Keywords
fibroblasts - fibrosis - mitomycin C - doxorubicin - 5-fluorouracil - cytostatic drugs
Introduction
Dacryocystorhinostomy is one of the most widespread surgeries performed on patients
with primary acquired nasolacrimal duct obstruction. The purpose of this surgical
intervention is to form stable epithelized anastomosis (ostium) between the lacrimal
sac cavity and the nasal cavity. Despite improvements in the surgical technique, data
show that the rate of recurrences of this intervention is as high as 13 to 17%.[1]
[2] One of the most common causes for a negative surgery outcome is excessive cicatrization
at the ostium site.[3]
[4] Recently, cytostatic drugs, which are administered either applicationally or by
injection into the mucous membrane of the nasal cavity or lacrimal sac at the final
stage of the surgery, have become relatively widespread to reduce the severity of
the fibrotization. Cytostatic drugs are known to reduce fibroblast proliferation,
which, in turn, causes a reduction in fibrotization in the region in which a cytostatic
drug is administered.[5]
Several authors have reported an accentuated decrease in the number of lacrimal passage
obliteration recurrences following the administration of cytostatic drugs,[6]
[7] while other authors did not report this effect.[8]
[9] The authors of the present study argue that the absence of clinically-significant
fibrotization inhibition was due to the fact that the required cytostatic concentration
had not yet been achieved in the tissues of the formed anastomosis region.[8] The present paper is a study on the dosage-dependent effect that cytostatic drugs
have on cultivated fibroblasts of the human nasal mucosa. Knowing a concentration
at which cytostatic drugs retain their toxicity against nasal mucosa fibroblasts will
enable the implementation of these techniques in the clinical practice to prevent
excessive cicatrization when performing various surgical interventions in the nasal
cavity, particularly dacryocystorhinostomy.
The purpose of the present paper is to determine the cytotoxic concentrations of mitomycin
С, doxorubicin and 5-fluorouracil that affect the nasal mucosa fibroblasts.
Methods
Isolation and Characterization of the Cell Culture
The authors obtained the histological material necessary for the study during an endonasal
endoscopic dacryocystorhinostomy. Following nasal mucosa decongestion with 0.1% xylometazoline
solution and local topical anesthesia with 10% lidocaine solution (Pharmstandard-Leksredstva,
Kursk, Kursk Oblast, Russia), a 1.5-mm deep horizontal incision (using a diamond-shaped
knife with an incision depth limitation) was made in the lacrimal fossa projection
region, and 4 × 2-mm section of mucous membrane was bluntly separated at that depth.
This biopsy specimen was soaked in 0.04% gentacimin solution (Dalhimfarm, Khabarovsk,
Khabarovskiy kray, Russia) for 30 minutes, and then transferred to the cell technology
laboratory for further treatment.
The specimen was sliced into 1 × 1-mm fragments using a surgical scalpel. The fragments
were placed in Petri dishes with a diameter and growth surface area of 3.5 cm and
10 cm2 respectively (Corning, Corning, NY, US) and cultured in Dulbecco's Modified Eagle's
Medium (DMEM), supplemented with 2 mM of glutamine, 100 U/ml of penicillin, 0.1 mg/ml
of streptomycin (Gibco, Thermo Fisher, Waltham, MA, US) and 10% Fetal bovine serum
(FBS) at 37°C, in a humidified atmosphere containing 5% of CO2. On the 4th to 5th days of cultivation, the cells started migrating from the fragment
tissue to the plastic. By the 14th day of cultivation, the explants were removed from
the dishes, and the remaining cells were dissociated with a 0.05% Trypsin-ethylenediaminetetraacetic
acid (EDTA) solution (Gibco) and passaged in T25 culture flask. Our previous experience
with explant cultivation showed that the proliferation of first-passage fibroblasts
is not sufficiently active. Due to this, a growth factor was added to the first passage
medium. First-passage cells were cultivated in DMEM using the aforementioned composition
and fibroblast growth factors (FGF, Sigma-Aldrich, St. Louis, MO, US) with a concentration
of 4 ng/ml, while the second-passage cells had no FGF treatment. The growth medium
was replaced every 3 to 4 days. The cells were subcultured at a 1:4 to 1:6 ratio.
Cell-growth monitoring and the morphology assessment were performed using a Zeiss
Axio Vert.A1 (Carl Zeiss, Oberkochen, Germany) inverted microscope. Second- to fourth-passage
cells were used in the experiments.
Immunocytochemical Analysis
To characterize the cell culture obtained, the cells were stained for fibroblast specific
markers: vimentin and cluster of differentiation 90 (CD90). The cells were grown in
Petri dishes for confocal microscopy (5 × 103 cells per dish). After attaining 50% of confluence, the cells were fixed with 4%
paraformaldehyde (10 minutes at 4°C), washed 3 times with Phosphate buffered saline
(PBS), and incubated for 30 minutes at room temperature in PBS containing 0.2% tween-20,
0.2% triton x-100, and 2% goat serum. Then the samples were incubated with primary
antibodies to vimentin (1:40; Abcam, Cambridge, United Kingdom,) in PBS with 0.2%
tween-20 and 0.2% goat serum (for 1 hour at 37°C), washed 3 times with PBS, and incubated
with second anti-mouse immunoglobulin antibodies (goat anti-mouse Alexa Fluor 555,
1 μg/ml, Invitrogen, Carlsbad, CA, US) for 1 hour at 37°C. The samples were washed
with PBS, and the cell nuclei were poststained with 4',6-diamidino-2-phenylindole
(DAPI) (1:400; Invitrogen). For CD90 visualization, the cells were incubated for 10 minutes
at 4° С with anti-CD90 primarily labeled antibodies (1:11; Miltenyi Biotec, Bergisch
Gladbach, North Rhine-Westphalia, Gernamy) carrying phycoerythrin fluorescent dye
([Fig. 1]).
Fig. 1 Immunocytochemical staining. Confocal laser scanning microscopy (Nikon A1R MP + ). Cell nuclei are stained with
DAPI (1:400; Invitrogen) (blue fluorescence). Bar scale: 50 µm. (A) Anti-vimentin primary monoclonal antibodies (1:40; Abcam) and antimouse immunoglobulin
secondary antibodies, conjugated with Alexa555 (1:750; Invitrogen) fluorescent dye
(red fluorescence). (B) Primarily labeled anti-cluster of differentiation 90 - phycoerythin antibodies (1:11;
Miltenyi Biotec) for CD90 visualization (red fluorescence).
Confocal Laser Scanning Microscopy
The scanning was performed using a A1R MP+ confocal laser scanning microscope (Nikon,
Shinagawa, Tokyo, Japan). The 405-nm and 561-nm emission lasers and the following
optics were used in the present study: Plan Apo 20x/0,75 Dic N, Apo IR 60x/1,27 WI
and Apo TIRF 60x/1,49 oil Dic lens (Nikon, Shinagawa, Tokyo, Japan). The cell contours
were visualized using differential interference contrast. The images obtained were
processed using the NIS-Elements AR software (Nikon).
Cell Viability Assay
the CellTiter 96 AQueous One Solution Reagent kit (Promega, Madison, WI, US) was used
for the MTS test. The MTS reagent was defrosted immediately before use. The test was
performed according to manufacturer's instructions (procedure TB245). The cells were
cultured in 96-well plates (3,000 cells/well) in DMEM containing 10% FBS, antibiotics
(100 units/mL of penicillin, 100 µg/ml of streptomycin, Gibco), and GlutaMax (2 mМ,
Gibco). After 24 hours of cultivation, drugs at various concentrations obtained by
serial dilutions in the growth medium were added inside the wells. After 24 hours
of incubation, 20 µl of MTS (3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)
reagent were added to each well containing 100 µl of growth medium. The cells incubated
with the culture medium were used as the negative control. The culture medium without
cells was used for the blank.
The optical density was measured at 490 nm after 4 hours of incubation with the MTS
reagent. The cell viability was calculated using the following formula:
Number of living cells = (Аs – Аb / Аc – Аb) × 100%
Аs
– mean value of the specimen's optical density (OD); mean OD sample;
Аb
– mean value of the blank sample's optical density; mean OD control blank;
Аc
– mean value of the control's optical density; mean OD control.
Values of IC50 were found with Hill equation using the GraphPad Prism 6 (GraphPad
Software, San Diego, CA, US) software.
Results
As a result of the study, a human nasal mucosa fibroblast cell culture positive for
specific markers (vimentin and CD90) was obtained.
The culture was used as a test system to determine the cytotoxicity effect of the
following cytostatic drugs: mitomycin с, doxorubicin and 5-fluorouracil. The MTS test
is a colorimetric method that enables the determination of the number of viable cells
when studying cell proliferation and the cytotoxicity of various drugs. Mitochondrial
NADH nicotinamide adenine dinucleotide (reduced)-dependent oxidoreductases are capable
of reducing the MTS reagent to formazan, whose absorption rate reaches its maximum
value at 490 nm to 500 nm.
[Table 1] shows the results of the analysis of the number of viable fibroblasts depending
on the concentration of the cytostatic drugs used. These data are shown in [Fig. 2]. The IC50 values for mitomycin c, doxorubicin and 5-fluorouracil were 113 mkg/ml,
392 mkg/ml and 19 mkg/ml respectively.
Table 1
Results of the cytotoxicity analysis
|
Mitomycin C
|
Doxorubicin
|
5-fluorouracil
|
|
Concentration (mg/ml)
|
Survival (%)
|
Mean ± standard deviation
|
Concentration (mg/ml)
|
Survival (%)
|
Mean ± standard deviation
|
Concentration (mg/ml)
|
Survival, (%)
|
Mean ± standard deviation
|
|
Test 1
|
Test 2
|
|
|
Test 1
|
Test 2
|
|
|
Test 1
|
Test 2
|
Test 3
|
|
|
1
|
0
|
0
|
0 ± 0
|
1
|
0
|
0
|
0 ± 0
|
25
|
0
|
14
|
0
|
5 ± 8
|
|
0.25
|
14
|
17
|
16 ± 2
|
0.25
|
33
|
46
|
39 ± 9
|
12.5
|
2
|
27
|
51
|
27 ± 25
|
|
0.006
|
53
|
55
|
54 ± 2
|
0.006
|
51
|
78
|
65 ± 19
|
6.25
|
63
|
67
|
60
|
64 ± 4
|
|
0.015
|
59
|
62
|
61 ± 2
|
0.015
|
71
|
77
|
74 ± 4
|
3.125
|
75
|
78
|
75
|
76 ± 2
|
|
0.003
|
71
|
72
|
71 ± 1
|
0.003
|
79
|
84
|
82 ± 3
|
1.5625
|
78
|
80
|
78
|
79 ± 1
|
|
0.0009
|
81
|
94
|
87 ± 9
|
0.0009
|
86
|
83
|
85 ± 2
|
0.7813
|
81
|
84
|
78
|
81 ± 3
|
Fig. 2 Cell viability and drug concentration. (A) Mitomycin C; (B) doxorubicin; (C) 5-fluorouracil.
Discussion
The present paper is a study on the effect of cytostatic drugs on cultivated fibroblasts
of the nasal mucosa. The cytostatic drugs studied are known to reduce fibroblast proliferation
by inhibiting DNA replication. The effect of doxorubicin is intercalation, that is,
integration of the nitrogen bases of the DNA, which inhibits the effect of topoisomerase
II, thus making the relaxation of super-spiralized DNA sections impossible, and disturbing
the transcription process. The effect of 5-fluororacil implies replacing uracil with
fluorouracil in a replicated RNA molecule, which make its further processes impossible.
The effect of mitomycin C leads to the formation of covalent bonds between complimentary
DNA strands, thus hampering its replication.
The use of mitomycin C for antifibrotic purposes has significantly increased in the
lacrimal surgery practice.[5]
[6]
[7]
[8]
[9] There are also several reports on the use of 5-fluorouracil as an antifibrotic agent
for dacryocystorhinostomy.[10]
[11]
[12] The authors found no evidence of the use of doxorubicin to prevent dacryocystitis
recurrence. However, in vitro studies regarding mucosal cells showed the effect of
doxorubicin on collagenogenesis.[13]
As the analysis of the results of the present study showed, the toxic effect that
the drugs in question had on the cultivated fibroblasts of the human nasal mucosa
was dosage-dependent.
The present study showed that the toxic effect that mitomycin C had on fibroblasts
of the human nasal mucosa was sufficient for to terminate fibroblast growth at a concentration
of 0.25 mg/ml, which corresponds to the data of a previous research.[5]
To date, no studies on the toxicity of doxorubicin and 5-fluorouracil against fibroblast
cultures of the nasal mucosa have been performed. In the present study, the authors
found that the optimal effective doxorubicin concentration for the termination of
fibroblast growth is 0.25 mg/ml; yet, at this concentration, its toxic effect on the
fibroblasts is smaller than that of mitomycin C at the same concentration. Thus, it
is plausible to expect a lower clinical effect of doxorubicin at the concentration
of 0.25 mg/ml, when compared to that of mitomycin C at the same concentration and
dosage.
The analysis of the toxic effect of 5-fluorouracil showed that 12.5 mg/ml is the optimal
concentration of this drug to terminate fibroblast growth; however, at this concentration
its toxic effect on nasal cavity fibroblasts is also lower than that of mitomycin
C when used at the concentration of 0.25 mg/ml.
In all cases, an increase in concentration made the toxicity of the drugs studied
get close to its absolute value, while a decrease inhibited the cell's viability to
a degree apparently insufficient for the development of the clinical effects.
Surgeons currently tend to use cytostatic drugs at empirically chosen concentrations,
which leads to negative outcomes and deviation from the technique: for example, in
the studies by Bakri et al[11] and Watts et al,[12] the authors used 5-fluorouracil at concentrations of 0.5 mg/ml and 25 mg/ml respectively.
The present study provides a clue to explain the reason behind the lack of effect
of the drug in the former study and the relatively poor results in the latter one.
The results obtained in the present study enable us to assume that the use of cytostatic
drugs at exactly the concentrations determined by the authors may enable the achievement
of the maximum antifibrotic effect, with a simultaneous reduction in the number of
undesirable side effects on the cells, which also affects postdacryocystorhinostomy
tissue regeneration processes. The authors argue that it is appropriate to use these
data when carrying out experiments using a more complex model. Regarding the fact
that the in vitro research could not be directly extrapolated to the clinical practice,
we suppose that an experimental trial with an animal model should be an appropriate
following step for the present research. The data obtained with an animal model-based
research could be translated into the clinical practice.
Conclusion
The cytostatic drugs studied have a toxic effect on cultivated fibroblasts of the
nasal mucosa. The authors showed that this effect is dosage-dependent. In terms of
reducing the level of tissue fibrotization in the nasal cavity and, particularly,
in the dacryocystorhinostomy-formed junction site, the most justified approach is
to carry out an experimental study on the effect of mitomycin C, doxorubicin and 5-fluorouracil
at the concentrations of 0.25 mg/ml, 0.25 mg/ml, and 12.5 mg/ml respectively.
The authors suppose it is inappropriate to use these cytostatic drugs to conduct studies
with the goal of analyzing their antifibrotic effect on the nasal mucosa at concentrations
that differ from the aforementioned ones, since at such concentrations the drugs either
cannot have a clinically-significant effect or, as the authors assume, can have an
undesirable effect by inhibiting the proliferation not only of fibroblasts, but also
of other cells affecting nasal mucosa reparation.
