The animal studies were approved by the Kunming Medical University’s laboratory animal ethics committee.
The X‑ray crystallographic structures used in the studies were obtained from the Protein Data Bank (PDB)[23, 24]. In addition to 5 structures of CDK4 and 8 structures of CDK6, we also collected 14 structures of CDK2. The chemical structures of a total of 3167 US Food and Drug Administration (FDA) approved drugs were gathered from the ZINC database.[25, 26]. We used the docking software idock v2.2.1[27, 28] to dock all of the compounds onto the CDK4/6 structures first. The binding conformations and the binding affinities were predicted and prioritized according to the average predicted binding affinity. Then the top 50 ranking candidate CDK4/6 inhibitors were docked onto the CDK2 structures to screen for CDK2/4/6 triple inhibitors. Nine top ranking commercially available compounds were selected and evaluated.
Monatepil, Fluazuron, Temafloxacin, Ketanserin, Talniflumate, Altanserin, Dutasteride, Mizolastine, Vanoxerine dihydrochloride, 5-Fu were purchased from Sigma‑Aldrich.
Cell lines, cell culture, and experimental conditions.
The human HCC cell lines QGY7703 and Huh7 were obtained from Cell bank of Chinese Academy of Sciences(Shanghai, China), and cultured in D-MEM/F-12 medium (GIBCO, USA) containing 8% FBS (Hyclone, Mexico) at 37˚C in 5% CO2 and 95% humidified air. Cells were plated in 96‑, 24‑plates (NEST, China) with medium containing 8% FBS and the test compounds at indicated concentrations (1, 3, 10 and 30μM), and then incubated for indicated times (6, 12, 24, 48 or 72 h).
Cell viability MTT and CCK-8 assays.
MTT assay were conducted as described in previous studies[20-22].QGY7703 and Huh7 cells were plated at an initial density of 9x103cells/well in 96‑well plates, incubated with MTT (Sigma) reagents and the absorbance measured at 570 nm with a microplate reader (Multiskan Spectrum, Thermo Scientific Microplate Reader, USA). CCK-8 assay was performed as described in the CCK-8 Kit (Dojindo Laboratories). Cells were seeded in 96-well plate, treated with various drugs for indicated time prior to the addition of CCK-8 solution and OD values were measured at 450 nm using a microplate reader.
Cell cycle analysis.
The cell cycle profile was determined by Flow cytometry analysis, as described previously[20-22].Briefly, QGY7703 and Huh7 cells (4x104) were seeded in 24‑well plates in D-MEM/F-12 medium. After 24 h culture, medium were replaced with D-MEM/F-12 containing 8% FBS and vanoxerine dihydrochloride(1, 3, 10 or 30μM) for indicated times (6, 12 or 24 h), fixed in ice‑cold ethanol, and stained in Coulter DNA‑Prep Reagents (Beyotime Coulter, BeyotimeInstitute of Biotechnology, Beijing). The cellular DNA content was determined by EPICS xL4 flow cytometer (BD FACSCalibu, USA), and cell cycle distribution determined by BD FACStation software (USA).
QGY7703 and Huh7 cells were seeded in 6-well platein D-MEM/F-12 medium. After 48hours culture, the medium were replaced with D-MEM/F-12 containing 8% FBS and vanoxerine dihydrochloride(1, 3, 10 or 30μM) for indicated times (6, 12 or 24 h). Apoptosis was measured by annexinV and propidium iodide (PI) staining (Beyotime Institute of Biotechnology, Beijing) as described in previous studies[20-22].
Western blot analysis.
Cells were lysed and Western blotting analysis were performed as described previously[20-22].QHY7703 and Huh7 cells were plated at 6-well plates, cultured in serum starved media (0.125% FBS) for 24 hours and then with 10%FBS medium containing various concentrations (3, 10, 30μM) of vanoxerine dihydrochloride. Cells were harvested after 6 hours incubation and proteins analyzed by Western blotting. Primary antibodies were purchased from Cell Signaling Technology, Inc. Danvers, MA, USA). They include anti‑cyclin D1 (no. 2978), anti‑cyclinE (no. 4129), anti‑CDK2/4/6 (no. 2546), anti‑Rb (no. 9313), anti‑phospho‑CDK4, anti‑phospho‑CDK2/4/6 (no. 2561), anti‑Rb (no. 9301), and anti‑GAPDH (no. 5174). As positive controls, three siRNAs targeting each of the CDK2/4/6were designed as described previously, and used to inhibit the expressions of each of the CDK2/4/6proteins in QGY7703 and Huh7 cells. The proteins were measured using enhanced chemiluminescence detection system (Thermo Fisher scientific, USA).
Synergy quantitation of the combination treatment
Synergy quantitation of the drug combination studies were performed according to the Chou--Talalay method. Huh7 cells were plated at an initial density of 5x103cells/well in 96‑well plates, and treated cells with various concentrations of vanoxerine dihydrochloride and 5-Fu. Cell viabitily was detected by CCK-8 after 72 hours treatment, and the absorbance values were measured at 450 nm using microplate reader. The combined effect was analyzed by CompuSyn software (www.combosyn. com), which performs multiple drug dose-effect calculations using the Median Effects methods described by Chou and Talalay to determine the combination index (CI). The drug combinations quantitative definition of CI for additive effect (CI = 1) ,for synergism is CI < 1, and for antagonism is CI > 1.(The formula of the combined index of the two drugs is: CI =(D)1/(Dx)1+(D)2/(Dx)2 , Single dose (D), combined dose (Dx) ).
Determine the anti-cancer efficacy in nude mice xenografted with Huh7 cancer cells.
Female BALB/C nude mice (4‑5 weeks old, weighing 15 g; Animals laboratoryof Kunming Medical University China), were housed and cared under standard conditions (pathogen‑free, 12 h light/dark cycle, 50‑80% humidity, and15‑27˚C) in accordance with guidelines from animal ethics committee in Kunming Medical University. For the preclinical animal model, Huh7 cells (1x106cells in 0.2 ml PBS) were subcutaneously injected into the right flank of the nude mice. The size of the tumor was measured every day by a caliper, and tumor volume calculated by the formula V=ab2/2 (a=longest axis; b=shortest axis).When the tumors grew to 80‑100 m3 (7 days after inoculation), mice were divided randomly into 4 groups (5 mice/group),and treated for 21 days by i.p. injection daily of (1) control PBS, (2) vanoxerine dihydrochloride (40mg/kg), (3) 5-Fu (10mg/kg), (4) vanoxerine dihydrochloride (40mg/kg) plus 5-Fu (10mg/kg). At the end of experiments, mice were sacrificed by cervical dislocation. The tumor tissues were excised, weighed, images captured, and immunohistochemistry analysis performed.
Tumor tissues were fixed in 10% formalin and embedded in paraffin, sliced into 4 μm sections, deparaffinized, dehydrated, antigen retrieved, blocked with 5% goat serum, and incubated in the primary antibodies: anti-RB1 (1: 500; CST), anti-CDK2 (1:50; Abcam), anti-CDK4 (1: 500; CST), anti-CDK6 (1: 100 Abcam). The slides were washed and incubated with biotinylated anti-mouse or anti-rabbit secondary antibodies. The peroxidase reaction was visualized using 3,3'-diaminobenzidine tetrahydrochloride (DAB) and counterstained with hematoxylin. To quantitate the staining intensity, 5 random fields were chosen, and the numbers of total cells and positive cells were counted in each section under a microscope at 400x magnification. The percentage of positive cell populations from the 5 random fields was analyzed for statistics.
Data were obtained from the triplicates of three different experiments. Values are expressed as the mean ± standard deviation. The dates were analyzed by SPSS software (version 16.0). P<0.05 was considered to indicate statistically significant difference between values.