Cafestol overcomes ABT-737 resistance in Mcl-1-overexpressed renal carcinoma Caki cells through downregulation of Mcl-1 expression and upregulation of Bim expression

Although ABT-737, a small-molecule Bcl-2/Bcl-xL inhibitor, has recently emerged as a novel cancer therapeutic agent, ABT-737-induced apoptosis is often blocked in several types of cancer cells with elevated expression of Mcl-1. Cafestol, one of the major compounds in coffee beans, has been reported to have anti-carcinogenic activity and tumor cell growth-inhibitory activity, and we examined whether cafestol could overcome resistance against ABT-737 in Mcl-1-overexpressed human renal carcinoma Caki cells. ABT-737 alone had no effect on apoptosis, but cafestol markedly enhanced ABT-737-mediated apoptosis in Mcl-1-overexpressed Caki cells, human glioma U251MG cells, and human breast carcinoma MDA-MB231 cells. By contrast, co-treatment with ABT-737 and cafestol did not induce apoptosis in normal human skin fibroblast. Furthermore, combined treatment with cafestol and ABT-737 markedly reduced tumor growth compared with either drug alone in xenograft models. We found that cafestol inhibited Mcl-1 protein expression, which is important for ABT-737 resistance, through promotion of protein degradation. Moreover, cafestol increased Bim expression, and siRNA-mediated suppression of Bim expression reduced the apoptosis induced by cafestol plus ABT-737. Taken together, cafestol may be effectively used to enhance ABT-737 sensitivity in cancer therapy via downregulation of Mcl-1 expression and upregulation of Bim expression.

then induce apoptosis. 21 Furthermore, we recently showed that cafestol increased apoptosis in several types of cancer cells through through downregulation of anti-apoptotic proteins (Bcl-2 and Mcl-1) and inhibition of Akt phosphorylation. 22 As downregulation of Mcl-1 expression has been known to increase the sensitivity to ABT-737 in multiple cancer cells, 10,12,23 treatment with cafestol may be a promising agent, which can overcome resistance of cancer cells to  In this study, we aimed to investigate whether induction of cafestol plus ABT-737 mediated apoptosis and to identify molecular mechanisms of cafestol to overcome resistance against ABT-737 in Mcl-1-overexpressed Caki cells.

Results
Effect of ABT-737-mediated apoptosis in Mcl-1-overexpressed Caki cells. ABT-737 directly binds to antiapoptotic Bcl-2 protein (Bcl-2, Bcl-xL, and Bcl-w) and induces apoptosis, 6,7 but cancer cells with high expression levels of Mcl-1 have been reported to be resistant to ABT-737. 8,9 In an attempt to develop the therapeutic strategy to overcome ABT-737 resistance, we first tested whether ABT-737 differently regulate apoptosis in Caki/vector, Bcl-2- Cafestol synergizes ABT-737-induced apoptosis. We next examined whether the resistance of cancer cells overexpressing Mcl-1 to ABT-737 could be overcome by co-treatment with well-known chemopreventive agents, including resveratrol, curcumin, or cafestol. As shown in Figures 1a and b, when Caki/Mcl-1 cells were treated with ABT-737 or chemopreventive agent alone, cell death rarely occurred. However, combined treatment with cafestol and ABT-737 resulted in induction of sub-G1 population and PARP cleavage in a dosedependent manner (Figures 1a-c). Next, we examined whether combined treatment with ABT-737 and cafestol have synergistic effects. The isobologram analysis suggested that combined treatment with ABT-737 and cafestol have synergistic effects (Figure 1d). In addition, co-treatment with ABT-737 and cafestol increased chromatin damage in the nuclei and cytoplasmic histone-associated DNA fragmentation, indicating the key characteristic of apoptosis (Figures 1e and f). Next, we investigated whether the combined treatment with ABT-737 and cafestol gave rise to the activation of caspase-3. Exposure of Caki/Mcl-1 cells to cafestol plus ABT-737 increased caspase-3 activity (Figure 1g). To confirm whether the cell death induced by ABT-737 plus cafestol is dependent on caspase-3, we examined the effect of pretreatment with z-VAD-fmk, a pan-caspase inhibitor. As shown in Figure 1h, z-VAD completely inhibited the apoptosis and also blocked the PARP cleavage. Furthermore, we examined the effect of ABT-737 plus cafestol on long-term survival using clonogenic assay. Treatment with ABT-737 alone and cafestol alone did not affect colony formation. In contrast, combined treatment with ABT-737 plus cafestol markedly inhibited colony formation ( Figure 1i). Therefore, these data indicated that cafestol increases the sensitivity to ABT-737-mediated apoptosis in Caki/Mcl-1 cells.
Effect of combined treatment with cafestol and ABT-737 on other cancer cells and normal cells. To further investigate whether combined treatment with cafestol and ABT-737 induces apoptosis in other cancer cell types, human breast carcinoma cells (MDA-MB231), and human glioma cells (U251MG), cells were transiently transfected with pFLAG-CMV4/Mcl-1. When MDA-MB231/Mcl-1 and U251MG/Mcl-1 cells were treated with cafestol and ABT-737, sub-G1 population and the cleaved form of PARP were markedly increased, compared with the treatment with cafestol or ABT-737 alone (Figures 2a and b). Furthermore, to investigate the anti-cancer effect of ABT-737 and cafestol on other carcinoma cells, we used other cancer cells (human colon carcinoma cells (HCT116), human leukemia cells (U937), and human prostate carcinoma (PC3) cells, and human ovarian carcinoma cells (A2780). As shown in Figure 2c, combined treatment with ABT-737 and cafestol induced apoptosis in HCT116, U937, PC3, and A2780 cells. On the other hand, combined treatment with cafestol and ABT-737 did not affect the morphology or viability of human skin fibroblasts (HSF) cells (Figures 2d and e). These data suggest that cafestol increases sensitivity to ABT-737-mediated apoptosis also in other cancer cells but not in normal cells.
Combined treatment with cafestol and ABT-737 inhibits tumor growth in vivo. Next, we investigated the anti-cancer effect of combined treatment with cafestol and ABT-737 in vivo xenograft model. Caki/Mcl-1 cells injected subcutaneously (s.c.) into the right flank were established. Mice bearing tumor were treated with cafestol alone, ABT-737 alone, or as a combined treatment with cafestol and ABT-737. As shown in Figures 3a and b, combined treatment markedly suppressed tumor growth compared with vehicle, ABT-737 alone, and cafestol alone. Terminal deoxynucleotide transferase (TdT)mediated dUTP nick-end labeling (TUNEL) analysis showed that combined treatment with cafestol and ABT-737 increased cell death (Figure 3c). Moreover, immunohistochemical staining of tumor tissues showed that combined treatment increased activated caspase-3 ( Figure 3d). These results suggest that combined treatment with cafestol and ABT-737 inhibits tumor growth and induces apoptosis in vivo.    Figure 4f, CHX and cafestol led to the rapid degradation of Mcl-1 proteins, and degradation of the Mcl-1 KR protein is slower than Mcl-1. Therefore these data indicated that ubiquitination is involved in degradation of Mcl-1, but ubiquitin-independent pathway might be also associated with degradation of Mcl-1 proteins. We further examined whether cafestol could modulate the expression of two important proteasome subunits, 20S proteasome subunit alpha type 5 (PSMA5) and 19S proteasome non-ATPase regulatory subunit 4 (PSMD4/S5a). 25 However, cafestol had no effect on the expression of both proteins (Figure 4g). Finally, Upregulation of Bim expression is involved in cafestolinduced ABT-737-mediated apoptosis. Next, we investigated whether cafestol regulated other apoptosis-related proteins. Although cafestol had no effect on other proteins, it dose-dependently increased the protein levels of PUMA and Bim (Figure 5a). Protein levels of PUMA were increased from 12 h of 30 μM cafestrol treatment and those of Bim were enhanced from 6 h (Figure 5b). Furthermore, cafestol also dose-dependently increased the protein levels of PUMA and Bim in MDA-MB-231 and U251MG cells (Figure 5c). Therefore, we investigated whether these proteins were related to ABT-737 plus cafestol-induced apoptosis. Downregulation of PUMA expression by siRNA did not block apoptosis ( Figure 5d). However, ABT-737 plus cafestol-induced apoptosis was markedly inhibited by downregulation of Bim expression by siRNA (Figure 5e). These data suggest that upregulation of Bim expression critically contributed to the sensitization of ABT-737-mediated apoptosis.

Discussion
In this study, we show that cafestol acts as a potent enhancer of ABT-737-induced apoptosis in cancer cells, which have Mcl-1-mediated resistance to ABT-737. In addition, combined treatment with cafestol and ABT-737 markedly reduced tumor formation and induced apoptosis in a xenograft model. The main mechanism of cafestol-mediated ABT-737-induced apoptosis may be downregulation of Mcl-1 expression and upregulation of Bim expression.
ABT-737 binds to Bcl-xL, Bcl-2, and Bcl-w with high affinity (K i ≤ 1 nM), while it binds to Mcl-1 with K i = 14μM. 7 Therefore ABT-737 could induce apoptosis in several cancer cells, which highly expressed anti-apoptotic Bcl-2 family. For example, ABT-737 markedly increased apoptosis in multiple myeloma, 26 acute myeloid leukemia, 8 chronic lymphocytic leukemia, 27 and small cell lung cancer. 28 However, resistance to ABT-737 in several types of cancer cell has been attributed to the high level of Mcl-1 expression. As ABT-737 does not bind Mcl-1 with low affinity, activity of Mcl-1 is not affected by ABT-737. Therefore many researchers have found drugs to overcome resistance to ABT-737-mediated apoptosis via downregulation of Mcl-1 expression. The synthetic cytotoxic retinoid N-(4-hydroxyphenyl) retinamide (4-HPR) decreased Mcl-1 expression via reactive oxygen species production, resulting in cytotoxicity in acute lymphoblastic leukemia. 29 In addition, multiple drugs, such as quercetin, 30 gemcitabine, 31 actinomycin D, 32 and histone deacetylase inhibitor entinostat, 33 potentiates cancer cell to ABT-737-induced apoptosis by targeting Mcl-1. In our study, we found that cafestol also decreased Mcl-1 expression, resulting in increased sensitivity to ABT-737-mediated apoptosis.
In addition, cafestol markedly increased Bim and PUMA expression, pro-apoptotic BH3-only proteins (Figure 5b). BH3only proteins, such as NOXA, PUMA, and Bim, bind to the antiapoptotic Bcl-2 proteins, resulting in inhibition of apoptosis. Bim and PUMA could bind to Bcl-2, Bcl-xL, Bcl-w, and Mcl-1. 34 Therefore, we thought that cafestol-induced Bim and PUMA expression was involved in cafestol plus ABT-737-mediated apoptosis. However, downregulation of Bim, but not downregulation of PUMA, reduced ABT-737 plus cafestol-induced apoptosis (Figures 5d and e). Upregulation of Bim expression was detected within 6 h, but PUMA was increased within 12 h. Therefore, upregulation of PUMA expression might be a result and not the cause. Our data suggested that cafestol could induce ABT-737-mediated apoptosis via two mechanisms; downregulation of Mcl-1 expression and upregulation of Bim expression.
Collectively, these results suggest that cafestol sensitizes ABT-737-mediated apoptosis through the downregulation of  Anti-PARP antibody was purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-XIAP and anti-Bax antibodies were purchased from BD Biosciences (San Jose, CA, USA). The anti-Bim antibody was purchased from Millipore Corporation (Billerica, MA, USA). Anti-c-FLIP(L) antibody was obtained from ALEXIS Corporation (San Diego, CA, USA). Anti-PSMD/S5a and anti-PSMA5 antibodies were purchased from Cell Signaling Technology. All other chemicals were purchased from Sigma-Aldrich.
Stable transfection in Caki cells. The Caki cells were transfected in a stable manner with the pFLAG-CMV4-Mcl-1, pcDNA 3.1-Bcl-2 plasmid, or control plasmid pcDNA 3.1 vector using Lipofectamine2000 as prescribed by the manufacturer (Invitrogen, Carlsbad, CA, USA). After 48 h of incubation, transfected cells were selected in primary cell culture medium containing 700 μg/ml G418 (Invitrogen). After 2 or 3 weeks, single independent clones were randomly isolated, and each individual clone was plated separately. After clonal expansion, cells from each independent clone were tested for expression levels of Mcl-1 and Bcl-2 by immunoblotting.
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. Stained cells were analyzed by fluorescent-activated cell sorting on a FACScan flow cytometer to determine relative DNA content, which was based on red fluorescence intensity.
Western blotting analysis. Cells were washed with cold PBS and lysed on ice in 50 μl of lysis buffer (50 mM Tris-HCl, 1 mM EGTA, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, pH 7.5). Lysates were centrifuged at 10 000 × g for 10 min at 4°C, and the supernatant fractions were collected. Proteins were separated by SDS-PAGE and transferred to an Immobilon-P membrane (Amersham, Uppsala, Sweden). 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 cafestol and ABT-737 was evaluated using the isobologram method. In brief, cells were treated with different concentrations of cafestol and ABT-737 alone or in combination. After 48 h, relative survival was assessed, and the concentration effect curves were used to determine the IC 50 (the half-maximal inhibitory concentration) values for each drug alone and in combination with a fixed concentration of the second agent. 41 4′,6′-Diamidino-2-phenylindole staining (DAPI) for condensation and fragmentation of nuclei. To examine cellular nuclei, cells were fixed with 1% paraformaldehyde on glass slides for 30 min at room temperature. After fixation, cells were washed with PBS and a 300 nM DAPI solution (Roche, Mannheim, Germany) was added to the fixed cells for 5 min. After the nuclei were stained, cells were examined by fluorescence microscopy.
Cell death assessment by DNA fragmentation assays. A cell death detection ELISA plus kit (Boehringer Mannheim, Indianapolis, IN, USA) was used for assessing apoptotic activity by detecting fragmented DNA within the nucleus in ABT-737-, cafestol-, and combination of ABT-737 and cafestol-treated cells. Briefly, each culture plate was centrifuged for 10 min at 200 × g, the supernatant was removed, and the pellet was lysed for 30 min. After centrifuging the plate again at 200 × g for 10 min, the supernatant that contained the cytoplasmic histoneassociated DNA fragments was collected and incubated with an immobilized antihistone antibody. The reaction products were incubated with a peroxidase substrate for 5 min and measured by spectrophotometry at 405 and 490 nm (reference wavelength) with a microplate reader (Tecan, Männedorf, Switzerland). The signals in the wells containing the substrate alone were subtracted as the background.
Clonogenic survival assay. Caki cells (0.5 × 10 5 ) were seeded in a 12-well culture plates and was followed by treatment with cafestol and ABT-737 for 3 days. Clonogenic survival was determined by staining colonies using 0.4% coomassie blue and were visualized by a digital camera (Canon, Melville, NY, USA).
Animal. Male BALB/c-nude mice, aged 5 weeks, were purchased from the Central Lab Animal Inc. (Seoul, Korea). All the mice were allowed 1 week to acclimatize to the surroundings before the experiments and were kept at 25 ± 2°C, with a relative humidity of 55 ± 5% and a 12-h light-dark cycle. The study protocol was approved by the IRB Keimyung University Ethics Committee. in vivo xenograft model. Each mouse was s.c. injected on each flank with Mcl-1-overexpressing Caki cells (1 × 10 6 ). After tumors had grown to at around 2 weeks, 28 mice were randomly divided into four treatment groups: (1) vehicle alone, (2) ABT-737 alone, (3) cafestol alone, and (4) ABT-737 plus cafestol. ABT-737 and cafestol were administered at 75 mg/kg, respectively. ABT-737 was prepared in 65% of 5% dextrose, 30% propylene glycol, and 5% Tween-80 (pH 3.5). Cafestol was prepared in corn oil. Mice received intraperitoneal (i.p.) injection of ABT-737 and cafestol. Treatment was administered twice a week for 2 weeks. Growth of the s.c. tumors was measured twice a week. Tumor size was measured twice a week using a Vernier's caliper (Mytutoyo Co., Tokyo, Japan) across its two perpendicular diameters, and tumor size was calculated using the equation (length × width 2 )/2. The animals were killed through cervical dislocation, and tumors were collected for histological analysis. The tumors were fixed in 30% formalin, embedded in OCT compound (Miles Inc., Elkhart, IN, USA), and cut into 20 μm using cryostat (SLEE International, Inc., New York, NY, USA).
TUNEL assay. Apoptosis in tumor cells was detected by TUNEL assay. It was performed using the ApopTag Fluorescein In Situ Apoptosis Detection Kit (Millipore) as per the manufacturer's protocol.
Immunohistochemistry. Sections were mounted on gelatin-coated slides, dried for 1 h, and washed twice in PBS. This was followed by blocking with 1% BSA and incubation with active caspase-3 antibody (Cell Signaling Technology). DAPI (Vector Laboratories, Burlingame, CA, USA) was used to detect nuclei. Sections were photographed using a Carl Zeiss microscope (Carl Zeiss, Jena, Germany).
Reverse transcription-PCR. Total RNA was isolated using the TriZol reagent (Life Technologies, Gaithersburg, MD, USA), and cDNA was prepared using M-MLV reverse transcriptase (Gibco-BRL, Gaithersburg, MD, USA) according to the manufacturer's instructions. The following primers were used for the amplification of human Mcl-1 and actin: Mcl-1 (forward) 5′-GCGACTGGCAAAGCTTGGCCTCAA-3′ and (reverse) 5′-GTTACAGCTTGGATCCCAACTGCA-3′; and actin (forward) 5′-GG CATCGTCACCAACTGGGAC-3′ and (reverse) 5′-CGATTTCCCGCTCGGCCGTGG -3′. PCR amplification was carried out using the following cycling conditions: 94°C for 3 min followed by 17 cycles for actin, or 25 cycles for Mcl-1 of 94°C for 40 s, 58°C for 40 s, 72°C for 40 s, and a final extension at 72°C for 5 min. The amplified products were separated by electrophoresis on a 1.5% agarose gel and detected under UV light by the addition of ethidium bromide. For qPCR, cDNA and forward/ reverse primers (200 nM) were added to 2 × KAPA SYBR Fast master mix, and reactions were performed on RG-6000 real-time amplification instrument (Corbett Research, Cambridge, UK). The following primers were used for the amplification of human Mcl-1 and actin: Mcl-1 (sense) 5′-ATGCTTCGGAAACTGGACAT-3′ and (antisense) 5′-TCCTGATGCCACCTTCTAGG-3′, and actin (sense) 5′-CTACAATGA GCTGCGTGTG-3′ and (anti-sense) 5′-TGGGGTGTTGAAGGTCTC-3′. Threshold cycle number (Ct) of each gene was calculated, and actin was used as reference genes. Delta-delta Ct values of genes were presented as relative fold induction.
Proteasome activity assay. Measurement of proteasome activity within cells was determined using ZsProSensor-1 (proteasome sensor vector) (BD Biosciences). The ZsProSensor-1 stable cell line was created by transfection of Caki cells with the ZsProsensor-1 plasmid using Lipofectamine (Invitrogen). Clones were selected in the presence of 700 μg/ml G418 (Gibco-BRL), and fluorescence was detected using FACS Canto (BD Biosciences).