|Year : 2012 | Volume
| Issue : 4 | Page : 549-554
Cannabinoid receptor 2 is upregulated in melanoma
Zigang Zhao1, Jie Yang2, Hua Zhao1, Xiangdong Fang3, Hengjin Li1
1 Department of Dermatology, Chinese People's Liberation Army General Hospital, Beijing, China
2 Department of Dermatology, The Affiliated Hospital of North China Coal Medical College, Tangshan, China
3 Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China
|Date of Web Publication||29-Jan-2013|
Department of Dermatology, The General Hospital of the People's Liberation Army, Beijing - 100853
Aims: To investigate the expression of CB 2 in normal skin, pigmented nevus, and malignant melanoma; analyze its relation with genesis; and development of malignant melanoma.
Materials and Methods: In our study, we detected the expression of CB 2 in 20 cases of melanoma, nevus tissue, and normal skin tissues, respectively, by using immunohistochemistry, in situ hybridization, and RT-PCR. Moreover, we investigated the localization and expression of CB 2 in human melanoma cell line A375 and human immortalized keratinocyte cell line HaCaT by using immunofluorescence and western blotting.
Results: Our data revealed that CB 2 is unregulated in melanoma.
Conclusions: CB 2 plays a role in the development of melanoma.
Keywords: Cannabinoid, melanoma, receptor
|How to cite this article:|
Zhao Z, Yang J, Zhao H, Fang X, Li H. Cannabinoid receptor 2 is upregulated in melanoma. J Can Res Ther 2012;8:549-54
Zigang Zhao and Jie Yang Contributed equally to the manuscript
| > Introduction|| |
Melanoma is one of the most common skin cancers. Despite intensive research, prevention and early detection are the only effective measures against melanoma; no effective therapies exist for advanced melanoma. Recently, cannabis has been found that it has pharmacological effects such as inhibiting cell growth and antiinflammation.  In the early 1990s, cannabinoid receptors CB 1 and CB 2 were successfully cloned.  CB 1 is expressed mainly in the central nervous system (CNS) and also in the lungs, liver, and kidneys. CB 2 is mainly expressed in the immune system. Further, cannabinoid receptors are overexpressed in cancers such as hepatocellular carcinoma (HCC), prostate cancer, nerve horny cell tumor, breast cancer, lung cancer, pancreatic cancer, lymphoma, and skin tumors.  However, the role CB 2 plays in melanoma is not known. In this study, we detected the expression of CB 2 in melanoma cases and melanoma cells.
| > Materials and Methods|| |
Tissue samples were obtained from 20 patients, who were admitted between 2009 and 2011, with histologically diagnosed melanoma without other metastasis. The control, nevus and normal skin, were also obtained by biopsy from 20 nonmelanoma patients, respectively, whose skin was completely normal. The research study was reviewed and approved by the institutional review board and patient consent was obtained to use the tissue for research. After resection, the samples were rapidly frozen in liquid nitrogen or fixed in 4% neutral formaldehyde.
The human melanoma cell line A375 and human immortalized keratinocyte cell line HaCaT were provided by Professor Tianwen Gao of the Dermatology Department of Xijing Hospital affiliated to the Fourth Military Medical University. The two cell lines were cultured in Dulbecco's modified Eagle medium (DMEM) medium supplemented with 10% fetal bovine serum (FBS) and penicillin (100 U/mL)/streptomycin (100 U/ mL). Cells were grown in culture plates at 37°C under a 10% CO 2 atmosphere, and the medium was renewed every 2-3 days.
Cell viability in the cultures was determined by trypan blue exclusion. Cell cycle analysis was performed by flow cytometry determination of nuclear DNA content. Cells were detached with trypsin-EDTA, collected by centrifugation, washed once, and incubated (1 h, room temperature) in phosphate buffered saline (PBS) containing 1% (w/v) bovine serum albumin (BSA), 30% ethanol and 5 g/ mL Hoechst.
Total RNA from tissues was prepared using a TRIzol-A + kit (Tiangen Biotech BEIJING Co., Ltd.). RNA was reverse transcribed into cDNA with random hexamer primers in a 20 μL reaction volume using a Reverse Transcription System Kit (Promega). A total of 2 μL cDNA was amplified using specific primers. Polymerase chain reaction (PCR) conditions were 30 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 60 s. β-Actin was used as the control and 26 cycles of amplification were performed at a denaturing temperature of 55°C. The specific primers used were as follows: CB 2 sense, 5′-GTTCATCGCCTTCCTCTT-3′; antisense, 5′-CTCGGGGCTTCTTCTTTTG-3′ (415 bp); β-actin sense, 5′-AGCTCTGCTGGTAGGTGCAC-3′; antisense, 5′-GCTACGACCATCTAGGCACG-3′ (164 bp). PCR was performed using the 9600 PCR System (Perkin Elmer). The amplified products were analyzed by electrophoresis through 2% agarose gels, and the ratios of the amounts of amplified CB 2 and those of β-actin were measured using a gel imaging analysis system (GIS-2008) to determine the relative expression level of CB 2 mRNA in melanoma and normal skin tissues.
Normal skin, nevus and malignant melanoma tissues were paraffin embedded to be processed into 3-μm serial sections. Two oligonucleotide probes for CB 2 mRNA were designed and labeled with digoxin at their 5′ ends. The sequences were 5′-AGGTATCGGTCAATGGCGGTCAGCAGGAG-3′ and 5′-GCCAACCTCACATCCAGCCTCATTCGGG-3′. After dewaxing and hydration, the sections were placed in cell drilling buffer for 10 min and blocked in peroxide hydrogen buffer for 10 min at room temperature. Compound digestion solution was then applied to the sections and allowed to react for 30 min at room temperature. Next, prehybridization solution was applied and allowed to react for 1 h at room temperature in a wet container. Next, the sections were washed using 0.2 × SSC. Next, a working hybridization solution was added and allowed to react for 4 h at room temperature in a wet container. The sections were washed using 2 × SSC and Tris buffer at 37°C. Next, digoxin-labeled antibody working solution and high-sensitivity peroxidase-labeled avidin working solution were added and allowed to react for 45 min at 37°C in a wet container. The sections were washed in PBS, and proteins were visualized by diaminobenzidine (DAB) staining. The sections were stained with hematoxylin and after dehydration, the slides were mounted. PBS replacing probe solution was used for the blank control. Prehybridization replacing probe solution was used for the negative control. (An in situ hybridization kit was purchased from Boster Company.)
Paraffin-embedded tissues were used for immunohistochemical analysis. After dewaxing, the sections were treated with 0.5% potassium permanganate for 3-5 min, washed in water, and bleached in 2% oxalic acid for 1-2 min for melanin discolor and antigen repair. The sections were then incubated in 3% H 2 O 2 at room temperature, followed by washing in PBS. Anti-CB 2 rabbit antibody (ABR Corporation) was incubated with the sections overnight at 4°C. PBS was used as the negative control. Goat-anti-rabbit secondary antibody conjugated with horseradish peroxidase was incubated with the sections for 30 min at 37°C (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.). Proteins were visualized using DAB staining. Finally, the sections were stained with hematoxylin, and after dehydration, the slides were mounted with cover slips.
Cells grown on coverslips were transfected, washed in PBS buffer, fixed in 4% paraformaldehyde, permeabilised, and incubated 1 h at room temperature with the following primary antibodies, anti-CB 2 rabbit antibody (1:100, ABR Corporation). Signals were visualized with goat-anti-rabbit IgG/TRITC secondary antibodies (1:200, Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.) was incubated with the sections for 1 h at 37°C. Washed in PBS buffer, fixed in Hoechst 33342 for 20 min. Fluorescence of mounted samples was examined with fluorescent microscope.
Particulate cells were subjected to SDS-PAGE, and proteins were transferred from the gels onto polyvinylidene difluoride (PVDF) membranes. The blots were incubated with polyclonal antibodies raised against the human CB 2 receptor (1:1000; Affinity Bioreagents, Golden, CO, USA) as described. Antigen preabsorption experiments were performed by preincubating (4°C, 8 h) 1μL of anti-CB 2 antibody (Ab) and 100μL PBS with or without 20μL of the corresponding immune peptide. β-actin (1:500, Sigma) was used as a loading control. In all cases, samples were subjected to luminography with an enhanced chemiluminescence detection kit.
The results of immunohistochemical staining and in situ hybridization were observed using CX40 (Olympus). We selected five representative fields (400×) on each slide and quantified the area of positive CB 2 staining and the accumulative optical density (IOD) using Image-pro Plus 6.0 software (Media Cybernetics, Silver Spring, USA). The basic steps to be followed when using ImagePro Plus are: Acquire the image, enhance and process the image as needed for measurement. Calibrate the software for the scale or magnification of the image. Mean density (gray-level), red density, green density, blue density, IOD, area, minor axis, perimeter rate and roundness were parameters used for discrimination, followed by their outlined presentation and counting in each image by the macro. Export the measurements to spreadsheets, graphing or stats software.
SPSS11.0 software was used to perform the statistical analysis. All results were reported as mean ± SD. Comparisons were made using the analysis of variance.
| > Results|| |
A 415-bp amplified PCR product for CB 2 was detected and its expression was higher in melanoma tissue than that in nevus and normal tissue [Figure 1]. We calculated the band intensities of CB 2 and β-actin from three independent experiments and found that the difference was statistically significant [P < 0.05, [Table 1], [Figure 2]].
|Figure 1: The mRNA expression of CB2 in tissues detected by RTPCR. RT-PCR assays of CB2 mRNA (lane 1– 3) and β-actin, normal skin (lane 1), nevus (lane 2) and melanoma (lane 3)|
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Next, we performed in situ hybridization analysis [Figure 3]. Consistent with the RT-PCR data, positive staining was significantly lower in the normal tissue and nevus than in the melanoma tissue. We calculated the area of positive CB 2 staining and the accumulative optical density from three independent experiments and found that the difference was statistically significant [P < 0.05, [Table 2], [Figure 4]]. These data collectively suggest that CB 2 mRNA is increased in melanoma tissue.
|Figure 3: CB2 mRNA expression in normal human skin (a), nevus (b), and melanoma (c) detected by in situ hybridization (400×). The positive signal was stained with antisense cRNA and brown-colored. The original pictures were taken at the same magnifi cation (400×)|
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|Figure 4: The area of positive CB2 staining and the accumulative optical density (IOD) detected by in situ hybridization|
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|Table 2: The area of CB2 staining and the accumulative optical density (IOD) detected by in situ hybridization (x±s)|
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We detected the expression of CB 2 and found that positive staining was significantly higher in melanoma tissue than in nevus and normal tissue. In the normal tissues, the cells had clear boundaries and CB 2 was mainly expressed in the prickle cell and basal cell layer [Figure 5]a. In the nevus tissues, CB 2 was expressed in the nevocyte [Figure 5]b. In the melanoma tissues, the cells were irregular and CB 2 was overexpressed [Figure 5]c. We calculated the area of positive CB 2 staining and the accumulative optical density from three independent experiments and found that the difference was statistically significant [P < 0.05, [Table 3], [Figure 6]]. By using immunofluorescence we found that CB 2 was expressed in cytoplasm and membrane in the human melanoma cell line A375 [Figure 7].
|Figure 5: CB2 expression by immunohischemistry (400×). Immunohistochemistry results show positive staining for human CB2 in the normal human skin (a), nevus (b) and melanoma (c)|
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|Figure 6: The area of positive CB2 staining and the accumulative optical density (IOD) detected by immunohistochemistry|
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The CB 2 band in 45 KD was significantly over expression in melanoma cell line A375 than in keratinocyte cell line HaCaT [Figure 8].
|Figure 8: CB2 protein expression in HaCat and A375 detected by western blotting. Equal amount total protein extract of HaCat cells (lane 1) and A375 cells were probed with CB2 and β-actin antibody|
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|Table 3: The area of positive CB2 staining and the accumulative optical density (IOD) detected by immunohistochemistry (x±s)|
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| > Discussion|| |
Human CB 2 comprises approximately 360 amino acids. Human CB 1 and CB 2 share approximately 44% amino acid similarity. As is commonly seen in G protein-coupled receptors, CB 2 has seven transmembrane spanning domains.  It has been found that CB 2 is expressed in the spleen, lymph nodes, and lymphocytes, suggesting that CB 2 plays a role in the nonnervous system.
The CB receptor is widely distributed in the CNS, peripheral immune system, cardiovascular system, digestive system, reproductive system, and skin. CB 2 is mainly distributed in the peripheral immune system, such as in the marginal zone of the spleen; tonsils; and immune cells such as neutrophils, lymphocytes, mast cells, macrophages, monocyte, and natural killer cells (NK cells). , Previous studies have reported that CB 2 mRNA is most abundantly expressed in the spleen and tonsils; however, it is only slightly expressed in cultured mouse cerebellar cells and rat microglial cells. In many other peripheral immune tissues such as in the thymus and bone marrow, CB 2 expression is very low. Ibrahim et al. detected the expression of CB 2 in the glabrous skin of rat hind paw and found that it is only expressed in epidermal tissue.  In normal human skin tissue, CB 2 is mainly expressed in the epidermal basal cell layer, macrophages, mast cells, infundibulum cells of undifferentiated hair follicles, epithelial cells of the sweat gland muscle, sweat gland ducts, undifferentiated cells of sebaceous glands, single epidermal nerve fibers, subcutaneous unmyelinated nerve fibers, myelinated nerve fibers in the skin, etc.  CB 2 participates in sensory conduction of the skin, skin immunity, skin cancer, cell differentiation, and other important physiological and pathological processes. Our study proved that CB 2 is expressed in the prickle and basal cell layers of the epidermis and is unevenly distributed. However, CB 2 expression in the subcutaneous tissues is not obvious. CB 2 was also expressed in keratinocyte cell lines, suggesting that CB 2 is expressed at some special stages. Basal cells are involved in differentiation, growing and migrating with the epidermis, and hair follicles. Therefore, further studies are needed to determine if CB 2 participates in cell differentiation.
CB 2 is expressed in breast cancer, pancreatic cancer, HCC,  prostate cancer, , neural tumor keratinocytes, ,, lung cancer,  thyroid cancer,  lymphoma,  and skin tumors, , including sweat glands nipple adenoma, basal cell carcinoma, squamous cell carcinoma, and malignant melanoma. According to previous reports, CB 2 is overexpressed in human HCC and this overexpression is associated with HCC tumor grade. In addition, patients with a higher CB 2 expression have a better survival rate.  It has been suggested that CB 2 is expressed in normal human skin keratinocytes, epidermal cells, squamous cell carcinoma, and basal cell carcinoma. Further, cannabinoids can inhibit skin tumor growth in vivo.  Blαzquez found that CB 1 and CB 2 are expressed in human melanoma tissues and cell lines; in addition, activation of CB receptors inhibits tumor growth, angiogenesis, and metastasis and promotes apoptosis of melanoma cells in mice exografted tumor models.  Melanoma is highly malignant and is predisposed to metastasis to blood vessels and lymph nodes with rapid development, poor prognosis, and high mortality. In our study, we found that CB 2 is overexpressed in human melanoma tissues and cell lines, suggesting that CB 2 is associated with melanoma development. It is noteworthy that cannabinoids act on tumor cells, specifically in vitro. We speculated that it is because of the different roles of CB receptors in normal cells, nevocyte and melanoma cells and their different distribution in normal and melanoma cells. It has been found that CB 2 is expressed in gliomas but not in normal astrocytes. Our study found that CB 2 is overexpressed in melanoma but not expressed in normal surrounding tissues.
The activation of CB receptors can significantly reduce the expression of vascular endothelial growth factor (VEGF) and angiopoietin-2 and can inhibit the migration and survival of vascular endothelial cells, blocking the formation of blood vessels and tumor growth.  In addition, the activation of CB receptors can induce the expression of p53, p27, and the key intermediate protein 1 (KIP1), resulting in cell arrest in the G0/G1 phase, which leads to increased apoptosis and suppression of tumor growth. Activated CB 2 can inhibit Akt and reduce the phosphorylation of Rb, suppressing tumor cell proliferation.  Activated CB 2 can also inhibit the activity and expression of matrix metalloproteinase 2 (MMP2) and reduce basal collagen digestion and tumor invasion.  CB 2 is more distributed in the peripheral skin and less distributed in the CNS, and it has been proven that drugs targeting CB 2 have little effect on the central nervous system and do not lead to addiction. Therefore, CB 2 is a candidate target for the treatment of skin cancer, and activators of CB 2 could be designed in the future.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
[Table 1], [Table 2], [Table 3]