A total of 44 X-ray crystallographic structures of CDK2 in complex with a ligand were collected from the Protein Data Bank (PDB) (14). The co-crystallized ligands and water molecules were manually removed. The structures of FDA-approved drugs were collected from the Drug Bank-approved (DBAP) and FDA catalogs of the ZINC database (15,16). The DBAP catalog (version 2014-03-19) comprising 1,738 compounds and the FDA catalog (version 2012-07-25) comprising 3,176 compounds were downloaded. The 44 CDK2 structures in PDB format and the 4,914 compounds in Mol2 format were then converted into PDBQT format using AutoDockTools (17). The free and open-source docking software idock v2.1.2 (9,10) developed by our group was then applied to dock all of the 4,914 compounds onto all of the 44 CDK2 structures, and to predict their binding conformations as well as their binding affinities. Finally, the compounds were sorted in an ascending order according to their predicted binding free energy averaged across the 44 CDK2 structures, and the top nine commercially available compounds were purchased (Sigma-Aldrich, St. Louis, MO, USA) and biologically evaluated.

ADA, oxaliplatin, nilotinib, LS-194959, estradiol benzoate, nandrolone phenylpropionate, vilazodone, azelastine hydrochloride, latuda and paliperidone were purchased from Sigma-Aldrich. Anti-cyclin D, -B1 and -E as well as anti-CDK2, -Rb, phosphorylated (pho)-CDK2 (Thr-160), pho-Rb (Ser-795) and GAPDH were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA).

The colorectal cancer cell lines LoVo and DLD1 were obtained from the American Type Culture Collection (Manassas, VA, USA). These cell lines were cultured in RPMI 1640 medium (GE Healthcare Life Sciences, Shanghai, China) containing 10% fetal bovine serum (FBS) (Invitrogen Life Technologies, Carlsbad, CA, USA) at 37°C in 5% CO₂ and 95% humidified air. The present study was approved by the ethics committee of the Kunming Medical University (Kunming, China).

Cells were plated in 96-, 24-, or six-well plates (Corning Incorporated, Corning, NY, USA) with medium containing 0.125% FBS for 24 h and then treated with medium containing 10% FBS and the test compounds at various concentrations as indicated (1, 3, 10 and 30 µM), and incubated for 6, 12, 24, 48 or 72 h.

For the MTT assay (Sigma-Aldrich), cells were plated at an initial density of 9×10³ cells/well in 96-well plates and incubated with 0.5 mg/ml MTT (Sigma-Aldrich) for 4 h. The medium was then discarded and 200 µl dimethyl-sulfoxide (Sigma-Aldrich) was added to dissolve the formed formazan crystals. The absorbance was measured at 570 nm with a Synergy 2 microplate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA) according the standard protocol.

LoVo or DLD1 cells (4×10⁴) were seeded in 24-well plates in RPMI 1640 medium containing 0.125% FBS, and cultured for 24 h. The cells were incubated in medium containing 10% FBS and various doses of ADA (1, 3, 10 or 30 µM) for 6, 12 or 24 h at 37°C, then fixed in ice-cold 70% ethanol and stained using a Coulter DNA-Prep Reagents kit (Beckman Coulter, Brea, CA, USA). Cellular DNA content of 1×10⁴ cells from each sample was determined using an EPICS xL4 flow cytometer (Beckman Coulter). The cell cycle phase distribution was analyzed using ModFit LT 2.0 software (Verity Software House, Topsham, ME, USA). All data were obtained from two separate experiments of which each was performed in triplicate.

Cells were lysed in radioimmunoprecipitation assay buffer (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) containing 1 mM phenylmethanesulfonylfluoride (Beijing Solarbio Science & Technology Co., Ltd.) and protease inhibitor cocktail (Beijing Solarbio Science & Technology Co., Ltd.) for 30 min at 4°C. After centrifugation for 15 min at 5,668 × g, the supernatants were recovered and the protein concentration was measured using a bicinchoninic acid Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of cell lysates were resolved using 10% SDS-PAGE and transferred onto nitrocellulose membranes (Sigma-Aldrich). After blocking, the membranes were incubated sequentially with the following primary antibodies (Cell Signaling Technology, Inc.) in 5% w/v bovine serum albumin (Sigma-Aldrich), 1X Tris-buffered saline, and 0.1% Tween^® 20 (MP Biomedicals, Illkirch, France) at 4°C overnight, with gentle agitation: Rabbit monoclonal anti-cyclin D1 (cat. no. 2978), rabbit monoclonal anti-cyclin B1 (cat. no. 12231), mouse monoclonal anti-cyclin E (cat. no. 4129), rabbit monoclonal anti-CDK2 (cat. no. 2546), mouse monoclonal anti-Rb (cat. no. 9313), polyclonal anti-phospho-CDK2 (cat. no. 2561), polyclonal anti-Rb (cat. no. 9301), rabbit monoclonal anti-GAPDH (cat. no. 5174; 1:1,000 dilution for all antibodies). The membranes were then incubated with the appropriate secondary antibodies, including goat anti-rabbit IgG horseradish peroxidase (HRP)-linked antibody (Cell Signalling Technology, Inc.; cat. no. 7074; 1:1,000–3,000 dilution) and horse anti-mouse IgG HRP-linked antibody (Cell Signalling Technology, Inc.; cat. no. 7076; 1:1,000–3,000 dilution). Proteins were detected using an enhanced chemiluminescence detection system (Amersham, Piscataway, NJ, USA). To ensure equal loading of the samples, the membranes were re-probed with an anti-GAPDH antibody (Cell Signalling Technology, Inc.).

Female BALB/C nude mice (n=50; weighing 15 g; 4–5 weeks old; Vital River Laboratory Technology Co. Ltd., Beijing, China), were housed under specific pathogen-free conditions with a 12 h light/dark cycle, in an environment containing 50–80% humidity at 15–27°C, and cared for in accordance with the guidelines of the laboratory animal ethics committee of Kunming University (Kunming, China). The cages, food, and water of the mice were sterilized. In order to establish the xenograft model, 1×10⁶ DLD1 cells in 0.2 ml phosphate-buffered saline were injected subcutaneously into the right flank of the mice (n=3) and the tumor size was measured every day using a caliper. One week after inoculation, when the tumors grew to a volume of 80–100 m³, the mice were randomly divided into groups (5 mice/group) and gavaged daily for 21 days with 0.5% carboxymethylcellulose (CMC)-NaCl (Sigma-Aldrich) containing various doses of ADA (15, 20, 65 and 100 mg/kg) and oxaliplatin (Sigma-Aldrich; 40 mg/kg). Mice were sacrificed by cervical dislocation, tumors were excised and weighed, and their images were captured. The tumor volume was calculated using the formula V=ab²/2 (a=longest axis; b=shortest axis).

Data were obtained from at least three experiments. Values are expressed as the mean ± standard deviation. Statistical analysis was performed by Student's t-test, and the results were analyzed using SPSS 16.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference between values.

A total of 4,914 FDA-approved drugs were extracted from the DBAP and FDA catalogs of the ZINC database. The drugs were docked onto CDK2 and ranked according to their average predicted binding affinity. Nine top-scoring compounds were selected based on their commercial availability and further examined (Table I) (18–26). The selected drugs were nilotinib, LS-194959, ADA, estradiol benzoate, nandrolone phenylpropionate, vilazodone, azelastine hydrochloride, latuda and paliperidone.

The present study first evaluated the anti-cancer effects of the nine compounds using MTT assays. The nine compounds all decreased the viability of LoVo and DLD1 cells. The IC₅₀-values were calculated using GraphPad Prime5 (GraphPad, Inc., La Jolla, CA, USA). Among them, ADA had the lowest IC₅₀ (4.43 µM for DLD1 and 7.135 µM for LoVo) (Fig. 1A). The growth inhibitory effect of ADA was dose- and time-dependent (P<0.05; Fig. 1B), with marked inhibition observed at concentrations >3 µM.

In order to assess whether ADA inhibited the activity of CDK2 in colorectal cancer cells, LoVo or DLD1 cells were treated with ADA (3, 10 or 30 µM) for 6, 12 or 24 h, and its effects on the cell cycle profile were assessed using flow cytometry. ADA significantly increased the G1-phase population as compared to that in the control group in a dose- and time-dependent manner (P<0.05) (Fig. 2A). After 24 h of incubation with ADA, increases in the G1-phase populations accompanied by simultaneous and significant decreases in the S- and G2/M-phase populations were apparent (Fig. 2B).

The present study investigated the effects of ADA on the expression of significant proteins involved in G1-to-S-phase transition, including CDK2, cyclin E, Rb, pho-CDK2 and pho-Rb by western blot analysis in LoVo and DLD1 cells. As shown in Fig. 3A and B, ADA reduced the expression of CDK2, Rb, pho-CDK2, pho-Rb and cyclin E (Fig. 3). By contrast, the expression of cyclin D1 and cyclin B1 was not affected. This observed activity pattern was typical for CDK2 inhibitors (27).

In order to assess the inhibitory potential of adapalene on the growth of colorectal carcinoma in vivo, DLD1 cell-derived xenograft tumors were established in BALB/C nude mice. Carcinoma volumes were measured every 3–4 days after the appearance of the tumors. At 7 days after tumor inoculation, the volume of the tumors reached 80–100 mm³, and animals were administered various doses of ADA (15, 65 or 100 mg/kg in 0.5% CMC-NaCl) daily for 21 days by oral gavage. In a separate experiment, the efficacy of ADA (20 mg/kg), oxaliplatin (40 mg/kg) and the combination of ADA (20 mg/kg) plus oxaliplatin (40 mg/kg) was compared.

The results showed that oral administration of ADA significantly inhibited tumor growth (P<0.05). Following 21 days of treatment a dose of ADA of as low as 15 mg/kg achieved a significant reduction in tumor weight and volume as compared with that in the control (P<0.05) (Fig. 4A–C). There was no significant difference between 15 and 65 mg/kg ADA treatment.

In addition, the potency of of ADA (20 mg/kg) was similar to that of oxaliplatin (40 mg/kg). Of note, combined administration produced the highest therapeutic effect (Fig. 5A–C). To the best of our knowledge, the present study was the first to demonstrate the anti-cancer activity of ADA in vivo, which may be a suitable CDK2-targeting drug for the treatment of human colorectal cancer.

Fig. 6A illustrates the conformation of CDK2 in complex with ADA in a three-dimensional manner predicted by iview (28). Fig. 6B shows the intermolecular interaction diagram in a two-dimensional manner using PoseView (29). ADA was predicted to reside in the adenosine triphosphate-binding site of CDK2 and interact with CDK2 mainly through hydrophobic contacts with Phe82, Ile10, Leu134, Lys33 and His84.