Abstract
Arginine biosynthesis and nitric oxide (NO) production are important for cancer homeostasis. Degradation of arginine may be used to inhibit liver tumors with low argininosuccinate synthetase (ASS) expression. In this report, we investigated an alternative therapeutic approach by targeting argininosuccinate lyase (ASL). ASL is transcriptionally induced by endoplasmic reticulum stress and is overexpressed in some human liver tumors. Knockdown of ASL expression by short hairpin RNA (shRNA) in three liver cancer cell lines, ML-1, HuH-7, and HepG2, decreased colony formation in vitro and tumor growth in vivo. Furthermore, lentiviral infection of ASL shRNA inhibited tumor growth in a therapeutic animal tumor model. Analysis of ASL shRNA on the cell-cycle progression revealed a G2-M delay. Among cell-cycle regulatory molecules, cyclin A2 expression was reduced. Reintroduction of exogenous cyclin A2 restored the cell growth in ASL-knockdown cells. Autophagy was observed in the cells treated with ASL shRNA, as shown by an increase in LC3-II levels and autophagosome formation. The total cellular arginine level was not altered significantly. Inhibition of autophagy further attenuated cell growth, suggesting that autophagy induced by ASL shRNA plays a feedback prosurvival function. Knockdown of ASL reduced NO content, and addition of NO donor partially recovered the growth inhibition by ASL shRNA. In summary, downregulation of ASL attenuated tumor growth and the inhibition was mainly mediated by a decrease of cyclin A2 and NO.
Highlights
Arginine is classified as a nonessential amino acid for mature and healthy animals but regarded as an essential amino acid for young and growing animals [1]
argininosuccinate lyase (ASL) expression is induced by endoplasmic reticulum (ER) stress and overexpressed in liver cancer To test whether ASL is induced by ER stress, the human hepatoma cell line HuH-7 and mouse hepatoma cell line ML-1 were incubated in the absence or presence of tunicamycin, an artificial ER stress inducer
We examined whether ASL expression is elevated in human hepatocellular carcinoma
Summary
Arginine is classified as a nonessential amino acid for mature and healthy animals but regarded as an essential amino acid for young and growing animals [1]. Arginine biosynthesis is initiated from glutamate or proline via an important intermediate, citrulline. The endogenous synthesis of arginine from citrulline may not be sufficient for humans in growth or diseased condition. Arginine plays a major role in producing nitric oxide Arginine is the precursor for many biologic molecules, including polyamines, urea, glutamate, proline, and agmatine [2]. Many of these biologic products have been implicated in tumor development, and feeding mice with arginine enhances tumor growth
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