J Liver Cancer.  2025 Mar;25(1):79-90. 10.17998/jlc.2024.12.27.

Synergistic effects of L-arginine and argininosuccinate synthetase 1 in inducing apoptosis in hepatocellular carcinoma

• Kim JS
• Choi WM
• Kim HI
• Chung SW
• Choi J
• Lee D
• Kim KM

• KMID: 2567066
• DOI: http://doi.org/10.17998/jlc.2024.12.27

Abstract

Backgrounds/Aims
Hepatocellular carcinoma (HCC) is a malignant cancer with an increasing incidence worldwide. Although numerous efforts have been made to identify effective therapies for HCC, current strategies have limitations. We present a new approach for targeting L-arginine and argininosuccinate synthetase 1 (ASS1).
Methods
ASS1 expression in HCC cell lines and primary hepatocytes was detected using polymerase chain reaction and western blotting. Proliferation, migration, signaling pathways, and nitric oxide production in HCC cell lines were measured using MTS, colony formation, wound healing, Western blot, and Griess assays.
Results
ASS1 expression varied among the HCC cell lines, and cisplatin cytotoxicity was ASS1-dependent. L-arginine alone induced apoptosis in HCC cell lines, regardless of ASS1 expression; however, its effect was enhanced in ASS1-expressing HCC cell lines. Cisplatin cytotoxicity also increased, suggesting that L-arginine acts as a sensitizer to cisplatin in HCC cell lines. ASS1 and L-arginine produced nitric oxide and inhibited key proliferation- and survival-related signaling pathways such as PI3K/Akt and MAPK. Additionally, ASS1 and L-arginine reduced the expression of PKM1 and PKM2 in the glycolysis pathway.
Conclusions
Our study revealed that ASS1 and L-arginine exhibited anticancer effects in HCC and sensitized cisplatin-resistant HCC cells to chemotherapy. The combination of ASS1 and L-arginine significantly enhanced the anticancer effects, even in HCC cell lines with low or absent ASS1 expression. These findings highlight the critical roles of arginine and ASS1 in HCC and suggest that increasing arginine availability could be a promising therapeutic strategy.

Keyword

Liver neoplasms; Arginine; ASS1; Nitric oxide; Cisplatin

Figure

• 

  Figure 1. ASS1 expression increases the sensitivity of HCC cell lines to cisplatin. (A) Endogenous ASS1 expression levels in HCC cell lines. The protein lanes are as follows: 1) F2N, 2) Huh6, 3) Huh7, 4) Hep3B, 5) SK-Hep1, 6) PLC/PRF/5, 7) SNU398, 8) SNU449, 9) SNU475, 10) AMCH1, 11) AMC-H2. (B) Endogenous ASS1 mRNA expression levels in HCC cell lines. The mRNA lanes are as follows: 1) HepG2, 2) Hep3B, 3) Huh7, 4) PLC/PRF/5, 5) SNU398, 6) SNU449, 7) SNU475, 8) AMC-H1, 9) AMC-H2, 10) A431. A431 was used as a positive control. (C) Higher en-dogenous ASS1 expression increases sensitivity to cisplatin. Cell viability in response to cisplatin was evaluated by overexpressing ASS1 in Huh7 and SNU475 cells or silencing its expression in Hep3B and PLC/PRF/5 cells using siRNA. Data are presented as mean± SEM. ASS1, argininosuccinate synthetase; GADPH, glyceraldehyde-3-phosphate dehydrogenase; CON, control; siRNA, small interfering RNA; HCC, hepatocellular carcinoma; SEM, standard error of the mean. **P<0.01 and ***P<0.001.

• 

  Figure 2. L-arginine cytotoxicity is enhanced by ASS1 expression. (A) ASS1 inhibits HCC cell proliferation. Relative viability is shown as fold change. (B) The effect of ASS1 and its end-product, L-arginine (A5, L-arginine 5 mM; A10, L-arginine 10 mM), on Huh7 cells was examined. (C) L-arginine cytotoxicity was related to endogenous ASS1 expression levels. Three ASS1-low cell lines (SNU398, SNU475, and Huh7) and two ASS1-high cell lines (Hep3B and SNU449) were used to assess the cytotoxic effects of L-arginine. (D) ASS1 expression enhances L-arginine cytotoxicity in ASS1-low Huh7 and SNU475 cells. After 24 hours of ASS1 transfection, cells were treated with L-arginine at the indicated concentrations for 72 hours, and viability was measured using the MTS assay. Data are presented as mean±SEM. CON, control; ASS1, argininosuccinate synthetase; HCC, hepatocellular carcinoma; SEM, standard error of the mean. *P<0.05, **P<0.01, and ***P<0.001.

• 

  Figure 3. L-Arginine sensitizes cells to cisplatin. (A) L-arginine enhances cisplatin cytotoxicity regardless of ASS1 expression. After overexpressing ASS1 in Huh7 and SNU475 cells, they were treated with CDDP and/or L-arginine (L-Arg) to assess cell viability. (B) L-Arg alone increases the cytotoxicity of CDDP in HCC cell lines with varying endogenous ASS1 expression levels. (C) L-Arg acts as a sensitizer to cisplatin in patient-derived primary HCC cells with different ASS1 expression levels. Primary HCC cells were treated with 10 mM L-Arg and varying concentrations of CDDP, and cell viability was measured using the MTS assay. (D) L-Arg exerts cytotoxicity in pancreatic cancer cell lines regardless of ASS1 expression. Four pancreatic cancer cell lines were treated with 10 μM CDDP and various concentrations of L-Arg for 72 hours, and cell viability was measured using the MTS assay. Data are presented as mean±SEM. CDDP, cisplatin; ASS1, argininosuccinate synthetase; GADPH, glyceraldehyde-3-phosphate dehydrogenase; HCC, hepatocellular carcinoma; SEM, standard error of the mean. *P<0.05, **P<0.01, and ***P<0.001.

• 

  Figure 4. NO as a key factor in L-arginine cytotoxicity. (A) ASS1 expression increases NO production. After altering ASS1 expression levels, NO was measured using the Griess assay. (B) ASS1 enhances NO synthesis by L-arginine in HCC cell lines. Following overexpression of ASS1 and treatment with L-arginine (A5, L-arginine 5 mM; A10, L-arginine 10 mM) in Huh7 cells, NO production was measured using the Griess assay in each group. (C) L-arginine-mediated NO production was influenced by endogenous ASS1 levels. One hour after treatment with L-arginine (A0, control; A5, L-arginine 5 mM; A10, L-arginine 10 mM), NO production was measured using the Griess assay. Data are presented as mean±SEM. CON, control; ASS1, argininosuccinate synthetase; NO, nitric oxide; HCC, hepatocellular carcinoma; SEM, standard error of the mean. ***P<0.001.

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  Figure 5. L-arginine modifies metastatic potential and survival in HCC. (A) L-arginine (L-Arg) inhibits the metastatic potential of HCC as examined by a migration assay. After modifying ASS1 expression, cells were scratched and treated with L-arginine for the indicated times. Migration was assessed in three different wound areas using light microscopy. (B) L-Arg inhibits the metastatic potential of HCC, measured by a colony formation assay. After altering ASS1 expression and treating with L-Arg (A5, L-Arg 5 mM; A10, L-Arg 10 mM), colonies were observed for 12-14 days, with the medium changed every other day. Colonies were stained with crystal violet and counted under light microscopy. (C, D) After transfection of ASS1 in Huh7 cells, cells were treated with L-Arg at the indicated concentrations for 48 hours and signaling pathways and glycolytic enzyme expression were examined using western blot analysis. (C) ASS1 helps L-arginine selectively inhibit the PI3K/Akt pathway in HCC. (D) L-arginine disrupts glycolysis by reducing glycolytic enzyme expression. CON, control; PARP, poly(ADPribose) polymerase; PFKP, phosphofructokinase, platelet; mTOR, mammalian target of rapamycin; AMPK, AMP-activated protein kinase; PKM1/2, pyruvate kinase M1/2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LDHA, lactate dehydrogenase A; ASS1, argininosuccinate synthetase; HCC, hepatocellular carcinoma.

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Reference

References

1. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. 2015; 136:E359–E386.

2. Kim DY. Changing etiology and epidemiology of hepatocellular carcinoma: Asia and worldwide. J Liver Cancer. 2024; 24:62–70.

3. Korean Liver Cancer Association (KLCA); National Cancer Center (NCC) Korea. 2022 KLCA-NCC Korea practice guidelines for the management of hepatocellular carcinoma. J Liver Cancer. 2023; 23:1–120.

4. Boroughs LK, DeBerardinis RJ. Metabolic pathways promoting cancer cell survival and growth. Nat Cell Biol. 2015; 17:351–359.

5. Tsun ZY, Possemato R. Amino acid management in cancer. Semin Cell Dev Biol. 2015; 43:22–32.

6. Suschek CV, Schnorr O, Hemmrich K, Aust O, Klotz LO, Sies H, et al. Critical role of L-arginine in endothelial cell survival during oxidative stress. Circulation. 2003; 107:2607–2614.

7. Patil MD, Bhaumik J, Babykutty S, Banerjee UC, Fukumura D. Arginine dependence of tumor cells: targeting a chink in cancer's armor. Oncogene. 2016; 35:4957–4972.

8. Morris SM Jr. Enzymes of arginine metabolism. J Nutr. 2004; 134 Suppl 10:2743S–2747S; discussion 2765S.

9. Morris SM Jr. Arginine metabolism revisited. J Nutr. 2016; 146:2579S2586S.

10. Giatromanolaki A, Harris AL, Koukourakis MI. The prognostic and therapeutic implications of distinct patterns of argininosuccinate synthase 1 (ASS1) and arginase-2 (ARG2) expression by cancer cells and tumor stroma in non-small-cell lung cancer. Cancer Metab. 2021; 9:28.

11. Silberman A, Goldman O, Assayag OB, Jacob A, Rabinovich S, Adler L, et al. Acid-induced downregulation of ASS1 contributes to the maintenance of intracellular pH in cancer. Cancer Res. 2019; 79:518–533.

12. Liu J, Ma J, Wu Z, Li W, Zhang D, Han L, et al. Arginine deiminase augments the chemosensitivity of argininosuccinate synthetase-deficient pancreatic cancer cells to gemcitabine via inhibition of NF-κB signaling. BMC Cancer. 2014; 14:686.

13. Shan YS, Hsu HP, Lai MD, Yen MC, Chen WC, Fang JH, et al. Argininosuccinate synthetase 1 suppression and arginine restriction inhibit cell migration in gastric cancer cell lines. Sci Rep. 2015; 5:9783.

14. McAlpine JA, Lu HT, Wu KC, Knowles SK, Thomson JA. Down-regulation of argininosuccinate synthetase is associated with cisplatin resistance in hepatocellular carcinoma cell lines: implications for PEGylated arginine deiminase combination therapy. BMC Cancer. 2014; 14:621.

15. Nicholson LJ, Smith PR, Hiller L, Szlosarek PW, Kimberley C, Sehouli J, et al. Epigenetic silencing of argininosuccinate synthetase confers resistance to platinum-induced cell death but collateral sensitivity to arginine auxotrophy in ovarian cancer. Int J Cancer. 2009; 125:1454–1463.

16. Battisti S, Valente D, Albonici L, Bei R, Modesti A, Palumbo C. Nutritional stress and arginine auxotrophy confer high sensitivity to chloroquine toxicity in mesothelioma cells. Am J Respir Cell Mol Biol. 2012; 46:498–506.

17. Allen MD, Luong P, Hudson C, Leyton J, Delage B, Ghazaly E, et al. Prognostic and therapeutic impact of argininosuccinate synthetase 1 control in bladder cancer as monitored longitudinally by PET imaging. Cancer Res. 2014; 74:896–907.

18. Delage B, Fennell DA, Nicholson L, McNeish I, Lemoine NR, Crook T, et al. Arginine deprivation and argininosuccinate synthetase expression in the treatment of cancer. Int J Cancer. 2010; 126:2762–2772.

19. Singh PK, Deorukhkar AA, Venkatesulu BP, Li X, Tailor R, Bomalaski JS, et al. Exploiting arginine auxotrophy with pegylated arginine deiminase (ADI-PEG20) to sensitize pancreatic cancer to radiotherapy via metabolic dysregulation. Mol Cancer Ther. 2019; 18:2381–2393.

20. Kim S, Lee M, Song Y, Lee SY, Choi I, Park IS, et al. Argininosuccinate synthase 1 suppresses tumor progression through activation of PERK/eIF2α/ATF4/CHOP axis in hepatocellular carcinoma. J Exp Clin Cancer Res. 2021; 40:127.

21. Kim JS, Choi GH, Jung Y, Kim KM, Jang SJ, Yu ES, et al. Downregulation of Raf-1 kinase inhibitory protein as a sorafenib resistance mechanism in hepatocellular carcinoma cell lines. J Cancer Res Clin Oncol. 2018; 144:1487–1501.

22. Strober W. Trypan blue exclusion test of cell viability. Curr Protoc Immunol. 2015; 111:A3.B.1–A3.B.3.

23. Rodriguez LG, Wu X, Guan JL. Wound-healing assay. Methods Mol Biol. 2005; 294:23–29.

24. Hu X, Yang F, Mei H. Pituitary tumor transforming gene 1 promotes proliferation and malignant phenotype in osteosarcoma via NF-κB signaling. J Orthop Sci. 2024; 29:306–314.

25. Long Y, Tsai WB, Chang JT, Estecio M, Wangpaichitr M, Savaraj N, et al. Cisplatin-induced synthetic lethality to arginine-starvation therapy by transcriptional suppression of ASS1 is regulated by DEC1, HIF-1α, and cMyc transcription network and is independent of ASS1 promoter DNA methylation. Oncotarget. 2016; 7:82658–82670.

26. Szefel J, Danielak A, Kruszewski WJ. Metabolic pathways of L-arginine and therapeutic consequences in tumors. Adv Med Sci. 2019; 64:104–110.

27. Ramírez-Patiño R, Avalos-Navarro G, Figuera LE, Varela-Hernández JJ, Bautista-Herrera LA, Muñoz-Valle JF, et al. Influence of nitric oxide signaling mechanisms in cancer. Int J Immunopathol Pharmacol. 2022; 36:3946320221135454.

28. Dios-Barbeito S, González R, Cadenas M, García LF, Victor VM, Padillo FJ, et al. Impact of nitric oxide in liver cancer microenvironment. Nitric Oxide. 2022; 128:1–11.

29. Platel V, Faure S, Corre I, Clere N. Endothelial-to-mesenchymal transition (EndoMT): roles in tumorigenesis, metastatic extravasation and therapy resistance. J Oncol. 2019; 2019:8361945.

30. Zhu Y, Yang R, Law JH, Khan M, Yip KW, Sun Q. Editorial: hallmark of cancer: resisting cell death. Front Oncol. 2022; 12:1069947.

31. Chan PY, Phillips MM, Ellis S, Johnston A, Feng X, Arora A, et al. A phase 1 study of ADI-PEG20 (pegargiminase) combined with cisplatin and pemetrexed in ASS1-negative metastatic uveal melanoma. Pigment Cell Melanoma Res. 2022; 35:461–470.

32. Wang H, Chen X, Calvisi DF. Hepatocellular carcinoma (HCC): the most promising therapeutic targets in the preclinical arena based on tumor biology characteristics. Expert Opin Ther Targets. 2021; 25:645–658.

33. Bednarz-Misa I, Fleszar MG, Fortuna P, Lewandowski Ł, MierzchałaPasierb M, Diakowska D, et al. Altered L-arginine metabolic pathways in gastric cancer: potential therapeutic targets and biomarkers. Biomolecules. 2021; 11:1086.

34. Cho Y, Choi JW, Kwon H, Kim KY, Lee BC, Chu HH, et al. Transarterial chemoembolization for hepatocellular carcinoma: 2023 expert consensus-based practical recommendations of the Korean Liver Cancer Association. J Liver Cancer. 2023; 23:241–261.

35. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011; 144:646–674.

36. Lohitesh K, Chowdhury R, Mukherjee S. Resistance a major hindrance to chemotherapy in hepatocellular carcinoma: an insight. Cancer Cell Int. 2018; 18:44.

37. Kremer JC, Prudner BC, Lange SES, Bean GR, Schultze MB, Brashears CB, et al. Arginine deprivation inhibits the Warburg effect and upregulates glutamine anaplerosis and serine biosynthesis in ASS1-deficient cancers. Cell Rep. 2017; 18:991–1004.

38. Sun EJ, Wankell M, Palamuthusingam P, McFarlane C, Hebbard L. Targeting the PI3K/Akt/mTOR pathway in hepatocellular carcinoma. Biomedicines. 2021; 9:1639.

39. Cheng SC, Quintin J, Cramer RA, Shepardson KM, Saeed S, Kumar V, et al. mTOR- and HIF-1α-mediated aerobic glycolysis as metabolic basis for trained immunity. Science. 2014; 345:1250684.

40. Hu X, Xu Q, Wan H, Hu Y, Xing S, Yang H, et al. PI3K-Akt-mTOR/PFKFB3 pathway mediated lung fibroblast aerobic glycolysis and collagen synthesis in lipopolysaccharide-induced pulmonary fibrosis. Lab Invest. 2020; 100:801–811.

41. Sun XG, Chu XH, Godje ISG, Liu SY, Hu HY, Zhang YB, et al. Aerobic glycolysis induced by mTOR/HIF-1α promotes early brain injury after subarachnoid hemorrhage via activating M1 microglia. Transl Stroke Res. 2024; 15:1–15.

42. Zhang H, Su X, Burley SK, Zheng XFS. mTOR regulates aerobic glycolysis through NEAT1 and nuclear paraspeckle-mediated mechanism in hepatocellular carcinoma. Theranostics. 2022; 12:3518–3533.

43. Zahra K, Dey T, Mishra SP, Pandey U. Pyruvate kinase M2 and cancer: the role of PKM2 in promoting tumorigenesis. Front Oncol. 2020; 10:159.

44. Yang C, Pataskar A, Feng X, Navarro JM, Paniagua I, Jacobs JJL, et al. Arginine deprivation enriches lung cancer proteomes with cysteine by inducing arginine-to-cysteine substitutants. Mol Cell. 2024; 84:1904–1916.e7.

45. Geiger R, Rieckmann JC, Wolf T, Basso C, Feng Y, Fuhrer T, et al. L-arginine modulates T cell metabolism and enhances survival and anti-tumor activity. Cell. 2016; 167:829–842.e13.