Fisetin enhances cisplatin sensitivity in renal cell carcinoma via the CDK6/PI3K/Akt/mTOR signaling pathway

Abstract

Cisplatin resistance is ubiquitous among patients with renal cell carcinoma (RCC). The present study assessed the role of fisetin in regulating cisplatin sensitivity and increasing the efficacy of chemotherapy for patients with RCC. Cell Counting Kit-8 and colony formation assays were used to assess the proliferation of RCC cells after fisetin and cisplatin treatment. The mRNA expression levels of cyclin-dependent kinase (CDK)6 were evaluated using reverse transcription-quantitative PCR. The expression levels of CDK6 and key proteins of the PI3K/Akt/mTOR signaling pathway were assessed using western blotting. The present study demonstrated that fisetin inhibited the proliferation and colony-forming ability of RCC cells, and induced apoptosis and cell cycle arrest in a dose-dependent manner. Additionally, fisetin enhanced the antineoplastic effects of cisplatin, as demonstrated by the increase in proliferation inhibition and apoptosis promotion after fisetin and cisplatin combination treatment. Furthermore, fisetin regulated the PI3K/Akt/mTOR signaling pathway through CDK6 inhibition, which enhanced cisplatin sensitivity. Overexpression of CDK6 neutralized the positive effects of fisetin on the improvement of cisplatin sensitivity in RCC cells. In conclusion, fisetin may enhance the sensitivity of RCC cells to cisplatin via the CDK6/PI3K/Akt/mTOR signaling pathway.

Keywords: cisplatin; drug resistance; fisetin; renal cell carcinoma.

Figure 1.

Fisetin decreases renal cell carcinoma cell proliferation and promotes apoptosis. (A) Cell Counting Kit-8 assay was used to assess the effects of different doses of fisetin on the proliferation of 786-O and Caki-1 cells at 0, 12, 24 and 48 h. Fisetin exhibited the greatest inhibitory effect at 60·M. (B) Colony formation assay was used to assess the effects of different doses of fisetin on the proliferation of 786-O and Caki-1 cells. The effects of fisetin were dose-dependent and the fewest colonies were formed at 60·M. (C) Cell cycle arrest assessed using flow cytometry. The number of cells in G₁ phase notably increased with the increase of fisetin concentration, whereas the number of cells in G₂/M phase was markedly reduced in a dose-dependent manner. (D) Expression levels of cell cycle-related proteins assessed using western blotting. Fisetin significantly promoted the expression of cyclin B1, p21 and p27. (E) Caki-1 and 786-O cell apoptosis was assessed using flow cytometry. The apoptosis rate was significantly elevated with an increase in fisetin dosage. (F) Expression levels of apoptosis marker proteins assessed after fisetin treatment using western blotting. Fisetin significantly upregulated Bax and cleaved-caspase-3/9 in both 786-O and Caki-1 cells, whereas the expression levels of Bcl2 were significantly decreased following fisetin treatment. OD, optical density. ***P<0.001 vs. 0 µM.

Figure 2.

Fisetin enhances cisplatin sensitivity in 786-O and Caki-1 RCC cells. (A) Effects of fisetin and cisplatin combination treatment on RCC cell proliferation. Fisetin or cisplatin alone significantly inhibited cell proliferation; however, the combination of these two drugs demonstrated a stronger inhibitory effect. (B) Colony formation assays after fisetin and cisplatin combination treatment. Combination therapy demonstrated marked proliferation suppression compared with cisplatin or fisetin alone treatment. (C) Effects of fisetin and cisplatin alone, compared with the combination, on the cell cycle progression of 786-O and Caki-1 cells. Fisetin and cisplatin in combination notably increased the proportion of cells in G₁ phase and decreased that in G₂/M phase. (D) Change in the expression levels of cell cycle-related proteins when fisetin and cisplatin were used in combination. The expression levels of cyclin B1, p21 and p27 exhibited the greatest increase following fisetin + cisplatin treatment. (E) Effects of fisetin and cisplatin alone and combined on apoptosis. Combination treatment resulted in the greatest increase in the proportion of apoptotic 786-O and Caki-1 cells. (F) Effects of combined fisetin and cisplatin treatment on apoptosis-associated protein expression. Fisetin + cisplatin upregulated Bax, cleaved-caspase 3/9, and downregulated Bcl2 to a larger extent than fisetin or cisplatin alone. RCC, renal cell carcinoma; OD, optical density. *P<0.05, **P<0.01, ***P<0.001, vs. Control; ^#P<0.05, ^##P<0.01, ^###P<0.001, vs. cisplatin.

Figure 3.

Fisetin enhances cisplatin sensitivity via CDK6 in renal cell carcinoma cells. Expression levels of CDK6 (A) protein and (B) mRNA were significantly decreased in the fisetin treatment group in both 786-O and Caki-1 cells. (C) Reverse transcription-quantitative PCR used to assess the transfection efficiency of pc-CDK6. CDK6, cyclin-dependent kinase 6; NC, negative control; pc, pcDNA. ***P<0.001, vs. control.

Figure 4.

Fisetin enhances cisplatin sensitivity in RCC cells via the PI3K/Akt/mTOR signaling pathway to inhibit cell proliferation, delay the cell cycle and promote apoptosis. (A) Expression levels of key proteins in the PI3K/Akt/mTOR signaling pathway assessed following fisetin and cisplatin treatment. Fisetin and cisplatin significantly decreased the protein expression levels of p-Akt, p-PI3K and p-mTOR, but CDK6 overexpression increased the levels of p-Akt, p-PI3K and p-mTOR. (B) RCC cell proliferation after CDK6 overexpression. Fisetin and cisplatin reduced 786-O and Caki-1 cell proliferation, whereas the effects were undermined by CDK6 overexpression. (C) Number of colonies formed significantly increased after CDK6 overexpression. Fisetin + cisplatin treatment inhibited RCC cell-forming colonies, whereas CDK6 overexpression demonstrated the opposite effect. (D) Number of cells remaining in G₁ phase notably increased, whereas they decreased in G₂/M phase after the combination of fisetin and cisplatin treatment. CDK6 overexpression counteracted effects of fisetin and cisplatin. (E) Overexpression of CDK6 neutralized the effects of fisetin and cisplatin on the expression levels of cell cycle-associated proteins. The expression levels of cyclin B1, p21 and p27 were significantly reduced after CDK6 overexpression. (F) Proportion of apoptotic RCC cells notably reduced after CDK6 overexpression, which undermined the proapoptotic role of fisetin and cisplatin. (G) Overexpression of CDK6 neutralized the effects of fisetin and cisplatin on the expression levels of apoptosis-associated proteins. CDK6 significantly downregulated Bax, cleaved-caspase 3 and cleaved-caspase 9 in both 786-O cells and Caki-1 cells, whereas the expression levels of Bcl2 were significantly increased after CDK6 overexpression. RCC, renal cell carcinoma; pc, pcDNA; NC, negative control; OD, optical density; p, phosphorylated. *P<0.05, **P<0.01, ***P<0.001, vs. Control; ^##P<0.01, ^###P<0.001, vs. cisplatin + fisetin + pc-NC.