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Pre-clinical effects of metformin and aspirin on the cell lines of different breast cancer subtypes

  • Maria Eduarda Azambuja Amaral
  • Laura Roesler Nery
  • Carlos Eduardo Leite
  • Walter Filgueira de Azevedo Junior
  • Maria Martha Campos
PRECLINICAL STUDIES

Summary

Background Breast cancer is highly prevalent among women worldwide. It is classified into three main subtypes: estrogen receptor positive (ER+), human epidermal growth factor receptor 2 positive (HER2+), and triple negative breast cancer (TNBC). This study has evaluated the effects of aspirin and metformin, isolated or in a combination, in breast cancer cells of the different subtypes. Methods The breast cancer cell lines MCF-7, MDA-MB-231, and SK-BR-3 were treated with aspirin and/or metformin (0.01 mM - 10 mM); functional in vitro assays were performed. The interactions with the estrogen receptors (ER) were evaluated in silico. Results Metformin (2.5, 5 and 10 mM) altered the morphology and reduced the viability and migration of the ER+ cell line MCF-7, whereas aspirin triggered this effect only at 10 mM. A synergistic effect for the combination of metformin and aspirin (2.5, 5 or 10 mM each) was observed in the TNBC cell subtype MDA-MB-231, according to the evaluation of its viability and colony formation. Partial inhibitory effects were observed for either of the drugs in the HER2+ cell subtype SK-BR-3. The effects of metformin and aspirin partly relied on cyclooxygenase-2 (COX-2) upregulation, without the production of lipoxins. In silico, metformin and aspirin bound to the ERα receptor with the same energy. Conclusion We have provided novel evidence on the mechanisms of action of aspirin and metformin in breast cancer cells, showing favorable outcomes for these drugs in the ER+ and TNBC subtypes.

Keywords

Breast cance Drug repurposing Metformin Aspirin 

Notes

Acknowledgments

We would like to thank Dr. Eduardo Filippi-Chiela for providing the MCF-7 cell line and Dr. Mônica Ryff Moreira Roca Vianna for sharing the laboratory facilities for the western blotting analyzes.

Funding

This study was supported by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brasil. MEAA is a master’s degree student in Cellular and Molecular Biology supported by the CAPES/PROEX Program. M.M.C. and W.F.A received grants from CNPq (Grant Numbers: 303842–2014-8 and 308883–2014-4, respectively).

Compliance with ethical standards

Conflict of interest

None of the authors have any conflict of interest to disclose regarding the publication of the present manuscript.

Ethical approval

This article has not featured any studies with human participants or animals whilst it was being performed by any of the authors.

Supplementary material

10637_2018_568_MOESM1_ESM.docx (11.9 mb)
ESM 1 (DOCX 12225 kb)

References

  1. 1.
    American Cancer Society (2017) Cancer facts & figures 2017. American Cancer Society Inc., AtlantaGoogle Scholar
  2. 2.
    American Cancer Society (2016) Breast cancer facts & figures 2015–2016. American Cancer Society Inc., AtlantaGoogle Scholar
  3. 3.
    Orecchioni S, Reggiani F, Talarico G, Mancuso P, Calleri A, Gregato G, Labanca V, Noonan DM, Dallaglio K, Albini A, Bertolini F (2015) The biguanides metformin and phenformin inhibit angiogenesis, local and metastatic growth of breast cancer by targeting both neoplastic and microenvironment cells. Int J Cancer 136(6):E534–E544CrossRefPubMedGoogle Scholar
  4. 4.
    Wang J, Li G, Wang Y, Tang S, Sun X, Feng X, Li Y, Bao G, Li P, Mao X, Wang M, Liu P (2014) Suppression of tumor angiogenesis by metformin treatment via a mechanism linked to targeting of HER2/HIF-1α/VEGF secretion axis. Oncotarget 6(42):1–14Google Scholar
  5. 5.
    Talarico G, Orecchioni S, Dallaglio K, Reggiani F, Mancuso P, Calleri A, Gregato G, Labanca V, Rossi T, Noonan DM, Albini A, Bertolini F (2016) Aspirin and atenolol enhance metformin activity against breast cancer by targeting both neoplastic and microenvironment cells. Sci Rep 6(1):18673CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Jacob L, Kostev K, Rathmann W, Kalder M (2016) Impact of metformin on metastases in patients with breast cancer and type 2 diabetes. J Diabetes Complicat 30(6):1056–1059CrossRefPubMedGoogle Scholar
  7. 7.
    Alimova IN, Liu B, Fan Z, Edgerton SM, Dillon T, Lind SE, Thor AD (2009) Metformin inhibits breast cancer cell growth, colony formation and induces cell cycle arrest in vitro. Cell Cycle 8(6):909–915CrossRefPubMedGoogle Scholar
  8. 8.
    Gao Z-Y, Liu Z, Bi M-H, Zhang J-J, Han Z, Han X, Wang H-Y, Sun G-P, Liu H (2016) Metformin induces apoptosis via a mitochondria-mediated pathway in human breast cancer cells in vitro. Exp Ther Med 11(5):1700–1706CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Fan C, Wang Y, Liu Z, Sun Y, Wang X, Wei G, Wei J (2015) Metformin exerts anticancer effects through the inhibition of the sonic hedgehog signaling pathway in breast cancer. Int J Mol Med 36(1):204–214CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Chen M, Zhang J, Hu F, Liu S, Zhou Z (2015) Metformin affects the features of a human hepatocellular cell line (HepG2) by regulating macrophage polarization in a co-culture microenviroment. Diabetes Metab Res Rev 31(8):781–789CrossRefPubMedGoogle Scholar
  11. 11.
    Jung YR, Kim EJ, Choi HJ, Park J-J, Kim H-S, Lee Y-J, Park M-J, Lee M (2015) Aspirin targets SIRT1 and AMPK to induce senescence of colorectal carcinoma cells. Mol Pharmacol 88(4):708–719CrossRefPubMedGoogle Scholar
  12. 12.
    Vaughan LE, Prizment A, Blair CK, Thomas W, Anderson KE (2016) Aspirin use and the incidence of breast, colon, ovarian, and pancreatic cancers in elderly women in the Iowa Women’s Health Study. Cancer Causes Control 27(11):1395–1402CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Zhang D, Bai B, Xi Y, Wang T, Zhao Y (2016) Is aspirin use associated with a decreased risk of ovarian cancer? A systematic review and meta-analysis of observational studies with dose-response analysis. Gynecol Oncol 142(2):368–377CrossRefPubMedGoogle Scholar
  14. 14.
    van Staalduinen J, Frouws M, Reimers M, Bastiaannet E, van Herk-Sukel MPP, Lemmens V, de Steur WO, Hartgrink HH, van de Velde CJH, Liefers G-J (2016) The effect of aspirin and nonsteroidal anti-inflammatory drug use after diagnosis on survival of oesophageal cancer patients. Br J Cancer 114(9):1053–1059CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Tseng C-H (2016) Metformin reduces gastric cancer risk in patients with type 2 diabetes mellitus. Aging (Albany NY) 8(8):1636–1649CrossRefGoogle Scholar
  16. 16.
    Kim HJ, Kwon H, Lee JW, Kim HJ, Lee SB, Park HS, Sohn G, Lee Y, Koh BS, Yu JH, Son BH, Ahn SH (2015) Metformin increases survival in hormone receptor-positive, HER2-positive breast cancer patients with diabetes. Breast Cancer Res 17(1):64CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Tewari D, Majumdar D, Vallabhaneni S, Bera AK (2017) Aspirin induces cell death by directly modulating mitochondrial voltage-dependent anion channel (VDAC). Sci Rep 7:45184CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Cheng R, Liu Y, Cui J, Yang M, Liu X, Li P (2017) Aspirin regulation of c-myc and cyclinD1 proteins to overcome tamoxifen resistance in estrogen receptor-positive breast cancer cells. 8(18):30252–30264Google Scholar
  19. 19.
    Yue W, Zheng X, Lin Y, Yang CS, Xu Q, Carpizo D, Huang H, DiPaola RS, Tan X-L (2015) Metformin combined with aspirin significantly inhibit pancreatic cancer cell growth in vitro and in vivo by suppressing anti-apoptotic proteins Mcl-1 and Bcl-2. Oncotarget 6(25):21208–21224CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Abdelmonsif DA, Sultan AS, El-Hadidy WF, Abdallah DM (2017) Targeting AMPK, mTOR and β-catenin by combined metformin and aspirin therapy in HCC: an appraisal in Egyptian HCC patients. Mol Diagn Ther 22(1):115–127CrossRefGoogle Scholar
  21. 21.
    Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65(1–2):55–63CrossRefPubMedGoogle Scholar
  22. 22.
    Liang C-C, Park AY, Guan J-L (2007) In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc 2(2):329–333CrossRefPubMedGoogle Scholar
  23. 23.
    With P, Stereochemistry R (2017) The crystal structure of aspirin acetylated human cyclooxygenase-2: insight into the formation of products with reversed stereochemistry. 55(8):1226–1238Google Scholar
  24. 24.
    Lecomte M, Laneuville O, Ji C, DeWitt DL, Smith WL (1994) Acetylation of human prostaglandin endoperoxide synthase-2 (cyclooxygenase-2) by aspirin. J Biol Chem 269(18):13207–13215PubMedGoogle Scholar
  25. 25.
    Gobbetti T, Ducheix S, Le Faouder P, Perez T, Riols F, Boue J, Bertrand-Michel J, Dubourdeau M, Guillou H, Perretti M, Vergnolle N, Cenac N (2015) Protective effects of n-6 fatty acids-enriched diet on intestinal ischaemia/reperfusion injury involve lipoxin a4and its receptor. Br J Pharmacol 172(3):910–923CrossRefPubMedGoogle Scholar
  26. 26.
    Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE (2000) The protein data bank. Nucleic Acids Res 28(1):235–242CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Thomsen R, Christensen MH (2006) MolDock: a new technique for high-accuracy molecular docking. J Med Chem 49(11):3315–3321CrossRefPubMedGoogle Scholar
  28. 28.
    Trott O, Olson AJ (2009) AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 31(2):455–461Google Scholar
  29. 29.
    Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, Olson AJ (2009) AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J Comput Chem 30(16):2785–2791CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Xavier MM, Pintro VO, Levin B, Pintro VO, Carvalho NL, De Azevedo WF (2016) SAnDReS a computational tool for statistical analysis of docking results and development of scoring functions. Comb Chem High Throughput Screen 19(10):801–812CrossRefPubMedGoogle Scholar
  31. 31.
    Falah RR, Talib WH, Shbailat SJ (2017) Combination of metformin and curcumin targets breast cancer in mice by angiogenesis inhibition, immune system modulation and induction of p53 independent apoptosis. Ther Adv Med Oncol 9(4):235–252CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Wang Z, Cheng Q, Tang K, Sun Y, Zhang K, Zhang Y, Luo S, Zhang H, Ye D, Huang B (2015) Lipid mediator lipoxin A4 inhibits tumor growth by targeting IL-10-producing regulatory B (Breg) cells. Cancer Lett 364(2):118–124CrossRefPubMedGoogle Scholar
  33. 33.
    Zong L, Li J, Chen X, Chen K, Li W, Li X, Zhang L, Duan W, Lei J, Xu Q, Shan T, Ma Q, Sun H (2016) Lipoxin A4 attenuates cell invasion by inhibiting ROS/ERK/MMP pathway in pancreatic cancer. Oxidative Med Cell Longev 2016:1–9CrossRefGoogle Scholar
  34. 34.
    Pamplona FA, Menezes-de-Lima O, Takahashi RN (2010) Aspirin-triggered lipoxin induces CB1-dependent catalepsy in mice. Neurosci Lett 470(1):33–37CrossRefPubMedGoogle Scholar
  35. 35.
    Niraula S, Dowling RJO, Ennis M, Chang MC, Done SJ, Hood N, Escallon J, Leong WL, McCready DR, Reedijk M, Stambolic V, Goodwin PJ (2012) Metformin in early breast cancer: a prospective window of opportunity neoadjuvant study. Breast Cancer Res Treat 135(3):821–830CrossRefPubMedGoogle Scholar
  36. 36.
    Kalinsky K, Zheng T, Hibshoosh H, Du X, Mundi P, Yang J, Refice S, Feldman SM, Taback B, Connolly E, Crew KD, Maurer MA, Hershman DL (2017) Proteomic modulation in breast tumors after metformin exposure: results from a ‘window of opportunity’ trial. Clin Transl Oncol 19(2):180–188CrossRefPubMedGoogle Scholar
  37. 37.
    Dowling RJO, Niraula S, Chang MC, Done SJ, Ennis M, McCready DR, Leong WL, Escallon JM, Reedijk M, Goodwin PJ, Stambolic V (2015) Changes in insulin receptor signaling underlie neoadjuvant metformin administration in breast cancer: a prospective window of opportunity neoadjuvant study. Breast Cancer Res 17(1):540CrossRefGoogle Scholar
  38. 38.
    Kim S, Shore DL, Wilson LE, Sanniez EI, Kim JH, Taylor JA, Sandler DP (2015) Lifetime use of nonsteroidal anti- inflammatory drugs and breast cancer risk : results from a prospective study of women with a sister with breast cancer. BMC Cancer 1–10Google Scholar
  39. 39.
    Elwood PC, Morgan G, Pickering JE, Galante J, Weightman AL, Morris D, Kelson M, Dolwani S (2016) Aspirin in the treatment of cancer: reductions in metastatic spread and in mortality: a systematic review and meta-analyses of published studies. PLoS One 11(4):e0152402CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Bailey CJ, Path MRC, Turner RC (1996) Metformin. N Engl J Med 334(9):574–579CrossRefPubMedGoogle Scholar
  41. 41.
    Chen X, Hu C, Zhang W, Shen Y, Wang J, Hu F, Yu P (2015) Metformin inhibits the proliferation, metastasis, and cancer stem-like sphere formation in osteosarcoma MG63 cells in vitro. Tumor Biol 36(12):9873–9883CrossRefGoogle Scholar
  42. 42.
    Chen G, Feng W, Zhang S, Bian K, Yang Y, Fang C, Chen M, Yang J, Zou X (2015) Metformin inhibits gastric cancer via the inhibition of HIF1α/PKM2 signaling. Am J Cancer Res 5(4):1423–1434PubMedPubMedCentralGoogle Scholar
  43. 43.
    Cai X, Hu X, Tan X, Cheng W, Wang Q, Chen X, Guan Y, Chen C, Jing X (2015) Metformin induced AMPK activation, G0/G1 phase cell cycle arrest and the inhibition of growth of esophageal squamous cell carcinomas in vitro and in vivo. PLoS One 10(7):1–14Google Scholar
  44. 44.
    Ling S, Tian Y, Zhang H, Jia K, Feng T, Sun D, Gao Z, Xu F, Hou Z, Li Y, Wang L (2014) Metformin reverses multidrug resistance in human hepatocellular carcinoma Bel-7402/5-fluorouracil cells. Mol Med Rep 10(6):2891–2897CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Obara A, Fujita Y, Abudukadier A, Fukushima T, Oguri Y, Ogura M, Harashima S, Hosokawa M, Inagaki N (2015) DEPTOR-related mTOR suppression is involved in metformin’s anti-cancer action in human liver cancer cells. Biochem Biophys Res Commun 460(4):1047–1052CrossRefPubMedGoogle Scholar
  46. 46.
    Scherbakov AM, Sorokin DV, Tatarskiy VV, Prokhorov NS, Semina SE, Berstein LM, Krasil’Nikov MA (2016) The phenomenon of acquired resistance to metformin in breast cancer cells: the interaction of growth pathways and estrogen receptor signaling. IUBMB Life 68(4):281–292CrossRefPubMedGoogle Scholar
  47. 47.
    Maity G, De A, Das A, Banerjee S, Sarkar S, Banerjee SK (2015) Aspirin blocks growth of breast tumor cells and tumor-initiating cells and induces reprogramming factors of mesenchymal to epithelial transition. Lab Investig 95(7):702–717CrossRefPubMedGoogle Scholar
  48. 48.
    Hsieh CC, Huang YS (2016) Aspirin breaks the crosstalk between 3T3-L1 adipocytes and 4T1 breast cancer cells by regulating cytokine production. PLoS One 11(1):1–17CrossRefGoogle Scholar
  49. 49.
    McCarthy K, Bustin SA, Ogunkolade B, Khalaf S, Laban CA, McVittie CJ, Carpenter R, Jenkins PJ (2006) Cyclo-oxygenase-2 (COX-2) mRNA expression and hormone receptor status in breast cancer. Eur J Surg Oncol 32(7):707–709CrossRefPubMedGoogle Scholar
  50. 50.
    Chew GL, Huo CW, Huang D, Hill P, Cawson J, Frazer H, Hopper JL, Haviv I, Henderson MA, Britt K, Thompson EW (2015) Increased COX-2 expression in epithelial and stromal cells of high mammographic density tissues and in a xenograft model of mammographic density. Breast Cancer Res Treat 153(1):89–99CrossRefPubMedGoogle Scholar
  51. 51.
    Tury S, Becette V, Assayag F, Vacher S, Benoist C, Kamal M, Marangoni E, Bièche I, Lerebours F, Callens C (2016) Combination of COX-2 expression and PIK3CA mutation as prognostic and predictive markers for celecoxib treatment in breast cancer. Oncotarget 7(51):85124–85141CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Romano M (2010) Lipoxin and aspirin-triggered lipoxins. Sci World J 10:1048–1064CrossRefGoogle Scholar
  53. 53.
    Clària J, Serhan CN (1995) Aspirin triggers previously undescribed bioactive eicosanoids by human endothelial cell-leukocyte interactions. Proc Natl Acad Sci U S A 92(21):9475–9479CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    Davies NM, Sharkey KA, Asfaha S, Ton WKMACN, Wallace JL (1997) Aspirin causes rapid up-regulation of cyclo-oxygenase-2 expression in the stomach of rats. Aliment Pharmacol Ther 11(6):1101–1108CrossRefPubMedGoogle Scholar
  55. 55.
    Gostomska K, Alicja P, Piotr O (2017) Protective effects of levamisole , acetylsalicylic acid , and α - tocopherol against dioxin toxicity measured as the expression of AhR and COX - 2 in a chicken embryo model. Histochem Cell Biol 147(4):523–536CrossRefGoogle Scholar
  56. 56.
    Duan Y, Chen F, Zhang A, Zhu B, Sun J, Xie Q, Chen Z (2014) Aspirin inhibits lipopolysaccharide-induced COX-2 expression and PGE2 production in porcine alveolar macrophages by modulating protein kinase C and protein tyrosine phosphatase activity. BMB Rep 47(1):45–50CrossRefPubMedPubMedCentralGoogle Scholar
  57. 57.
    Xu XM, Sansores-Garcia L, Chen XM, Matijevic-Aleksic N, Du M, Wu KK (1999) Suppression of inducible cyclooxygenase 2 gene transcription by aspirin and sodium salicylate. Proc Natl Acad Sci U S A 96:5292–5297CrossRefPubMedPubMedCentralGoogle Scholar
  58. 58.
    Shtivelband MI, Juneja HS, Lee S, Wu KK (2003) Aspirin and salicylate inhibit colon cancer medium- and vegf-induced endothelial tube formation: correlation with suppression of cyclooxygenase-2 expression. J Thromb Haemost 1(10):2225–2233CrossRefPubMedGoogle Scholar
  59. 59.
    Wang ZS, Liu XH, Wang M, Jiang GJ, Qiu T, Chen ZY, Wang L (2015) Metformin attenuated the inflammation after renal ischemia / reperfusion and suppressed apoptosis of renal tubular epithelial cell in rats. Acta Cir Bras 30(9):617–623CrossRefPubMedGoogle Scholar
  60. 60.
    Tong D, Liu Q, Liu G, Xu J, Lan W, Jiang Y, Xiao H, Zhang D, Jiang J (2017) Metformin inhibits castration-induced EMT in prostate cancer by repressing COX2/PGE2/STAT3 axis. Cancer Lett 389:23–32CrossRefPubMedGoogle Scholar
  61. 61.
    Liu Q, Yuan W, Tong D, Liu G, Lan W (2016) Metformin represses bladder cancer progression by inhibiting stem cell repopulation via COX2 / PGE2 / STAT3 axis. Oncotarget 7(19):28235–28246PubMedPubMedCentralGoogle Scholar
  62. 62.
    Das UN (2012) Radiation resistance, invasiveness and metastasis are inflammatory events that could be suppressed by lipoxin A 4. Prostaglandins Leukot Essent Fat Acids 86(1–2):3–11CrossRefGoogle Scholar
  63. 63.
    Marginean A, Sharma-Walia N (2015) Lipoxins exert antiangiogenic and anti-inflammatory effects on Kaposi’s sarcoma cells. Transl Res 166(2):111–133CrossRefPubMedGoogle Scholar
  64. 64.
    Vethakanraj HS, Sesurajan BP, Padmanaban VP, Jayaprakasam M, Murali S, Sekar AK (2017) Anticancer effect of acid ceramidase inhibitor ceranib-2 in human breast cancer cell lines MCF-7, MDA MB-231 by the activation of SAPK/JNK, p38 MAPK apoptotic pathways, inhibition of the Akt pathway, downregulation of ERα. Anticancer Drugs 29(1):50–60CrossRefGoogle Scholar
  65. 65.
    Tao X, Xu L, Yin L, Han X, Qi Y, Xu Y, Song S, Zhao Y, Peng J (2017) Dioscin induces prostate cancer cell apoptosis through activation of estrogen receptor-β. Cell Death Dis 8(8):e2989CrossRefPubMedPubMedCentralGoogle Scholar
  66. 66.
    Kim T, Kim HI, An JY, Lee J, Lee NR, Heo J, Kim JE, Yu J, Lee YS, Inn KS, Kim NJ (2016) Identification of novel estrogen receptor (ER) agonists that have additional and complementary anti-cancer activities via ER-independent mechanism. Bioorg Med Chem Lett 26(7):1844–1848CrossRefPubMedGoogle Scholar

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Authors and Affiliations

  • Maria Eduarda Azambuja Amaral
    • 1
    • 2
  • Laura Roesler Nery
    • 3
  • Carlos Eduardo Leite
    • 2
  • Walter Filgueira de Azevedo Junior
    • 1
    • 4
  • Maria Martha Campos
    • 1
    • 2
    • 5
  1. 1.Programa de Pós-Graduação em Biologia Celular e Molecular, Escola de CiênciasPontifícia Universidade Católica do Rio Grande do Sul (PUCRS)Porto AlegreBrazil
  2. 2.Centro de Pesquisa em Toxicologia e Farmacologia, Escola de Ciências da SaúdePontifícia Universidade Católica do Rio Grande do Sul (PUCRS)Porto AlegreBrazil
  3. 3.ZebLab & Laboratório de Biologia e Desenvolvimento do Sistema Nervoso, Escola de CiênciasPontifícia Universidade Católica do Rio Grande do Sul (PUCRS)Porto AlegreBrazil
  4. 4.Laboratório de Biologia de Sistemas Computacionais, Escola de CiênciasPontifícia Universidade Católica do Rio Grande do Sul (PUCRS)Porto AlegreBrazil
  5. 5.Programa de Pós-Graduação em Odontologia, Escola de Ciências da SaúdePontifícia Universidade Católica do Rio Grande do Sul (PUCRS)Porto AlegreBrazil

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