Robert Ramer

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

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.

Cannabidiol inhibits lung cancer cell invasion and metastasis via intercellular adhesion molecule-1

Cannabinoids inhibit cancer cell invasion via increasing tissue inhibitor of matrix metalloproteinases‐1 (TIMP‐1). This study investigates the role of intercellular adhesion molecule‐1 (ICAM‐1) within this action. In the lung cancer cell lines A549, H358, and H460, cannabidiol (CBD; 0.001‐3 μM) elicited concentration‐dependent ICAM‐1 up‐regulation compared to vehicle via cannabinoid receptors, transient receptor potential vanilloid 1, and p42/44 mitogen‐activated protein kinase. Up‐regulation of ICAM‐1 mRNA by CBD in A549 was 4‐fold at 3 μM, with significant effects already evident at 0.01 μM. ICAM‐1 induction became significant after 2 h, whereas significant TIMP‐1 mRNA increases were observed only after 48 h. Inhibition of ICAM‐1 by antibody or siRNA approaches reversed the anti‐invasive and TIMP‐1‐up‐regulating action of CBD and the likewise ICAM‐1‐inducing cannabinoids Δ9‐tetrahydrocannabinol and R(+)‐methanandamide when compared to isotype or nonsilencing siRNA controls. ICAM‐1‐dependent anti‐invasive cannabinoid effects were confirmed in primary tumor cells from a lung cancer patient. In athymic nude mice, CBD elicited a 2.6‐ and 3.0‐fold increase of ICAM‐1 and TIMP‐1 protein in A549 xenografts, as compared to vehicle‐treated animals, and an antimetastatic effect that was fully reversed by a neutralizing antibody against ICAM‐1 [% metastatic lung nodules vs. isotype control (100%): 47.7% for CBD + isotype antibody and 106.6% for CBD + ICAM‐1 antibody]. Overall, our data indicate that cannabinoids induce ICAM‐1, thereby conferring TIMP‐1 induction and subsequent decreased cancer cell invasiveness.—Ramer, R., Bublitz, K., Freimuth, N., Merkord, J., Rohde, H., Haustein, M., Borchert, P., Schmuhl, E., Linnebacher, M., Hinz, B. Cannabidiol inhibits lung cancer cell invasion and metastasis via intercellular adhesion molecule‐1. FASEB J. 26, 1535‐1548 (2012). www.fasebj.org

COX-2 and PPAR-γ Confer Cannabidiol-Induced Apoptosis of Human Lung Cancer Cells

The antitumorigenic mechanism of cannabidiol is still controversial. This study investigates the role of COX-2 and PPAR-γ in cannabidiols proapoptotic and tumor-regressive action. In lung cancer cell lines (A549, H460) and primary cells from a patient with lung cancer, cannabidiol elicited decreased viability associated with apoptosis. Apoptotic cell death by cannabidiol was suppressed by NS-398 (COX-2 inhibitor), GW9662 (PPAR-γ antagonist), and siRNA targeting COX-2 and PPAR-γ. Cannabidiol-induced apoptosis was paralleled by upregulation of COX-2 and PPAR-γ mRNA and protein expression with a maximum induction of COX-2 mRNA after 8 hours and continuous increases of PPAR-γ mRNA when compared with vehicle. In response to cannabidiol, tumor cell lines exhibited increased levels of COX-2–dependent prostaglandins (PG) among which PGD2 and 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) caused a translocation of PPAR-γ to the nucleus and induced a PPAR-γ–dependent apoptotic cell death. Moreover, in A549-xenografted nude mice, cannabidiol caused upregulation of COX-2 and PPAR-γ in tumor tissue and tumor regression that was reversible by GW9662. Together, our data show a novel proapoptotic mechanism of cannabidiol involving initial upregulation of COX-2 and PPAR-γ and a subsequent nuclear translocation of PPAR-γ by COX-2–dependent PGs. Mol Cancer Ther; 12(1); 69–82. ©2012 AACR.

Antitumorigenic effects of cannabinoids beyond apoptosis

According to the World Health Organization, the cases of death caused by cancer will have been doubled until the year 2030. By 2010, cancer is expected to be the number one cause of death. Therefore, it is necessary to explore novel approaches for the treatment of cancer. Over past years, the antitumorigenic effects of cannabinoids have emerged as an exciting field in cancer research. Apart from their proapoptotic and antiproliferative action, recent research has shown that cannabinoids may likewise affect tumor cell angiogenesis, migration, invasion, adhesion, and metastasization. This review will summarize the data concerning the influence of cannabinoids on these locomotive processes beyond modulation of cancer cell apoptosis and proliferation. The findings discussed here provide a new perspective on the antitumorigenic potential of cannabinoids.

Latanoprost induces matrix metalloproteinase-1 expression in human nonpigmented ciliary epithelial cells through a cyclooxygenase-2-dependent mechanism

Prostaglandins (PGs) have been implicated in the regulation of intraocular pressure (IOP) by facilitating the remodeling of tissues involved in aqueous humor outflow. A contribution of cyclooxygenase‐2 (COX‐2)‐dependent PGs to this process was emphasized by a recent study showing an impaired COX‐2 expression in the nonpigmented ciliary epithelium (NPE) of patients with primary open‐angle glaucoma. With the use of human NPE cells (ODM‐2), the present study therefore investigated the effect of the antiglaucomatous drug latanoprost (PGF2α analog) on the expression of COX‐2 and its association with the induction of matrix metalloproteinases (MMPs). In NPE cells, latanoprost led to a concentration‐ and time‐dependent increase of COX‐2 mRNA levels. Up‐regulation of COX‐2 expression was accompanied by phosphorylations of p38 mitogen‐activated protein kinase (MAPK) and p42/44 MAPK and was abrogated by specific inhibitors of both pathways. PGE2 formation by latanoprost was abolished by the selective COX‐2 inhibitor NS‐398 and by the F‐prostaglandin receptor antagonist AL‐8810. Moreover, latanoprost led to a delayed up‐regulation of MMP‐1 mRNA, whereas the expression of MMP‐2, MMP‐9, TIMP‐1, and TIMP‐2 remained unchanged. Latanoprost‐induced MMP‐1 mRNA and protein expression was abolished by NS‐398 and by COX‐2‐silencing small‐interfering RNA. In line with this finding, MMP‐1 expression was also induced by PGE2, a major COX‐2 product. As a whole, our results show that MMP‐1 expression by latanoprost requires prior up‐regulation of COX‐2. Induction of COX‐2‐ and subsequent MMP‐1 expression in the NPE may represent a potential mechanism underlying the IOP‐lowering and antiglaucomatous action of latanoprost.

Aceclofenac spares cyclooxygenase 1 as a result of limited but sustained biotransformation to diclofenac

The mechanism of action of aceclofenac is currently unclear. This study investigated whether biotransformation to metabolites (4′‐hydroxy‐aceclofenac, diclofenac, 4′‐hydroxy‐diclofenac) contributes to inhibitory effects on the cyclooxygenase (COX) isozymes in vitro and ex vivo.

Up-regulation of cyclooxygenase-1 in neuroblastoma cell lines by retinoic acid and corticosteroids

Cyclooxygenases‐1 and ‐2 are both expressed in neuronal cells in vivo. In the neuroblastoma cell lines NG108 and N2a, however, only cyclooxygenase‐1 was detectable. Differentiation of the cells with retinoic acid increased cyclooxygenase‐1 mRNA and protein expression within 24 and 48 h, respectively. A further increase was observed when the cells were concomitantly treated with the glucocorticoid dexamethasone (a 2–3‐fold increase compared with retinoic acid alone). In the absence of retinoic acid, dexamethasone only slightly up‐regulated cyclooxygenase‐1 expression. The inhibitor of protein synthesis cycloheximide abrogated the effect of dexamethasone, indicating the involvement of newly synthesised proteins. Retinoic acid increased the transcription of cyclooxygenase‐1 mRNA, determined with a luciferase‐coupled promoter construct. Dexamethasone only slightly augmented cyclooxygenase‐1‐promoter activity but increased cyclooxygenase‐1 mRNA stability. Other corticosteroids, hydrocortisone and aldosterone, also up‐regulated cyclooxygenase‐1 whereas neurosteroids or oestrogen were ineffective. Up‐regulation was mediated primarily by the glucocorticoid receptor, because the receptor antagonist RU486 strongly reduced the effects of all corticosteroids. This indicated that in NG108 cells, the mineralocorticoid aldosterone may bind to the glucocorticoid receptor. Treatment of NG108 or N2a cells with corticosteroids did not alter the morphological phenotype obtained during differentiation. We thus show that corticosteroids, which down‐regulate cyclooxygenase expression in most cell types, up‐regulate cyclooxygenase‐1 during neuronal differentiation.

R(+)-Methanandamide and Other Cannabinoids Induce the Expression of Cyclooxygenase-2 and Matrix Metalloproteinases in Human Nonpigmented Ciliary Epithelial Cells

Prostaglandins (PGs) and matrix metalloproteinases (MMP) have been implicated in lowering intraocular pressure (IOP) by facilitating aqueous humor outflow. A possible role of cyclooxygenase-2 (COX-2) in this process was emphasized by findings showing an impaired COX-2 expression in the nonpigmented ciliary epithelium (NPE) of patients with primary open-angle glaucoma. Using human NPE cells, the present study therefore investigated the effect of the IOP-lowering cannabinoid R(+)-methanandamide [R(+)-MA] on the expression of COX-2 and different MMPs and tissue inhibitors of MMPs (TIMPs). R(+)-MA led to a concentration- and time-dependent increase of COX-2 mRNA expression. R(+)-MA-induced COX-2 expression was accompanied by time-dependent phosphorylations of p38 mitogen-activated protein kinase (MAPK) and p42/44 MAPK and was abrogated by inhibitors of both pathways. Moreover, R(+)-MA increased the mRNA and protein expression of MMP-1, MMP-3, MMP-9, and TIMP-1 but not that of MMP-2 and TIMP-2. Inhibition of COX-2 activity with NS-398 [N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide] was associated with a virtually complete suppression of R(+)-MA-induced MMP-9 and TIMP-1 expression. Consistent with these data, MMP-9 and TIMP-1 expression was also induced by PGE2, a major COX-2 product. Two other COX-2-inducing cannabinoids, anandamide and Δ9-tetrahydrocannabinol, caused the same pattern of MMP and TIMP expression as R(+)-MA both in the absence and presence of NS-398. Altogether, cannabinoids induce the production of several outflow-facilitating mediators in the human NPE. Our results further imply an involvement of COX-2-dependent PGs in MMP-9 and TIMP-1 expression. In conclusion, stimulation of intraocular COX-2 and MMP expression may represent a potential mechanism contributing to the IOP-lowering action of different cannabinoids.

R(+)-Methanandamide-Induced Apoptosis of Human Cervical Carcinoma Cells Involves A Cyclooxygenase-2-Dependent Pathway

PurposeCannabinoids have received renewed interest due to their antitumorigenic effects. Using human cervical carcinoma cells (HeLa), this study investigates the role of cyclooxygenase-2 (COX-2) in apoptosis elicited by the endocannabinoid analog R(+)-methanandamide (MA).MethodsCOX-2 expression was assessed by RT-PCR and Western blotting. PGE2/PGD2 levels in cell culture supernatants and DNA fragmentation were measured by ELISA.ResultsMA led to an induction of COX-2 expression, PGD2 and PGE2 synthesis. Cells were significantly less sensitive to MA-induced apoptosis when COX-2 was suppressed by siRNA or the selective COX-2 inhibitor NS-398. COX-2 expression and apoptosis by MA was also prevented by the ceramide synthase inhibitor fumonisin B1, but not by antagonists to cannabinoid receptors and TRPV1. In line with the established role of peroxisome proliferator-activated receptor γ (PPARγ) in the proapoptotic action of PGs of the D and J series, inhibition of MA-induced apoptosis was also achieved by siRNA targeting lipocalin-type PGD synthase (L-PGDS) or PPARγ. A role of COX-2 and PPARγ in MA-induced apoptosis was confirmed in another human cervical cancer cell line (C33A) and in human lung carcinoma cells (A549).ConclusionThis study demonstrates COX-2 induction and synthesis of L-PGDS-derived, PPARγ-activating PGs as a possible mechanism of apoptosis by MA.

Decrease of plasminogen activator inhibitor-1 may contribute to the anti-invasive action of cannabidiol on human lung cancer cells.

ABSTRACTPurposeUsing human lung cancer cells, we evaluated the involvement of plasminogen activator inhibitor-1 (PAI-1) in the anti-invasive action of cannabidiol, a non-psychoactive cannabinoid.MethodsInvasion was quantified by a modified Boyden chamber assay. PAI-1 protein in cell culture media and PAI-1 mRNA were determined by immunoblotting and RT-PCR, respectively.ResultsCannabidiol caused a profound inhibition of A549 cell invasion, accompanied by a decreased expression and secretion of PAI-1. Cannabidiols effects on PAI-1 secretion and invasion were suppressed by antagonists to CB1 and CB2 receptors as well as to transient receptor potential vanilloid 1. Recombinant human PAI-1 and PAI-1 siRNA led to a concentration-dependent up- and down-regulation of invasiveness, respectively, suggesting a crucial role of PAI-1 in A549 invasiveness. Evidence for a causal link between cannabidiols effects on PAI-1 and invasion was provided by experiments showing a reversal of its anti-invasive action by addition of recombinant PAI-1 at non-proinvasive concentrations. Key data were confirmed in two other human lung cancer cell lines (H460, H358). In vivo, a significant downregulation of PAI-1 protein by cannabidiol was demonstrated in A549 xenografts.ConclusionOur data provide evidence for a hitherto unknown mechanism underlying the anti-invasive action of cannabidiol on human lung cancer cells.