A total of three CCA cell lines, KKU-055, KKU-100 and KKU-213, were established as previously described (28). Cells were obtained from the Japanese Collection of Research Bioresources Cell Bank. Cells were maintained in Dulbecco's modified Eagle's medium (Wako Pure Chemical Industries, Ltd.) containing 10% fetal bovine serum (HyClone; GE Healthcare), 100 U/ml penicillin and 100 µg/ml streptomycin. CCA cells were cultured in a humidified incubator at 37°C with 5% CO₂.

CMA3 was purchased from Abcam, MTA was from Sigma-Aldrich; Merck KGaA, MTT and propidium iodide (PI) were from Sigma-Aldrich; Merck KGaA, RNase A was from Takara Bio, Inc., and Pacific Blue™ Annexin V was from BioLegend, Inc.

Sources of antibodies were as follows: Rat anti-Hsc70 (1B5 clone; cat. no. ADI-SPA-815B) monoclonal antibody (mAb) was from EnZo Life Sciences, Inc.; rabbit anti-Bim (C34C5 clone; cat. no. 2933) mAb, rabbit anti-cleaved caspase-3 (Asp175; 5A1E clone; cat. no. 9664) mAb, rabbit anti-cleaved caspase-8 (Asp391; 18C8 clone; cat. no. 9496) mAb and rabbit anti-cleaved caspase-9 (Asp315; D8I9E clone; cat. no. 20750) mAb, horseradish peroxidase (HRP)-linked anti-rabbit IgG (cat. no. 7071), and HRP-linked anti-mouse IgG (cat. no. 7076) were from Cell Signaling Technology, Inc.; mouse anti-FLIP short and long forms (FLIP_S/L; G-11 clone; cat. no. sc-5276) mAb, mouse anti-Mcl-1 (22 clone; cat. no. sc-12756) mAb, mouse anti-Noxa (114C307 clone; cat. no. sc-56169) mAb, mouse anti-survivin (D8 clone; cat. no. sc-17779) mAb, rabbit anti-cIAP2 (H-85 clone; cat. no. sc-7944) polyclonal antibody (pAb), rabbit anti-Sp1 (PEP 2 clone; cat. no. sc-59) pAb, and rabbit anti-XIAP (H-202 clone; cat. no. sc-11426) pAb, were from Santa Cruz Biotechnology, Inc.; mouse anti-BAX (2D2 clone; cat. no. 633601) mAb was from Biolegend, Inc.; mouse anti-α-smooth muscle actin (α-SMA; 1A4 clone; cat. no. M0851) mAb, and rabbit anti-rat (cat. no. P0450) pAb were from Dako; Agilent Technologies, Inc.; mouse anti-cytokeratin (AE1&AE3 clone; cat. no. 313M) cocktail was from Cell Marque™ (Sigma-Aldrich; Merck KGaA); rabbit anti- cytokeratin 19 (CK19; cat. no. HPA002465) was from Sigma-Aldrich (Merck KGaA); rabbit anti-BAX (E63 clone; cat. no. ab32503) pAb, and rabbit anti-caspase-9 (cat. no. ab2014) pAb were from Abcam and the rabbit anti-Ki-67 (30-9 clone; cat. no. 790-4286) mAb was from Ventana Medical Systems, Inc.

The anti-proliferative effects of CMA3 on KKU-055, KKU-100 and KKU-213 CCA cells were determined by MTT assay. CCA cells were seeded at 4×10³ cells per well into a 96-well plate. At 24 h after seeding, CMA3 or MTA were added at different concentrations (0, 2.5, 5, 10, 20, 40, 80 and 160 nM for CMA3 and 0, 12.5, 25, 50, 100 and 200 nM for MTA) and cells were incubated for 24 and 48 h. MTT solution was added to obtain a final concentration of 0.5 mg/ml. Then formazan crystals were dissolved by acidified isopropanol (0.04 N HCl in isopropanol). The absorbance was measured at 570 nm using a microplate reader (iMark; Bio-Rad Laboratories, Inc.). The data were analyzed using the GraphPad Prism 8 (GraphPad Software, Inc.). The optical density₅₇₀ of the control was set to 100%.

Cells were treated at indicated concentrations of CMA3 for 24 h. After that cells were harvested and fixed with cold 70% ethanol overnight at 4°C. Before analysis, cells were washed with PBS and incubated with 0.1 mg/ml of RNase A at 37°C for 1 h, followed by PI staining (50 µg/ml) at 4°C for 30 min in the dark. Samples were analyzed by LSR II flow cytometer (BD Biosciences; Becton, Dickinson and Company) and data were analyzed by FlowJo software version 10.4 (Tree Star, Inc.).

Cells were treated with various concentrations of CMA3 for 24 h. Then cells were collected and incubated with Pacific Blue™ Annexin V at room temperature for 30 min in the dark. PI was added at the final concentration of 1 µg/ml before flow cytometry analysis. Annexin V and PI-stained cells were determined by flow cytometry. Annexin V-bound and PI-stained cells were analyzed by FlowJo software.

KKU-213 cells were treated with CMA3 at indicated concentrations and times. Protein lysate was prepared as described elsewhere (29). Protein concentrations were determined by the bicinchoninic acid protein assay (Thermo Fisher Scientific, Inc.). A total of 20 µg of protein was separated by SDS-PAGE (10-15%) and transferred to a PVDF membrane (GE Healthcare Japan). The membrane was blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) at room temperature for 30 min and was incubated with specific primary antibodies (1:1,000) at 4°C overnight. Membranes were washed with TBST 3 times prior to the incubation with the corresponding HRP-conjugated secondary antibody (1:2,000) for 1 h at room temperature. Signals were detected using Chemi-Lumi One Super reagents (Nacalai Tesque, Inc.) and visualized by ImageQuant LAS 4000 system (GE Healthcare Japan). The quantitative analyses of blots were quantified by Image J (30). Hsc70 was used as an internal control.

KKU-213 cells were treated with 40 nM CMA3 or 200 nM MTA for 0, 6, 12, 18 and 24 h. RNA preparation and RT-PCR were performed as previously described (31). Gene expression was presented as relative expression (fold, control at 0 h=1). The oligonucleotide primers used in this study were previously reported elsewhere; Mcl-1 (32), XIAP (33), β-actin (34).

To demonstrate the toxic effects of CMA3 in the mouse model, a total of eight 6-8 week-old male Balb/c Rag-2/Jak3 double deficient mice (35) were randomly separated into 4 groups (n=2/group; body weight ~22-25 g/mouse); group 1 was intravenously injected with a vehicle, dimethyl sulfoxide (DMSO) once a week, group 2 was injected with 0.1 mg/kg CMA3 once a week, group 3 was injected with 0.1 mg/kg CMA3 twice a week and group 4 was injected with 0.5 mg/kg CMA3 once a week. The vehicle or CMA3 was given for 3 weeks. The toxicity was monitored by observation of the general appearance and determination of the body weight.

KKU-213 cells (1×10⁵ cells/site) were subcutaneously injected into both flanks of 6-8 week-old male Balb/c Rag-2/Jak3 double deficient mice. At 3 days after CCA injection, 14 mice were randomly divided into 2 groups (n=7/group; body weight ~22-25 g/mouse). CMA3 was administered to the treatment group at 0.5 mg/kg intravenously, once a week for 3 weeks. DMSO was given to the control group. Body weight and tumor volume were monitored every 3 days. On day 22, mice were sacrificed and tumors were collected for the immunohistochemistry staining.

Mice were housed and monitored in the animal research facility according to the institutional guidelines. All protocols were approved by the Institutional Animal Care and Use Committee of Kumamoto University. All of the animal experiments were conducted according to the Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology, Japan. In brief, mice were housed in a strictly control environment (22±2°C, 50±10% humidity and 12-h light/dark cycle). Food and water were provided ad libitum. Health and behavior of those mice were monitored every 3 days to evaluate humane endpoints. If mice showed loss of appetite, severe weight loss (>20% of the original weight) or signs of pain associated behavior, mice would be euthanized using isoflurane. Death was verified as follows: Lack of respiration, heartbeat and corneal reflex. A total of 5% inhaled isoflurane was used as an anesthetic.

Paraffin embeded xenograft tumor tissues were prepared according to a standard protocol (36) and sectioned at 3-µm thickness. Expression of cytokeratin, Ki-67 and α-SMA were detected using BenchMark XT automated staining system (Ventana Medical Systems, Inc.). EZ prep, Cell conditioning (CC1) and Inhibitor CM (Ventana Medical Systems, Inc.) were used according to manufacturer's protocol. Images were taken by an Eclipse Ni-E light microscope (Nikon Corporation) using NIS Elements D software version 4.5 (Nikon Corporation). Ki-67 positive nuclei per cytokeratin areas were quantified by ImageJ (n=4/group, 3 images/tumor).

Caspase-9, CK19 and Bax expression was determined by immunohistochemistry as previously described (37). The caspase-9 or Bax-positive areas were determined by ImageJ (n=5/group, 5 images/tumor). The expression of caspase-9 or Bax was presented as the marker-positive area/CK19-positive nuclei/cancer area.

The data are presented as the mean ± standard deviation from three independent experiments unless otherwise specified. The statistical differences between the control and experimental groups were determined by Student's t-test. For CMA3 toxicity testing, mean percentage of body weights among the groups were analyzed using one-way analysis of variance with Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference. Statistical analyses were performed using the GraphPad Prism software version 8 (GraphPad Software, Inc.) and SPSS software version 19.0 (IBM, Corp.).

Effects of CMA3 and the prototype drug, MTA, on cell proliferations of 3 CCA cell lines, KKU-213, KKU-055 and KKU-100, were determined by MTT assay. Cells were treated with increasing concentrations of CMA3 or MTA for 24 and 48 h. The dose-response curves revealed CMA3 and MTA suppressed CCA cell proliferation in dose- and time-dependent manners (Fig. 1). The half maximal inhibitory concentrations (IC_50s) of CMA3 on KKU-213 at 24 and 48 h were 22.48±4.08 and 9.79±1.15 nM; IC_50s of KKU-055 were 21.14±2.24 and 13.34±1.28 nM; and IC_50s of KKU-100 were 30.52±2.91 and 14.74±1.34 nM. The IC_50s of MTA were higher than those of CMA3; IC_50s of KKU-213 at 24 and 48 h were >200 and 46.08±1.63 nM; IC_50s of KKU-055 were >200 and 76.44±11.70 nM; and IC_50s of KKU-100 were >200 and 104.77±4.22 nM (Table I).

CMA3 is a DNA-binding agent that binds to DNA at a minor groove and inhibits DNA replication and transcription (26). To verify the effects of CMA3 on cell cycle distribution, KKU-213 and KKU-055 were treated with 0, 2.5, 5, and 10 nM CMA3 for 24 h. Cells were stained with PI and the cell cycle distributions were analyzed by flow cytometry. The results showed increased concentrations of CMA3 significantly increased cells in the S phase. Fig. 2A shows the representative histograms. CMA3 caused the accumulation of cells in S phase, while the reduction of cells in G1 phase was observed (Fig. 2A). A total of 42.5±1.7, 45.8±0.9, 50.4±5.9 and 62.8±2.4% of KKU-213 and 50.0±2.6, 53.2±1.9, 64.9±8.1 and 74.6±3.3% of KKU-055 were in S phase of the cell cycle when cells were treated with 0, 2.5, 5, and 10 nM CMA3 (P<0.05 and P<0.001; Fig. 2B).

To investigate the effect of CMA3 on apoptotic induction, Annexin V/PI staining was performed in KKU-213, KKU-055 and KKU-100 cells after treatment with 0, 10, 20 and 40 nM of CMA3 for 24 h. Annexin V and PI stained cells were evaluated by flow cytometry. Annexin V-positive and PI-negative cells were early apoptotic cells, whereas Annexin V and PI double-positive cells were late apoptotic. The percentages of early and late apoptotic cells were dramatically increased when cells were treated with higher concentrations of CMA3 (Fig. 3A). The percentages of Annexin V-positive cells were quantified by combining of percentages of early and late apoptotic cells and are presented in Fig. S1A and B (P<0.05). The results showed increased percentages of apoptotic cells in all three CCA cell lines in a dose-dependent manner. To elucidate the mechanism of CMA3-induced apoptosis, caspase activations were investigated. Caspase-8, -9 and -3 were selected to be the representatives of the extrinsic, intrinsic and common apoptotic pathways (38). The results demonstrated CMA3 induced caspase-dependent apoptosis in KKU-213 through both extrinsic (cleaved caspase-8), intrinsic (cleaved caspase-9) and eventually common (cleaved caspase-3) pathways in dose- and time-dependent manners (Fig. 3B and 3C). Similar results of CMA3-induced caspase-dependent apoptosis were observed in KKU-055 (Fig. S2A and B). Increased cleaved caspase-8, -9 and -3 levels were observed when cells were treated with 10-20 nM CMA3, with the highest levels observed following 12 h of 20 nM CMA3 treatment.

CMA3 is a structural analog of MTA. GC-rich, Sp1/Sp3-specific binding of MTA and CMA3 are established in neurons (20). Moreover, the roles of MTA on inhibition of Sp1 binding to the XIAP promoter and caspase-dependent apoptosis induction have been demonstrated in various cancer cells (17). Functions of CMA3 on Sp1-related anti-apoptotic protein expression were of interest. Sp1-related anti-apoptotic proteins including FLIP, cIAP2, Mcl-1, survivin and XIAP were selected (13,39,40). The results showed FLIP_s, cIAP2, Mcl-1, survivin and XIAP were reduced in KKU-213 cells treated with 40 nM CMA3 (Fig. 4A). CMA3 treatment had no effects on either Sp1 or Sp1-related Bcl-2 family pro-apoptotic proteins, e.g., Bcl-2-associated X-protein, Bax, Bcl-2-like protein 11, Bim and Noxa (Fig. 4B and 4C). The quantitative analysis of Sp1, Sp1-related proteins was reported in Fig. 4D. Similar effects of CMA3-suppressed Sp1-related anti-apoptotic proteins were observed in KKU-055 when treated with 20 nM CMA3 (Fig. S2C). The inhibitory effects of CMA3 and MTA on Mcl-1 and XIAP expression were confirmed by RT-PCR. KKU-213 cells were treated with 40 nM CMA3 or 200 nM MTA for the indicated times and then the expression levels of Mcl-1 and XIAP were measured. The results showed CMA3 and MTA efficiently inhibited Mcl-1 and XIAP expression and the reductions of expression were observed at 6 h onward. Moreover, CMA3 possessed a stronger effect on Mcl-1 expression (Fig. S3).

To determine the therapeutic potential of CMA3 on CCA in vivo, KKU-213 cells were injected subcutaneously into flanks of Balb/c RJ mice. Mice were randomly assigned to the control and CMA3-treated groups and the treatments were given for 3 weeks. No noticeable toxicity of CMA3 was observed during the experiment (Fig. S4C), which corresponded with the results of the CMA3 toxicity test (Fig. S5). Tumor volumes were measured. The results demonstrated that on day 22, the tumor volumes of the treatment group were significantly smaller than those of the control group (P<0.001; 178.3±89.4 vs. 476.0±174.7 mm³; Fig. 5A). All mice were subsequently euthanized and xenograft tumors were weighed. The average tumor weights of the CMA3-treated group were significantly decreased compared with control (P<0.05; 0.34±0.22 vs. 0.56±0.20 g; Fig. S4A and B). Moreover, when tumors were removed, they were then examined for the densities of cancer and stromal cell compartments. Ki-67 was used as proliferative marker, while pancytokeratin and α-SMA were used as epithelial (CCA) and active stromal markers. Ki-67 nuclei/cytokeratin-positive areas were assessed and compared between groups. The results demonstrated that the Ki-67/cytokeratin-positive areas were smaller in the CMA3-treated group when compared with the control group [P<0.001; 1.02±0.17 vs. 0.75±0.15 nuclei/arbitrary units (AU); Fig. 5B]. Representative images of Ki-67, pancytokeratin and α-SMA staining are shown in Fig. 5C.

The effects of CMA3 on the apoptotic proteins, caspase-9 and Bax, were determined in xenograft tumors. The results revealed that CMA3 treatment potentiated caspase-9 and Bax expression (Fig. 5D). The expression of caspase-9 and Bax in the CMA3-treated group were significantly increased compared with DMSO-treated control; caspase-9 expression were 3.25±1.10 vs. 2.14±0.76 in control and Bax were 3.60±0.95 vs. 2.86±1.16 in control (P<0.05 and P<0.001; AU/cells/field; Fig. S6).