWH-015 for 10 days. As shown in Figure 8a , cannabinoid treatment almost completely prevented hepatomegaly and ascites. Moreover, levels of the HCC tumor marker α -fetoprotein (AFP) were dramatically reduced in the livers of animals treated with Δ 9 -THC or JWH-015 ( Figure 8b ). Analysis of tumor samples revealed that cannabinoid treatment enhanced AMPK phosphorylation and inhibited Akt and S6 phosphorylation. Furthermore, THC and JWH-015 enhanced autophagy and apoptosis in these tumors ( Figure 8a ).

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 100 IU/ml penicillin G sodium, 100  μ g/ml streptomycin sulfate, 0.25  μ g/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.

In addition, it has been recently shown that AMPK binds to and directly phosphorylates the Ser/Thr kinase ULK1, the mammalian ortholog of the yeast protein kinase Atg1, and that this phosphorylation is required for ULK1-mediated autophagy. 35, 36 Thus, under certain cellular settings, mTORC1 inhibition and AMPK activation may cooperate to trigger autophagy. 36, 37, 38 Although future research is needed to completely clarify this point, our data suggest that this could be the mechanism by which cannabinoids trigger autophagy in HCC cells.

Δ 9 -Tetrahydrocannabinol (Δ 9 -THC) and JWH-015 activate adenosine monophosphate-activated kinase AMPK via CaMKK β . ( a ) Left panel: Effect of Δ 9 -THC (8  μ M) or JWH-015 (8  μ M) 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 Δ 9 -THC or JWH-015 (24 h) on the AMPK, ACC, Akt, S6 phosphorylation and microtubule-associated protein 1 light chain 3 α (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 Δ 9 -THC (8  μ M) or JWH-015 (8  μ M) on the viability (48 h, as determined by the MTT test) of HepG2 cells transfected with siC- or CaMKK β -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: CaMKK β 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 Δ 9 -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 Δ 9 -THC (8  μ M) or JWH-015 (8  μ M) on the viability (48 h, as determined by the MTT test) of HepG2 cells incubated in the presence or absence of the 10  μ M STO609 (STO; a CaMKK α / 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 Δ 9 -THC, JWH-015 and STO (10  μ M) 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)