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Kim, Choi, Kim, Chung, Choi, Lee, and Kim: Synergistic effects of L-arginine and argininosuccinate synthetase 1 in inducing apoptosis in hepatocellular carcinoma
Original Article

J Liver Cancer 2025;25(1):79-90.

Published online: 14 January 2025

DOI: https://doi.org/10.17998/jlc.2024.12.27

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

Jin Sun Kim*, Won-Mook Choi*, †, Ha-Il Kim, Sung Won Chung, Jonggi Choi, Danbi Lee, Kang Mo Kim†

Department of Gastroenterology, Liver Center, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea

Corresponding author: Kang Mo Kim, Department of Gastroenterology, Liver Center, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Korea E-mail: kimkm70@amc.seoul.kr

Corresponding author: Won-Mook Choi, Department of Gastroenterology, Liver Center, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Korea E-mail: dr.choi85@gmail.com

* These two authors contributed equally to this work as first authors.

† These two authors contributed equally to this work as corresponding author.

Received 9 October 2024       Revised 20 December 2024       Accepted 27 December 2024

© 2025 The Korean Liver Cancer Association.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

Keywords: Liver neoplasms, Arginine, ASS1, Nitric oxide, Cisplatin

GRAPHICAL ABSTRACT

INTRODUCTION

Hepatocellular carcinoma (HCC), a primary liver cancer, is the sixth most common malignant tumor and the second leading cause of cancer-related deaths worldwide.1 The highest incidence and mortality rates of HCC have been observed in Asia, including South Korea. Chronic hepatitis B and alcohol use have been identified as the major causes of HCC. Recently, metabolic alterations such as metabolic dysfunction-associated steatotic liver disease and type 2 diabetes have also been recognized as contributors to HCC development.2 Owing to the complexity of HCC progression, its prognosis is typically poor. Various therapeutic drugs, including receptor tyrosine kinase inhibitors, have been developed to treat HCC and improve overall survival rates. However, these treatments are expensive, and none are entirely effective against HCC.3 Therefore, an urgent need exists to identify novel therapeutic targets for HCC treatment.
Metabolic reprogramming has been recognized as a hallmark of tumorigenesis and tumor progression.4 Recent studies have shown that cancer cells exhibit altered amino acid metabolism.5 Since amino acid metabolism is significantly upregulated in many cancers, its dysfunction can lead to an addiction to specific amino acids. L-arginine is a multifunctional amino acid required for various biological processes relevant to tumorigenesis, including nitric oxide (NO), creatine, and polyamine synthesis, which are essential for cell proliferation and survival.6-9
Argininosuccinate synthetase 1 (ASS1) is a key enzyme that acts at the rate-limiting step in arginine synthesis. Altered ASS1 expression can influence the tumor microenvironment, drug efficacy, and defective arginine metabolism.7,10 The downregulation of ASS1 reportedly makes cancer cells dependent on arginine supplementation for survival in various cancers.11-14 This phenomenon, known as arginine auxotrophy, is a notable metabolic defect observed in HCC.15,16 Furthermore, ASS1 downregulation and arginine auxotrophy contribute to chemoresistance and poor clinical outcomes in HCC.17-19 While many studies have explored the mechanisms regulating ASS1 expression, few studies exist on its practical implications in clinical settings, particularly in HCC therapy. Our previous study showed that low ASS1 expression significantly reduced the overall survival (OS) of HCC patients,20 suggesting that ASS1 and altered arginine metabolism could be promising therapeutic targets for HCC.
Here, we investigated the effects of ASS1 and arginine in overcoming cisplatin resistance, which is frequently observed in HCC. Our findings demonstrate that L-arginine, similar to ASS1, sensitizes cisplatin-resistant HCC cell lines to chemotherapy. Additionally, we observed that L-arginine had a cytotoxic effect on HCC cell lines with low or absent ASS1 expression, and that its combination with ASS1 further increased apoptotic cell death. We also found that the cytotoxicity of ASS1 and L-arginine was mediated by NO production and alterations in key signaling pathways.

METHODS

Tumor cell lines and patient-derived primary cultured HCC cells

Human HCC cell lines were obtained from the Korean Cell Line Bank. The cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Welgene, Gyeongsan, Korea) supplemented with 10% fetal bovine serum (FBS) (GIBCO, Gaithersburg, MD, USA) and maintained at 37℃ in a humidified 5% CO2 chamber. We also performed primary cell cultures from specimens resected from HCC patients newly diagnosed at the Asan Medical Center, Korea. All tissues samples were obtained after receiving written informed consent from the patients. This study was approved by the Institutional Review Board of Asan Medical Center (No. 2007-0332). After blood removal, primary hepatocytes were isolated, purified, and cultured as previously described.21

Cell viability assay

To examine the sensitivity of HCC cell lines and patient-derived HCC primary cells to cisplatin and L-arginine (Sigma-Aldrich, St. Louis, MO, USA), cytotoxicity was measured using the MTS assay (Promega, Madison, WI, USA). Cells were plated in 96-well plates at 2×103 cells/well for each cell line. The cells were then exposed to different drug concentrations for 72 hours. Twenty microliters of MTS solution were added to each well containing 100 µL of culture medium, and the cells were incubated for 2 hours at 37℃. Absorbance at 490 nm was measured using a SunriseTM microplate reader with MagellanTM software (Tecan, Männedorf, Switzerland). Cell viability was expressed as a percentage of the viability of untreated cells. The concentration of sorafenib that resulted in 50% growth inhibition was calculated using Prism 5 software (GraphPad, Boston, MA, USA). The trypan blue exclusion assay, another viability assay, was performed as described.22

mRNA extraction and reverse transcription polymerase chain reaction (RT-PCR)

Total cellular RNA was extracted using TRIzol (ThermoFisher Scientific, Waltham, MA, USA) according to the instructions of the manufacturer. The isolated RNA was resuspended in RNase-free water and the RNA concentration was measured at 260 nm using a NanoDrop 2000 UV-VIS spectrophotometer (ThermoFisher Scientific). Reverse transcription was performed in a 20 µL reaction mixture using a first-strand cDNA synthesis kit (Invitrogen, Waltham, MA, USA) according to the instructions of the manufacturer. RT-PCR was performed using AccuPower PCR premix (BIONEER, Daejeon, Korea) with the addition of first-strand cDNA and thermocycled in a Perkin-Elmer 9600 thermal cycler (Perkin-Elmer, Waltham, MA, USA). The ASS primer sequences were as follows: 5’-GCTGAAGGAACAAGGCTATGACG-3’ (sense) and 5’-GCCAGATGAACTCCTCCACAAAC-3’ (antisense).

Transfection with ASS1 plasmid and siRNA

Huh7 cells were transfected with pCMV3 or ASS1-Flag and seeded (1×106 cells/well) in DMEM with 10% FBS in 6-well plates. After seeding 5×105 cells in a 10 cm dish, siRNA targeting ASS1 was purchased from L-004819-00-0005 (Dharmacon, Lafayette, CO, USA), SMARTpool ON-TARGETplus ASS1 siRNA (Horizon Discovery, Waterbeach, UK), siRNA ID 445-1, 445-2, and 445-3 (BIONEER), and sc-45810 (Santa Cruz Biotechnology, Dallas, TX, USA). siRNA was transfected into HCC cell lines using Lipofectamine RNAiMAX (Invitrogen) reagent with Opti-MEM, and the cells were incubated at 37℃ for 48 hours.

NO concentration detection

To determine NO production levels in HCC cell lines expressing modified ASS1, the cells were seeded in 96-well plates. NO levels were measured using the Griess assay (Promega) following the instructions of the manufacturer. The absorbance of the final product, nitrite, was measured within 30 minutes using a plate reader with a filter between 520 and 550 nm.

Wound healing and colony formation assays

Metastatic potential was measured by wound healing23 and colony forming assays.24 Huh7 cells transfected with pCMV3 or ASS1-Flag were seeded at 1×106 cells/well in DMEM with 10% FBS in 6-well plates. After 24 hours, the cells were grown to full confluence on the plates and scratched using a pipette tip. The cells were washed with phosphate-buffered saline (PBS) to remove cellular debris and allowed to migrate for 48 hours. Cell migration images were captured using light microscopy and three fields in the wound area were analyzed.
For the colony formation assay, Huh7 cells transfected with pCMV3 or ASS1-Flag were seeded at 1×10 cells/well in 6-well plates. Starting on the day after L-arginine treatment, the medium was replaced every other day. The cells were cultured for 12 days, after which the plates were washed with PBS and stained with 4% crystal violet (Sigma-Aldrich). The number of colonies was counted and analyzed.

Western blot assay

Cell pellets were lysed using RIPA buffer (Curebio, Seoul, Korea) for 30 minutes at 4℃, and lysates were collected by centrifugation at 20,000×g for 25 minutes. The protein concentration of the cell lysate of each group was measured using the PierceTM BCA Protein Assay Kit (ThermoFisher Scientific). Equal amounts of protein were separated using SDS-PAGE. The proteins were transferred to a PVDF membrane using a Bio-Rad transfer system (Hercules, CA, USA) and blocked with 5% skim milk.
The primary antibodies used for hybridization included those against PARP, pAMPK, p-mTOR, pAkt, pERK, Mcl-1, cyclin D1, GAPDH, and glycolytic enzymes, and were purchased from Cell Signaling Technology (Danvers, MA, USA). The ASS1 antibody was purchased from Abcam (Cambridge, UK), and the β-actin antibody was purchased from Sigma-Aldrich. Secondary antibodies conjugated with horseradish peroxidase were then applied to the blots. Target protein bands were detected using ECL reagent (GE Healthcare, Chicago, IL, USA).

RESULTS

ASS1 expression enhanced cisplatin sensitivity in HCC cell lines

ASS1 is the rate-limiting enzyme for arginine synthesis and has been shown to influence cisplatin sensitivity in various cancer cell lines.25 In studies using colorectal and breast cancer cell lines, the anticancer effect of cisplatin was found to be ASS1-dependent. To explore this relationship in HCC, we first examined the endogenous ASS1 expression at the mRNA and protein levels (Fig. 1A, B). Therefore, we selected four HCC cell lines with varying ASS1 expression levels to assess their response to cisplatin (Fig. 1C). In Huh7, SNU475 (low ASS1), Hep3B, and PLC/PRF/5 (high ASS1) cell lines, we compared the inhibitory effect of cisplatin on proliferation after modifying ASS1 expression. Overexpression of ASS1 increased cisplatin sensitivity, while downregulation increased the resistance of high ASS1-expressing cells. These findings suggested that ASS1 expression significantly affects cisplatin sensitivity in HCC cell lines.

L-arginine exhibited cytotoxicity and sensitized HCC cell lines to cisplatin

We observed that ASS1 enhanced cisplatin cytotoxicity in HCC cell lines, suggesting that it may act as a tumor suppressor. To explore this, we examined the anti-tumor effects of ASS1 by altering its expression levels. Increased ASS1 expression increased cell death (Fig. 2A), supporting its role as a tumor suppressor. ASS1 is a key enzyme in L-arginine synthesis,26 and its effects may be driven by L-arginine production. We found that L-arginine exhibited concentration-dependent cytotoxicity comparable to that of ASS1 overexpression (Fig. 2B), indicating that the inhibitory effects of ASS1 may be linked to increased L-arginine levels.
Next, we treated HCC cell lines with different endogenous ASS1 expression levels with L-arginine and found that higher ASS1 expression increased reactivity to L-arginine and reduced cell viability (Fig. 2C). Overexpression of ASS1 in ASS1-low HCC cell lines combined with L-arginine treatment significantly enhanced cytotoxicity (Fig. 2D), suggesting that the effect of L-arginine is amplified by ASS1.
We then assessed whether L-arginine enhanced cisplatin sensitivity. L-arginine increased the cytotoxic effect of cisplatin, especially in ASS1-overexpressing cells (Fig. 3A). In ASS1-high HCC cell lines, L-arginine further potentiated cisplatin sensitivity. In ASS1-low cells, L-arginine exhibited increased sensitivity, albeit to a lesser extent (Fig. 3B). These findings suggest that L-arginine sensitizes HCC cell lines to cisplatin, regardless of ASS1 expression, and the effect is greater at higher ASS1 levels.
To explore its clinical relevance, we examined primary hepatocytes from HCC patients with varying ASS1 levels. Cisplatin response was closely associated with ASS1 expression, with higher ASS1 levels leading to increased sensitivity. L-arginine also showed enhanced cytotoxicity in ASS1-high hepatocytes, mirroring the results from established HCC cell lines (Fig. 3C). Additionally, we tested L-arginine in pancreatic cancer cell lines and found that it exerted a stronger cytotoxic effect than in HCC cell lines (Fig. 3D), suggesting that L-arginine may be effective beyond HCC. These results indicate that ASS1 and L-arginine play key roles in overcoming cisplatin resistance in HCC and may have broader anticancer potential.

ASS1 and L-arginine induced cell death via NO production

L-arginine is the sole substrate for NO synthesis, a key factor in both tumor development and suppression.27 NO production by L-arginine may underlie its cytotoxicity. To assess the role of NO in ASS1- and L-arginine-mediated cytotoxicity in HCC cell lines, we first modified ASS1 expression and measured NO levels in the cell supernatant (Fig. 4A). ASS1 overexpression significantly increased NO production, whereas ASS1 knockdown inhibited it. As ASS1, NO synthase (NOS), and L-arginine are essential for NO synthesis,28 forced ASS1 expression enhances this pathway, whereas downregulation of ASS1 disrupts it. We examined whether exogenous L-arginine could similarly promote NO production in HCC cell lines and patient-derived primary hepatocytes with varying ASS1 expression levels (Fig. 4B, C). L-arginine significantly increased NO synthesis in both cell types, with higher NO levels observed in ASS1-high cells. NO synthesis showed no difference between 5 mM and 10 mM L-arginine, likely due to enzyme saturation, beyond which additional substrates had no effect.

L-arginine inhibited HCC metastatic potential

Metastatic potential is critical in tumor progression.29 Our previous research showed that ASS1 acts as a tumor suppressor by inhibiting metastatic potential.20 Building on this, we investigated whether L-arginine, the end-product of ASS1, also inhibits HCC metastasis across varying ASS1 expression levels. We conducted wound healing and colony formation assays after modifying ASS1 expression and treating cells with L-arginine.
In the wound-healing assay (Fig. 5A), L-arginine inhibited Huh7 cell migration in a concentration-dependent manner, with greater inhibition observed in ASS1-overexpressing cells. Similarly, in the colony formation assay (Fig. 5B), L-arginine suppressed colony formation, with more pronounced effects observed in ASS1-overexpressing Huh7 cells. At 5 mM L-arginine, colony formation was significantly reduced in both ASS1-high and ASS1-low cells. However, at 10 mM L-arginine, the inhibitory effect was similar regardless of ASS1 expression. These results suggest that L-arginine can suppress the metastatic potential of HCC cell lines independently of ASS1 expression, with greater inhibition observed when ASS1 was overexpressed.

L-arginine modified survival and metabolic pathway

We observed that ASS1 and its end product, L-arginine, act as tumor suppressors and exert cytotoxic effects in HCC. We then investigated how L-arginine modifies the signaling pathways related to cancer proliferation and survival. After altering ASS1 expression, we examined changes in these pathways and compared the differences based on ASS1 levels (Fig. 5C). Cleaved PARP confirmed that L-arginine induced apoptosis in both ASS1-high and ASS1-low Huh7 cells, with significantly greater apoptosis observed in ASS1-overexpressing cells. To understand the differential cytotoxicity, we analyzed the key proliferation and survival pathways. L-arginine reduced ERK activity (MAPK pathway) regardless of ASS1 expression but significantly decreased Akt activity (PI3K/Akt pathway) in ASS1-high cells. Activity of mTOR, a downstream target of Akt, was markedly reduced by L-arginine, particularly in ASS1-overexpressing cells, and was inhibited by ASS1 expression alone. Other survival factors, such as cyclin D1 and Mcl-1, were inhibited by L-arginine, regardless of ASS1 levels (Fig. 5C). Given the role of mTOR in the regulation of glycolysis, we explored whether L-arginine affects glycolytic enzymes (Fig. 5D). L-arginine reduced mTOR phosphorylation, an effect enhanced by ASS1 overexpression. Additionally, L-arginine specifically downregulated the expression of PKM1, PKM2, and pyruvate dehydrogenase, suggesting that L-arginine modulates aerobic glycolysis through enzyme regulation.

DISCUSSION

HCC, the most common type of primary liver cancer, can be caused by various factors, such as hepatitis or aflatoxin B. Cancer progression, including HCC, is often driven by the reprogramming of cellular metabolism, a phenomenon first described by Otto Warburg. This shift in metabolism, often characterized by increased glycolysis even in the presence of oxygen (Warburg effect), is now recognized as a hallmark of cancer.30 However, the exact role of metabolism and its intermediates in HCC initiation and progression remains unclear. Our study focused on arginine metabolism due to the frequent loss or downregulation of ASS1, a key enzyme in arginine biosynthesis, in HCC. Previous studies, including ours, have demonstrated that low ASS1 expression is closely linked to poor prognosis in HCC patients.20 Moreover, ASS1 downregulation has been associated with resistance to platinum-based chemotherapy, which is commonly used to treat various cancers, including breast and ovarian cancers.25,31
To address the urgent need for effective therapies for HCC, researchers have been developing new drugs such as sorafenib and lenvatinib and exploring novel therapeutic targets.32 Our study focused on arginine metabolism as a potential target for HCC therapy. Evidence suggests that higher ASS1 expression correlates with better prognosis and longer OS in HCC patients.33 In the current study, we found that ASS1, along with its metabolic product L-arginine, can sensitize HCC cell lines to cisplatin, a chemotherapy drug, by enhancing its cytotoxic effects. Furthermore, L-arginine demonstrated significant anticancer activity even in HCC cell lines with low or no ASS1 expression, suggesting that L-arginine exerts its effects independently of ASS1 in some cases. Using HCC patient-derived hepatocytes with varying ASS1 expression levels, we confirmed that both ASS1 and L-arginine inhibited cell proliferation and induced apoptosis. Interestingly, we observed a similar anticancer effect of L-arginine in pancreatic cancer cells, which are known for their low ASS1 expression. This suggests that L-arginine may have broader therapeutic potential beyond HCC, as it effectively inhibited cancer cell growth regardless of ASS1 expression in both HCC and pancreatic cancer cells. However, one of the limitations of our study is that cisplatin was less frequently selected for transarterial chemoembolization in the treatment of HCC,34 highlighting the need for further research to determine whether similar results can be achieved with other chemotherapeutic agents, such as doxorubicin.
We also explored the mechanism underlying the cytotoxic effects of ASS1 and L-arginine by focusing on NO production, which is known to play a role in both tumor progression and suppression. Our data showed that L-arginine, through its involvement in the NO pathway, induces significant NO production, particularly in cells overexpressing ASS1. However, the instability of NO, especially after treatment with high concentrations of L-arginine, suggests that its production may peak and then quickly decline owing to rapid decomposition. Further studies on the involvement of NOS and on shorter reaction times are necessary to fully understand the role of NO in this process.35
Metastasis and recurrence remain the primary challenges in the successful treatment of HCC because they are strongly associated with poor clinical outcomes.36 Our study demonstrated that both ASS1 and L-arginine inhibit HCC metastasis, as evidenced by wound healing and colony formation assays. The metastasis-inhibitory effect of L-arginine was observed at different ASS1 expression levels, although this effect was more pronounced in ASS1-overexpressing cells. Notably, at 10 mM L-arginine, we observed a similar inhibition of metastatic potential, regardless of ASS1 levels. If this concentration is safe in clinical settings, L-arginine could be a valuable therapeutic option for inhibiting metastasis in HCC patients. Further studies are warranted to validate the potential therapeutic effects of L-arginine in treating HCC in in vivo preclinical or clinical settings.
ASS1, a rate-limiting enzyme in intracellular arginine biosynthesis, regulates arginine availability in the tumor microenvironment. In ASS1-deficient cancers, arginine auxotrophy is exploited by deprivation therapies to induce metabolic stress and apoptosis.37 However, our study demonstrated that L-arginine induces apoptosis in HCC cell lines by modulating key signaling pathways. Specifically, L-arginine in combination with ASS1 overexpression effectively inhibited the PI3K/Akt/mTOR pathway, which regulates cell proliferation, lipogenesis, autophagy, and metabolism.38 This inhibition disrupts these processes and enhances the cytotoxic effects of chemotherapy. Furthermore, ASS1 overexpression downregulated mTOR activity, impacting energy metabolism and glycolysis.39-42 We observed significant reductions in the glycolytic enzymes PKM1 and PKM2, indicating that L-arginine directly inhibits aerobic glycolysis,43 targeting metabolic vulnerabilities of HCC.
This discrepancy with previous studies that focused on ASS1- deficient cancers and arginine deprivation-induced apoptosis likely reflects differences in the metabolic context. In ASS1-expressing cells, increased intracellular arginine may drive proapoptotic signaling and enhance cisplatin sensitivity via NO production and oxidative stress. This aligns with the findings of Yang et al.,44 who highlighted the role of amino acid balance and metabolic adaptation in cancer. Additionally, within the tumor microenvironment, arginine supplementation, which enhances T-cell function and anti-tumor immunity,45 may serve as a promising therapeutic strategy, particularly for ASS1-expressing cancers.
In summary, this study demonstrated that L-arginine has significant anticancer effects in HCC, both as a standalone treatment and in combination with ASS1 overexpression. L-arginine induces apoptosis, inhibits metastasis, and modulates key signaling pathways, making it a promising candidate for HCC therapy. Future studies should focus on validating the synergistic anti-tumor effects of L-arginine and ASS1 against HCC in preclinical or clinical settings and further elucidate the mechanisms underlying the selective inhibition of the PI3K/Akt/mTOR pathway by L-arginine, as well as its potential applications in other cancers, such as pancreatic cancer. Developing strategies to upregulate ASS1 expression or combining L-arginine with current therapies may improve treatment outcomes for HCC patients, particularly those resistant to platinum-based chemotherapy.

Notes

Conflicts of Interest

Won-Mook Choi is an editorial board member of Journal of Liver Cancer , and was not involved in the review process of this article. Otherwise, the authors have no conflicts of interest to disclose.

Ethics Statement

This study was approved by the Institutional Review Board of Asan Medical Center (IRB No. 2016-0805). Studies including all human participants were performed after receiving written informed consent.

Funding Statement

This study was supported by the Korean Liver Cancer Association Research Award (2023), the National Research Foundation of Korea (NRF-2016R1D1A1B03936277), and Research funding from the Asan Institute for Life Science of Asan Medical Center (2014-399).

Data Availability

All relevant data are within the paper. Further enquiries can be directed to the corresponding author.

Author Contributions

Conceptualization: JSK

Data curation: JSK, HIK

Formal analysis: JSK, HIK

Funding acquisition: KMK

Investigation: KMK

Methodology: JSK, WMC, KMK

Project administration: WMC, KMK

Resources: WMC, SWC, JC, DL, KMK

Software: WMC, KMK

Supervision: WMC, KMK

Validation: SWC, JC, DL

Visualization: JSK

Writing - original draft: JSK

Writing - review & editing: WMC, KMK

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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.
jlc-2024-12-27f1.tif
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.
jlc-2024-12-27f2.tif
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.
jlc-2024-12-27f3.tif
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.
jlc-2024-12-27f4.tif
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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