Novel hexahydrocannabinol analogs as potential anti-cancer agents inhibit cell proliferation and tumor angiogenesis

European Journal of Pharmacology 650 (2011) 64–71

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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r

Molecular and Cellular Pharmacology

Novel hexahydrocannabinol analogs as potential anti-cancer agents inhibit cell proliferation and tumor angiogenesis Dinesh Thapa a, Jong Suk Lee a, Se-Woong Heo a, Yong Rok Lee b,⁎, Keon Wook Kang c, Mi-Kyoung Kwak a, Han Gon Choi a,1, Jung-Ae Kim a,⁎ a b c

College of Pharmacy, Yeungnam University, Gyeongsan 712-749, South Korea School of Chemical Engineering and Technology, Yeungnam University, Gyeongsan 712-749, South Korea College of Pharmacy, Chosun University, Gwangju 501-759, South Korea

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Article history: Received 25 May 2010 Received in revised form 16 September 2010 Accepted 23 September 2010 Available online 13 October 2010 Keywords: Synthetic hexahydrocannabinol Anti-angiogenesis Cell proliferation NF-κB Tamoxifen-resistant MCF-7 breast cancer

a b s t r a c t Both natural and synthetic cannabinoids have been shown to suppress the growth of tumor cells in culture and in animal models by affecting key signaling pathways including angiogenesis, a pivotal step in tumor growth, invasion, and metastasis. In our search for cannabinoid-like anticancer agents devoid of psychoactive side effects, we synthesized and evaluated the anti-angiogenic effects of a novel series of hexahydrocannabinol analogs. Among these, two analogs LYR-7 [(9S)-3,6,6,9-tetramethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol] and LYR-8 [(1-((9S)-1-hydroxy-6,6,9-trimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-2-yl)ethanone)] were selected based on their anti-angiogenic activity and lack of binding affinity for cannabinoid receptors. Both LYR-7 and LYR-8 inhibited VEGF-induced proliferation, migration, and capillary-like tube formation of HUVECs in a concentration-dependent manner. The inhibitory effect of the compounds on cell proliferation was more selective in endothelial cells than in breast cancer cells (MCF-7 and tamoxifen-resistant MCF-7). We also noted effective inhibition of VEGF-induced new blood vessel formation by the compounds in the in vivo chick chorioallantoic membrane (CAM) assay. Furthermore, both LYR analogs potently inhibited VEGF production and NF-κB transcriptional activity in cancer cells. Additionally, LYR-7 or LYR-8 strongly inhibited breast cancer cellinduced angiogenesis and tumor growth. Together, these results suggest that novel synthetic hexahydrocannabinol analogs, LYR-7 and LYR-8, inhibit tumor growth by targeting VEGF-mediated angiogenesis signaling in endothelial cells and suppressing VEGF production and cancer cell growth. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Cannabinoids exert anti-proliferative actions on a wide spectrum of tumor cells in culture and in animal models by inducing direct growth arrest and death of tumor cells and by inhibiting tumor angiogenesis and metastasis (Galve-Roperh et al., 2000; Guzman et al., 2002; Guzman, 2003). However, the potential development of cannabinoids as anti-cancer drugs has been severely restricted because of their undesired psychoactive properties. In particular, activation of the central cannabinoid receptors (cannabinoid CB1 receptors), which are primarily found in the brain, is linked to psychoactivity. On the other hand, peripheral cannabinoid receptors (cannabinoid CB2 receptors) are almost exclusively found in the immune system. Selective CB2 agonists without psychoactivity exhibit other side-effects such as immune suppression (Pertwee, 2005; Zhu et al., 2000). Therefore, the alternative use of such cannabinoids in ⁎ Corresponding authors. Tel.: +82 53 810 2816; fax: +82 53 810 4654. E-mail addresses: [email protected] (Y.R. Lee), [email protected] (J.-A. Kim). 1 Current address: College of Pharmacy, Hanyang University, 1271, Sa-3-Dong, Ansan 426-791, South Korea. 0014-2999/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2010.09.073

cancer treatment is best supported by the use of cannabinoids with very weak binding or no affinity to CB receptors. Angiogenesis is a crucial regulator of tumor growth and metastases (Folkman, 1995). Tumor angiogenesis is regulated by the production of angiogenic stimulators including vascular endothelial growth factor (VEGF), which is a key regulatory factor in the prognosis of various cancers. Therefore, inhibition of VEGF production is a promising therapeutic approach for cancer treatment. One of the recent major clinical advances in cancer treatment is the use of antiangiogenic drugs such as bevacizumab, sorafenib, and sunitinib. Bevacizumab, the monoclonal anti-VEGF antibody, combined with taxane has been approved for the first-line treatment of metastatic breast cancer (Kerbel, 2009). Sunitinib, VEGF receptor tyrosine kinase inhibitor, is another approach for anti-angiogenic therapy and acts directly on endothelial cells. Several class of cannabinoids have been shown to suppress tumor growth either by inhibiting proangiogenic factor production (Casanova et al., 2003; Blazquez et al., 2004; Preet et al., 2008) or by directly inducing apoptosis of vascular endothelial cells (Kogan et al. 2006). In the present study, we examined whether novel synthetic hexahydrocannabinol analogs could inhibit tumor-angiogenesis

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through the suppression of VEGF in cancer cells and VEGF-mediated signaling in endothelial cells. Since tamoxifen-resistant MCF-7 (TAMR-MCF-7) cells have shown a strong association between enhanced VEGF production and more aggressive phenotype (Kim et al., 2008; Kim et al., 2009), we used this cell line as the model cancer cell line for the tumor-angiogenesis study. The cancer cell-inoculated CAM assay was used to mimic the tumor microenvironment; this assay has been widely used to evaluate not only angiogenesis, but also tumor-induced angiogenesis and metastasis (Ribatti et al., 2001; Tufan and Satiroglu-Tufan, 2005). 2. Materials and methods 2.1. Reagents and antibodies Recombinant human vascular endothelial growth factor (VEGF) was purchased from R&D Systems (Minneapolis, MN, USA). Endothelial growth medium (EGM)-2 bullet kit, which contains endothelial cell basal medium (EBM)-2 and EGM-2 SingleQuots (hydrocortisone, hFGF, VEGF, R3-IGF-1, ascorbic acid, hEGF, heparin, gentamicin and FBS), was purchased from Cambrex (San Diego, CA, USA). Matrigel was obtained from BD Biosciences (San Jose, CA, USA). HEPESbuffered saline solution, Trypsin/EDTA, and Trypsin Neutralizing solution (TNS) were purchased from Clonetics, Inc. (Walkersville, MD, USA). Triton-X-100, bovine serum albumin (BSA), 3-(4,5dimethylthiazol-2-yl)-2,5-di-phenyl tetrazolium bromide (MTT), mitomycin C, sodium pyruvate, dimethyl sulfoxide (DMSO), protease inhibitor cocktail, sodium dodecyl sulfate (SDS) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Cannabinoid CB1 receptor antagonist AM281 [N-(morpholin-4-yl)-5-(4-iodophenyl)-1-(2,4dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] and cannabinoid CB2 receptor antagonist AM630 (6-iodo-pravadoline) were purchased from Tocris Bioscience (Bristol, UK). The bicinchoninic acid (BCA) protein assay reagents and the chemiluminescent substrate (ECL kit) for horseradish peroxidase were from Pierce Biotechnology (Rockford, IL, USA). Primary antibodies specific for VEGF and actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 2.2. Chemical synthesis and treatment of LYR analogs LYR analogs were synthesized according to the method described previously (Lee and Xia, 2008). The chemical structures are shown in Fig. 1A. A 50 mM stock solution of LYRs were prepared in DMSO, stored at −20 °C, and then diluted as needed. For in vitro incubations, LYRs were directly applied at a final DMSO concentration of 0.1–0.2% (v/v). For in vivo experiments (CAM tumor implantation), LYRs were prepared at 1% (v/v) DMSO in phosphate buffered saline (PBS) supplemented with 0.1% BSA. No significant influence of the vehicle was observed on any of the parameters assessed. 2.3. Cell culture Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza Walkersville, Inc. HUVECs were maintained in cell culture flasks coated with 0.2% gelatin and cultured with EBM-2 containing hydrocortisone, human basic fibroblast growth factor, VEGF, R3-IGF-1, ascorbic acid, human epidermal growth factor, heparin, gentamicin, and 2% FBS. Confluent cultures of HUVECs were serially passaged and were used between passages 2 and 6. MCF-7 (human breast cancer cell line) cells were obtained from American Type Culture Collection (Manassas, VA, USA). Tamoxifen (TAM)-resistant breast cancer cell line (MCF-7/TAMR cells) was developed as described earlier (Kim et al., 2009). Both of these cell lines were grown in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. HT29 human colon cancer cells were maintained in RPMI 1640 medium. All cells were maintained at 37 °C in a

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5% CO2-humidified atmosphere. The culture medium was replaced every other day. 2.4. Tube formation assay Tube formation assays were performed on 48-well plates coated with 100 μl of Matrigel per well and polymerized at 37 °C for 30 min. HUVECs were suspended in EBM-2 containing 1% FBS, plated on Matrigel at a density of 1 × 104 cells per well, and then co-treated with VEGF (20 ng/ml) and indicated concentrations of LYR analogs. After 14 h, four fields of each culture well were randomly selected and photographed with a microscope attached to a CCD camera (TE2000-U; Nikon). Digital images were analyzed with an image analysis system (ImageInside Ver 3.32) for the quantitation of tube length. 2.5. Migration assays Two types of cell migration assays were performed with HUVECs. First, cell migration was examined in scratch assays as described previously (Park et al., 2007). Confluent HUVECs were pretreated with mitomycin C (25 μg/ml) for 30 min and then a scratch line was made in the cell monolayer. After being rinsed with Dulbecco's phosphate buffered saline (DPBS), the cells were further incubated in EBM-2 medium (1% FBS) supplemented with 20 ng/ml VEGF in the presence of LYR-7 or LYR-8 for 10 h. Pictures of the scratches were taken using a digital camera system (Nikon, Japan) connected to a light microscope (Olympus). For modified Boyden chamber migration assay, HUVECs were cultured onto gelatin-coated 8-μm pore size chambers (Corning, NY, USA), and the bottom well was filled with EBM-2 medium containing VEGF (20 ng/ml) as the chemoattractant. 8 h after incubating chambers, cells were fixed with methanol and stained with H&E. Migrating cells were imaged (100×) and counted using a microscope connected to a digital camera. 2.6. Cell viability assay The cytotoxicity of LYR analogs in MCF-7 and TAMR-MCF-7 breast cancer cells was measured using the MTT assay (Carmichael et al., 1987). Briefly, cells (1 × 104 cells/well) were seeded in 96-well microtiter plates (Nunc, Denmark). After 24 h, fresh medium (DMEM supplemented with 10% FBS) containing indicated concentrations of LYR analogs or DMSO vehicle was replaced and incubated for 48 h. Relative cell viability was determined by the amount of MTT converted to formazan salt and expressed as a percent of the control culture. 2.7. HUVEC proliferation assay HUVECs plated at a density of 1 × 104 cells/cm2 were incubated in serum-reduced (0.2% FBS) EBM-2 for 24 h and then co-treated with VEGF (20 ng/ml) in the absence or presence of LYR-7 or LYR-8 for 48 h. After incubation, the viable cell number was determined by MTT assay. 2.8. VEGF enzyme-linked immunosorbent assay Secreted VEGF levels were determined by using a Quantikine human VEGF ELISA kit (R&D Systems, Minneapolis, MN, USA) as described previously (Kim et al., 2008; Park et al., 2009). In brief, MCF-7 cells were seeded in 24-well plates and grown to 80–90% confluence. The cells were switched to fresh serum-free medium in the presence or absence of LYR analogs and incubated for 18 h. After the treatment, the supernatants were collected and the cells were subjected to the MTT assay to measure relative cell viability. The concentration of VEGF in the unknown samples was then determined by comparing the optical

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Fig. 1. LYR analogs inhibit tube formation of HUVECs. (A) LYR analogs (LYR-1 to LYR-8) are structurally related to classical cannabinoid Δ9-tetrahydrocannabinol (THC). Chemical structure of Δ9-THC is shown in the box. (B) HUVECs (1 × 104) were plated in a well coated with Matrigel basement membrane matrix. Cells were treated with 5 μM LYR analogs in the presence of VEGF (20 ng/ml). After 14 h, cells were photographed with a digital camera under a phase contrast microscope at 100× magnification. (C) Experiment was carried out as described above with the indicated concentrations of LYR-7 or LYR-8 for 14 h. (D) Total tube length per field was measured by ImageInside software. The bar graph shows the means ± S.E.M. of the experiment carried out in triplicate. *P b 0.05, compared to VEGF-stimulated control group.

density of the samples to the standard curve and normalized to the cell viability in each well. 2.9. Protein extraction and Western blotting Whole cell lysates were prepared using RIPA buffer. Protein content was measured with BCA protein assay reagent (Pierce, Rockford, IL, USA). Equal amounts of total protein were separated by SDS-PAGE and transferred onto Hybond ECL nitrocellulose membranes (Amersham Life Science, Buckinghamshire, UK) at 200 mA for an hour. The membranes were blocked in 5% skim milk in Tris-buffered saline (TBS)-Tween 20 (TBS-T) at room temperature for 1 h followed by incubation with specific antibodies in skim milkTBS at 4 °C overnight. Then, the membrane was washed three times

with TBS-T and incubated with horseradish peroxidase-conjugated secondary antibody in skim milk-TBS for 1 h at room temperature. The immunoreactive proteins were visualized using an ECL kit (Pierce) and digitally processed using LAS-4000 mini (Fuji, Japan). Membranes were stripped and reprobed with an actin antibody for loading control. Densitometric analysis of the blots was performed with Multi Gauge Ver 3.2 imaging software in a Fuji Image Station. 2.10. Transient transfection and luciferase assay Transient transfections were performed using the GeneJammer transfection reagent (Stratagene, La Jolla, CA, USA) as described previously (Thapa et al., 2008). Briefly, HT29 cells were transfected with plasmid mixtures containing 0.6 μg of NF-κB-luc (Panomics,

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Fremont, CA, USA) and 0.05 μg of pRL-TK. The transfected cells were pretreated with LYR analogs for 1 h, followed by tumor necrosis factor (TNF)-α for 3 h. The cell lysates were used for luciferase assay using a dual luciferase reporter assay kit (Promega, Madison, WI, USA), and the emitted light was measured with a luminometer (Turner BioSystems, Sunnyvale, CA, USA). 2.11. In vivo chicken chorioallantoic membrane (CAM) assay Fertilized eggs were purchased from Baek-ja Farm (Cheongsong, Korea) and the CAM was prepared as described previously (Park et al., 2009). VEGF was used to stimulate vessel growth on the CAMs of 9day-old chick embryos. Sterile filter disks absorbed with VEGF (20 ng/ disk) dissolved in PBS containing 0.1% BSA were placed on the growing CAMs. Test compounds or a vehicle was then added directly to CAMs topically. After 72 h, CAM tissue directly beneath the disk were resected from the embryo and harvested under light microscopy (Leica, Wetzlar, Germany). The number of vessel branch points contained in a circular region equal to the area of filter disk was then counted for each section. In the tumor angiogenesis experiments, all the procedures were the same as above except that TAMR-MCF-7 or MCF-7 human breast cancer cells (1.5 × 106 cells/CAM) were inoculated onto the CAM instead of VEGF (Kim et al., 2008, 2009). The number of vessel branch points contained in a tumor region was counted by two observers in a double-blind manner. 2.12. Statistical analysis The data are presented as means ± S.E.M. Statistical analysis was done with a Student's t test or one-way ANOVA followed by the Student–Newman–Keuls comparison (GraphPad Prism4.0 software,

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San Diego, CA, USA) to calculate differences between groups. P values of b0.05 were considered statistically significant. 3. Results 3.1. Anti-angiogenic activities of hexahydrocannabinol analogs: inhibition of HUVEC tube formation, migration, and invasion From a novel series of synthetic hexahydrocannabinols that are structurally similar to natural cannabinoid THC, eight analogs named LYR-1 to LYR-8 (Fig. 1A) were screened for anti-angiogenic properties. The tube formation assay was employed for preliminary screening because it is one of the most popular in vitro angiogenesis tests (Vernon et al., 1992). Endothelial cells were plated on a Matrigel-coated surface in the presence of VEGF, a well-known factor that induces endothelial cell growth and is widely expressed in most cancers (Ferrara and Kerbel, 2005). As shown in Fig. 1B, the cells differentiate and align to form a network of tubes, i.e., capillary-like structures. All the LYR analogs (5 μM) tested caused a blockage of the in vitro endothelial tube formation. Among these, LYR-7 and LYR-8 were chosen for further experiments because they strongly inhibited angiogenesis while having very little or no affinity for cannabinoid receptor CB1 or CB2 (Supplementary Fig. S1; Thapa et al., 2010), thus eliminating the concern about potential psychoactive side effects. Further study with LYR-7 and LYR-8 showed that such inhibitory effects of the two analogs were concentration-dependent (Fig. 1C and D). As endothelial cell migration is another important step of angiogenesis (Park et al., 2006), wound-healing migration assay was performed to determine the effects of LYR-7 and LYR-8 on HUVEC migration. LYR analogs significantly delayed VEGF-induced migration at 10 h after HUVEC wound injury (Fig. 2A). These effects were concentration-dependent, and significant inhibitory activity was seen at 1 μM (Fig. 2A, right panel). Similarly, in a subsequent Transwell

Fig. 2. LYR-7 and LYR-8 inhibit migration and invasion of HUVECs. (A) Scratch wound migration of HUVECs: inhibitory effect of LYR-7 and LYR-8 on VEGF-induced HUVEC migration. Inactivated HUVECs were subjected to wound-healing migration assays and the migrating cells were counted using ImageInside software. Experiment was performed in triplicate. (B) Chemotactic migration in Transwell: effect of LYR analogs on VEGF-induced HUVEC migration in the Transwell assay. Red cells with irregular shape were migrating cells attached on the outside surface of the top chamber. (A–B) The bar graphs in the right panel show the summary of quantitative results of the average number of migrating HUVECs ± S.E.M. *P b 0.05, compared to VEGF-stimulated control group.

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assay (Fig. 2B), both analogs significantly inhibited chemotactic migration of HUVECs. 3.2. Anti-proliferative activities of LYR-7 and LYR-8 against endothelial and cancer cells We also examined the inhibitory action of the analogs against the proliferation of endothelial cells. LYR-7 and LYR-8 significantly inhibited VEGF-induced HUVEC proliferation with an IC50 of 8 μM (Fig. 3A). Since primary HUVECs are reported to express CB receptors (Blazquez et al., 2003) and the analog LYR-7 shows little affinity for CB receptors (Supplementary Fig. S1), we examined the involvement of

CB receptors in such action of the two analogs. Preincubation of HUVECs with AM281, a selective cannabinoid CB1 receptor antagonist (1 μM), or AM630, a selective cannabinoid CB2 receptor antagonist (1 μM), did not alter the inhibitory effects of LYR analogs on VEGFstimulated HUVEC proliferation (Fig. 3B). Next, we examined the selectivity of the LYR analogs toward endothelial cells by comparing cell viability changes in the analog-treated endothelial cells and cancer cells. Under normal HUVEC culture conditions (2% serum and other growth supplements), LYR analogs inhibited endothelial proliferation in a concentration-dependent manner (Fig. 3C and D). Much higher concentrations of the LYR analogs were needed to inhibit the proliferation of MCF-7 breast cancer cells than of endothelial cells.

Fig. 3. Inhibitory effects of LYR-7 and LYR-8 on growth factor-induced cell proliferation and TNF-α-induced NF-κB transcriptional activity. (A) HUVECs (1 × 104 per well in a 48 wellplate) were starved with 0.2% FBS medium and then treated with VEGF (20 ng/ml) and different concentrations of LYR analogs for 48 h. Cell viability was quantified by MTT assay. Values are means ± S.E.M. of eight measurements. *P b 0.05 versus VEGF alone. (B) HUVECs were cultured and stimulated with VEGF as described above. AM281 (1 μM) and AM630 (1 μM) were pre-treated for 1 h before the LYRs (5 μM) and VEGF co-treatment. Cell viability was quantified by MTT assay. Values are means ± S.E.M. of eight measurements. # P b 0.05 versus vehicle-treated control. *P b 0.05 versus VEGF alone. (C–D) The effects of LYR analogs on serum-treated proliferation of HUVECs, MCF-7 and TAMR-MCF-7 cells: The treatment is the same as in A and B. The data points represent three experiments performed in triplicate. (E) NF-κB-luc and pRL-TK were introduced into HT29 cells using GeneJammer reagent. Cells were then pre-treated with or without LYR analogs (5 μM) for 1 h, followed by TNF-α (10 ng/ml) for 3 h. Luciferase activity was measured and expressed as relative luciferase units (RLU) (firefly luciferase/Renilla luciferase). Bar graphs show the means ± S.E.M. of three experiments. #P b 0.05 versus vehicle-treated control. *P b 0.05 versus TNF-α-stimulated group.

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More importantly, the effects of the two compounds on chemoresistant TAMR-MCF-7 cell proliferation were similar to those observed in parent MCF-7 cells (Fig. 3C and D). Since there are many reports highlighting the potential role of NF-κB in angiogenesis and its blockade resulted in impaired tumor angiogenesis (Huang et al., 2001; Rius et al., 2008; Schmidt et al., 2007), we also examined whether the compounds had NF-κB inhibitory activity using NF-κB-luciferase reporter assay in HT29 cancer cells. NF-κB transcriptional activity, which was stimulated by TNF-α, was strongly inhibited by LYR-7 and LYR-8 among the other analogs (Fig. 3E). 3.3. LYR analogs inhibit angiogenesis in vivo To confirm the anti-angiogenic effects of LYR-7 and LYR-8 in vivo, CAM assays were performed at different concentrations. As shown in Fig. 4A and B, LYR-7 or LYR-8 at 0.5 μg/CAM strongly inhibited VEGFinduced angiogenesis, and 5 μg/CAM LYR-7 or LYR-8 almost completely abolished VEGF-induced angiogenesis in the CAM assays, indicating that these analogs effectively inhibited angiogenesis in vivo. 3.4. LYR analogs inhibit VEGF expression in breast cancer cells VEGF production by tumor cells under hypoxic conditions is believed to be one of the most specific and critical regulators of angiogenic signaling cascades (Ferrara, 2002). In breast cancer cells, increased levels of VEGF are correlated with aggressive, metastatic, and drug-resistant phenotypes (Marson et al., 2001). TAMR-MCF-7 is one of such phenotypes (Kim et al., 2009). In the cells, LYR analogs significantly suppressed VEGF secretion (Fig. 5A) and protein expression (Fig. 5B and C) in a concentration-dependent manner. 3.5. LYR analogs inhibit tumor angiogenesis and breast cancer growth To investigate whether LYR analogs inhibit tumor-induced angiogenesis in vivo, the cancer-implanted CAM model was used. TAMR-MCF-7 and MCF-7 cancer cell suspensions with or without LYR analogs were seeded onto the chorioallantoic membrane of 9-day-old chick embryos. New blood vessel formation and tumor growth on the membrane after 5 days of incubation were analyzed. As shown in Fig. 6A and B, the cancer cell-implanted tumors induced profuse vasculature radiating from cell masses. Notably, TAMR-MCF-7 cellinduced angiogenesis was about 5-fold higher than that in the control group treated with PBS and about 2-fold higher than that in the recombinant human VEGF-treated group (Fig. 6C). Consistent with the previous finding (Kim et al., 2009), tumor vasculature density was significantly higher in TAMR-MCF-7 cell-implanted CAMs than in MCF-7 cell-implanted CAMs. Such cancer cell-induced angiogenesis was significantly suppressed by LYR-7 or LYR-8 treatment. Although we did not dissect out the tumor mass from the membranes, the visible tumor masses were notably diminished by LYRs treatment (Fig. 6A and B; indicated by dot circles). 4. Discussion In this study, hexahydrocannabinols, novel synthetic analogs that are structurally related to the cannabinoids, were identified as potent angiogenesis inhibitors. We have demonstrated that, among the hexahydrocannabinol analogs, LYR-7 and LYR-8 most effectively (a) inhibit endothelial and tumor cell growth; (b) inhibit VEGFinduced angiogenesis in vitro and in vivo, and the growth of tumors inoculated on chick chorioallantoic membrane; and (c) block the secretion of VEGF in cancer cells. Angiogenesis is a complex multistep process involving endothelial cell proliferation, invasion, chemotactic migration, differentiation into tube-like structures, and the production of a basement membrane around the vessel (Folkman, 1995; Kesisis et al., 2007). Using various

Fig. 4. LYR-7 and LYR-8 inhibit the VEGF-induced angiogenesis in vivo. (A) VEGF (20 ng/CAM) or vehicle (0.1% BSA in PBS) was loaded onto a dried cortisone-saturated filter disk placed on an avascular area of the CAM. The disks were treated with various concentrations of LYR-7 or LYR-8. After 72 h incubation, the CAM membrane was resected and imaged under microscope. (B) Quantitation of new branches formed from existing blood vessels: Photographs were imported into imaging software to quantitate the number of new branches formed. The bar graph represents the mean number of branch points±S.E.M. of at least six chick embryos. #Pb 0.05, compared to PBS-treated control. *Pb 0.05, compared to VEGF-treated group.

in vitro and in vivo experiments, we clearly showed that LYR analogs could inhibit these multistep processes of angiogenesis. Unlike other conventional cannabinoids, which have been linked to potential psychoactive properties via activation of cannabinoid CB1 receptor, our previous (Thapa et al., 2010) and the present study (Supplementary Fig. S1) revealed that these two compounds have little or no affinity for CB receptors in a receptor binding assay. The CB receptor involvement in the anti-cancer action of cannabinoids depends on the nature of cannabinoid. Cannabidiol (CBD), one of the bioactive constituents of marijuana, exhibits anti-tumor activities through a cannabinoid receptor-independent mechanism (Massi et al., 2004; Vaccani et al., 2005), whereas the anti-tumor activities of THC are associated with both cannabinoid CB1 and CB2 receptors (Sanchez et al., 2001; Ramer and Hinz, 2008; Sarfaraz et al., 2008). In the present study, neither the CB1 antagonist AM281 nor the CB2 antagonist AM630 prevented the LYR-7- or LYR-8-induced inhibition of cell proliferation (Fig. 3B) and tube formation (data not shown). These findings along with poor or non-receptor affinity of both LYR analogs indicate that the anti-angiogenic activity of both LYR analogs seems to be mediated through a cannabinoid receptor-independent mechanism. Since LYR-7 and LYR-8 strongly inhibit NF-κB transcriptional activity and NF-κB plays a well-known role in VEGF regulation

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Fig. 5. LYR-7 and LYR-8 inhibit VEGF expression in TAMR-MCF-7 cells. (A) TAMR-MCF-7 cells were treated with the indicated concentrations of LYR analogs for 18 h, and supernatants were collected. The secreted level of VEGF was quantified by ELISA as described in Materials and methods. The bar graph represents the means ± S.E.M. of six analyses. *P b 0.05, compared to vehicle-treated control. (B) The TAMR-MCF-7 cells were treated as above and whole cell lysates were used to determine cellular VEGF levels by western blot analysis. (C) The band densities were measured and presented in a graph showing relative density ± S.E.M. of three independent experiments. *P b 0.05, compared to vehicle-treated control.

and angiogenesis, we suggest that NF-κB is a possible target of LYR action. Although most breast cancers are initially responsive to anti-estrogen therapy with tamoxifen, they ultimately develop resistance to tamoxifen (Clemons et al., 2002). In TAM-resistant breast tumor tissues, microvessel counts are significantly higher than those in TAM-responsive tumor tissues (Marson et al., 2001). Consistent with this, we have also shown that angiogenic potential and VEGF production are higher in TAMR-MCF-7 than in MCF-7 cells (Kim et al., 2008, 2009). VEGF is therefore an important therapeutic target for tamoxifen-resistant breast cancer phenotypes. Our results showed that LYR-7 and LYR-8 inhibit VEGF production and suppress proliferation to a similar degree in both chemoresistant TAMR-MCF-7 cells and responsive MCF-7 cells. We therefore hypothesize that LYR-7 and LYR-8 may have great potential as valuable anti-cancer drugs in the treatment of drug-resistant breast cancers. This notion was further supported by the results from the breast cancer-inoculated CAM model in which cancer cell-induced neovascularization and tumor growth were suppressed by LYR-7 and LYR-8. There are several reports showing cell density-dependent toxicity for cannabinoids (Ramer and Hinz, 2008), and other therapeutic drugs such as tamoxifen and doxorubicin (Brandt et al., 2004; Kobayashi et al., 1992). In our study, 1 × 104 cells were used for cell proliferation assay in vitro whereas 1.5 × 106 cells (1500× higher) in mass was used for tumor growth onto CAM assay in vivo. Based on the reports that increasing cell density is associated with decreased cytotoxicity, in the

Fig. 6. LYR-7 and LYR-8 inhibit cancer-induced angiogenesis and tumor growth. (A) For tumor implantation on CAM, 1.5 × 106 TAMR-MCF-7 cells were loaded onto each CAM and a single dose (20 μM) of LYR-7 or LYR-8 was given at the time of implantation. After 5 days of incubation, the CAM tissues were resected and digital images were captured. (B) A parallel experiment was carried out with MCF-7 cells. As shown in the photographs, LYR-7 and LYR-8 inhibited both angiogenesis and tumor growth (size). (C) New blood vessels were quantified and expressed as percentage of PBS-treated control. #P b 0.05, compared to PBS- or VEGF-treated groups. $P b 0.05 versus vehicletreated MCF-7 cell-implanted group. *P b 0.05, compared to vehicle-treated cancer cell (TAMR-MCF-7 or MCF-7)-implanted group.

present study, the inhibition of tumor-induced angiogenesis in CAM assay was not probably due to cytotoxicity. In addition, the inhibitory effect of LYR analogs on TAMR-MCF-7 cell-induced angiogenesis was also noted at low non-toxic doses, 1–10 μM (data not shown). Besides, concomitant observations of chick embryo viability and morphology of resected membranes confirmed that LYR analogs were non-toxic at the test doses (1–20 μM). In summary, these findings suggest that LYR analog-mediated anti-angiogenic effects are not likely due to cell toxicity, but largely due to modulation of angiogenic process and VEGF-production. On the other hand, based on the result that LYR analogs at 20 μM caused significant inhibition of the tumor cell proliferation in vitro, the direct action of LYR analogs on tumor cell proliferation cannot be ruled out as one of the possible mechanisms in reduced tumor growth and tumor-induced angiogenesis.

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In conclusion, our results indicate that at least two mechanisms may contribute in this anti-cancer action of synthetic hexahydrocannabinols: direct action in cancer cells (inhibition of growth and VEGF secretion) and indirect action through endothelial cells (inhibition of endothelial cell proliferation, migration, invasion, and tube formation). These results further suggest that hexahydrocannabinol and similar analogs may be excellent leads for the development of antiangiogenic and anti-cancer drugs. Acknowledgements This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology [2010-0001661] and by the Regional Technology Innovation Program of the Ministry of Commerce, Industry, and Energy (MOCIE) [grant No. RTI04-01-04]. Appendix A. Supplementary data Supplementary data to this article can be found online at doi:10.1016/j.ejphar.2010.09.073. References Blazquez, C., Casanova, M.L., Planas, A., Gomez Del Pulgar, T., Villanueva, C., FernandezAcenero, M.J., Aragones, J., Huffman, J.W., Jorcano, J.L., Guzman, M., 2003. Inhibition of tumor angiogenesis by cannabinoids. FASEB J. 17, 529–531. Blazquez, C., Gonzalez-Feria, L., Alvarez, L., Haro, A., Casanova, M.L., Guzman, M., 2004. Cannabinoids inhibit the vascular endothelial growth factor pathway in gliomas. Cancer Res. 64, 5617–5623. Brandt, S., Heller, H., Schuster, K.D., Grote, J., 2004. Tamoxifen induces suppression of cell viability and apoptosis in the human hepatoblastoma cell line HepG2 via downregulation of telomerase activity. Liver Int. 24, 46–54. Carmichael, J., Degraff, W.G., Gazdar, A.F., Minna, J.D., Mitchell, J.B., 1987. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res. 15, 936–942. Casanova, M.L., Blazquez, C., Martinez-Palacio, J., Villanueva, C., Fernandez-Acenero, M.J., Huffman, J.W., Jorcano, J.L., Guzman, M., 2003. Inhibition of skin tumor growth and angiogenesis in vivo by activation of cannabinoid receptors. J. Clin. Invest. 111, 43–50. Clemons, M., Danson, S., Howell, A., 2002. Tamoxifen (“Nolvadex”): a review. Cancer Treat. Rev. 28, 165–180. Ferrara, N., 2002. VEGF and the quest for tumour angiogenesis factors. Nat. Rev. Cancer. 2, 795–803. Ferrara, N., Kerbel, R.S., 2005. Angiogenesis as a therapeutic target. Nature 438, 967–974. Folkman, J., 1995. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat. Med. 1, 27–31. Galve-Roperh, I., Sanchez, C., Cortes, M.L., Gomez del Pulgar, T., Izquierdo, M., Guzman, M., 2000. Anti-tumoral action of cannabinoids: involvement of sustained ceramide accumulation and extracellular signal-regulated kinase activation. Nat. Med. 6, 313–319. Guzman, M., 2003. Cannabinoids: potential anticancer agents. Nat. Rev. Cancer 3, 745–755. Guzman, M., Sanchez, C., Galve-Roperh, I., 2002. Cannabinoids and cell fate. Pharmacol. Ther. 95, 175–184. Huang, S., Pettaway, C.A., Uehara, H., Bucana, C.D., Fidler, I.J., 2001. Blockade of NFkappaB activity in human prostate cancer cells is associated with suppression of angiogenesis, invasion, and metastasis. Oncogene 20, 4188–4197. Kerbel, R.S., 2009. Issues regarding improving the impact of antiangiogenic drugs for the treatment of breast cancer. Breast 18, S41–S47. Kesisis, G., Broxterman, H., Giaccone, G., 2007. Angiogenesis inhibitors. Drug selectivity and target specificity. Curr. Pharm. Des. 13, 2795–2809. Kim, J.A., Cho, K.B., Kim, M.R., Park, B.C., Kim, S.K., Lee, M.Y., Kang, K.W., 2008. Decreased production of vascular endothelial growth factor in adriamycin-resistant breast cancer cells. Cancer Lett. 268, 225–232.

71

Kim, M.R., Choi, H.S., Yang, J.W., Park, B.C., Kim, J.A., Kang, K.W., 2009. Enhancement of vascular endothelial growth factor-mediated angiogenesis in tamoxifen-resistant breast cancer cells: role of Pin1 overexpression. Mol. Cancer Ther. 8, 2163–2171. Kobayashi, H., Takemura, Y., Ohnuma, T., 1992. Relationship between tumor cell density and drug concentration and the cytotoxic effects of doxorubicin or vincristine: mechanism of inoculum effects. Cancer Chemother. Pharmacol. 31, 6–10. Kogan, N.M., Blazquez, C., Alvarez, L., Gallily, R., Schlesinger, M., Guzman, M., Mechoulam, R., 2006. A cannabinoid quinone inhibits angiogenesis by targeting vascular endothelial cells. Mol. Pharmacol. 70, 51–59. Lee, Y.R., Xia, L., 2008. Efficient one-pot synthetic approaches for the cannabinoid analogues and their application to biologically interesting (−)-hexahydrocannabinol and (+)-hexahydrocannabinol. Tetrahedron Lett. 49, 3283–3287. Marson, L.P., Kurian, K.M., Miller, W.R., Dixon, J.M., 2001. The effect of tamoxifen on breast tumour vascularity. Breast Cancer Res. Treat. 66, 9–15. Massi, P., Vaccani, A., Ceruti, S., Colombo, A., Abbracchio, M.P., Parolaro, D., 2004. Antitumor effects of cannabidiol, a nonpsychoactive cannabinoid, on human glioma cell lines. J. Pharmacol. Exp. Ther. 308, 838–845. Park, H.J., Zhang, Y., Georgescu, S.P., Johnson, K.L., Kong, D., Galper, J.B., 2006. Human umbilical vein endothelial cells and human dermal microvascular endothelial cells offer new insights into the relationship between lipid metabolism and angiogenesis. Stem Cell Rev. 2, 93–102. Park, B.C., Thapa, D., Lee, Y.S., Kwak, M.K., Lee, E.S., Choi, H.G., Yong, C.S., Kim, J.A., 2007. 1-furan-2-yl-3-pyridin-2-yl-propenone inhibits the invasion and migration of HT1080 human fibrosarcoma cells through the inhibition of proMMP-2 activation and down regulation of MMP-9 and MT1-MMP. Eur. J. Pharmacol. 567, 193–197. Park, B.C., Park, S.Y., Lee, J.S., Mousa, S.A., Kim, J.T., Kwak, M.K., Kang, K.W., Lee, E.S., Choi, H.G., Yong, C.S., Kim, J.A., 2009. The anti-angiogenic effects of 1-furan-2-yl-3pyridin-2-yl-propenone are mediated through the suppression of both VEGF production and VEGF-induced signaling. Vasc. Pharmacol. 50, 123–131. Pertwee, R.G., 2005. Pharmacological actions of cannabinoids. Handb. Exp. Pharmacol. 168, 1–51. Preet, A., Ganju, R.K., Groopman, J.E., 2008. Delta9-Tetrahydrocannabinol inhibits epithelial growth factor-induced lung cancer cell migration in vitro as well as its growth and metastasis in vivo. Oncogene 27, 339–346. Ramer, R., Hinz, B., 2008. Inhibition of cancer cell invasion by cannabinoids via increased expression of tissue inhibitor of matrix metalloproteinases-1. J. Natl Cancer Inst. 100, 59–69. Ribatti, D., Nico, B., Vacca, A., Roncali, L., Burri, P.H., Djonov, V., 2001. Chorioallantoic membrane capillary bed: a useful target for studying angiogenesis and antiangiogenesis in vivo. Anat. Rec. 264, 317–324. Rius, J., Guma, M., Schachtrup, C., Akassoglou, K., Zinkernagel, A.S., Nizet, V., Johnson, R.S., Haddad, G.G., Karin, M., 2008. NF-kappaB links innate immunity to the hypoxic response through transcriptional regulation of HIF-1alpha. Nature 453, 807–811. Sanchez, C., de Ceballos, M.L., Gomez del Pulgar, T., Rueda, D., Corbacho, C., Velasco, G., Galve-Roperh, I., Huffman, J.W., Ramon Y Cajal, S., Guzman, M., 2001. Inhibition of glioma growth in vivo by selective activation of the CB2 cannabinoid receptor. Cancer Res. 61, 5784–5789. Sarfaraz, S., Adhami, V.M., Syed, D.N., Afaq, F., Mukhtar, H., 2008. Cannabinoids for cancer treatment: progress and promise. Cancer Res. 68, 339–342. Schmidt, D., Textor, B., Pein, O.T., Licht, A.H., Andrecht, S., Sator-Schmitt, Fusenig, N.E., Angel, P., Schorpp-Kistner, M., 2007. Critical role for NF-kappaB-induced JunB in VEGF regulation and tumor angiogenesis. EMBO J. 26, 710–719. Thapa, D., Lee, J.S., Park, S.Y., Bae, Y.H., Bae, S.K., Kwon, J.B., Kim, K.J., Kwak, M.K., Park, Y.J., Choi, H.G., Kim, J.A., 2008. Clotrimazole ameliorates intestinal inflammation and abnormal angiogenesis by inhibiting interleukin-8 expression through a nuclear factor-kappaB-dependent manner. J. Pharmacol. Exp. Ther. 327, 353–364. Thapa, D., Babu, D., Park, M.A., Kwak, M.K., Lee, Y.R., Kim, J.M., Kwon, T.K., Kim, J.A., 2010. Induction of p53-independent apoptosis by a novel synthetic hexahydrocannabinol analog is mediated via Sp1-dependent NSAID-activated gene-1 in colon cancer cells. Biochem. Pharmacol. 80, 62–71. Tufan, A.C., Satiroglu-Tufan, N.L., 2005. The chick embryo chorioallantoic membrane as a model system for the study of tumor angiogenesis, invasion and development of anti-angiogenic agents. Curr. Cancer Drug Targets 5, 249–266. Vaccani, A., Massi, P., Colombo, A., Rubino, T., Parolaro, D., 2005. Cannabidiol inhibits human glioma cell migration through a cannabinoid receptor-independent mechanism. Br. J. Pharmacol. 144, 1032–1036. Vernon, R.B., Angello, J.C., Iruela-Arispe, M.L., Lane, T.F., Sage, E.H., 1992. Reorganization of basement membrane matrices by cellular traction promotes the formation of cellular networks in vitro. Lab. Invest. 66, 536–547. Zhu, L.X., Sharma, S., Stolina, M., Gardner, B., Roth, M.D., Tashkin, D.P., Dubinett, S.M., 2000. Delta-9-tetrahydrocannabinol inhibits antitumor immunity by a CB2 receptor-mediated, cytokine-dependent pathway. J. Immunol. 165, 373–380.