Anti-proliferative and anti-migratory properties of coffee diterpenes kahweol acetate and cafestol in human renal cancer cells

Abstract

Despite improvements in systemic therapy options for renal cancer, it remains one of the most drug-resistant malignancies. Interestingly, reports have shown that kahweol and cafestol, natural diterpenes extracted from coffee beans, exhibit anti-cancer activity. However, the multiple potential pharmacological actions of both have yet to be fully understood. This study therefore investigated the effects of kahweol acetate and cafestol on human renal cancer ACHN and Caki-1 cells. Accordingly, the combination of kahweol acetate and cafestol administration synergistically inhibited cell proliferation and migration by inducing apoptosis and inhibiting epithelial-mesenchymal transition. Mechanistic dissection revealed that kahweol acetate and cafestol inhibited Akt and ERK phosphorylation. Moreover, kahweol acetate and cafestol downregulated the expression of not only C-C chemokine receptors 2, 5, and 6 but also programmed death-ligand 1, indicating their effects on the tumor microenvironment. Thus, kahweol acetate and cafestol may be novel therapeutic candidates for renal cancer considering that they exert multiple pharmacological effects.

Figure 1

Anti-proliferative and migration effects of kahweol acetate and cafestol on renal cancer cells. (a–d) ACHN (a,c) and Caki-1 (b,d) cells were seeded in 12-well plates (5 × 10⁴ cells/well) with RPMI containing 10% FBS. Each cell was treated with or without pre-determined concentrations of kahweol acetate and cafestol for 24 and 48 h. (e,f) Anti-proliferative effects of the combination of kahweol acetate and cafestol on ACHN (e) and Caki-1 (f) cells for 24 and 48 h. (g,h) Anti-migration effect of the combination of kahweol acetate and cafestol on ACHN (g) and Caki-1 (h) cells for 12 h. Bar = 500 µm. All experiments were performed in triplicate. Data are presented as means ± standard error of the mean. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. ^#Synergistic effects were observed.

Figure 2

Kahweol acetate and cafestol treatment induced apoptosis by downregulating anti-apoptotic proteins in renal cancer cells. (a,b) TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay (a) and statistics (b) showing apoptosis of ACHN or Caki-1 cells under kahweol acetate and cafestol treatment. ACHN and Caki-1 cells (3 × 10⁴ cells) were exposed to predetermined concentrations of kahweol acetate and cafestol for 12 h on sterile slide coverslips, after which TUNEL assays were performed. All experiments were performed in triplicate. Data are presented as means ± standard error of the mean (SEM). Bar = 50 µm. (c–f) Protein levels of apoptosis-associated genes, p/tSTAT3 in ACHN (c) and Caki-1 (d) cells, Bcl-2/Bcl-xL in ACHN (e) and Caki-1 (f) cells, treated for 24 h were measured using Western blot analysis and quantitatively analyzed using densitometry with ImageJ software as shown by bar graphs. Equal amounts of cell lysates (10 µg) were subjected to electrophoresis. Data are presented means ± SEM (n = 4). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 3

Kahweol acetate and cafestol treatment promoted downregulation of epithelial–mesenchymal transition (EMT)-related proteins in renal cancer cells. (a–d), Protein levels of EMT-associated genes, Snail in ACHN (a) and Caki-1 (b) cells, Twist in ACHN (c) and Caki-1 (d) cells, treated for 24 h were measured using Western blot analysis and quantitatively analyzed using densitometry with ImageJ software as shown by bar graphs. Soluble cell lysates (30 µg in Snail, 10 µg in Twist) were subjected to electrophoresis. Data were presented as means ± standard error of the mean (n = 4). *p < 0.05; **p < 0.01.

Figure 4

The combination of 30 µM kahweol acetate and 30 µM cafestol inhibited Akt and ERK signaling in renal cancer cells. (a–d) Phosphorylation of Akt in ACHN (a) and Caki-1 (b) cells, phosphorylation of ERK in ACHN (c) and Caki-1 (d) cells, at the indicated times after combination treatment were assessed using Western blot analysis and quantitatively analyzed using densitometry with ImageJ software as shown by bar graphs. Equal amounts of cell lysates (10 µg) were subjected to electrophoresis. Data are presented as means ± standard error of the mean (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 5

Kahweol acetate and cafestol treatment promoted the downregulation of immune signaling molecules in renal cancer cells. (a–d) Protein levels of C–C chemokine receptors (CCRs; CCR2/5/6) in ACHN (a) and Caki-1 (b) cells, and programmed death-ligand 1 (PD-L1) in ACHN (c) and Caki-1 (d) cells treated for 24 h were measured using Western blot analysis and quantitatively analyzed using densitometry with ImageJ software as shown by bar graphs. The soluble cell lysates (10 µg in CCR2/5 and PD-L1, 20 µg in CCR6) were subjected to electrophoresis. Data were presented as means ± standard error of the mean (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 6

Schematic illustration of the anticancer mechanisms of kahweol acetate and cafestol. Kahweol acetate and cafestol inhibited cell proliferation and migration by inducing apoptosis and inhibiting epithelial–mesenchymal transition (orange and yellow colored molecules, respectively). Kahweol acetate and cafestol also inhibited CCR2/5/6, may potentially target the chemokine axis, and may affect the tumor immune environment by downregulating PD-L1 (blue colored molecules). Moreover, kahweol acetate and cafestol also inhibited the phosphorylation of Akt and ERK, which play central roles in tumor progression (gray colored molecules).