Inhibition of l -type amino acid transporter 1 activity as a new therapeutic target for cholangiocarcinoma treatment

Abstract

Unlike normal cells, cancer cells undergo unlimited growth and multiplication, causing them to require massive amounts of amino acid to support their continuous metabolism. Among the amino acid transporters expressed on the plasma membrane, l-type amino acid transporter-1, a Na⁺-independent neutral amino acid transporter, is highly expressed in many types of human cancer including cholangiocarcinoma. Our previous study reported that l-type amino acid transporter-1 and its co-functional protein CD98 were highly expressed and implicated in cholangiocarcinoma progression and carcinogenesis. Therefore, this study determined the effect of JPH203, a selective inhibitor of l-type amino acid transporter-1 activity, on cholangiocarcinoma cell inhibition both in vitro and in vivo. JPH203 dramatically suppressed [¹⁴C]l-leucine uptake as well as cell growth in cholangiocarcinoma cell lines along with altering the expression of l-type amino acid transporter-1 and CD98 in response to amino acid depletion. We also demonstrated that JPH203 induced both G2/M and G0/G1 cell cycle arrest, as well as reduced the S phase accompanied by altered expression of the proteins in cell cycle progression: cyclin D1, CDK4, and CDK6. There was also cell cycle arrest of the related proteins, P21 and P27, in KKU-055 and KKU-213 cholangiocarcinoma cells. Apoptosis induction, detected by an increase in trypan blue–stained cells along with a cleaved caspase-3/caspase-3 ratio, occurred in JPH203-treated cholangiocarcinoma cells at the highest concentration tested (100 µM). As expected, daily intravenous administration of JPH203 (12.5 and 25 mg/kg) significantly inhibited tumor growth in KKU-213 cholangiocarcinoma cell xenografts in the nude mice model in a dose-dependent manner with no statistically significant change in the animal’s body weight and with no differences in the histology and appearance of the internal organs compared with the control group. Our study demonstrates that suppression of l-type amino acid transporter-1 activity using JPH203 might be used as a new therapeutic strategy for cholangiocarcinoma treatment.

Introduction

Increased essential nutrient uptake, such as glucose and amino acids, is required for proliferating cells, especially in cancer cells which have lost their normal control of proliferation. ¹ Many studies have revealed that numerous nutrient transporters are upregulated in cancer cells to support their massive growth. Among the known amino acid transport systems, system L is a major Na⁺-independent transport agency responsible for the transport of neutral amino acids, including several essential amino acids. ² System L has four isoforms: l-type amino acid transporter 1 (LAT1), LAT2, LAT3, and LAT4. While LAT1 and LAT2 require CD98, a heavy chained cell surface antigen forming a heterodimer for the obligatory amino acid exchange on the plasma membrane, LAT3 and LAT4 are facilitated diffusers of amino acid substrates.^3–7

LAT1 is one of the most actively studied amino acid transporters in basic research and drug development in human cancers. Many studies have demonstrated that LAT1 is overexpressed and plays a critical role in various human cancers, including cholangiocarcinoma (CCA).^8–12 CCA is a usually fatal cancer arising from the epithelial cells of the biliary tract. The highest incidence of CCA has been reported from northeast Thailand, ¹³ where it is associated with the highest prevalence of liver fluke (Opisthorchis viverrini) infection.^14,15 There is strong evidence indicating that chronic inflammation during liver fluke infection is a key event of CCA carcinogenesis. ¹⁶ The lack of effective medical treatment makes radical surgical resection the only chance of cure; ¹⁷ however, patients with CCA typically present at an advanced stage of the disease with non-resectable tumors, resulting in a very poor prognosis and only limited therapeutic possibilities. ¹⁸ Thus, new approaches to therapy are required to improve patient survival.

JPH203 (also known as KYT0353) is a novel tyrosine analog that selectively inhibits LAT1 transport activity.^19,20 To date, only three studies have shown that JPH203 has anti-tumor activities in human cancers. JPH203 was highly effective against [¹⁴C]l-leucine uptake and cell growth in human colon cancer cells, ¹⁹ human oral cancer cells, ²¹ and leukemic cells. ²² Moreover, this specific LAT1 inhibitor showed statistically significant growth inhibition against xenografted human colon cancer cells ¹⁹ and murine T-lymphoma cells with tPTEN^−/− xenografts in the nude mouse model. ²² Furthermore, JPH203 was nontoxic to normal murine thymocytes and human peripheral blood lymphocytes, ²² suggesting that inhibition of LAT1 activity using JPH203 might be used as a novel therapeutic strategy for cancer treatment.

Our previous study has shown that the expression of LAT1 and CD98 was increased in CCA development during oxidative stress due to O. viverrini infection, which might be regulated by the oncogenic signaling pathway, phosphatidylinositol-3-kinase (PI3K)/AKT. We also showed that CCA tissues exhibited strong LAT1 immunostaining compared to normal bile duct tissues. Furthermore, LAT1 plays an important role as a tumor prognostic factor for CCA patients. ¹¹

Based on our previous report, we hypothesized that LAT1 is the main system l-amino acid transporter in CCA, potentially making it a target for CCA cell inhibition. In this study, we investigated the expression of system l-amino acid transporters and CD98 in CCA cell lines. Moreover, we determined, for the first time, the effects of a novel selective LAT1 inhibitor, JPH203, on cell growth and its mechanism for cell growth inhibition in the CCA model. Our results from the in vitro model clearly demonstrated that LAT1 is the main system l-amino acid transporter in CCA cells and that it is expressed together with CD98. Inhibition of LAT1 transport activity using JPH203 could dramatically suppress amino acid uptake and CCA cell growth through altering the cell cycle distribution patterns as well as in inducing apoptosis. Furthermore, JPH203 has anti-tumor activity against CCA cell growth in the in vivo model without general toxicity. This result indicates the possibility of using JPH203 for CCA treatment.

Materials and methods

Cell lines and cell culture

The human CCA cell lines, KKU-055, KKU-213, and KKU-100, were obtained from CCA patients and established at the Liver Fluke and Cholangiocarcinoma Research Center, Khon Kaen University. All of the cell lines were cultured in Ham’s F-12 medium (Sigma-Aldrich, St Louis, MO, USA) supplemented with inactivated 10% fetal bovine serum (FBS), 1% penicillin–streptomycin, and NaHCO₃ in a humidified atmosphere containing 5% CO₂.

Antibodies and inhibitor

Antibodies used in this study were as follows: anti-cyclin D1 (#2926D), CDK4 (#2906P), CDK6 (#3136P), P21 (#2946), and P27 (#2552P), purchased from Cell Signaling Technology (Danvers, MA, USA). Antibody against CD98 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-caspase-3 (ab32351) and anti-Ki67 were purchased from Abcam (Cambridge, UK). Anti-LAT1 antibody and JPH203 was kindly supplied by J-Pharma (Tokyo, Japan).

Reverse transcription polymerase chain reaction analysis

Reverse transcription polymerase chain reaction (RT-PCR) analysis was performed to determine the expression of the LATs and CD98 at the messenger RNA (mRNA) level. Total RNAs were prepared from the CCA cells maintained in the growth medium at 37°C in 10 cm Petri dishes using an RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) in accordance with the manufacturer’s instruction. RT-PCR analysis was performed with the PrimeScript^® RT reagent Kit (Takara Bio Inc., Shiga, Japan) under the conditions recommended by the manufacturer and used as a template for PCR amplification. PCR amplification was performed using the Promega PCR Master Mix (Promega, Madison, WI, USA) following the protocol: 95°C for 2 min; followed by 20 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s; with a final extension step of 72°C for 5 min. For LAT1, the forward and reverse primers used were 5′-TGCCTGTGTTCTTCATCCTG-3′ and 5′-CCTCCTGGCTATGTCTCCTG-3′, respectively. For LAT2, the forward and reverse primers were 5′-GCCCTCACCTTCTCCAACTA-3′ and 5′-AATGCATTCTTTGGCTCCAG-3′, respectively. For LAT3, the forward and reverse primers were 5′-CACGCTACTGCAAGATCCAA-3′ and 5′-AGAAGGGCTCTCCTTTCAGG-3′, respectively; and for LAT4, the forward and reverse primers were 5′-AAATTTGGCCTTCACTGTGG-3′ and 5′-ACGACGATGAAGGAGACACC-3′, respectively. For CD98, the forward and reverse primers were 5′-CAGAAGGATGATGTCGCTCA-3′ and 5′-CCAGTGGCGGATATAGGAGA-3′, respectively. A pair of primers, 5′-GCTG CTTT TAAC TCTG GTAA-3′ and 5′-CGCGGCCATCAC GCCACAGT-3′, was used for the PCR amplification of glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

Quantitative western blotting

To confirm the expression of LAT1 and CD98 in CCA cell lines, the cells were grown in 10 cm Petri dishes before harvesting and total protein extraction. To investigate the effects of JPH203, CCA cell lines were plated at 4 × 10⁵ cells in 10 cm Petri dishes, cultured overnight, and then treated with JPH203 at 1–100 µM and 0.1% dimethyl sulfoxide (DMSO) for 48 h. After incubation, the cells were harvested and subjected to protein extraction and western blotting. Briefly, total proteins were isolated by ice-cold radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris–HCl, pH 7.4, 1% Triton X-100, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 1 mM phenylmethylsulfonyl fluoride (PMSF), 2 mM sodium orthovanadate, and 5 mM sodium fluoride) supplied as Completed Protease Inhibitor Cocktails (1 tablet/10 mL of buffer; Roche, Basel, Switzerland). Protein concentrations were calculated using a Pierce® BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). A volume of 40 µg of proteins were then loaded onto the gels, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto polyvinylidene fluoride (PVDF) membranes (EMD Millipore Corp., Bedford, MA, USA) using the Bio-Rad western blot system (Bio-Rad Laboratories, Berkeley, CA, USA). After blocking with 5% skim milk in Tris-buffered saline (TBS), the membranes were incubated overnight at 4°C with appropriate primary antibody diluted in blocking buffer. The specific horseradish peroxidase–conjugated secondary antibody incubation was performed for 1 h at room temperature with gentle shaking. The signal was visualized using Pierce ECL Plus Western Blotting Substrate (Thermo Scientific), and the protein densitometric values were analyzed by Multi Gauge software (Fujifilm, Tokyo, Japan). The experiment was replicated three times, and densitometry values for each protein were normalized with the densitometry value of β-actin, which was used as internal control.

Amino acid uptake

To determine the inhibitory effect of JPH203 on amino acid transport in CCA cells, [¹⁴C]l-leucine (Moravek Biochemicals Inc., Brea, CA, USA) was used as the prototypical LAT1 substrate in uptake experiments evaluated via radioactivity. Briefly, the indicated cells were seeded at 2.5 × 10⁴ cells/well in 24-well plates and incubated at 37°C in 5% CO₂ for 2 days. After incubation, the adherent cells were washed three times and pre-incubated for 10 min at 37°C with 0.5 mL of sodium free Hanks’ balanced salt solution (Na⁺-free HBSS). Then, the uptake of 1 µM [¹⁴C]l-leucine was measured in the presence of JPH203 (0.01, 0.1,1, 3, and 10 µM) or 0.1% DMSO in Na⁺-free HBSS at 37°C for 1 min and was terminated by washing with cold Na⁺-free HBSS. Next, the cells were solubilized with 0.5 mL of 0.1 N NaOH for 20 min, and the cell lysate mixed with 3 mL of scintillation liquid and radioactivity was measured using an LSC-7200 β-scintillation counter (Hitachi Aloka Medical, Tokyo, Japan). An aliquot of the cell lysate was used to determine the protein concentration using a Pierce BCA Protein Assay kit (Thermo Scientific). All experiments were replicated three times. The IC₅₀ values of [¹⁴C]l-leucine uptake were calculated using GraphPad Prism5 (GraphPad Software Inc., San Diego, CA, USA).

Cell growth inhibition assay

A sulforhodamine B (SRB; Sigma-Aldrich) assay was performed to determine the effect of JPH203 on cell growth inhibition. The CCA cells were seeded at a density of 2 × 10³ cells in 100 µL of medium/well in 96-well plates and maintained at 37°C in 5% CO₂ overnight. Then, the cells were treated with different concentrations of JPH203 (0.1–100 µM) in 100 µL of medium, and 0.1% DMSO was used as a control at 24, 48, and 72 h. Later, the cell numbers were estimated using an SRB assay, as previously described. ²³ In brief, the adherent cells were fixed with 100 µL of 10% trichloroacetic acid (TCA, Wako Pure Chemical Industries Ltd., Tokyo, Japan) for 1 h at 4°C. Then, they were washed with deionized water and stained with 0.4% SRB dye in 1% acetic acid for 30 min. After removal of the SRB dye, the cells were washed with 1% acetic acid and were allowed to air dry. Thereafter, SRB-stained cellular proteins were dissolved in 200 µL of 10 mM Tris base (pH 10.5). The optical densities were measured at 540 nm in an enzyme-linked immunosorbent assay (ELISA) plate reader, Varioskan Flash (Thermo Fisher Scientific, Vantaa, Finland). The percent viability of the cells in each well was calculated. The experiments were replicated three times, and the IC₅₀ values were calculated using GraphPad Prism 5 (GraphPad Software Inc.).

Cell cycle analysis

CCA cells were seeded and treated with JPH203 as described in section “Quantitative western blotting.” After incubation, 1 × 10⁶ cells per experimental condition were harvested and washed with cold phosphate-buffered saline (PBS). Thereafter, the cells were fixed with 70% cold ethanol and kept at −20°C until analysis. After fixation, the cells were washed with cold PBS and incubated with 100 µg/mL RNaseA (Applichem Inc., Cheshire, CT, USA), and the cell cycle distribution was determined by staining the DNA with 40 µg/mL of propidium iodide (PI; Invitrogen, Paisley, UK) for 30 min. The results were analyzed by flow cytometry (FACS Calibur, BD Biosciences, San Jose, CA, USA). All experiments were done in triplicate.

Trypan blue dye exclusion assay

Six-well culture plates were used for cell culture. After plating the CCA cells at 1 × 10⁵ cells/well and culturing for overnight, designed concentrations of JPH203 were added for 48 h. After the treatment period, the cells were trypsinized and washed with PBS. The cells were then mixed thoroughly and 10 µL of cell suspension was added to 10 µL of 0.4% trypan blue dye (Bio-Rad Laboratories, Inc., Watford, UK) solution. Dead cells were characterized by the uptake of the dye and expressed as the percentage of cells staining blue. The results were detected using a TC20^™ Automated Cell Counter (Bio-Rad Laboratories, Inc., Berkeley, CA, USA). All experiments were done in triplicate.

Experimental animals

Six-week-old male athymic BALB/c nude mice (Clea Japan, Inc., Tokyo, Japan) were housed and monitored under pathogen-free conditions in accordance with institutional principals. Food and tap water were provided ad libitum. Mice were subcutaneously injected with 2 × 10⁶ KKU-213 cells in 0.5 mL of medium into the dorsal area. After 3 days, the mice were randomly divided into four groups: a control group (n = 5), JPH203 6.3 mg/kg treatment group (n = 5), JPH203 12.5 mg/kg treatment group (n = 5), and JPH203 25 mg/kg treatment group (n = 5). On the day of grouping (day 0), body weights and diameters of the tumor (length and width) were measured. The tumor volumes were calculated using the standard formula: tumor volume (mm³) = longer diameter × (shorter diameter)²/2. After grouping, the mice were intravenously injected with 0.2 mL of normal saline solution (NSS) daily for the control group and JPH203 for the treatment groups for 20 days. Tumor volumes and body weights were assessed twice a week. The tumor volumes were expressed relative to the initial tumor volume (day 0). After treatment, all mice were sacrificed for the collection of tumor samples as well as the liver, lungs, spleen, and kidneys. Formalin-fixed paraffin-embedded sections from the internal organs were analyzed by hematoxylin and eosin (H&E) staining according to standard methods. All slides were reviewed by a pathologist and were photographed using an Olympus CKX41 Microscope (Olympus, Tokyo, Japan) with CellSens standard software. The in vivo study was approved by the local animal welfare committee and was carried out in accordance with the local regulations.

Immunohistochemistry

The formalin-fixed and paraffin-embedded tumors were sliced into 4 µm sections and were deparaffinized in xylene and then rehydrated in graded alcohol. Antigen retrieval was accomplished by incubating the slides in Tris–EDTA Buffer (10 mM Tris base, 1 mM EDTA solution, and 0.05% Tween 20; pH 9.0) for 3 min in a pressure cooker. Then, the slides were incubated in 0.3% H₂O₂ for 30 min to suppress endogenous peroxidase activity and then washed with PBS. Next, the slides were blocked with 10% skim milk in PBS for 30 min. The rabbit polyclonal Ki67 antibody was used as primary antibody at a 1:300 dilution in PBS overnight at 4°C. The following day, after several washes with PBS, the slides were incubated with peroxidase-conjugated EnVision^™ secondary antibody (Dako, Glostrup, Denmark) for 90 min, and a peroxidase-labeled polymer, Diaminobenzidine tetrahydrochloride (DAB) solution, was used for signal development for 5 min. The sections were counterstained with hematoxylin followed by dehydrating and mounting. Ki67 positive cells of each tumor sections were counted in least five of ×400 power fields.

Statistical analysis

Statistical analysis was performed using GraphPad Prism version 5 (GraphPad Software Inc.). The data are expressed as mean ± standard deviation (SD) or mean ± standard error of the mean (SEM). The results of the western blotting were analyzed with one-way analysis of variance (ANOVA) followed by a Tukey’s multiple-comparison test. The results of cell cycle analysis, dye exclusion assay, and percent Ki67 positive cells were analyzed by Student’s t test. The results of the relative tumor volume were analyzed using a two-way ANOVA. A value of p < 0.05 was considered to indicate a statistically significant difference.

Results

Expression of the system l-amino acid transporters and CD98 in CCA cells

The PCR products for LAT1 and CD98 were clearly detected in all cells studied, whereas LAT2, LAT3, and LAT4 were hardly detected (Figure 1(a)). The presence of LAT1 and CD98 in all cells studied was further confirmed by quantitative western blotting. Consistent with the RT-PCR analysis, the CCA cells clearly expressed LAT1 and CD98 (Figure 1(b)). For the functional expression at the plasma membrane, LAT1, a non-glycosylated light chain with an apparent molecular mass of 40 kDa, forms a heterodimeric complex via disulfide bond with a glycosylated heavy chain or CD98 with an apparent molecular mass around 85 kDa. The heterodimeric complex of LAT1–CD98 is approximately 125 kDa. The results from the RT-PCR analysis and western blotting indicate that LAT1 is the main system l-transporter in our CCA cell lines and occurs together with CD98.

Figure 1.

Expression of LAT1 and CD98 and the functional effects of LAT1 inhibition on [¹⁴C]l-leucine uptake and cell growth in CCA cells. (a) Detection of system l-amino acid transporters (LAT1-LAT4) and CD98 by RT-PCR in CCA cells (KKU-055, KKU-213, and KKU-100). (b) Expression of LAT1 and CD98 levels in the indicated cells detected by quantitative western blotting. The densitometry value of LAT1 and CD98 was normalized to β-actin. The bar graphs show the average densitometry value ± SEM from three independent experiments. (c–e) Inhibition of [¹⁴C]l-leucine uptake by JPH203 in CCA cells. [¹⁴C]l-leucine uptake was measured for 1 min in the presence of various concentrations of JPH203. (f–h) JPH203 suppressed cell growth in CCA cells. The indicated cells were treated with various concentrations of JPH203 for 24, 48, and 72 h. The cell viabilities were determined by SRB assays and the growth curves are presented as percentage of untreated control. Each data point represents the mean ± SEM from three independent experiments.

JPH203 inhibits [¹⁴C]l-leucine uptake and cell growth in CCA cell lines

The inhibitory effects of JPH203 on [¹⁴C]l-leucine uptake and cell growth in CCA cells are summarized in Figure 1 and Table 1. At the lowest concentration of JPH203 (0.01 µM), [¹⁴C]l-leucine uptake in all CCA cells was slightly increased when compared to the untreated control. However, 0.1–10 µM JPH203 dramatically inhibited [¹⁴C]l-leucine uptake in a dose-dependent manner in all CCA cells studied with low IC₅₀ values. The KKU-213 cell line was more sensitive to JPH203 than KKU-055 and KKU-100 cell lines (Figure 1(c)–(e) and Table 1). The IC₅₀ values (mean ± SD) were 0.20 ± 0.03 µM for KKU-055, 0.12 ± 0.02 µM for KKU-213 cells, and 0.25 ± 0.04 µM for KKU-100 (Table 1).

Table 1.

Inhibitory effects of JPH203 on [¹⁴C]l-leucine uptake and cell growth inhibition in CCA cells.

Cell line	IC50 (µM) value for [14C]l-leucine uptake inhibition	IC50 (µM) value for growth inhibition
		24 h	48 h	72 h
KKU-055	0.20 ± 0.03	31.95 ± 1.15	6.16 ± 1.17	5.78 ± 1.15
KKU-213	0.12 ± 0.02	32.95 ± 1.16	5.98 ± 1.16	2.47 ± 1.19
KKU-100	0.25 ± 0.04	48.74 ± 1.22	21.51 ± 1.25	3.00 ± 1.28

To determine whether blocking [¹⁴C]l-leucine uptake using JPH203 suppresses CCA cell growth, an SRB assay was performed after treatment of the CCA cells with JPH203 at various concentrations for 24, 48, and 72 h in 96-well plates. As shown in Figure 1 and Table 1, JPH203 could inhibit the proliferation of CCA cells and IC₅₀ values were decreased in a time-dependent manner. However, the drug response for each CCA cell line to JPH203 showed different patterns, as indicated in Figure 1(f). At 0.1 and 1 µM JPH203, there were hardly any inhibition of KKU-055 CCA cell proliferation compared with KKU-213 and KKU-100 (Figure 1(g) and (h)). Consistent with the result for amino acid uptake, KKU-213 was more sensitive to JPH203 than KKU-055 and KKU-100 (IC₅₀ at 24–72 h for KKU-213 was lower than that of KKU-055 and KKU-100; Table 1).

JPH203 altered LAT1 and CD98 expression

A previous study demonstrated that inhibition system L activity in KKU-213 CCA cells using a broad system L inhibitor, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), can upregulate the expression of LAT1 and CD98. ⁹ Likewise, upregulated amino acid transporters, including LAT1, CD98, xCT, ASCT1, and ASCT2, were reported in human prostate cancer cell lines treated with BCH and leucine-free media. ²⁴ We, therefore, investigated the impact of JPH203 on LAT1 and CD98 expression. In accordance with previous reports, after the treatment of KKU-055 and KKU-213 cell lines with JPH203 (0–100 µM) for 48 h, both LAT1 and CD98 levels increased in a dose-dependent manner as detected by quantitative western blotting (Figure 2(a)–(f)). These results indicate that upregulated LAT1 and CD98 expression is a feedback effect related to amino acid deprivation caused by the suppression of LAT1 activity using JPH203.

Figure 2.

The effect of JPH203 on LAT1 and CD98 expression in CCA cells detected by quantitative immunoblot. (a–c) LAT1 and CD98 expressions were increased in a dose-dependent manner following treatment with JPH203 for 48 h in KKU-055 cells. (d–f) LAT1 and CD98 expressions were increased in a dose-dependent manner following treatment with JPH203 for 48 h in KKU-213 cells. The results show a representative western blot for LAT1 and CD98 expression. Bar graphs show the densitometry values normalized to β-actin and were presented as the percentage compared to the untreated control: They show the mean ± SEM from three independent experiments.

JPH203 induced cell cycle arrest through regulating the cell cycle regulators

To determine the mechanism of the anti-proliferative effect of JPH203, cell cycle analysis was performed using flow cytometric analysis after staining the cells with PI. As shown in Figure 3(a), exposure of the KKU-055 cells to 10–100 µM JPH203 for 48 h resulted in a statistically significant increase in G2/M-phase (p < 0.01) cells compared with the control. This was accompanied by a significant decrease in G0/G1 phase cells at 10 µM (p < 0.05) and a significant decrease in S-phase cells at 100 µM (p < 0.05). Unlike KKU-055, the results for KKU-213 showed that the number of cells in the G0/G1 phase was significantly increased in response to 10–100 µM JPH203 (p < 0.01 at 10 µM and p < 0.001 at 100 µM). Concomitantly, 10–100 µM of JPH203 significantly decreased the number of S-phase cells compared to the control (p < 0.001). However, a slight decrease of G2/M-phase cells was detected in a dose-dependent manner, but this was not statistically significant compared with the control (Figure 4(a)).

Figure 3.

JPH203 induced cell cycle arrest in KKU-055 cells by regulating cell cycle–related proteins. (a) A volume of 10–100 µM JPH203 significantly increased G2/M-phase cells (p < 0.01) which was accompanied by significantly decreased G0/G1 phase at 10 µM (p < 0.05) and decreased S phase at 100 µM (p < 0.01) after treatment for 48 h, detected by flow cytometry. Data are expressed as the mean ± SD of the percentage of cells in each cycle phase from three independent experiments. Results were analyzed by Student’s t test. (b) A representative western blot for the expression of cell cycle–related proteins, including cyclin D1, CDK4, CDK6, P21, and P27 in KKU-055 cells after treatment with JPH203 for 48 h. (c–g) The densitometry values of each protein were normalized to β-actin and were presented as the percentage compared to the untreated control. The mean ± SEM from three independent experiments are presented (***p < 0.001 versus control, one-way ANOVA followed by a Tukey’s multiple-comparison test).

Figure 4.

JPH203 induced cell cycle arrest in KKU-213 cells by regulating cell cycle–related proteins. (a) A volume of 10–100 µM JPH203 increased G0/G1-phase cells (p < 0.01 and p < 0.001 at the dose of 10 and 100 µM, respectively) and decreased S-phase cells (p < 0.001) after treatment for 48 h, detected by flow cytometry. Data are expressed as the mean ± SD of the percentage of cells in each cycle phase from three independent experiments. Results were analyzed by Student’s t test. (b) A representative western blot for the expression of cell cycle–related proteins, including cyclin D1, CDK4, CDK6, P21, and P27 in KKU-213 cells after treatment with JPH203 for 48 h. (c–g) The densitometry values of each protein were normalized to β-actin and were present as percentage compared to the untreated control. The mean ± SEM from three independent experiments are presented (*p < 0.05, **p < 0.01 vs control, one-way ANOVA followed by a Tukey’s multiple-comparison test).

To gain insight into the mechanism of cell cycle arrest on treatment with JPH203, we further investigated the expression levels of cell cycle–regulator proteins which function in the G0/G1 phase, including cyclin D1, CDK4, and CDK6. ²⁵ The expression of the inhibitors of CDKs, P21, and P27, which affect both G1/S and the G2/M,^26–28 was also investigated in this experiment. The CCA cells were exposed to JPH203 for 48 h and prepared for western blotting. The results in KKU-055 showed that JPH203 increased the levels of cyclin D1 and CDK4, except that the level of CDK4 at 100 µM was decreased by JPH203 (Figure 3(b)–(d)). In addition, 10–100 µM JPH203 could strongly suppress CDK6 levels (p < 0.001) as well as induce higher P21 levels when compared with control (Figure 3(b), (e), and (f)). Moreover, 100 µM of JPH203 markedly increased P27 expression as shown in Figure 3(g). This result indicates that the induction of growth inhibition by JPH203 in KKU-055 cells was caused by G2/M arrest mainly through elevated P21 levels. However, inhibition of KKU-055 cell growth at the highest JPH203 concentration (100 µM) is caused by both G2/M arrest and reduced S-phase cells due to increasing P21 and P27 levels and decreasing CDK4 and CDK6 levels.

As expected, the results for KKU-213 showed that the levels of cyclin D1, CDK4, and CDK6 which are required for G1/S phase transition were remarkably decreased in a dose-dependent manner when compared to the control, with significant differences being observed at 10–100 µM JPH203 for CDK4 levels (p < 0.05 and p < 0.01, respectively; Figure 4(b)–(e)). In addition, the levels of P21 increased in a dose-dependent manner, but this was not the case for the CDK inhibitor P27 (Figure 4(b), (f), and (g)). These results indicate that, in KKU-213 cells, the growth inhibition of JPH203 caused by cell cycle arrest at the G0/G1-phase and reduced S phase occurs via an increase in the CDK inhibitor P21. This may further inhibit the activity of cyclin D1–CDK4/CDK6, which is downregulated by this selective LAT1 inhibitor.

Apoptosis induction in response to JPH203 in CCA cells

Since JPH203-induced apoptosis has been demonstrated in human oral cancer ²¹ and leukemic cells, ²² we investigated the effect of this specific LAT1 inhibitor on apoptosis induction in our CCA model. To validate the induction of apoptosis in KKU-055 and KKU-213 CCA cells, a trypan blue dye exclusion assay was conducted to measure the percentage of cell death after 48 h of JPH203 (0–100 µM) treatment. In addition, the apoptotic-related protein, caspase-3, and its cleaved form were assessed by immunoblot. Our results showed that JPH203 could induce cell death in both CCA cell lines, as shown in Figure 5(a) and (b). Cell death was less than 15% in KKU-055 and 30% in KKU-213 after treatment with 100 µM JPH203. This was a significant change, p < 0.001, compared with the control. The percentage of cell death decreased slightly at 1 µM and increased slightly at 10 µM of JPH203 in both CCA cell lines compared to the control. The results from quantitative western blotting showed that the treatment of the CCA cells with JPH203 at 100 µM for KKU-055 and 10–100 µM for KKU-213 increased the cleaved caspase-3/caspase-3 ratio when compared to the control (Figure 5(c)–(e)). A statistically significant difference in the cleaved caspase-3/caspase-3 ratio was observed between KKU-213 treated with 100 µM JPH303 and the control (p < 0.05). These results suggest that JPH203-induced apoptosis is regulated by the activation of the caspase cascade under the highest concentration tested (100 µM), and that the KKU-213 cell line was more sensitive to JPH203 than the KKU-055 cell line.

Figure 5.

The effects of JPH203 on apoptosis induction in CCA cells. (a and b) CCA cells were treated with various concentrations of JPH203 (0–100 µM) for 48 h. The percentage of cell death was measured by trypan blue staining. The data represent the mean ± SEM from three independent experiments (***p < 0.001 by using a Student’s t test in comparison with the control). (c) Expression and activation of the apoptotic protein caspase-3 by JPH203 in CCA cells. The indicated cells were stimulated with JPH203 (0–100 µM) for 48 h. The cell lysate was prepared and analyzed by quantitative immunoblot. (d and e) Densitometry values of caspase-3 and its cleaved form were normalized to β-actin and expressed as ratio of the cleaved caspase-3/caspase-3. The mean ± SEM are shown from three independent experiments and analyzed by one-way ANOVA followed by a Tukey’s multiple-comparison test (*p < 0.05 compared to the untreated control).

JPH203 suppressed CCA cell growth in the in vivo model

Our in vitro models demonstrated that the KKU-213 cell line was the most sensitive to JPH203 when compared to the other cell lines. Thus, we evaluated the anti-tumor activity of JPH203 in a nude mouse xenograft model derived from KKU-213 CCA cells. JPH203 was administered intravenously daily for 20 days at three different doses (6.3, 12.5, and 25.0 mg/kg) starting at day 3 after the injection of cancer cells. On the days 18 and 21, JPH203 showed dose-dependent inhibition on tumor growth with significantly inhibited tumor growth in the groups of JPH203 at 12.5 mg/kg (on day 18, p < 0.05, and on day 21, p < 0.01) and 25 mg/kg (on day 18 and day 21, p < 0.001) when compared to the control group (Figure 6(a) and (b)). To confirm our findings on this growth suppression mechanism, immunohistochemical analysis was conducted to identify the expression of a proliferation marker Ki67 in the tumor tissues. Reduction of the cells positive for Ki67 was found in JPH203 treatment groups at 12.5 and 25 mg/kg (p < 0.01) compared with control group (Figure 6(c) and (d)). In contrast to the effects on tumor growth, the animals did not show any clinical signs of toxicity, changes in general behavior, or changes in physical activity in the JPH203-treated animals compared to the controls. Mice treated with JPH203 were healthy and had similar body weights to the control mice (Supplementary Figure 1). In addition, a histopathological review of the internal organs, including in the liver, lungs, spleen, and kidneys, analyzed by H&E staining showed no discernible toxicity of JPH203 (Supplementary Figure 1). There were no differences in the weight or appearance of the internal organs of mice between the control and treated groups. These results indicate that JPH203 was safe for the animals studied and that it should be considered for use in CCA patients.

Figure 6.

Anti-tumor activity of JPH203 in CCA-inoculated athymic BALB/c nude mice. (a) CCA tumor tissues were obtained from vehicle and JPH203-treated mice (n = 5/group). (b) The tumor volume was expressed relative to the initial tumor volume. The data were analyzed by two-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001 compared to the control group). (c) IHC to identify expression of Ki67 in tumor tissues, magnification ×400. (d) Bar graphs illustrate the proportion of Ki67 positive cells. The data of Ki67 positive cells were expressed as mean ± SEM (**p < 0.01 analyzed by Student’s t test).

Discussion

Although suppressed LAT1 activity using BCH has been reported in CCA,^8,9 BCH is a broad system L inhibitor that targets all members of LAT family. Therefore, BCH lacks selectivity for both LAT1 and cancer cells, which results in low cytotoxicity for several cancer cell lines, which is undesirable in clinical trials.^29,30 Our previous study ¹¹ showed that LAT1 expression is significantly associated with O. viverrini-induced cholangiocarcinogenesis and a shorter survival time in CCA patients. Moreover, the expression of LAT1 and CD98 in CCA is possibly regulated through the oncogenic PI3K/AKT signaling pathway. Collectively, these data led us to investigate, for the first time, the effects of a selective LAT1 inhibitor in our CCA model.

Our results show that LAT1 is the main system l-transporter, along with CD98, in CCA cells, including the KKU-055, KKU-213, and KKU-100 cell lines, which conforms to a previous study from Janpipatkul et al. ⁹ who reported a lower expression of LAT2, LAT3, and LAT4 than LAT1 in KKU-213 cells. Interestingly, our results indicate that JPH203 possesses a higher efficacy for inhibiting CCA cell proliferation than for BCH as shown in previous reports.^9,8 This phenomenon is possible due to the specificity of JPH203 to LAT1 in CCA cells being much higher than BCH.

The treatment of the CCA cells with JPH203 showed that the IC₅₀ values for [¹⁴C]l-leucine uptake inhibition were lower than those for cell growth inhibition. This is consistent with two previous studies reporting the effect of JPH203 on human colon cancer ¹⁹ and human oral cancer. ²¹ Oda et al. ¹⁹ suggested that JPH203 inhibits LAT1 by competing with its substrates; therefore, the differences in the IC₅₀ values would be caused by the concentrations of LAT1 substrates (neutral amino acids) in the incubation medium. Moreover, we also demonstrated that amino acid deprivation caused by JPH203 treatment for 48 h results in increasing LAT1 and CD98 expressions. This is likely to require a higher concentration of JPH203 to target LAT1 in the cell culture experiments that were performed for 24–74 h when compared to amino acid uptake experiments that were performed with a short incubation period (1 min). Furthermore, the increase in LAT1 and CD98 expressions may function as a compensatory survival mechanism under conditions of amino acid starvation. This result suggests that LAT1 and CD98 expression are regulated by intracellular amino acid levels, which is supported by previous studies in prostate cancer ²⁴ and also CCA. ⁹

Previous studies have also shown that amino acid depletion mediated by BCH contributes to cell cycle arrest at the G1 phase. This is regulated by cell cycle–related proteins such as cyclin D3, CDK6, and P27 in human oral cancer, ³¹ and cyclin D3 along with P27 in human prostate cancer. ³² These data are supported by our results; we show that JPH203 clearly induced cell cycle arrest at the G0/G1 phase and reduced S-phase progression in KKU-213 cells via the regulation of cell cycle–related proteins, including cyclin D1, CDK4, CDK6, and P21. Our results also reveal the different growth inhibition mechanism of JPH203 in KKU-055. This specific LAT1 inhibitor induced G2/M cell cycle arrest and reduced S-phase cells via increasing P21 and P27 concentrations in KKU-055. The increase in P21 and P27 during G2/M arrest by anti-cancer agents has been reported in many types of human cancer.^33–36 For example, gallic acid can induce cell cycle arrest at the G2/M phase through increased P21 and P27 expression in breast cancer. ³⁶ However, from our results, the different pattern of cell cycle distribution caused by JPH203 treatment in our CCA cells indicates that JPH203 can selectively target several components of the cell cycle machinery to arrest the cell cycle. This is dependent on both cell type and drug concentration.

Caspase proteins play a critical role in apoptosis and are responsible for many of the biochemical and morphological changes associated with this phenomenon. Therefore, an increased level of activated caspase proteins is one of the most common apoptosis markers that has been used to indicate apoptosis phenotype of cell.^37,38 Two previous studies reported that JPH203 can induce caspase-dependent apoptosis in cancer cells.^21,22 The previous study demonstrated that JPH203 can induce apoptosis of human oral cancer revealed by an increase of apoptosis markers included the cleaved form of caspase-3, caspase-7, and caspase-9, as well as cleaved poly (ADP-ribose) polymerase (PARP) together with 16.5% of Annexin V–FITC-positive cells. These were observed after treatment of the cells with 3 mM of JPH203 for 24 h. ²¹ The next study on leukemia reported that JPH203 induced type ІІ cell death, autophagy, followed by caspase-3-dependent apoptosis at 48 h after treatment. ²² Consistent with these studies, our data showed that the treatment of CCA cells with JPH203 (100 µM) for 48 h could increase the ratio of cleaved caspase-3/preform along with an increased number of dead cells, more than 10% and 20% for KKU-055 and KKU-213, respectively. These results indicate that JPH203 induced apoptosis in CCA cells via caspase-3 activation. However, the molecular mechanisms underlying apoptosis caused by JPH203 in the CCA model require more study.

To date, there is only one study on the effect of a LAT1 inhibitor using the CCA xenograft model. Kaira et al. ⁸ demonstrated the anti-tumor efficacy of BCH in HuCCT1 CCA xenograft nude mice; however, as indicated above, BCH does not specifically target LAT1 but also other system l-amino acid transporters. Kaira et al. ⁸ reported that daily intravenous administration of BCH, up to 200 mg/kg for 14 days, caused a statistically significant delay in tumor growth for 3 weeks after treatment. Compared to Kaira et al.’s study, daily intravenous administration of JPH203 at amounts substantially lower than for BCH (16-fold, 12.5 mg/kg; 8-fold, 25 mg/kg) for 20 days could significantly inhibit KKU-213 CCA cell growth in nude mice starting at day 18. This was confirmed by the decreasing percent of Ki67 positive cells found. Our results indicate that JPH203 is considerably more effective than BCH against CCA cell growth, conforming with the IC₅₀ values in inhibiting [¹⁴C]l-leucine uptake and cell growth in the in vitro model.^8,9 Furthermore, our results support the study of Oda et al., the first report of JPH203 in an in vivo model, using intravenously administered JPH203 (12.5 and 25.0 mg/kg). This showed a statistically significant inhibition of growth of HT-29 tumors transplanted to nude mice with only a slight decrease in body weight. ¹⁹ However, in our study, there was no statistically significant change that could be attributed to general toxicity, such as changes in the animals’ body weight, or the histology or appearance of the internal organs.

Taken together, this study demonstrates that the inhibition of LAT1 activity using JPH203 in CCA cells which show high LAT1 and CD98 expression leads to an intracellular decrease in essential neutral amino acids. This results in an altered expression of LAT1 and CD98, inhibiting cell growth and inducing cell cycle arrest and apoptosis in the in vitro model. Moreover, JPH203 shows anti-tumor efficacy in nude mice bearing human CCA cell xenografts without general toxicity. This study is the first to demonstrate the effects of a selective LAT1 inhibitor in the CCA model, thus providing useful information for the development of JPH203 as a therapeutic strategy for CCA patients.