Advertisement

Apoptosis

, Volume 20, Issue 3, pp 327–335 | Cite as

p27kip1 overexpression regulates VEGF expression, cell proliferation and apoptosis in cell culture from eutopic endometrium of women with endometriosis

  • G. A. Gonçalves
  • C. M. Camargo-Kosugi
  • T. C. S. Bonetti
  • A. L. Invitti
  • M. J. B. C. Girão
  • I. D. C. G. Silva
  • E. Schor
Original Paper

Abstract

We hypothesized that p27kip1 overexpression can regulate endometriosis cell proliferation, apoptosis and vascular endothelial growth factor (VEGF) expression in the endometrium. The overexpression of p27kip1 was obtained by transduction of p27kip1 in primary cultures of endometrium obtained from women with endometriosis tissue with gene therapy technology. First generation bicistronic adenovirus: AdCMVhp27IRESEGFP (Adp27) and AdCMVNull (AdNull) were engineered in order to induce p27kip1 expression in endometrial cells primary culture. The effect of p27kip1 overexpression was elucidated through the cell proliferation evaluation and the expression of the cell cycle-related proteins p16, p21, p27, and p53. Cell cycle and apoptosis in endometrial cells from women with and without endometriosis were also evaluated. The VEGF levels were evaluated 1 and 7 days after transduction. The experiments were performed using Immunofluorescence stainings and flow cytometry technique. The cell proliferation statistically diminished markedly following p27kip1 overexpression in the endometriosis group. This process was accompanied, however, by a statistically significant modulation of the cell cycle-related proteins p16, p21, p27 and p53 markedly increase following p27kip1 overexpression in the endometriosis group (p < 0.001) and an increase in apoptotic cells was observed. In the endometriosis group, significant downregulation of VEGF expression was observed 7 days after p27kip1 overexpression, attaining levels strikingly similar to those observed in the control endometrial cells. The findings of this study showed a link between the cell cycle control protein (p27kip1) and angiogenesis (VEGF). Our results, also reinforces the background of endometrial dysfunction as part of the origin of endometriosis. We believe that better knowledge of endometrium milieu and the establishment of the link between different, previously describe, altered pathways in this tissue can facilitate future genetic cell therapy.

Keywords

p27kip1 VEGF Endometriosis Endometrium 

Notes

Acknowledgments

The authors gratefully acknowledge the contributions of the Endometriosis Unit team of the Department of Gynecology, Federal University of Sao Paulo – Escola Paulista de Medicina (UNIFESP-EPM), São Paulo, Brazil. This study was supported by Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP #09/53000-2), and Coordenação de Aperfeiçoamento de Pessoa de Nível Superior (CAPES # 33009015014P5).

Conflict of interest

The authors have disclosed any commercial or financial interests or any other conflict of interest associated with publication of the article.

References

  1. 1.
    Giudice LC, Kao LC (2004) Endometriosis. Lancet 364(9447):1789–1799CrossRefPubMedGoogle Scholar
  2. 2.
    Gazvani R, Templeton A (2002) New considerations for the pathogenesis of endometriosis. Int J Gynaecol Obstet 76(2):117–126CrossRefPubMedGoogle Scholar
  3. 3.
    Missmer SA, Cramer DW (2003) The epidemiology of endometriosis. Obstet Gynecol Clin North Am 30(1):1–19, viiCrossRefPubMedGoogle Scholar
  4. 4.
    Carter JE (1994) Combined hysteroscopic and laparoscopic findings in patients with chronic pelvic pain. J Am Assoc Gynecol Laparosc 2(1):43–47CrossRefPubMedGoogle Scholar
  5. 5.
    Carter JE (1994) Laparoscopic treatment for chronic pelvic pain: results from three-year follow-up. J Am Assoc Gynecol Laparosc 1(4, Part 2):S6–S7CrossRefGoogle Scholar
  6. 6.
    Sampson JA (1927) Peritoneal endometriosis due to the menstrual dissemination of endometrial tissue into the peritoneal cavity. Am J Obstet Gynecol 14:422–469Google Scholar
  7. 7.
    Kianpour M, Nematbakhsh M, Ahmadi SM, Jafarzadeh M, Hajjarian M, Pezeshki Z, Safari T, Eshraghi-Jazi F (2013) Serum and peritoneal fluid levels of vascular endothelial growth factor in women with endometriosis. Int J Fertil Steril. 7(2):96–99PubMedCentralPubMedGoogle Scholar
  8. 8.
    McLaren J (2000) Vascular endothelial growth factor and endometriotic angiogenesis. Hum Reprod Update 6(1):45–55CrossRefPubMedGoogle Scholar
  9. 9.
    May KE, Villar J, Kirtley S et al (2011) Endometrial alterations in endometriosis: a systematic review of putative biomarkers. Hum Reprod Update 17(5):637–653CrossRefPubMedGoogle Scholar
  10. 10.
    Braun-Dullaeus RC, Mann MJ, Dzau VJ (1998) Cell cycle progression: new therapeutic target for vascular proliferative disease. Circulation 98(1):82–89CrossRefPubMedGoogle Scholar
  11. 11.
    Machado DE, Berardo PT, Palmero CY et al (2010) Higher expression of vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 (Flk-1) and metalloproteinase-9 (MMP-9) in a rat model of peritoneal endometriosis is similar to cancer diseases. J Exp Clin Cancer Res 29:4CrossRefPubMedCentralPubMedGoogle Scholar
  12. 12.
    Sherr CJ, Roberts JM (1999) CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev 13(12):1501–1512CrossRefPubMedGoogle Scholar
  13. 13.
    Tsihlias J, Kapusta L, Slingerland J (1999) The prognostic significance of altered cyclin-dependent kinase inhibitors in human cancer. Annu Rev Med 50:401–423CrossRefPubMedGoogle Scholar
  14. 14.
    Besson A, Gurian-West M, Schmidt A et al (2004) p27Kip1 modulates cell migration through the regulation of RhoA activation. Genes Dev 18(8):862–876CrossRefPubMedCentralPubMedGoogle Scholar
  15. 15.
    Velarde MC, Aghajanova L, Nezhat CR et al (2009) Increased mitogen-activated protein kinase kinase/extracellularly regulated kinase activity in human endometrial stromal fibroblasts of women with endometriosis reduces 3’,5’-cyclic adenosine 5’-monophosphate inhibition of cyclin D1. Endocrinology 150(10):4701–4712CrossRefPubMedCentralPubMedGoogle Scholar
  16. 16.
    Charnock-Jones DS, Sharkey AM, Rajput-Williams J et al (1993) Identification and localization of alternately spliced mRNAs for vascular endothelial growth factor in human uterus and estrogen regulation in endometrial carcinoma cell lines. Biol Reprod 48(5):1120–1128CrossRefPubMedGoogle Scholar
  17. 17.
    Russo AA, Jeffrey PD, Patten AK et al (1996) Crystal structure of the p27Kip1 cyclin-dependent-kinase inhibitor bound to the cyclin A-Cdk2 complex. Nature 382(6589):325–331CrossRefPubMedGoogle Scholar
  18. 18.
    Chu IM, Hengst L, Slingerland JM (2008) The Cdk inhibitor p27 in human cancer: prognostic potential and relevance to anticancer therapy. Nat Rev Cancer 8(4):253–267CrossRefPubMedGoogle Scholar
  19. 19.
    Hong YB, Kang HJ, Kim HJ et al (2009) Inhibition of cell proliferation by a resveratrol analog in human pancreatic and breast cancer cells. Exp Mol Med 41(3):151–160CrossRefPubMedCentralPubMedGoogle Scholar
  20. 20.
    Chen J, Ding WH, Lu GX et al (2009) Impact of p27mt gene on transplantation model of human colorectal cancer in nude mice. World J Gastroenterol 15(3):369–372CrossRefPubMedCentralPubMedGoogle Scholar
  21. 21.
    Chen X, Wang W, Zhang L et al (2010) Effects of Ad-p27mt gene transfer on the expression of Bax, Bcl-2, VEGF and MMP-9 in the transplanted liver tumors in nude mice. J Huazhong Univ Sci Technol Med Sci 30(5):611–614CrossRefPubMedGoogle Scholar
  22. 22.
    ASRM (1997) Revised American Society for Reproductive Medicine classification of endometriosis: 1996. Fertil Steril 67(5):817–821CrossRefGoogle Scholar
  23. 23.
    Noyes RW, Hertig AT, Rock J (1975) Dating the endometrial biopsy. Am J Obstet Gynecol 122(2):262–263PubMedGoogle Scholar
  24. 24.
    D’Amora P, Maciel TT, Tambellini R et al (2009) Disrupted cell cycle control in cultured endometrial cells from patients with endometriosis harboring the progesterone receptor polymorphism PROGINS. Am J Pathol 175(1):215–224CrossRefPubMedCentralPubMedGoogle Scholar
  25. 25.
    Khorram O, Taylor RN, Ryan IP et al (1993) Peritoneal fluid concentrations of the cytokine RANTES correlate with the severity of endometriosis. Am J Obstet Gynecol 169(6):1545–1549CrossRefPubMedGoogle Scholar
  26. 26.
    Griffioen AW, Molema G (2000) Angiogenesis: potentials for pharmacologic intervention in the treatment of cancer, cardiovascular diseases, and chronic inflammation. Pharmacol Rev 52(2):237–268PubMedGoogle Scholar
  27. 27.
    Li A, Dubey S, Varney ML et al (2003) IL-8 directly enhanced endothelial cell survival, proliferation, and matrix metalloproteinases production and regulated angiogenesis. J Immunol 170(6):3369–3376CrossRefPubMedGoogle Scholar
  28. 28.
    Seli E, Arici A (2003) Endometriosis: interaction of immune and endocrine systems. Semin Reprod Med 21(2):135–144CrossRefPubMedGoogle Scholar
  29. 29.
    Seli E, Berkkanoglu M, Arici A (2003) Pathogenesis of endometriosis. Obstet Gynecol Clin North Am 30(1):41–61CrossRefPubMedGoogle Scholar
  30. 30.
    Crosignani P, Olive D, Bergqvist A et al (2006) Advances in the management of endometriosis: an update for clinicians. Hum Reprod Update 12(2):179–189CrossRefPubMedGoogle Scholar
  31. 31.
    Taylor RN, Yu J, Torres PB et al (2009) Mechanistic and therapeutic implications of angiogenesis in endometriosis. Reprod Sci 16(2):140–146CrossRefPubMedCentralPubMedGoogle Scholar
  32. 32.
    Laschke MW, Menger MD (2007) In vitro and in vivo approaches to study angiogenesis in the pathophysiology and therapy of endometriosis. Hum Reprod Update 13(4):331–342CrossRefPubMedGoogle Scholar
  33. 33.
    Hur SE, Lee JY, Moon HS et al (2006) Angiopoietin-1, angiopoietin-2 and Tie-2 expression in eutopic endometrium in advanced endometriosis. Mol Hum Reprod 12(7):421–426CrossRefPubMedGoogle Scholar
  34. 34.
    Abramov Y, Barak V, Nisman B et al (1997) Vascular endothelial growth factor plasma levels correlate to the clinical picture in severe ovarian hyperstimulation syndrome. Fertil Steril 67(2):261–265CrossRefPubMedGoogle Scholar
  35. 35.
    Tischer E, Mitchell R, Hartman T et al (1991) The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing. J Biol Chem 266(18):11947–11954PubMedGoogle Scholar
  36. 36.
    Sharkey AM, Day K, McPherson A et al (2000) Vascular endothelial growth factor expression in human endometrium is regulated by hypoxia. J Clin Endocrinol Metab 85(1):402–409PubMedGoogle Scholar
  37. 37.
    Folkman J, Klagsbrun M (1987) Vascular physiology. A family of angiogenic peptides. Nature 329(6141):671–672CrossRefPubMedGoogle Scholar
  38. 38.
    Cho S, Choi YS, Jeon YE et al (2012) Expression of vascular endothelial growth factor (VEGF) and its soluble receptor-1 in endometriosis. Microvasc Res 83(2):237–242CrossRefPubMedGoogle Scholar
  39. 39.
    Wegiel B, Ekberg J, Talasila KM, Jalili S, Persson JL (2009) The role of VEGF and a functional link between VEGF and p27Kip1 in acute myeloid leukemia. Leukemia 23(2):251–261CrossRefPubMedGoogle Scholar
  40. 40.
    Schor E, da Silva ID, Sato H et al (2009) P27Kip1 is down-regulated in the endometrium of women with endometriosis. Fertil Steril 91(3):682–686CrossRefPubMedGoogle Scholar
  41. 41.
    Yamamoto S, Tsuda H, Miyai K et al (2009) Aberrant expression of p27(Kip1)-interacting cell-cycle regulatory proteins in ovarian clear cell carcinomas and their precursors with special consideration of two distinct multistage clear cell carcinogenetic pathways. Virchows Arch 455(5):413–422CrossRefPubMedGoogle Scholar
  42. 42.
    Zhang W, Tong Q, Wu Q et al (2009) Upregulated p27kip1 can downregulate survivin expression and inhibit telomerase activity in gastric carcinoma cells. Cancer Invest 27(9):898–900CrossRefPubMedGoogle Scholar
  43. 43.
    Nguyen MT, Beck J, Lue H et al (2003) A 16-residue peptide fragment of macrophage migration inhibitory factor, MIF-(50-65), exhibits redox activity and has MIF-like biological functions. J Biol Chem 278(36):33654–33671CrossRefPubMedGoogle Scholar
  44. 44.
    Yamamoto S, Tsuda H, Miyai K et al (2010) Cumulative alterations of p27-related cell-cycle regulators in the development of endometriosis-associated ovarian clear cell adenocarcinoma. Histopathology 56(6):740–749CrossRefPubMedGoogle Scholar
  45. 45.
    Camargo-Kosugi CM, da Silva ID, Sato H et al (2009) The V109G polymorphism in the p27 gene is associated with endometriosis. Eur J Obstet Gynecol Reprod Biol 145(2):180–183CrossRefPubMedGoogle Scholar
  46. 46.
    da Silva CM, Vilaça Belo A, Passos Andrade S, Peixoto Campos P, Cristina França Ferreira M, Lopes da Silva-Filho A, Mendonça Carneiro M (2014) Identification of local angiogenic and inflammatory markers in the menstrual blood of women with endometriosis. Biomed Pharmacother. doi: 10.1016/j.biopha.2014.08.005 Google Scholar
  47. 47.
    Rathore N, Kriplani A, Yadav RK, Jaiswal U, Netam R (2014) Distinct peritoneal fluid ghrelin and leptin in infertile women with endometriosis and their correlation with interleukin-6 and vascular endothelial growth factor. Gynecol Endocrinol 30(9):671–675CrossRefPubMedGoogle Scholar
  48. 48.
    Huang F, Wang H, Zou Y, Liu Q, Cao J, Yin T (2013) Effect of GnRH-II on the ESC proliferation, apoptosis and VEGF secretion in patients with endometriosis in vitro. Int J Clin Exp Pathol 6(11):2487–2496PubMedCentralPubMedGoogle Scholar
  49. 49.
    Huang F, Cao J, Liu Q, Zou Y, Li H, Yin T (2013) MAPK/ERK signal pathway involved expression of COX-2 and VEGF by IL-1β induced in human endometriosis stromal cells in vitro. Int J Clin Exp Pathol 6(10):2129–2136PubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • G. A. Gonçalves
    • 1
    • 2
  • C. M. Camargo-Kosugi
    • 1
  • T. C. S. Bonetti
    • 1
  • A. L. Invitti
    • 1
  • M. J. B. C. Girão
    • 1
    • 2
  • I. D. C. G. Silva
    • 1
  • E. Schor
    • 1
  1. 1.Molecular and Proteomics Gynecology Cellular and Viral Vectors Laboratory, Department of GynecologyFederal University of São Paulo, Escola Paulista de Medicina (UNIFESP-EPM)São PauloBrazil
  2. 2.Charitable Association of Blood Collection-COLSANSão PauloBrazil

Personalised recommendations