Abstract
L-γ-Glutamyl-p-nitroanilide (GPNA) is widely used to inhibit the glutamine (Gln) transporter ASCT2, but recent studies have demonstrated that it is also able to inhibit other sodium-dependent and independent amino acid transporters. Moreover, GPNA is a well known substrate of the enzyme γ-glutamyltransferase (GGT). Our aim was to evaluate the effect of GGT-mediated GPNA catabolism on cell viability and Gln transport. The GGT-catalyzed hydrolysis of GPNA produced cytotoxic effects in lung cancer A549 cells, resulting from the release of metabolite p-nitroaniline (PNA) rather than from the inhibition of Gln uptake. Interestingly, compounds like valproic acid, verapamil and reversan were able to increase the cytotoxicity of GPNA and PNA, suggesting a key role of intracellular detoxification mechanisms. Our data indicate that the mechanism of action of GPNA is more complex than believed, and further confirm the poor specificity of GPNA as an inhibitor of Gln transport. Different factors may modulate the final effects of GPNA, ranging from GGT and ASCT2 expression to intracellular defenses against xenobiotics. Thus, other strategies - such as a genetic suppression of ASCT2 or the identification of new specific inhibitors - should be preferred when inhibition of ASCT2 function is required.
Highlights
Glutamine (Gln) is a nonessential aminoacid that plays a critical role in cell growth and proliferation
The human lung cancer cell line A549 is highly sensitive to Gln starvation
When cells were incubated with 250 μM GPNA, a time-dependent increase of absorbance at 405 nm, corresponding to the release of the chromogen PNA from GPNA, was observed (Fig. 2a,b)
Summary
Glutamine (Gln) is a nonessential aminoacid that plays a critical role in cell growth and proliferation. The role played by Gln in tumor growth under hypoxic conditions and in autophagy-mediated prosurvival pathways was demonstrated[1,2,3,4,5] In this context, different studies have described an upregulation of high affinity glutamine transporters in cancer cells[4,6,7], which is permissive for high rates of uptake and metabolism of the amino acid often observed in human cancers. It has been suggested that depriving cancer cells of Gln would be a feasible approach to limit tumor growth and to enhance the effects of some antitumor drugs[8] In this perspective, several studies focused on the ASCT2 transporter as a potential therapeutic target, and different approaches, including its inhibition, silencing or degradation upon pharmacologically-induced endoplasmic reticulum (ER) stress, were used[4,9,10]. The mutagenicity and/or cytotoxicity of some gamma-glutamyl derivatives, including PNA, was demonstrated many years ago in Salmonella typhymurium strains[21,22]
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