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Cannabidiol inhibits cancer cell invasion via upregulation of tissue inhibitor of matrix metalloproteinases-1.

By October 31, 2013No Comments

pm2Cannabidiol inhibits cancer cell invasion via upregulation of tissue inhibitor of matrix metalloproteinases-1.

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Institute of Toxicology and Pharmacology, University of Rostock, Schillingallee 70, D-18057 Rostock, Germany.

Abstract

Although cannabinoids exhibit a broad variety of anticarcinogenic effects, their potential use in cancer therapy is limited by their psychoactive effects. Here we evaluated the impact of cannabidiol, a plant-derived non-psychoactive cannabinoid, on cancer cell invasion. Using Matrigel invasion assays we found a cannabidiol-driven impaired invasion of human cervical cancer (HeLa, C33A) and human lung cancer cells (A549) that was reversed by antagonists to both CB(1) and CB(2) receptors as well as to transient receptor potential vanilloid 1 (TRPV1). The decrease of invasion by cannabidiol appeared concomitantly with upregulation of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1). Knockdown of cannabidiol-induced TIMP-1 expression by siRNA led to a reversal of the cannabidiol-elicited decrease in tumor cell invasiveness, implying a causal link between the TIMP-1-upregulating and anti-invasive action of cannabidiol. P38 and p42/44 mitogen-activated protein kinases were identified as upstream targets conferring TIMP-1 induction and subsequent decreased invasiveness. Additionally, in vivo studies in thymic-aplastic nude mice revealed a significant inhibition of A549 lung metastasis in cannabidiol-treated animals as compared to vehicle-treated controls. Altogether, these findings provide a novel mechanism underlying the anti-invasive action of cannabidiol and imply its use as a therapeutic option for the treatment of highly invasive cancers.
Copyright 2009 Elsevier Inc. All rights reserved.
PMID:

 19914218
[PubMed – indexed for MEDLINE]

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Abbreviations

  • AM-251, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide;
  • AM-630, (6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl) (4-methoxyphenyl)methanone;
  • cannabidiol, 2-((1S,6S)-3-methyl-6-(prop-1-en-2-yl) cyclohex-2-enyl)-5-pentylbenzene-1,3-diol;
  • CB1,cannabinoid receptor 1;
  • CB2, cannabinoid receptor 2;
  • DMSO, dimethyl sulfoxide;
  • EDTA,ethylenediaminetetraacetic acid;
  • HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;
  • MAPK,mitogen-activated protein kinase;
  • MMP, matrix metalloproteinase;
  • PMSF, phenylmethylsulfonyl fluoride;
  • RT-PCR, reverse transcriptase-polymerase chain reaction;
  • siRNA, small-interfering RNA;
  • THC, Δ9-tetrahydrocannabinol;
  • TIMP, tissue inhibitor of matrix metalloproteinase;
  • TRPV1, transient receptor potential vanilloid 1;
  • WST-1, 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1.6-benzene disulfonate

Keywords

  • Cannabidiol;
  • Matrigel cell invasion;
  • Tissue inhibitor of matrix metalloproteinases-1;
  • Human cancer cells;
  • Experimental lung metastasis

Figures and tables from this article:

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Fig. 1.

Influence of cannabidiol (CBD) on HeLa cell invasion. (A) Time-dependency of Matrigel invasion of HeLa cells following stimulation of cells with 10 μM cannabidiol or vehicle over a 72-h incubation period. (B) Migration of HeLa cells through uncoated Boyden chambers after a 48- and 72-h stimulation with 10 μM cannabidiol or vehicle. (C) Concentration-dependency of cannabidiol’s anti-invasive action (black bars) and its impact on cell viability (open bars) after a 72-h incubation period. (D) Effect of a 1-h pretreatment with AM-251 (AM1; CB1 antagonist; 1 μM), AM-630 (AM2; CB2antagonist; 1 μM) and capsazepine (capsa; TRPV1 antagonist; 1 μM) on the anti-invasive action of cannabidiol (10 μM) after a 72-h incubation. Values are means ± SEM of n = 4 (A and B), n = 3–4 (C), and n = 7–8 (D) experiments. *P < 0.05; **P < 0.01; ***P < 0.001, vs. corresponding vehicle control; ###P < 0.001 vs. cannabidiol-treated cells, Student’s t-test.

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Fig. 2.

Impact of cannabidiol (CBD) on TIMP-1 expression. (A) Time-course of TIMP-1 mRNA normalized to β-actin. Cells were incubated with 10 μM cannabidiol or its vehicle over a 48-h incubation period. (B) Concentration-dependent effect of cannabidiol (0.01–10 μM) or its vehicle on TIMP-1, MMP-2 and MMP-9 protein levels following a 72-h incubation of cells. (C) Influence of a 1-h preincubation with AM-251 (AM1; CB1 antagonist; 1 μM), AM-630 (AM2; CB2 antagonist; 1 μM) and capsazepine (capsa; TRPV1 antagonist; 1 μM) on cannabidiol-mediated TIMP-1 induction after a 72-h incubation period. (D) Influence of receptor antagonists on TIMP-1 expression without cannabidiol treatment after a 72-h incubation period. mRNA data (A) are means ± SEM of n = 3 experiments. *P < 0.05; **P < 0.01, vs. corresponding vehicle control (Student’s t-test). Values above selected blots are means ± SEM obtained from densitometric analysis of n = 4 (B and C) or n = 3 (D) blots and represent percent control in comparison with vehicle-treated cells (100%) in the absence of test substance. Protein staining of supernatants is shown as loading control (LC).

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Fig. 3.

Role of TIMP-1 in cannabidiol’s (CBD) effect on HeLa cell invasion. (A) Effect of TIMP-1 siRNA (0.25 μg/ml) on cannabidiol’s action on cell invasion and TIMP-1 protein expression. HeLa cells were transfected with TIMP-1 siRNA at a final concentration of 0.25 μg/ml siRNA (si) or with non-silencing siRNA (nonsi) for 24 h. Subsequently, cells were placed into invasion chambers, retransfected with the indicated type of siRNA or suspension buffer to provide constant knockdown conditions and incubated with 10 μM cannabidiol or vehicle for a further 72 h. Protein staining of supernatants is shown as loading control (LC). (B) Microscopy of HeLa cells from different treatment groups that invaded through Matrigel-coated membranes. Cells were stained with Diff-Quick® (Medion Diagnostics GmbH, Büdingen, CH) and documented under a 200× magnification. Percent control (A) represents comparison with vehicle-treated cells (100%) in the absence of test substance. Values are means ± SEM of n = 4 experiments. ***P < 0.001, vs. corresponding vehicle control; ###P < 0.001, vs. cannabidiol (Student’s t-test).

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Fig. 4.

Role of p38 and p42/44 mitogen-activated protein kinase (MAPK) pathways in the effects of cannabidiol (CBD) on HeLa cell invasion and TIMP-1 expression. (A) Effect of a 1-h pretreatment with 10 μM SB203580 (SB; inhibitor of p38 MAPK activity) and 10 μM PD98059 (PD; inhibitor of p42/44 MAPK activation) on the anti-invasive action of cannabidiol (10 μM) after a 72-h incubation period. (B) Effect of the indicated treatment on TIMP-1 protein levels after a 72-h incubation period. Percent control represents comparison with vehicle-treated cells (100%) in the absence of test substance. Values are means ± SEM of n = 3–4 experiments (A), ***P < 0.001, vs. corresponding vehicle control; ###P < 0.001, vs. cannabidiol (Student’s t-test). Values above the representative blot are means ± SEM obtained from densitometric analysis of n = 7 blots (B) and represent percent control in comparison with vehicle-treated cells (100%) in the absence of test substance. Protein staining of supernatants is shown as loading control (LC). (C) Effect of cannabidiol on activation of p38 and p42/44 MAPK after 0.25–12 h. (D) Effect of a 1-h pretreatment with AM-251 (AM1; 1 μM), AM-630 (AM2; 1 μM), and capsazepine (Capsa; 1 μM) on phosphorylation of p38 and p42/44 MAPKs after a 2-h incubation with 10 μM cannabidiol. To analyze MAPK phosphorylations, immunoblots were probed with antibodies directed against the phosphorylated form of p38 or p42/44. Equal loading of lysates was ensured by probing membranes with antibodies against the nonphosphorylated forms of p38 and p42/44 MAPKs.

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Fig. 5.

Involvement of cannabinoid receptors, TRPV1, p38 and p42/44 MAPKs in the TIMP-1-dependent anti-invasive action of cannabidiol (CBD) on A549 cells. (A) Effect of a 1-h pretreatment of cells with AM-251 (AM1; CB1 antagonist; 1 μM), AM-630 (AM2; CB2 antagonist; 1 μM) and capsazepine (capsa; TRPV1 antagonist; 1 μM) on the anti-invasive (A, upper panel) and TIMP-1-inducing action of 10 μM cannabidiol (A, lower panel) after a 72-h incubation period. (B) Effect of a 1-h pretreatment with 10 μM SB203580 (SB; inhibitor of p38 MAPK activity) and 10 μM PD98059 (PD; inhibitor of p42/44 MAPK activation) on the anti-invasive action of 10 μM cannabidiol (B, upper panel) and on TIMP-1 protein levels (B, lower panel) after a 72-h incubation period. (C) Effect of TIMP-1 siRNA (0.25 μg/ml) on cannabidiol’s action on A549 cell invasion (C, upper panel) and TIMP-1 expression (C, lower panel) after a 72-h incubation period. Percent control represents comparison with vehicle-treated cells (100%) in the absence of test substance of n = 3–4 (A), and n = 4 (B and C) experiments. ***P < 0.001, vs. corresponding vehicle control; ###P < 0.001; ##P < 0.01, vs. cannabidiol (Student’st-test). Values above the representative TIMP-1 blots are means ± SEM obtained from densitometric analysis of n = 4 (A) or n = 5 (B) blots and represent percent control in comparison with vehicle-treated cells (100%) in the absence of test substance. Protein staining of supernatants is shown as loading control (LC).

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Fig. 6.

Involvement of cannabinoid receptors, TRPV1, p38 and p42/44 MAPKs in the TIMP-1-dependent anti-invasive action of cannabidiol (CBD) on C33A cells. (A) Effect of a 1-h pretreatment of cells with AM-251 (AM1; CB1 antagonist; 1 μM), AM-630 (AM2; CB2 antagonist; 1 μM) and capsazepine (capsa; TRPV1 antagonist; 1 μM) on the anti-invasive (A, upper panel) and TIMP-1-inducing action of 10 μM cannabidiol (A, lower panel) after a 72-h incubation period. (B) Effect of a 1-h pretreatment with 10 μM SB203580 (SB; inhibitor of p38 MAPK activity) and PD98059 (PD; inhibitor of p42/44 MAPK activation) on the anti-invasive action of 10 μM cannabidiol (B, upper panel) and on TIMP-1 protein levels (B, lower panel) after a 72-h incubation period. (C) Effect of TIMP-1 siRNA (0.25 μg/ml) on cannabidiol’s action on C33A cell invasion (C, upper panel) and TIMP-1 expression (C, lower panel) after a 72-h incubation period. Percent control represents comparison with vehicle-treated cells (100%) in the absence of test substance of n = 4 experiments. ***P < 0.001, vs. corresponding vehicle control; ###P < 0.001, vs. cannabidiol (Student’s t-test). Values above the representative TIMP-1 blots are means ± SEM obtained from densitometric analysis of n = 5 (A) or n = 3 (B) blots and represent percent control in comparison with vehicle-treated cells (100%) in the absence of test substance. Protein staining of supernatants is shown as loading control (LC).

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Fig. 7.

In vivo action of cannabidiol (CBD) on tumor metastasis. A549 cells were injected intravenously in athymic nude mice. Mice were given cannabidiol (5 mg/kg body weight) all 72 h for 28 days starting 24 h after injection of the cells. At the end of the experiment, the animals were sacrificed and one lung per animal was evaluated for metastatic lesions. (A) Left panel: number of metastatic nodules in vehicle- and cannabidiol-treated mice. Data are means ± SEM obtained fromn = 5 mice per group. Right panel: illustration of metastatic lesions in murine lungs from different experimental groups as indicated above. (B) Illustration of metastatic lesions in hematoxylin and eosin stained paraffine sections in murine lungs from vehicle- and cannabidiol-treated mice.

Table 1.Influence of cellular density on the viability of cannabidiol (CBD)-treated HeLa, A549 and C33A cells. Cells were seeded with the indicated number of cells per well of a 48-well plate in serum-free DMEM and treated with 10 μM cannabidiol or vehicle, respectively. After a 72-h incubation time, cellular viability was measured by WST-1. Values are means ± SEM of n = 4 experiments vs. corresponding vehicle control.
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Table 2.Involvement of cannabinoid receptors, TRPV1, p38 and p42/44 MAPKs and TIMP-1 in the anti-invasive action of 0.025 μM cannabidiol (CBD) on HeLa cells. Effect of a 1-h pretreatment of cells with AM-251 (CB1 antagonist; 1 μM), AM-630 (CB2 antagonist; 1 μM) and capsazepine (TRPV1 antagonist; 1 μM) on the anti-invasive action of 0.025 μM cannabidiol (upper set). Effect of a 1-h pretreatment with 10 μM SB203580 (inhibitor of p38 MAPK activity) and PD98059 (inhibitor of p42/44 MAPK activation) on the anti-invasive action of 0.025 μM cannabidiol (middle set), and effect of TIMP-1 siRNA (0.25 μg/ml) or non-silencing siRNA (0.25 μg/ml) on the anti-invasive action of 0.025 μM cannabidiol (lower set). Cells were incubated with cannabidiol for 72 h. Percent control represents comparison with vehicle-treated cells (100%) in the absence of test substance of n = 4 experiments.
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This study was supported by grants from the Deutsche Krebshilfe e. V. (Bonn, Germany), Deutsche Forschungsgemeinschaft (SFB 539 TP BI.6) and the FORUN programme of the Medical Faculty of the University of Rostock.
Corresponding author contact information
Corresponding author. Tel.: +49 381 4945770; fax: +49 381 4945772.

Copyright © 2009 Elsevier Inc. All rights reserved.
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