August 2007
Volume 48, Issue 8
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Glaucoma  |   August 2007
Interferon-α and Interferon-γ Sensitize Human Tenon Fibroblasts to Mitomycin-C
Author Affiliations
  • Xiao Yang Wang
    From the Center for Vision Research, University of Sydney, Westmead, Hospital Sydney, NSW, Australia; the
  • Jonathan G. Crowston
    From the Center for Vision Research, University of Sydney, Westmead, Hospital Sydney, NSW, Australia; the
    Center for Eye Research Australia, University of Melbourne, East Melbourne, Australia; and the
  • Hans Zoellner
    Department of Oral Pathology and Oral Medicine, Cellular and Molecular Research Unit, University of Sydney, Sydney, Australia.
  • Paul R. Healey
    From the Center for Vision Research, University of Sydney, Westmead, Hospital Sydney, NSW, Australia; the
Investigative Ophthalmology & Visual Science August 2007, Vol.48, 3655-3661. doi:10.1167/iovs.06-1121
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      Xiao Yang Wang, Jonathan G. Crowston, Hans Zoellner, Paul R. Healey; Interferon-α and Interferon-γ Sensitize Human Tenon Fibroblasts to Mitomycin-C. Invest. Ophthalmol. Vis. Sci. 2007;48(8):3655-3661. doi: 10.1167/iovs.06-1121.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To investigate the effect of interferon (IFN)-α and IFN-γ pretreatment on mitomycin C (MMC)–induced cell death in human Tenon fibroblasts (HTFs) and the mechanisms by which IFN-α and IFN-γ modulate the susceptibility of HTFs to MMC.

methods. HTFs were pretreated with IFN-α and IFN-γ for 48 hours before 5-minute application of 0.4 mg/mL MMC. Cell death after 48 hours was determined by Annexin V/propidium iodide (PI) staining and lactate dehydrogenase (LDH) release assay. Fas, Fas-ligand, and Bcl-2 expression were determined by flow cytometry. Fas associated death domain (FADD), Bax, cytochrome c, and caspase expression were determined by Western blot analysis and immunofluorescence staining.

results. MMC treatment increased cell death and upregulated Fas and FADD expression, but had no effect on Fas-Ligand, Bax, Bcl-2, or cytochrome c. Neither IFN-α nor IFN-γ alone induced HTF death, but each increased cell death 2 days after MMC treatment in a dose-dependent fashion. Combination IFN-α and IFN-γ had a synergistic effect. IFN-α and IFN-γ pretreatment increased Fas expression. Fas upregulation was associated with increased sensitivity to MMC. IFN pretreatment increased procaspase-8, procaspase-9, and procaspase-3 expression, and caspase-3 activation. Caspase-8, caspase-3, and broad caspase inhibitors, but not caspase-9 inhibitor, inhibited MMC-induced cell death in nonpretreated and IFN-pretreated cells.

conclusions. IFN-α and IFN-γ enhance the susceptibility of HTFs to MMC-induced cell death through a Fas-mediated and a caspase-3–dependent pathway. Pretreatment with IFN primed HTFs to MMC, providing a potential means for initially slowing the healing response with IFN and subsequently terminating fibroblast activity through MMC-induced cell death.

Tenon fibroblasts are the key players in postoperative subconjunctival scarring, the major cause of trabeculectomy failure. 1 Mitomycin-C (MMC) functionally inhibits and kills Tenon fibroblasts, providing a potent inhibitory effect on the scarring response. 2 Recent evidence suggests that fibroblast apoptosis mediates the reduction in fibroblast number at the end of the normal wound healing response and heralds the transition of active granulation tissue to an inactive scar. 3 Failure to terminate fibroblast activity because of defective apoptosis can lead to prolonged healing and excess scar formation. 4 Clinically relevant concentrations of MMC induce apoptosis in human Tenon fibroblasts (HTFs). 2 5 Although the precise mechanism is not fully understood, it is proposed that MMC increases the success of trabeculectomy, at least in part through the induction of fibroblast apoptosis and premature termination of the healing response. 5  
Apoptosis differs fundamentally from necrosis in that it is amenable to regulation. MMC alters the expression of a number of gene products known to alter the susceptibility of cells to apoptotic triggers. 5 Furthermore, the ability of MMC to induce fibroblast apoptosis can be modulated by altering the constituents of culture media that nourish the fibroblasts in vitro. We have shown that factors present in human and bovine serum inhibit the ability of MMC to induce fibroblast apoptosis. 6 Conversely, activating antibodies that signal through Fas/CD95 augments MMC-induced apoptosis. 5 The ability of exogenous factors to alter the susceptibility of fibroblasts to MMC may not only explain the varied outcomes seen in clinical practice to identical MMC treatment strategies, it may suggest that the susceptibility of discrete fibroblast populations to apoptotic triggers can be modulated. We hypothesize that the postoperative healing response may be better titrated by a “first-hit” intraoperative treatment that inhibits fibroblast activity only mildly but that specifically primes fibroblasts to subsequent postoperative treatment (second hit), which terminates the scarring response by inducing apoptosis once the healing process is completed. 
Interferon-α and IFN-γ are cytokines known to reduce fibrosis in various tissues. 7 8 9 10 Clinically, IFN-α and IFN-γ have been used with varying success to reduce scarring in patients with keloid and burns. 7 11 A randomized phase II trial showed similar success rates between postoperative subconjunctival IFN-α 2b and 5-fluorouracil (5-FU) after trabeculectomy. 12 Mechanisms by which IFNs modulate wound healing are thought to include inhibition of fibroblast proliferation 13 and collagen synthesis, antagonism of the fibrogenic effect of TGF-β, 9 14 15 and induction of fibroblast apoptosis. 16 IFN-α and IFN-γ can regulate the expression of apoptosis-associated genes and can sensitize some types of cells to apoptotic stimuli. 17 18 19 20 21 In search of candidate agents with the potential to slow the scarring response and prime cells for subsequent MMC-induced apoptosis, this study determined the effect of IFN-α and IFN-γ pretreatment on MMC-induced apoptosis. We also investigated the mechanisms by which these cytokines modulate the susceptibility of HTFs to MMC. 
Methods
Fibroblast Culture
HTF cell lines were generated from subconjunctival Tenon biopsy specimens obtained from glaucoma patients during surgery, as previously described. 5 The tenets of the Declaration of Helsinki were observed, and institutional human experimentation committee approval was granted. Fibroblasts were cultured in RPMI medium with l-glutamine supplemented with 10% heat-inactivated newborn calf serum and 50,000 U/L penicillin–streptomycin. Medium and chemicals were from Invitrogen (Life Technologies, Grand Island, NY). 
IFN and MMC Treatment
HTFs were seeded into culture wells and incubated with recombinant human IFN-α 2b (1000–100,000 U; Intron A; Schering-Plough, Kenilworth, NJ) or IFN-γ (50–500 U; Roche Applied Science, Mannheim, Germany) in complete RPMI culture medium for 48 hours. For induction of cell death, the fibroblast monolayer was treated with single applications of MMC (Kyowa Hakko Kogyo Ltd., Tokyo, Japan) for 5 minutes at a concentration of 0.4 mg/mL. This dose was selected to reflect clinical practice in glaucoma filtration surgery. MMC was reconstituted in phosphate-buffered saline (PBS). Controls were treated with 5-minute applications of PBS only. After treatment, the monolayers were washed three times with RPMI and were cultured in RPMI culture medium for 2 days before analysis of cell death. 
Quantitative Analysis of Cell Death
Annexin V-FITC/Propidium Iodide FACS Analysis.
Annexin V-FITC staining and propidium iodide (PI) uptake were determined in nonpermeabilized cells with the use of an apoptosis detection kit (R&D Systems, Minneapolis, MN) according to manufacturer’s instructions. Two days after MMC treatment, detached and adherent fibroblasts were harvested and mixed. Aliquots of 1 × 105 cells were resuspended in 100 μL binding buffer (10 mM HEPES-NaOH, 140 mM NaCl, 2.5 mM CaCl2) containing 5 μL Annexin V-FITC and 10 μL propidium iodide and were incubated for 15 minutes at room temperature. After two washes with PBS, 400 μL binding buffer was added, and samples were immediately analyzed with appropriate software (FACScan and CellQuest; Becton Dickson, San Jose, CA). Scatter profiles for fluorescence intensities in FL-1 (Annexin-V) and FL-2 (PI) were plotted. Quadrants were set to differentiate positive and negative staining. The percentage of cells in each quadrant was determined with WinMDI version 2.8. 
Lactate Dehydrogenase Release Assay.
Cells dying by apoptosis or necrosis released LDH into the supernatant. The amount of LDH in the supernatant was measured with a cytotoxicity detection kit (Roche). Briefly, HTFs seeded into 96-well plates, at 5 × 103 cells/well, were pretreated with IFN for 48 hours After treatment with 0.4 mg MMC for 5 minutes, cells were washed three times in PBS and were cultured in 200 μL phenol-free RPMI containing 0.1% BSA for 2 days. In inhibition experiments, the blocking effect of antagonizing anti–Fas antibody ZB4 (500 ng/mL; Upstate Ltd., Hampshire, UK) or caspase inhibitor peptides (R&D Systems) was assessed by adding these agents to the culture medium 1 hour before exposure to MMC. For analysis, 100 μL supernatant was extracted from each well and was placed in separate wells of a new 96-well plate, and 100 μL catalyst solution was added to each well and incubated at 37°C for 15 minutes. Absorbance was measured using a microplate reader with a 490-nm filter. Low-absorbance control cultures contained untreated fibroblasts, and high-absorbance control cultures equating to 100% cell death were obtained by lysing fibroblasts with 0.1% Triton X-100 in 200 μL RPMI medium. The percentage of cell death was calculated using a formula previously described 5 : % cell death = (experimental cells – low-absorbance control cells)/(high-absorbance control cells – low-absorbance control cells). 
Flow Cytometry for Fas, Fas-Ligand, and Bcl-2
Floating HTFs were harvested and mixed with adherent cells after trypsinization. Aliquots of 1 × 105 fibroblasts were incubated with 100 μL FITC-conjugated mouse anti–Fas (DX2 1:100, BD PharMingen, San Diego, CA), mouse anti–Fas-ligand (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), and mouse anti–Bcl-2 (1:100, Dako, Carpinteria, CA) for 30 minutes at 4°C. FITC-conjugated mouse IgG1 isotype (1:100; BD PharMingen) antibody was used as a negative control. For the detection of intracellular marker, cells were fixed with 2% paraformaldehyde for 60 minutes at 4°C and subsequently were permeabilized with 0.2% Tween-20 for 15 minutes at 37°C before incubation with antibody. Stained cells were washed twice with PBS buffer, resuspended in 200 μL 1% paraformaldehyde, and analyzed by FACScan. 
Western Immunoblotting
HTFs were harvested and lysed in lysis buffer (200 mM Tris, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, 1% sodium deoxycholate, 0.2 mM Na3VO4, 2 mM NaF, 10 μg/mL leupeptin, 10 μg/mL aprotinin, and 10 μg/mL pepstatin) on ice for 1 hour. Protein concentration of lysates was determined by Bradford assay (Bio-Rad, Hercules, CA). Equal volumes of protein (30–40 μg) were run on 8% to 16% gradient polyacrylamide gels (Bio-Rad) and then were transferred to nitrocellulose membranes (Protran; PerkinElmer Life Sciences, Boston, MA). Membranes were incubated for 1 hour with mouse anti–cytochrome c (1:1000), mouse anti–FADD (1:250), rabbit anti–caspase-8 (1:1000), rabbit anti–caspase-9 (1:2000; BD PharMingen), rabbit anti–caspase 3 (1:1000; Santa Cruz Biotechnology), and rabbit anti–Bax (1:1000; R&D Systems) and then were incubated with horseradish peroxidase–labeled goat anti–mouse antibody (1:5000; Biosource, Camarillo CA) or donkey anti–rabbit antibody (1:5000; Chemicon, Temecula, CA) for 1 hour. Bands were visualized by enhanced chemiluminescence (PerkinElmer). 
Immunofluorescence Staining
HTFs with or without IFN pretreatment were subjected to treatment with 0.4 mg/mL MMC for 5 minutes. Detached and adherent fibroblasts were harvested before and 1 or 2 days after MMC treatment. Cells were fixed and permeabilized with cytofix/cytoperm solution (BD PharMingen) for 1 hour at 4°C, followed by incubation of FITC-conjugated monoclonal rabbit anti–active caspase-3 antibody (BD PharMingen) for 1 hour. After two washes, cells were centrifuged onto glass slides and were mounted with mounting medium (Vectashield; Vector Laboratories, Burlingame, CA) containing DAPI. Stained cells were analyzed by fluorescence microscopy, and the percentage of caspase-3–positive cells were counted in three random areas per slide. 
Statistical Analysis
Quantitative experiments were analyzed with the use of unpaired Student’s t tests. Data are presented as mean ± SEM. Statistical significance was accepted with P < 0.05. 
Results
HTF Death in Response to MMC Treatment
Our previously published data show that MMC induces HTF death by apoptosis. 2 5 In this study, to quantitate the effect of MMC on HTF death, cultured monolayers were treated for 5 minutes with 0.4 mg/mL MMC or PBS (as a control). After 2 days, fibroblasts were collected, and apoptosis was quantified by flow cytometry with Annexin V-FITC/PI staining. As shown in Figure 1A , there were few apoptotic cells, which were positive for annexin in control, and the total cell death rate was 5.4%. MMC induced a sixfold increase in apoptosis, with total cell death increasing to 32.2% (P < 0.001). 
HTFs constitutively expressed Fas receptor, and expression was increased 2 days after MMC treatment compared with control (61.1% ± 9.2% vs. 20.3% ± 0.6%; P < 0.05; Fig. 1B ). Cultured HTFs expressed little Fas-ligand, and MMC had no effect on Fas-ligand expression (Fig. 1C) . To test whether the Fas receptor was functional, agonistic anti–Fas antibody (CH11; 50 ng/mL) was added to the HTF culture immediately after MMC treatment, and cell death was determined at day 2 by LDH assay. CH11 potentiated MMC-induced cell death, increasing cell death from 41% ± 2% for MMC treated alone to 67% ± 8% with the addition of CH11 (P < 0.01; Fig. 1D ). MMC treatment significantly increased FADD expression in HTFs but had no effect on Bcl-2 and Bax expression or on the release of cytochrome c to the cytosol (Figs. 1E 1F)
Effect of IFN on Fas and Fas-Ligand Expression
Forty-eight–hour pretreatment with 5000 U IFN-α slightly increased Fas expression (26% ± 1.8% vs.19% ± 0.2% in control; P < 0.05). Fas expression was increased to 41% ± 4.2% of total cells after 48-hour exposure to 100 U IFN-γ (P < 0.01). Combined incubation with IFN-α and IFN-γ at these concentrations resulted in a further increase, with 64% ± 6.2% of HTFs positive for Fas (P < 0.001; Fig. 2 ). Neither IFN-α nor IFN-γ altered Fas-ligand expression after 48-hour incubation (data not shown). 
Effect of IFN on Mitomycin-C–Induced Cell Death
As shown in Figure 3 , IFN-α and IFN-γ alone did not induce cell death in cultured HTFs but enhanced susceptibility to MMC, leading to a significant increase in cell death in a dose-dependent fashion. MMC induced 24% to 31% cell death in the absence of the cytokine compared with 40% to 100% cell death in HTFs pretreated with IFN-α or IFN-γ. A synergistic effect was observed after HTFs were pretreated with a combination of 5000 U IFN-α and 100 U IFN-γ, which induced a significant additional increase in MMC-induced cell death (Fig. 4A) . The increased sensitivity of HTFs to MMC was consistent with the level of Fas upregulation after IFN-α and IFN-γ pretreatment. However, application of ZB4, a blocking antibody against Fas, did not prevent cell death in IFN and MMC-treated fibroblasts (Fig. 4A) . In a parallel experiment, ZB4 completely inhibited agonistic anti-Fas (CH11)–induced cell death (Fig. 4B) , suggesting that interaction between Fas receptor and Fas-ligand was not critical for IFN and MMC-induced cell death. 
Caspase Expression after IFN/MMC Treatment
Expression of caspase-8, -9, and -3 was analyzed before and 1 or 2 days after MMC treatment in nonpretreated or IFN-pretreated HTFs. Western blot analysis revealed that MMC increased procaspase-8, procaspase-9, and procaspase-3 expression. IFN-α pretreatment for 48 hours enhanced the expression of procaspase-9 and procaspase-3, whereas IFN-γ pretreatment enhanced procaspase-8, procaspase-9, and procaspase-3 expression. Combinination IFN-α and IFN-γ resulted in further increases in the expression of procaspase-8 and -3 the day after exposure to MMC compared with treatment with IFN or MMC alone (Fig. 5A) . All three procaspases declined on day 2 after MMC treatment, possibly because of reduced viable fibroblasts at this stage or cleavage of the caspases to active subunits. 
Immunofluorescence staining using a monoclonal antibody specific for active caspase-3 showed little (8%) active caspase-3 in untreated HTFs. MMC treatment increased to 15% and 25%, respectively, of active caspase-3–positive cells on day 1 and day 2 (P < 0.05 vs. control). IFN pretreatment induced the activation of caspase-3 with a 2.5-fold increase in cells positive for active caspase-3 (P < 0.05% vs. control). Additional increases of active caspase-3–positive cells were found after 1 or 2 days of MMC treatment (31% and 44%; P < 0.05% vs. control; Fig. 5B ). The increased caspase-3 activation was consistent with the increased amount of cell death observed in HTFs treated with IFN and MMC. 
Effect of Caspase Inhibitors on IFN and MMC-Induced Apoptosis
To identify the specific caspases required for IFN and MMC-induced cell death, we used noncompetitive synthetic peptide inhibitors with different anticaspase specificities: z-IETD-fmk (caspase-8 inhibitor), z-LEHD-fmk (caspase-9 inhibitor), z-DEVD-fmk (caspase-3 inhibitor), and z-VAD-fmk (broad caspase inhibitor). Results are shown in Figure 6 . In fibroblasts treated with MMC alone, 100 μM z-IETD-fmk resulted in a 62.84% reduction of cell death, and 200 μM z-IETD-fmk resulted in a 47.66% reduction compared with controls (P < 0.001). Application of 100 μM caspase-9 inhibitor z-LEHD-fmk caused an apparent reduction of 29.16%, though this did not reach a level of statistical significance (P = 0.058). Inhibition of caspase-3 with 100 μM or 200 μM z-DEVD-fmk resulted in 44.56% and 77% reductions in cell death, respectively (P < 0.0001). MMC-induced cell death was reduced by 44.6% with 100 μM broad caspase inhibitor z-VAD-fmk and was completely inhibited at a concentration of 200 μM (P < 0.0001), indicating that MMC-induced cell death occurs by apoptosis on activation of the caspase cascade. In IFN-pretreated fibroblasts, with the exception of z-LEHD-fmk, these caspase inhibitors also reduced MMC-induced cell death, but to a lesser extent than with nonpretreated cells. Cell death was inhibited by 18.28% with 100 μM z-IETD-fmk (P < 0.05) and by 15.92% with 200 μM z-IETD-fmk (P = 0.084). Adding 100 μM or 200μM z-DEVD-fmk resulted in 29.4% and 43.5% inhibition of cell death (P < 0.0001); 100 μM or 200 μM z-VAD-fmk resulted in 27.5% and 49.9% inhibition of cell death (P < 0.0001). 
Discussion
This study demonstrated that IFN-α and IFN-γ prime HTFs to subsequent MMC treatment, resulting in extensive apoptosis. Combined IFN-α and IFN-γ had a synergistic effect and further increased the susceptibility of HTFs to MMC. Sensitization was associated with an upregulation of Fas receptor and increased caspase-8 and caspase-3 expression and activation. 
Current intraoperative anti–fibrosis treatments in trabeculectomy are titrated against a prediction of the anticipated postoperative wound healing response. Errors in estimation can lead to clinical failure from excess wound healing in undertreated or late bleb leak, hypotony maculopathy, and endophthalmitis from excessive inhibition of scar formation. An alternative treatment strategy would be to initially slow wound healing by reducing fibroblast activity and later terminating the healing response by the induction of fibroblast apoptosis in a way that spares the adjacent tissues. To address this, we propose a two-hit treatment strategy, whereby a first hit inhibits fibroblast activity and specifically primes fibroblasts to subsequent treatment (second hit), which induces apoptosis in primed cells. 
There are several advantages to using IFN-α and IFN-γ. First, these cytokines have been used as anti–scarring treatments for keloid, burns, and glaucoma filtration surgery. 7 11 12 Second, these cytokines have been reported to inhibit HTF proliferation and to reduce collagen synthesis and production. 13 22 23 24 Further, both interferons increase the expression of death receptors of the tumor necrosis receptor family and other apoptosis-related genes known to alter the susceptibility of cells to apoptotic triggers. By combining IFNs and MMC, HTFs become more sensitive to MMC-induced apoptosis. These pretreatments may thus provide a method for improving anti–scarring treatment during and after trabeculectomy. 
Previous studies have demonstrated that MMC-induced HTF death by apoptosis is an important mechanism for MMC anti–scarring activity, though the pathway by which MMC induces apoptosis in Tenon fibroblasts has not been fully elucidated. MMC has been reported to upregulate Fas expression in HTFs. 5 The application of agonistic anti–Fas antibody potentiated MMC apoptotic activity. In this study, we have confirmed these findings and have shown that MMC treatment increases FADD recruitment in HTFs. Furthermore, we have shown that the increased sensitivity of HTFs to MMC is consistent with the increased level of Fas expression after IFN pretreatment. These results support the notion that Fas signaling is involved in MMC-induced apoptosis. However, blocking anti–Fas antibody failed to prevent MMC-induced apoptosis or IFN sensitization of HTFs to MMC. In addition, there was no change in Fas-ligand expression after exposure to MMC or IFN, suggesting that despite an apparent role of Fas, interaction between Fas and Fas-ligand is not critical to MMC-induced apoptosis. A precedent for such Fas-ligand–independent, Fas-mediated apoptosis has been reported after exposure to cytotoxic agents. 25 26 27 It has been demonstrated that some anticancer drugs induce clustering and translocation of Fas receptor at the cell surface, resulting in the recruitment of FADD and the activation of caspase-8, thus activating the Fas pathway independently of Fas ligand. It seems reasonable to suggest that MMC might trigger the Fas pathway in this manner, consistent with the absence of an effect of blocking antibody for Fas in the activity under study. This possibility is also consistent with recent studies demonstrating that ultraviolet light irradiation can directly activate the Fas pathway by directly inducing the recruitment of FADD to Fas in the absence of Fas-ligand cross linking. 28 29  
Caspases are a family of cysteine proteases that play a crucial role in the process of apoptosis. They are divided into initiator caspases (such as caspase-8 and caspase-9) and executioner caspases (including caspase-3 and caspase-7) according to their function and their sequence of activation. In death receptor–mediated apoptosis, defined as the extrinsic pathway, caspase-8 is activated, whereas in the mitochondrial or intrinsic pathway, caspase-9 activation is involved. Both initiator caspases lead to the activation of executioner caspase-3 and caspase-7, which in turn cleaves specific proteins, resulting in the typical hallmarks of apoptosis. Our results showed that a broad caspase inhibitor almost completely abrogated MMC-induced apoptosis, confirming a central role for caspases. Seong et al. 30 reported MMC-induced apoptosis in HTFs through the activation of intrinsic and extrinsic caspase pathways, with increased activity of caspase-9 and caspase-3. Our data showed that an increase of procaspase-9 was not critical to the apoptotic process because a caspase-9 inhibitor failed to prevent MMC-induced apoptosis. In addition, MMC treatment did not alter the expression of Bcl-2 and Bax and had little effect on cytochrome c release. Taken together, it appears that the intrinsic caspase pathway is dispensable with regard to MMC-induced apoptosis in HTFs. In contrast, application of a peptide inhibitor specific to caspase-8 inhibited apoptosis in IFN and MMC-treated cells, further suggesting a role for Fas and the extrinsic pathway. 
Our results demonstrate that IFN enhances the susceptibility of HTFs to MMC through multiple pathways. These cytokines increase the expression of Fas and procaspase-8, both of which are components of the death-inducing signaling complex (DISC) in the Fas pathway. In addition, IFN increases caspase-3 production and activation. Caspase-3 activity has been described as essential for cytotoxic drug-induced apoptosis. Transfection of procaspase-3 can render cells more sensitive to cytotoxic drugs and can overcome acquired drug resistance. 31 The enhanced susceptibility to drug-induced apoptosis in procaspase-3–overexpressing cells can be explained by the recent finding that caspase-3 is involved in caspase amplification loops, thereby multiplying the impact of given drugs. 32 33 The biological significance of caspase-3 activation in our model was confirmed by the use of caspase-3 inhibitor, which significantly inhibited MMC-induced apoptosis, indicating that upregulation and activation of caspase-3 are important for IFN sensitization. 
In conclusion, IFN-α and IFN-γ pretreatment sensitize HTFs to MMC-induced apoptosis. The mechanism involves a Fas-mediated, caspase-3–dependent pathway. Data from this study support the biological feasibility of two-hit combination therapy through which IFN initially slows the healing process and primes treated fibroblasts to MMC-induced apoptosis, which may serve to switch off the wound healing response. 3  
 
Figure 1.
 
(A) HTF apoptosis detected by Annexin V and PI labeling in the absence (left) or presence (right) of 0.4 mg/mL MMC treatment. Cells in the lower left quadrant, which were negative for annexin V and PI, were viable. Cells in the lower right quadrant were annexin V-FITC positive and PI negative, indicating that the cells were in an early stage of apoptosis. Cells in the upper right quadrant, which stained positively for annexin V-FITC and PI, were secondary necrotic fibroblasts. Total percentage of cell death is shown in bold for each condition studied. (B) Flow cytometric analysis for Fas expression in HTFs with or without MMC treatment compared with isotype control. (C) Flow cytometric analysis for Fas-ligand expression. (D) LDH assay for cell death in HTFs after CH11 and MMC treatment (mean ± SEM; n = 8). (E) Flow cytometric analysis for Bcl-2 expression in control (gray line plot) and MMC-treated (black line plot) cells. (F) Western blot for FADD, cytochrome c, and Bax. Data are representative of three experiments.
Figure 1.
 
(A) HTF apoptosis detected by Annexin V and PI labeling in the absence (left) or presence (right) of 0.4 mg/mL MMC treatment. Cells in the lower left quadrant, which were negative for annexin V and PI, were viable. Cells in the lower right quadrant were annexin V-FITC positive and PI negative, indicating that the cells were in an early stage of apoptosis. Cells in the upper right quadrant, which stained positively for annexin V-FITC and PI, were secondary necrotic fibroblasts. Total percentage of cell death is shown in bold for each condition studied. (B) Flow cytometric analysis for Fas expression in HTFs with or without MMC treatment compared with isotype control. (C) Flow cytometric analysis for Fas-ligand expression. (D) LDH assay for cell death in HTFs after CH11 and MMC treatment (mean ± SEM; n = 8). (E) Flow cytometric analysis for Bcl-2 expression in control (gray line plot) and MMC-treated (black line plot) cells. (F) Western blot for FADD, cytochrome c, and Bax. Data are representative of three experiments.
Figure 2.
 
Flow cytometric analysis of Fas receptor expression by HTFs 48 hours after IFN-α (5000 U) or IFN-γ (100 U) treatment, or both. Gray solid plot presents cells labeled with isotype control antibody. Fas receptor in control cells is shown by the profiles illustrated with dashed line, while solid line plot presents the distribution of Fas in the cells treated with IFNs. Data are representative of three separate experiments.
Figure 2.
 
Flow cytometric analysis of Fas receptor expression by HTFs 48 hours after IFN-α (5000 U) or IFN-γ (100 U) treatment, or both. Gray solid plot presents cells labeled with isotype control antibody. Fas receptor in control cells is shown by the profiles illustrated with dashed line, while solid line plot presents the distribution of Fas in the cells treated with IFNs. Data are representative of three separate experiments.
Figure 3.
 
IFN-α and IFN-γ dose–response of cell death in MMC-treated HTFs. Fibroblasts were pretreated with IFN-α or IFN-γ for 48 hours before the application of 0.4 mg/mL MMC. Cell death was analyzed by LDH assay 2 days after MMC treatment. Data are mean ± SEM and are representative of three experiments.
Figure 3.
 
IFN-α and IFN-γ dose–response of cell death in MMC-treated HTFs. Fibroblasts were pretreated with IFN-α or IFN-γ for 48 hours before the application of 0.4 mg/mL MMC. Cell death was analyzed by LDH assay 2 days after MMC treatment. Data are mean ± SEM and are representative of three experiments.
Figure 4.
 
(A) HTFs were pretreated with 5000 U IFN-α, 100 U IFN-γ, or a combination of both for 48 hours before the application of 0.4 mg/mL MMC for 5 minutes. Cells were further cultured for 2 days in the presence or absence of 500 ng/mL antagonistic anti–Fas (ZB4). (B) Cells were stimulated with CH11 (50 ng/mL) for 2 days, with or without the application of ZB4. Cell death was analyzed by LDH assay. Data are representative of three experiments and are presented as mean ± SEM. *P < 0.01 compared with non-IFN–pretreated (control) cells. **P < 0.01 compared with IFN-α– or IFN-γ–pretreated cells.
Figure 4.
 
(A) HTFs were pretreated with 5000 U IFN-α, 100 U IFN-γ, or a combination of both for 48 hours before the application of 0.4 mg/mL MMC for 5 minutes. Cells were further cultured for 2 days in the presence or absence of 500 ng/mL antagonistic anti–Fas (ZB4). (B) Cells were stimulated with CH11 (50 ng/mL) for 2 days, with or without the application of ZB4. Cell death was analyzed by LDH assay. Data are representative of three experiments and are presented as mean ± SEM. *P < 0.01 compared with non-IFN–pretreated (control) cells. **P < 0.01 compared with IFN-α– or IFN-γ–pretreated cells.
Figure 5.
 
Expression of caspase-8, -9, and -3. HTFs with or without IFN pretreatment were treated with 0.4 mg/mL MMC for 5 minutes. Attached and floating cells were harvested before and 1 or 2 days after MMC treatment. (A) Western blot for procaspase-8, -9, and -3. (B) Immunofluorescence staining for active caspase-3; positive cells stained green. Cell nuclei stained blue. Data are representative of three to four experiments.
Figure 5.
 
Expression of caspase-8, -9, and -3. HTFs with or without IFN pretreatment were treated with 0.4 mg/mL MMC for 5 minutes. Attached and floating cells were harvested before and 1 or 2 days after MMC treatment. (A) Western blot for procaspase-8, -9, and -3. (B) Immunofluorescence staining for active caspase-3; positive cells stained green. Cell nuclei stained blue. Data are representative of three to four experiments.
Figure 6.
 
Effect of caspase inhibitors on IFN and MMC-induced cell death. HTFs with or without combined IFN-α (5000 U/mL) and INF-γ (100 U/mL) pretreatment were treated with 0.4 mg/mL MMC for 5 minutes. Cells were further cultured for 2 days after MMC treatment in the presence or absence of 100 μM or 200 μM inhibitors specific for caspase-8 (z-IETD-fmk), caspase-9 (z-LEHD-fmk), caspase-3 (z-DEVD-fmk), and broad caspase (z-VAD-fmk). Cell death was analyzed by LDH assay. Data are representative of three experiments and are presented as mean ± SEM. *P < 0.05. **P < 0.01 compared with MMC.
Figure 6.
 
Effect of caspase inhibitors on IFN and MMC-induced cell death. HTFs with or without combined IFN-α (5000 U/mL) and INF-γ (100 U/mL) pretreatment were treated with 0.4 mg/mL MMC for 5 minutes. Cells were further cultured for 2 days after MMC treatment in the presence or absence of 100 μM or 200 μM inhibitors specific for caspase-8 (z-IETD-fmk), caspase-9 (z-LEHD-fmk), caspase-3 (z-DEVD-fmk), and broad caspase (z-VAD-fmk). Cell death was analyzed by LDH assay. Data are representative of three experiments and are presented as mean ± SEM. *P < 0.05. **P < 0.01 compared with MMC.
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Figure 1.
 
(A) HTF apoptosis detected by Annexin V and PI labeling in the absence (left) or presence (right) of 0.4 mg/mL MMC treatment. Cells in the lower left quadrant, which were negative for annexin V and PI, were viable. Cells in the lower right quadrant were annexin V-FITC positive and PI negative, indicating that the cells were in an early stage of apoptosis. Cells in the upper right quadrant, which stained positively for annexin V-FITC and PI, were secondary necrotic fibroblasts. Total percentage of cell death is shown in bold for each condition studied. (B) Flow cytometric analysis for Fas expression in HTFs with or without MMC treatment compared with isotype control. (C) Flow cytometric analysis for Fas-ligand expression. (D) LDH assay for cell death in HTFs after CH11 and MMC treatment (mean ± SEM; n = 8). (E) Flow cytometric analysis for Bcl-2 expression in control (gray line plot) and MMC-treated (black line plot) cells. (F) Western blot for FADD, cytochrome c, and Bax. Data are representative of three experiments.
Figure 1.
 
(A) HTF apoptosis detected by Annexin V and PI labeling in the absence (left) or presence (right) of 0.4 mg/mL MMC treatment. Cells in the lower left quadrant, which were negative for annexin V and PI, were viable. Cells in the lower right quadrant were annexin V-FITC positive and PI negative, indicating that the cells were in an early stage of apoptosis. Cells in the upper right quadrant, which stained positively for annexin V-FITC and PI, were secondary necrotic fibroblasts. Total percentage of cell death is shown in bold for each condition studied. (B) Flow cytometric analysis for Fas expression in HTFs with or without MMC treatment compared with isotype control. (C) Flow cytometric analysis for Fas-ligand expression. (D) LDH assay for cell death in HTFs after CH11 and MMC treatment (mean ± SEM; n = 8). (E) Flow cytometric analysis for Bcl-2 expression in control (gray line plot) and MMC-treated (black line plot) cells. (F) Western blot for FADD, cytochrome c, and Bax. Data are representative of three experiments.
Figure 2.
 
Flow cytometric analysis of Fas receptor expression by HTFs 48 hours after IFN-α (5000 U) or IFN-γ (100 U) treatment, or both. Gray solid plot presents cells labeled with isotype control antibody. Fas receptor in control cells is shown by the profiles illustrated with dashed line, while solid line plot presents the distribution of Fas in the cells treated with IFNs. Data are representative of three separate experiments.
Figure 2.
 
Flow cytometric analysis of Fas receptor expression by HTFs 48 hours after IFN-α (5000 U) or IFN-γ (100 U) treatment, or both. Gray solid plot presents cells labeled with isotype control antibody. Fas receptor in control cells is shown by the profiles illustrated with dashed line, while solid line plot presents the distribution of Fas in the cells treated with IFNs. Data are representative of three separate experiments.
Figure 3.
 
IFN-α and IFN-γ dose–response of cell death in MMC-treated HTFs. Fibroblasts were pretreated with IFN-α or IFN-γ for 48 hours before the application of 0.4 mg/mL MMC. Cell death was analyzed by LDH assay 2 days after MMC treatment. Data are mean ± SEM and are representative of three experiments.
Figure 3.
 
IFN-α and IFN-γ dose–response of cell death in MMC-treated HTFs. Fibroblasts were pretreated with IFN-α or IFN-γ for 48 hours before the application of 0.4 mg/mL MMC. Cell death was analyzed by LDH assay 2 days after MMC treatment. Data are mean ± SEM and are representative of three experiments.
Figure 4.
 
(A) HTFs were pretreated with 5000 U IFN-α, 100 U IFN-γ, or a combination of both for 48 hours before the application of 0.4 mg/mL MMC for 5 minutes. Cells were further cultured for 2 days in the presence or absence of 500 ng/mL antagonistic anti–Fas (ZB4). (B) Cells were stimulated with CH11 (50 ng/mL) for 2 days, with or without the application of ZB4. Cell death was analyzed by LDH assay. Data are representative of three experiments and are presented as mean ± SEM. *P < 0.01 compared with non-IFN–pretreated (control) cells. **P < 0.01 compared with IFN-α– or IFN-γ–pretreated cells.
Figure 4.
 
(A) HTFs were pretreated with 5000 U IFN-α, 100 U IFN-γ, or a combination of both for 48 hours before the application of 0.4 mg/mL MMC for 5 minutes. Cells were further cultured for 2 days in the presence or absence of 500 ng/mL antagonistic anti–Fas (ZB4). (B) Cells were stimulated with CH11 (50 ng/mL) for 2 days, with or without the application of ZB4. Cell death was analyzed by LDH assay. Data are representative of three experiments and are presented as mean ± SEM. *P < 0.01 compared with non-IFN–pretreated (control) cells. **P < 0.01 compared with IFN-α– or IFN-γ–pretreated cells.
Figure 5.
 
Expression of caspase-8, -9, and -3. HTFs with or without IFN pretreatment were treated with 0.4 mg/mL MMC for 5 minutes. Attached and floating cells were harvested before and 1 or 2 days after MMC treatment. (A) Western blot for procaspase-8, -9, and -3. (B) Immunofluorescence staining for active caspase-3; positive cells stained green. Cell nuclei stained blue. Data are representative of three to four experiments.
Figure 5.
 
Expression of caspase-8, -9, and -3. HTFs with or without IFN pretreatment were treated with 0.4 mg/mL MMC for 5 minutes. Attached and floating cells were harvested before and 1 or 2 days after MMC treatment. (A) Western blot for procaspase-8, -9, and -3. (B) Immunofluorescence staining for active caspase-3; positive cells stained green. Cell nuclei stained blue. Data are representative of three to four experiments.
Figure 6.
 
Effect of caspase inhibitors on IFN and MMC-induced cell death. HTFs with or without combined IFN-α (5000 U/mL) and INF-γ (100 U/mL) pretreatment were treated with 0.4 mg/mL MMC for 5 minutes. Cells were further cultured for 2 days after MMC treatment in the presence or absence of 100 μM or 200 μM inhibitors specific for caspase-8 (z-IETD-fmk), caspase-9 (z-LEHD-fmk), caspase-3 (z-DEVD-fmk), and broad caspase (z-VAD-fmk). Cell death was analyzed by LDH assay. Data are representative of three experiments and are presented as mean ± SEM. *P < 0.05. **P < 0.01 compared with MMC.
Figure 6.
 
Effect of caspase inhibitors on IFN and MMC-induced cell death. HTFs with or without combined IFN-α (5000 U/mL) and INF-γ (100 U/mL) pretreatment were treated with 0.4 mg/mL MMC for 5 minutes. Cells were further cultured for 2 days after MMC treatment in the presence or absence of 100 μM or 200 μM inhibitors specific for caspase-8 (z-IETD-fmk), caspase-9 (z-LEHD-fmk), caspase-3 (z-DEVD-fmk), and broad caspase (z-VAD-fmk). Cell death was analyzed by LDH assay. Data are representative of three experiments and are presented as mean ± SEM. *P < 0.05. **P < 0.01 compared with MMC.
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