Journal of Zhejiang University SCIENCE B

, Volume 13, Issue 12, pp 997–1005 | Cite as

Effect of mitomycin on normal dermal fibroblast and HaCat cell: an in vitro study

  • Yao-wen Wang
  • Ji-hao Ren
  • Kun Xia
  • Shu-hui Wang
  • Tuan-fang Yin
  • Ding-hua Xie
  • Li-hua Li



To evaluate the effects of mitomycin on the growth of human dermal fibroblast and immortalized human keratinocyte line (HaCat cell), particularly the effect of mitomycin on intracellular messenger RNA (mRNA) synthesis of collagen and growth factors of fibroblast.


The normal dermal fibroblast and HaCat cell were cultured in vitro. Cell cultures were exposed to 0.4 and 0.04 mg/ml of mitomycin solution, and serum-free culture medium was used as control. The cellular morphology change, growth characteristics, cell proliferation, and apoptosis were observed at different intervals. For the fibroblasts, the mRNA expression changes of transforming growth factor (TGF)-β1, basic fibroblast growth factor (bFGF), procollagen I, and III were detected by reverse transcription polymerase chain reaction (RT-PCR).


The cultured normal human skin fibroblast and HaCat cell grew exponentially. A 5-min exposure to mitomycin at either 0.4 or 0.04 mg/ml caused marked dose-dependent cell proliferation inhibition on both fibroblasts and HaCat cells. Cell morphology changed, cell density decreased, and the growth curves were without an exponential phase. The fibroblast proliferated on the 5th day after the 5-min exposure of mitomycin at 0.04 mg/ml. Meanwhile, 5-min application of mitomycin at either 0.04 or 0.4 mg/ml induced fibroblast apoptosis but not necrosis. The apoptosis rate of the fibroblast increased with a higher concentration of mytomycin (p<0.05). A 5-min exposure to mitomycin at 0.4 mg/ml resulted in a marked decrease in the mRNA production of TGF-β1, procollagen I and III, and a marked increase in the mRNA production of bFGF.


Mitomycin can inhibit fibroblast proliferation, induce fibroblast apoptosis, and regulate intracellular protein expression on mRNA levels. In additon, mitomycin can inhibit HaCat cell proliferation, so epithelial cell needs more protecting to avoid mitomycin’s side effect when it is applied clinically.

Key words

Mitomycin Fibroblast HaCat cell Apoptosis TGF-β1 bFGF Procollagen 

CLC number



Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Bank, H.L., 1988. Rapid assessment of islet viability with acridine orange and propidium iodide. In Vitro Cel. Dev. Biol., 24(4):266–269. [doi:10.1007/BF02628826]CrossRefGoogle Scholar
  2. Banthia, V., Selesnick, S.H., 2003. Mitomycin-C in the Post Surgical Ear Canal. American Academy of Otolaryngology/Head and Neck Surgery. Annual Meeting, Orlando, Florida, USA, p.882–886.Google Scholar
  3. Battelino, S., Hocevar-Boltezar, I., Zargi, M., 2005. Intraoperative use of mitomycin C in fibrous atresia of the external auditory canal. Ear Nose Throat J., 84(12):776–779.PubMedGoogle Scholar
  4. Chen, T., Kunnavatana, S.S., Koch, R.J., 2006. Effects of mitomycin-C on normal dermal fibroblasts. Laryngoscope, 116(4):514–517. [doi:10.1097/01.MLG.0000205 590.62824.0A]PubMedCrossRefGoogle Scholar
  5. Crowston, J.G., Chang, L.H., Constable, P.H., Daniels, J.T., Akhar, A.N., Khaw, P.T., 2002. Apoptosis gene expression and death receptor signaling in mitomycin C-treated human Tenon capsule fibroblasts. Invest. Ophthalmol. Vis. Sci., 43(3):692–699.PubMedGoogle Scholar
  6. Ferguson, B., Gray, S.D., Thibeault, S., 2005. Time and dose effects of mitomycin C on extracellular matrix fibroblasts and proteins. Laryngoscope, 115(1):110–115. [doi:10. 1097/01.mlg.0000150694.08259.80]PubMedCrossRefGoogle Scholar
  7. Gospodarowicz, D., 1991. Biological activities of fibroblast growth factors. Ann. N. Y. Acad. Sci., 638:1–8. [doi:10.1111/j.1749-6632.1991.tb49012.x]PubMedCrossRefGoogle Scholar
  8. Gray, S.D., Tritle, N., Li, W., 2003. The effect of mitomycin on extracellular matrix proteins in a rat wound model. Laryngoscope, 113(2):237–242. [doi:10.1097/00005537-200302000-00008]PubMedCrossRefGoogle Scholar
  9. Hong, R., Lum, J., Koch, R.J., 1999. Growth of keloid-producing fibroblasts in commercially available serum-free media: a comparative study. Otolaryngol. Head Neck Surg., 121(4):469–473. [doi:10.1016/S0194-5998(99) 70239-1]PubMedCrossRefGoogle Scholar
  10. Kikuchi, K., Kakano, T., Takehara, K., 1995. Effects of various growth factors and histamine on cultured keloid fibroblasts. Dermatology, 190(1):4–8. [doi:10.1159/000246625]PubMedCrossRefGoogle Scholar
  11. Kim, T.I., Tchah, H., Lee, S., Cho, B.J., Kook, M.S., 2003. Apoptosis in keratocytes caused by mitomycin C. Invest. Ophthalmol. Vis. Sci., 44(5):1912–1917. [doi:10.1167/iovs.02-0977]PubMedCrossRefGoogle Scholar
  12. Kim, T.I., Tchah, H., Cho, E.H., Kook, M.S., 2004. Evaluation of safety of cultured corneal fibroblasts with cotreatment of alcohol and mitomycin C. Invest. Ophthalmol. Vis. Sci., 45(1):86–92. [doi:10.1167/iovs.03-0520]PubMedCrossRefGoogle Scholar
  13. Moses, H.L., Yang, E.Y., Peitenpol, J.A., 1990. TGF-β stimulation and inhibition of cell proliferation: new mechanistic insights. Cell, 63(2):245–257. [doi:10.1016/0092-8674(90)90155-8]PubMedCrossRefGoogle Scholar
  14. Occleston, N.L., Daniels, J.T., Tarnuzzer, R.W., Sethi, K.K., Alexander, R.A., Bhattacharya, S.S., Schultz, G.S., Khaw, P.T., 1997. Single exposures to antiproliferatives: long-term effects on ocular fibroblasts wound-healing behavior. Invest. Ophthalmol. Vis. Sci., 38(10):1998–2007.PubMedGoogle Scholar
  15. Peltonen, J., Hsiao, L.L., Jaakkola, S., Sollberg, S., Aumailley, M., Timpl, R., Chu, M.L., Uitto, J., 1991. Activation of collagen gene expression in keloids: co-localization of type I and VI collagen and transforming growth factor-β1 mRNA. J. Invest. Dermatol., 97(2):240–248. [doi:10. 1111/1523-1747.ep12480289]PubMedCrossRefGoogle Scholar
  16. Prasad, M., Ward, R.F., April, M.M., Bent, J.P., Froehlich, P., 2002. Topical mitomycin as an adjunct to choanal atresia repair. Arch. Otolaryngol. Head Neck Surg., 128(4):398–400.PubMedGoogle Scholar
  17. Ragab, S., 2005. The effect of radiofrequency and mitomycin C on the closure rate of human. tympanostomy. Otol. Neurotol., 26(3):355–360. [doi:10.1097/01.mao.0000 169760.70302.f1]PubMedCrossRefGoogle Scholar
  18. Rahbar, R., Jones, D.T., Nuss, R.C., Roberson, D.W., Kenna, M.A., McGill, T.J., Healy, G.B., 2002. The role of mitomycin in the prevention and treatment of scar formation in the pediatric aerodigestive tract. Arch. Otolaryngol. Head Neck Surg., 128(4):401–406.PubMedGoogle Scholar
  19. Schwartzman, R.A., Cidlowski, J.A., 1993. Apoptosis: the biochemistry and molecular biology of programmed cell death. Endocr. Rev., 14(2):133–151. [doi:10.1210/edrv-14-2-133]PubMedGoogle Scholar
  20. Seong, G.J., Park, C., Kim, C.Y., Hong, Y.J., So, H.S., Kim, S.D., Park, R., 2005. Mitomycin-C induced the apoptosis of human Tenon’s capsule fibroblast by activation of c-Jun N-terminal kinase 1 and caspase-3 protease. Invest. Ophthalmol. Vis. Sci., 46(10):3545–3552. [doi:10.1167/iovs.04-1358]PubMedCrossRefGoogle Scholar
  21. Simman, R., Hashim, A., Frances, W., 2003. Effect of mitomycin-C on keloid fibroblasts: an in vitro study. Ann. Plast. Surg., 50(1):71–76. [doi:10.1097/00000637-200301000-00012]PubMedCrossRefGoogle Scholar
  22. Simpson, C.B., James, J.C., 2006. The efficacy of mitomycin-C in the treatment of laryngotracheal-stenosis. Laryngoscope, 116(10):1923–1925. [doi:10.1097/01.mlg.0000 235934.27964.88]PubMedCrossRefGoogle Scholar
  23. Stewart, C.E.IV, Kim, J.Y., 2006. Application of mitomycin-C for head and neck keloids. Otolaryngol. Head Neck Surg., 135(6):946–950. [doi:10.1016/j.otohns.2005.07.026]PubMedCrossRefGoogle Scholar
  24. Tahery, M.M., Lee, D.A., 1989. Review: pharmacologic control of wound healing in glaucoma filtration surgery. J. Ocul. Pharmacol., 5(2):155–179. [doi:10.1089/jop. 1989.5.155]PubMedCrossRefGoogle Scholar
  25. Younai, S., Nichter, L.S., Wellisz, T., Reinisch, J., Nimni, M.E., Tuan, T.L., 1994. Modulation of collagen synthesis by transforming growth factor-beta in keloid and hypertrophic scar fibroblasts. Ann. Plast. Surg., 33(2):148–151. [doi:10.1097/00000637-199408000-00005]PubMedCrossRefGoogle Scholar

Copyright information

© Zhejiang University and Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Yao-wen Wang
    • 1
  • Ji-hao Ren
    • 2
  • Kun Xia
    • 3
  • Shu-hui Wang
    • 2
  • Tuan-fang Yin
    • 2
  • Ding-hua Xie
    • 2
  • Li-hua Li
    • 2
  1. 1.Otolaryngology-Head and Neck Surgery DepartmentNingbo First HospitalNingboChina
  2. 2.Otolaryngology-Head and Neck Surgery Department, the Second Xiangya HospitalCentral South UniversityChangshaChina
  3. 3.State Key Laboratory of Medical GeneticsCentral South UniversityChangshaChina

Personalised recommendations