Korean Journal of Clinical Laboratory Science (대한임상검사과학회지)

Korean Society for Clinical Laboratory Science (대한임상검사과학회)
권호 표지
DOI QR코드

DOI QR Code

Effect of Adefovir Dipivoxil on the Inhibition of Osteogenic Differentiation of Mesenchymal Stem Cells and Osteoblasts

아데포비어가 중간엽 줄기세포와 조골세포의 골형성 분화 억제에 미치는 영향

  • Ho PARK (Department of Clinical Laboratory Science, Wonkwang Health Science University)
  • 박호 (원광보건대학교 임상병리학과)
  • Received : 2023.11.09
  • Accepted : 2023.12.05
  • Published : 2023.12.31
Download PDF
⟨ Previous Next ⟩

Abstract

Adefovir dipivoxil (ADV) is used for the treatment of hepatitis and acquired immunodeficiency syndrome, but long-term use can cause osteoporosis. In this study, the effect of ADV on the osteocyte maturation process was evaluated at the level of undifferentiated cells using mesenchymal stem cells (MSCs) and osteoblasts (MG63). First, MSCs and MG63 cells were treated with ADV at different concentrations, and then a Cell Counting Kit-8 analysis was performed to determine the effect on the proliferation of each cell. Additionally, crystal violet and Hoechst staining were performed for the morphological analysis of each cell and nucleus. To determine the cause of cell hypertrophy, the transforming growth factor-beta (TGF-β) expression was investigated, and alkaline phosphatase (ALP) staining and activity were measured to determine the degree of differentiation of the MSCs and MG63 cells into mature osteocytes. The results confirmed that the ADV increases the expression of TGF-β in MSCs and MG63 cells, causing cellular and nuclear hypertrophy, and can cause osteoporosis by inhibiting cell proliferation and affecting the differentiation of mature osteocytes. Therefore, it is believed that these results can be used as a basis for understanding the adverse effects of ADV at a cytological level in basic medicine and clinical research.

Adefovir dipivoxil (ADV)은 간염 바이러스 및 에이즈 치료제로 사용되고 있으나 장기간 복용 시 부작용으로 골다공증을 유발할 수 있다. 골다공증은 골밀도 감소를 특징으로 하는 질환으로 ADV와 골 분화 억제의 상관성에 대한 연구가 필요하다. 이에 대한 연구를 위해, 미분화 세포인 중간엽 줄기세포(mesenchymal stem cells. MSCs)와 골아세포(MG63)를 이용하여 ADV가 미분화 세포 수준에서 골세포 성숙과정에 미치는 영향력을 평가하였다. 먼저, MSCs와 MG63 세포에 ADV를 농도별로 처리한 후 각 세포의 증식에 미치는 영향을 확인하기 위해 Cell Counting Kit-8 분석을 시행하였다. 또한 각 세포와 핵의 형태학적 분석을 위해 crystal violet과 Hoechst 염색을 시행하였다. 세포의 비대 현상의 원인을 규명하기 위해 TGF-β 발현을 조사하였고, 이에 따른 MSCs와 MG63 세포의 성숙한 골세포로의 분화도를 확인하고자 ALP 염색과 활성도를 측정하였다. 그 결과, ADV는 MSCs와 MG63 세포에서 핵과 세포질의 비대 현상, 세포의 증식능 억제, 성숙 골세포로의 분화 능력의 감소를 유발 할 수 있음을 확인하였다. 이러한 현상은 ADV가 TGF-β 발현을 증가시키는 것과 관련이 있었고, TGF-β의 증가는 MSCs와 MG63 세포부터 성숙한 골세포로의 분화 억제에 관여하고 있음을 시사하고 있다. 결론적으로, ADV 약물은 MSCs와 MG63 세포의 TGF-β 발현을 증가하여 세포 형태와 핵의 비대증을 유발하며, 세포의 증식억제 및 성숙한 골세포 분화능에 영향을 주어 골다공증을 유발할 수 있음을 확인하였다. 따라서 ADV 복용에 따른 부작용을 규명하기 위해 세포학적 수준에서 골다공증 유발에 미치는 생물학적 상관성과 원인을 이해하기 위한 근거자료로서 기초의학과 임상연구에 이용 될 수 있을 것으로 사료된다.

Keywords

Acknowledgement

This paper was supported by Wonkwang Health Science University in 2023.

References

  1. Murata K, Tsukuda S, Suizu F, Kimura A, Sugiyama M, Watashi K, et al. Immunomodulatory mechanism of acyclic nucleoside phosphates in treatment of hepatitis B virus infection. Hepatology. 2020;71:1533-1545. https://doi.org/10.1002/hep.30956
  2. Shahabadi N, Falsafi M. Experimental and molecular docking studies on DNA binding interaction of adefovir dipivoxil: advances toward treatment of hepatitis B virus infections. Spectrochim Acta A Mol Biomol Spectrosc. 2014;125:154-159. https://doi.org/10.1016/j.saa.2014.01.066
  3. Park H. Cytological study on the cause of the osteoporotic side effects of adefovir dipivoxil. Korean J Clin Lab Sci. 2019;51:379-385. https://doi.org/10.15324/kjcls.2019.51.3.379
  4. Makita T, Kanzaki H, Onishi H, Ikeda A, Takaki A, Wada N, et al. [Adefovir dipivoxil-induced Fanconi's syndrome and osteomalacia following multiple bone fractures in a patient with chronic hepatitis B]. Yakugaku Zasshi. 2019;139:641-645. Japanese. https://doi.org/10.1248/yakushi.18-00170
  5. Wei Z, He JW, Fu WZ, Zhang ZL. Osteomalacia induced by long-term low-dose adefovir dipivoxil: clinical characteristics and genetic predictors. Bone. 2016;93:97-103. https://doi.org/10.1016/j.bone.2016.09.017
  6. Lv Y, Li X, Liang S, Liang D, Xu F, Zhu X, et al. The clinical and pathological features of adefovir dipivoxil-related renal impairment. Clin Nephrol. 2019;91:180-186. https://doi.org/10.5414/cn109574
  7. Eguchi H, Tsuruta M, Tani J, Kuwahara R, Hiromatsu Y. Hypophosphatemic osteomalacia due to drug-induced Fanconi's syndrome associated with adefovir dipivoxil treatment for hepatitis B. Intern Med. 2014;53:233-237. https://doi.org/10.2169/internalmedicine.53.1213
  8. Zhu S, Yang YH, Gao RW, Li R, Zou YZ, Feng L, et al. Clinical features of hypophosphatemic osteomalacia induced by long-term low-dose adefovir dipivoxil. Drug Des Devel Ther. 2017;12:41-45. https://doi.org/10.2147/dddt.s140988
  9. Johnston CB, Dagar M. Osteoporosis in older adults. Med Clin North Am. 2020;104:873-884. https://doi.org/10.1016/j.mcna.2020.06.004
  10. Aspray TJ, Hill TR. Osteoporosis and the ageing skeleton. Subcell Biochem. 2019;91:453-476. https://doi.org/10.1007/978-981-13-3681-2_16
  11. Eastell R, Szulc P. Use of bone turnover markers in postmenopausal osteoporosis. Lancet Diabetes Endocrinol. 2017;5:908-923. https://doi.org/10.1016/s2213-8587(17)30184-5
  12. Li Z, Li D, Chen R, Gao S, Xu Z, Li N. Cell death regulation: a new way for natural products to treat osteoporosis. Pharmacol Res. 2023;187:106635. https://doi.org/10.1016/j.phrs.2022.106635
  13. Li H, Ghazanfari R, Zacharaki D, Lim HC, Scheding S. Isolation and characterization of primary bone marrow mesenchymal stromal cells. Ann N Y Acad Sci. 2016;1370:109-118. https://doi.org/10.1111/nyas.13102
  14. Iaquinta MR, Lanzillotti C, Mazziotta C, Bononi I, Frontini F, Mazzoni E, et al. The role of microRNAs in the osteogenic and chondrogenic differentiation of mesenchymal stem cells and bone pathologies. Theranostics. 2021;11:6573-6591. https://doi.org/10.7150/thno.55664
  15. Meyer MB, Benkusky NA, Sen B, Rubin J, Pike JW. Epigenetic plasticity drives adipogenic and osteogenic differentiation of marrow-derived mesenchymal stem cells. J Biol Chem. 2016; 291:17829-17847. https://doi.org/10.1074/jbc.m116.736538
  16. De Clercq E. Current treatment of hepatitis B virus infections. Rev Med Virol. 2015;25:354-365. https://doi.org/10.1002/rmv.1849
  17. An JH, Jin F, Kim HS, Ryu HC, Kim JS, Kim HM, et al. Application of ionic liquid to polymorphic transformation of anti-viral/HIV drug adefovir dipivoxil. Arch Pharm Res. 2016;39:646-659. https://doi.org/10.1007/s12272-016-0721-0
  18. Murray JM, Stancevic O, Lutgehetmann M, Wursthorn K, Petersen J, Dandri M. Variability in long-term hepatitis B virus dynamics under antiviral therapy. J Theor Biol. 2016;391:74-80. https://doi.org/10.1016/j.jtbi.2015.12.005
  19. Takagi J, Morita H, Ito K, Ohashi T, Hirase S, Ito T, et al. Urinary β-2 microglobulin levels sensitively altered in an osteomalacia patient receiving add-on adefovir dipivoxil therapy for hepatitis B virus infection. Intern Med. 2016;55:1599-1603. https://doi.org/10.2169/internalmedicine.55.6301
  20. Wang BF, Wang Y, Wang BY, Sun FR, Zhang D, Chen YS. Osteomalacia and Fanconi's syndrome caused by long-term low-dose adefovir dipivoxil. J Clin Pharm Ther. 2015;40:345-348. https://doi.org/10.1111/jcpt.12259
  21. Lin Y, Pan F, Wang Y, Chen Z, Lin C, Yao L, et al. Adefovir dipivoxil-induced Fanconi syndrome and its predictive factors: a study of 28 cases. Oncol Lett. 2017;13:307-314. https://doi.org/10.3892/ol.2016.5393
  22. Gopinath V. Osteoporosis. Med Clin North Am. 2023;107: 213-225. https://doi.org/10.1016/j.mcna.2022.10.013
  23. Zhong L, Huang X, Karperien M, Post JN. The regulatory role of signaling crosstalk in hypertrophy of MSCs and human articular chondrocytes. Int J Mol Sci. 2015;16:19225-19247. https://doi.org/10.3390/ijms160819225
  24. Wang X, Guan Y, Xiang S, Clark KL, Alexander PG, Simonian LE, et al. Role of canonical Wnt/β-catenin pathway in regulating chondrocytic hypertrophy in mesenchymal stem cell-based cartilage tissue engineering. Front Cell Dev Biol. 2022;10:812081. https://doi.org/10.3389/fcell.2022.812081
  25. Crane JL, Cao X. Bone marrow mesenchymal stem cells and TGF-β signaling in bone remodeling. J Clin Invest. 2014;124:466-472. https://doi.org/10.1172/jci70050
  26. Kim P, Park J, Lee DJ, Mizuno S, Shinohara M, Hong CP, et al. Mast4 determines the cell fate of MSCs for bone and cartilage development. Nat Commun. 2022;13:3960. https://doi.org/10.1038/s41467-022-31697-3
  27. Du X, Cai L, Xie J, Zhou X. The role of TGF-beta3 in cartilage development and osteoarthritis. Bone Res. 2023;11:2. https://doi.org/10.1038/s41413-022-00239-4
  28. Choi H, Ahn YH, Kim TH, Bae CH, Lee JC, You HK, et al. TGF-β sinaling regulates cementum formation through osterix expression. Sci Rep. 2016;6:26046. https://doi.org/10.1038/srep26046
  29. Erlebacher A, Derynck R. Increased expression of TGF-beta 2 in osteoblasts results in an osteoporosis-like phenotype. J Cell Biol. 1996;132:195-210. https://doi.org/10.1083/jcb.132.1.195
  30. Sun X, Xie Z, Ma Y, Pan X, Wang J, Chen Z, et al. TGF-β inhibits osteogenesis by upregulating the expression of ubiquitin ligase SMURF1 via MAPK-ERK signaling. J Cell Physiol. 2018;233:596-606. https://doi.org/10.1002/jcp.25920