CKD-602 (7-[2-(N-isopropylamino) ethyl]-(20S)-camptothecin, belotecan) is a synthetic water-soluble camptothecin derivative and topoisomerase I inhibitor that has been shown to exert a clinical anticancer effect on various types of tumor. In the present study, the anticancer effects of CKD-602 on the following three human oral squamous cell carcinoma (OSCC) cell lines originating from Korean cancer patients: YD-8 (tongue), YD-9 (buccal mucosa) and YD-38 (lower gingiva) were analyzed. The apoptotic proportion of the cells and cell cycle position were analyzed using flow cytometry. The expression of cell cycle regulatory proteins was detected by western blot analysis. CKD-602 was demonstrated to exert a time- and dose-dependent antiproliferative effect in all cell lines
Squamous cell carcinoma is the most common type of malignant neoplasm of the oral mucosa and accounts for >90% of all intraoral malignant tumors, in Korea (
Head and neck SCC cell lines in cultures are widely used to understand therapeutic development. However, the establishment of SCC cell lines is considered difficult and low success rates have been reported. Cell lines have previously been derived from patients who had received radiotherapy, with or without chemotherapy (
DNA topoisomerase I (Top1), an essential nuclear enzyme that controls and modifies the topological state of DNA in numerous cellular metabolic processes (
CKD-602 (Chong Kun Dang Pharmaceutical Corp., Seoul, Korea) was dissolved in distilled water at 1 μg/ml, and stored as a stock solution in aliquots at −20°C until use. Final concentrations between 0.01 and 10 μg/ml CKD-602 were obtained by appropriate dilutions of the stock solution with RPMI 1640 medium (Gibco-BRL, Grand Island, NY, USA).
Three OSCC cell lines, YD-8 (60501; tongue), YD-9 (60502; buccal mucosa) and YD-38 (60508; lower gingiva) were used (
Each cell line was maintained in RPMI-1640 medium (Gibco-BRL), supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco-BRL), 100 μg/ml streptomycin (Gibco-BRL) and 100 IU/ml penicillin (Gibco-BRL), as a monolayer under standard conditions (37°C, and in a humidified atmosphere of 5% CO2). To transfer or passage the cell lines, each confluent monolayer was washed with phosphate-buffered saline (PBS; Welgene, Daegu, Korea) and detached with a 0.05% trypsin/0.02% EDTA solution (Gibco-BRL).
Cells at a density of 2×104 cells/well in 100 μl RPMI with 10% FBS were added to the wells of a 96-well plate. The cells were treated with different concentrations (0.01, 0.1, 0.5, 1, 5 and 10 μg/ml) of CKD-602 for 24, 48 and 72 h. Control samples of each cell line were treated with medium only. For the viability assay, 20 μl/well CellTiter 96® AQueous One Solution Reagent (MTS; Promega Corporation, Madison, WI, USA) was added. After 1 h incubation at 37°C in a humidified atmosphere of 5% CO2, the absorbance at 490 nm was recorded using an ELISA plate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA) The assay was performed in triplicate with three independent experiments for each condition. The data from the treatment groups were normalized to those of the control samples and are presented as the mean ±standard error of the mean. The half maximal (50%) inhibitory concentration (IC50) values were calculated from the dose-response curve.
Apoptosis was quantified using fluorescein isothiocyanate (FITC)-Annexin V Apoptosis Detection kit I (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions. Briefly, the cells were plated at a density of 1×106 cells/well in a 100 mm culture dish, treated with 0.1 and 0.5 μg/ml CKD-602 for 48 h, and harvested by centrifugation at 500 × g for 3 min. The cell pellets were resuspended in Annexin V binding buffer containing 140 mM CaCl2, 10 mM HEPES/NaOH and 2.5 mM MgCl2. FITC-conjugated Annexin V (5 μl) and propidium iodide (PI; 5 μl) were added to the cells, and the mixtures were incubated for 15 min at room temperature in the dark. The analyses were performed using a Fluorescence-Activated Cell Sorting (FACScan) instrument (Becton-Dickinson, Franklin Lakes, CA, USA). Annexin V- and PI-positive cells were considered to be apoptotic.
The cells were plated at a density of 1×106 cells/well in 100 mm culture dishes. The cells were treated with 0.02 μg/ml CKD-602 for 48 h, harvested by centrifugation at 500 × g for 3 min, washed twice in ice-cold PBS, fixed in 70% ethanol and stored at −20°C for a minimum of 1 h; subsequently, cells were washed with ice-cold PBS and resuspended in 500 μl PI/RNase Staining Buffer (BD Biosciences). The cell cycle position was evaluated by FACScan using an excitation laser set at 480 nm and a detection wavelength of 575 nm. A minimum of 10,000 events/sample was analyzed.
CKD-602-treated and non-treated cells were suspended in RIPA buffer (Rockland, Gilbertsville, PA, USA) containing 5 μM AEBSF, 1.5 μM aprotinin, 10 μM E-64, 0.01 μM leupeptin and phosphatase inhibitors [1 mM sodium orthovanadate (Na2VO4; S6508; 1 mM sodium molybdate (Na2MoO4; M1003) 4 mM sodium tartrate dihydrate (S4797); 2 mM imidazole (I0125) all purchased from Sigma-Aldrich, (St. Louis, MO, USA)], and were placed on ice for 20 min. Following centrifugation at 4°C at 10,000 × g for 20 min, the cell supernatant was collected. The protein concentration was determined using a bicinchoninic protein assay kit (Pierce, Rockford, IL, USA). The whole lysate (20 μg) was resolved on a 10% or 12.5% SDS-PAGE gel, transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA, USA) by electroblotting, and probed with polyclonal rabbit anti-human β-actin (4967), monoclonal mouse anti-human p53 (9826), monoclonal mouse anti-human phospho-H3 (Ser 10; 3377), polyclonal rabbit anti-human cyclin B1 (4138), monoclonal rabbit anti-human cyclin A2 (4656), polyclonal rabbit anti-human cdc2 (9112), polyclonal rabbit anti-human phopho-cdc2 (Tyr 15; 9111), polyclonal rabbit anti-human Myt1 (4282) or polyclonal rabbit anti-human phospho H2AX (Ser 130; 9718) antibodies (1:1,000; Cell Signaling Technology, Inc. (Danvers, MA, USA) overnight at 4°C. The membrane was washed three times with 1× PBS and 0.05% Tween-20 for 15 min, and incubated for 1 h with a horseradish peroxidase-conjugated polyclonal horse anti-rabbit (7074) or polyclonal goat anti-mouse (7076) antibody. (1:2,000; Cell Signaling Technology, Inc.). The blot was developed using an enhanced chemiluminescence kit (Intron Biotechnology Inc., Seongnam, Korea).
Using the cell viability assay, CKD-602 was revealed to exert a significant cytotoxic effect on all cell lines in a time- and dose-dependent manner (
Cell cycle analysis was performed following treatment with 0.02 μg/ml CKD-602 for 48 h and 72 h in each cell line. CKD-602 induced G2/M phase cell accumulation in all cell lines (
As CKD-602 induced G2/M phase arrest in the OSCC cell lines, whether alterations in the cell cycle regulatory proteins occurred following treatment with a non-cytotoxic dose of CKD-602 (0.02 μg/ml) was subsequently investigated. The phosphorylation of histone H2AX (γH2AX) Ser 139 is one of the early events initiated by double-stranded DNA breaks. Immunoblotting analysis revealed that the phosphorylation of histone H2AX was increased in the CKD-602 treatment cells. In the cell cycle, distinct cyclin/cyclin-dependent kinase complexes are activated to regulate cell cycle progression. Cyclin A- and cyclin B-associated cdc2 regulates the G2/M phases. During G2 phase, the cdc2/cyclin B complex is maintained as inactive by the phosphorylation of cdc2 Tyr 15 and Thr 14 by the Myt1 kinases (
As the phosphorylation of histone H3 is a molecular checkpoint for entering mitosis (
Annexin V/FITC staining, along with flow cytometry, enables the quantitative assessment of living (Annexin V-FITC-negative/PI-negative), early apoptotic (Annexin V-FITC-positive/PI-negative), late apoptotic/necrotic (Annexin V-FITC-positive/PI-positive) and dead (Annexin V-FITC-negative/PI-positive) cells. The effects of 24 h CKD-602 treatment on YD cell apoptosis are shown in
Topoisomerase inhibitors are a class of agents that target topoisomerase specifically by intercalating inside the topoisomeares cleavage complexes and mediate the changes in DNA structure during the normal cell cycle. Recently, topoisomerases have become widely investigated targets for cancer chemotherapy treatment. Topoisomerase inhibitors are hypothesized to suppress the regulatory step, which normally reseals the parent strand of DNA following passage of the daughter strand (
CKD-602 is a potent Top1 inhibitor that successfully overcomes the poor water solubility and toxicity of the corresponding parent drug, camptothecin. Clinical trials for the treatment of various types of cancer with CKD-602 are ongoing and have shown promising results (
In the present study, CKD-602 induced cytotoxicity in OSCC cell lines, causing apoptosis in a dose- and time-dependent manner. The cytotoxic effect of CKD-602 was more prominent in the YD-9 and YD-38 cell lines, which did not possess p53 mutations, than in the YD-8 cell line, which did have a p53 mutation (
As over half of human tumors exhibit a p53 mutation or deficiency, the investigation of cell cycle checkpoints in tumor cells with various p53 statuses provides a potential basis for developing novel tumor therapeutics. In the present study, three OSCC cell lines were used with genetically different p53 statuses. The YD-8 cell line had a point mutation at codon 273 of exon 8 and the GGT sequence was altered to CAT, which resulted in a change from arginine to histidine. The levels of p53 protein were detected in YD-8 and YD-9 by immunoblotting (
Treatment of the cells with CKD-602 for 48 h and 72 h resulted in cell cycle arrest at the G2/M phase (
In conclusion, in the present study, CKD-602 was demonstrated to exert an
This study was supported by the SNUBH Research Fund (grant no. 02-2011-001).
Cell viability following CKD-602 treatment. The cells were incubated with various concentrations of CKD-602 (0, 0.001, 0.01, 0.1, 1, 3, 5 and 10 μg/ml) for 24, 48 and 72 h. Cell viability was determined using an MTS assay. All cell lines exhibited a significant reduction in viability, dependent on CKD-602 treatment time and dose. (A) YD-8, (B) YD-9 and (C) YD-38 cell lines. The data are presented as the mean percentage of viable cells ± standard deviation (n=3).
CKD-602 induces G2/M phase arrest in oral squamous cell cancer cells. The three YD cell lines were incubated with 0.02 μg/ml CKD-602 for 48 and 72 h, fixed and stained with propidium iodide, and analyzed for DNA content.
Effects of CKD-602 on cell cycle regulating molecules in YD cell lines. The cells were treated with 0.02 μg/ml CKD-602 and the protein expression levels at 24 and 48 h following treatment were examined by western blot analysis.
Induction of apoptosis in CKD-602-treated YD cell lines. The cells were harvested following 24 h incubation with 0.1 and 0.5 μg/ml CKD-602. Apoptosis was determined by staining the cells with Annexin V-FITC and PI labeling. The percentage figure in each window indicates the proportion of Annexin V-FITC positive cells. FITC, fluorescein isothiocyanate; PI, propidium iodide.
Growth inhibition (IC50, μg/ml) of oral squamous cell cancer cell lines by CKD-602.
IC50, μg/ml | |||
---|---|---|---|
| |||
Treatment duration | YD-8 | YD-9 | YD-38 |
48 h | - | ≥0.3 | ≥0.24 |
72 h | ≥2.4 | ≥0.18 | ≥0.05 |
Results of FACS analysis evaluating apoptotic effects following treatment with CKD-602 for 48 h.
Fraction of cells, % | ||||||
---|---|---|---|---|---|---|
| ||||||
YD-8 | YD-9 | YD-38 | ||||
|
|
| ||||
CKD-602 | Live | Apoptotic | Live | Apoptotic | Live | Apoptotic |
Control | 94.06±0.13 | 4.78±0.91 | 92.14±1.91 | 6.57±1.63 | 88.03±5.04 | 7.15±3.62 |
0.1 μg/ml | 87.97±3.80 | 10.24±3.07 | 67.58±4.12 | 30.91±4.04 | 82.56±6.87 | 14.56±3.96 |
0.5 μg/ml | 83.73±4.19 | 13.77±2.77 | 60.16±5.46 | 38.74±14.6 | 70.85±3.51 | 26.31±1.20 |