Corosolic acid (analytical standard, 89067) was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany) and dissolved in dimethyl sulfoxide (DMSO). RPMI-1640 medium, DMEM/F12 medium, fetal bovine serum (FBS) and penicillin-streptomycin solution were obtained from Gibco (Grand Island, NY, USA). DMSO, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI) and glutamine were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Lipofectamine 2000 was obtained from Invitrogen (Carlsbad, CA, USA). The kit for apoptosis was obtained from BD Biosciences (San Jose, CA, USA). The antibodies used in the present study were as follows: p53 (cat. no. 48818; Cell Signaling Technology, Inc., Danvers, MA, USA), p21 (cat. no. ab109520; Abcam, Cambridge, MA, USA), cyclin B1 (cat. no. 4135; Cell Signaling Technology, Inc. Danvers, MA, USA), Cdc25B (cat. no. ab124819; Abcam), Bax (cat. no. 2774; Cell Signaling Technology, Inc.), Bcl-2 (cat. no. 15071; Cell Signaling Technology, Inc.), caspase-9 (cat. no. ab32539; Abcam), caspase-3 (cat. no. 9662), ASK1 (cat. no. 3762), p-JNK (cat. no. 9255), JNK (cat. no. 9252), p-p38 (cat. no. 9216), p38 (cat. no. 9212), FoxM1 (cat. no. 5436), MELK (cat. no. 2274), β-actin (cat. no. 4970; all were from Cell Signaling Technology, Inc.) and the HRP conjugated goat anti-mouse (sc-2031)/rabbit(sc-2030) secondary antibodies (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). All other chemicals and reagents were purchased from Beyotime Institute of Biotechnology (Nantong, China).

The human retinoblastoma cell line Y-79 and the human RPE cell line ARPE-19 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Y-79 cells were cultured in RPMI-1640 medium with 10% FBS and 1% penicillin-streptomycin (P/S). ARPE-19 cells were cultured in DMEM/F12 medium with 10% FBS, 2 mM glutamine and 1% P/S. Cells were maintained at 37ºC in a humidified atmosphere containing 5% CO2 and passaged once every 3 days. For the analysis of function of MELK-FoxM1 signaling, cells were transiently transfected with the indicated plasmids [FoxM1-luc reporter vector (2 µg, provided by Dr Fanfan Zhou's Laboratory, University of Sydney), FoxM1 expression vector (2 µg, pCW57.1-FOXM1b plasmid; cat. no. 68811; Addgene, Cambridge, MA, USA) or MELK expression vector (2 µg,provided by Dr Fanfan Zhou's Laboratory, University of Sydney)] or small interfering RNA for MELK (100 pmol MELK siRNA; cat. no. sc-61016; Santa Cruz Biotechnology, Inc.) using Lipofectamine 2000. Cells were cultured for 24 h post transfection and the expressions of the related molecules were assessed by western blot analysis before treatment with the indicated agents.

To assess the effect of CA on cell viability, MTT assay was used as previously mentioned (23). After treatment, 10 µl of MTT (5 mg/ml stock in PBS) was added per well and the incubation was continued for 2 h. Then, the culture medium was removed and 100 µl DMSO was added to dissolve the formazan crystals. The absorbance at 570 nm was determined with an ELISA reader (Bio-Rad Laboratories, Hercules, CA, USA).

To assess the effect of CA on cell cycle distribution, PI staining was used as previously mentioned (24). After treatment, cells were collected by centrifugation, fixed in 70% ethanol, re-suspended in PBS containing RNase (1 mg/ml) and PI (50 µg/ml), incubated for 30 min in the dark and then analyzed using flow cytometry (Becton-Dickinson; BD Biosciences).

To assess the effect of CA on cell apoptosis, double staining with Annexin V-FITC and PI was used as previously described (24). After treatment, cells were collected by centrifugation, washed twice with cold PBS, re-suspended in binding buffer containing 10 µl of Annexin V-FITC stock and 10 µl of PI, and then analyzed using flow cytometry (Becton-Dickinson; BD Biosciences).

To assess the effect of CA on the expression profiles of related proteins, western blot analysis was used as previously described (25). After treatment, cells were lysed for 20 min in lysis buffer and the concentration of protein sample was determined with the Bradford method. Samples (50 µg) were separated on SDS-PAGE gel (10%) and electrophoretically transferred onto polyvinylidene fluoride (PVDF) membrane. After blocking, the membrane was incubated with the primary antibody at 4°C for overnight and horseradish peroxidase (HRP)-conjugated secondary antibody at 37°C for 2 h. The protein bands were visualized by ECL detection kit (Beyotime Institute of Biotechnology).

To assess the effect of CA on the transcriptional activity of FoxM1, luciferase assay was used as previously described (26). After transfection, cells were treated with the indicated agents for 24 h and the luciferase activity was measured using luciferase assay system. Relative luciferase activity was expressed as percentage induction of promoter activity by the FoxM1 expression vector, where the promoter activity resulting from transfection with FoxM1 was set at 100%.

Biostatistical analyses were conducted with Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA) and SPSS 16.0 software package (SPSS, Inc., Chicago, IL, USA). All experiments were conducted in triplicate and the results were indicative of three independent studies. Data are expressed as means ± SD and statistical comparisons were made using the Student's t-test and the one-way ANOVA. A P<0.05 was considered to indicate a statistically significant result.

In order to analyze the biological effect of CA, human retinoblastoma Y-79 cells were treated with various concentrations of CA for 24 or 48 h and cell viability was assessed by MTT assay. The results demonstrated that CA significantly inhibited cell proliferation of Y-79 cells in a dose- and time-dependent manner (Fig. 1B). The value of IC₅₀ was calculated using the GraphPad Prism software. The results indicated that treatment of 4.15 µM CA for 24 h or 3.37 µM for 48 h resulted in a reduced cell proliferation by 50% in the Y-79 cells. However, CA treatment had little impact on untransformed cells such as the human retinal pigment epithelial cell line ARPE-19 (Fig. 1C). Then, treatment with concentrations of 2, 5 and 10 µM CA for 24 h was selected to continue this study. The data suggest that CA has an inhibitory effect against human retinoblastoma Y-79 cells.

In order to identify whether CA induces proliferation inhibition via triggering cell cycle arrest, human retinoblastoma Y-79 cells were treated with CA (0, 2, 5 and 10 µM) for 24 h, and cell cycle distribution was assessed by flow cytometric analysis. The results indicated that CA treatment (10 µM) significantly increased the population of cells in the G2/M phase to 32.14±1.37 compared to the non-treatment cells (12.53±1.18) (Fig. 2). Furthermore, the change in cell cycle regulators upon CA treatment was analyzed using western blot analysis. As shown in Fig. 3, CA treatment dose-dependently affected the cell phase distribution via inducing upregulation of p53 and p21WAF1 as well as downregulation of cyclin B1, Cdc25B and Aurora B in the Y-79 cells.

In order to identify whether CA induces proliferation inhibition via triggering cell apoptosis, human retinoblastoma Y-79 cells were treated with CA (0, 2, 5 and 10 µM) for 24 h, and cell apoptosis was assessed by flow cytometric analysis. The results indicated that CA treatment (10 µM) significantly increased the percentage of apoptotic cells to 54.58±4.46 compared to the non-treatment cells (1.18±0.25%) (Fig. 4). Furthermore, the change in cell apoptosis-related signaling pathways upon CA treatment was analyzed. First, as shown in Fig. 5A, CA treatment dose-dependently affected the mitochondrial pathway via inducing upregulation of Bax, downregulation of Bcl-2 and cleavage of caspase-9 and caspase-3. Then, as shown in Fig. 5B, CA treatment dose-dependently affected the MAPK pathway via inducing upregulation of ASK1 and phosphorylation of JNK and p38, but not ERK (data not shown).

A previous study estimated that FoxM1 is the direct target of UA (22). First, we examined the inhibitory effect of CA on the expression profiles of MELK and FoxM1. The results revealed that CA treatment for 24 h significantly suppressed the expression levels of MELK and FoxM1 in a dose-dependent manner in Y-79 cells (Fig. 6A). In addition, MELK overexpression did not affect the inhibitory effect of CA on FoxM1 expression; likewise FoxM1 overexpression did not affect the inhibitory effect of CA on MELK expression (Fig. 6B). Then, we examined the inhibitory effect of CA on the transcriptional activity of FoxM1. As shown in Fig. 7, CA treatment for 24 h significantly suppressed the transcriptional activity of FoxM1 in cells transfected with FoxM1 + MELK (−) or FoxM1 in a dose-dependent manner, indicating that CA abrogated FoxM1 activity driven by FoxM1 itself or MELK, and such a compound abrogated MELK-dependent FoxM1 activity possibly by inhibiting MELK expression.

Several lines of evidence have suggested that MELK-FoxM1 signaling plays a key role in the process of cell cycle and cell apoptosis (27–29). To examine whether CA induces cell cycle arrest and cell apoptosis via regulating MELK-FoxM1 signaling, Y-79 cells were transfected with FoxM1 alone, MELK alone or MELK + FoxM1, and then treated with CA (10 µM) for 24 h. The results indicated that overexpression of MELK + FoxM1, rather than FoxM1 alone, significantly reversed the inductive effect of CA on cell cycle arrest and cell apoptosis (Figs. 8A and 9A). However, overexpression of MELK alone slightly attenuated the inductive effect of CA compared to the other groups, indicating that MELK may exert its effect mainly through the FoxM1 pathway. In addition, the changes in related molecules were further investigated, and the results were consistent with the changes in cell cycle distribution and cell apoptosis (Figs. 8B and 9B and C).