Mitomycin C in dacryocystorhinostomy: the search for the right concentration and duration--a fundamental study on human nasal mucosa fibroblasts.
OBJECTIVE
To establish primary cultures of human nasal mucosal fibroblasts (HNMFs) and to test the effect of varying concentrations of mitomycin C (MMC) and treatment durations on cellular proliferation and viability of the fibroblasts.


METHODS
Laboratory investigation.


METHODS
Nasal mucosa harvested from patients undergoing a dacryocystorhinostomy was used to establish primary cultures by explant culture method. Cells were expanded and frozen at every passage, and passage 3 cells were used for further experiments. The cells were then treated with different concentrations of mitomycin C (0.1-0.5 mg/ml) for different time periods (3, 5, and 10 minutes). Cell viability was checked by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Cellular proliferation index was determined with bromodeoxyuridine immunostaining. Apoptotic index was measured using annexin A5 affinity assay, propidium iodide staining, and 4',6-diamidino-2-phenylindole counterstaining. The actin cytoskeletons of fibroblasts were studied using phalloidin staining.


RESULTS
The doubling time of cultured HNMFs is approximately 24 hours. Similarly, 0.4 mg/ml beyond 5 minutes and 0.5 mg/ml concentration at all time points were lethal and caused extensive cell death when compared with controls. A concentration of 0.2 mg/ml for 3 minutes of exposure prevented cell proliferation of HNMF cells by inducing cell cycle arrest, without causing extensive apoptosis.


CONCLUSIONS
The minimum effective concentration appears to be 0.2 mg/ml for 3 minutes. This in vitro study could be the starting point for further clinical and histopathologic studies to validate its clinical usefulness.
*Dacryology Service, Ophthalmic Plastics Surgery; †Sudhakar and Sreekant Stem Cell Biology Laboratory; and ‡Center for Epidemiology and Biostatistics, LV Prasad Eye Institute, Banjara Hills, Hyderabad, Andhra Pradesh, India.
Mohammad Javed
MJ
Ali
Indumathi
I
Mariappan
Savitri
S
Maddileti
Md Hasnat
MH
Ali
Milind N
MN
Naik
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003608:Dacryocystorhinostomy / Q000379:methods*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects*; D006801:Humans; D016685:Mitomycin / Q000494:pharmacology*; D009297:Nasal Mucosa / Q000166:cytology*; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology*
D000903:Antibiotics, Antineoplastic; D019384:Nucleic Acid Synthesis Inhibitors; D016685:Mitomycin