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Cannabinoid CB1 receptor is expressed in chromophobe renal cell carcinoma and renal oncocytoma
Clinical Biochemistry 46 (2013) 638–641
Contents lists available at SciVerse ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Cannabinoid CB1 receptor is expressed in chromophobe renal cell carcinoma and renal oncocytoma Gorka Larrinaga a, b,⁎, Begoña Sanz a, Lorena Blanco a, Itxaro Perez a, María L. Candenas c, Francisco M. Pinto c, Amaia Irazusta b, Javier Gil a, José I. López d a
Department of Physiology, Faculty of Medicine and Dentistry, University of the Basque Country (UPV/EHU), Leioa, Bizkaia, Spain Department of Nursing I, Nursing School, University of the Basque Country (UPV/EHU), Leioa, Bizkaia, Spain Institute for Chemical Research, CSIC, Sevilla, Spain d Department of Anatomic Pathology, Hospital Universitario Cruces, BioCruces Research Institute, University of the Basque Country (UPV/EHU), Barakaldo, Bizkaia, Spain b c
a r t i c l e
i n f o
Article history: Received 11 June 2012 Received in revised form 4 December 2012 Accepted 29 December 2012 Available online 12 January 2013 Keywords: Chromophobe renal cell carcinoma Renal oncocytoma Cannabinoid receptors CB1 RT-PCR Immunohistochemistry
a b s t r a c t Objective: To analyze the mRNA and protein expression of cannabinoid receptors CB1 and CB2 in chromophobe renal cell carcinoma (ChRCC) and renal oncocytoma (RO). Design and methods: Fresh and formalin-fixed tissue samples of ChRCC and RO were analyzed by using real-time quantitative RT-PCR and immunohistochemical techniques (n = 40). Results: Quantitative RT-PCR analysis showed that CB1 mRNA was underexpressed by 12-fold in ChRCC and had a variable expression in RO. CB1 protein showed intense positive immunostaining in both neoplasms. Both CB2 mRNA and protein were not expressed in tumor and non tumor renal tissue. Conclusion: This distinct immunoprofile may eventually be used as an additional tool with practical interest in the differential diagnosis of renal tumors. © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Cannabinoids and their endogenous analogues exert important pharmacological and physiological actions by activating CB1 and CB2 receptors in mammals [1–3]. The endocannabinoid system (ECS) is widely distributed in mammalian tissues and regulates nervous, cardiovascular, digestive, reproductive, immune and metabolic functions [4]. There is increasing evidence that endocannabinoids, such as anandamide and 2-arachidonoylglycerol, modulate the activity of enzymes and nuclear factors involved in the control of fundamental processes of cell homeostasis and in neoplastic transformation by activating CB1 and CB2 receptors [5–8]. Recent works have reported distinct changes in the expression of endocannabinoids and their receptors in neoplasms of diverse origins and have proposed antitumoral properties to natural and synthetic cannabinoids [6,8]. Despite these advances, studies about the distribution of CB1 and CB2 receptors in the human kidneys and their dysregulation in renal tumors are still scarce. We begun to clarify this question demonstrating recently the presence of CB1 receptors in human fetal and adult kidneys [9], thus suggesting that these receptors may play some role in human kidney development and in the regulation of tubular functions. Further, we ⁎ Corresponding author at: Department of Physiology, Medical School, University of the Basque Country (UPV/EHU), 48940 Leioa, Bizkaia, Spain. E-mail address: [email protected] (G. Larrinaga).
showed in a subsequent study that CB1 protein expression is downregulated in clear cell renal cell carcinoma (CCRCC) [10], the most frequent kidney neoplasm [11,12]. The current WHO classification of renal tumors in adults [12] links the diverse histological neoplastic entities of the kidney to a distinct site of origin along the nephron and to specific molecular anomalies. This classification has also prognostic connotations and is widely used by pathologists nowadays. The differential diagnosis in some cases may be difficult in routine practice. In this context, the present paper aims to analyze the expression of CB receptors in ChRCC and RO in order to define the specific CB receptors profile of these tumors and to evaluate if our findings could be used as an additional diagnostic tool in the differential diagnosis of kidney tumors. Material and methods The authors declare that all the experiments carried out in this study comply with current Spanish and European Union laws and conform to the principles outlined in the Declaration of Helsinki. RNA extraction and real-time quantitative polymerase chain reaction (qPCR) The expression of the genes encoding the CB1 (CNR1) and CB2 (CNR2) receptors was analyzed in fresh renal tissue obtained from
0009-9120/$ – see front matter © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clinbiochem.2012.12.023
G. Larrinaga et al. / Clinical Biochemistry 46 (2013) 638–641 Table 1 Primer sequences for CNR1, CNR2, TBP, PPIA, ACTB and SDHA. Gene
CNR1 CNR2 TBP PPIA ACTB SDHA
Primer sequences Forward
Reverse
5′-AGGGGATGCGAAGGGATT-3′ 5′-CACCCACAACACAACCCAAA-3′ 5′-GGATAAGAGAGCCACGAACCAC-3′ 5′-GGTCCCAAAGACAGCAGAAAA-3′ 5′-TCCCTGGAGAAGAGCTACGA-3′ 5′-TCTGCCCACACCAGCACT-3′
5′-AGTGGTGATGGTGCGGAAG-3′ 5′-AGCCATCCTTGGAGCCATT-3′ 5′-TTAGCTGGAAAACCCAACTTCTG-3′ 5′-TCACCACCCTGACACATAAACC-3′ 5′-ATCTGCTGGAAGGTGGACAG-3′ 5′-CCTCTCCACGACATCCTTCC-3′
surgical specimens of 8 ChRCC and 6 RO. Patient consent and Hospital Ethics Committee approval were obtained a priori. Tissue samples from neoplasms and surrounding normal tissues were immersed in RNAlater (Ambion, Huntingdon, UK) immediately after dissection and stored in RNAlater at −80 °C until use. RT-PCR reactions were performed essentially as described previously [9,10,13–15]. Total RNA of approximately 30 mg of human renal tissue was isolated in every case according to the method described by Chomczynski and Sacchi [16]. In addition, a pool of RNAs from twenty different human tissues (Human total RNA master panel, BD Biosciences, Clontech, CA, USA) was used as a positive control of amplification. Complementary cDNAs were synthesized from 25 μg of total RNA of each human sample using the first-strand cDNA synthesis kit (Amersham Biosciences, Essex, UK). The resulting cDNA samples were amplified by qPCR using specific oligonucleotide primer pairs designed with the analysis software Primer 3 [17]. The sequences of the primer pairs used for human CNR1 and CNR2 are shown in Table 1. The human genes TATA box binding protein (TBP), peptidylprolyl isomerase A (PPIA), β-actin (ACTB) and succinate dehydrogenase complex, subunit A (SDHA) were chosen as internal quality controls of RT-PCR reactions on the basis of previous experiments on human renal cell carcinoma [15,18,19]. The primer sequences of these four housekeeping genes are also displayed in Table 1. All primers were synthesized and purified by Sigma-Genosys (Cambridge, UK). The expression of CNR1, CNR2 and the housekeeping genes, was quantified in all cDNAs by real-time qPCR using the iCycler iQ real-time detection apparatus (BioRad Laboratories, Hercules, CA, USA). Dilutions of the cDNA template were prepared from each tissue and amplified in triplicate using SensiMix Plus SYBR + FLUORESCEIN (Quantace Ltd., London, UK). The parameters used for PCR amplification were: 10 s at 94 °C, 20 s at 60 °C and 30 s at 72 °C, for 50 cycles. The identity of each product was established by DNA sequence analysis and the specificity of PCR reactions was confirmed by melting curve analysis of the products and by size verification of the amplicon in a conventional agarose gel stained with ethidium bromide. The fold change in gene expression was calculated by the 2 −ΔΔCT method, as described previously [13,19]. Real-time qPCR data were expressed as the fold change of the target gene expression relative to
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the geometric mean (g.m.) mRNA expression of the housekeeping genes in each sample [20]. For each type of renal tumor, paired malignant (tumor) and uninvolved surrounding samples (normal) from the same patient were always measured in the same analytical run to exclude between-run variations. Each assay was performed in triplicate and three negative controls (with no template, no reverse transcriptase and no RNA in the reverse transcriptase reaction) were included in each plate to detect any possible contamination. Immunohistochemistry Surgical specimens were submitted in fresh within the first 30 min after removal to prevent autolysis, and fixed in formalin for no more than 24 h. Representative material for immunohistochemical analysis was obtained prospectively from the same 8 ChRCC and 6 RO selected for mRNA expression, and retrospectively in 12 ChRCC and 14 RO additional cases selected from our files (total cases: 20 ChRCC and 20 RO). On the other hand, 10 CCRCCs were also added to the study. Consecutive histological slides of the selected blocks in every case were immunostained with cannabinoid receptors CB1 and CB2 following routine methods in an automatized immunostainer (Envision FLEX, Dako Autostainer Plus). Tris-EDTA was used for antigen retrieval. CB1 polyclonal antibody (ABR Affinity BioReagents, catalog ref. PA1-743, Golden, CO, USA, working dilution 1:1000) and CB2 polyclonal antibody (Cayman Chemical, catalog ref. 101550, Ann Arbor, MI, USA, working dilution 1:200) were used. The specificity of these antibodies in human cells was previously assessed by our group [9,10,21]. Negative control slides were not exposed to the primary antibody and were incubated in PBS and then processed in the same conditions as test slides. A second pathologist blindly evaluated selected immunohistochemical slides for accuracy. Statistical analyses Data from quantitative RT-PCR were analyzed statistically using SPSS® 19.0. Mann–Whitney test was performed to detect differences between uninvolved tissues and tumors. Statistically significant differences were considered at p b 0.05. Results mRNA expression of cannabinoid receptors RT-PCR revealed the presence of a single transcript, corresponding to the expected product size encoding the cannabinoid CB1 receptor (131 bp) (Fig. 1). The identity of the amplified fragment was confirmed by DNA sequence analysis. The CNR1 transcript could be observed in both tumor and surrounding normal tissues after amplification of cDNA. However, the pattern of expression was different in both neoplasms. In ChRCC, the expression of the CB1 receptor was 12-fold lower in tumor than in normal tissue
Fig. 1. mRNA expression of the cannabinoid CB1 receptor in kidney tumor (T) and surrounding normal tissue (N) from two different patients. Agarose gel showing the presence of a single transcript, corresponding to the expected product size encoding the CB1 receptor (131 bp, CNR1). The mRNA encoding the 110 bp product expected for the cannabinoid CB2 receptor (CNR2) was undetectable. The expression of SHDA, one of the genes used as internal control, is also shown. M, molecular size standards; +, positive control showing the expression of all genes in a pool of cDNAs from 20 different human tissues; and −, negative control with no RNA in the reverse transcriptase reaction.
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G. Larrinaga et al. / Clinical Biochemistry 46 (2013) 638–641
Discussion
Fig. 2. mRNA levels of CNR1 measured in tumor and nontumor tissues (normal) from ChRCC (n = 8) and RO (n = 6) patients. qRT-PCR data for each analyzed sample are recorded as relative units (×10−5). *Statistically significant (p b 0.05).
(Mann–Whitney test p = 0.011) (Fig. 2). The decrease in CNR1 mRNA in tumor versus adjacent normal tissue was observed in all samples assayed. Conversely, in RO, the CNR1 expression in tumor and normal tissue showed high variations between patients. The mean value was 2-fold lower in RO than in the adjacent normal tissue, however, it was not statistically significant (Mann–Whitney test p= 0.746) (Fig. 2). The mRNA encoding the 110 bp product expected for the cannabinoid CB2 receptor was undetectable in cDNA from tumor or normal renal tissues, even after real-time PCR amplification for 50 cycles (Table 1). The mRNAs of CNR1, CNR2 and the four housekeeping genes were all detected in the cDNA pool used as positive control (Fig. 1, showing only SDHA among the housekeeping genes).
Immunohistochemical expression of cannabinoid receptors Fig. 3 and Table 2 show the pattern of immunohistochemical expression of cannabinoid receptors in the normal kidney and renal tumors. Focusing specifically on the distal nephron, CB1 receptor immunostaining was intense and selective in intercalated cells of collecting ducts (Fig. 3B), as it was both in ChRCC and RO (Figs. 3D and F). CB1 immunostaining was negative in CCRCC (Fig. 3H), whereas CB2 was absent both in ChRCC, RO and CCRCC and in normal tissue (data not shown).
CB1 receptors are highly expressed in central nervous system and are considered principally responsible for the cognitive, behavioral, motor and antinociceptive effects of cannabinoids [22]. However, these proteins and/or their mRNAs are also present at relevant levels in many human peripheral tissues. This ubiquity allows these receptors to participate in several physiological processes, and its imbalance has been implicated in several pathological conditions [3,23,24]. With this regard, several studies suggested that the cannabinoid receptor system is altered during carcinogenesis [5,6,8], however, the regulation of their expression in human renal cancer tissues is a barely investigated topic. Previous studies in human kidneys have demonstrated the presence of CB1 receptors and the absence of CB2 receptors at the mRNA and protein levels [9,23,25]. CB1 receptor is expressed in the maturing proximal and distal tubules of fetal kidneys and it is specifically expressed in the proximal convoluted tubules and in the intercalated cells of the collecting ducts of adult renal parenchyma [9]. The current WHO classification of renal tumors in adults considers that CCRCC, the most frequent renal neoplasm, originates in proximal tubule cells, and that ChRCC and its benign counterpart, RO, derives from the intercalated cells of the collecting ducts [11]. The distinction of CCRCC from ChRCC based on histological criteria is not always easy for pathologists, especially when CCRCC are composed of eosinophilic cells. However, the distinction matters since the clinical experience demonstrates that CCRCC behave significantly more aggressively than ChRCC after stratification by stage [26]. Recent reviews stress the difficulties in the differential diagnosis between the diverse subtypes of renal cell carcinomas, and the prognostic importance of its correct identification based on immunohistochemistry [27,28]. Since distal nephron cells are enriched in CB1 receptors [9], we investigated whether tumors from this origin maintain CB1 expression, and then be of additional help in difficult cases. Thus, in this and in a previous study [10], we demonstrated that CB1 protein expression is markedly down-regulated in CCRCC. However, in the present work, an intense CB1 receptor staining was observed in ChRCC and RO. This distinct immunoprofile may eventually be used as an additional tool with practical interest in the differential diagnosis of tumors from proximal (CCRCC) and distal nephron (ChRCC and RO). With regard to the CB1 receptor mRNA expression in kidney neoplasms, it was not correlated with the protein expression, particularly
Fig. 3. Microscopic detail of representative areas of chromophobe renal cell carcinoma, renal oncocytoma and clear cell renal cell carcinoma showing non neoplastic collecting ducts (A), chromophobe renal cell carcinoma (C), renal oncocytoma (E) and clear cell renal cell carcinoma (G) (×400). CB1 immunostaining showing selective staining of intercalated cells (B) and diffuse and intense immunostaining of chromophobe renal cell carcinoma (D) and renal oncocytoma (F), and negative staining in clear cell renal cell carcinoma (H) (original magnification, ×400).
G. Larrinaga et al. / Clinical Biochemistry 46 (2013) 638–641 Table 2 Semi-quantitative evaluation of renal tissues. Immunostaining intensities: (−) negative, (+) mild, (++) moderate and (+++) intense. Tissue/cell type
Intercalated cells ChRCC RO CCRCC
CB1 immunostaining
CB2 immunostaining
Staining-intensity
Staining-intensity
+++ +++ Diffuse +++ Diffuse −
− − − −
in malignant tumors. In CCRCC, CNR1 mRNA expression was not altered when compared to normal surrounding tissue [10]. CNR1 mRNA was underexpressed in ChRCC and variably expressed in RO when compared with non tumor renal tissue. These data suggest that modification of CB1 protein levels could occur at a postranscriptional stage in these neoplasms. A similar discrepancy between protein and mRNA expression has been described recently in renal carcinomas and other non-neoplastic kidney diseases when receptors and enzymes of the renin–angiotensin system were measured [29,30]. This finding illustrates the importance of not relying only on mRNA level the evaluation of any protein change [31]. The literature of the past ten years shows that the expression of CB1 receptors in human malignancies is cancer-type dependant. While increased expression of CB1 has been reported in prostatic adenocarcinoma [32], hepatocellular carcinoma [33], pancreatic ductal adenocarcinoma [34,35] and breast cancer [36], down-regulations have been demonstrated in colorectal cancer [37,38]. Instead, CB1 expression was similar to normal tissues in non-melanoma skin cancer [39,40], and Kaposi's sarcoma [41]. However, regardless of these tumor-specific changes, most of these studies have suggested that endogenous and exogenous cannabinoids have proapoptotic, antiproliferative, antimetastatic and antiangiogenic properties [5–8]. In conclusion, this study shows that ChRCC and RO express the CB1 receptor with similar intensity than in non tumor renal tissue, suggesting that this protein is regulated in a different way in tumors from the proximal and distal nephron. The precise role of CB1 in these neoplasms still remains to be clarified. Nevertheless, the CB1 receptor should not be disregarded as drug target in view of the potential antitumor properties of natural and synthetic cannabinoids [5–8]. Acknowledgment The authors also thank Ana Abascal, Alicia Esteve and Mar Gonzalez, lab technicians at the Department of Anatomic Pathology, Cruces University Hospital, for their excellent immunohistochemical work. This work was supported by grants from the Jesús Gangoiti-Barrera Foundation, Gobierno Vasco (GIC07/84), MEC (CTQ2007-61024/BQU) and SAIOTEK (SA-2008/00046). References [1] Matsuda LA, Lolait SJ, Brownstein MJ, Young AC, Bonner TI. Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature 1990;346:561–4. [2] Munro S, Thomas KL, Abu Shaar M. Molecular characterization of a peripheral receptor for cannabinoids. Nature 1993;365:661–5. [3] Mackie K. Cannabinoid receptors: where they are and what they do. J Neuroendocrinol 2008;20:10–4. [4] Graham ES, Ashton JC, Glass MC. Cannabinoid receptors: a brief history and “what's hot”. Front Biosci 2009;14:944–57. [5] Flygare J, Sander B. The endocannabinoid system in cancer-potential therapeutic target? Clin Oral Investig 2008;12:291–302. [6] Pisanti S, Bifulco M. Endocannabinoid system modulation in cancer biology and therapy. Pharmacol Res 2009;60:107–16. [7] Alexander A, Smith PF, Rosengren RJ. Cannabinoids in the treatment of cancer. Cancer Lett 2009;285:6–12.
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Contents lists available at SciVerse ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Cannabinoid CB1 receptor is expressed in chromophobe renal cell carcinoma and renal oncocytoma Gorka Larrinaga a, b,⁎, Begoña Sanz a, Lorena Blanco a, Itxaro Perez a, María L. Candenas c, Francisco M. Pinto c, Amaia Irazusta b, Javier Gil a, José I. López d a
Department of Physiology, Faculty of Medicine and Dentistry, University of the Basque Country (UPV/EHU), Leioa, Bizkaia, Spain Department of Nursing I, Nursing School, University of the Basque Country (UPV/EHU), Leioa, Bizkaia, Spain Institute for Chemical Research, CSIC, Sevilla, Spain d Department of Anatomic Pathology, Hospital Universitario Cruces, BioCruces Research Institute, University of the Basque Country (UPV/EHU), Barakaldo, Bizkaia, Spain b c
a r t i c l e
i n f o
Article history: Received 11 June 2012 Received in revised form 4 December 2012 Accepted 29 December 2012 Available online 12 January 2013 Keywords: Chromophobe renal cell carcinoma Renal oncocytoma Cannabinoid receptors CB1 RT-PCR Immunohistochemistry
a b s t r a c t Objective: To analyze the mRNA and protein expression of cannabinoid receptors CB1 and CB2 in chromophobe renal cell carcinoma (ChRCC) and renal oncocytoma (RO). Design and methods: Fresh and formalin-fixed tissue samples of ChRCC and RO were analyzed by using real-time quantitative RT-PCR and immunohistochemical techniques (n = 40). Results: Quantitative RT-PCR analysis showed that CB1 mRNA was underexpressed by 12-fold in ChRCC and had a variable expression in RO. CB1 protein showed intense positive immunostaining in both neoplasms. Both CB2 mRNA and protein were not expressed in tumor and non tumor renal tissue. Conclusion: This distinct immunoprofile may eventually be used as an additional tool with practical interest in the differential diagnosis of renal tumors. © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Cannabinoids and their endogenous analogues exert important pharmacological and physiological actions by activating CB1 and CB2 receptors in mammals [1–3]. The endocannabinoid system (ECS) is widely distributed in mammalian tissues and regulates nervous, cardiovascular, digestive, reproductive, immune and metabolic functions [4]. There is increasing evidence that endocannabinoids, such as anandamide and 2-arachidonoylglycerol, modulate the activity of enzymes and nuclear factors involved in the control of fundamental processes of cell homeostasis and in neoplastic transformation by activating CB1 and CB2 receptors [5–8]. Recent works have reported distinct changes in the expression of endocannabinoids and their receptors in neoplasms of diverse origins and have proposed antitumoral properties to natural and synthetic cannabinoids [6,8]. Despite these advances, studies about the distribution of CB1 and CB2 receptors in the human kidneys and their dysregulation in renal tumors are still scarce. We begun to clarify this question demonstrating recently the presence of CB1 receptors in human fetal and adult kidneys [9], thus suggesting that these receptors may play some role in human kidney development and in the regulation of tubular functions. Further, we ⁎ Corresponding author at: Department of Physiology, Medical School, University of the Basque Country (UPV/EHU), 48940 Leioa, Bizkaia, Spain. E-mail address: [email protected] (G. Larrinaga).
showed in a subsequent study that CB1 protein expression is downregulated in clear cell renal cell carcinoma (CCRCC) [10], the most frequent kidney neoplasm [11,12]. The current WHO classification of renal tumors in adults [12] links the diverse histological neoplastic entities of the kidney to a distinct site of origin along the nephron and to specific molecular anomalies. This classification has also prognostic connotations and is widely used by pathologists nowadays. The differential diagnosis in some cases may be difficult in routine practice. In this context, the present paper aims to analyze the expression of CB receptors in ChRCC and RO in order to define the specific CB receptors profile of these tumors and to evaluate if our findings could be used as an additional diagnostic tool in the differential diagnosis of kidney tumors. Material and methods The authors declare that all the experiments carried out in this study comply with current Spanish and European Union laws and conform to the principles outlined in the Declaration of Helsinki. RNA extraction and real-time quantitative polymerase chain reaction (qPCR) The expression of the genes encoding the CB1 (CNR1) and CB2 (CNR2) receptors was analyzed in fresh renal tissue obtained from
0009-9120/$ – see front matter © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clinbiochem.2012.12.023
G. Larrinaga et al. / Clinical Biochemistry 46 (2013) 638–641 Table 1 Primer sequences for CNR1, CNR2, TBP, PPIA, ACTB and SDHA. Gene
CNR1 CNR2 TBP PPIA ACTB SDHA
Primer sequences Forward
Reverse
5′-AGGGGATGCGAAGGGATT-3′ 5′-CACCCACAACACAACCCAAA-3′ 5′-GGATAAGAGAGCCACGAACCAC-3′ 5′-GGTCCCAAAGACAGCAGAAAA-3′ 5′-TCCCTGGAGAAGAGCTACGA-3′ 5′-TCTGCCCACACCAGCACT-3′
5′-AGTGGTGATGGTGCGGAAG-3′ 5′-AGCCATCCTTGGAGCCATT-3′ 5′-TTAGCTGGAAAACCCAACTTCTG-3′ 5′-TCACCACCCTGACACATAAACC-3′ 5′-ATCTGCTGGAAGGTGGACAG-3′ 5′-CCTCTCCACGACATCCTTCC-3′
surgical specimens of 8 ChRCC and 6 RO. Patient consent and Hospital Ethics Committee approval were obtained a priori. Tissue samples from neoplasms and surrounding normal tissues were immersed in RNAlater (Ambion, Huntingdon, UK) immediately after dissection and stored in RNAlater at −80 °C until use. RT-PCR reactions were performed essentially as described previously [9,10,13–15]. Total RNA of approximately 30 mg of human renal tissue was isolated in every case according to the method described by Chomczynski and Sacchi [16]. In addition, a pool of RNAs from twenty different human tissues (Human total RNA master panel, BD Biosciences, Clontech, CA, USA) was used as a positive control of amplification. Complementary cDNAs were synthesized from 25 μg of total RNA of each human sample using the first-strand cDNA synthesis kit (Amersham Biosciences, Essex, UK). The resulting cDNA samples were amplified by qPCR using specific oligonucleotide primer pairs designed with the analysis software Primer 3 [17]. The sequences of the primer pairs used for human CNR1 and CNR2 are shown in Table 1. The human genes TATA box binding protein (TBP), peptidylprolyl isomerase A (PPIA), β-actin (ACTB) and succinate dehydrogenase complex, subunit A (SDHA) were chosen as internal quality controls of RT-PCR reactions on the basis of previous experiments on human renal cell carcinoma [15,18,19]. The primer sequences of these four housekeeping genes are also displayed in Table 1. All primers were synthesized and purified by Sigma-Genosys (Cambridge, UK). The expression of CNR1, CNR2 and the housekeeping genes, was quantified in all cDNAs by real-time qPCR using the iCycler iQ real-time detection apparatus (BioRad Laboratories, Hercules, CA, USA). Dilutions of the cDNA template were prepared from each tissue and amplified in triplicate using SensiMix Plus SYBR + FLUORESCEIN (Quantace Ltd., London, UK). The parameters used for PCR amplification were: 10 s at 94 °C, 20 s at 60 °C and 30 s at 72 °C, for 50 cycles. The identity of each product was established by DNA sequence analysis and the specificity of PCR reactions was confirmed by melting curve analysis of the products and by size verification of the amplicon in a conventional agarose gel stained with ethidium bromide. The fold change in gene expression was calculated by the 2 −ΔΔCT method, as described previously [13,19]. Real-time qPCR data were expressed as the fold change of the target gene expression relative to
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the geometric mean (g.m.) mRNA expression of the housekeeping genes in each sample [20]. For each type of renal tumor, paired malignant (tumor) and uninvolved surrounding samples (normal) from the same patient were always measured in the same analytical run to exclude between-run variations. Each assay was performed in triplicate and three negative controls (with no template, no reverse transcriptase and no RNA in the reverse transcriptase reaction) were included in each plate to detect any possible contamination. Immunohistochemistry Surgical specimens were submitted in fresh within the first 30 min after removal to prevent autolysis, and fixed in formalin for no more than 24 h. Representative material for immunohistochemical analysis was obtained prospectively from the same 8 ChRCC and 6 RO selected for mRNA expression, and retrospectively in 12 ChRCC and 14 RO additional cases selected from our files (total cases: 20 ChRCC and 20 RO). On the other hand, 10 CCRCCs were also added to the study. Consecutive histological slides of the selected blocks in every case were immunostained with cannabinoid receptors CB1 and CB2 following routine methods in an automatized immunostainer (Envision FLEX, Dako Autostainer Plus). Tris-EDTA was used for antigen retrieval. CB1 polyclonal antibody (ABR Affinity BioReagents, catalog ref. PA1-743, Golden, CO, USA, working dilution 1:1000) and CB2 polyclonal antibody (Cayman Chemical, catalog ref. 101550, Ann Arbor, MI, USA, working dilution 1:200) were used. The specificity of these antibodies in human cells was previously assessed by our group [9,10,21]. Negative control slides were not exposed to the primary antibody and were incubated in PBS and then processed in the same conditions as test slides. A second pathologist blindly evaluated selected immunohistochemical slides for accuracy. Statistical analyses Data from quantitative RT-PCR were analyzed statistically using SPSS® 19.0. Mann–Whitney test was performed to detect differences between uninvolved tissues and tumors. Statistically significant differences were considered at p b 0.05. Results mRNA expression of cannabinoid receptors RT-PCR revealed the presence of a single transcript, corresponding to the expected product size encoding the cannabinoid CB1 receptor (131 bp) (Fig. 1). The identity of the amplified fragment was confirmed by DNA sequence analysis. The CNR1 transcript could be observed in both tumor and surrounding normal tissues after amplification of cDNA. However, the pattern of expression was different in both neoplasms. In ChRCC, the expression of the CB1 receptor was 12-fold lower in tumor than in normal tissue
Fig. 1. mRNA expression of the cannabinoid CB1 receptor in kidney tumor (T) and surrounding normal tissue (N) from two different patients. Agarose gel showing the presence of a single transcript, corresponding to the expected product size encoding the CB1 receptor (131 bp, CNR1). The mRNA encoding the 110 bp product expected for the cannabinoid CB2 receptor (CNR2) was undetectable. The expression of SHDA, one of the genes used as internal control, is also shown. M, molecular size standards; +, positive control showing the expression of all genes in a pool of cDNAs from 20 different human tissues; and −, negative control with no RNA in the reverse transcriptase reaction.
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Discussion
Fig. 2. mRNA levels of CNR1 measured in tumor and nontumor tissues (normal) from ChRCC (n = 8) and RO (n = 6) patients. qRT-PCR data for each analyzed sample are recorded as relative units (×10−5). *Statistically significant (p b 0.05).
(Mann–Whitney test p = 0.011) (Fig. 2). The decrease in CNR1 mRNA in tumor versus adjacent normal tissue was observed in all samples assayed. Conversely, in RO, the CNR1 expression in tumor and normal tissue showed high variations between patients. The mean value was 2-fold lower in RO than in the adjacent normal tissue, however, it was not statistically significant (Mann–Whitney test p= 0.746) (Fig. 2). The mRNA encoding the 110 bp product expected for the cannabinoid CB2 receptor was undetectable in cDNA from tumor or normal renal tissues, even after real-time PCR amplification for 50 cycles (Table 1). The mRNAs of CNR1, CNR2 and the four housekeeping genes were all detected in the cDNA pool used as positive control (Fig. 1, showing only SDHA among the housekeeping genes).
Immunohistochemical expression of cannabinoid receptors Fig. 3 and Table 2 show the pattern of immunohistochemical expression of cannabinoid receptors in the normal kidney and renal tumors. Focusing specifically on the distal nephron, CB1 receptor immunostaining was intense and selective in intercalated cells of collecting ducts (Fig. 3B), as it was both in ChRCC and RO (Figs. 3D and F). CB1 immunostaining was negative in CCRCC (Fig. 3H), whereas CB2 was absent both in ChRCC, RO and CCRCC and in normal tissue (data not shown).
CB1 receptors are highly expressed in central nervous system and are considered principally responsible for the cognitive, behavioral, motor and antinociceptive effects of cannabinoids [22]. However, these proteins and/or their mRNAs are also present at relevant levels in many human peripheral tissues. This ubiquity allows these receptors to participate in several physiological processes, and its imbalance has been implicated in several pathological conditions [3,23,24]. With this regard, several studies suggested that the cannabinoid receptor system is altered during carcinogenesis [5,6,8], however, the regulation of their expression in human renal cancer tissues is a barely investigated topic. Previous studies in human kidneys have demonstrated the presence of CB1 receptors and the absence of CB2 receptors at the mRNA and protein levels [9,23,25]. CB1 receptor is expressed in the maturing proximal and distal tubules of fetal kidneys and it is specifically expressed in the proximal convoluted tubules and in the intercalated cells of the collecting ducts of adult renal parenchyma [9]. The current WHO classification of renal tumors in adults considers that CCRCC, the most frequent renal neoplasm, originates in proximal tubule cells, and that ChRCC and its benign counterpart, RO, derives from the intercalated cells of the collecting ducts [11]. The distinction of CCRCC from ChRCC based on histological criteria is not always easy for pathologists, especially when CCRCC are composed of eosinophilic cells. However, the distinction matters since the clinical experience demonstrates that CCRCC behave significantly more aggressively than ChRCC after stratification by stage [26]. Recent reviews stress the difficulties in the differential diagnosis between the diverse subtypes of renal cell carcinomas, and the prognostic importance of its correct identification based on immunohistochemistry [27,28]. Since distal nephron cells are enriched in CB1 receptors [9], we investigated whether tumors from this origin maintain CB1 expression, and then be of additional help in difficult cases. Thus, in this and in a previous study [10], we demonstrated that CB1 protein expression is markedly down-regulated in CCRCC. However, in the present work, an intense CB1 receptor staining was observed in ChRCC and RO. This distinct immunoprofile may eventually be used as an additional tool with practical interest in the differential diagnosis of tumors from proximal (CCRCC) and distal nephron (ChRCC and RO). With regard to the CB1 receptor mRNA expression in kidney neoplasms, it was not correlated with the protein expression, particularly
Fig. 3. Microscopic detail of representative areas of chromophobe renal cell carcinoma, renal oncocytoma and clear cell renal cell carcinoma showing non neoplastic collecting ducts (A), chromophobe renal cell carcinoma (C), renal oncocytoma (E) and clear cell renal cell carcinoma (G) (×400). CB1 immunostaining showing selective staining of intercalated cells (B) and diffuse and intense immunostaining of chromophobe renal cell carcinoma (D) and renal oncocytoma (F), and negative staining in clear cell renal cell carcinoma (H) (original magnification, ×400).
G. Larrinaga et al. / Clinical Biochemistry 46 (2013) 638–641 Table 2 Semi-quantitative evaluation of renal tissues. Immunostaining intensities: (−) negative, (+) mild, (++) moderate and (+++) intense. Tissue/cell type
Intercalated cells ChRCC RO CCRCC
CB1 immunostaining
CB2 immunostaining
Staining-intensity
Staining-intensity
+++ +++ Diffuse +++ Diffuse −
− − − −
in malignant tumors. In CCRCC, CNR1 mRNA expression was not altered when compared to normal surrounding tissue [10]. CNR1 mRNA was underexpressed in ChRCC and variably expressed in RO when compared with non tumor renal tissue. These data suggest that modification of CB1 protein levels could occur at a postranscriptional stage in these neoplasms. A similar discrepancy between protein and mRNA expression has been described recently in renal carcinomas and other non-neoplastic kidney diseases when receptors and enzymes of the renin–angiotensin system were measured [29,30]. This finding illustrates the importance of not relying only on mRNA level the evaluation of any protein change [31]. The literature of the past ten years shows that the expression of CB1 receptors in human malignancies is cancer-type dependant. While increased expression of CB1 has been reported in prostatic adenocarcinoma [32], hepatocellular carcinoma [33], pancreatic ductal adenocarcinoma [34,35] and breast cancer [36], down-regulations have been demonstrated in colorectal cancer [37,38]. Instead, CB1 expression was similar to normal tissues in non-melanoma skin cancer [39,40], and Kaposi's sarcoma [41]. However, regardless of these tumor-specific changes, most of these studies have suggested that endogenous and exogenous cannabinoids have proapoptotic, antiproliferative, antimetastatic and antiangiogenic properties [5–8]. In conclusion, this study shows that ChRCC and RO express the CB1 receptor with similar intensity than in non tumor renal tissue, suggesting that this protein is regulated in a different way in tumors from the proximal and distal nephron. The precise role of CB1 in these neoplasms still remains to be clarified. Nevertheless, the CB1 receptor should not be disregarded as drug target in view of the potential antitumor properties of natural and synthetic cannabinoids [5–8]. Acknowledgment The authors also thank Ana Abascal, Alicia Esteve and Mar Gonzalez, lab technicians at the Department of Anatomic Pathology, Cruces University Hospital, for their excellent immunohistochemical work. This work was supported by grants from the Jesús Gangoiti-Barrera Foundation, Gobierno Vasco (GIC07/84), MEC (CTQ2007-61024/BQU) and SAIOTEK (SA-2008/00046). References [1] Matsuda LA, Lolait SJ, Brownstein MJ, Young AC, Bonner TI. Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature 1990;346:561–4. [2] Munro S, Thomas KL, Abu Shaar M. Molecular characterization of a peripheral receptor for cannabinoids. Nature 1993;365:661–5. [3] Mackie K. Cannabinoid receptors: where they are and what they do. J Neuroendocrinol 2008;20:10–4. [4] Graham ES, Ashton JC, Glass MC. Cannabinoid receptors: a brief history and “what's hot”. Front Biosci 2009;14:944–57. [5] Flygare J, Sander B. The endocannabinoid system in cancer-potential therapeutic target? Clin Oral Investig 2008;12:291–302. [6] Pisanti S, Bifulco M. Endocannabinoid system modulation in cancer biology and therapy. Pharmacol Res 2009;60:107–16. [7] Alexander A, Smith PF, Rosengren RJ. Cannabinoids in the treatment of cancer. Cancer Lett 2009;285:6–12.
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