Induction and Mechanism of Apoptosis by Hydroxycamptothecin in Human Tenon's Capsule Fibroblasts

Abstract

Purpose.: We investigated apoptosis induced by hydroxycamptothecin (HCPT) in human Tenon's capsule using fibroblasts cultured from human Tenon's capsule (HTFs), and the mechanism of induction.

Methods.: HTFs were treated with 0–4 mg/L HCPT for 24 hours. Cell proliferation was measured by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay, and apoptotic cells were analyzed by Hoechst 33258 stain. The mRNA and protein levels of caspase-3, -8, and -9 were detected by RT-PCR and Western blotting.

Results.: By MTT assay, HCPT induced apoptosis in HTFs in a concentration- and time-dependent manner. Hoechst 33258 staining and transmission electron microscopy showed typical apoptotic morphology, such as condensed chromatin, irregular nuclei, and apoptotic body formation. The mRNA and protein levels of caspase-3 and caspase-9 were upregulated, while caspase-8 was unchanged. Z-VAD-FMK, a caspase inhibitor, inhibited the apoptosis of fibroblasts induced by HCPT. The expression levels of caspase-3 and caspase-9 were down-regulated after Z-VAD-FMK treatment.

Conclusions.: Caspase-3 and caspase-9 are important elements in regulating HCPT-induced apoptosis in HTFs.

Introduction

Glaucoma is the world's second leading cause of irreversible blindness. ^1,2 The estimated number of people with bilateral blindness from glaucoma was 8.4 million in 2010 and will be 11.2 million in 2020. ³ Filtration surgery (trabeculectomy) is the most frequent procedure used to decrease the intraocular pressure (IOP) in cases of glaucoma. This operation opens a channel for drainage of the aqueous humor from the anterior chamber to the subconjunctival space. However, the reported failure rate is approximately 15% to 30%. ^4–6 The main reason for surgical failure is the proliferation of fibroblasts leading to scarring of the channel in the filtration area. To control postoperative scarring, antiproliferative agents, such as mitomycin C or 5-fluorouracil, are used to prevent fibroblast growth and scar formation. ^7,8 However, side effects after the administration of antiproliferative agents include wound leakage, corneal erosion, corneal stromal neovascularization, scleral ulceration, and necrosis of the iris. ^9,10  

Human DNA topoisomerase I (Topo I) is a 765 amino acid nuclear enzyme, ¹¹ involved in topologic changes in DNA structure. ¹² It has key roles in DNA replication, transcription, and recombination. The identification of eukaryotic Topo I as the cellular target of camptothecin (CPT) is more recent. ¹³ A natural CPT analog, 10-hydroxycamptothecin (HCPT), has a strong antitumor activity against colorectal cancer, gastric cancer, hepatoma, and human lung cancer cells. ^14–17 HCPT has been shown to be more active and less toxic than other natural camptothecins. ^18,19 Moreover, it can inhibit noncancerous cells by affecting their biologic functions. HCPT can inhibit fibroblast proliferation, reducing scar formation after laminectomy ²⁰ and systemic sclerosis. ²¹  

The aim of our study was to determine whether HCPT induced cell death that exhibited the typical biochemical characteristics of apoptosis in human Tenon's capsule fibroblasts (HTFs), and to identify the pathway through which this is mediated. Therefore, we investigated the mRNA and protein levels of caspases-3, -8, and -9 in human Tenon's capsule, and in HTF cells after incubation with HCPT and/or Z-VAD-FMK (carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone). Our results indicated that HCPT inhibited growth and induced apoptosis in human Tenon's capsule cells, and Z-VAD-FMK inhibited apoptosis induced by HCPT in HTFs. We found that HCPT induced nuclear condensation, DNA fragmentation, and caspase-3 and caspase-9 activation, but did not activate caspase-8. Our results might be useful in future human trials before clinical application. 

Materials and Methods

Cell Culture and Maintenance

Human Tenon's capsule was obtained from fresh eyes donated to the eye bank of Jiangsu Province in Jiangsu Province Hospital affiliated with Nanjing Medical University. All fibroblasts were divided into five groups by HCPT treatment: 0.06, 0.25, 1, and 4 mg/L, and control (0.9% NaCl only). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal bovine serum (FBS), 0.1 U/L penicillin, and 50 μg/mL streptomycin at 37°C in 5% CO₂. The study was conducted in accordance with the tenets of the Declaration of Helsinki. 

Reagents and Antibodies

The 10-hydroxycamptothecin was obtained from Lishizhen Pharmaceutical Co. Ltd. (Lishizhen, Hubei, China). Trypsin was obtained from Gibco (Grand Island, NY).Hoechst 33258 stain and caspase inhibitor Z-VAD-FMK, caspase inhibitor, were from Beyotime Institute of Biotechnology. Mouse polyclonal anti-keratin and anti-vimentin were from Kangchen Biotechnology (Jiangsu, China). Mouse monoclonal GAPDH and actin antibodies were from Sigma Biotechnology (Sigma-Aldrich, St. Louis, MO). Rabbit polyclonal caspase-3, -8, and -9 antibodies were from Bioworld Biotechnology Pharmingen (Bioworld Technology, St. Louis Park, MN). Horseradish peroxidase (HRP)-labeled anti-mouse and anti-rabbit secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Chemiluminescent ECL substrate was from Santa Cruz Biotechnology. Reverse transcriptase was from Tiangen Biotech Co., Ltd. (Beijing, China) and Taq enzyme was from Takara Biotechnology (Dalian, Liaoning, China). 

Apoptosis Analysis

After 3 to 6 generations of logarithmic growth, fibroblasts were digested with 0.25% trypsin and transferred to six-well plates. At 80% to 90% confluence, fibroblasts were starved for 24 hours and treated with 0.06 to 4 mg/L HCPT for 24 hours. Treated cells were fixed with 4% paraformaldehyde at room temperature for 20 minutes, treated with 0.2% Triton X-100 for 5 minutes, and incubated with 1% bovine BSA at room temperature for 1 hour. Fixed cells were incubated with Hoechst for 5 minutes and mounted for fluorescent microscopy. 

Cell Viability Assay

Cell proliferation assays of HTFs were performed by standard 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay as described previously. ²² In brief, cells were seeded into 96-well plates at an initial density of 6 × 10³ cells/well and serum starved. After 24 hours, cells were incubated in DMEM containing HCPT (0, 0.06, 0.25, 1, or 4 mg/L) and/or caspase-inhibitor Z-VAD-FMK. At predetermined times, cultures were washed and incubated in DMEM containing 1 mg/mL MTT. Formazan crystals that formed at 4 hours were dissolved in dimethylsulfoxide (DMSO). Relative concentrations of formazan (as indicators of cell number) were determined by quantifying optical absorbance at 490 nm against a control wavelength of 590 nm in an automatic plate reader (Elx800, Bio-Tek; Bio-Rad, Richmond, CA). 

Western Blot Analysis

For Western blots to detect caspase-3, -8, and -9, HTFs were starved overnight in serum-free medium and incubated with HCPT (0, 0.06, 0.25, 1, or 4 mg/L) for 24 hours. Cells were lysed with ice-cold radio immunoprecipitation assay lysis buffer (50 mmol/L Tris, 150 mmol/L NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mmol/L sodium orthovanadate, 1 mmol/L sodium fluoride, 1 mmol/L EDTA, and 1 mmol/L phenylmethanesulfonyl fluoride [PMSF], 1% cocktail, pH 7.4). Lysates were clarified by centrifugation at 12,000g for 20 minutes at 4°C. 

Total protein content was determined using BCA assay as described previously, ²³ and equal amounts of samples were separated using 12% SDS-PAGE. Samples were transferred to polyvinylidene difluoride membranes with a Bio-Rad transfer unit at 20 V for 1 hour at room temperature. Membranes were blocked in blocking buffer for 14 hours followed by incubation with primary antibody (dilution 1:500–1:1000) and incubated with HRP-conjugated secondary antibody (dilution 1:2000). Immunoblotted proteins were visualized with ECL reagents for 4 minutes. GAPDH or actin was used as protein loading controls. 

RNA Preparation and RT-PCR

Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA) and 2 μg of RNA was used as a template for the synthesis of cDNA using the RevertAidFirst Strand cDNA Synthesis Kit (Invitrogen) according to the manufacturer's instructions. PCR was performed in 25 μL using PCR Master Mix (Invitrogen). 

Human caspase-3, -8, and -9, and GAPDH cDNA fragments were amplified using the primer pairs 5′-AGAAGATCACAGCAAAAGGAGC-3′ and 5′-TCAAGCTTGTCGGCATACTG-3′; 5′-GCAAAAGCACGGGAGAAAG-3′ and 5′-GGATACAGCAGATGAAGCAG-3′; 5′-TAACAGGCAAGCAGCAAA-3′ and 5′-TCTTGGCAGTCAGGTCGC-3′; and 5′-TGAACGGGAAGCTCACTGG-3′ and 5′-TCCACCACCCTGTTGCTGTA-3′, respectively. PCR was performed at 95°C for 4 minutes, followed by 28 cycles at 94°C for 30 seconds, 55°C for 45 seconds, and 72°C for 40 seconds. After the last cycle, extension was for 10 minutes. PCR products were separated on 1.0% agarose gels, stained with ethidium bromide and photographed. 

Statistical Analysis

Statistical analysis was performed using SPSS 13.0 software. Student's t-test was used to analyze differences between groups. For comparisons between multiple groups, one-way ANOVA was followed by SNK tests. Statistical significance was considered at P < 0.05. 

Results

Fibroblasts in Culture and Identification

Adherent human Tenon's tissue showed small amounts of long, spindle-shaped, radial, or spiral-shaped cells (Fig. 1A). After 2 to 3 days, most cells were stellate, curved, and spindle-shaped, and showed other irregular forms with more abundant cytoplasm and large nuclei. After passage, cell bodies were slightly longer, and cells migrated to the surrounding space when possible, growing longitudinally when space was limited. After passage, cells would confluence approximately 80% to 90% after 5 to 7 days (Fig. 1B). 

Figure 1. 

 

(A) 0 generation of cells migrated from a human Tenon's capsule. (B) Fifth generation of purified human Tenon's fibroblasts. All pictures were taken at 100× magnification.

To identify fibroblasts, immunofluorescence staining was used. Vimentin antibody staining showed cytoplasm as green fluorescence, with the majority of fibroblasts spindle-shaped with blue-stained nuclei (Fig. 2A). Keratin antibody staining was negative, showing cells with blue-stained nuclei (Fig. 2B). 

Figure 2. 

 

Identification of human Tenon's fibroblasts cells incubated with vimentin and keratin antibodies. (A) Immunofluorescence with vimentin-positive staining (green), and nuclei (blue) labeled with DAPI. (B) Negative keratin staining (green), and nuclear staining with DAPI (blue). All pictures were taken at 200× magnification.

HCPT Effect on Fibroblast Apoptosis and Viability

Hoechst Staining for Apoptosis.

Third-generation HTFs were used for Hoechst staining. The nuclei of normal cells stained blue, while apoptotic cells demonstrated a decrease in volume, density reduction, and chromatin condensation. Apoptotic cells showed shiny white nuclei by fluorescence microscopy. The numbers of apoptotic cells increased gradually with increasing concentrations of HCPT (Fig. 3). 

Figure 3. 

 

Detection of apoptotic cells by Hoechst 33258 staining. HTFs were control cells without any treatment (A), or 0.06, 0.25, 1, or 4 mg/L HCPT (B–E). Apoptotic nuclei were marginated distinctively and fragmented under the fluorescent microscope (A–E). Quantitative analysis of apoptotic cells after treatments (bar graphs) are average results from three different experiments (F). #, *P ≤ 0.05; # #, **P ≤ 0.01.

Cell Viability Measured by MTT.

HTFs were incubated with HCPT at 0, 0.06, 0.25, 1, or 4 mg/L for 24, 48, and 72 hours. A slight increase in OD values for cell proliferation was detected between the 0.06 mg/L HCPT and control groups, while higher concentrations of HCPT inhibited HTF proliferation in a dose-dependent way, compared to untreated controls. Differences were significant between the 24, 48, and 72-hour HCPT groups and the controls, reflecting time-dependence. Incubation with Z-VAD-FMK for 24 hours inhibited apoptosis induced by HCPT, although this was not significant between the control group and the Z-VAD-FMK + HCPT group (P > 0.05). However, a significant difference was detected between the HCPT and HCPT + Z-VAD-FMK groups (P < 0.05, Fig. 4). 

Figure 4. 

 

Detection of HTF cell viability by MTT assay. HCPT was used at four different concentrations. (A–E) HCPT inhibited the OD values of HTFs. (B–D) HCPT at 0.25 to 4 mg/L caused a significant inhibition of proliferation. (E) Significant differences were detected between HCPT and HCPT + Z-VAD-FMK groups (P < 0.05). Results are means of three experiments. #, *P ≤ 0.05; # #, **P ≤ 0.01.

Protein Levels for Caspase-3, -8, and -9

HTFs were starved overnight in serum-free medium and incubated with HCPT 0, 0.06, 0.25, 1, or 4 mg/L for 24 hours. Incubation with higher concentrations of HCPT caused protein levels of caspase-3 and caspase-9 to be higher in a dose-dependent way compared to controls, especially at HCPT 0.25 mg/L. However, caspase-8 was not changed obviously compared to controls (Fig. 5). 

Figure 5. 

 

Immunoblot for caspases-3, -8, and -9. HTFs were exposed to various concentrations of HCPT for 24 hours. Total cell lysates were analyzed by Western blot with anti-caspase-3, anti-caspase-8, or anti-caspase-9. β-Actin staining and GAPDH were performed to ensure equal loading. (A) Immunoblot for caspase 3; positions of the 35 kDa proenzyme of caspase-3 and of the 17 kDa cleavage product are indicated. (B) No change in caspase-8 was observed. (C) Expression of caspase-9 was upregulated dose dependently. #, *P ≤ 0.05; # #, **P ≤ 0.01.

Influence of Z-VAD-FMK on Caspase-3, -8, and -9

To investigate whether the increase in caspase-3 and caspase-9 was induced by HCPT or by apoptosis, we tested the mRNA and protein levels of caspase-3, -8, and -9 in cells incubated with Z-VAD-FMK for 24 hours. Z-VAD-FMK decreased the mRNA and protein levels of caspase-3 and caspase-9 after inducing with HCPT. In addition, Z-VAD-FMK inhibited the mRNA and protein expression of caspase-8, but there was no significant difference between the HCPT and control groups (Fig. 6). 

Figure 6. 

 

Z-VAD-FMK effect on caspase-3, -8, and -9. Lane 1: control HTFs. Lane 2: apoptotic stimuli alone (0.25 mg/L). Lane 3: apoptotic stimuli and Z-VAD-FMK (20 mM). Semiquantitative RT-PCR (A) and Western blot (B–D) showed the expression of caspase-3 and -9 were upregulated, while caspase-8 was not obvious in lane 2, compared to the control. Z-VAD-FMK inhibited the mRNA and protein levels of caspase-3, -8, and -9 exposed to apoptotic stimuli. #, *P ≤ 0.05; # #, **P ≤ 0.01.

Discussion

Apoptosis is a form of cellular suicide characterized by well-defined ultrastructural changes, including membrane blebbing, condensation of the chromatin and cytoplasm, and ultimately internucleosomal cleavage of DNA. ²⁴ Apoptotic cell death occurs in two phases, an initial commitment phase followed by an execution phase characterized by dramatic stereotypic changes in cell morphology. ²⁵ Apoptosis could be a potential general mechanism for the antiproliferative effects of HCPT. Apoptosis, or programmed cell death, is characterized by cell shrinkage, chromatin condensation, DNA fragmentation and the activation of specific cysteine proteases known as caspases. Two pathways that converge on caspase-3, one involving caspase-8 and the other involving mitochondrial release of cytochrome c and activation of caspase-9, have been described. ²⁶ Z-VAD-FMK is a potent inhibitor of apoptosis induced by a wide range of stimuli in a number of different systems, including human monocytic THP.1 cells, in which Z-VAD-FMK inhibits apoptosis at an early stage. ^27,28  

A family of at least 10 related cysteine proteases has been identified as caspases. ²⁹ These proteins are important in apoptosis. ³⁰ Caspase-3, which is central to the apoptotic cascade, is considered to be the executor of apoptosis. ³¹ In tumor cells undergoing apoptosis, multiple species of caspase-3 constitute the major pool of activated caspases regardless of the initial apoptotic stimulus. ³² However, in the cells of caspase-3-deficient mice, apoptosis occurs normally, demonstrating that other caspases with overlapping substrate specificity can replace caspase-3. ³³ Caspases-3, -6, and -7 are the major effector caspases. Activator caspases, such as caspase-8, are proposed to act near or at the apex of the hypothetical caspase cascade, activating effector caspases. The identity of the activator caspases is less clear, although evidence suggests that caspase-8 is recruited to the Fas signaling complex and initiates the caspase cascade involved in Fas-mediated apoptosis. ^34–37 Caspase-8 and caspase-10 can process and activate all known pro-caspases as well as each other. ^36,38  

We demonstrated that HCPT induced apoptosis in human Tenon's capsule in a dose-dependent and time-dependent manner. Apoptotic HTF cells were observed by Hoechst 33258 staining. Higher concentrations of HCPT led to clear enhancement of apoptosis in HTF cells. 

We demonstrated that HCPT inhibits the growth of human Tenon's capsule cells, suggesting that the antiproliferative effects of HCPT are mediated through induction of apoptosis. Moreover, to our knowledge for the first time in human Tenon's capsule cells, we showed that HCPT-induced apoptosis might act through the caspase pathway. Consistent with our results, HCPT inhibited fibroblast proliferation and reduced epidural scar adhesion after laminectomy in a concentration-dependent manner. ²⁰ As a cell cycle-specific agent, HCPT mainly blocks the G1/S transition of the cell cycle ³⁹ and inhibits the proliferation of many types of tumor cells. ^14–17 Recently, HCPT was demonstrated to exert potent cytotoxic effects on B16-F10 cells through the induction of apoptosis. ⁴⁰ Similar to our results, HCPT induced apoptosis in a pancreatic cancer cell line, involving the mitochondria and activation of caspase-3/caspase-9. ⁴¹ Interestingly, we found that HCPT treatment activated the caspase-8 pathway to induce apoptosis in human lung cancer cells. ¹⁷ Further studies are needed to clarify whether the HCPT-mediated apoptotic response varies in cells of different tissue origin. Although our data supported the involvement of caspases, further studies are in progress to understand the details of the mitochondrial pathway stimulated by HCPT in HTFs. Nevertheless, our experiments provided evidence that HCPT is an effective inducer of apoptosis in HTFs through the activation of the caspase-3 and caspase-9 cascade, but independent of caspase-8 activation. 

In summary, we determined that HCPT inhibited proliferation and induced apoptosis in HTFs through a mitochondria-dependent pathway. The exact mechanism of intracellular signal transduction remains to be determined. In the future, HCPT might be a feasible therapeutic approach to reduce excessive postoperative scarring and prevent failure of glaucoma filtration surgery. 

Footnotes

 Disclosure: W. Tang, None; Y. Zhang, None; C. Qian, None; Z. Yuan, None; J. Du, None