Kenalog is a synthetic glucocorticoid drug used to treat various cancers including ocular and choroidal melanoma. However, the drug achieves rarely sustainable results for patients. To overcome this difficulty, the structure of Kenalog was altered by ionizing radiation (IR) to develop a more effective anticancer agent for treatment of various skin cancers. The anticancer effect of modified Kenalog (Kenalog-IR) was
Glucocorticoids have been used for the treatment of various disorders including skin-related diseases and cancers (
In recent years, several approaches have been devised to increase the bioactivity of glucocorticoids to overcome the problems related to low efficacy and increased adverse effects (
To elucidate the contribution of IR in IMD approaches, the
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
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
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
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 (
As observed in
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 (
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 (
Kenalog is a synthetic glucocorticoid used to treat various cancers and skin diseases. Treatment with Kenalog rarely achieves sustainable results for patients and has adverse effects. To improve its efficacy, ionizing radiation was used to modify the structure of Kenalog to develop a potential anticancer drug. As analyzed by LC-MS, Kenalog-IR formed four peaks (
Generation of apoptosis and apoptosis-related markers in drug treatment trials is a superlative approach in cancer treatment. Apoptosis is an important phenomenon in the mechanism of homeostasis, it balances several processes including cell division and cell demise (
However, initiation and execution of apoptosis are mediated by cysteine-dependent aspartate such as caspase-3 in association with the activation of cleaved poly(ADP-ribose) polymerase PARP (
Collectively, the findings of the present study elucidated the mechanism of cell death induced by Kenalog-IR during melanoma cancer treatment, however the use of such a therapeutic approach especially in skin cancers still remains challenging. Several corticosteroids and glucocorticoids have been reported to have low solubility, low bioavailability and longer duration of action (
In conclusion, the present study, elucidated the importance of an IR-modified drug in cancer treatment. Kenalog-IR inhibited melanoma cancer cell proliferation and caused cell death by the intrinsic apoptosis pathway. Moreover, Kenalog-IR-induced cell death was associated with increased production of ROS modulated by the release of caspases and activation of cleaved PARP. In general, the results of the present study, justify the hypothesis that incrementally modified drugs by IR may potentially be used as anticancer candidates in various cancer treatment strategies. However, further studies will have to be conducted in the future to determine the structures of Kenalog generated by IR and the stability of these compounds before being subjected to clinical trials.
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This research was funded by the Ministry of Science and ICT through the Basic Science Research Program by the Korean government.
The analyzed datasets generated during the study are available from the corresponding author upon reasonable request.
RAK, EHL and HWB conceived, designed and wrote the study. RAK, FJR, HJC and CHP performed the experiments and analyzed data. BYC and HWB were involved in the conception of the study, supervised the experimental work and revised the manuscript. All authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.
Not applicable.
Not applicable.
The authors state that they have no competing interests.
ionizing radiation
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
reactive oxygen species
incrementally modified drugs
Effects of ionizing radiation on Kenalog. The chromatograms of Kenalog (upper panel) and Kenalog-IR (lower panel) of crude fractions. Kenalog (1 g) was dissolved in a liter of methanol and subjected to γ irradiation of 50 kGy. The white powder suspension changed to a yellow syrup after irradiation and exhibited four major peaks of Kenalog-IR at different retention times (lower panel) and one peak of Kenalog (upper panel) as assessed by LC-MS and HPLC. The arrows indicate the retention time of each peak. IR, ionizing radiation; LC-MS, liquid chromatography coupled with mass spectrometry; HPLC, liquid chromatography.
Kenalog-IR inhibits proliferation and induces cytotoxic effects in SK-Mel-5 cells. Cell viability and cell death of Kenalog and Kenalog-IR-treated cells. The viability of cells was assessed by MTT assay and was observed to be (A) decreased in a dose-dependent manner both at 24 and 48 h following Kenalog-IR treatment in SK-Mel-5 cells (a1=0.003, a2=1×10−11, a3=2×10−10, *=1.1×10−9 at 24 h) and (B) less decreased in normal skin fibroblast cells, CCD-986sk (a1=0.0004, a2=0.0004, n.s=0.87). (C) Cell proliferation was determined by trypan blue exclusion assay. SK-Mel-5 cells were treated with 100 µg/ml of Kenalog and Kenalog-IR for 24 h (a1=6×10−6, *=0.01 and n.s=0.251). (D) Indicates the reduction of the total number of cells (a2=8.8×10−6 and *=0.01). A greater number of dead cells was observed in Kenalog-IR treated group. (E) Morphological appearance SK-Mel-5 following Kenalog and Kenalog-IR treatment for 24 h observed under a microscope. The arrows reveal white patches. Data are represented as the mean ± SEM of three independent experiments (*P<0.05 between treatment groups, ‘NS’ indicates not significant between treatment groups and a small letter ‘a’ indicates a significant difference between treatments and the control). IR, ionizing radiation.
Kenalog-IR induces apoptosis with aggravating DNA integrity. Apoptosis and cell cycle analysis of Kenalog- and Kenalog-IR-treated cells was determined by Muse Annexin V-FITC/PI and a Muse cell cycle flow cytometer. (A) Percentage of live, early, late and total apoptosis. The population of positively stained propidium iodine cells was used to calculate the percentage of viable cells (a1=2.5×10−12, a2=1.6×10−12, b=0.9, *=2.5×10−11). (B) Quadrants indicating Annexin V-FITC/PI stained SK-Mel-5 cells exhibiting live, early apoptosis, late apoptosis, and dead cells. (C and D) Cells were pretreated with 40 µM of Z-VAD-FMK for 1 h and further treated with treatments + Z-VAD-FMK for 24 h. (C) Cells were harvested for percentage apoptosis analysis (a=1.09×10−5, b=0.84 and n.s=0.73) and (D) Annexin V-FITC/PI quadrants. (E) DNA fragmentation assay. Cells were treated with treatments as indicated for 24 h and DNA was harvested for 1.2% gel electrophoresis. In addition, a DNA ladder (1 Kb) was used as the marker at 100 V/cm for 30 min. (F) Verification of extrinsic apoptosis. Cells were treated as indicated with Z-VAD-FMK for western blot assay. The anti-caspase-8 antibody was used to detect the extrinsic apoptosis pathway and GAPDH was used as a control. (G and H) Cell cycle analysis after 24 h of treatment. Nocodazole (400 nM) was used to induce G2/M cell cycle arrest. (G) The cell cycle summary (a1=0.01, a2=0.003, a3=2.5×10−6, *=1.8×10−5, **=1.3×10−7), and the phases indicated (H) the DNA content index profiles in each group and cell cycle phases G1/0, S and G2/M, expressed as the percentage of positively-stained cells (G). Data are presented as the mean ± SEM of three independent experiments (*P<0.05 between treatment groups, ‘NS’ indicates not significant between treatment groups, small letters ‘a’ and ‘b’ indicate significant and not significant differences between treatments and the control respectively). IR, ionizing radiation.
Kenalog-IR induces apoptosis through the intrinsic mitochondrial pathway. Analysis of apoptotic protein markers. Whole cell-lysates were extracted by RIPA and an equal amount of protein was analyzed by western blotting. (A) Dose-dependent expression of Bcl-2 family proteins, caspase-3 and PARP gradually determined at 100 µg/ml of Kenalog-IR treatment (a1=1.2×10−7, a2=3.4×10−12, a3=0.0065 and b=0.072). (B) Analysis of pro- and anti-apoptotic Bcl-2 family proteins 24 h after Kenalog-IR treatment (a1=0.041, a2=0.0042, a3=4×10−8, n.s=0.18 and *=0.00017). (C) The Kenalog-IR activated expression of intrinsic apoptosis pathways proteins (a1=1.1×10−13, a2=2.8×10−10 and a3=2.4×10−11) and (D) Kenalog-IR induced the expression of cleaved PARP 24 h of treatment (a=0.00006 and *=1×10−7). (E) SK-Mel-5 cells were cultured in absence (line 1) or presence of Kenalog (line 2), Kenalog-IR (line 3) and Doxo as a positive control (line 4). Cytosolic and mitochondria fractions were separated by 12% SDS-PAGE. The histograms shown in the right panel of parts A-D indicate ImageJ extracted intensities as normalized against the control protein (GAPDH) or the total form of respective proteins. Data are presented as the mean ± SEM of three independent experiments [*P<0.05 between treatment groups, ‘NS’ indicates not significant between treatment groups, a small letter ‘a’ indicates significant differences between treatments and the control or calibrator (GAPDH) or the total form of the protein of interest]. IR, ionizing radiation; Doxo, doxorubicin.
Kenalog-IR increases production of ROS which in turn increases mitochondria-mediated apoptosis. Intracellular ROS levels of SK-Mel-5 cells. Cells treated with Kenalog and Kenalog-IR for 24 h were assessed by flow cytometer. (A) The ROS-negative (M1) and ROS-positive (M2) cells were expressed as a percentage in M1 and M2 gates respectively (a=3.7×10−7, b1=0.99, b2=0.065, *=2.9×10−9, n.s=0.175). (B) The population of positive ROS-stained cells was used to calculate the percentage of ROS-stained cells as shown in the histogram. (C) Hypothetical illustration of Kenalog-IR-induced ROS. (D) Mitochondrial ROS determination by flow cytometer. Cells were pre-labeled with 5 µM MitoSOX reagent for 1 h and exposed to treatment for 18 h. Cells were harvested, washed and further incubated with MitoSOX reagent for 10 min at 37°C in the dark. Quadrants indicate ROS-positive cells. (E) Analysis of the ROS-mediated pathway. Cells were treated with Kenalog or Kenalog-IR and cell lysates were subjected to 10 or 15% SDS-PAGE. SOD1 was used to denote increased levels of ROS while GAPDH was used as a control. The histograms shown in the right panel indicate ImageJ extracted intensities as normalized against the total protein (a1=2.86×10−9, a2=2×10−5, *=1.04×10−8, **=0.002). Data are presented as the mean ± SEM of three independent experiments (*P<0.05 between treatment groups, ‘NS’ indicates not significant between treatment groups, small letters ‘a’ and ‘b’ indicate significant and not significant differences between treatments and the control respectively). IR, ionizing radiation; ROS, reactive oxygen species.