Inhibition of Hyaluronic Acid Synthesis Decreases Endometrial Cell Attachment, Migration, and Invasion

To characterize the effects of 4-methylumbelliferone (4-MU) on expression of the hyaluronic acid (HA) system and on attachment, migration, and invasion of endometrial epithelial (EECs) and stroma cells (ESCs) to peritoneal mesothelial cells (PMCs), this in vitro study was performed in an Academic Center. De-identified endometrial tissue samples used were from reproductive-aged women. EECs and ESCs isolated from menstrual endometrial biopsies were treated with 4-MU or vehicle. Real-time polymerase chain reaction and western blot were used to assess expression of HA synthases (HAS), hyaluronidase, and standard CD44. Established in vitro assays were used to assess attachment, migration, and invasion with and without treatment with 4-MU. Chi square and Student’s t-test were used to analyze the results as appropriate. The addition of 4-MU decreased mRNA and protein expression of HAS 2, HAS 3, and CD44 in EECs and ESCs compared to control. Treatment with 4-MU also decreased attachment, migration, and invasion of EECs and ESCs to PMCs compared to control. 4-MU decreases endometrial cell adhesion, migration, and invasion to PMCs. This effect appears to be mediated by a decrease in HAS 2, HAS 3, and CD44. 4-MU is a potential treatment for endometriosis. Future in vivo studies are needed to evaluate 4-MU as a therapeutic agent for endometriosis. Similar content being viewed by others

Sampson JA. Metastatic or embolic endometriosis, due to the menstrual dissemination of endometrial tissue into the venous circulation. Am J Pathol. 1927;3(2):93–110 143. Griffith JS, Liu YG, Tekmal RR, Binkley PA, Holden AE, Schenken RS. Menstrual endometrial cells from women with endometriosis demonstrate increased adherence to peritoneal cells and increased expression of CD44 splice variants. Fertil Steril. 2010;93(6):1745–9. Witz CA, Cho S, Centonze VE, Montoya-Rodriguez IA, Schenken RS. Time series analysis of transmesothelial invasion by endometrial stromal and epithelial cells using three-dimensional confocal microscopy. Fertil Steril. 2003;79(Suppl 1):770–8. Laurent TC, Fraser JR. Hyaluronan. FASEB J. 1992;6(7):2397–404. Dechaud H, Witz CA, Montoya-Rodriguez IA, Degraffenreid LA, Schenken RS. Mesothelial cell-associated hyaluronic acid promotes adhesion of endometrial cells to mesothelium. Fertil Steril. 2001;76(5):1012–8. Knudtson JF, Tekmal RR, Santos MT, Binkley PA, Krishnegowda N, Valente P, et al. Impaired development of early endometriotic lesions in CD44 knockout mice. Reprod Sci. 2016;23(1):87–91. Urakawa H, Nishida Y, Wasa J, Arai E, Zhuo L, Kimata K, et al. Inhibition of hyaluronan synthesis in breast cancer cells by 4-methylumbelliferone suppresses tumorigenicity in vitro and metastatic lesions of bone in vivo. Int J Cancer. 2012;130(2):454–66. Piccioni F, Malvicini M, Garcia MG, Rodriguez A, Atorrasagasti C, Kippes N, et al. Antitumor effects of hyaluronic acid inhibitor 4-methylumbelliferone in an orthotopic hepatocellular carcinoma model in mice. Glycobiology. 2012;22(3):400–10. Kultti A, Pasonen-Seppanen S, Jauhiainen M, et al. 4-Methylumbelliferone inhibits hyaluronan synthesis by depletion of cellular UDP-glucuronic acid and downregulation of hyaluronan synthase 2 and 3. Exp Cell Res. 2009;315(11):1914–23. Fontaine L, Grand M, Molho D, Chabert MJ, Boschetti E. Choleretic, spasmolytic and general pharmacologic activities of 4-methylumbelliferone. Therapie. 1968;23(1):51–62. Olivares CN, Alaniz LD, Menger MD, Baranao RI, Laschke MW, Meresman GF. Inhibition of hyaluronic acid synthesis suppresses angiogenesis in developing endometriotic lesions. PLoS One. 2016;11(3):e0152302. Knudtson JF, McLaughlin JE, Santos MT, Binkley PA, Tekmal RR, Schenken RS. The hyaluronic acid system is intact in menstrual endometrial cells in women with and without endometriosis. Reprod Sci. 2018;1933719118766257. Nair HB, Luthra R, Kirma N, Liu YG, Flowers L, Evans D, et al. Induction of aromatase expression in cervical carcinomas: effects of endogenous estrogen on cervical cancer cell proliferation. Cancer Res. 2005;65(23):11164–73. Tekmal RR, Liu YG, Nair HB, et al. Estrogen receptor alpha is required for mammary development and the induction of mammary hyperplasia and epigenetic alterations in the aromatase transgenic mice. J Steroid Biochem. 2005;95(1–5):9–15. Lucidi RS, Witz CA, Chrisco M, Binkley PA, Shain SA, Schenken RS. A novel in vitro model of the early endometriotic lesion demonstrates that attachment of endometrial cells to mesothelial cells is dependent on the source of endometrial cells. Fertil Steril. 2005;84(1):16–21. Liu J, Sareddy GR, Zhou M, Viswanadhapalli S, Li X, Lai Z, et al. Differential effects of estrogen receptor beta isoforms on glioblastoma progression. Cancer Res. 2018;78(12):3176–89. Liang CC, Park AY, Guan JL. In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc. 2007;2(2):329–33. Nair AS, Nair HB, Lucidi RS, Kirchner AJ, Schenken RS, Tekmal RR, et al. Modeling the early endometriotic lesion: mesothelium-endometrial cell co-culture increases endometrial invasion and alters mesothelial and endometrial gene transcription. Fertil Steril. 2008;90(4 Suppl):1487–95. Kakizaki I, Kojima K, Takagaki K, et al. A novel mechanism for the inhibition of hyaluronan biosynthesis by 4-methylumbelliferone. J Biol Chem. 2004;279(32):33281–9. Lokeshwar VB, Lopez LE, Munoz D, Chi A, Shirodkar SP, Lokeshwar SD, et al. Antitumor activity of hyaluronic acid synthesis inhibitor 4-methylumbelliferone in prostate cancer cells. Cancer Res. 2010;70(7):2613–23. Nagase H, Kudo D, Suto A, Yoshida E, Suto S, Negishi M, et al. 4-Methylumbelliferone suppresses hyaluronan synthesis and tumor progression in SCID mice intra-abdominally inoculated with pancreatic Cancer cells. Pancreas. 2017;46(2):190–7. Nagy N, Kuipers HF, Frymoyer AR, et al. 4-methylumbelliferone treatment and hyaluronan inhibition as a therapeutic strategy in inflammation, autoimmunity, and cancer. Front Immunol. 2015;6:123. Funding The project described was supported by the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant KL2 TR001118 (JFK). The project described was supported by the Eunice Kennedy Shriver National Institute for Children Health and Development, National Institutes of Health, through Grant K23 HD097307 (JFK). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. The project was also supported by the American Society for Reproductive Medicine grant (JFK). Author information Authors and Affiliations Corresponding author Ethics declarations Conflict of Interest The authors declare that they have no conflict of interest. Additional information Capsule 4-Methylumbelliferone (4-MU) inhibits hyaluronic acid synthesis and its receptor. The addition of 4-MU to endometrial cells decreases endometrial cell attachment, migration, and invasion to peritoneal mesothelial cells. The material contained in the manuscript has not been published, has not been submitted, or is not being submitted elsewhere for publication. All authors are in agreement to submission of this manuscript. Presentation This work was presented on October 10, 2018 at the American Society of Reproductive Medicine annual meeting in Denver, Colorado. Rights and permissions About this article Cite this article McLaughlin, J.E., Santos, M.T., Binkley, P.A. et al. Inhibition of Hyaluronic Acid Synthesis Decreases Endometrial Cell Attachment, Migration, and Invasion. Reprod. Sci. 27, 1058–1063 (2020). https://doi.org/10.1007/s43032-019-00100-w Received: Accepted: Published: Version of record: Issue date: DOI: https://doi.org/10.1007/s43032-019-00100-w