Dig Dis Sci DOI 10.1007/s10620-014-3175-6
RAPID COMMUNICATIONS
Adiponectin Inhibits Murine Pancreatic Cancer Growth Motohiko Kato • Kenji Watabe • Masahiko Tsujii Tohru Funahashi • Iichiro Shimomura • Tetsuo Takehara
•
Received: 22 December 2013 / Accepted: 16 April 2014 Ó Springer Science+Business Media New York 2014
Abstract Background Adiponectin is an adipose tissue-derived secretory hormone whose plasma concentrations are lower in obese individuals. Obesity is a risk factor for the development and growth of pancreatic cancer, and hypoadiponectinemia was suggested to be involved in the growth of Pan02 murine pancreatic cancer cells that were inoculated into the flanks of congenitally obese mice. Aim The aim of this study was to clarify the role of adiponectin in the growth of pancreatic cancer cells. Methods We examined the effect of adiponectin on the growth of Pan02 cells using recombinant adiponectin and adiponectin knockout mice. Results The in vitro treatment of Pan02 cells with adiponectin inhibited cellular proliferation that was accompanied by increased apoptosis and caspase-3 and caspase-7 activities. Transplantation of Pan02 cells into the pancreas of knockout mice resulted in a larger tumor volume with fewer terminal deoxyribonucleotidyl transferase-
Motohiko Kato and Kenji Watabe contributed equally to this work. M. Kato K. Watabe M. Tsujii T. Takehara (&) Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan e-mail: [email protected] T. Funahashi Department of Metabolism and Atherosclerosis, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan I. Shimomura Department of Metabolic Medicine, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
mediated dUTP nick end labeling (TUNEL) positive cells compared with wild-type mice. Conclusions The results indicate that adiponectin directly suppresses the proliferation of Pan02 cells. Keywords Apoptosis
Pancreatic cancer Obesity Adiponectin
Introduction Adiponectin is an adipose tissue-derived secretory hormone. Compared with other adipose-derived hormones, adiponectin circulates abundantly throughout the body, and its plasma concentrations are lower in obese individuals [1, 2]. Adiponectin has antidiabetic, antiatherosclerotic, and anti-inflammatory properties in various organs [3–7]. Additionally, low levels of plasma adiponectin have been associated with common forms of cancer [8]. Two isoforms of adiponectin receptors, Adipo-R1 and Adipo-R2, have been identified, and these receptors may mediate some of the actions of adiponectin through the activation of intracellular signaling pathways [9]. Obesity is a risk factor for the development and growth of numerous cancers, including pancreatic cancer [10, 11]. The mechanisms underlying the association between obesity and pancreatic cancer have been investigated through hormonal, inflammatory, and immunological changes in obesity. Recently, it was reported that serum adiponectin negatively correlated with tumor weight in a mouse model of pancreatic cancer using congenitally obese mice and flank-implanted Pan02 murine pancreatic cancer cells [12]. In this study, we investigated the role of adiponectin in the growth of Pan02 cells using recombinant adiponectin and adiponectin knockout (APN-KO) mice.
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Materials and Methods
caspase-7 (Caspase-Glo assay, Promega, Madison, WI) according to the manufacturer’s protocol.
Cell Culture Animals Pan02 is a murine pancreatic adenocarcinoma cell line that is syngeneic to C57BL/6 and was obtained from the Division of Cancer Treatment Tumor Repository (National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD). The cells were maintained in RPMI 1640 medium (Sigma Chemical, St. Louis, MO) containing 10 % FBS (Sigma Chemical, St. Louis, MO) and 1 % antibiotic–antimycotic (Invitrogen, Carlsbad, CA) in a humidified atmosphere of 95 % air with 5 % CO2 at 37 °C. Real-Time RT-PCR Analysis Total RNA was extracted using QIAshredder and RNeasy Mini kits (Qiagen, Hilden, Germany), and samples (0.3 lg of total RNA) were reverse-transcribed in 20 lL of a reaction mixture that contained 200 U of Superscript II (GIBCO-BRL, Grand Island, NY) and 0.2 lg of oligo (dT) primer. Quantitative real-time PCR was performed using a QuantiFast SYBR Green PCR kit with specific primers (Qiagen) on a LightCycler (Roche Diagnostics, Indianapolis, IN). Primers used were GAPDH (QT00199388, Qiagen), Adipo-R1 (forward, 50 -ACAGAGTCTGAAGCAAGAGCA-30 ; reverse 50 -GGTG ATGGTACCGTCCGTGAG-30 ), and Aipo-R2 (forward 50 AAGACTCGGGCCTACAATGGACAGCCATGG-30 ; reverse 50 -CAATGTTGATACGTCCACAATTAC-30 ). The mRNA expression levels were normalized relative to that of GAPDH and expressed in arbitrary units. Adiponectin Treatment of Pan02 Cells Recombinant human adiponectin and bovine serum albumin were purchased from R&D systems (Minneapolis, MN) and Takara Bio Inc. (Otsu, Japan), respectively. For the adiponectin assay, Pan02 cells were serum-starved for 24 h followed by adiponectin treatment in fresh media as indicated. Cell proliferation was determined by WST assay using Cell Count Reagent SF (Nacalai Tesque, Kyoto, Japan). Apoptotic cells were detected using the Annexin V-FITC Apoptosis Detection Kit (BioVision, Milpitas, CA) according to the manufacturer’s instructions. Briefly, cells were trypsinized, pooled together with spontaneously detached cells, and stained with Annexin-V and propidium iodide. Labeled cells were analyzed using a FACSAria flow cytometer (BD Biosciences, San Jose, CA). Apoptotic cells were counted as those cells that were double-positive for Annexin-V and propidium iodide. Caspase activity was measured in the supernatant of the cultured Pan02 cells using a luminescent substrate assay for caspase-3 and
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APN-KO mice were generated as described previously and backcrossed with the C57BL/6 J strain for five generations [13]. C57BL/6 J mice were purchased as wild-type (WT) controls from Clea Japan (Tokyo, Japan). The ethics committee for animal experimentation of Osaka University Graduate School of Medicine approved the animal experiments that were reported in this study. Orthotopic Implantation Model of Pancreatic Cancer Pan02 cells were transplanted to the pancreases of mice following the method described by Vonlaufen with minor modifications [14]. Eight- to ten-week-old WT or APN-KO male mice were anesthetized, and an incision was made in the left flank. The spleen and tail of the pancreas were exteriorized, and 5 9 105 Pan02 cells were injected into the tail of the pancreas. Five weeks after transplantation, the mice were killed, the pancreas was removed with spleen and duodenum, and the tumor size was measured. The tumor volume was calculated as (length 9 width2)/2. All tumors were processed for histological examination. Immunohistochemistry Apoptotic cells were identified using terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL; ApopTag, Chemicon International, Temecula, CA) according to the manufacturer’s protocol followed by nuclear staining with methylene blue. The proportion of TUNEL-positive cells in the tumor was calculated from 10 randomly selected high-power fields. CD34 staining was performed using an antibody purchased from Cedarlane Laboratories (Ontario, Canada), and staining was detected using the respective biotinylated secondary IgGs (BD Biosciences), streptavidin/horseradish peroxidase (Vector Labs, Burlingame, CA), and 3,30 -diaminobenzidine (Dako, Carpinteria, CA). Nuclear staining was performed using methylene blue. Microvessel density of the tumor was defined as the number of microvessel per microscopic field and calculated from 10 randomly selected high-power fields. Statistical Analysis Results are expressed as the mean ± SEM. Comparisons between groups were performed using the Wilcoxon rank test for nonparametric data, and more than three groups were compared using Tukey’s honestly significant difference (HSD) test. A P value less than 0.05 was considered statistically
Dig Dis Sci
Fig. 1 The effect of adiponectin on proliferation of the murine pancreatic cancer cell line Pan02. a Real-time RT-PCR analysis of the mRNA expression of the adiponectin receptors Adipo-R1 and AdipoR2 in Pan02 cells and mouse liver tissue (n = 4). Data are expressed as the mean ± SEM. Significant difference is shown. ***p \ 0.001.
b Pan02 cells were serum-starved for 24 h followed by the treatment with various concentrations of adiponectin, and cell proliferation was assessed using the WST assay (n = 6). Data are expressed as the mean ± SEM. Significant differences to no adiponectin are shown. *p \ 0.05
Fig. 2 Adiponectin is proapoptotic for the murine pancreatic cancer cell line Pan02. Pan02 cells were serum-starved for 24 h followed by the treatment with various concentrations of adiponectin and assayed for apoptosis and caspase activity. a Apoptotic cells were presented as the percentage of cells that were positive for Annexin-V and propidium iodide using flow cytometry (n = 3). Data are expressed
as the mean ± SEM. Significant difference to no adiponectin is shown. ***p \ 0.001. b Caspase activity was measured using a luminescent substrate assay for caspase-3 and caspase-7 (n = 3). Data are expressed as the mean ± SEM. Significant differences to no adiponectin are shown. *p \ 0.05, ***p \ 0.001
significant. Data analysis was performed using the JMP 9 statistical package (Statistical Analysis Systems, Cary, NC).
between the two groups (Fig. 1a). We then examined the effect of recombinant adiponectin on the proliferation of pancreatic cancer cells. Adiponectin treatment at concentrations over 5 lg/mL inhibited the proliferation of Pan02 cells (Fig. 1b). In contrast, bovine serum albumin as control protein did not affect the proliferation of the cells over 5 lg/mL (data not shown).
Results Adiponectin Inhibits the Proliferation of the Murine Pancreatic Cancer Cell Line Pan02
Adiponectin Induces Apoptosis in Pan02 Cells Because adiponectin demonstrates its biological functions through binding to its membrane receptors, we first examined the expression of these receptors in pancreatic cancer cells in comparison with mouse liver tissue as control. While Adipo-R1 was expressed 3 times more abundantly in murine pancreatic cell line Pan02 compared with liver tissue, Adipo-R2 was expressed similarly
Previously, adiponectin was shown to inhibit the cellular proliferation of hepatocellular carcinoma cells, and one of the targets for adiponectin action was cyclin D1 [15]. In our study, adiponectin did not affect the expression of either cyclin D1 or a marker for proliferation, proliferating cell nuclear antigen, in Pan02 cells (data not shown). We then examined whether
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Fig. 3 Orthotopic implantation of the murine pancreatic cancer cell line Pan02. Pan02 cells were transplanted into the pancreas of WT and APN-KO mice. Five weeks later, the mice were killed and analyzed. a The pancreas was removed along with the spleen and duodenum. The arrowhead indicates the tumor. b The tumor volume was plotted (n = 5, 6). *p \ 0.05. c–f Immunohistochemistry was
performed using TUNEL staining (c) and a CD34 antibody (e) followed by nuclear staining with methylene blue. d Apoptotic cells were reduced in APN-KO mice compared with those of WT mice. Data are presented as the mean ± SEM (n = 5, 6) *p \ 0.05. f Microvessel density was comparable between WT and APN-KO mice. Data are presented as the mean ± SEM (n = 5, 6)
adiponectin was proapoptotic for Pan02 cells. Treatment of Pan02 cells with adiponectin at concentrations over 10 lg/mL increased apoptosis (Fig. 2a). Caspases are effectors and executioners of cell death, and caspase activation is a crucial step in the induction of apoptosis. We then examined caspase activity after the treatment of Pan02 cells with adiponectin and found that adiponectin at concentrations over 5 lg/mL increased caspase activity (Fig. 2b).
growth of Pan02 cells that were transplanted into the pancreases of APN-KO mice. The body weight of the mice was comparable between WT and APN-KO on normal diet (WT, 23.8 ± 0.2 g, n = 6; APN-KO, 24.1 ± 0.3 g, n = 5; P = 0.46). Five weeks after transplantation, the tumor volume of APN-KO mice was significantly larger than that of WT mice (Fig. 3a, b). TUNEL staining of the tumor revealed that apoptosis was reduced in APN-KO mice compared with that of WT mice (Fig. 3c, d). Previously, adiponectin deficiency was shown to reduce tumor vascularization in a mouse model of mammary cancer [16]. In our model, however, CD34 staining of the tumor revealed that the microvessel density was comparable between APN-KO and WT mice (Fig. 3e, f).
Tumor Growth of Pan02 Cells Was Accelerated in APN-KO Mice To investigate the physiological relevance of our in vitro results, we evaluated the effect of adiponectin on the
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Discussion In this study, we observed the inhibitory effect of adiponectin on the proliferation of Pan02 cells in vitro. A subsequent in vivo study using the orthotopic implantation mouse model demonstrated that adiponectin deficiency promoted the growth of Pan02 cells. Taken together, our data provide novel evidence for a direct inhibitory effect of adiponectin on the growth of a murine pancreatic cancer cell line. Recently, two prospective cohort studies demonstrated that the circulating plasma adiponectin level was inversely associated with pancreatic cancer risk [17, 18]. Therefore, adiponectin may mediate the effect of obesity on the development and growth of pancreatic cancer. The effects of adiponectin on apoptosis are complicated. Some reports demonstrated the antiapoptotic activity of adiponectin during ischemia/perfusion injury, whereas others showed the apoptotic activity in cancer cell lines [19]. In this study, the inhibitory effect of adiponectin on the proliferation of Pan02 cells was associated with increased apoptosis in vitro and in vivo. In a mouse model of mammary cancer, adiponectin deficiency reduced the growth rate of tumors that was accompanied by reduced vascularization and increased apoptosis [16]. In our model, vascularization of the tumor was comparable between APN-KO and WT mice. Thus, adiponectin seems to downregulate the growth of pancreatic cancer through the induction of apoptosis. In conclusion, we demonstrated that adiponectin exerted an antiproliferative effect on murine pancreatic cancer cells in vitro and in vivo, and this activity might be mediated by inducing apoptosis. The therapeutic application of adiponectin has been suggested by studies showing that the pharmacologic enhancement of adiponectin and an adiponectin analog increased survival of myeloma-bearing mice and ameliorated type 2 diabetes in mice, respectively [20, 21]. Our results suggest that adiponectin is a potential therapeutic target in pancreatic cancer. Further studies are required to clarify the molecular mechanisms that underlie the antiproliferative activity of adiponectin in pancreatic cancer.
2. 3.
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6.
7.
8. 9.
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12.
13.
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16.
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18. Acknowledgments The authors thank Wei Li (Osaka University) for her excellent technical assistance. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.
19. 20.
Conflict of interest
None. 21.
References
(AdiPose Most abundant Gene transcript 1). Biochem Biophys Res Commun. 1996;221:286–289. Matsuzawa Y. Adiponectin: a key player in obesity related disorders. Curr Pharm Des. 2010;16:1896–1901. Nishihara T, Matsuda M, Araki H, et al. Effect of adiponectin on murine colitis induced by dextran sulfate sodium. Gastroenterology. 2006;131:853–861. Araki H, Nishihara T, Matsuda M, et al. Adiponectin plays a protective role in caerulein-induced acute pancreatitis in mice fed a high-fat diet. Gut. 2008;57:1431–1440. Kato M, Watabe K, Hamasaki T, et al. Association of low serum adiponectin levels with erosive esophagitis in men: an analysis of 2405 subjects undergoing physical check-ups. J Gastroenterol. 2011;46:1361–1367. Yamamoto S, Watabe K, Araki H, et al. Protective role of adiponectin against ethanol-induced gastric injury in mice. Am J Physiol Gastrointest Liver Physiol. 2012;302:G773–G780. Kishida K, Funahashi T, Shimomura I. Molecular mechanisms of diabetes and atherosclerosis: role of adiponectin. Endocr Metab Immune Disord Drug Targets. 2012;12:118–131. Housa D, Housova J, Vernerova Z, et al. Adipocytokines and cancer. Physiol Res. 2006;55:233–244. Yamauchi T, Kamon J, Ito Y, et al. Cloning of adiponectin receptors that mediate antidiabetic metabolic effects. Nature. 2003;423:762–769. Calle EE, Rodriguez C, Walker-Thurmond K, et al. Overweight, obesity, and mortality from cancer in a prospectively studied cohort of U.S. adults. N Engl J Med. 2003;348:1625–1638. Bracci PM. Obesity and pancreatic cancer: overview of epidemiologic evidence and biologic mechanisms. Mol Carcinog. 2012;51:53–63. White PB, Ziegler KM, Swartz-Basile DA, et al. Obesity, but not high-fat diet, promotes murine pancreatic cancer growth. J Gastrointest Surg. 2012;16:1680–1685. Maeda N, Shimomura I, Kishida K, et al. Diet-induced insulin resistance in mice lacking adiponectin/ACRP30. Nat Med. 2002; 8:731–737. Vonlaufen A, Phillips PA, Xu Z, et al. Pancreatic stellate cells: partners in crime with pancreatic cancer cells. Cancer Res. 2008; 68:2085–2093. Saxena NK, Fu PP, Nagalingam A, et al. Adiponectin modulates C-jun N-terminal kinase and mammalian target of rapamycin and inhibits hepatocellular carcinoma. Gastroenterology. 2010;139:1762–1773. Denzel MS, Hebbard LW, Shostak G, et al. Adiponectin deficiency limits tumor vascularization in the MMTV-PyV-mT mouse model of mammary cancer. Clin Cancer Res. 2009;15:3256–3264. Grote VA, Rohrmann S, Dossus L, et al. The association of circulating adiponectin levels with pancreatic cancer risk: a study within the prospective EPIC cohort. Int J Cancer. 2012;130: 2428–2437. Bao Y, Giovannucci EL, Kraft P, et al. A prospective study of plasma adiponectin and pancreatic cancer risk in five US cohorts. J Natl Cancer Inst. 2013;105:95–103. Sun Y, Chen X. Effect of adiponectin on apoptosis: proapoptosis or antiapoptosis? Biofactors. 2010;36:179–186. Fowler JA, Lwin ST, Drake MT, et al. Host-derived adiponectin is tumor-suppressive and a novel therapeutic target for multiple myeloma and the associated bone disease. Blood. 2011;118:5872–5882. Okada-Iwabu M, Yamauchi T, Iwabu M, et al. A small-molecule AdipoR agonist for type 2 diabetes and short life in obesity. Nature. 2013;503:493–499.
1. Maeda K, Okubo K, Shimomura I, et al. cDNA cloning and expression of a novel adipose specific collagen-like factor, apM1
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RAPID COMMUNICATIONS
Adiponectin Inhibits Murine Pancreatic Cancer Growth Motohiko Kato • Kenji Watabe • Masahiko Tsujii Tohru Funahashi • Iichiro Shimomura • Tetsuo Takehara
•
Received: 22 December 2013 / Accepted: 16 April 2014 Ó Springer Science+Business Media New York 2014
Abstract Background Adiponectin is an adipose tissue-derived secretory hormone whose plasma concentrations are lower in obese individuals. Obesity is a risk factor for the development and growth of pancreatic cancer, and hypoadiponectinemia was suggested to be involved in the growth of Pan02 murine pancreatic cancer cells that were inoculated into the flanks of congenitally obese mice. Aim The aim of this study was to clarify the role of adiponectin in the growth of pancreatic cancer cells. Methods We examined the effect of adiponectin on the growth of Pan02 cells using recombinant adiponectin and adiponectin knockout mice. Results The in vitro treatment of Pan02 cells with adiponectin inhibited cellular proliferation that was accompanied by increased apoptosis and caspase-3 and caspase-7 activities. Transplantation of Pan02 cells into the pancreas of knockout mice resulted in a larger tumor volume with fewer terminal deoxyribonucleotidyl transferase-
Motohiko Kato and Kenji Watabe contributed equally to this work. M. Kato K. Watabe M. Tsujii T. Takehara (&) Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan e-mail: [email protected] T. Funahashi Department of Metabolism and Atherosclerosis, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan I. Shimomura Department of Metabolic Medicine, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
mediated dUTP nick end labeling (TUNEL) positive cells compared with wild-type mice. Conclusions The results indicate that adiponectin directly suppresses the proliferation of Pan02 cells. Keywords Apoptosis
Pancreatic cancer Obesity Adiponectin
Introduction Adiponectin is an adipose tissue-derived secretory hormone. Compared with other adipose-derived hormones, adiponectin circulates abundantly throughout the body, and its plasma concentrations are lower in obese individuals [1, 2]. Adiponectin has antidiabetic, antiatherosclerotic, and anti-inflammatory properties in various organs [3–7]. Additionally, low levels of plasma adiponectin have been associated with common forms of cancer [8]. Two isoforms of adiponectin receptors, Adipo-R1 and Adipo-R2, have been identified, and these receptors may mediate some of the actions of adiponectin through the activation of intracellular signaling pathways [9]. Obesity is a risk factor for the development and growth of numerous cancers, including pancreatic cancer [10, 11]. The mechanisms underlying the association between obesity and pancreatic cancer have been investigated through hormonal, inflammatory, and immunological changes in obesity. Recently, it was reported that serum adiponectin negatively correlated with tumor weight in a mouse model of pancreatic cancer using congenitally obese mice and flank-implanted Pan02 murine pancreatic cancer cells [12]. In this study, we investigated the role of adiponectin in the growth of Pan02 cells using recombinant adiponectin and adiponectin knockout (APN-KO) mice.
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Materials and Methods
caspase-7 (Caspase-Glo assay, Promega, Madison, WI) according to the manufacturer’s protocol.
Cell Culture Animals Pan02 is a murine pancreatic adenocarcinoma cell line that is syngeneic to C57BL/6 and was obtained from the Division of Cancer Treatment Tumor Repository (National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD). The cells were maintained in RPMI 1640 medium (Sigma Chemical, St. Louis, MO) containing 10 % FBS (Sigma Chemical, St. Louis, MO) and 1 % antibiotic–antimycotic (Invitrogen, Carlsbad, CA) in a humidified atmosphere of 95 % air with 5 % CO2 at 37 °C. Real-Time RT-PCR Analysis Total RNA was extracted using QIAshredder and RNeasy Mini kits (Qiagen, Hilden, Germany), and samples (0.3 lg of total RNA) were reverse-transcribed in 20 lL of a reaction mixture that contained 200 U of Superscript II (GIBCO-BRL, Grand Island, NY) and 0.2 lg of oligo (dT) primer. Quantitative real-time PCR was performed using a QuantiFast SYBR Green PCR kit with specific primers (Qiagen) on a LightCycler (Roche Diagnostics, Indianapolis, IN). Primers used were GAPDH (QT00199388, Qiagen), Adipo-R1 (forward, 50 -ACAGAGTCTGAAGCAAGAGCA-30 ; reverse 50 -GGTG ATGGTACCGTCCGTGAG-30 ), and Aipo-R2 (forward 50 AAGACTCGGGCCTACAATGGACAGCCATGG-30 ; reverse 50 -CAATGTTGATACGTCCACAATTAC-30 ). The mRNA expression levels were normalized relative to that of GAPDH and expressed in arbitrary units. Adiponectin Treatment of Pan02 Cells Recombinant human adiponectin and bovine serum albumin were purchased from R&D systems (Minneapolis, MN) and Takara Bio Inc. (Otsu, Japan), respectively. For the adiponectin assay, Pan02 cells were serum-starved for 24 h followed by adiponectin treatment in fresh media as indicated. Cell proliferation was determined by WST assay using Cell Count Reagent SF (Nacalai Tesque, Kyoto, Japan). Apoptotic cells were detected using the Annexin V-FITC Apoptosis Detection Kit (BioVision, Milpitas, CA) according to the manufacturer’s instructions. Briefly, cells were trypsinized, pooled together with spontaneously detached cells, and stained with Annexin-V and propidium iodide. Labeled cells were analyzed using a FACSAria flow cytometer (BD Biosciences, San Jose, CA). Apoptotic cells were counted as those cells that were double-positive for Annexin-V and propidium iodide. Caspase activity was measured in the supernatant of the cultured Pan02 cells using a luminescent substrate assay for caspase-3 and
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APN-KO mice were generated as described previously and backcrossed with the C57BL/6 J strain for five generations [13]. C57BL/6 J mice were purchased as wild-type (WT) controls from Clea Japan (Tokyo, Japan). The ethics committee for animal experimentation of Osaka University Graduate School of Medicine approved the animal experiments that were reported in this study. Orthotopic Implantation Model of Pancreatic Cancer Pan02 cells were transplanted to the pancreases of mice following the method described by Vonlaufen with minor modifications [14]. Eight- to ten-week-old WT or APN-KO male mice were anesthetized, and an incision was made in the left flank. The spleen and tail of the pancreas were exteriorized, and 5 9 105 Pan02 cells were injected into the tail of the pancreas. Five weeks after transplantation, the mice were killed, the pancreas was removed with spleen and duodenum, and the tumor size was measured. The tumor volume was calculated as (length 9 width2)/2. All tumors were processed for histological examination. Immunohistochemistry Apoptotic cells were identified using terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL; ApopTag, Chemicon International, Temecula, CA) according to the manufacturer’s protocol followed by nuclear staining with methylene blue. The proportion of TUNEL-positive cells in the tumor was calculated from 10 randomly selected high-power fields. CD34 staining was performed using an antibody purchased from Cedarlane Laboratories (Ontario, Canada), and staining was detected using the respective biotinylated secondary IgGs (BD Biosciences), streptavidin/horseradish peroxidase (Vector Labs, Burlingame, CA), and 3,30 -diaminobenzidine (Dako, Carpinteria, CA). Nuclear staining was performed using methylene blue. Microvessel density of the tumor was defined as the number of microvessel per microscopic field and calculated from 10 randomly selected high-power fields. Statistical Analysis Results are expressed as the mean ± SEM. Comparisons between groups were performed using the Wilcoxon rank test for nonparametric data, and more than three groups were compared using Tukey’s honestly significant difference (HSD) test. A P value less than 0.05 was considered statistically
Dig Dis Sci
Fig. 1 The effect of adiponectin on proliferation of the murine pancreatic cancer cell line Pan02. a Real-time RT-PCR analysis of the mRNA expression of the adiponectin receptors Adipo-R1 and AdipoR2 in Pan02 cells and mouse liver tissue (n = 4). Data are expressed as the mean ± SEM. Significant difference is shown. ***p \ 0.001.
b Pan02 cells were serum-starved for 24 h followed by the treatment with various concentrations of adiponectin, and cell proliferation was assessed using the WST assay (n = 6). Data are expressed as the mean ± SEM. Significant differences to no adiponectin are shown. *p \ 0.05
Fig. 2 Adiponectin is proapoptotic for the murine pancreatic cancer cell line Pan02. Pan02 cells were serum-starved for 24 h followed by the treatment with various concentrations of adiponectin and assayed for apoptosis and caspase activity. a Apoptotic cells were presented as the percentage of cells that were positive for Annexin-V and propidium iodide using flow cytometry (n = 3). Data are expressed
as the mean ± SEM. Significant difference to no adiponectin is shown. ***p \ 0.001. b Caspase activity was measured using a luminescent substrate assay for caspase-3 and caspase-7 (n = 3). Data are expressed as the mean ± SEM. Significant differences to no adiponectin are shown. *p \ 0.05, ***p \ 0.001
significant. Data analysis was performed using the JMP 9 statistical package (Statistical Analysis Systems, Cary, NC).
between the two groups (Fig. 1a). We then examined the effect of recombinant adiponectin on the proliferation of pancreatic cancer cells. Adiponectin treatment at concentrations over 5 lg/mL inhibited the proliferation of Pan02 cells (Fig. 1b). In contrast, bovine serum albumin as control protein did not affect the proliferation of the cells over 5 lg/mL (data not shown).
Results Adiponectin Inhibits the Proliferation of the Murine Pancreatic Cancer Cell Line Pan02
Adiponectin Induces Apoptosis in Pan02 Cells Because adiponectin demonstrates its biological functions through binding to its membrane receptors, we first examined the expression of these receptors in pancreatic cancer cells in comparison with mouse liver tissue as control. While Adipo-R1 was expressed 3 times more abundantly in murine pancreatic cell line Pan02 compared with liver tissue, Adipo-R2 was expressed similarly
Previously, adiponectin was shown to inhibit the cellular proliferation of hepatocellular carcinoma cells, and one of the targets for adiponectin action was cyclin D1 [15]. In our study, adiponectin did not affect the expression of either cyclin D1 or a marker for proliferation, proliferating cell nuclear antigen, in Pan02 cells (data not shown). We then examined whether
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Fig. 3 Orthotopic implantation of the murine pancreatic cancer cell line Pan02. Pan02 cells were transplanted into the pancreas of WT and APN-KO mice. Five weeks later, the mice were killed and analyzed. a The pancreas was removed along with the spleen and duodenum. The arrowhead indicates the tumor. b The tumor volume was plotted (n = 5, 6). *p \ 0.05. c–f Immunohistochemistry was
performed using TUNEL staining (c) and a CD34 antibody (e) followed by nuclear staining with methylene blue. d Apoptotic cells were reduced in APN-KO mice compared with those of WT mice. Data are presented as the mean ± SEM (n = 5, 6) *p \ 0.05. f Microvessel density was comparable between WT and APN-KO mice. Data are presented as the mean ± SEM (n = 5, 6)
adiponectin was proapoptotic for Pan02 cells. Treatment of Pan02 cells with adiponectin at concentrations over 10 lg/mL increased apoptosis (Fig. 2a). Caspases are effectors and executioners of cell death, and caspase activation is a crucial step in the induction of apoptosis. We then examined caspase activity after the treatment of Pan02 cells with adiponectin and found that adiponectin at concentrations over 5 lg/mL increased caspase activity (Fig. 2b).
growth of Pan02 cells that were transplanted into the pancreases of APN-KO mice. The body weight of the mice was comparable between WT and APN-KO on normal diet (WT, 23.8 ± 0.2 g, n = 6; APN-KO, 24.1 ± 0.3 g, n = 5; P = 0.46). Five weeks after transplantation, the tumor volume of APN-KO mice was significantly larger than that of WT mice (Fig. 3a, b). TUNEL staining of the tumor revealed that apoptosis was reduced in APN-KO mice compared with that of WT mice (Fig. 3c, d). Previously, adiponectin deficiency was shown to reduce tumor vascularization in a mouse model of mammary cancer [16]. In our model, however, CD34 staining of the tumor revealed that the microvessel density was comparable between APN-KO and WT mice (Fig. 3e, f).
Tumor Growth of Pan02 Cells Was Accelerated in APN-KO Mice To investigate the physiological relevance of our in vitro results, we evaluated the effect of adiponectin on the
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Discussion In this study, we observed the inhibitory effect of adiponectin on the proliferation of Pan02 cells in vitro. A subsequent in vivo study using the orthotopic implantation mouse model demonstrated that adiponectin deficiency promoted the growth of Pan02 cells. Taken together, our data provide novel evidence for a direct inhibitory effect of adiponectin on the growth of a murine pancreatic cancer cell line. Recently, two prospective cohort studies demonstrated that the circulating plasma adiponectin level was inversely associated with pancreatic cancer risk [17, 18]. Therefore, adiponectin may mediate the effect of obesity on the development and growth of pancreatic cancer. The effects of adiponectin on apoptosis are complicated. Some reports demonstrated the antiapoptotic activity of adiponectin during ischemia/perfusion injury, whereas others showed the apoptotic activity in cancer cell lines [19]. In this study, the inhibitory effect of adiponectin on the proliferation of Pan02 cells was associated with increased apoptosis in vitro and in vivo. In a mouse model of mammary cancer, adiponectin deficiency reduced the growth rate of tumors that was accompanied by reduced vascularization and increased apoptosis [16]. In our model, vascularization of the tumor was comparable between APN-KO and WT mice. Thus, adiponectin seems to downregulate the growth of pancreatic cancer through the induction of apoptosis. In conclusion, we demonstrated that adiponectin exerted an antiproliferative effect on murine pancreatic cancer cells in vitro and in vivo, and this activity might be mediated by inducing apoptosis. The therapeutic application of adiponectin has been suggested by studies showing that the pharmacologic enhancement of adiponectin and an adiponectin analog increased survival of myeloma-bearing mice and ameliorated type 2 diabetes in mice, respectively [20, 21]. Our results suggest that adiponectin is a potential therapeutic target in pancreatic cancer. Further studies are required to clarify the molecular mechanisms that underlie the antiproliferative activity of adiponectin in pancreatic cancer.
2. 3.
4.
5.
6.
7.
8. 9.
10.
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12.
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15.
16.
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18. Acknowledgments The authors thank Wei Li (Osaka University) for her excellent technical assistance. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.
19. 20.
Conflict of interest
None. 21.
References
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1. Maeda K, Okubo K, Shimomura I, et al. cDNA cloning and expression of a novel adipose specific collagen-like factor, apM1
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