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

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.

. 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 × 10 4 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. www.nature.com/scientificreports/ proliferation assay of proximal tubular cells from normal adult human kidney (HK-2) was performed. Since the combination of 30 µM kahweol acetate and 30 µM cafestol did not affect viability of HK-2 cells at all, 30 µM of kahweol acetate and cafestol was thought as non-cytotoxic concentrations of kahweol acetate and cafestol (Supplementary Figure S1). These results indicate that medium concentrations (10-30 µM) of kahweol acetate and cafestol exert an anti-proliferative effect on renal cancer cells without impairment of normal kidney cells.

Synergistic inhibition of human renal cancer cell proliferation and migration following combined kahweol acetate and cafestol treatment.
Given that kahweol acetate and cafestol are both derived from coffee, these diterpenes can be ingested concurrently when drinking coffee 11,12 . In fact, we had also reported that the combination of these diterpenes synergistically affected prostate cancer cell viability 10 . The anti-cancer effects of combined kahweol acetate and cafestol treatment on human renal cancer cell proliferation and migration was evaluated by calculating the combination index (CI) 13 . Accordingly, the combination of 30 µM kahweol acetate and 30 µM cafestol promoted greater inhibition of ACHN and Caki-1 proliferation after 24 and 48 h compared to 30 µM kahweol acetate or 30 µM cafestol alone (CI < 1; synergism) ( Fig. 1e,f). Even low-dose combination treatment (i.e., 10 µM kahweol acetate and 10 µM cafestol for 24 and 48 h) promoted inhibition of cell proliferation comparable to 30 µM of each diterpene treatment alone (CI < 1). Given studies showing that diterpenes had multifunctional anti-cancer effects 11,12,14 , cell migration assays were also conducted to verify the effects of kahweol acetate and cafestol on ACHN and Caki-1 cells. Similar to results of proliferation assays, the combination of 30 µM kahweol acetate and 30 µM cafestol promoted significantly greater inhibition of cell migration compared to 30 µM kahweol acetate or 30 µM cafestol alone (CI < 1). In addition, we performed wound healing assay to further confirm above results from transwell migration assay. The combination of 30 µM kahweol acetate and 30 µM cafestol promoted significantly greater inhibition of cell migration compared to 30 µM kahweol acetate or 30 µM cafestol alone (Supplementary Figure S2). Importantly, the combination of 30 µM of diterpenes showed comparable effects to 100 µM of each diterpene alone (Fig. 1g,h).

Apoptosis and downregulation of anti-apoptotic proteins in human renal cancer cells following kahweol acetate and cafestol treatment.
TdT-mediated dUTP-biotin nick end labeling (TUNEL) assays of ACHN and Caki-1 cells were performed to assess whether kahweol acetate and cafestol could induce apoptosis. Consistent with the results of proliferation assays, kahweol acetate and cafestol strongly induced apoptosis, even with 30 µM of each diterpene alone (Fig. 2a,b). Next, western blot analyses were performed to determine changes in key proteins involved in cancer cell proliferation, including apoptosis-related proteins. Accordingly, the combination of 30 µM kahweol acetate and 30 µM cafestol hampered STAT3 activation (Fig. 2c,d). Moreover, the combination of 30 µM kahweol acetate and 30 µM cafestol promoted significantly lower expression of Bcl-2 and Bcl-xL, downstream of STAT3, compared to untreated cells (Fig. 2e,f). In addition, Bcl-2-associated X protein (Bax) increased by kahweol acetate and cafestol (Supplementary Figure S3). We further checked caspase-related apoptosis proteins to examine whether apoptosis induced by kahweol acetate and cafestol was caspase-dependent or not, there were no fragmentation of caspase-3 and PARP observed regardless of the concentration of kahweol acetate and cafestol (Supplementary Figure S4). The combination of 30 µM diterpenes inhibited the aforementioned apoptosis-related proteins to an extent comparable to high concentrations (100 µM) of each diterpene alone.
Downregulation of epithelial-mesenchymal transition-related proteins following kahweol acetate and cafestol treatment. Western blot analyses of epithelial-mesenchymal transition (EMT)related proteins were performed to trace the mechanism whereby cell migration was inhibited. Accordingly, the combination of 30 µM kahweol acetate and 30 µM cafestol, as well as high concentrations (100 µM) of each diterpene alone, clearly reduced Snail expression in ACHN and Caki-1 cells (Fig. 3a,b). The combination of 30 µM kahweol acetate and 30 µM cafestol, as well as high concentrations (100 µM) of each diterpene alone, also clearly reduced expression of Twist, which had been reported to interact with Snail 15 , in ACHN and Caki-1 cells (Fig. 3c,d). Among other EMT-related proteins we examined, although E-cadherin and Slug did not express at all regardless of the concentration of kahweol acetate and cafestol, N-cadherin and β-catenin showed the tendency to decrease by kahweol acetate and cafestol in ACHN and Caki-1 cells (Supplementary Figure S5). The aforementioned results indicated that kahweol acetate and cafestol decreased migration ability of human renal cancer cells through the inhibition of EMT.
Downregulation of Akt and ERK signaling following kahweol acetate and cafestol treatment. Studies have reported Akt and ERK as factors strongly associated with the acceleration of renal cancer cell aggressiveness in terms of both growth and metastasis 16,17 . Akt and ERK, which promote EMT, are activated by STAT3 through downstream signaling 18,19 . Accordingly, the combination of 30 µM kahweol acetate and 30 µM cafestol rapidly inhibited Akt (Fig. 4a, b) and ERK phosphorylation (Fig. 4c,d) in ACHN and Caki-1 cells.

Inhibition of immune signaling proteins after kahweol acetate and cafestol treatment.
Cancer progression can be regulated by not only internal cancer cell signals but also external factors. Some immune cells that infiltrate into cancer tissues promote cancer progression by secreting chemokines that often activate cancer cells 20,21 . We thus investigated the influence of kahweol acetate and cafestol on C-C chemokine receptors (CCR2, 5, and 6) of representative chemokines activating renal cancer cells [22][23][24][25][26] . Accordingly, the combination of 30 µM kahweol acetate and 30 µM cafestol or 100 µM kahweol acetate administration reduced the expression of CCR2, CCR5, and CCR6 in both ACHN and Caki-1 cells (Fig. 5a,b). Moreover, with the emergence of immune checkpoint inhibitors as the prominent treatment method for renal cancer nowadays, the role of programmed www.nature.com/scientificreports/  www.nature.com/scientificreports/ death-ligand 1 (PD-L1) has also become a significant concern 7 . Accordingly, the combination of 30 µM kahweol acetate and 30 µM cafestol or 100 µM kahweol acetate administration reduced PD-L1 expression in both ACHN and Caki-1 cells (Fig. 5c,d). The aforementioned results indicated that kahweol acetate and cafestol may also affect the tumor microenvironment by inhibiting the immunological tolerance of renal cancer cells.

Discussion
Recently, several studies have shown that kahweol or cafestol can regulate tumor cell activity and apoptosis-related proteins through multiple targets, individually contributing to the inhibition of renal cancer cell proliferation [27][28][29][30] . The present study examined the anti-cancer properties of coffee kahweol acetate and cafestol and their synergistic effects on two renal cancer cells, following our previous study focusing on prostate cancer cells 10 . Accordingly, our results showed that kahweol acetate and cafestol inhibited the proliferation and migration of both ACHN and Caki-1 cells, with their synergistic effects apparent at relatively low concentrations. Moreover, the combination of kahweol acetate and cafestol induced downregulation in not only anti-apoptotic proteins (Bcl-2 and Bcl-xL) www.nature.com/scientificreports/ but also EMT-related proteins. Inhibition of STAT3, Akt, and ERK signaling pathway, which play pivotal roles in tumor cell proliferation, invasion, and migration, had also been observed. Among apoptosis-related proteins, although the fragmentation of caspase-3 and PARP was not observed, Bax was increased by kahweol acetate and cafestol. Recently, Bax is reported to induce caspase-independent apoptosis 31 . The diterpenes may induce apoptosis through the caspase-independent apoptotic pathway: however, the role and the mechanism of this pathway in renal cell cancer should be further investigated. The aforementioned results therefore suggest that kahweol acetate and cafestol, which have been identified as potential anti-cancer agents, can directly suppress renal cancer cell activities. Immunotherapy, such as IFN-α and IL-2, had been the mainstream renal cancer therapy until the 1990s given the immunogenic property of renal cancer 32 . Recently, however, immune checkpoint inhibitors have become the prominent treatment method for renal cancer, with immune tolerance again becoming the main concern in the treatment of renal cancer 7 . Our previous studies had revealed that chemokines and their receptors were strong mediators of prostate and renal cancer progression with their effects on the tumor microenvironment [33][34][35][36][37] . Macrophage-like cells prepared from human monocytic leukemia cell line THP-1 promoted renal cancer cell migration, while CCL20, a specific ligand of CCR6, was more involved in macrophage-induced renal cancer cell migration than other cytokines 37 . Furthermore, AKT activation was involved in the promotion of renal cancer cell migration through the CCL20-CCR6 axis 37 . Some studies have already reported ERK activation is also induced by CCL20-CCR6 axis in cancer cells 38,39 . Therefore, it is interesting to note that kahweol acetate and cafestol treatment reduced CCR2, CCR5, and CCR6. The CCL2-CCR2 axis was reported to promote both cancer cell proliferation and migration by paracrine and by autocrine 40 . The CCL5-CCR5 axis also can arrange an immune-suppressive microenvironment, especially, BRCA1-associated protein 1-mutant clear cell renal cancer 41 . Moreover, studies have shown that PD-L1 expression was a negative prognostic factor and was associated with more advanced clinical features in patients with renal cancer 42,43 . Accordingly, our results showed that kahweol acetate and cafestol treatment also significantly inhibited PD-L1. T lymphocytes express PD-1 which is the receptor of PD-L1 and regulates the activity of T lymphocytes 44 . Therefore, inhibition of PD-L1 by kahweol acetate and cafestol treatment may increase the activity of T lymphocytes attacking renal cancer cells. An exploratory clinical study showed coffee consumption did not affect the number of T lymphocytes 45 . In addition, kahweol acetate and cafestol did not damage DNA of human peripheral lymphocytes 46 . Hence, although we did not clarify the direct effect of kahweol acetate and cafestol on T lymphocytes in this study, the diterpenes may not have significant effects on T lymphocytes. The aforementioned results thus indicate that kahweol acetate and cafestol can indirectly suppress renal cancer cell activities through by controlling the immunological tolerance of renal cancer cells. As shown in Fig. 6, kahweol acetate and cafestol exhibit multifunctional direct and indirect anti-tumor effects in renal cancer cells. Moreover, previous studies have suggested kahweol and cafestol could exhibit anti-angiogenic properties mainly by downregulating VEGF receptor-2, a primary mediator of 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 epithelialmesenchymal 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). www.nature.com/scientificreports/ the pro-angiogenic effect of VEGF 14 , with VEGF receptor inhibitor plus kahweol showing a synergistic effect in inducing renal cancer cell apoptosis 30 . Hence, the combination of kahweol acetate and cafestol may enhance the anti-cancer effects of conventional therapeutic agents, including tyrosine kinase inhibitors. The current study revealed that kahweol acetate and cafestol may serve as not only therapeutic agents but also promising natural ingredients exhibiting anti-tumor effects against renal cancer cells. Although kahweol and cafestol are diterpenes included in unfiltered coffees, such as French press, espresso, and boiled coffees, concentrations of both diterpenes vary depending on the quality/blend and process of coffee preparation 47 . Studies have shown that 17.2 and 19.7 mg of kahweol and cafestol levels were present per cup (150 mL) of Arabica or Robusta coffee brewed using French press, respectively 12 . Assuming that an adult has a blood volume of 5000 mL and considering that approximately 70% of the consumed kahweol and cafestol can be absorbed in small intestine 11 , kahweol and cafestol concentrations may reach 30 µM each with three or four cups of coffee. Hence, achieving a concentration of 30 µM for both kahweol and cafestol, at which both exerted anti-cancer effects against renal cancer cells in our experiment, seems to be practicable.
In conclusion, the current study suggested that kahweol acetate and cafestol synergistically contributed to the inhibition of not only renal cancer proliferation and migration, by inducing apoptosis and inhibiting EMT, but also tumor microenvironment-related pathways, such as CCRs and PD-L1. Therefore, the combined use of kahweol acetate and cafestol may be a potentially effective therapeutic modality against renal cancer. Cell proliferation assay. ACHN and Caki-1 cells were seeded in 12-well plates (5 × 10 4 cells/well) with RPMI containing 10% FBS. Each was cell treated with or without predetermined concentrations of kahweol acetate and cafestol for 24 and 48 h. After the cells were harvested, cell numbers were counted using a hemocytometer.

Reagents and antibodies.
Cell migration assay. ACHN and Caki-1 cells (1 × 10 4 cells/well) were seeded onto the upper chamber of transwell plates (cell culture inserts with 8.0-μm pore sizes for 24-well plates) with RPMI containing 0.1% FBS, while the lower compartment was filled with predetermined concentrations of kahweol acetate and cafestol in RPMI containing 10% FBS. Cells were then incubated for 12 h at 37 °C in a humidified incubator containing 5% CO 2 . Thereafter, the cells on the filter of cell culture inserts were fixed with 4% paraformaldehyde in phosphatebuffered saline (PBS) for 10 min. The cells on top of the filter were carefully removed with a cotton swab, while those on the back of the filter were stained with 0.1% crystal violet for 20 min. The stained filter was then microscopically photographed, after which migrated cells in two random fields were counted.
Western blot analyses. Cell lysates were prepared using RIPA lysis buffer (FUJIFILM Wako Pure Chemical Corporation, Japan) containing 1% protease inhibitor cocktail and phosphatase inhibitor cocktail (Sigma-Aldrich). Soluble lysates (10-30 µg) were mixed with a lithium dodecyl sulfate sample buffer and sample reducing agent, both obtained from Thermo Fisher Scientific (Waltham, MA, USA), and separated through sodium dodecyl sulfate polyacrylamide gel electrophoresis. Separated proteins were then transferred to nitrocellulose membranes. The membranes were blocked with 1% gelatin and 0.05% Tween in Tris-buffered saline for 1 h at room temperature and then incubated overnight at 4 °C with primary antibody according to the manufacturer's instructions. After washing, the membranes were incubated with HRP-conjugated anti-rabbit or anti-mouse secondary antibody for 1 h at room temperature. Protein bands were detected using the Super Signal West Femto maximum sensitivity substrate (Thermo Fisher Scientific). Full length images of cropped blots presented in Figs www.nature.com/scientificreports/