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-upregulating 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.

Besides their palliative benefits in cancer therapy, accumulating evidence suggests a potential advance of cannabinoids as anticancer agents. Accordingly, several investigations revealed antitumorigenic cannabinoid actions, such as inhibition of tumor cell proliferation (1, 2) and angiogenesis (3,–,5), as well as induction of apoptosis and autophagy (6,–,8). A possible clinical use of cannabinoids for the treatment of highly invasive cancer types is further supported by recent findings showing a decrease of tumor cell invasion by the phytocannabinoids Δ9-tetrahydrocannabinol (THC; refs. 9,–,11) and 2-[(1S,6S)-3-methyl-6-(prop-1-en-2-yl) cyclohex-2-enyl]-5-pentylbenzene-1,3-diol (CBD; cannabidiol; refs. 12,–,14), as well as by the hydrolysis stable anandamide analog R(+)-methanandamide (MA; ref. 11), the CB2 agonist JWH-133 (9), the endocannabinoid 2-arachidonyl glycerol (15), and the synthetic cannabinoid WIN55,212–2 (16).

Among cannabinoid-based drugs, CBD has raised particular interest due to its lack of adverse psychoactive effects that limit the clinical use of classic cannabinoids. Besides its beneficial effects on inflammation, pain, and spasticity when used for the treatment of multiple sclerosis (17), CBD has been reported to exert inhibitory effects on tumor angiogenesis (18) and metastasis (12, 13, 19) and to induce cancer cell apoptosis (20, 21). Concerning its anti-invasive mechanism, one study demonstrated an inhibitor of basic helix-loop-helix transcription factors, inhibitor of differentiation-1 (Id-1), to be involved in the anti-invasive effect of CBD on breast cancer cells (12). Recently, we were able to demonstrate an anti-invasive action of CBD on human lung and cervical carcinoma cells causally linked to up-regulation of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) via a mechanism involving activation of cannabinoid receptors and transient receptor potential vanilloid 1 (TRPV1) (13). In another study, this anti-invasive pathway was likewise elicited by THC and MA (11).

The present study focuses on the role of the intercellular adhesion molecule-1 (ICAM-1) in the antimetastatic action of CBD in vivo and the TIMP-1-inducing and anti-invasive action of CBD and other cannabinoids in vitro. ICAM-1, also referred to as CD54, is an inducible 80- to 110-kDa transmembrane glycoprotein belonging to the immunoglobulin superfamily. ICAM-1 is traditionally known to play a crucial role as an adhesion molecule in trafficking of inflammatory cells and in cell-to-cell interactions during antigen presentation (22). In addition, increasing evidence suggests a function for ICAM-1 in cellular signal transduction pathways by eliciting outside-in signaling (22). The presently available literature is controversial on whether up-regulation or suppression of ICAM-1 expression contributes to tumor progression and metastasis. On the one hand, increasing numbers of data suggest that enhanced ICAM-1 levels on cancer cells may be involved in tumor suppression via an immuno-surveillance mechanism. Accordingly, several studies revealed increased tumor susceptibility to lymphocyte adhesion and cell-mediated cytotoxicity following transfection (23, 24) or up-regulation of ICAM-1 (25, 26) or even under conditions of basal ICAM-1 expression (27). Vice versa, down-regulation of ICAM-1 by transforming growth factor β1 has been demonstrated to decrease both lymphocyte adhesion to cancer cells as well as cancer cell cytotoxicity (28). In line with this notion, ICAM-1 expression has been reported to be negatively correlated to metastasis of several cancer types in clinical studies (29,–,31). On the other hand, some studies using tumor necrosis factor (TNF) as ICAM-1 inductor or ICAM-1-overexpressing cells have suggested proinvasive properties for ICAM-1 (32,–,34). In addition, one investigation reported that the enhanced metastatic ability of TNF-treated malignant melanoma cells is reduced by ICAM-1 antisense oligonucleotides (35).

The current study was initiated on the basis of accidental findings showing a profound up-regulation of ICAM-1 in different non-small-cell lung cancer (NSCLC) cells by CBD, the above-mentioned anticarcinogenic properties of ICAM-1, and accumulating evidence pointing to a signaling function of ICAM-1 in eliciting cellular events, such as activation of mitogen-activated protein kinases (MAPKs) and activator protein-1 (AP-1) eventually leading to the production of various cytokines and adhesion molecules (22, 36,–,39). Consequently, an inductive action of ICAM-1 on the pivotal anti-invasive factor TIMP-1 that has likewise been shown to be induced on MAPK activation (11, 13) and to contain an AP-1 binding site in its promoter region (40, 41) appeared feasible.

Here, we demonstrate for the first time that increased ICAM-1 levels elicited by CBD result in a decrease of tumor cell invasion and metastasis. ICAM-1 up-regulation by CBD was observed in a panel of lung tumor cells, lung tumor xenografts, and primary tumor cells obtained from a brain metastasis of a patient with NSCLC. The functional importance of this finding was proven by experiments showing that de novo synthesis of ICAM-1 may represent a pivotal link between upstream cannabinoid-activated receptors and receptor-elicited p42/44 MAPK activation and downstream TIMP-1-dependent inhibition of invasion. Furthermore, to the best of our knowledge, this is the first study on the anti-invasive action of cannabinoids using human primary tumor cells and the first to provide an inhibitor-based antimetastatic mechanism of a cannabinoid in an in vivo model.


Materials
(−)-CBD was purchased from Tocris (Bad Soden, Germany). N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM-251), (6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl) (4-methoxyphenyl)methanone (AM-630), capsazepine, PD98059, and SB203580 were bought from Alexis Deutschland (Grünberg, Germany). Dulbecco's modified Eagle's medium (DMEM) with 4 mM L-glutamine and 4.5 mg/ml glucose was from Cambrex Bio Science Verviers S.p.r.l. (Verviers, Belgium). Fetal calf serum (FCS) and penicillin-streptomycin were obtained from PAN Biotech (Aidenbach, Germany) and Invitrogen (Karlsruhe, Germany), respectively. Dimethyl sulfoxide (DMSO), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), NaCl, ethylenediaminetetraacetic acid (EDTA), Triton X-100, and glycerol were bought from Applichem (Darmstadt, Germany). Phenylmethylsulfonyl fluoride (PMSF), leupeptin, and aprotinin were obtained from Sigma (Taufkirchen, Germany). Isotype control and neutralizing ICAM-1 antibodies for in vitro and in vivo experiments were purchased from R&D Systems (Minneapolis, MN, USA).

Cell culture
A549, H358, and H460 cells were maintained in DMEM with 4 mM L-glutamine and 4.5 mg/ml glucose supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were grown in a humidified incubator at 37°C and 5% CO2. All incubations were performed in serum-free medium. Phosphate-buffered saline (PBS) was used as a vehicle for the tested substances with a final concentration of 0.1% (v/v) ethanol (for all cannabinoids) or 0.1% (v/v) DMSO (for AM-251, AM-630, capsazepine, PD98059, and SB203580). Primary metastasizing lung tumor cells were obtained from resections of brain metastases of 2 patients with NSCLC (see Fig. 8 and Table 2). Samples from brain metastasis of lung carcinoma were excised, stored at 4°C in PBS, and immediately transferred to the laboratory. Samples were minced, and single-cell suspensions were generated. Data for patient 1 (see Fig. 8 and Table 2) were obtained from experiments with cells from a resection of a brain metastasis of a 67-yr-old male Caucasian with NSCLC. For these experiments, cells were passaged 5–7 times without intermediate freezing steps in DMEM containing 20% FCS and 100 U/ml penicillin and 100 μg/ml streptomycin. Experiments were performed using cells from passage 5–7 in serum-free DMEM after cells reached ∼70% confluence. Results for patient 2 (see Table 2) were obtained from Matrigel invasion assays using cells from a brain metastasis of a 47-yr-old female Caucasian with NSCLC at passage 0. All patients were informed about the establishment of cellular models from their individual tumors and gave informed consent in written form. The procedure was approved by the institutional ethics committee at the University of Rostock.

Matrigel invasion assay
The invasiveness of cells was quantified using a modified Boyden chamber technique with Matrigel-coated membranes (BD Biosciences, Oxford, UK), according to the manufacturer's instructions, as described recently (11, 13, 42). In this assay, cells must overcome a reconstituted basement membrane by proteolytic degradation of a Matrigel layer and active migration. In brief, the upper sides of the transwell inserts (8-μm pore size) were coated with 28.4 μg Matrigel/insert in a 24-well plate format. Cells were used at a final concentration of 5 × 105 cells/well in a volume of 500 μl serum-free DMEM in each insert and treated with test substances or vehicles for the indicated times. DMEM containing 10% FCS was used as a chemoattractant in the companion plate. Following incubation in a humidified incubator at 37°C and 5% CO2 for the indicated times, the noninvading cells on the upper surface of the inserts were removed with a cotton swab, and viability of invaded cells on the lower surface was measured by the colorimetric 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,6-benzene disulfonate (WST-1) test (Roche Diagnostics, Mannheim, Germany). For calculation of migration, the viability of cells on the lower side of uncoated invasion chambers was determined by the WST-1 test. Invasion was expressed as the invasion index, which is calculated as the absorbance at 490 nm of cells that invaded through Matrigel-coated Boyden chambers divided by absorbance of cells that migrated through uncoated control inserts with equal treatment [(invasion/migration)×100%].

Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis
Cells were seeded into 24-well plates at a density of 1 × 105 cells/well. Following incubation with the respective cannabinoid or its vehicle for the indicated times, cell culture media were removed, and cells were lysed for subsequent RNA isolation using the RNeasy total RNA Kit (Qiagen, Hilden, Germany). β-Actin (internal standard), ICAM-1, and TIMP-1 mRNA levels were determined by quantitative real-time RT-PCR, as described recently (43). Primers and probes for human β-actin, ICAM-1, and TIMP-1 were TaqMan Gene Expression Assays (Applied Biosystems, Darmstadt, Germany).

Western blot analysis
For determination of TIMP-1 protein levels (see Fig. 3), cells grown to confluence in 24-well-plates were incubated with test substances or vehicles. Afterward, cell culture media were centrifuged at 500 g and used for Western blot analysis. For all other blots, TIMP-1 was determined in cell culture media collected from the upper Boyden chambers at the end of the respective invasion experiment. Total protein in the cell culture medium was measured using the bicinchoninic acid assay (Pierce, Rockford, IL, USA). For analysis of ICAM-1 expression, cells grown in 24-well plates (see Figs. 1A, 4, and 6) or 6-well-plates (see Figs. 1B, C, 2, and 7) were incubated with test substances or vehicles. For Western blot analysis of p42/44 and phospho-p42/44 MAPK, cells grown to confluence in 6-well-plates were incubated with test substances or vehicle for the indicated times. In case of lysate analyses, cells were washed, harvested, lysed in solubilization buffer (50 mM HEPES, pH 7.4; 150 mM NaCl; 1 mM EDTA; 1% (v/v) TritonX-100; 10% (v/v) glycerol; 1 mM PMSF; 1 μg/ml leupeptin; and 10 μg/ml aprotinin), homogenized by sonication, and centrifuged at 10,000 g for 5 min. Supernatants were used for Western blot analysis.

All proteins were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel (Applichem). Following transfer to nitrocellulose and blocking of the membranes with 5% milk powder, blots were probed with specific antibodies raised to TIMP-1 (Oncogene Research Products, San Diego, CA, USA), ICAM-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p42/44 MAPK, or phospho-p42/44 MAPK (Cell Signaling Technology, Danvers, MA, USA). Membranes were probed with horseradish peroxidase-conjugated Fab-specific anti-mouse IgG for detection of TIMP-1 and ICAM-1 (New England BioLabs, Frankfurt, Germany) or anti-rabbit IgG (Cell Signaling Technology) for analysis of p42/44 MAPK activation. Densitometric analysis of band intensities was achieved by optical scanning and quantifying using the Quantity One 1-D Analysis Software (Bio-Rad, Munich, Germany). Vehicle controls were defined as 100% for evaluation of changes in protein expression. To ensure that equal amounts of protein in cell culture medium used for protein analysis of TIMP-1 had been transferred to the membrane, proteins on Western blot membranes were stained with Ponceau Red (Carl Roth, Karlsruhe, Germany). To ascertain equal protein loading in Western blots of cell culture medium, a band with a size of ∼65 kDa that appeared unregulated is shown as a loading control for protein analysis of cell culture medium. To ascertain equal protein loading in Western blots of cell lysates, membranes were probed with an antibody raised to β-actin (Calbiochem, Bad Soden, Germany).

siRNA transfections
Cells were transfected with small interfering RNA (siRNA) targeting the indicated sequence of ICAM-1 using RNAiFect as transfection reagent (Qiagen) or nonsilencing negative control RNA (Eurogentec, Seraing, Belgium). The target sequence of the ICAM-1 siRNA (Qiagen) was 5′-CGGCCAGCTTATACACAAGAA-3′. A BLAST search revealed that the sequence selected did not show any homology to other known human genes. Transfections were performed according to the manufacturer's instructions. For invasion assays, cells grown to confluence were transfected with 1.25 μg/ml siRNA or nonsilencing siRNA as negative control with an equal ratio (w/v) of RNA to transfection reagent for 24 h in medium supplemented with 10% FCS. Subsequently, cells were trypsinized, centrifuged at 200 g, resuspended to a final density of 5 × 105 cells in 500 μl of serum-free DMEM containing the same amounts of siRNA or nonsilencing siRNA to provide constant transfection conditions, and seeded for invasion analysis, as described above.

Induction of A549 xenografts in nude mice
All animal experiments were approved by the Animal Ethics Committee at the University of Rostock. Tumors were induced in NMRI mice (nu/nu; Charles River Laboratories, Frederick, MD, USA) by subcutaneous flank inoculation of 1 × 107 A549 lung tumor cells. Animals were assigned randomly to a vehicle and a CBD group and were injected intraperitoneally every 72 h with vehicle or CBD (5 mg/kg body wt). The treatment started 7 d after subcutaneous injection of tumor cells into the dorsal right side. After 29 d, animals were sacrificed, and tumors were explanted for protein analysis. Therefore, tissue parts were quick-frozen in liquid nitrogen. For Western blot analysis, homogenates were treated as described previously (14).

Mouse model of tumor metastasis
Athymic nude mice (NMRI-nu/nu; Charles River Laboratories) were given injections of A549 cells (1×106 cells/100 μl in PBS) through the lateral tail vein (d 1) and, after 24 h (d 2), were treated intraperitoneally with CBD (5 mg/kg body wt), vehicle, isotype control, or ICAM-1 antibody. Treatment protocols for the evaluation of the antimetastatic action of CBD in vivo revealed 5 mg/kg CBD as an appropriate dose for this purpose (13). CBD or its vehicle was administered every 72 h. Isotype control and ICAM-1 antibodies were used at a dose of 5 μg/mouse and were administered 1×/wk. Mice were sacrificed on d 28, and total lungs were evaluated for metastases nodules. To contrast lung nodules, lungs were fixed in Bouin's fluid (15 ml saturated picrinic acid, 5 ml formaldehyde, and 1 ml glacial acetic acid), and metastatic nodes were scored under a stereoscopic microscope in an investigator-blinded fashion. For histopathological examination, lung samples were fixed in 4% formalin. Paraffin sections were stained with hematoxylin and eosin. All experimental protocols for animal experiments were conducted in accordance with the policies of the Animal Ethics Committee at the University of Rostock.

Statistical analysis
Invasion indices and, mRNA and protein levels are indicated as means ± SE. Numbers of metastatic nodules are shown as box plots (whiskers: 5–95 percentile). Comparisons between groups were performed with Student's 2-tailed t test or with 1-way ANOVA plus Bonferroni test using GraphPad Prism 5.00 (GraphPad Software, San Diego, CA, USA). Results were considered to be statistically significant at values of P < 0.05.

Concentration dependence of CBD's action on ICAM-1 mRNA and protein expression
Treatment of A549, H358, and H460 cells with CBD caused a concentration-dependent increase of ICAM-1 mRNA levels following a 24-h incubation period (Fig. 1A–C, left panels) that was significant at concentrations as low as 0.01 μM in A549 (Fig. 1A, left panel), 0.001 in H358 (Fig. 1B, left panel) and 1 μM in H460 cells (Fig. 1C, left panel). Western blot analyses revealed a corresponding concentration-dependent ICAM-1 protein up-regulation by CBD in all three cell lines tested after a 48-h incubation (Fig. 1A–C, right panels).

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