Abstract
Endogenous S-nitrosothiols, including S-nitrosoglutathione (GSNO), mediate nitric oxide (NO)-based signaling, inflammatory responses, and smooth muscle function. Reduced GSNO levels have been implicated in several respiratory diseases, and inhibition of GSNO reductase, (GSNOR) the primary enzyme that metabolizes GSNO, represents a novel approach to treating inflammatory lung diseases. Recently, an association between decreased GSNOR expression and human lung cancer risk was proposed in part based on immunohistochemical staining using a polyclonal GSNOR antibody. GSNOR is an isozyme of the alcohol dehydrogenase (ADH) family, and we demonstrate that the antibody used in those studies cross reacts substantially with other ADH proteins and may not be an appropriate reagent. We evaluated human lung cancer tissue arrays using monoclonal antibodies highly specific for human GSNOR with minimal cross reactivity to other ADH proteins. We verified the presence of GSNOR in ≥85% of specimens examined, and extensive analysis of these samples demonstrated no difference in GSNOR protein expression between cancerous and normal lung tissues. Additionally, GSNOR and other ADH mRNA levels were evaluated quantitatively in lung cancer cDNA arrays by qPCR. Consistent with our immunohistochemical findings, GSNOR mRNA levels were not changed in lung cancer tissues, however the expression levels of other ADH genes were decreased. ADH IB mRNA levels were reduced (>10-fold) in 65% of the lung cancer cDNA specimens. We conclude that the previously reported results showed an incorrect association of GSNOR and human lung cancer risk, and a decrease in ADH IB, rather than GSNOR, correlates with human lung cancer.
Highlights
S-nitrosoglutathione (GSNO) is an endogenous nitric oxide donor that serves as a depot for nitric oxide (NO) in the body and plays an integral role in communicating NO mediated signaling functions
Because the protein sequence of GSNOR is highly conserved with other alcohol dehydrogenase (ADH) isozymes, we determined its specificity for GSNOR compared with its specificity toward other ADHs
We demonstrate no consistent differences in the expression of GSNOR when comparing lung cancer tissue to normal lung tissue
Summary
S-nitrosoglutathione (GSNO) is an endogenous nitric oxide donor that serves as a depot for nitric oxide (NO) in the body and plays an integral role in communicating NO mediated signaling functions. This high amino acid sequence identity creates challenges in developing specific antibodies to GSNOR. Marozkina and colleagues used commercially available polyclonal antibodies against human GSNOR to suggest that decreased GSNOR activity from a therapeutic GSNOR inhibitor could leave the lung vulnerable to oncogenic effects from nitrosative stress [12]. We demonstrate that these polyclonal antibodies do not have sufficient specificity to conclude that the signal observed was due to GSNOR rather than other ADH isozymes. Monoclonal antibodies to GSNOR provide a more appropriate tool for characterizing GSNOR protein expression
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