The coffee diterpene kahweol enhances sensitivity to sorafenib in human renal carcinoma Caki cells through down-regulation of Mcl-1 and c-FLIP expression

Sorafenib is approved for the treatment of hepatocellular carcinoma (HCC) and advanced renal cell carcinoma (RCC). However, low tumor response and side effects have been widely reported. Therefore, to improve the efficacy of sorafenib, we investigated whether combined treatment with sorafenib and kahweol, the coffee-specific diterpene, has a synergistic effect on apoptotic cell death. Combined treatment with sorafenib and kahweol markedly induced caspase-mediated apoptosis in renal carcinoma Caki cells. Combined treatment with sorafenib and kahweol induced down-regulation of Mcl-1 and c-FLIP expression. We found down-regulation of Mcl-1 and c-FLIP expression was modulated by the ubiquitin-proteasome pathway. Ectopic expression of Mcl-1 inhibited sorafenib plus kahweol-induced apoptosis. Interestingly, combined treatment with sorafenib and kahweol induced apoptotic cell death in c-FLIP overexpressed cells. In addition, combined treatment with sorafenib and kahweol markedly induced apoptosis in human lung carcinoma (A549) and breast carcinoma (MDA-MB-361) cells, but not in human normal mesangial cells and human skin fibroblast cells (HSF). Collectively, our study demonstrates that combined treatment with sorafenib and kahweol induces apoptotic cell death through down-regulation of Mcl-1 expression.


INTRODUCTION
Sorafenib, a tyrosine kinase inhibitor, possesses potential inhibitory activity against several receptor tyrosine kinases including vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor-β (PDGFR-β) [1]. It is an effective chemotherapeutic drug in human hepatocellular, renal, colon and breast cancer [2][3][4][5]. However, the efficacy of sorafenib is limited, as it improves only survival rate by three months in hepatocellular carcinoma patients [1,2]. Furthermore, the anti-cancer effect of sorafenib is inhibited by development of multiple drug resistance mechanisms [6]. Sorafenib resistance was caused by several molecular mechanisms including activation of the epidermal growth factor receptor (EGFR), induction of epithelial-mesenchymal transition (EMT), overexpression of hypoxia inducing factor 1α (HIF1-α) [7][8][9]. Combination therapy with other less toxic agents could improve the therapeutic efficacy and reduce the drug resistance of sorafenib.
Kahweol, a diterpene molecule from coffee beans, has a variety of bioactivities, including anticarcinogenesis, anti-tumor, and anti-inflammation properties [10][11][12][13]. Anti-carcinogenic properties of kahweol are correlated with the induction of phase II detoxifying and antioxidant enzymes [10]. Our group reported that anti-tumor properties of kahweol may in part be due to inhibition of Akt phosphorylation and activation of JNK signal pathway [14]. Furthermore, kahweol has sensitizing effects of anti-cancer drugs-induced apoptosis. For examples, kahweol sensitizes TRAIL-induced apoptosis through down-regulation of Bcl-2 and c-FLIP expression [15]. Combined treatment with melatonin and kahweol induces apoptosis through up-regulation of PUMA expression [16].
The aim of the present study was to investigate the synergistic effects of kahweol on sorafenib-mediated apoptosis in renal carcinoma Caki cells. The low-dose combination of sorafenib and kahweol enhanced the cytotoxicity by down-regulation of anti-apoptotic protein Mcl-1 and c-FLIP expression.

Combined treatment with sorafenib and kahweol induces apoptosis
Sorafenib has anti-cancer effects through inhibiting the RAF-MEK-ERK pathway and receptor tyrosine kinases. However, sorafenib has a many side effects at full dose. Combined sorafenib and other agents reduce dosage of sorafenib, thereby alleviates its side effects. Therefore, we examined whether combined treatment with natural compounds and sorafenib (sub-lethal dosage) induces cell death. As shown in Figure 1A and 1B, among natural compounds, kahweol markedly induced apoptosis dosedependently in sorafenib-treated cells.
The combination of sorafenib with kahweol induced typical apoptotic morphologies, including blebbing, formation of apoptotic bodies, cell shrinkage, cell detachment on the plate, and chromatin condensation (Figure 2A and 2B). Combined treatment with sorafenib and kahweol markedly induced sub-G1 population and PARP cleavage, which is a substrate of caspase-3 ( Figure  2C). Next, we quantified the synergy between the two drugs using Isobologram analysis. Isoboles suggested that sorafenib plus kahweol had synergistic effects ( Figure  2D). In addition, combined treatment with sorafenib and kahweol induced cytoplasmic histone-associated DNA fragments ( Figure 2E). To further address the caspase activation in kahweol-mediated sensitization to sorafenibinduced apoptosis, we used a pan-caspase inhibitor (z-VAD). Combined treatment markedly increased caspase-3 activation ( Figure 2F), and z-VAD blocked sorafenib plus kahweol-induced apoptosis as well as PARP cleavage ( Figure 2G). To determine the molecular mechanisms underlying combined treatment-induced apoptosis, we investigated expression levels of apoptosisrelated proteins. Both c-FLIP and Mcl-1 expression were down-regulated ( Figure 2H). In contrast, other apoptosisrelated proteins did not differ in control and combination treated Caki cells. In addition, down-regulation of c-FLIP and Mcl-1 expression was independent of caspase activation ( Figure 2I). Collectively, these results indicated that combined treatment with sorafenib and kahweol induces caspase-dependent apoptosis and down-regulation of Mcl-1 and c-FLIP expression.

Ectopic expression of Mcl-1 overcomes apoptosis in sorafenib plus kahweol-treated cells
To evaluate the significance of Mcl-1downregulation in sorafenib plus kahweol-induced apoptosis, we used Mcl-1 overexpressing cells. When Mcl-1 was overexpressed, the apoptosis population and PARP cleavage caused by sorafenib plus kahweol were markedly inhibited ( Figure 3A). Combined treatment with sorafenib and kahweol gradually decreased Mcl-1 expression over 2 h, but the Mcl-1 mRNA expression was not changed ( Figure 3B and 3C). Therefore, we investigated whether sorafenib plus kahweol modulates the protein stability of Mcl-1. Caki cells were treated with cycloheximide (CHX) in the presence or absence of sorafenib plus kahweol. As shown in Figure 3D, CHX in the presence of sorafenib plus kahweol rapidly down-regulated expression of Mcl-1 compared with CHX alone. Because Mcl-1 protein stability is well known to be regulated by the ubiquitinproteasome pathways [17], we examined whether proteasome inhibitors (MG132 and lactacystin) reverse sorafenib plus kahweol-induced Mcl-1 downregulation. Proteasome inhibitors prevented Mcl-1 down-regulation ( Figure 3E). These data suggested that the combined treatment induces down-regulation of Mcl-1 expression by the ubiquitin-proteasome pathways and down-regulation of Mcl-1 expression plays a critical role in sorafenib plus kahweol-induced cell death.

Combined treatment with sorafenib and kahweol induces c-FLIP down-regulation
As shown in Figure 2H, c-FLIP expression was down-regulated by combined treatment. Combined treatment with sorafenib and kahweol did not effect on c-FLIP mRNA expression, but c-FLIP protein levels were rapidly down-regulated after 1 h treatment ( Figure  4A). As shown in Figure 4B, CHX alone gradually reduced c-FLIP expression, but combined treatment with CHX and sorafenib plus kahweol more rapidly reduced c-FLIP protein expression. In addition, both proteasome inhibitors also prevented c-FLIP down-regulation ( Figure  4C). To evaluate the functional importance of the c-FLIP proteins in sorafenib plus kahweol-induced apoptosis, we used c-FLIP overexpressing cells. In contrast to Mcl-1, overexpression of c-FLIP, surprisingly, did not inhibit sorafenib plus kahweol-induced apoptosis and PARP cleavage ( Figure 4D). However, anti-FAS (FAS ligand)induced apoptosis and PARP cleavage were prevented by ectopic expression of c-FLIP ( Figure 4E). Interestingly, these data suggested that combined treatment with sorafenib and kahweol induces apoptotic cell death in c-FLIP overexpressed Caki cells.

Combined treatment with sorafenib and kahweol increases proteasome activity
Combined treatment with sorafenib and kahweol reduced Mcl-1 and c-FLIP at the post-transcriptional levels in a proteasome dependent manner ( Figures 3E and  4C). Next, we investigated whether combined treatments induced proteasome subunits expression, 19S proteasome non-ATPase regulatory subunit 4 (PSMD4/S5a), and 20S proteasome subunit alpha type 5 (PSMA5) and beta type 5 (PSMB5). However, combined treatment with sorafenib and kahweol did not alter the expression levels of these proteins ( Figure 5A). Next, we investigated whether combined treatment specifically modulate E3 ligase of c-FLIP and Mcl-1. However, as shown in Figure      E3 ligase (β-TrCP) and c-FLIP E3 ligase (Cbl and Itch) was not induced in combined treated cells. Furthermore, deubiquitinase of Mcl-1, USP9x, was not altered by sorafenib and kahweol treatment ( Figure 5C). In addition, we investigated whether reactive oxygen species (ROS) is involved in combined treatment-induced apoptosis. As shown in Figure 5D, although high concentrations of sorafenib induced ROS production, combined treatment with sorafenib and kahweol did not induce ROS production. Furthermore, ROS scavengers (NAC and trolox) had no effect on combined treatment-induced apoptosis ( Figure 5E). Therefore, combined treatment with sorafenib and kahweol-induced apoptosis is independent of ROS signaling.

Combined treatment with sorafenib and kahweol induces apoptosis in other cancer cells but not in normal cells
We next investigated the effect of sorafenib plus kahweol on apoptosis in other renal cancer cells (A498 and ACHN cells) and other type cancer cells, including human lung carcinoma (A549) and breast carcinoma (MDA-MB-361) cells. We found that combined treatment with sorafenib and kahweol induced apoptotic cell death and cleavage of PARP in A498, ACHN, A549, and MDA-MB-361 cells ( Figure 6A and 6B). Furthermore, sorafenib plus kahweol induced down-regulation of Mcl-1 and c-FLIP expression ( Figure 6A and 6B). By contrast, combined treatment with sorafenib and kahweol did not induce morphological changes, apoptosis, and expression of Mcl-1 and c-FLIP expression in normal human mesangial cells (MC) and normal human skin fibroblast cells (HSF) ( Figure 6C-6F).

DISCUSSION
Combination of natural compounds and conventional anti-cancer drug at their lower individual concentrations markedly enhances efficacy in induction of apoptotic cell death. Recent studies reported that sorafenib combination treatment with resveratrol, indol-3-carbinol, bufalin, fisetin, and triptolide effectively induced apoptosis in various carcinoma cells by different mechanisms [18][19][20][21][22]. In this study, the combined treatment with low dose sorafenib and kahweol induced apoptotic cell death in cancer cells, but not in normal cells. Combined treatment with sorafenib and kahweol potentiated the cytotoxicity in renal carcinoma cells through down-regulation of Mcl-1 and c-FLIP protein expression in a proteasome-dependent manner. Our study is the first to show the combined treatment with sorafenib and kahweol may be effective in overcoming resistance to anti-cancer drugs in renal carcinoma cells.
Numerous investigations have been reported that generation of intracellular ROS plays a critical a role in apoptosis [23]. Interestingly, a high concentration of kahweol (75 μM) induced ROS production in breast carcinoma MDA-MB-231 cells [24]. However, a low concentration of kahweol (25 μM) did not increase ROS production in our experiment. In addition, as shown in Figure 5E, pretreatment with NAC or trolox did not prevent combined treatment-induced sub-G1 population and cleavage of PARP. Therefore, ROS is not critical for the induction of apoptosis by combined treatment with sorafenib and kahweol.
Mcl-1 and c-FLIP protein expression are regulated at the transcriptional and post-translational levels [17,[25][26][27]. Combined treatment with sorafenib and kahweol did not alter Mcl-1 and a c-FLIP mRNA level, suggesting that down-regulation of Mcl-1 and c-FLIP protein is likely to be a post-transcriptional event. Interestingly, inhibition of proteasome with MG132 or lactacystin rescued Mcl-1 and c-FLIP down-regulation ( Figures 3E and 4C). Combined treatment with sorafenib and kahweol induced downregulation of c-FLIP and Mcl-1 expression by proteasomal degradation (Figures 3E and 4C), but combined treatment with sorafenib and kahweol did not increase proteasome activity (Data not shown; negative data). Furthermore, combined treatment did not change expression levels of subunits of proteasome catalytic core proteins ( Figure 5A).
Collectively, these results suggest that sorafenib plus kahweol induced apoptosis through down-regulating Mcl-1 expression at post-translational levels. Therefore, it uncovered a critical role of kahweol in enhancing the anti-cancer effect of sorafenib, suggesting that combined treatment with sorafenib and kahweol offers a rationale for a new therapeutic combination for the treatment of renal carcinoma.

Cell culture and materials
Human renal carcinoma (Caki, ACHN and A498), human lung carcinoma (A549), and human breast carcinoma (MDA-MB-361) cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Primary cultured human mesangial cells (MC) (Cryo NHMC) were purchased from Clonetics (San Diego, CA). The normal human skin fibroblasts (HSFs) cells were purchased form Korea Cell Line Bank (Seoul, Korea). The culture medium used throughout these experiments was Dulbecco's modified Eagle's medium (DMEM) or RPMI containing 10% fetal bovine serum (FBS), 20 mM HEPES buffer and 100 μg/mL gentamycin. The PCR primers were purchased from Macrogen (Seoul, Korea). The z-VADfmk was purchased from R&D system (MN, USA), and N-acetyl-L-cysteine (NAC) and Trolox was obtained from Calbiochem (San Diego, CA, USA). Sorafenib was purchased from selleck chemicals (Houston, TX, USA).

Flow cytometry analysis
For flow cytometry, the cells were resuspended in 100 μl of phosphate-buffered saline (PBS), and 200 μl of 95% ethanol was added while the cells were being vortexed. Then, the cells were incubated at 4°C for 1 h, washed with PBS, resuspended in 250 μl of 1.12% sodium citrate buffer (pH 8.4) with 12.5 μg of RNase and incubated for an additional 30 min at 37°C. The cellular DNA was then stained by adding 250 μl of a propidium iodide solution (50 μg/ml) to the cells for 30 min at room temperature. The stained cells were analyzed by fluorescent-activated cell sorting on a FACScan flow cytometer to determine the relative DNA content, which was based on the red fluorescence intensity.

4′,6′-Diamidino-2-phenylindole staining (DAPI) for nuclei condensation and fragmentation
To examine the cellular nuclei, the cells were fixed with 1% paraformaldehyde on glass slides for 30 min at room temperature. After fixation, the cells were washed with PBS and a 300 nM 4′,6′-diamidino-2-phenylindole solution (Roche, Mannheim, Germany) was added to the fixed cells for 5 min. After the nuclei were stained, the cells were examined by fluorescence microscopy.

Western blot analysis
For the Western blot experiments, the cells were washed with cold PBS and lysed on ice in modified RIPA buffer (50 mM Tris-HCl pH 7.4, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM Na 3 VO 4 , and 1 mM NaF) containing protease inhibitors (100 μM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml pepstatin, and 2 mM EDTA). The lysates were centrifuged at 10,000 x g for 10 min at 4°C, and the supernatant fractions were collected. The proteins were separated by SDS-PAGE electrophoresis and transferred to Immobilon-P membranes. The specific proteins were detected using an enhanced chemiluminescence (ECL) Western blotting kit according to the manufacturer's instructions.

Determination of synergy
The possible synergistic effect of sorafenib and kahweol was evaluated using the isobologram method. In brief, cells were treated with different concentrations of sorafenib and kahweol alone or in combination. After 24 h, relative survival was assessed, and the concentration effect curves were used to determine the IC50 (the halfmaximal inhibitory concentration) values for each drug alone and in combination with a fixed concentration of the second agent. The XTT assay was employed to measure cell viability using a WelCount Cell Viability Assay Kit (WelGENE, Daegu, Korea). In brief, the reagent was added to each well and was then measured with a multiwell plate reader (at 450 nm/690 nm).

The DNA fragmentation assay
A cell death detection ELISA plus kit (Boehringer Mannheim; Indianapolis, IN) was used to determine the level of apoptosis by detecting fragmented DNA within the nuclei of kahweol-treated cells, sorafenib-treated cells, or cells that were treated with a combination of sorafenib and kahweol. Briefly, each culture plate was centrifuged for 10 min at 200 × g, the supernatant was removed, and the cell pellet was lysed for 30 min. Then, the plate was centrifuged again at 200 × g for 10 min and the supernatant, which contained the cytoplasmic histoneassociated DNA fragments, was collected and incubated with an immobilized anti-histone antibody. The reaction products were incubated with a peroxidase substrate for 5 min and were measured by spectrophotometry at 405 and 490 nm (reference wavelength) with a microplate reader. The signals in the wells containing the substrate alone were subtracted as the background.