A role of ghrelin in canine mammary carcinoma cells proliferation, apoptosis and migration

BMC Veterinary Research

Magdalena Król³  Tomasz Motyl³  Joanna Mucha³  Izabella Dolka²  Emilia J Orzechowska¹  Karol M Pawłowski³  Kinga Majchrzak³ 

[1] Department of Molecular Biology, Faculty of Biology, Warsaw University, Miecznikowa 1, Warsaw, Poland;Department of Pathology and Veterinary Diagnostics, Faculty of Veterinary Medicine, Warsaw University of Life Sciences - WULS, Nowoursynowska 159, Warsaw, 02-776, Poland;Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences - WULS, Nowoursynowska 159, Warsaw, 02-776, Poland

关键词: Growth hormone secretagogue receptor;    Ghrelin;    Canine mammary carcinoma;   

Others  :  1119717
DOI  :  10.1186/1746-6148-8-170

 received in 2012-05-11, accepted in 2012-09-20,  发布年份 2012

【 摘 要 】

Background

Ghrelin is a natural ligand of the growth hormone secretagogue receptor (GHS-R). They are often co-expressed in multiple human tumors and related cancer cell lines what can indicate that the ghrelin/GHS-R axis may have an important role in tumor growth and progression. However, a role of ghrelin in canine tumors remains unknown. Thus, the aim of our study was two-fold: (1) to assess expression of ghrelin and its receptor in canine mammary cancer and (2) to examine the effect of ghrelin on carcinoma cells proliferation, apoptosis, migration and invasion. The expression of ghrelin and its receptor in canine mammary cancer tissues and cell lines (isolated from primary tumors and their metastases) was examined using Real-time qPCR and immunohistochemistry. For apoptosis analysis the Annexin V and propidium iodide dual staining was applied whereas cell proliferation was evaluated by MTT assay and BrdU incorporation test. The influence of ghrelin on cancer cells migration and invasion was assessed using Boyden chamber assays and wound healing assay.

Results

The highest expression of ghrelin was observed in metastatic cancers whereas the lowest expression of ghrelin receptor was detected in tumors of the 3^rd grade of malignancy. Higher expression of ghrelin and its receptor was detected in cancer cell lines isolated from metastases than in cell lines isolated from primary tumors. In vitro experiments demonstrated that exposure to low doses of ghrelin stimulates cellular proliferation, inhibits apoptosis and promotes motility and invasion of canine mammary cancer cells. Growth hormone secretagogue receptor inhibitor ([D-Lys³]-GHRP6) as well as RNA interference enhances early apoptosis.

Conclusion

The presence of ghrelin and GHS-R in all of the examined canine mammary tumors may indicate their biological role in cancer growth and development. Our experiments conducted in vitro confirmed that ghrelin promotes cancer development and metastasis.

【 授权许可】

   
2012 Majchrzak et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

Figure 9.

Figure 10.

Figure 11.

Figure 12.

Figure 13.

Figure 14.

【 参考文献 】

• [1]Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K: Ghrelin is a growth hormone-releasing acylated peptide from stomach. Nature 1999, 402:656-660.
• [2]Howard AD, Feighner SD, Cully DF, Arena JP, Liberator PA, Rosenblum CI, Hamelin M, Hreniuk DL, Palyha OC, Anderson J, Paress PS, Diaz C, Chou M, Liu KK, McKee KK, Pong SS, Chaung LM, Elbrecht A, Dashkevicz M, Heavens R, Rigby M, Sirinathsinghji DJ, Dean DC, Melillo DG, Patchett AA, Nargund R, Griffin PS, DeMartino JA, Gupta SK, Schaeffer JM, Smith RG, Van der Ploeg LH: A receptor in pituitary and hypothalamus that functions in growth hormone release. Science 1996, 273:974-977.
• [3]Gnanapavan S, Kola B, Bustin SA, Morris DG, McGee P, Fairclough P, Bhattacharya S, Carpenter R, Ashley B, Korbonits G, Korbonits M: The tissue distribution of the mRNA of ghrelin and subtypes of its receptor, GHS-R, in humans. J Clin Endocrinol Metab 2002, 87:2988-2991.
• [4]Kojima M, Hosoda H, Matsuo H, Kangawa K: Ghrelin: discovery of the natural endogenous ligand for the growth hormone secretagogue receptor. Trends Endocrinol Metab 2001, 12:118-122.
• [5]Date Y, Kojima M, Hosoda H, Sawaguchi A, Mondal MS, Suganuma T, Matsukura S, Kangawa K, Nakazato M: Ghrelin, a novel growth hormone-releasing acylated peptide, is synthesized in a distinct endocrine cell type in the gastrointestinal tracts of rats and humans. Endocrinology 2000, 141:4255-4266.
• [6]Leite-Moreira AF, Soares JB: Physiological, pathological and potential therapeutic roles of ghrelin. Drug Discov Today 2007, 12:276-288.
• [7]Yokoyama M, Nakahara K, Kojima M, Hosoda H, Kangawa K, Murakami N: Influencing the between-feeding and endocrine responses of plasma ghrelin in healthy dogs. Eur J Endocrinol 2005, 152:155-160.
• [8]Bhatti SF, Hofland LJ, van Koetsveld PM, Van Ham LM, Duchateau L, Mol JA, van der Lely AJ, Kooistra HS: Effects of food intake and food withholding on plasma ghrelin concentrations in healthy dogs. Am J Vet Res 2006, 67:1557-1563.
• [9]Jeusette IC, Lhoest ET, Istasse LP, Diez MO: Influence of obesity on plasma lipid and lipoprotein concentrations in dogs. Am J Vet Res 2005, 66:81-86.
• [10]Majchrzak K, Szyszko K, Pawłowski KM, Motyl T, Król M: A role of ghrelin in cancerogenesis. Pol J Vet Sci 2012, 15:181-189.
• [11]Cassoni P, Papotti M, Catapano F, Ghe C, Deghenghi R, Ghigo E, Muccioli G: Specific binding sites for synthetic growth hormone secretagogues in non-tumoral and neoplastic human thyroid tissue. J Endocrinol 2000, 165:139-146.
• [12]Cassoni P, Papotti M, Ghe C, Catapano F, Sapino A, Graziani A, Deghenghi R, Reissman T, Ghigo E, Muccioli G: Identification, characterization and biological activity of specific receptors for natural (ghrelin) and synthetic growth hormone secretagogues and analogs in human breast carcinoma and cell lines. J Clin Endocrinol Metab 2001, 86:1738-1745.
• [13]Cassoni P, Ghe C, Marrocco T, Tarabra E, Allia E, Catapano F, Deghenghi R, Ghigo E, Papotti M, Muccioli G: Expression of ghrelin and biological activity of specific receptors for ghrelin and des-acyl ghrelin in human prostate neoplasms and related cell lines. Eur J Endocrinol 2004, 150:73-184.
• [14]Ghe C, Cassoni P, Catapano F, Marrocco T, Deghenghi R, Ghigo E, Muccioli G, Papotti M: The antiproliferative effect of synthetic peptidyl GH secretagogues in human CALU-1 lung carcinoma cells. Endocrinology 2002, 143:484-491.
• [15]Jeffery PL, Herington AC, Chopin LK: Expression and action of the growth hormone releasing peptide ghrelin and its receptor in prostate cancer cell lines. J Endocrinol 2002, 172:R7-R11.
• [16]Jeffery PL, Murray RE, Yeh AH, McNamara JF, Duncan RP, Francis GD, Herington AC, Chopin LK: Expression and function of the ghrelin axis, including a novel preproghrelin isoform, in human breast cancer tissues and cell lines. Endocr Relat Cancer 2005, 12:839-850.
• [17]Murata M, Okimura Y, Iida K, Matsumoto M, Sowa H, Kaji H, Kojima M, Kangawa K, Chihara K: Ghrelin modulates the downstream molecules of insulin signaling in hepatoma cells. J Biol Chem 2002, 277:5667-5674.
• [18]Duxbury MS, Waseem T, Ito H, Robinson MK, Zinner MJ, Ashley SW, Whang EE: Ghrelin promotes pancreatic adenocarcinoma cellular proliferation and invasiveness. Biochem Biophys Res Commun 2003, 309:464-468.
• [19]Volante M, Allia E, Fulcheri E, Cassoni P, Ghigo E, Muccioli G, Papotti M: Ghrelin in fetal thyroid and follicular tumors and cell lines: expression and effect on tumor growth. Am J Pathol 2003, 162:645-654.
• [20]Nanzer AM, Khalaf S, Mozid AM, Fowkes RC, Patel MV, Burrin JM, Grossman AB, Korbonits M: Ghrelin exerts a proliferative effect on a rat pituitary somatotroph cell line via the mitogen-activated protein kinase pathway. Eur J Endocrinol 2004, 151:233-240.
• [21]Yeh AH, Jeffery PL, Duncan RP, Herington AC, Chopin LK: Ghrelin and a novel preproghrelin isoform are highly expressed in prostate cancer and ghrelin activates mitogen-activated protein kinase in prostate cancer. Clin Cancer Res 2005, 11:8295-8303.
• [22]Delhanty PJ, van Koetsveld PM, Gauna C, van de Zande B, Vitale G, Hofland LJ, van der Lely AJ: Ghrelin and its unacylated isoform stimulate the growth of adrenocortical tumor cells via an anti-apoptotic pathway. Am J Physiol Endocrinol Metab 2007, 293:E302-E309.
• [23]Król M, Polańska J, Pawłowski KM, Turowski P, Skierski J, Majewska A, Ugorski M, Morty RE, Motyl T: Transcriptomic signature of cell lines isolated from canine mammary adenocarcinoma metastases to lungs. J Appl Genet 2010, 51:37-50.
• [24]Król M, Pawłowski KM, Skierski J, Turowski P, Majewska A, Polańska J, Ugorski M, Morty RE, Motyl T: Transcriptomic “portraits” of canine mammary cancer cell lines with various phenotypes. J Appl Genet 2010, 51:169-183.
• [25]Pawłowski KM, Popielarz D, Szyszko K, Gajewska M, Motyl T, Król M: Growth hormone receptor (ghr) RNAi decreases proliferation and enhances apoptosis in CMT-U27 canine mammary carcinoma cell line. Vet Comp Oncol 2011, 10:2-15.
• [26]Misdorp W, Else RW, Hellemen A, Lipscomb TP: Histological classification of mammary tumors of the dog and cat. Armed Forces Institute of Pathology 1999, 7:11-29.
• [27]Miasdorp W: Tumors of the mammary gland. Iowa State Press, Iowa, USA; 2002:575-606.
• [28]Król M, Pawłowski KM, Majchrzak K, Gajewska M, Majewska A, Motyl T: Global gene expression profiles of canine macrophages and canine mam mary cancer cells grown as a co-culture in vitro. BMC Vet Res 2012, 8:16. BioMed Central Full Text
• [29]Król M, Pawłowski KM, Szyszko K, Maciejewski H, Dolka I, Manuali E, Jank M, Motyl T: Gene expression profiles of canine mammary cancer cells grown with carcinoma-associated fibroblasts (CAFs) as a co-culture in vitro. BMC Veterinary Research 2012, 8:35. BioMed Central Full Text
• [30]Schmittgen TD, Livac KJ: Analyzing real-time PCR data by the comparative Ct method. Nat Prot 2008, 3:1101-1108.
• [31]Katugampola SD, Pallikaros Z, Davenport AP: [125I-His(9)]-ghrelin, a novel radioligand for localizing GHS orphan receptors in human and rat tissue: up-regulation of receptors with athersclerosis. Br J Phyarmacol 2001, 134:143-149.
• [32]Rodriguez LG, Wu X, Guan JL: Wound-healing assay. Methods Mol Biol 2005, 294:23-29.
• [33]Waseem T, Javaid-Ur-Rehman , Ahmad F, Azam M, Qureshi MA: Role of ghrelin axis in colorectal cancer: a novel association. Peptides 2008, 29:1369-1376.
• [34]Wang Z, Wang W, Qiu W, Fan Y, Zhao J, Wang Y, Zheng Q: Involvement of ghrelin-growth hormone secretagogue receptor system in pathoclinical profiles of digestive system cancer. Acta Biochim Biophys Sin 2007, 39:992-998.
• [35]He XT, Fan XM, Zha XL: Ghrelin inhibites 5-fluorouracil-induced apoptosis in colonic cancer cells. J Gastroenterol Hepatol 2011, 26:1169-1173.
• [36]Kim MS, Yoon CY, Jang PG, Park YS, Shin CS, Park HS, Ryu JW, Pak YK, Park JY, Lee KU, Kim SY, Lee HK, Kim YB, Park KS: The mitogenic and antiapoptotic actions of ghrelin in 3T3-L1 adipocytes. Mol Endocrinol 2004, 18:2291-2301.
• [37]Kim SW, Her SJ, Park SJ, Kim D, Park KS, Lee HK, Han BH, Kim MS, Shin CS, Kim SY: Ghrelin stimulates proliferation and differentiation and inhibits apoptosis in osteoblastic MC3T3-E1 cells. Bone 2005, 37:359-369.
• [38]Xiang Y, Li Q, Li M, Wang W, Cui C, Zhang J: Ghrelin inhibits AGEs-induced apoptosis in human endothelial cells involving ERK1/2 and PI3K/Akt pathways. Cell Biochem Funct 2011, 29:149-155.
• [39]Dixit VD, Weeraratna AT, Yang H, Bertak D, Cooper-Jenkins A, Riggins GJ, Eberhart CG, Taub DD: Ghrelin and the growth hormone secretagogue receptor constitute a novel autocrine pathway in astrocytoma motility. J Biol Chem 2006, 281:16681-16690.
• [40]Chen JH, Huang SM, Chen CC, Tsai CF, Yeh WL, Chou SJ, Hsieh WT, Lu DY: Ghrelin induces cell migration through GHS-R, CaMKII, AMPK, and NF-ĸB signaling pathway in glioma cells. J Cell Biochem 2011, 112:2931-2941.