The Kenalog drug was obtained from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany) and 1 g of the stock was dissolved in a liter of methanol. The suspension was irradiated at 50 kGy at a dose rate of 10 kGy/h generated by a ⁶⁰Co irradiator (MDS Nordion, Ottawa, ON, Canada) at the Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute. Irradiated samples were evaporated in vacuo, and crude compounds were analyzed by liquid chromatography coupled with mass spectrometry (LC-MS) and liquid chromatography (HPLC). To determine the chromatograms of the Kenalog-IR, irradiated samples were evaporated in vacuo and analyzed by high performance HPLC or LC-MS (Agilent Technologies, Inc., Palo Alto, CA, USA) in a YMC-Pack ODS-A-302 column (4.6 mm i.d. × 150 mm; YMC Co., Ltd., Kyoto, Japan). The mobile phase comprised of linear gradient starting with 0.1% (v/v) HCOOH at 40°C and increased to 50% (v/v) MeCN in 0.1% (v/v) CHOOH/H₂O over 20 min, and then increased to 100% MeCN for a further 20 min (detection, UV 254 nm; flow rate, 1.0 ml/min). This irradiated solution was named Kenalog-IR and a final concentration of 1 mg/ml was used for analysis.

The cell lines used for the present study, SK-Mel-5 was purchased from the American Type Culture Collection (ATCC; Rockville, MD, USA) and CCD-986sk skin fibroblast was obtained from the Korean Cell Line Bank (KCLB; Seoul, Korea). SK-Mel-5 cells were prepared in Dulbecco's modified Eagle's medium (DMEM) and CCD-986sk in Roswell Park Memorial Institute (RPMI)-1640 medium with 100 U/ml penicillin and 100 µg/ml streptomycin and 10% heat-inactivated fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Cells were maintained at 37°C, in an incubator until 80–90% confluence was reached, and an equal number of cells were incubated without or with various concentrations of Kenalog and Kenalog-IR (0, 5, 10, 25, 50 and 100 µg/ml) for each set of experimental conditions. Cells were washed with 1X phosphate-buffered saline pH 7.4 (PBS) and harvested with 0.5% trypsin-0.2% EDTA (Gibco; Thermo Fisher Scientific, Inc.) and were either used directly for analysis or stored at −80°C for further analysis.

Cell viability was measured by determining mitochondrial function using MTT assay kit [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Roche Diagnostics, Indianapolis, IN, USA)] and cell toxicity was assessed by trypan blue staining. The SK-Mel-5 cells were seeded at a density of 0.3×10⁴ cells/well in a 96-well flat bottom plate. After 24 h, the cells were treated with various concentrations of Kenalog or Kenalog-IR for 24 and 48 h. After treatment, 20 µl of 5 mg/ml MTT solution was added to each well and incubated for 2.5 h at 37°C, in a 5% CO₂ atmosphere. The supernatant was removed and solubilized with dimethyl sulfoxide (DMSO) for 10 min to dissolve the formazan produced. The absorbance was measured at 570 nm using a microplate reader (Tecan Group Ltd., Männedorf, Switzerland). Cell viability was expressed as the percentage of difference from the control at the corresponding concentration points. For the cytotoxic (cell death) assay, the cells were treated with various concentrations of compounds as aforementioned at a density of 0.3×10⁶ in a 65-mm culture dish. The staining was carried out by trypan blue dye-exclusion using a counting chamber and dead and live cells were counted by an Olympus IX71fluorescence microscope (Olympus Corp., Tokyo, Japan). To assess the non-specific cytotoxic effect of Kenalog-IR, normal skin fibroblast cells CCD-986sk were used. Briefly, 0.3×10⁶ cells/well were seeded into a 96-well plate for 96 h and subsequently treated with Kenalog or Kenalog-IR for 24 and 48 h. Then, cell viability was assessed by MTT assay as aforementioned.

For quantitative analysis of apoptotic and necrotic dead cells, Muse Annexin V and Dead Cell Assay kit (MCH100105; EMD Millipore, Billerica, MA, USA) was used. SK-Mel-5 cells (3×10⁵) were seeded in a 65-mm culture dish for 24 h and then treated with 100 µg/ml of Kenalog or Kenalog-IR for 24 h at 37°C, in a 5% CO₂ atmosphere. Cells were harvested and washed with PBS pH 7.4 as aforementioned. Furthermore, the cells were stained with Annexin V and Dead Cell reagent for 20 min and flow cytometric assessment was performed by Muse™ Cell Analyzer (EMD Millipore). The live and apoptotic cells were distinguished from necrotic cells as follows: Live cells (Annexin V-FITC⁻/PI⁻) called double negative, early apoptotic cells (Annexin V-FITC⁺/PI⁻), late apoptotic cells (Annexin V-FITC⁺/PI⁺) called double positive and necrotic cells (Annexin V-FITC⁻/PI⁺). The apoptotic cells were expressed as the percentage of live cells, early/late apoptotic cells, and dead cells determined by Muse analysis software (Muse 1.1.2; EMD Millipore).

The chromosomal DNA fragments were identified using agarose gel electrophoresis. Briefly, 3×10⁵ SK-Mel-5 cells were seeded and treated with 100 µg/ml Kenalog or Kenalog-IR and 2 µM doxorubicin for 18 h. The 0.1% DMSO contained media and the fresh media-treated cells were used as vehicle and control, respectively. After treatment, the cells were washed two times with PBS and cell lysates were harvested by DNA lysis buffer-1 (10 mM EDTA, 0.25% Triton X-100 and 2.5 mM Tris-HCl at pH 8) and incubated at room temperature (RT) for 15 min. Cells were centrifuged at 13,000 × g for 20 min at 4°C and an equal volume of supernatant and isopropanol were mixed and incubated at −80°C for 1 h. After cold incubation, the samples were centrifuged at 13,000 × g for 20 min at 4°C and the pellets were washed three times with cold 75% ethanol by centrifugation at 13,000 × g for 20 min at 4°C. Pellets were then left to dry at RT and suspended with 100 µl of DNA lysis buffer-2 (10 mM EDTA, and 2.5 mM Tris-HCl at pH 8). The samples were further incubated with 0.1 mg/ml RNase A for 30 min at RT and mixed with 0.25 mg/ml proteinase K for 1 h at RT. Then, the samples were mixed with 6X loading dye to a final concentration of 1X, and loaded in 1.2% agarose gel containing 1X gel red stain. Electrophoresis was run for 30 min at 100 V/cm.

For cell cycle assessment, Muse cell cycle reagent (EMD Millipore) was used. The analysis of differential DNA content in each phase of the cell cycle (G₀/G₁, S, and G2/M) was determined in melanoma cells. Briefly, 3×10⁵ SK-Mel-5 cells were seeded and treatment was carried out as described in the apoptosis assay aforementioned. Nocodazole (400 nM) was used to induce G2/M phase cell arrest. Cells were then stained and incubated for 30 min with Muse cell cycle reagent at 4°C and flow cytometric assessment was performed by Muse™ Cell Analyzer (EMD Millipore). The DNA content was expressed as the percentage of cells in the respective cell cycle phase.

Intracellular ROS produced by stressed cells were assessed by Muse oxidative stress reagent assay (EMD Millipore). SK-Mel-5 cells (1×10⁵) were seeded in a 65-mm culture dish for 24 h and then treated with 100 µg/ml of Kenalog and Kenalog-IR or 2 µM doxorubicin as the positive control. Cells were harvested and the cell suspension was incubated with assay reagent for 30 min and the oxidized red dihydroethidium (DHE) fluorescence intensity was assessed by flow cytometer using Muse™ Cell Analyzer (EMD Millipore). The ROS-positive and ROS-negative cells were expressed as a percentage of the ROS gated profile. For the intracellular source of ROS determination, MitoSOX assay (Invitrogen; Thermo Fisher Scientific, Inc.) was used. Cells were pre-labeled with MitoSOX reagent before treatment. Then cells were treated as indicated above for 18 h and harvested with trypsin. The cells were harvested, washed and further incubated with a buffer containing 5 µM MitoSOX for 10 min at 37°C in the dark. After the incubation time, the cells were washed twice and suspended for measurements with a flow cytometer.

Treated and untreated SK-Mel-5 cells were harvested, lysed with radioimmunoprecipitation assay buffer (RIPA; Rockland Immunochemicals, Inc., Limerick, PA, USA) and cytosolic and mitochondria fractions were separated by Cytochrome c Release Apoptosis Assay kit (cat. no. ab65311; Abcam, Cambridge, UK) following the manufacturer's instructions. Cell debris was removed by centrifugation and the protein concentration was determined by bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Cell lysates containing an equal amount of protein (40 µg) were prepared and separated by 10 or 15% SDS-PAGE and 10 µg of cytosolic fractions and 15 µg of both mitochondria and debris fractions were loaded onto 12% SDS-PAGE and transferred to polyvinylidene difluoride membranes (PVDF; Merck Millipore). The membranes were then blocked with 5% non-fat milk in Tris-buffered saline containing Tween-20 (TBST) for 1 h at RT. The membranes were subsequently probed overnight at 4°C with primary antibodies at dilution of 1:1,000 for anti-Bcl-2 (cat. no. 2870), anti-Bax (cat. no. 5023), anti-Bad (cat. no. 9292), anti-caspase-9 and cleaved caspase-9 (cat. no. 9502), anti-caspase-7 (cat. no. 9492) and cleaved caspase-7 (cat. no. 9491), anti-caspase-3 (cat. no. 9662) and cleaved caspase-3 (cat. no. 9664), anti-PARP (cat. no. 9532) and cleaved PARP (cat. no. 9541), anti-AIF, anti-AKT (cat. no. 4691) and p-AKT (cat. no. 4060), anti-mTOR (cat. no. 2983) and p-mTOR (cat. no. 5536), anti-β-actin (cat. no. 4970) and anti-GAPDH (cat. no. 2118) (all primary antibodies; Cell Signaling Technology, Danvers, MA, USA) and sodium dismutase-1 (cat. no. SC-11407) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The blots were then incubated with secondary antibody horseradish peroxidase (HRP)-conjugated anti-mouse (cat. no. 7076 at 1:2,500) or anti-rabbit IgG (cat. no. 7074 at 1:3,000) both from Cell Signaling Technology (Danvers, MA, USA) for 1 h at RT. The membranes were incubated with the chemiluminescence (ECL) reagent (Thermo Fisher Scientific, Inc.) for protein band detection.

All data were evaluated by one-way ANOVA, followed by Tukey's post hoc test and results were considered statistically significant when the P-value was <0.05. Experiments were performed at least of three times independently.

To investigate the radiolytic effects of ionizing radiation on Kenalog, 50 kGy of γ radiation was used to modify the chemical structures of the drug. Sample solutions of Kenalog and Kenalog-IR were analyzed by LC-MS and as revealed in Fig. 1, one major peak was observed in the chromatogram of Kenalog (Fig. 1, upper panel) and four peaks in the Kenalog-IR chromatogram at a various retention times (Fig. 1, lower panel). These newly generated peaks indicated that ionizing radiation caused alterations to Kenalog and that the changes may contribute to the cytotoxic effects of Kenalog-IR.

In order to investigate the anticancer activity of Kenalog-IR, MTT and trypan blue assays were used to evaluate the cytotoxic effects in the SK-Mel-5 cell line. It has been previously reported that the clinically available form of Kenalog has less cytotoxic effects compared to other corticosteroids (9). Thus, we first compared the cytotoxic effect of Kenalog and Kenalog-IR. As anticipated, Kenalog-treated cells exhibited a lesser effect on the viability of SK-Mel-5 cells in a concentration and time-dependent manner compared to Kenalog-IR. At 24 and 48 h, 100 µg/ml Kenalog-IR significantly reduced cell viability to 29% (P=1×10⁻¹¹) and 35% (P=2×10⁻¹⁰), respectively (Fig. 2A) compared to the control and 100 µg/ml of Kenalog. In addition to the enhanced specific desired cytotoxic effect of the drug, attaining a platform that will not induce toxic effects in normal cells is very important in cancer treatment. The non-specific cytotoxic effects of Kenalog-IR in the skin fibroblast cell line CCD-986sk was observed. At 24 h, Kenalog-IR significantly reduced cell viability to 86% (P=0.004) but no significant differences (P=0.87) were observed between Kenalog and Kenalog-IR-treated groups (Fig. 2B). This indicated that treatment of SK-Mel-5 cells with Kenalog-IR induced less toxicity to normal cells. Furthermore, trypan blue assay also revealed that Kenalog-IR significantly induced 79% (P=6×10⁻⁷) of cell death as compared to 8% (P=0.01) observed in Kenalog-treated cells (Fig. 2C). For the total number of cells, a similar trend was observed where Kenalog-IR reduced the number of cells to approximately half (Fig. 2D) when compared to the control and Kenalog-treated cells. In addition, the cytotoxic effects related to apoptosis were also observed by morphological changes in Kenalog-IR-treated cells. The morphological assessment revealed that, 100 µg/ml of Kenalog-IR caused plasma membrane protrusion and changes in pigmentation as observed with white patches (Fig. 2E). Collectively the results revealed that Kenalog-IR exhibited a potential effect in anticancer treatment compared to the original drug, Kenalog.

As observed in Fig. 2, Kenalog-IR treated cells exhibited morphological changes and cell death when compared to Kenalog-treated cells. To understand the nature of Kenalog-IR-induced cell death, apoptosis and the cell cycle were assessed in SK-Mel-5 cells for 24 h. As indicated by flow cytometry, Kenalog-IR treated cells resulted in 62% (P=2.5×10⁻¹²) of total apoptosis and 37% (P=1.6×10⁻¹²) of live cells (Fig. 3A and B). In comparison to the live cells in the control group, the reduction of a number of live cells in Kenalog-IR treated cells indicated that at 24 h many cells had progressed into apoptosis. Since apoptosis is known to be triggered through the intrinsic (caspase-mediated) pathway or extrinsic (death receptor-mediated) pathway, it was of interest to determine which pathway was involved in Kenalog-IR-induced cell death. To investigate this phenomenon, we blocked the intrinsic apoptosis pathway with caspase pan-inhibitor Z-VAD FMK and treated the cells as aforementioned. The results revealed that blocking caspase activation decreased total apoptosis to 28% (P=1.09×10⁻⁵) in Kenalog-IR-treated cells (Fig. 3C and D) which indicated the involvement of intrinsic apoptosis in cell death. Furthermore, DNA fragmentation analysis revealed that Kenalog-IR induced cell death as confirmed by DNA fragments observed in a gel electrophoresis result (Fig. 3E, lane 5). To assess that no extrinsic apoptosis was involved, we investigated caspase-8, a key molecule in the extrinsic pathway. The result revealed no activation of caspase-8 in Kenalog-IR treated cells (Fig. 3F). Furthermore, it was of interest to reveal whether Kenalog-IR-induced apoptosis was related to cell cycle arrest. As revealed (Fig. 3G and H), Kenalog-IR induced 36.3% (P=0.003) cell arrest in the G2/M phase even when combined with nocodazole a known inducer of G2/M arrest, the same effect was observed (38.6%, P=2.5×10⁻⁴). In sum, these data indicated that Kenalog-IR induced intrinsic apoptosis with a compromised cell cycle.

To further examine the apoptosis pathway involved in Kenalog-IR-induced cell death, the expression of several programmed cell death markers was examined by immunoblotting. Apoptosis is induced either by the intrinsic pathway based on caspase activation or the extrinsic pathways involving death receptors (18). The expression of pro- and anti-Bcl-2 family proteins, caspase-3 and PARP was gradually determined at 100 µg/ml (Fig. 4A). Further analysis of the Bcl-2 family 24 h after Kenalog-IR treatment, revealed increased levels of pro-apoptotic members Bax (P=0.0042) and Bad (P=4×10⁻⁸) and decreased expression levels of anti-apoptotic member Bcl-2 (P=0.041) (Fig. 4B). Executioner caspases, markers of the intrinsic pathway were also found to be activated. Kenalog-IR activated the expression of cleaved caspase-7 (P=2.8×10⁻¹⁰) and cleaved caspase-3 (P=2.4×10⁻¹¹) while it decreased the expression of both the total and the cleaved form of caspase-9 (Fig. 4C) at 24 h of treatment. The activation of caspase-7 and −3 indicated the involvement of the intrinsic pathway. Furthermore, the mediator of the intrinsic pathway involved in the release of the PARP enzyme was investigated. PARP is involved in the release of mitochondrial proteins such as cytochrome c or apoptosis-inducing factor (AIF) and regulates its translocation to the cytoplasm (19). Kenalog-IR decreased AIF protein levels in the mitochondria fraction (Fig. 4E, mitochondria fraction lane 3) and induced the expression of cleaved PARP at 24 h of treatment (Fig. 4D). These results demonstrated that Kenalog-IR induced apoptosis through activation of the intrinsic mitochondrial pathway in SK-Mel-5 cells.

Reactive oxygen species (ROS) is the hallmark of apoptosis and a significant indicator of cells undergoing oxidative stress. Normally, the induction of the intrinsic pathway is linked with the leakage of mitochondrial proteins which pass through the distracted electron transport chain (ETC) and stimulate further production of ROS and executor proteins. Based on our aforementioned results, caspase activation in particular caspase-3 and −7 were regarded as key molecules revealing impaired mitochondrial function (20,21). Given these facts, investigation of whether apoptotic induction was linked with ROS production was performed. Using intracellular detection of superoxide radicals by dihydroethidium (DHE), it was revealed that Kenalog-IR significantly induced 84% (P=3.7×10⁻⁷) of ROS positive cells (Fig. 5A, lane 4) compared to 31% (P=0.990) of Kenalog-treated cells (Fig. 5A, lane 3). Furthermore, blocking of ROS by N-acetyl-Cysteine (NAC) revealed a reduction of ROS positive cells to 27% (P=0.06) (Fig. 5A, lane 5). These results indicated that Kenalog-IR-induced apoptosis was mediated by ROS production. It is well known that accumulation of superoxide and other oxidative stress molecules are associated with increased levels of ROS which cause increased expression of the enzyme sodium dismutase (SOD) and subsequently a downstream pathway related to ROS induction (Fig. 5C). To ascertain this, the source of ROS induced by Kenalog-IR was first revealed. The MitoSOX assay results revealed that the majority of ROS were generated from the mitochondria (Fig. 5D). To establish whether the increased levels of apoptosis were related to ROS, western blot analysis was performed for the ROS-related pathway. The results revealed that the expression level SOD1 was increased in Kenalog-IR-treated cells (Fig. 5E, lane 3) demonstrating that ROS was increased and scavenged by this enzyme. Furthermore, decreased expression of both phosphorylated AKT (P=2.86×10⁻⁹) and phosphorylated mTOR (P=2×10⁻⁵) molecules (Fig. 5E) was observed indicating the involvement of the AKT/mTOR pathway in the induction of apoptosis. The experiments in Fig. 5, revealed that ROS production was the source of Kenalog-IR-induced apoptosis in SK-Mel-5 cells.