Salivary α-amylase exhibits antiproliferative effects in primary cell cultures of rat mammary epithelial cells and human breast cancer cells

Journal of Experimental & Clinical Cancer Research

Wolfgang Löscher¹  Catharina Bertram²  Ralf Hass²  Maren Fedrowitz¹ 

[1] Department of Pharmacology, Toxicology, and Pharmacy, University of Veterinary Medicine, Buenteweg 17, Hannover, 30559, Germany;Biochemistry and Tumor Biology Lab, Gynecology Research Unit, Department of Obstetrics and Gynecology, Carl-Neuberg-Str. 1, Medical University, Hannover, 30625, Germany

关键词: mammary gland;    primary cell culture;    breast cancer;    cell proliferation;    amylase;   

Others  :  827116
DOI  :  10.1186/1756-9966-30-102

 received in 2011-08-11, accepted in 2011-10-25,  发布年份 2011

【 摘 要 】

Background

Breast cancer is one of the most diagnosed cancers in females, frequently with fatal outcome, so that new strategies for modulating cell proliferation in the mammary tissue are urgently needed. There is some, as yet inconclusive evidence that α-amylase may constitute a novel candidate for affecting cellular growth.

Methods

The present investigation aimed to examine if salivary α-amylase, an enzyme well known for the metabolism of starch and recently introduced as a stress marker, is able to exert antiproliferative effects on the growth of mammary gland epithelial cells.

For this purpose, primary epithelial cultures of breast tissue from two different inbred rat strains, Fischer 344 (F344) and Lewis, as well as breast tumor cells of human origin were used. Treatment with human salivary α-amylase was performed once daily for 2 days followed by cell counting (trypan blue assay) to determine alterations in cell numbers. Cell senescence after α-amylase treatment was assessed by β-galactosidase assay. Endogenous α-amylase was detected in cells from F344 and Lewis by immunofluorescence.

Results

Salivary α-amylase treatment in vitro significantly decreased the proliferation of primary cells from F344 and Lewis rats in a concentration-dependent manner. Noticeably, the sensitivity towards α-amylase was significantly higher in Lewis cells with stronger impact on cell growth after 5 and 50 U/ml compared to F344 cells. An antiproliferative effect of α-amylase was also determined in mammary tumor cells of human origin, but this effect varied depending on the donor, age, and type of the cells.

Conclusions

The results presented here indicate for the first time that salivary α-amylase affects cell growth in rat mammary epithelial cells and in breast tumor cells of human origin. Thus, α-amylase may be considered a novel, promising target for balancing cellular growth, which may provide an interesting tool for tumor prophylaxis and treatment.

【 授权许可】

   
2011 Fedrowitz et al; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Jemal A, Siegel R, Xu J, Ward E: Cancer Statistics, 2010. CA Cancer J Clin 2010, 60:277-300.
• [2]Karnoub AE, Dash AB, Vo AP, Sullivan A, Brooks MW, Bell GW, Richardson AL, Polyak K, Tubo R, Weinberg RA: Mesenchymal stem cells within tumor stroma promote breast cancer metastasis. Nature 2007, 449:557-563.
• [3]Finak G, Bertos N, Pepin F, Sadekova S, Souleimanova M, Zhao H, Chen H, Omeroglu G, Meterissian S, Omeroglu A, Hallett A, Park M: Stromal gene expression predicts clinical outcome in breast cancer. Nature Med 2008, 14:518-527.
• [4]Fedrowitz M, Löscher W: Effects of magnetic field exposure in the mammary gland tissue of female Fischer 344 rats and the role of amylase. Eur J Cancer 2007, 5(Suppl):77.
• [5]Fedrowitz M, Löscher W: Alterations in amylase activity in the mammary gland of female Fischer 344 rats after exposure to 50 Hertz magnetic fields. Naunyn Schmiedeberg´s Arch Pharmacol 2008, 377(Suppl 1):83.
• [6]Zakowski JJ, Bruns DE: Biochemistry of human alpha amylase isoenzymes. Crit Rev Clin Lab Sci 1985, 21:283-322.
• [7]Moridani MJ, Bromberg IL: Lipase and pancreatic amylase versus total amylase as biomarkers of pancreatitis: an analytical investigation. Clin Biochem 2003, 36:31-33.
• [8]Brown RC, Chalmers DM, Rowe VL, Kelleher J, Littlewood JM, Losowsky MS: Comparison of the diagnostic value of serum pancreatic isoamylase and immunoreactive trypsin measurement in patients with cystic fibrosis. J Clin Pathol 1982, 35:547-549.
• [9]Zakowski JJ, Gregory MR, Bruns DE: Amylase from human serous ovarian tumors: purification and characterization. Clin Chem 1984, 30:62-68.
• [10]Gregory MR, Gregory WW, Bruns DE, Zakowski JJ: Amylase inhibits Neisseria gonorrhoeae by degrading starch in the growth medium. J Clin Microbiol 1983, 18:1366-1369.
• [11]Chaudhuri B, Rojek J, Vickerman MM, Tanzer JM, Scannapieco FA: Interaction of salivary alpha-amylase and amylase-binding-protein A (AbpA) of Streptococcus gordonii with glucosyltransferase of S. gordonii and Streptococcus mutans. BMC Microbiology 2007, 7:60. BioMed Central Full Text
• [12]Groot PC, Bleeker MJ, Pronk JC, Arwert F, Mager WH, Planta RJ, Eriksson AW, Frants RR: The human α-amylase multigene family consists of haplotypes with variable numbers of genes. Genomics 1989, 5:29-42.
• [13]Heitlinger LA, Lee PC, Dillon WP, Lebenthal E: Mammary amylase: a possible alternate pathway of carbohydrate digestion in infancy. Pediatr Res 1983, 17:15-18.
• [14]Skerlavay M, Epstein JA, Sobrero AJ: Cervical mucus amylase levels in normal menstrual cycles. Fertil Steril 1968, 19:726-730.
• [15]Hokari S, Miura K, Koyama I, Kobayashi M, Matsunaga T, Iino N, Komoda T: Expression of α-amylase isoenzymes in rat tissues. Comp Biochem Physiol Part B 2003, 135:63-69.
• [16]Yanagitani N, Kaira K, Sunaga N, Naito Y, Koike Y, Ishihara S, Ishizuka T, Saito R, Mori M: Serum amylase is a sensitive tumor marker for amylase-producing small cell lung cancer? Int J Clin Oncol 2007, 12:231-233.
• [17]Tomita N, Matsuura N, Horii A, Emi M, Nishide T, Ogawa M, Mori T, Doi O, Matsubara K: Expression of α-amylase in human lung cancers. Cancer Res 1988, 48:3288-3291.
• [18]Coyne JD, Dervan PA: Primary acinic cell carcinoma of the breast. J Clin Pathol 2005, 55:545-547.
• [19]Tanahashi C, Yasuki S, Akamine N, Yatabe Y, Ichihara S: Pure acinic cell carcinoma of the breast in an 80-year-old Japanese woman. Pathol Int 2007, 57:43-46.
• [20]Beard J: The cancer problem. Lancet 1905, 4:281-283.
• [21]Novak JF, Trnka F: Proenzyme therapy of cancer. Anticancer Res 2005, 25:1157-1178.
• [22]Nagasawa H, Kusakawa S: Comparison of plasma component levels in four strains of female mice with different mammary tumour potentials. In Vivo 2001, 15:139-144.
• [23]Simickova M, Pecen L, Eben K, Nekulova M, Vermousek I, Stratil P, Rejthar A, Cernoch M, Lang B, Sakalova J: Biochemical analysis of breast cyst fluid as a possible predictor of breast carcinoma development. Neoplasma 1994, 41:245-252.
• [24]Saez Mdel C, Barriga C, Garcia JJ, Rodriguez AB, Ortega E: Exercise-induced stress enhances mammary tumor growth in rats: Beneficial effect of the hormone melatonin. Mol Cell Biochem 2007, 294:19-24.
• [25]Rohleder N, Nater UM, Wolf JM, Ehlert U, Kirschbaum C: Psychosocial stress-induced activation of salivary alpha-amylase: An indicator of sympathetic activity? Ann NY Acad Sci 2004, 1032:258-263.
• [26]van Stegeren A, Rohleder N, Everaerd W, Wolf OT: Salivary alpha amylase as marker for adrenergic activity during stress: effect of betablockade. Psychoendocrinology 2006, 31:137-141.
• [27]Nater UM, Rohleder N: Salivary alpha-amylase as a non-invasive biomarker for the sympathetic nervous system: Current state of research. Psychoendocrinology 2009, 34:486-496.
• [28]Dhabhar FS, McEwen BS, Spencer RL: Stress response, adrenal steroid receptor levels and corticosteroid-binding globulin levels - a comparison between Sprague-Dawley, Fischer 344 and Lewis rats. Brain Res 1993, 616:89-98.
• [29]Sternberg EM, Hill JM, Chrousos GP, Kamilaris T, Listwak SJ, Gold PW, Wilder RL: Inflammatory mediator-induced hypothalamic-pituitary-adrenal axis activation is defective in streptococcal cell wall arthritis-susceptible Lewis rats. Proc Natl Acad Sci 1989, 86:2374-2378.
• [30]Dhabhar FS, Miller AH, McEwen BS, Spencer RL: Differential activation of adrenal steroid receptors in neural and immune tissues of Sprague-Dawley, Fischer 344, and Lewis rats. J Neuroimmunology 1995, 56:77-90.
• [31]Haag JD, Newton MA, Gould MN: Mammary carcinoma suppressor and susceptibility genes in the Wistar-Kyoto rat. Carcinogenesis 1992, 13:1933-1935.
• [32]Hass R, Bertram C: Characterization of human breast cancer epithelial cells (HBCEC) derived from long term cultured biopsies. J Exp Clin Cancer Res 2009, 28:127-139. BioMed Central Full Text
• [33]Novaro V, Roskelley CD, Bissell MJ: Collagen-IV and laminin-1 regulate estrogen receptor α expression and function in mouse mammary epithelial cells. J Cell Sci 2003, 116:2975-2986.
• [34]Lavrentieva A, Majore I, Kasper C, Hass R: Effects of hypoxic culture conditions on umbilical cord-derived human mesenchymal stem cells. Cell Commun Signal 2010, 8:18. BioMed Central Full Text
• [35]Gilles C, Polette M, Zahm JM, Tournier JM, Volders L, Foidart J, Birembaut P: Vimentin contributes to human mammary epithelial cell migration. J Cell Sci 1999, 112:4615-4625.
• [36]Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, Linskens M, Rubeli I, Pereira-Smith O, Peacocke M, Campisi J: A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci 1995, 92:9363-9367.
• [37]Matoso A, Easley SE, Gnepp DR, Mangray S: Salivary gland acinar-like differentiation of the breast. Histopathology 2009, 54:262-263.
• [38]Lindberg T, Skude G: Amylase in human milk. Pediatrics 1982, 70:235-238.
• [39]Hall FF, Ratliff CR, Hayakawa T, Culp TW, Hightower NC: Substrate differentiation of human pancreatic and salivary alpha-amylases. Am J Dig Dis 1970, 15:1031-1038.
• [40]Stiefel DJ, Keller PJ: Comparison of human pancreatic and parotid amylase activities on different substrates. Clin Chem 1975, 21:343-346.
• [41]Dhabhar FS, McEwen BS, Spencer RL: Adaptation to prolonged or repeated stress - comparison between rat strains showing intrinsic differences in reactivity to acute stress. Neuroendocrinology 1997, 65:360-368.
• [42]Fedrowitz M, Löscher W: Power-frequency magnetic fields increase cell proliferation in the mammary gland of female Fischer 344 rats but not various other rat strains or substrains. Oncology 2005, 69:486-498.
• [43]Ossenkopp KP, Kavaliers M, Lipa S: Increased mortality in land snails (Cepaea nemoralis) exposed to powerline (60-Hz) magnetic fields and effects of the light-dark cycle. Neurosci Lett 1990, 114:89-94.
• [44]Pipkin JL, Hinson WG, Young JF, Rowland KL, Shaddock JG, Tolleson WH, Duffy PH, Casciano DA: Induction of stress proteins by electromagnetic fields in cultured HL-60 cells. Bioelectromagnetics 1999, 20:347-357.
• [45]Yoshikawa T, Tanigawa M, Tanigawa T, Imai A, Hongo H, Kondo M: Enhancement of nitric oxide generation by low frequency electromagnetic field. Pathophysiology 2000, 7:131-135.
• [46]Maffini MV, Soto AM, Calabro JM, Ucci AA, Sonnenschein C: The stroma as a crucial target in rat mammary gland carcinogenesis. J Cell Sci 2004, 117:1495-1502.
• [47]Medina D: Stromal fibroblasts influence human mammary epithelial cell morphogenesis. Proc Natl Acad Sci 2004, 101:4723-4724.
• [48]Zangani D, Darcy KM, Shoemaker S, Ip MM: Adipocyte-epithelial interactions regulate the in vitro development of normal mammary epithelial cells. Exp Cell Res 1999, 247:399-409.
• [49]Frisch SM, Screaton RA: Anoikis mechanisms. Curr Opin Cell Biol 2001, 13:555-562.
• [50]Rennebeck G, Martelli M, Kyprianou N: Anoikis and survival connections in the tumor. Microenvironment: Is there a role in prostate cancer metastasis? Cancer Res 2005, 65:11230-11235.
• [51]Hahnel R, Twaddle E, Brindle L: The influence of enzymes on the estrogen receptors of human uterus and breast carcinoma. Steroids 1974, 24:489-506.
• [52]Kasperczyk S, Brzoza Z, Kasperczyk A, Beck B, Duliban H, Mertas A: The changes of alpha-amylase activity in serum and different tissues of female rat during sex cycle - isoelectrofocusing studies of alpha-amylase. Med Sci Monit 2001, 7:49-53.
• [53]Bruzzone A, Pinero PC, Castillo LF, Sarappa MG, Rojas P, Lanari C, Lüthy IA: α2-Adrenoceptor action on cell proliferation and mammary tumour growth in mice. Brit J Pharmacol 2008, 155:494-504.
• [54]Marchetti B, Spinola PG, Pelletier G, Labrie F: A potential role for catecholamines in the development and progression of carcinogen-induced tumors: hormonal control of beta-adrenergic receptors and correlation with tumor growth. J Steroid Biochem Molec Biol 1991, 38:307-320.
• [55]Marchetti B, Spinola PG, Plante M, Poyet P, Follea N, Pelletier G, Labrie F: Beta-adrenergic receptors in DMBA-induced rat mammary tumors: correlation with progesterone receptor and tumor growth. Breast Cancer Res Treat 1989, 13:251-263.
• [56]Lüthy IA, Bruzzone A, Pinero PC, Castillo LF, Chiesa IJ, Vazquez SM, Sarappa MG: Adrenoceptors: non conventional target for breast cancer? Curr Med Chemistry 2009, 16:1850-1862.