Colorectal adenocarcinoma cell lines DLD-1 (ATCC CCL-221TM), HCT-15 (ATCC CCL-225TM) and HT-29 (ATCC HTB-38TM) were obtained from the American Type Culture Collection and cultured at 37°C in a humidified 5% CO₂ incubator in either RPMI (DLD-1, HCT-15) or DMEM (HT-29) medium supplemented with 10% FBS, 10 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (all media and supplements from Sigma-Aldrich; Merck KGaA).

The 5-FU-resistant colon cancer cell line was developed as previously reported (6). Briefly, parental colon cancer cell lines were treated with gradually increasing concentrations of 5-FU (Sigma-Aldrich; Merck KGaA). The initial 5-FU concentration added to cells was 10% of the IC₅₀; this concentration was gradually increased. Colon cancer cell lines resistant to 16 µM of continuous 5-FU administered continuously were established according to a previous study (7).

Each colon cancer cell line was treated for 72 h with 5-FU at the indicated concentrations in 96-well plates (Thermo Fisher Scientific, Inc.). Subsequently, cell proliferation was determined using the Premix WST-1 Cell Proliferation Assay (Takara Bio Inc., Kusatsu, Shiga, Japan) and an Infinite M1000 PRO microplate reader (Tecan Japan, Kawasaki, Kanagawa, Japan). The half-maximal inhibitory concentration (IC₅₀) of 5-FU was defined as the drug concentration resulting in 50% cell survival relative to that of untreated cells. Triplicate wells were treated with various drug concentrations and average IC₅₀ values were determined. As known antagonists of BCL2 and BCLXL in in vitro studies, ABT-199 (Selleck Chemicals) and WEHI-539 hydrochloride (MedChem Express) were used and the IC₅₀ values of each drug were obtained, respectively.

Parental and 5-FU-resistant colon cancer cells were allowed to adhere to 6-well plates for 24 h and cells were treated with either 5-FU or WEHI-539 hydrochloride as indicated. Cells were then stained with a phycoerythrin-conjugated Annexin V antibody and 7-AAD (BD Pharmingen; BD Biosciences). Apoptotic cells were analyzed using a BD FACSCanto II flow cytometer (BD Biosciences) with FACSDiva software (BD Biosciences). The percentage of apoptotic cells was calculated by dividing the percentage of either Annexin V-positive or 7-AAD positive cells by the total cells. Apoptosis was also assessed using the Caspase-Glo^® 3/7 Assay (Promega). Five thousand cells were plated in white-walled 96-well round plates (Thermo Fisher Scientific, Inc.) and treated with the drugs as indicated. After incubation, 100 µl of Caspase-Glo^® reagent was added to each well and the contents of the well were gently mixed with a plate shaker at 50 × g for 30 sec; this was followed by incubation at 20°C room temperature for 1 h. The luminescence of each sample was measured using an Infinite M1000 PRO microplate reader. The caspase inhibitor Q-VD-OPH (Bay Bioscience, Kobe, Hyogo, Japan) was also used.

Western blotting was performed as previously described (8). Briefly, separated proteins were transferred to polyvinylidene difluoride membranes and blotted with specific antibodies to detect BCL2 (at dilution of 1:500; Thermo Fisher Scientific, Inc.; #13-8800), BCLW (1:1,000; Cell Signaling Technology; cat. no. 2724), BCLXL (1:1,000; Cell Signaling Technology; cat. no. 2764), MCL1 (1:1,000; Cell Signaling Technology; cat. no. 5453), BAK (1:1,000; Cell Signaling Technology; cat. no. 12105), BAX (1:1,000; Cell Signaling Technology; cat. no. 5023), BIM (1:1,000; Cell Signaling Technology; cat. no. 2933), BID (1:1,000; Cell Signaling Technology; #2002), BAD (1:1,000; Cell Signaling Technology; cat. no. 9292), NOXA (1:1,000; Cell Signaling Technology; cat. no. 14766), PUMA (1:1,000; Cell Signaling Technology; cat. no. 12450), BMF [1:1,000; Abcam; cat. no. EPR10930 (2)], HRK (1:200; R&D Systems; cat. no. AF851), and actin (1:3,000; Santa Cruz Biotechnology; cat. no. sc1615). After incubation with either horseradish peroxidase-linked anti-rabbit IgG (1:2,000; Cell Signaling Technology; cat. no. 7074S) or anti-mouse IgG (1:2,000; Cell Signaling Technology; cat. no. 7076S), the membranes were stained with ECL Select Western Blotting Detection Reagent (GE Healthcare UK Ltd.). Finally, the bands were imaged either by exposing membranes to BIOMAX XAR film (Sigma-Aldrich; Merck KGaA) and developing the images using a Kodak X-OMAT 1000 Processor (Kodak via Thermo Fisher Scientific, Inc.) or using an LAS-4000UV mini (GE Healthcare UK Ltd.) and MultiGauge software (Fujifilm, Tokyo, Japan).

We conducted fluorescence activated cell sorting (FACS)-based BH3 profiling as previously described (9,10). Nine BH3 peptides were obtained as HPLC-purified products from Sigma-Aldrich; Merck KGaA (Table I). All peptides were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck KGaA) as 1 mM stock solutions and stored at −80°C. As a control for mitochondrial depolarization, p-trifluoromethoxy carbonyl cyanide phenyl hydrazine (FCCP) was used. Two hundred thousand parental and 5-FU-resistant colon cancer cells were suspended in TE-B buffer [300 mM trehalose, 10 mM HEPES-KOH (pH 7.7), 80 mM KCl, 1 mM EDTA, 1 mM EGTA, 0.1% bovine serum albumin (BSA), and 5 mM succinate; all from Sigma-Aldrich; Merck KGaA] containing 0.001% digitonin (Sigma-Aldrich; Merck KGaA) and 20 µg/ml oligomycin (Sigma-Aldrich; Merck KGaA), followed by incubation with each BH3 peptide at a final concentration of 10 µM for 30 min. After staining the cells with 25 nM tetramethylrhodamine ethyl ester (Invitrogen/Thermo Fisher Scientific, Inc.), fluorescence intensities were analyzed using a BD FACSCanto II flow cytometer (BD Biosciences) with FACSDiva software (BD Biosciences). The percentage of relative mitochondrial depolarization was calculated using the following equation:

where DMSO(fv) is the mean fluorescence value as the negative control, X(fv) indicates the fv as the tested BH3 peptide, and FCCP(fv) indicates the fv as the positive control.

One million parental and 5-FU-resistant HT-29 cells in 6-well plates were transfected with either siRNAs targeting human BCLXL (Dharmacon; siBCLXL#1: D-003458-03, siBCLXL#2: D-003458-30) or a non-targeting siRNA (Dharmacon; siNT: D-001210-03-05) using RNAi Max reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions.

BCLXL immunoprecipitation experiments were conducted using the ImmunoCruz™ IP/WB Optima F System (Santa Cruz Biotechnology; #SC-45043). Separated total proteins (1,000 µg) were incubated with a complex of BCLXL antibody and IP matrix overnight at 4°C. After incubation and centrifugation, the eluted supernatant was subjected to western blotting for BID, BIM, and BCLXL.

Fifteen female BALB/c nu/nu mice (six weeks of age, 18–20 g/each) were purchased from Sankyo Labo Service Corporation, Inc. (Tokyo, Japan). Five million 5-FU-resistant HT-29 cells suspended in 100 µl DMEM and 100 µl Matrigel (BD Biosciences) were injected subcutaneously into the left flank. In place of WEHI-539, which has a labile and potentially toxic hydrazone moiety, we used another BCLXL inhibitor, A-1155463, which is known to be efficacious on tumor growth suppression in vivo (11). Mice were divided into three experimental groups: Control (vehicle-treated), 5-FU, and A-1155463. When tumor volumes reached 100 mm³, administration of either intravenous 5-FU (50 mg/kg) injections once a week or intraperitoneal A-1155463 (5 mg/kg) injections every day was conducted for four weeks as previously reported (11,12). Tumor size and body weight were measured throughout the experimental period. Tumor volumes were calculated with the following formula:

Percent volume was calculated with the following formula:

Mice were sacrificed when the tumor diameter reached 20 mm or when the study period finished. Tumors and other organs including the lung and liver were isolated, followed by hematoxylin and eosin staining. Apoptotic cells were analyzed by TUNEL staining using an in situ apoptosis detection kit (Takara Bio Inc.). The percentage of TUNEL-positive cells was calculated by counting cells in 5 different fields. The animal study was conducted according to protocols approved by the Animal Ethics Committee of the Sapporo Medical University School of Medicine (Protocol no. 17-142). Euthanasia was conducted by high concentration carbon dioxide and all efforts were made to minimize suffering.

All data represent the mean ± SD. For comparisons between two groups, two-tailed unpaired Student's t-tests were performed. For multiple comparisons of in vitro experiments, one way analysis of variance followed by Tukey test was conducted. IC₅₀ values for each drug tested and dose-response curves were analyzed using Graph-Pad PRISM 5 (GraphPad Software, Inc., La Jolla, CA, USA). For in vivo experiments, one way analysis of variance followed by Dunnett's post hoc test was adopted for comparison with the control group. All statistical analyses were performed using SPSS software version 21 (IBM, Armonk, NY, USA).

We established three colon cancer cell lines which were resistant to 16 µM continuous 5-FU treatment. The 5-FU IC₅₀ values of resistant colon cancer cells were markedly higher than those of the parental cells (DLD-1: 5-FU-resistant 776.0 µM, parental 10.3 µM; HCT-15: 5-FU-resistant 44.2 µM, parental 4.1 µM; HT-29: 5-FU-resistant >1,000 µM, parental 7.2 µM, respectively) (Fig. 1A-C).

To evaluate the mechanisms of 5-FU resistance, we first verified the occurrence of apoptosis in parental cells and three 5-FU-resistant colon cancer cell lines. Each 5-FU-resistant colon cancer cell line contained significantly fewer Annexin-positive apoptotic cells compared with the parental cells, suggesting that apoptotic resistance had been acquired by 5-FU-resistant colon cancer cell lines (Figs. 2A and S1). We then examined expression alterations in both anti-apoptotic and apoptotic proteins between parental and 5-FU-resistant colon cancer cells (Fig. 2B and C). Western blot analysis showed that BCLXL and MCL1 protein expression levels were similar between the parental and 5-FU-resistant cells in the three colon cancer cell lines. Whereas BCL2 protein expression in 5-FU-resistant DLD-1 cells was decreased, both BCLW expression in DLD-1 and BCL2 protein expression in HT-29 was increased in the 5-FU-resistant cells. The expression of apoptosis-related proteins, e.g., BAX was decreased whereas BIM, BAD, and NOXA expression was increased in the 5-FU-resistant HCT-15 cells. Whereas NOXA expression was decreased in the 5-FU-resistant HT-29 cells, BIM expression was increased, suggesting that the expression patterns of both anti-apoptotic and apoptotic proteins were altered during the acquisition of 5-FU resistance in the three colon cancer cell lines. From this analysis of apoptosis-related protein expression, it may be difficult to identify specific apoptotic responses to 5-FU resistance solely by quantifying protein expression.

To further clarify the relationship between 5-FU resistance and apoptosis-related proteins, we conducted a functional analysis via BH3 profiling (Fig. 3A). When treated with BIM and BID BH3 peptides, which interact directly with BAK and BAX proteins resulting in the release of cytochrome c into the cytoplasm, strong mitochondrial depolarization was observed in the parental and all 5-FU-resistant cells. We then treated cells with BAD, NOXA, PUMA, BMF, and HRK BH3 peptides, which have variable affinities for anti-apoptotic proteins, and compared the mitochondrial depolarization between parental and 5-FU-resistant colon cancer cells. In DLD-1 and HCT-15 cells, no significant differences were observed between parental and 5-FU-resistant cells when treated with these BH3 peptides. However, greater mitochondrial depolarization was observed in 5-FU-resistant HT-29 cells when treated with BAD (Fig. 3B), PUMA (Fig. 3C), and BMF (Fig. 3D) BH3 peptides, which was correlated with dependency on BCL2, BCLXL or BCLW. Furthermore, treatment with HRK peptide, which binds specifically to BCLXL, caused a small increase in mitochondrial depolarization in the 5-FU-resistant HT-29 cells (Fig. 3E mitochondrial depolarization: Parental, 0.7%; 5-FU-resistant, 20.3%). To further clarify the dependency on BCLXL protein, HT-29 cells were treated with the XXA1 peptide, which has been structurally identified (13) as a specific inhibitor of BCLXL. Significant differences in mitochondrial depolarization were observed between the parental and 5-FU-resistant HT-29 cells (Fig. 3F mitochondrial depolarization: Parental, 1.5%; 5-FU-resistant, 50.0%). As for DLD-1 and HCT-15, there were no differences in mitochondrial depolarization between parental and 5-FU-resistant cells when treated with XXA1 peptide. We also treated cells with MS1 peptide as a specific MCL inhibitor (14); no changes in mitochondrial depolarization were observed in both parental and 5-FU-resistant colon cancer cells. Taken together, results of the BH3 profiling of 5-FU-resistant HT-29 cells revealed strong dependency on BCLXL for cell survival compared with parental cells, while no relationship to apoptosis-related protein expression profiles was determined, especially BCLXL protein expression. We then focused on the BCLXL dependence of 5-FU-resistant HT-29 colon cancer cells.

To confirm the BCLXL dependency in 5-FU-resistant HT-29 cells, we first treated parental and 5-FU-resistant colon cancer cells with the BCLXL-specific inhibitor WEHI-539 and assessed the effect of this inhibition on cell survival. As expected, the IC₅₀ of WEHI-539 in 5-FU-resistant HT-29 cells was markedly lower compared to that in parental cells (Fig. 4A: 5-FU-resistant, 0.66 µM; parental, >100 µM), whereas no difference in IC₅₀ values between parental and 5-FU-resistant cells was observed in DLD-1 (Fig. 4B: 5-FU-resistant, 9.94 µM; parental, 7.12 µM) and HCT-15 (Fig. 4C: 5-FU-resistant, 9.83 µM; parental, 6.31 µM) cells. To rule out any possibility of the contribution of induced BCL2 expression to 5-FU resistance in HT-29 cells as shown in Fig. 2B, we also treated HT-29 cells with the BCL2-specific inhibitor ABT-199 (Fig. 4D). No marked differences in IC₅₀ values were observed between the parental and 5-FU-resistant HT-29 cells (IC₅₀: Parental, 16.5 µM; 5-FU-resistant, 13.5 µM), suggesting that these inhibitory data support the result of the BH3 profiling rather than that of the anti-apoptotic protein expression profiles. We then examined the effect of WEHI-539 on apoptosis in 5-FU-resistant HT-29 cells to examine how BCLXL inhibition could kill these cells. An significantly increased percentage of apoptotic cells was observed in the 5-FU-resistant HT-29 cells, whereas no additional Annexin V-positive cells were noted in the parental cells (Fig. 4E). Furthermore, the activities of caspase-3 and caspase-7, both of which are key effectors of apoptosis, were upregulated in the 5-FU-resistant HT-29 cells when treated with WEHI-539 and were completely inhibited through co-treatment with the caspase inhibitor Q-VD-OPH (Fig. 4F). These results indicate that BCLXL, but not BCL2, exerts substantial anti-apoptotic functions in 5-FU-resistant HT-29 cells.

We then inhibited BCLXL protein expression in 5-FU-resistant HT-29 cells through siRNA transfection (Fig. 5A), followed by treatment with 5-FU. The IC₅₀ of 5-FU in the resistant HT-29 cells was markedly decreased following BCLXL inhibition compared to that in both cells transfected with non-targeting siRNA (siNT) and untransfected HT-29 cells (Fig. 5B: siBCLXL#1 transfected, 174.9 µM; siBCLXL#2 transfected, 236.5 µM; siNT transfected, >1,000 µM; untransfected, >1,000 µM). Furthermore, a significantly increased percentage of apoptotic cells was observed following BCLXL inhibition during 5-FU treatment (Fig. 5C), suggesting that BCLXL downregulation restored 5-FU sensitivity in the resistant HT-29 cells by enhancing their vulnerability to apoptosis.

To examine whether an interaction between BCLXL and an apoptotic protein would account for functional BCLXL dependency, we performed immunoprecipitation on cell extracts of parental and 5-FU-resistant HT-29 cells using a BCLXL antibody and examined the binding of apoptotic BIM and BID proteins. As shown in Fig. 6A and B, BIM protein preferentially bound to BCLXL in the 5-FU-resistant HT-29 cells compared to the parental cells. In summary, BCLXL dependence in 5-FU-resistant HT-29 cells was mediated through the sequestration of BIM by BCLXL.

We further tested the effect of BCLXL inhibition on the growth of 5-FU-resistant HT-29 cells using a tumor xenograft model. Prior to our in vivo study, we found that the A-1155643 IC₅₀ values of 5-FU-resistant HT-29 cells were markedly lower compared to those in parental cells (5-FU-resistant, 1.2 µM; parental, >100 µM). After transplantation of these cells into the left flank, mice were treated for four weeks with either 5-FU or BCLXL inhibitor A-1155643, respectively. Whereas tumor volume gradually increased in the mice treated with 5-FU, similar to vehicle-treated mice, BCLXL inhibitor-treated mice showed stable tumor size as well as a significantly different size compared with vehicle-treated mice (Figs. 7A and B and S2). A significant increase in TUNEL-positive apoptotic tumor cells was also observed in mice treated with A-1155643 (Fig. 7C and D). A-1155643 treatment was well tolerated, without significantly decreasing body weight (day 28 body weight: Vehicle, 17.9±1.8 g; 5-FU treatment, 18.3±1.7 g; A-1155643 treatment, 17.7±1.9 g, respectively; Fig. S3). Also, no severe bleeding in the lung and liver (Fig. S4), which usually occurs due to thrombocytopenia, was observed in the BCLXL inhibitor-treated mice.