Enhanced Antitumor Effect in Liver Cancer by Amino Acid Depletion-Induced Oxidative Stress

Keiichiro Okuda,Yoshito Itoh,Michihisa Moriguchi,Atsushi Umemura,Aya Takahashi,Hiroyoshi Taketani,Shinya Okishio,Yu Liu,Yuya Seko,Kota Yano,Kanji Yamaguchi,Hayato Nakagawa,Seita Kataoka,Yasuhide Mitsumoto,Takeshi Okanoue,Taichiro Nishikawa,Toshihide Shima,Yoshihiro Kanbara

doi:10.3389/fonc.2021.758549

Abstract

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer. HCC cells consume large amounts of glutamine to survive, but can adapt to glutamine depletion in the presence of an exogenous asparagine. L-asparaginase (ASNase) converts glutamine and asparagine to glutamate and aspartate, respectively, and has been used to treat leukemia. Here we examined the effects of ASNase treatment on HCC cells and explored the potential impact of combining ASNase with the tyrosine kinase inhibitor lenvatinib (Len) for HCC treatment. Cell viability and death of HCC cell lines treated with either Len or ASNase alone or with Len and ASNase combined were determined. We assessed mRNA and protein expression levels of glutamine synthetase (GS) and asparagine synthetase (ASNS) by real-time quantitative PCR and immunoblotting. The antitumor effect of the combination therapy relative to Len or ASNase monotherapy was also evaluated in a xenograft tumor mouse model. ASNase treatment inhibited growth of SNU387 and SNU398 HCC cells, which have low GS and high ASNS expression levels, respectively, but did not clearly inhibit growth of the other cell lines. Len plus ASNase combination therapy synergistically inhibited proliferation and induced oxidative stress leading to cell death of some HCC cells lines. However, cell death of Huh7 cells, which express ASCT2, an important glutamine transporter for cancer cells, was not affected by the combination treatment. In a xenograft model, Len combined with ASNase significantly attenuated tumor development relative to mice treated with Len or ASNase alone. ASNase-mediated targeting of two amino acids, glutamine and asparagine, which are indispensable for HCC survival, induces oxidative stress and can be a novel cancer treatment option that exerts a synergistic effect when used in combination with Len.

Highlights

Hepatocellular carcinoma (HCC) is the sixth most common malignant tumor and the fourth leading cause of cancerrelated death worldwide [1]
We first examined the effect of Len, a tyrosine kinase inhibitors (TKIs) that is commonly used for HCC treatment, and ASNase on the survival of six liver cancer cell lines carrying specific mutations
Glutamine is a nonessential amino acid, many cancer cells, including HCC cells, cannot survive without extracellular glutamine, and are thought to be dependent upon glutamine uptake [18]. In both sarcoma cells [26] and HCC cells [9], glutamine synthetase (GS), the rate-limiting enzyme required in glutamine synthesis, is needed to adapt to glutamine deprivation and for survival

Summary

Introduction

Hepatocellular carcinoma (HCC) is the sixth most common malignant tumor and the fourth leading cause of cancerrelated death worldwide [1]. Many HCC patients are diagnosed at advanced stages of disease, and local treatment options, including curative hepatic resection, tumor ablation, or transarterial therapy, are not suitable. Systemic treatments for advanced HCC are urgently needed. For systemic treatment of HCC, tyrosine kinase inhibitors (TKIs) such as sorafenib, regorafenib, and lenvatinib (Len) are widely used. The efficacy of TKIs is largely insufficient, Len did exhibit a significantly higher response rate relative to sorafenib as a first-line therapeutic option for advanced HCC in the phase III REFLECT trial [2]. Recent studies revealed that tumor cells, including HCC cells, take advantage of metabolic alterations to advance proliferation and survival [3]. Previous studies convincingly demonstrated that glutamine is consumed by proliferating tumor cells with preference compared to other amino acids. Glutamine levels are substantially lower in the tumor core relative to peripheral regions [5]

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Conclusion