Enoxacin with UVA Irradiation Induces Apoptosis in the AsPC1 Human Pancreatic Cancer Cell Line Through ROS Generation

Materials and Methods

Cell culture and reagents. The human pancreatic cancer cell line, AsPC1, was obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in the recommended RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum (Gibco, Invitrogen, Carlsbad, CA, USA), 100 U/ml penicillin, and 100 pg/ml streptomycin (Sigma, St. Louis, MO, USA) in 95% humidified air and 5% carbon dioxide at 37°C. Enoxacin was purchased from LKT Laboratories, Inc. (St. Paul, MN, USA). Histidine, mannitol, and superoxide dismutase (SOD) were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Sodium azide (NaN3) was purchased from Sigma. The other chemicals used were purchased from commercial suppliers.

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Figure 1.

Induction of apoptosis in AsPC1 cells by enoxacin combined with UVA irradiation as quantified using a MUSE cell analyzer. A: Cells were treated with UVA irradiation with or without 100 μM enoxacin for 10, 20 30 or 40 min. Apoptotic cells were quantified at 4 h after UVA irradiation. B: Cells were treated with or without 100 μM enoxacin under UVA irradiation for 20 min. Apoptotic cells were quantified at 0, 2, 4, and 6 h after UV irradiation. C: Cells were treated without or with 12.5, 25, 50, and 100 μM enoxacin under 20 min UVA irradiation. Apoptotic cells were quantified at 4 h after UV irradiation. Ds. Western blot of the effects of enoxacin (0, 12.5, 25, 50, and 100 μM) with UVA irradiation on poly (ADP-ribose) polymerase (PARP) cleavage. AsPC1 cells were treated with 100 μM enoxacin and 20 min UVA irradiation, and then quantified at 4 h after UV irradiation. Cell lysates were then analyzed by western blotting to detect levels of total and cleaved PARP, as described in the Materials and Methods. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was detected to indicate the relative amounts of loaded proteins. The results shown are representative of three independent experiments. Statistically different at ^#p<0.05, ^##p<0.01, and ^###p<0.005 compared to the control, and at *p<0.05, **p<0.05 and ***p<0.001 compared to values at 12.5, 25 and 50 μM enoxacin, respectively.

UVA irradiation. UVA irradiation of cells was performed using six parallel FL20SBLB fluorescent lamps (Toshiba, Tokyo, Japan) with a peak emission frequency of 352 nm at a distance of approximately 20 cm. The irradiation intensity was measured using a radiometer (UVR-305/365; Toshiba, Kanagawa, Japan), which was placed at the same distance from the UVA source as the cells. UVA irradiation was applied for 10 min to 40 min in the presence of 100 μM enoxacin.

Annexin V and dead cell assay. Live and apoptotic cell numbers were determined using the MUSE Annexin V and Dead Cell Kit (Merck Millipore KGaA, Darmstadt, Germany) according to the manufacturer's instructions. Briefly, cells were seeded at a density of 2.0×10⁵ cells/well in 6-well plates. After 12 h, enoxacin was added to each well to a final concentration of 100 μM and the cells were then exposed to UVA for 10 to 40 min or not, then incubated in a CO₂ incubator for different periods of time (0, 2, 4 and 6 h) before analysis. The cells were then washed twice with phosphate-buffered saline, trypsinized, and mixed with reagents from the Muse Annexin V and Dead Cell Assay Kit. A MUSE Cell Analyzer was used for data acquisition and analysis; assays were conducted in triplicate.

Western blot analysis. Cells were seeded at a density of 5.0×10⁵ cells/well in 6-well plates. After 12 h, 100 μM enoxacin was added to each well and then cells were exposed to UVA or not then incubated in a CO₂ incubator for 4 h. The cells were washed twice with phosphate-buffered saline and then lysed with RIPA buffer (Thermo Fisher Scientific, San Jose, CA, USA) containing a protease/phosphatase inhibitor cocktail (Cell Signaling Technology, Beverly, MA, USA). Aliquots of protein (30-40 μg) were then resolved on 12% gels, transferred to a polyvinylidene difluoride membrane, and incubated with primary antibodies specific to poly (ADP-ribose) polymerase (PARP, 1:1,000 dilution), cleaved PARP (1:1000 dilution), and to glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:10,000 dilution) (Cell Signaling Technology Inc., Beverly, MA, USA) for 1 h. Then the membrane was washed and incubated with enzyme-conjugated secondary antibodies for 1 h. Finally, proteins were detected using a chemiluminescence reagent (GE Healthcare, Piscataway, NJ, USA).

Effects of reactive oxygen species (ROS) scavengers. To determine whether ROS, including singlet oxygen, superoxide radicals and hydroxyl radicals, were involved in the induction of apoptosis by enoxacin combined with UVA irradiation, the effects of ROS scavengers on apoptosis were investigated(11, 12). Briefly, cells were seeded at a density of 2.0×10⁵ cells/well in 6-well plates. After 12 h, 100 μM enoxacin and individual scavenger (100 μM histidine, 100 μM NaN₃, 150 unit/ml SOD and 100 μM mannitol) were added to each well and the cells were then exposed to UVA or not then incubated in a CO₂ incubator for 4 h. The cells were then washed twice with phosphate-buffered saline, trypsinized, and mixed with reagents from the Muse Annexin V and Dead Cell Assay Kit. A MUSE Cell Analyzer was used for data acquisition and analysis; assays were conducted in triplicate.

Statistical analysis. Statistical analyses of differences were performed by one-way ANOVA followed by a modified Fisher's least square difference method.