Apigenin was purchased from Sigma-Aldrich/Merck KGaA and a Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Laboratories. Apigenin was dissolved in dimethylsulfoxide (DMSO) and subsequently diluted in culture medium. The DMSO concentration did not exceed 0.1% during treatment.

The human RCC cell lines ACHN, Caki-1, and NC65 (ATCC) were used. Primary RCC cells were separated from surgical specimens obtained from five patients with untreated RCC, as previously described (24). Pathologic stage and grade were consistent with the 2004 WHO criteria (https://www.patologi.com/WHO%20kidney%20testis.pdf), as follows: T2N0M0 grade 1 in patient 1 (70 years, male); T3bN0M0 grade 2 in patient 2 (68 years, female); T2N0M0 grade 1 in patient 3 (70 years, male); T3bN0M1b grade 2 in patient 4 (76 years, female); and T2N0M0 grade 2 in patient 5 (63 years, male) at Hyogo College of Medicine Hospital between March 1997 and February 1998. All cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin and streptomycin, and then maintained at 37°C in 5% CO₂.

Ethical approval for the use of human tissue was granted by the Hyogo College of Medicine (Hyogo, Japan). All patients provided individual written informed consent for the use of their sampled tissues.

Cytotoxicity was determined based on colorimetric assay findings of cell viability obtained with a CCK-8 Kit (Dojindo Laboratories). Briefly, a 100-µl suspension of 0.5×10⁴ cells was seeded into a 96-well flat bottom microtiter plate. After incubation for 24 h, a drug solution or medium alone (control) was added to the plates in triplicate, and then each plate was incubated for an additional 24 h. Subsequently, 10 µl of CCK-8 solution was added for 3 h. Absorbance (A) in each well was measured using a SPECTRAmax PLUS384 (Molecular Devices, LLC) at 450 nm as the reference, and cell viability was determined based on the percentage of control cells using the following formula: [Percent cell viability = (A of treated wells/A of control wells) ×100] (25).

Cell viability and morphologic changes were evaluated using trypan blue dye exclusion test and phase-contrast microscopy findings, respectively. Cells were seeded into a 6-well plate at 1.5×10⁵ cells per well and cultured for 24 h, and then treated in duplicate with apigenin for 24 h. After treatment, the cells were harvested and viability was assessed after 0.5% trypan blue dye (Sigma-Aldrich/Merck KGaA) staining for 1 min at room temperature, and then counted using a hemocytometer under a phase contrast microscope as previously reported (26). Cell death was observed and photographs of adherent cells were obtained using phase-contrast microscopy after removal of medium containing floating cells.

Cells were treated with apigenin for 24 h, then harvested, washed twice with cold assay buffer, and processed for cell cycle analysis. Briefly, the cells were fixed in cell cycle phase determination fixative at room temperature and stored at −20°C overnight for later analysis according to the protocol of the manufacturer (cell cycle phase determination kit, no. 10009349, Cayman Chemical). Fixed cells were centrifuged at 1,500 rpm and washed with cold PBS twice. Next, RNase A (20 µg/ml final concentration) and propidium iodide (PI) staining solution (20 µg/ml final concentration) were added, and the cells were incubated for 30 min at room temperature in the dark. Analysis was performed using a LSRFortessa^™ X-20 instrument (BD Biosciences) equipped with BD FACSDiva software. Furthermore, FlowJo v10.7.1 (FlowJo LLC) trial cell cycle analysis software was used to determine the percentage of cells in each of the different cell cycle phases.

Cells were plated in 10-cm plates for 24 h and then treated with apigenin for 6–24 h in a cell culture incubator at 37°C. Following the indicated treatment, cells were lysed for 30 min on ice in lysis buffer with a protease inhibitor cocktail, and then protein concentrations were determined using a Bradford Assay Kit (Bio-Rad Laboratories). Next, 20 µg of protein was separated using 10% SDS-PAGE and transferred to a PVDF membrane. After blocking nonspecific binding sites for 2 h at room temperature with 5% skim milk in TBS with 0.1% Tween-20, the membranes were incubated overnight at 4°C with the following primary antibodies: cyclin-dependent kinase 1 (CDK1; bs-0542R, Bioss Inc.), cyclin A (sc-271682), cyclin B1 (sc-245), cyclin D1 (sc-6281), cyclin D3 (sc-6283), and cyclin E (sc-247) from Santa Cruz Biotechnology, Inc. at 1:200 dilution, and β-actin mouse polyclonal [E4D9Z, Cell Signaling Technology, Inc. (CST)] at 1:2,000 dilution. The membranes were washed three times by TBST buffer for 30 min at room temperature and then incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG secondary antibody (code. 330, MBL) at 1:2,000 dilution and HRP-conjugated goat anti-rabbit IgG secondary antibody (sc-2004, Santa Cruz Biotechnology, Inc.) at 1:1,000 dilution for 1 h at room temperature. Finally, the membranes were washed three times with TBST buffer for 30 min at room temperature and signals were detected using chemiluminescence ECL kit (GE Healthcare) with an ImageQuant LAS 4010 system (GE Healthcare).

All determinations were conducted at least three times, and the results are expressed as the mean ± SD of three experiments. All analyses were performed using Graphpad Prism V8 for Mac (GraphPad Software, Inc.). A two-tailed value of P<0.05 was considered to indicate statistical significance. Differences between treatment groups and controls were analyzed by one-way ANOVA analysis of variance, followed by Dunnett's multiple comparison test.

First, the effects of different concentrations of apigenin on cell viability were examined using Caki-1, a human RCC cell line. For cells treated for 24 h, apigenin inhibited proliferation in a dose-dependent manner with a 50% inhibition concentration (IC₅₀) value of 27.02 µM. Similar antiproliferative effects were also noted with the RCC cell lines ACHN and NC65 (Fig. 1A), which showed IC₅₀ values of 50.40 and 23.34 µM, respectively. Furthermore, apigenin inhibited proliferation of Caki-1 cells in a time-dependent manner (Fig. 1B). This antiproliferative activity of apigenin was further confirmed by findings obtained with a trypan blue dye-exclusion test (Fig. 1C). Furthermore, a marked decrease in cell numbers, cell swelling, and destruction of cells were also observed using phase-contrast microscopy when the cells were treated with apigenin (Fig. 1D).

To further examine the antiproliferative effect of apigenin on RCC cells, we examined primary RCC cells obtained from five patients. In all patient samples, a marked dose-dependent antiproliferative effect was achieved, regardless of the varying sensitivity of the RCC cells (Fig. 2). The IC₅₀ values for apigenin with cells from those five cases (patient 1–5) were 73.02, 43.74, 35.63, 26.80, and 53.51 µM, respectively.

Taken together, these findings clearly demonstrated that treatment of human RCC lines as well as primary RCC cells with apigenin provides an antiproliferative effect.

In order to better understand the mechanism of cell proliferation inhibition, cell cycle distribution in different phases of the cell cycle was analyzed following apigenin treatment. There were marked changes in the cell cycle shown by Caki-1 cells treated with apigenin including an increase in percentage of cells in the G₂/M phase, along with a concomitant decrease in those in the G₀/G₁ and S phases as compared with untreated cells (Fig. 3A). Following treatment with apigenin at 20, 30, and 50 µM, the percentage of Caki-1 cells in the G₂/M phase was 22.4, 34.8, and 48.9%, respectively, while only 24% of the control cells were found to be in the G₂/M phase (Fig. 3B).

To further assess the molecular mechanisms related to inhibition of cell proliferation, the effects of apigenin on expression levels of cyclin A, B1, D1, D3, and E, as well as CDK1 in RCC cells were evaluated. Apigenin significantly reduced cyclin A, B1, D3 and E expression levels in Caki-1 cells (Fig. 4A-D), whereas there was no effect on cyclin D1 or CDK1 expression noted. Additionally, downregulation of cyclin A, B1, D3, and E expression was also observed in the primary RCC cells (Fig. 4E and F).