Korean J Physiol Pharmacol.  2017 Nov;21(6):599-607. 10.4196/kjpp.2017.21.6.599.

JPH203, a selective L-type amino acid transporter 1 inhibitor, induces mitochondria-dependent apoptosis in Saos2 human osteosarcoma cells

Affiliations
  • 1Department of Pharmacology, Chonnam National University Medical School, Gwangju 61469, Korea. jkkim57@jnu.ac.kr
  • 2Department of Oral Physiology, Chosun University School of Dentistry, Gwangju 61452, Korea.
  • 3Department of Bio-system Pharmacology, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan.
  • 4Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Denver Anschutz Medical Campus, Aurora, Colorado 80045, USA.
  • 5Department of Pharmacology and Toxicology, Kyorin University School of Medicine, Tokyo 181-8611, Japan.
  • 6J-Pharma Co., Ltd., Yokohama, Kanagawa 230-0046, Japan.

Abstract

Most normal cells express L-type amino acid transporter 2 (LAT2). However, L-type amino acid transporter 1 (LAT1) is highly expressed in many tumor cells and presumed to support their increased growth and proliferation. This study examined the effects of JPH203, a selective LAT1 inhibitor, on cell growth and its mechanism for cell death in Saos2 human osteosarcoma cells. FOB human osteoblastic cells and Saos2 cells expressed LAT1 and LAT2 together with their associating protein 4F2 heavy chain, but the expression of LAT2 in the Saos2 cells was especially weak. JPH203 and BCH, a non-selective L-type amino acid transporter inhibitor, potently inhibited L-leucine uptake in Saos2 cells. As expected, the intrinsic ability of JPH203 to inhibit L-leucine uptake was far more efficient than that of BCH in Saos2 cells. Likewise, JPH203 and BCH inhibited Saos2 cell growth with JPH203 being superior to BCH in this regard. Furthermore, JPH203 increased apoptosis rates and formed DNA ladder in Saos2 cells. Moreover, JPH203 activated the mitochondria-dependent apoptotic signaling pathway by upregulating pro-apoptotic factors, such as Bad, Bax, and Bak, and the active form of caspase-9, and downregulating anti-apoptotic factors, such as Bcl-2 and Bcl-xL. These results suggest that the inhibition of LAT1 activity via JPH203, which may act as a potential novel anti-cancer agent, leads to apoptosis mediated by the mitochondria-dependent intrinsic apoptotic signaling pathway by inducing the intracellular depletion of neutral amino acids essential for cell growth in Saos2 human osteosarcoma cells.

Keyword

Anti-cancer therapy; Apoptosis; JPH203; L-type amino acid transporter 1; Osteosarcoma cells

MeSH Terms

Amino Acid Transport Systems*
Amino Acids, Neutral
Antigens, CD98 Heavy Chain
Apoptosis*
Caspase 9
Cell Death
DNA
Humans*
Leucine
Osteoblasts
Osteosarcoma*
Amino Acid Transport Systems
Amino Acids, Neutral
Antigens, CD98 Heavy Chain
Caspase 9
DNA
Leucine

Figure

  • Fig. 1 Chemical structure of JPH203.

  • Fig. 2 Detection of LAT1, LAT2, and 4F2hc by qRT-PCR in Saos2 and FOB cells. Detection of LAT1, LAT2, and 4F2hc in Saos2 (A) and FOB cells (B). The qRT-PCR was performed as described in METHODS. The qRT-PCR results, which were recorded as threshold cycle numbers (Ct), were normalized against an internal control (GAPDH). The deviations in the results represent four separate experiments.

  • Fig. 3 Concentration-dependent inhibition of [14C]L-leucine uptake by JPH203 and BCH in Saos2 and FOB cells. [14C]L-leucine uptake (1 µM) was measured for 1 min in the presence of various JPH203 (A) and BCH (B) concentrations in Saos2 and FOB cells. The results were expressed as a percentage of the control L-leucine uptake in the absence of JPH203 or BCH. The control cells were measured in the absence of JPH203 or BCH treatment. Each data point represents the mean±S.E.M. for four experiments. *p<0.05 vs. control, **p<0.01 vs. control and ***p<0.001 vs. control.

  • Fig. 4 Effect of JPH203 and BCH on cell viability in Saos2 and FOB cells. Saos2 and FOB cells were treated with various concentrations of JPH203 (A, Saos2 cells; B, FOB cells) or BCH (C, Saos2 cells; D, FOB cells) for 1-4 days. The cell viabilities were determined by MTT assays. The percentage cell viability was calculated as a ratio of the optical density at 570 nm of JPH203 or BCH treated cells and untreated control cells. The control cells were measured in the absence of JPH203 or BCH treatment. Each data point represents the mean±S.E.M. of four experiments. *p<0.05 vs. control, **p<0.01 vs. control and ***p<0.001 vs. control.

  • Fig. 5 Inhibition of colony formation by JPH203 in Saos2 cells. (A) Colony formation was assessed in Saos2 cells treated with 100 µM JPH203 for 10 days, and colonies were counted. (B) Each data point represents the mean±S.E.M. for four experiments. *p<0.05 vs. control.

  • Fig. 6 JPH203-induced cell death in Saos2 cells through apoptosis. (A) Apoptotic cell population induction of Saos2 cells by JPH203. To identify the JPH203-induced Saos2 cell apoptosis, FACS analysis were performed following Annexin-V and PI staining. Saos2 cells were cultured in complete medium, and treated with 3 mM JPH203 for 24 h. After treatment, the cells were analyzed by flow cytometry. LL, lower left (normal); UL, upper left (necrosis); LR, lower right (early phase of apoptosis); UR, upper right (late phase of apoptosis). (B) Fragmentation of genomic DNA by JPH203. Saos2 cells were treated with 3 mM JPH203 for 24 h and the genomic DNA was subjected to agarose gel electrophoresis.

  • Fig. 7 JPH203-induced apoptosis in Saos2 cells mediated by the mitochondria-mediated intrinsic apoptotic pathway. Saos2 cells were treated with 3 mM JPH203 for 24 h. Cell lysate was prepared and analyzed by immunoblotting as described in METHODS.


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