The human cervical carcinoma cell lines, HeLa and ME-180, were obtained from the Korean Cell Line Bank (Seoul, Korea). The HeLa and ME-180 cells were maintained in Dulbecco's modified Easgle's medium (DMEM) and Roswell Park Memorial Institute (RPMI)-1640 medium (Welgene Inc., Korea), supplemented with 10% fetal bovine serum (FBS) (Capricorn Scientific GmbH, Germany) and 100 units of penicillin and streptomycin (Welgene). The cells were cultured in a humidified incubator containing 5% CO₂ at 37°C. SNS-032 and AT7519 were purchased from Selleck Chemicals (Houston, TX, USA).

The HeLa and ME-180 cells (2–5×10³ cells/well) were plated in 96-well plates and allowed to attach for 24 h prior to treatment. The cells were exposed to 0.001, 0.01, 0.1, 0.5, 1, or 10 µM AT7519 and SNS-032 for 48 h, and cell viability was measured using the resazurin reduction ratio (21). Resazurin solution was added to a final concentration of 50 µM, followed by incubation for 2–4 h and spectroscopy at A600 (Epoch, BioTek Instruments, Inc, Winooski, VT, USA). IC₅₀ values were calculated using the ED50 Plus v1.0 online (http://www.sciencegateway.org/protocols/cellbio/drug/data/ed50v10.xls).

The HeLa cells (1×10⁴) were plated in 35-mm culture plates and treated with various concentrations of 0.05 or 0.1 µM of AT7519 and SNS-032 for 3 days. The cells were fixed in 2% formaldehyde/0.2% glutaraldehyde for 15 min at room temperature, and SA β-galactosidase staining was performed as previously described (22).

Delivery of γ-radiation was achieved using a dual-source ¹³⁷Cs unit at a dose rate of 3.2 Gy/min with a GC-3000 Elan irradiator (MDS Nordion, Ottawa, Canada).

The cells were lysed in radioimmunoprecipitation assay (RIPA) buffer [50 mM Tris-Cl (pH 8.0), 150 mM NaCl, 0.1% SDS, 0.5% deoxycholic acid, 1% NP-40] containing a protease inhibitor and a phosphatase inhibitor cocktail and briefly sonicated. The protein content was measured using the Coomassie (Bradford) Protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). A total of 10–40 µg of cell lysates were separated on 8, 10 12, or 15% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were immunoblotted with antibodies against poly(ADP-ribose) polymerase (PARP)-1 (#SC-8007, 1:2,000), p53 (#SC-126, 1:2,000), Chk1 (#SC-8408, 1:2,000), cyclin A (#SC-239, 1:1,000), cyclin B1 (#SC-245, 1:1,000), cyclin D1 (#SC20044, 1:500), cyclin E (#SC-247, 1:500), β-actin (#SC-47778, 1:3,000) (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA), caspase 3 (#9662, 1:1,000), cleaved caspase 3 (#4199, 1:1,000), phospho-p53 (ser15) (#9284, 1:2,000), phospho-Chk1 (ser345) (#2341, 1:1,000), phospho-Chk2 (thr68) (#2661, 1:2,000) (all from Cell Signaling Technology, Danvers, MA, USA), γ-H2AX (ser139) (#05-636, 1:2,000), Chk2 (#07-057, 1:2,000), phospho-ATM (ser1981) (#05-740, 1:5,000) (Millipore, USA) and ATM (#1549-1, 1:2,000) (Epitomics, Burlingame, CA, USA). HRP-conjugated secondary antibodies were obtained from Enzo Life Sciences (Farmingdale, NY, USA; ADI-SAB-100-J, ADI-SAB-300-J, 1:20,000). Chemiluminescence was detected using enhanced chemiluminescence detection reagents (Western Bright™ ECL kit, Advansta CO, USA).

The HeLa and ME-180 cells (1×10⁶ cells) were plated in 100-mm plates and exposed 1 µM of CDK inhibitors for 16 h. For combination treatments with γ-irradiation, 1 µM AT7519 and 1 µM SNS-032 were added to the cells 1 h prior to irradiation, and the cells were further incubated for 4 h following irradiation. BrdU was pulsed for 30 min before cells were harvested and fixed in 70% ethanol at −20°C for 16 h. The fixed cells were rinsed with phosphate-buffered saline (PBS) 3 times and incubated in 1.5 M HCl for 30 min at room temperature. After rinsing with PBS, the cells were resuspended in 100 µl of 0.5% bovine serum albumin (BSA) in PBS and incubated for 10 min. Fluorescein isothiocyanate (FITC)-conjugated BrdU antibody (#11-5071-41, eBioscience/Thermo Fisher Scientific, Waltham, MA, USA) was added to cells, followed by 1 h of incubation prior to the analysis of the BrdU-positive cell profile using a flow cytometer [BD FACSCalibur (SN. E97501075), BD Biosciences, San Jose, CA, USA].

Eight-micrometer pore size Transwell filters (Corning Inc., Corning, NY, USA) were placed into 24-well plates and the upper chambers were covered with Matrigel for the invasion assay (BD Biosciences). Following treatment with 0.5 µM AT7519 or 0.2 µM SNS-032 for 24 h, HeLa (6×10⁴ cells/well) and ME-180 (1.5×10⁵ cells/well) cells were suspended in 150 µl FBS-free medium and seeded onto the filters. The lower chambers were filled with 500 µl medium containing 10 or 20% FBS. At 16–48 h after seeding, the cells were fixed with ice-cold methanol for 5 min, and stained with 0.5% crystal violet (Sigma-Aldrich, St. Louis, MO, USA) in 20% methanol for 10 min at room temperature. After washing with distilled water, the cells on the top side of the filter were removed with a cotton swab, then the number of migrated cells to the lower side were counted.

A total of 50,000 human umbilical vein endothelial cells (HUVECs; #C-12203, Promo Cell, Heidelberg, Germany) were seeded into each well of a 24-well plate pre-coated with Matrigel; and added CDK inhibitors simultaneously. The cells were incubated overnight to allow the formation of tube-like structures. Endothelial cell tube formation was assessed using an IN Cell Analyzer imaging device (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Tubular structures were quantified by counting the number of branches in each field 11 h after seeding.

A total of 96 female BALB/c nude mice (5 weeks old, weighing 15 g) were purchased from Orient Bio Co. (Seongnam, Korea) and allowed to acclimate to the new environment for 1 week before use. The room temperature and relative humidity were maintained at 22±3°C and 50±20%, respectively. The mice were allowed access to water and food (Purina) ad libitum. For tumor generation, exponentially growing 1×10⁶ ME-180 cells were injected subcutaneously into the right hind leg of each male BALB/c nude mouse. Tumor diameters were measured using a caliper, and tumor volumes were calculated with the following formula: V = 0.523×AxB², where 'A' is the longest diameter and 'B' is the shortest diameter of the tumors. All animal experiments were conducted following a protocol approved by the Korea Institute of Radiological and Medical Sciences (KIRAMS) Animal Care and Use Committee (Reference no. KIRAMS 201400400). The weight of the mice upon sacrifice was 25 g.

The tumor-bearing mice were randomly divided into 6 groups of 8 mice per group and treated as follows: i) Control; ii) irradiation; iii) AT7519; iv) irradiation and AT7519; v) SNS-032; and vi) irradiation and SNS-032. When the tumors were 6–7 mm in mean diameter, the mice were treated with AT7519 (15 mg/kg once a day for 5 days for a 2-week duration) or SNS-032 (15 mg/kg injected intraperitoneally every 2 days for a 2-week duration). For the administration of radiation, the mice were lightly anesthetized with 5 mg/kg tiletamine/zolazepam (Virbac ZoletilTM 50; Virbac Lab., Carros, France), and the tumor-bearing legs were irradiated with a ⁶⁰Co irradiator (Thermatron 780, Atomic Energy of Canada) at a dose rate of 1.3 Gy/min.

The anti-metastatic potential in the 3 experimental groups: [i) control; ii) AT7519; and iii) SNS-032 (16 mice per group)] was tested using the spontaneous lung metastasis model. Briefly, 1×10⁶ ME-180 cells per mouse were administered to the right thighs of 5-week-old male BALB/c nude mice. When the tumors reached a diameter of 6–7 mm, the mice were randomly assigned to one of the three groups. Mouse lungs were removed on days 45 and 60 following treatment and fixed with Bouin's solution to count lung nodules under a polarizing light microscope with a 4X objective lens.

Data were analyzed using the Kruskal-Wallis non-parametric statistical test followed by the Mann-Whitney U test using Bonferroni correction to adjust the probability. Statistical analyses were performed using IBM SPSS Statistics version 20.

To determine whether the CDK inhibitors, AT7519 and SNS-032, exert anticancer effects, cell viability assays were performed in two cervical cancer cell lines, HeLa and ME-180. As shown in Fig. 1A, cell growth was markedly inhibited in a dose-dependent manner. AT7519 inhibited HeLa and ME-180 cell growth with IC₅₀ values of 0.352 and 0.599 µM, respectively, whereas SNS-032 inhibited HeLa and ME-180 cell growth with IC₅₀ values of 0.183 and 0.206 µM, respectively. To determine the anticancer mechanisms of action of AT7519 and SNS-032, we first analyzed the cell cycle following treatment with either AT7519 or SNS-032. As shown in Fig. 1B, these CDK inhibitors dysregulated cell cycle progression and increased the sub-G1 cell population, particularly in the HeLa cells. Thus, we next examined whether these drugs induced the apop-tosis of cervical cancer cells. As shown Fig. 1C, the cleavage of PARP-1 and caspase 3, classical markers of apoptosis, was increased following treatment with either AT7519 or SNS-032, in a dose-dependent manner. The HeLa cells were more susceptible to apoptosis by these CDK inhibitors. As CDK inhibition disrupts cell cycle progression, we investigated whether prolonged exposure to AT7519 or SNS-032 induces premature cellular senescence. The results of an SA β-galactosidase assay revealed that the numbers of senescent cells increased 2.5- and 3.5-fold following treatment with 0.05 or 0.1 µM AT7519, respectively, for 3 days. Similar to AT7519, the number of SA β-galactosidase-stained cells increased by 2.4- and 3.7-fold following treatment with 0.05 or 0.1 µM SNS-032, respectively (Fig. 1D). SA β-gal positivity was also increased in the ME-180 cells following treatment with the CDK inhibitors. Finally, we measured the actively proliferating cell populations by BrdU incorporation assay. As shown in Fig. 1E, the population of BrdU-positive cells decreased from 18–4% following treatment with 1 µM AT7519 and to 3% with 1 µM SNS-032, respectively. The inhibition of cellular proliferation was dependent on the dose of the inhibitor applied (data not shown). As cyclins are the crucial cofactors of CDKs and seem to be deregulated in various cases of cervical cancer, we examined the levels of several key cyclins by western blot analysis. The levels of all the cyclins, (namely cyclin D1, E, A and B1) investigated were decreased by AT7519 and SNS-032 treatment (Fig. 1F). These results suggest that AT7519 and SNS-032 inhibited the growth of cervical cancer cells by inducing cell cycle deregulation, apoptosis, cellular senescence and cytostasis.

To confirm the anticancer effects of these inhibitors in vivo and to examine their radiosensitizing effects, a human xenograft tumor was established. As shown in Fig. 2, the growth of subcutaneous ME-180 xenograft tumors in the legs of BALB/c nude mice were examined following exposure to various CDK inhibitors. The treatments commenced when the tumor volume reached 164–180 mm³. The volume of the control tumors (CON) progressively increased after reaching 400–500 mm³. The irradiation of the tumors of mice treated with a single exposure of 9 Gy (RT) suppressed tumor growth. The growth of the tumors of the mice treated with AT7519 or SNS-032 was slower than that of tumors of the controls or the tumors from the mice treated only with irradiation. The growth of the tumors of the mice that were both irradiated and treated with AT7519 or SNS-032 was significantly slower than that of the tumors of the mice treated with irradiation alone (P<0.05). Consequently, while the volume of the tumors of the untreated controls increased 2-fold in approximately 5 days, the volume of the tumors from the mice treated with irradiation doubled in 10 days. When irradiation was combined with treatment with AT7519 or SNS-032, the tumor-doubling time was delayed to 33 or 15 days, respectively.

As AT7519 and SNS-032 were shown to sensitize ME-180 xenograft tumors to radiation in vivo, we examined the mechanisms through which CDK inhibitors radiosensitize ME-180 cells. We first assessed the activation of the DNA damage signaling pathway. The ME-180 cells were treated with 1 µM AT7519 or 1 µM SNS-032 for 1 h, followed by γ-irradiation. We found that the levels of γ-H2AX, a DNA double-strand break marker, were slightly increased in the CDK inhibitor-treated cells than in the cells treated with radiation alone (Fig. 3A). Although the phosphorylation levels of ATM and Chk2 were similar, p53 was markedly activated 4 h following both irradiation and treatment with AT7519 or SNS-032. In addition, AT7519 and SNS-032 completely abolished Chk1 phosphorylation induced by IR. These results suggest that AT7519 and SNS0-032 enhance cellular radiosensitivity via p53 activation and Chk1 inhibition. To determine whether AT7519 or SNS-032 accelerate apoptosis induced by radiation, the levels of apoptotic markers were examined by western blot analyses (Fig. 3B). The combination of AT7519 or SNS-032 with radiation increased the population of apoptotic cells. We then assessed the effects of the combination of radiation and CDK inhibitor treatment on S-phase cell cycle progression by BrdU incorporation assay (Fig. 3C). Although there was no inhibition of proliferation following treatment with radiation alone under our experimental conditions, the combination of AT7519 or SNS-032 and IR significantly decreased the population of cells in the S phase by 50%.

To evaluate the effects of AT7519 and SNS-032 on cancer metastasis, we performed cell migration and invasion assays. For the migration assay, the HeLa and ME-180 cells were seeded in Transwell^® chambers and treated with 0.5 µM AT7519 or 0.2 µM SNS-032 for 24 h. Both AT7519 and SNS-032 inhibited cervical cancer cell migration (Fig. 4A). The number of migratory HeLa cells was significantly reduced following treatment with AT7519 and SNS-032 to 0.38±0.08 and 0.393±0.059, respectively. The number of migratory ME-180 cells were also reduced following treatment with AT7519 (0.220±0.045) and SNS-032 (0.111±0.010). The inhibitory effects of AT7519 and SNS-032 on cell invasion were also confirmed (Fig. 4B). The number of invasive HeLa cells was reduced following treatment with 0.5 µM AT7519 (0.273±0.063) and 0.2 µM SNS-032 (0.334±0.045). The number of invasive ME-180 cells was also reduced following treatment with AT7519 (0.087±0.063) and SNS-032 (0.070±0.004).

As angio-genesis accelerates tumor metastasis, the anti-angiogenic activities of the CDK inhibitors were measured. As shown in Fig. 5, HUVECs formed a well-organized tubular structure on Matrigel^®, representing functional activity of endothelial cells. However, both AT7519 and SNS-032 inhibited tube formation. Compared to 83.75±2.56 branch points in the control group, the number of branch points per field decreased to 28±3.56 (P<0.001) and 20±3.58 (P<0.001) in the presence of AT7519 and SNS-032, respectively. From this result, CDK inhibitors appeared to suppress tumor angiogenesis, in addition to suppressing the invasion and migration of cervical cancer cells.

To confirm the anti-metastatic activity of CDK inhibitors in vivo, a spontaneous lung metastasis model was employed using an ME-180 xenograft tumor model. Forty-five days after treatment, the average number of lung nodules of the control group was 12.83±1.89. The number of lung nodules decreased to 7.75±0.94 in the AT7519 group and to 9.12±0.79 in the SNS-032 group. The anti-metastatic effects were more prominent after 60 days. Compared to 15.5±0.99 in the control group, the number of lung nodules was 8.88±1.16 (P<0.05) and 9.63±1.08 (P<0.05) in the AT7519 and SNS-032 groups, respectively (Fig. 6). These results confirm that both AT7519 and SNS-032 were able to suppress tumor metastasis to the lungs in vivo.