Cancer Letters

Cancer Letters

Volume 241, Issue 2, 28 September 2006, Pages 309-319
Cancer Letters

Argpyrimidine-modified Heat Shock Protein 27 in human non-small cell lung cancer: A possible mechanism for evasion of apoptosis

https://doi.org/10.1016/j.canlet.2005.10.042Get rights and content

Abstract

Tumors generally display a high glycolytic rate. One consequence of increased glycolysis is the non-enzymatic glycation of proteins leading to the formation of advanced glycation end-products (AGEs). Therefore, we studied the presence of AGEs in non-small cell lung cancer and consequences thereof. We show the presence of two AGEs, i.e. the major AGE Nε-(carboxymethyl)lysine (CML) and the methylglyoxal–arginine adduct argpyrimidine, in human non-small cell lung cancer tissues by immunohistochemistry. We found in squamous cell carcinoma and adenocarcinoma tissues a strong CML positivity in both tumour cells and tumour-surrounding stroma. In contrast, argpyrimidine positivity was predominantly found in tumor cells and was strong in squamous cell carcinomas, but only weak in adenocarcinomas (2.6±0.5 vs. 1.2±0.4, respectively; P<0.005). In accordance, argpyrimidine was found in the human lung squamous carcinoma cell line SW1573, while it was almost absent in the adenocarcinoma cell line H460. Heat shock protein 27 (Hsp27) was identified as a major argpyrimidine-modified protein. In agreement with a previously described anti-apoptotic activity of argpyrimidine-modified Hsp27, the percentage of active caspase-3 positive tumor cells in squamous cell carcinomas was significantly lower when compared to adenocarcinomas. In addition, incubation with cisplatin induced almost no caspase-3 activation in SW1573 cells while a strong activation was seen in H460 cells; which was significantly reduced by incubation with an inhibitor of glyoxalase I, the enzyme that catalyzes the conversion of methylglyoxal. These findings suggest that a high level of argpyrimidine-modified Hsp27 is a mechanism of cancer cells for evasion of apoptosis.

Introduction

Tumors display altered metabolic patterns compared to normal cells, with an increase in anaerobic metabolism of glucose. The inefficiency of this pathway is compensated by a high rate of glucose uptake and glycolysis, a phenomenon also known as the Warburg effect. One consequence of increased glycolysis is the formation of advanced glycation end-products (AGEs) [1]. AGEs are a heterogeneous family of compounds, which arise mainly from the non-enzymatic reaction of sugar ketone or aldehyde groups with free amino groups on proteins. One example of a glucose-derived AGE is Nε-(carboxymethyl)lysine (CML), which has been identified as a major AGE in vivo [2] and is described to be a ligand for the receptor of AGE (RAGE) [3]. Next to glucose, reactive dicarbonyl compounds, such as methylglyoxal, are also important precursors in the formation of AGEs. Methylglyoxal is a side-product of glycolysis, mainly formed by the non-enzymatic fragmentation of triose phosphates [4]. The formation of AGEs has been linked to a variety of detrimental processes associated with ageing [5], Alzheimer's disease [6], atherosclerosis [7] and diabetes [8].

Cells with a high glycolytic rate have been shown to increase intracellular levels of methylglyoxal [9] and to accumulate methylglyoxal–protein modifications [10]. On proteins, methylglyoxal primarily modifies arginine residues [2], [11]. In addition, methylglyoxal causes covalent changes of nucleic acids [12] and histones [13], events which may induce protein degradation and mutagenesis. It has been demonstrated that cells defend themselves against methylglyoxal by the enzyme glyoxalase I, which uses reduced glutathione to convert methylglyoxal to S-d-lactoylglutathione, a compound that is further degraded by glyoxalase II to d-lactate [14]. Accordingly, it was shown that a glyoxalase I inhibitor increased intracellular methylglyoxal levels and consequently induced apoptosis in tumor cells [15], [16]. Altogether, these findings suggest that methylglyoxal could play an important role in tumor biology.

We recently demonstrated the presence of AGEs in patient-derived cancer tissues [17]. We have now examined in human non-small cell lung cancer tissues by immunohistochemistry the expression of the major AGE CML [18] and the expression of the methylglyoxal–arginine modification argpyrimidine (Nδ-(5-hydroxy-4, 6-dimethylpyrimidine-2-yl)-l-ornithine) [2], [19]. In addition, the expression of these AGEs was tested in human lung squamous carcinoma cell line SW1573 and adenocarcinoma cell line H460. It was previously found that in cancer cells endogenous heat shock protein 27 (Hsp27) is a major argpyrimidine-modified protein and that this modification is involved in Hsp27 oligomerization to prevent cytochrome c-mediated caspase activation [10]. Hence, we studied whether argpyrimidine and Hsp27 co-localized in the lung cancer tissues and determined the correlation with active caspase-3 positivity. Finally, we investigated the correlation of argpyrimidine-modified Hsp27 levels with the sensitivity of the lung cancer cell lines for cisplatin-induced apoptosis and the reversal of this induction with an effective glyoxalase I inhibitor.

Section snippets

Preparation and characterization of antibodies

Monoclonal mouse anti-CML IgG was prepared and characterized as recently described [17], [20]. Monoclonal mouse anti-argpyrimidine IgG was a generous gift from Dr K. Uchida (University of Nagoya, Japan), and was characterized before [2]. Monoclonal mouse anti-Hsp27 IgG was purchased from Cell Signaling and polyclonal goat anti-Hsp27 IgG was from Santa Cruz. The use of the polyclonal rabbit anti-active caspase-3 IgG (Pharmingen) for the detection of active caspase-3 in tissues was described

Expression of CML and argpyrimidine in human non-small cell lung cancer tissues

To determine whether AGEs are expressed in tissues of non-small cell lung cancer patients, we analyzed the expression of CML, previously described as a major AGE [18], and of the methylglyoxal–arginine adduct argpyrimidine on sections of human lung squamous cell carcinoma (n=5) and adenocarcinoma tissues (n=5). We found a moderate to strong, but inhomogeneous cytoplasmic CML staining of tumor cells, which was of the same order of magnitude in both the squamous cell carcinoma (Fig. 1A) and the

Discussion

Tumors generally display a high rate of glycolysis and one consequence of high glycolytic activity is the non-enzymatic glycation of proteins and the formation of AGEs. The purpose of this study was to investigate the formation of AGEs in relation to cancer, a subject that is still largely unexplored. We demonstrate the presence of two AGEs in human non-small cell lung tumors; the presence of CML was of the same order of magnitude between human lung squamous cell carcinoma and adenocarcinoma

Acknowledgements

We gratefully thank Dr Uchida for providing us with the anti-argpyrimidine antibody and Dr Creighton for the glyoxalase I inhibitor HCCG diester. The authors want to thank Jan van Bezu and Anke Hardebol for technical support. Casper G. Schalkwijk is supported by a grant from the Diabetes Fonds Nederland.

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