The Combination of Arginine Deprivation and 5-Fluorouracil Improves Therapeutic Efficacy in Argininosuccinate Synthetase Negative Hepatocellular Carcinoma
Abstract
:1. Introduction
2. Results
2.1. Determination of ASS Expression Level among Cancer Cell Types
2.2. The Levels of ASS Expression Dictate the Sensitivity to Arginine Deprivation
2.3. The Anti-Tumor Effect of 5-FU in Combination with ADI-PEG20
2.4. ADI-PEG20 and 5-FU Combination Effectively Increase Cell Death
2.5. Effects of ADI-PEG20 on the Key Enzymes Involved in 5-FU Sensitivity
2.6. Reconstitution of ASS Expression Affect the Sensitivity to Combination Treatment with ADI-PEG20 Plus 5-FU Treatment as Well as the Enzyme Involved in the De Novo Pyrimidine Synthesis
2.7. ASS Expression in HCC Specimens from Thai Patients
3. Discussion
4. Materials and Methods
4.1. Cell Lines and Culture
4.2. Reagents
4.3. Growth Inhibitory Assay
4.4. Reverse Transcription and Real Time PCR
4.5. Western Blotting
4.6. Apoptosis Assay
4.7. ASS Overexpression
4.8. Immunohistochemistry
4.9. Patient and Tissue Samples
4.10. Statistical Analysis
5. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
Abbreviations
ADI-PEG20 | Pegylated arginine deiminase |
dUMP | Deoxyuridine monophosphate |
FDUMP | Fluorodeoxyuridine monophosphase |
TS | Thymidylate synthase |
DPYD | Dihydropyrimidine dehydrogenase |
CPS I | Carbamoyl phosphate synthase I |
OTC | Ornithine transcabamoylase |
AAT | Aspartate aminotransferase |
MDH 1 | Malate dehydrogenase 1 |
MDH 2 | Malate dehydrogenase 2 |
CAD | Carbamoyl-phosphate synthase 2, aspartate trancarbamylase and dihydrooratase complex |
Ph-CAD | Phosphorylated-carbamoyl-phosphate synthase 2, aspartate trancarbamylase and dihydrooratase complex |
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Cancer | Level of ASS Expression (Arbitrary Unit) | ||
---|---|---|---|
Normal Liver | HCC | Ratio (Tumor/Normal) | |
HCC | 57.07 ± 1.47 a | 43.94 ± 1.46 * | 0.79 ± 0.03 |
(N = 40) | 56.48 (39.38 − 83.44) b | 43.88 (18.21 − 75.23) | 0.81 (0.27 − 1.13) |
Variables | Patients | ASS Expression Ratio a | p-Value b | |
---|---|---|---|---|
Low | High | |||
All case | 40 | 18 | 22 | - |
Gender | - | - | - | 0.673 |
Male | 37 | 17 | 20 | - |
Female | 3 | 1 | 2 | - |
Age (years) | - | - | - | 0.251 |
<60 | 25 | 13 | 12 | - |
≥60 | 15 | 5 | 10 | - |
Viral status (Hepatitis B or C) | - | - | - | 0.165 |
Yes | 29 | 15 | 14 | - |
No | 11 | 3 | 8 | - |
Tumor differentiation | - | - | - | 0.032 |
Grade 1 | 7 | 1 | 6 | - |
Grade 2 | 14 | 4 | 10 | - |
Grade 3 | 14 | 9 | 5 | - |
Grade 4 | 5 | 4 | 1 | - |
Lympho-vascular invasion | - | - | - | 0.533 |
Yes | 6 | 2 | 4 | - |
No | 34 | 16 | 18 | - |
Lymph node metastasis | - | - | - | 0.386 |
Yes | 14 | 5 | 9 | - |
No | 26 | 13 | 13 | - |
TNM stage | - | - | - | 0.072 |
I–II | 14 | 9 | 5 | - |
III–IV | 26 | 9 | 17 | - |
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Thongkum, A.; Wu, C.; Li, Y.-Y.; Wangpaichitr, M.; Navasumrit, P.; Parnlob, V.; Sricharunrat, T.; Bhudhisawasdi, V.; Ruchirawat, M.; Savaraj, N. The Combination of Arginine Deprivation and 5-Fluorouracil Improves Therapeutic Efficacy in Argininosuccinate Synthetase Negative Hepatocellular Carcinoma. Int. J. Mol. Sci. 2017, 18, 1175. https://doi.org/10.3390/ijms18061175
Thongkum A, Wu C, Li Y-Y, Wangpaichitr M, Navasumrit P, Parnlob V, Sricharunrat T, Bhudhisawasdi V, Ruchirawat M, Savaraj N. The Combination of Arginine Deprivation and 5-Fluorouracil Improves Therapeutic Efficacy in Argininosuccinate Synthetase Negative Hepatocellular Carcinoma. International Journal of Molecular Sciences. 2017; 18(6):1175. https://doi.org/10.3390/ijms18061175
Chicago/Turabian StyleThongkum, Angkana, Chunjing Wu, Ying-Ying Li, Medhi Wangpaichitr, Panida Navasumrit, Varabhorn Parnlob, Thaniya Sricharunrat, Vajarabhongsa Bhudhisawasdi, Mathuros Ruchirawat, and Niramol Savaraj. 2017. "The Combination of Arginine Deprivation and 5-Fluorouracil Improves Therapeutic Efficacy in Argininosuccinate Synthetase Negative Hepatocellular Carcinoma" International Journal of Molecular Sciences 18, no. 6: 1175. https://doi.org/10.3390/ijms18061175