Human RCC Caki-1 (BFN60700344, ATCC, Shanghai) and 786-O (cat. no. BFN60700343, ATCC, Shanghai) cell lines, derived from two Caucasian males with different stages of RCC, were selected based on our previous research (11,22). The cell lines were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.), supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (Nanjing KeyGen Biotech Co., Ltd.). The cells were maintained at 37°C in an incubator (SANYO Electric Co., Ltd.) with 5% CO₂. Fisetin, with a purity of >98%, was obtained from Nanjing Best Biotechnology Co., Ltd (cat. no. D50546; 500 mg). The cisplatin-resistant cells were established by repeated subculturing with gradual increase of Cisplatin(Best Biotechnology Co., Ltd.; D50445-1 ml; 1, 2, 4, 6, 8 and 10 µM) over 6 months, and finally 10 µM cisplatin was used in the cisplatin and fisetin + cisplatin treatment group.

CDK6 cDNA was cloned into pcDNA3.0 (Invitrogen™; Thermo Fisher Scientific, Inc.). A total of 1.5×10⁵ cells/well were seeded into 24-well plates 24 h before plasmid transfection. Transfection was performed using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) in accordance with the manufacturer's instructions. Caki-1 cells and 786-O cells were transfected with 4.5 µg pcDNA3.1-CDK6 or pcDNA3.1-entry at 37°C for 48 h. An empty vector (pcDNA3.1-entry) was used as the negative control. At 48 h post-transfection, reverse transcription (RT)-quantitative (q)PCR was used to detect the efficiency.

The CCK-8 assay kit (Beyotime Institute of Biotechnology) was used to assess RCC cell proliferation. A total of 5×10³ cells/well (100 µl/well) were seeded in a 96-well plate and were routinely cultured for 24 h. Subsequently, 100 µl fisetin-containing medium was added to each well to adjust the final concentration of fisetin to 20, 40 and 60 µM. These concentrations were chosen based on previous studies (23,24). After incubation for 0, 12, 24 and 48 h at 37°C, 10 µl CCK-8 solution was added to each well and maintained for 2 h at 37°C. Finally, the optical density values were read at 450 nm.

RCC cells were placed at a concentration of 1,000 cells/well in a 6-well plate and cultured at 37°C. After 14 days, RCC cells were washed and fixed in 4% paraformaldehyde, followed by staining with leuco crystal violet (Wuhan Servicebio Technology Co., Ltd.). After washing and drying, the number of colonies >0.3 mm in diameter was counted by naked eye.

A total of 5×10⁶ cells were collected and washed with cold PBS, followed by overnight fixation with 70% ethanol at 4°C. Cells were centrifuged at 300 × g for 10 min at 4°C, washed with cold PBS and then the supernatant was discarded. RNase A (100 µl) was then added to resuspend the cells and maintained at 37°C for 30 min. PI solution (400 µl) was added and thoroughly mixed with the cells, and the mixture was placed in the dark at 4°C for 30 min. Flow cytometry (Attune NxT flow cytometer, Thermo Fisher. Scientific, Inc.; FlowJo V10.10.0, link: http://www.flowjo.com/) was then used to analyze cells in different cell cycle phases.

RIPA buffer (Nanjing Best Biotechnology Co., Ltd.) supplemented with 1% PMSF (Nanjing Best Biotechnology Co, Ltd.) was used to isolate total proteins from RCC cells. The concentration of proteins was detected using the BCA kit (Beyotime Institute of Biotechnology), and then SDS-PAGE was used to separate the proteins (50 micrograms per lane. Due to the wide range of molecular weights of proteins involved in this study, the gel concentrations were divided into the following: 6% for more than 200 kDa; 8% for 100–200 kDa; 10% for 40–60 kDa; 12% was used for 20 to 40 kDa), which were transferred to PVDF membranes (MilliporeSigma). Blocking was performed using 5% skim milk powder at room temperature for 1 h. Overnight at 4°C, the membranes were incubated with primary antibodies against cyclin B1 (4138, Cell Signaling Technology, Inc.; dilution: 1:1,000), p21 (2947, Cell Signaling Technology, Inc.; 1:1,000), p27 (3686, Cell Signaling Technology, Inc.; dilution: 1:1,000), Bax (41162; Cell Signaling Technology, Inc.; dilution: 1:1,000), Bcl2 (sc-7382, Santa Cruz Biotechnology, Inc.; dilution: 1:800), Cleaved caspase −3 (9664, Cell Signaling Technology, Inc.; 1:1,000), Cleaved caspase −9 (20750, Cell Signaling Technology, Inc.; dilution: 1:1,000), CDK6 (sc-7961, Santa Cruz Biotechnology, Inc.; dilution: 1:800), phosphorylated (p)-Akt (sc-377556, Santa Cruz Biotechnology, Inc.; 1:800), Akt (sc-5298, Santa Cruz Biotechnology, Inc.; dilution: 1:800), p-PI3K (ab278545, Abcam; dilution: 1:200), PI3K (sc-365290, Santa Cruz Biotechnology, Inc.; dilution: 1:800), p-mTOR (sc-293133, Santa Cruz Biotechnology, Inc.; 1:800), mTOR (sc-517464; Santa Cruz Biotechnology, Inc.; dilution: 1:800) and β-actin (sc-81178, Santa Cruz Biotechnology, Inc.; dilution: 1:800). After three washes with TBST (2% Tween), membranes were hybridized with HRP-conjugated secondary antibodies (ab131368, ab99697, ab190369; all Abcam; dilution: 1:1,000) for 90 min at room temperature. Finally, protein bands were detected using the ECL Assay Kit (Shanghai Yeasen Biotechnology Co., Ltd.). Semi-quantitative analysis of WB bands was performed using ImageJ (V 1.8.0, link: http://imagej.net/software/imagej/).

RCC cell apoptosis was assessed by applying the Annexin V-FITC kit (BioLegend, Inc.). Briefly, 1×10⁵ cells were suspended in 500 µl binding buffer, then 5 µl Annexin V-FITC was added and incubated for 10 min at 4°C. Subsequently, 5 µl PI solution was added and incubated for another 15 min at 25°C. Flow cytometry (Attune NxT flow cytometer, Thermo Fisher. Scientific, Inc.; FlowJo V10.10.0) was then performed to count the number of apoptotic cells.

A total of 1×10⁷ cells were lysed in 1 ml TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.) and mixed with 0.2 ml chloroform. Subsequently, 0.5 ml isopropanol was added to the supernatant after centrifugation at 12,000 × g for 8 min at 4°C. The mixture was centrifuged at 12,000 × g for 10 min at 4°C. The precipitate was collected, washed with 75% ethanol and dissolved in diethyl pyrocarbonate water. cDNA was then synthesized using the 1st Strand cDNA Synthesis Kit (Shanghai Yeasen Biotechnology Co., Ltd.) according to the manufacturer's protocol. The cDNA levels were then detected using the Applied Biosystems^® 7500 (Thermo Fisher Scientific, Inc.) with the qPCR SYBR Green Master Mix (Shanghai Yeasen Biotechnology Co., Ltd.). Pre-denaturation was performed at 95°C for 30 sec. Denaturation was performed at 95°C for 10 sec and annealing at 60°C for 30 sec for 40 cycles. GAPDH was used as an internal reference. The primers were as follows: CDK6, forward (F) 5′-CGACTGACACTCGCAGCC-3′ and reverse (R) 5′-AGTCCAGAATCATTGCACCTGAG-3′ and GAPDH, F 5′-TCATTTCCTGGTATGACAACGA-3′ and R 5′-GGTCTTACTCCTTGGAGGC-3′. Gene expression was calculated using GADPH and the 2^−ΔΔCq method (25).

All of the data in the present study were analyzed using GraphPad 8(Dotmatics) and are presented as the mean ± standard deviation. To compare groups, one-way ANOVA or the Student's unpaired t test were used. Tukey's HSD (Honestly Significant Difference) test was used for post hoc testing. P<0.05 was considered to indicate a statistically significant difference. Each individual experiment was performed in triplicate.

The effects of fisetin on the development of RCC cells were assessed using different concentrations of fisetin. First, the CCK-8 assay was used to evaluate the viability of the 786-O and Caki-1 cells. The results revealed that fisetin significantly inhibited the proliferation of 786-O and Caki-1 cells in a dose-dependent manner (Fig. 1A). Furthermore, tumor colony formation was significantly inhibited by fisetin treatment, and the strongest inhibitory effects were achieved in response to 60 µM fisetin (Fig. 1B). Moreover, with an increase in fisetin concentration, the cells exhibited a G₁ phase block, indicating that fisetin could deter the proliferation of 786-O and Caki-1 cells and the proliferation inhibition was most marked in response to 60 µM fisetin (Fig. 1C). The expression levels of cell cycle-related proteins cyclin B1, p21 and p27 were significantly increased in the fisetin treatment group compared with those in the control group (Fig. 1D), with the cell cycle arrest in the G₂/M phase. Apoptosis was also observed in the 786-O and Caki-1 cells. The findings revealed that fisetin significantly increased the proportion of apoptotic cells and promoted RCC cell apoptosis, in comparison with non-treated cells (Fig. 1E). Furthermore, the expression levels of the apoptotic activators Bax and cleaved-caspase 3/9 were significantly increased, whereas the expression levels of the apoptotic inhibitor Bcl2 were significantly decreased after fisetin treatment compared with those in the control group (Fig. 1F). These findings indicated that fisetin impeded RCC cell proliferation and enhanced apoptosis. Additionally, the effects of fisetin on RCC cell development were dose-dependent.

The role of fisetin in cisplatin drug resistance was assessed and it was demonstrated that although fisetin or cisplatin treatment alone could inhibit 786-O and Caki-1 cell proliferation, the combination of these two drugs displayed significantly stronger anti-proliferative effects, in comparison with the control (Fig. 2A). Therefore, it was hypothesized that fisetin may enhance cisplatin sensitivity in RCC cells. To evaluate this hypothesis, the ability of the cells to form tumorspheres after combination treatment was assessed. The results demonstrated that the number and size of tumorspheres were markedly decreased when cells were treated with cisplatin supplemented with fisetin (Fig. 2B). Furthermore, an augmented inhibitory effect was also demonstrated in the cell cycle of the two RCC cell lines, as shown in Fig. 2C, where the proportion of cells in the G₁ phase notably increased in the cisplatin + fisetin combination treatment group compared with cisplatin treatment group. The expression levels of cycle-related proteins cyclin B1, p21 and p27 were also significantly increased when treated with this drug combination compared with cisplatin treatment group (Fig. 2D), inducing the cell cycle arrest in the G₂/M phase. Fisetin also significantly enhanced the inhibitory impacts of cisplatin on 786-O and Caki-1 cell apoptosis; the proportion of apoptotic cells was significantly higher in the group treated with cisplatin combined with fisetin compared with that treated with cisplatin or fisetin alone (Fig. 2E), as were the expression levels of apoptosis activators, Bax and cleaved-caspase 3/9 (Fig. 2F). Conversely, the expression levels of the apoptotic inhibitor Bcl2 were significantly reduced when cisplatin was combined with fisetin compared with cisplatin (Fig. 2F). Fisetin and cisplatin in combination enhanced the chemosensitivity of RCC cells and thus demonstrated marked antitumor effects on the development of RCC cells.

To elucidate the mechanisms underlying the effects of fisetin, the targets of fisetin were assessed and it was demonstrated that the protein expression levels of CDK6 were significantly decreased after treating 786-O and Caki-1 cells with fisetin compared with those in the control group (Fig. 3A). Subsequently, the mRNA expression levels of CDK6 were assessed using RT-qPCR and the results demonstrated that the mRNA expression levels of CDK6 were also significantly decreased by fisetin treatment compared with those in the control group (Fig. 3B). Therefore, we hypothesized that fisetin enhanced the sensitivity of cisplatin to RCC cells via the inhibition of CDK6 expression. RT-qPCR results demonstrated that the expression levels of CDK6 in both cell lines were significantly increased post-transfection with pcDNA3.1-CDK6 compared with those in the pc-negative control cells (Fig. 3C).

To assess our hypothesis, CDK6 was overexpressed in 786-O and Caki-1 cells. The findings revealed that the expression levels of p-Akt, p-PI3K and p-mTOR were significantly reduced when cells were treated with cisplatin and fisetin compared with cisplatin but were significantly increased after CDK6 overexpression (Fig. 4A). These results suggested that fisetin may inhibit the PI3K/Akt signaling pathway by targeting CDK6, thereby enhancing the sensitivity of RCC cells to cisplatin. Treatment with fisetin combined with cisplatin significantly inhibited cell proliferation, whereas overexpression of CDK6 partially reversed the proliferation inhibition caused by fisetin (Fig. 4B). Moreover, fisetin and cisplatin treatment significantly reduced tumor colony formation compared with control and CDK6 overexpression significantly undermined the inhibitory effect caused by fisetin compared with cisplatin + fisetin + pc-NC group (Fig. 4C). In addition, combination treatment notably arrested the cell cycle in the G2 phase for a longer time and could not normally enter the M phase, whereas overexpression of CDK6 counteracted the effects of cisplatin and fisetin combination (Fig. 4D). Treatment with fisetin and cisplatin also significantly enhanced the expression levels of cyclin B1, p21 and p27 compared with control group, and overexpression of CDK6 significantly decreased the expression levels of these three proteins compared with cisplatin + fisetin + pc-NC group (Fig. 4E), decreasing the proportion of cells in G₂/M phase. Furthermore, the cisplatin + fisetin treatment group demonstrated a significant increase in apoptosis compared with control group, whereas overexpression of CDK6 significantly reduced the apoptosis-inducing effect caused by this drug combination compared with cisplatin + fisetin + pc-NC group (Fig. 4F). Cisplatin + fisetin treatment also significantly increased the expression levels of the proapoptotic proteins Bax and cleaved-caspase 3/9, and it significantly reduced the expression levels of the anti-apoptotic protein Bcl2 compared with control group, whereas overexpression of CDK6 partially reversed these effects compared with cisplatin + fisetin + pc-NC group (Fig. 4G).