Abstract
Lomefloxacin (LFX) is a widely used fluoroquinolone antimicrobial agent that plays an important role in the treatment of human and animal infections; however, it has been reported to cause phototoxicity. In this study, we investigated the induction of apoptosis due to ultraviolet A (UVA) light in the presence and absence of LFX in HL-60 human promyelocytic leukemia cells. HL-60 cells were exposed to UVA at an intensity of 1.1 mW/cm2 for 20 min in the presence and absence of LFX, and the induction of apoptosis was examined by analyzing cell morphology, DNA fragmentation, and caspase-3 activity. Cells treated with 100 μM LFX and UVA clearly showed membrane blebbing and cell shrinkage. The proportion of apoptotic cells was significantly higher in cells treated with both UVA and LFX than in those treated with UVA or LFX alone. In addition, DNA ladder formation and caspase-3 activation were observed in cells treated with both UVA and LFX. A significant reduction in the number of UVA-induced apoptotic cells and caspase-3 activation was observed when histidine was present, which suggested that photodynamically-generated singlet oxygen is an important mediator of apoptosis. These results indicate that the combination of UVA and LFX induces apoptosis in HL-60 cells.
The ultraviolet (UV) radiation from sunlight is the most prominent physical carcinogen in our natural environment, and its role in skin carcinogenesis has been established (1, 2). UVA (320-400 nm) is the predominant UV band that reaches the earth's surface, and penetrates the skin more deeply than UVB, causing the generation of reactive oxygen species (ROS), which are responsible for oxidative damage, in both the epidermal and dermal skin layers. Studies have reported that UVA causes ROS-mediated oxidative damage to the skin through lipid peroxidation, protein peroxidation, and oxidative DNA damage (3, 4).
Fluoroquinolones are widely used broad-spectrum antibiotics that play an important role in the treatment of human and animal infections (5, 6). In mammalian cells, they are strong inhibitors of bacterial DNA gyrase and type II DNA topoisomerase, which are enzymes responsible for DNA supercoiling and are involved in chromosome condensation, DNA replication, transcription, and recombination (7, 8).
Necrosis and apoptosis are the two major processes of cell death. Necrosis is a passive process that is most commonly induced by severe external damage, which causes cytoplasmic swelling and disruption of the cell membrane. As a consequence, lysosomal enzymes are released, usually inducing an inflammatory reaction. In contrast, apoptosis is an active process that consists of a suicide program for the removal of unnecessary, aged, or damaged cells. Cells undergoing apoptosis exhibit characteristic morphological changes, including initial shrinkage, followed by widespread membrane blebbing, chromatin condensation, and DNA fragmentation. The cells further disassemble into membrane-enclosed vesicles called apoptotic bodies that are rapidly taken up and digested by neighboring cells and phagocytes (9-11). A well-known physical agent that triggers the apoptotic process in cells is UV light, which directly damages DNA and leads to high ROS production (12, 13). The induction of apoptosis following UVA exposure appears to be a protective mechanism for removing severely damaged cells that bear the risk of malignant transformation. Since leukemia cells easily undergo apoptosis, the capacity of chemicals to evoke apoptosis in them may determine the feasibility of the sensitivity response. In addition, understanding the apoptosis-inducing properties of photosensitizing agents may be useful not only for the evaluation of photoallergenicity for preventing adverse effects, but also for the application of photosensitization as a cancer treatment.
In this study, we investigated the apoptosis-inducing ability of lomefloxacin (LFX), a widely used fluoroquinolone antimicrobial agent that plays an important role in the treatment of human and animal infections but which causes photocontact dermatitis and drug photosensitivity.
Materials and Methods
Chemicals. LFX, histidine, superoxide dismutase (SOD), mannitol, and ethidium bromide were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents used were of analytical grade.
Cell culture. HL-60 Human promyelocytic leukemia cells were obtained from the Riken Gene Bank (Tokyo, Japan). Cells were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Gibco BRL, Tokyo, Japan), 100 U/ml penicillin G, 100 μg/ml streptomycin, and 2 mM glutamine (Sigma-Aldrich) in a humidified atmosphere of 5% CO2 at 37°C. Before exposure, cells were harvested and washed twice in phosphate-buffered saline (PBS; pH 7.4). HL-60 cells were washed, re-suspended at a density of 1×106 cells/ml in 2.5 ml of serum-free RPMI-1640 and transferred into a 35×10-mm polystyrene tissue culture dish (Corning, Corning, NY, USA) for exposure to UVA. Immediately before exposure, LFX was added to the cell suspension at a concentration of 100 μM .
UVA irradiation. UVA experiments were performed using six parallel FL20SBLB lamps (Toshiba, Tokyo, Japan) with a peak emission frequency of 352 nm. Cells were irradiated at a distance of approximately 20 cm. The irradiation intensity was measured using a radiometer (UVR-305/365; Toshiba) that was placed at the same distance from the UVA source as the cells. Total UVA irradiation dose applied throughout the 20-min exposure was 0.84 J/cm2. Samples treated with LFX alone were maintained in the same position for the same period as cells receiving UVA exposure. After treatment, the cells were once again incubated in a humidified atmosphere of 5% CO2 at 37°C for 6 h.
Evaluation of apoptosis and cell damage. HL-60 cells were examined using a phase-contrast inverted microscope (Olympus, Tokyo, Japan) under 400× magnification. The integrity of the treated cells was determined by staining the cells with Trypan blue immediately after treatment. The proportion of apoptotic cells was calculated by calculating the ratio of the number of unstained cells with morphological changes to the number of total unstained cells on a hemocytometer glass. The integrity of intact cells was also checked immediately prior to each series of treatments, with cell suspensions exhibiting >99% integrity being used in the experiments. The proportion of intact cells before treatment was regarded as the baseline for the integrity determination.
Electrophoretic analysis for DNA fragmentation. Treated cells were harvested, washed in phosphate-buffered saline (pH 7.4), and lysed in 100 μl of 0.1 M phosphate-citrate buffer. Following lysis, the samples were centrifuged at 16,000 × g for 5 min. The supernatants were then treated with 200 μg/ml DNase-free RNase at 37°C for 1 h, followed by 1 μg/ml proteinase K at 50°C for 1 h. The samples were separated by electrophoresis on 1.5% (w/v) agarose gels containing 1 μg/ml ethidium bromide. The DNA fragments (DNA ladders) were visualized using a UV transilluminator (ATTO, Tokyo, Japan). The sizes of the DNA fragments were determined by comparison to DNA molecular weight markers (100-bp DNA ladder; Invitrogen, Carlsbad, CA, USA).
Proportion of Trypan blue-negative HL-60 cells following UVA exposure in the presence or absence of 100 μM lomefloxacin (LFX). ●, 100 μM LFX alone; □, UVA alone; ▪, 100 μM LFX + UVA. Values represent the mean±S.D. of four independent experiments.
Measurement of caspase-3 activity. Caspase-3 activity was assayed using the specific fluorogenic substrate N-acetyl-Asp-Glu-Val-Asp-7-amido-4-trifluoromethylcoumarin (Ac-DEVD-AFC; MBL, Tokyo, Japan). After incubation, treated cells were washed with 50 mM phosphate-buffered saline (pH 7.4) and resuspended in buffer containing 50 mM Tris-HCL (pH 7.4), 1 mM EDTA, and 10 mM EGTA. The cell lysates were then centrifuged at 800 × g for 5 min, and the supernatant was incubated with peptide substrate (50 μM) at 37°C for 2 h. The formation of 7-amino-4-trifluoromethylcoumarin was measured using a fluorescence spectrophotometer (F-3000; Hitachi, Tokyo, Japan) with an excitation wavelength of 400 nm and emission wavelength of 505 nm. The enzymic activity measured immediately prior to each experiment was used as the control value. The caspase activity was expressed as the ratio of released 7-amino-4-trifluoromethylcoumarin under the experimental condition to that of the untreated control.
Effect of ROS scavengers. To determine whether ROS, including singlet oxygen, superoxide radicals and hydroxyl radicals, participate in the induction of apoptosis by UVA, the effect of reactive oxygen scavengers tryptophan (10 mM), histidine (10 mM), 100 μg/ml SOD and 100 mM mannitol was investigated on the proportion of cells exhibiting morphological changes associated with apoptosis and the activation of caspase-3.
Analysis of cellular morphology using phase-contrast microscopy 4 h after treatment with the following conditions: (a) untreated; (b) 100 μM lomefloxacin (LFX) alone; (c) UVA alone; or (d) 100 μM LFX + UVA.
Statistical analysis. The results were expressed as the mean±standard deviation (S.D.). The values were compared by one-way analysis of variance with p=0.05 as the minimum level of significance.
Results
Cell damage. We first exposed HL-60 cells to UVA at 1.1 mW/cm2 in the presence and absence of 100 μM LFX and we examined cellular integrity by staining with Trypan blue. Figure 1 shows the proportion of unstained HL-60 cells versus the incubation time after UVA exposure. The results showed that the unstained (intact) fraction decreased exponentially with increasing incubation time. After 2 h of incubation following exposure to UVA for 20 min, the fraction of unstained cells was 80% and 11% in the group treated with LFX plus UVA and that treated with UVA only, respectively. LFX alone did not cause cell damage.
Morphological changes. We next assessed the induction of apoptosis by examining the morphology of the cells using phase-contrast microscopy after a 4-h incubation period following LFX-only, UVA-only, and LFX plus UVA treatments (Figure 2). There was no significant morphological change in the cells treated with LFX alone (Figure 2B) and UVA alone (Figure 2C). In contrast, membrane blebbing and cell shrinkage were clearly seen in the group treated with LFX plus UVA (Figure 2D).
Induction of apoptosis. We determined the proportion of apoptotic cells from phase-contrast microscopy images. Figure 3 shows the proportion of apoptotic HL-60 cells versus the duration of incubation after treatment. Under all conditions, the proportion of apoptotic cells was less than 2% immediately following initiation of the treatment. In the LFX plus UVA group, there was a significant increase in the proportion of apoptotic cells over time, with the proportion of apoptotic cells reaching a maximum after 4 h, and subsequently decreasing. No significant increase in the proportion of apoptotic cells was observed in the UVA-only and LFX-only groups.
Proportion of apoptotic HL-60 cells following UVA exposure in the presence or absence of 100 μM lomefloxacin (LFX). ○, Untreated; ●, 100 μM LFX alone; □, UVA alone; ▪, 100 μM LFX + UVA. Values represent the mean±S.D. of four independent experiments. *Significantly different at p<0.05 in comparison to the untreated controls.
DNA fragmentation. To further explore the induction of apoptosis in these cells, we performed agarose gel electrophoresis using DNA samples from the HL-60 cells (Figure 4). No distinct DNA ladder was observed for cells treated with LFX alone and UVA alone (Figure 4, lanes 2 and 3, respectively). In contrast, a typical DNA ladder was observed 4 h after exposure to UVA for cells treated with 100 and 200 μM LFX plus UVA (Figure 4, lanes 5 and 6 respectively).
Caspase-3 activation. To investigate whether caspases were activated by UVA and LFX in the HL-60 cells, we measured the enzymatic activity of caspase-3 using a fluorescent peptide substrate (Figure 5). We found that caspase-3 activity increased, peaking at 1 h after treatment, in cells treated with UVA in the presence of 100 μM LFX. No increase in caspase-3 activity was observed in cells treated with UVA or LFX alone.
DNA ladder formation in HL-60 cells 4 h after treatment with lomefloxacin (LFX) with/without UVA. Lane 1, DNA molecular size markers; lane 2, untreated; lane 3, 200 μM LFX alone; lane 4, UVA alone; lane 5, 100 μM LFX + UVA; lane 6, 200 μM LFX + UVA.
Effect of ROS scavengers. Next, we examined the effect of reactive oxygen scavengers on the induction of apoptosis (Figure 6A) and the activation of caspase-3 (Figure 6B). Tryptophan and histidine significantly reduced the induction of apoptosis and caspase-3 activation in LFX plus UVA-treated cells. In contrast, SOD and mannitol did not appear to affect the induction of apoptosis and caspase-3 activation.
Discussion
In our initial study, we explored whether UVA exposure in the presence of LFX induce apoptosis in HL-60 cells. We found that UVA-induced apoptosis was greatly enhanced by LFX as shown by morphological indicators, namely membrane blebbing and cell shrinkage, which were clearly observed when cells were treated with the combination of UVA and LFX, while no significant morphological changes were observed in cells exposed to either UVA or LFX alone. The proportion of apoptotic cells in the LFX plus UVA-treated group increased by more than one order of magnitude when compared to cells treated with UVA or LFX alone. These results clearly demonstrate the synergistic effect of LFX and UVA on apoptosis.
Caspase-3 activity in HL-60 cells following UVA exposure in the presence or absence of 100 μM lomefloxacin (LFX). □ 100 μM LFX alone; ●, UVA alone; ▪, 100 μM LFX + UVA. Values represent the mean±S.D. of four independent experiments. *Significantly different at p<0.05 in comparison to the untreated controls.
The fragmentation of DNA at linker regions between nucleosomes into fragments that are multiples of 180-200 base pairs in length is a hallmark of apoptosis (22-24). In the present study, the LFX plus UVA treatment resulted in the formation of a characteristic DNA ladder on agarose gel electrophoresis. This was not observed immediately after exposure (data not shown), but was clearly observed 4 h later, indicating that the DNA fragmentation was caused by an enzymatic process rather than by direct photochemical damage to the HL-60 cells.
Caspase-3 is an important enzyme that is required for the execution of the final phase of apoptosis, and is activated in cells undergoing apoptotic death (25-27). We observed a significant activation of caspase-3 after the LFX plus UVA treatment, indicating the induction of apoptosis. Both the proportion of apoptotic cells and the activity of caspase-3 gradually increased, reaching a peak 4 h after treatment before subsequently decreasing, which suggested that caspase-3 acted as the executor caspase responsible for the induction of apoptosis following the combination treatment. However, the mechanism by which caspase-3 is activated by LFX plus UVA treatment remains to be determined. Some structure–activity relationship studies have been performed for quinolone-induced phototoxicity (4, 5), and it appears that the phototoxicity of quinolones is determined by the nature of their 8-position substituent; halogens produced the greatest photoreactive activity, while hydrogen and methoxy substituents had little effect. In the pathogenesis of various skin diseases, a number of signaling pathways are activated as a result of UVA-mediated ROS generation. ROS are believed to activate proliferative and cell survival signaling that can alter apoptotic pathways involved in the pathogenesis of a number of skin disorders, including photosensitivity (28-31).
Effect of reactive oxygen scavengers on UVA-induced apoptosis (A) and caspase 3 activation (B) in the absence (○) and presence (●) of 100 μM lomefloxacin (LFX). Values represent the mean±S.D. of four independent experiments. *Significantly different at p<0.05 in comparison to cells with no scavenger treatment (none).
The free-radical scavengers used in this study included tryptophan and histidine, which are known to scavenge singlet oxygen and possibly hydroxyl radicals, mannitol at a concentration (100 mM) that should scavenge photodynamically induced hydroxyl radicals, and SOD, which catalyzes the elimination of superoxide radicals (32-35). Histidine caused a significant reduction in UVA-induced apoptosis and caspase-3 activation, suggesting that singlet oxygen is more important than hydroxyl radicals or superoxide in the induction of apoptosis due to UVA and LFX. These results also suggest that ROS stimulate apoptotic signaling pathways via caspase-3 activation.
Conclusion
In conclusion, we demonstrated the occurrence of photodynamically-induced apoptosis in HL-60 cells, as evidenced by morphological changes, DNA ladder formation, and caspase-3 activation. We also observed a significant reduction in the number of UVA-induced apoptotic cells and caspase-3 activation when histidine was present, which suggests that photodynamically generated singlet oxygen is an important mediator of apoptosis. Further studies on the mechanism of apoptosis induction are expected to provide useful information for better evaluating phototoxicity and photoallergenicity.
- Received August 21, 2017.
- Revision received September 20, 2017.
- Accepted September 22, 2017.
- Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
