Role of L-Type Amino Acid Transporter 1 (LAT1) for the Selective Cytotoxicity of Sesamol in Human Melanoma Cells
Abstract
:1. Introduction
2. Results
2.1. Sesamol Distribution into SK-MEL-2 and Vero Cells
2.2. Uptake Kinetics of Sesamol in SK-MEL-2 and Vero Cells
2.3. LAT1 Activity and Equilibrium Distribution of Sesamol
2.4. Inhibition of Sesamol Uptake Kinetics in SK-MEL-2 and Vero Cells
2.5. Anti-Proliferation in SK-MEL-2 and Vero Cells
2.6. mRNA Expression of LAT1 Protein in SK-MEL-2 and Vero Cells
3. Discussion
4. Materials and Methods
4.1. Materials
4.2. Cell Lines and Cell Culture
4.3. Cellular Uptake of Sesamol
4.3.1. Time-Course Study
4.3.2. Uptake Kinetics
4.3.3. LAT1 Function
4.3.4. Competitive Uptake between Sesamol and the other LAT1 Substrates
4.3.5. Uptake Kinetics of Sesamol via LAT1-Mediated Transport
4.4. HPLC Analysis
4.5. Antiproliferative Effect
4.6. mRNA Expression of LAT1 and LAT2
4.7. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Sample Availability: Not available. |
Conditions | Sesamol Uptake Kinetic Parameters | |||||
---|---|---|---|---|---|---|
SK-MEL-2 | Vero | |||||
Vmax (nmol/min/mg) | Km (µM) | Pd (µL/min/mg) | Vmax (nmol/min/mg) | Km (µM) | Pd (µL/min/mg) | |
37 °C | 4.2 ± 0.1a | 224.6 ± 19.3a | 2.3 ± 0.2b | 0.9 ± 0.0a | 159.9 ± 18.4a | 2.1 ± 0.1a |
4 °C | 0.3 ± 0.1b | 26.8 ± 9.8b | 2.7 ± 0.1a | 0.4 ± 0.1b | 66.1 ± 20.0b | 2.4 ± 0.3a |
Compounds | Percent Inhibition of L-Dopa Distribution Volume (L-Dopa Distribution Volume; µL/mg Protein) | |
SK-MEL-2 | Vero Cells | |
Control | 0% (370.6 ± 13.0)a | 0% (155.6 ± 17.8)a |
1 mM BCH | 82% (67.3 ± 14.0)c | 69% (47.9 ± 3.7)b,c,d |
1 mM L-tyrosine | 72% (105.0 ± 5.2)c | 69% (48.7 ± 11.0)c,d |
2 mM L-leucine | 75% (94.1 ± 13.5)c | 75% (39.1 ± 2.3)d |
2 mM L-Alanine | 67% (121.2 ± 3.6)b | 35% (101.3 ± 4.0)a,b |
0.05 mM Melphalan | 47% (196.6 ± 20.3)a | 39% (95.5 ± 11.2)a,b,c |
1 mM Sesamol | 44% (207.6 ± 31.5)a | 33% (103.9 ± 9.2)a,b |
Compounds | Percent Inhibition of Sesamol Distribution Volume (Sesamol Distribution Volume; µL/mg Protein) | |
SK-MEL-2 | Vero Cells | |
Control | 0% (23.2 ± 2.1)a | 0% (17.9 ± 1.7)a |
1 mM BCH | 55% (10.3 ± 0.9)c,d | 42% (10.4 ± 1.9)c |
1 mM L-tyrosine | 75% (5.8 ± 0.2)e | 41% (10.5 ± 0.7) c |
2 mM L-leucine | 50% (11.7 ± 0.7)c | 23% (13.8 ± 1.4)b |
2 mM L-Alanine | 44% (13.1 ± 0.3)b | 25% (13.3 ± 2.0)b,c |
0.05 mM Melphalan | 60% (9.4 ± 0.7)c,d | 35% (11.7 ± 0.7)b,c |
1 mM L-Dopa | 68% (7.4 ± 0.8)d | 40% (10.8 ± 2.0)b,c |
Compounds | Sesamol Uptake Kinetic Parameters | |||||
---|---|---|---|---|---|---|
SK-MEL-2 | Vero | |||||
Vmax (nmol/min/mg) | Km (µM) | Pd (µL/min/mg) | Vmax (nmol/min/mg) | Km (µM) | Pd (µL/min/mg) | |
Sesamol | 4.2 ± 0.1a | 224.6 ± 19.3a | 2.3 ± 0.2b | 0.9 ± 0.0a | 159.9 ± 18.4a | 2.1 ± 0.1a |
Sesamol + BCH | 0.2 ± 0.0d | 6.0 ± 1.1d | 0.5 ± 0.1d | 0.3 ± 0.1b | 38.2 ± 10.2b | 0.4 ± 0.2b |
Sesamol + L-Leucine | 0.6 ± 0.2c | 62.1 ± 20.8b | 1.7 ± 0.2c | 0.4 ± 0.1b | 8.6 ± 2.4c | 2.5 ± 0.4a |
Sesamol + L-Alanine | 1.4 ± 0.2b | 34.9 ± 3.5c | 2.9 ± 0.0a | 0.7 ± 0.0b | 5.6 ± 1.7c | 2.4 ± 0.0a |
Compounds | IC50 (mM) | Selective Index | |
---|---|---|---|
SK-MEL-2 | Vero Cells | ||
Sesamol | 2.0 ± 0.1b | 6.7 ± 0.2a | 3.4 |
Sesamol + 1 mM BCH | 3.0 ± 0.0a | 4.8 ± 0.1b | 1.6 |
Melphalan | 0.1 ± 0.0d | 0.6 ± 0.3b | 6.0 |
Melphalan + 1 mM BCH | 0.2 ± 0.0c | 0.5 ± 0.1b | 2.5 |
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Srisongkram, T.; Weerapreeyakul, N.; Kärkkäinen, J.; Rautio, J. Role of L-Type Amino Acid Transporter 1 (LAT1) for the Selective Cytotoxicity of Sesamol in Human Melanoma Cells. Molecules 2019, 24, 3869. https://doi.org/10.3390/molecules24213869
Srisongkram T, Weerapreeyakul N, Kärkkäinen J, Rautio J. Role of L-Type Amino Acid Transporter 1 (LAT1) for the Selective Cytotoxicity of Sesamol in Human Melanoma Cells. Molecules. 2019; 24(21):3869. https://doi.org/10.3390/molecules24213869
Chicago/Turabian StyleSrisongkram, Tarapong, Natthida Weerapreeyakul, Jussi Kärkkäinen, and Jarkko Rautio. 2019. "Role of L-Type Amino Acid Transporter 1 (LAT1) for the Selective Cytotoxicity of Sesamol in Human Melanoma Cells" Molecules 24, no. 21: 3869. https://doi.org/10.3390/molecules24213869