Expression of Cytoplasmic 8-oxo-Gsn and MTH1 Correlates with Pathological Grading in Human Gastric Cancer

The accumulation of oxidative damage in nucleic acids is one of the major causes for mutagenesis and cell death (Nakabeppu et al., 2004). Among the cytoplasmic oxidative nucleic acids, 8-oxo-7, 8-dihydroguanine (8-oxoGsn) of RNA and 8-oxo-7, 8-dihydro-2’deoxyguanosine (8-oxodGsn) of Mt-DNA are the major types, while oxidative NTP pools, such as 8-oxo-dGTP, which is a product of dGTP oxidation and can be inserted into opposite dA or dC residues of template DNA at almost identical efficiencies, causing G:C to T:A or T:A to G:C transversion mutations. While, MTH1 is an oxidized purine nucleoside triphosphatase that upregulates to prevent cellular DNA or RNA oxidative damage, when 8-oxodGsn/Gsn increase (Shimura-Miura et al., 1999, Kosuke et al., 2006). Recently, Helge et al. (2014) and Huber et al. (2014) reported in nature that cancer cells required MTH1 activity to avoid incorporation of oxidized NTPs, resulting in nucleic-acid damage and cell death. Overexpression of MTH1 has been reported (Okamoto et al., 1996; Kennedy et al., 2003; Koketsu et al., 2004) in various tumors except gastric cancer. To explore the correlation between oxidation, MTH1 expression and


Introduction
The accumulation of oxidative damage in nucleic acids is one of the major causes for mutagenesis and cell death (Nakabeppu et al., 2004). Among the cytoplasmic oxidative nucleic acids,  of  of Mt-DNA are the major types, while oxidative NTP pools, such as 8-oxo-dGTP, which is a product of dGTP oxidation and can be inserted into opposite dA or dC residues of template DNA at almost identical efficiencies, causing G:C to T:A or T:A to G:C transversion mutations. While, MTH1 is an oxidized purine nucleoside triphosphatase that upregulates to prevent cellular DNA or RNA oxidative damage, when 8-oxodGsn/Gsn increase (Shimura-Miura et al., 1999, Kosuke et al., 2006. Recently, Helge et al. (2014) and Huber et al. (2014) reported in nature that cancer cells required MTH1 activity to avoid incorporation of oxidized NTPs, resulting in nucleic-acid damage and cell death.
Overexpression of MTH1 has been reported (Okamoto et al., 1996;Kennedy et al., 2003;Koketsu et al., 2004) in various tumors except gastric cancer. To explore the correlation between oxidation, MTH1 expression and

Expression of Cytoplasmic 8-oxo-Gsn and MTH1 Correlates with Pathological Grading in Human Gastric Cancer
Wen-Jie Song 1 , Ping Jiang 2 , Jian-Ping Cai 2 , Zhi-Qiang Zheng 1 * human gastric adenocarcinoma, we investigated the expression pattern of MTH1 and 8-oxoGsn/dGsn in this form of cancer. To the best of our knowledge, this is the first report describing the expression of MTH1 and 8-oxoG in gastric cancer.

Clinical data collection
In this study, 51 cases were selected.The patients had been confirmed histologically gastric cancer and received cancer radical operation at the second affiliated hospital of Wenzhou Medical University, during 2009-2013. Histological stages were graded according to the TNM classification (UICC/AJCC 2010).

Immunohistochemistry experimental procedures.
Resected tumor tissue of all cases were fixed in 4% formaldehyde solution (pH7.0) for periods not exceeding 24 h. The tissues were processed routinely for paraffin embedding, and 3ul thick sections were cut within a cryostat and placed on cationic anti-slip glass slides (ZSGB BIO., Beijing, China) for immunohistochemistry. Tissue samples were stained with hematoxylin and eosin to determine histological type and grade of tumors.
For the detection of 8-oxo-dGsn and 8-oxoGsn (Zheng et al..2009;Song et al., 2011). The sections were de-waxed using xylene and gradually dehydrated with gradient ethanol and washed with PBS. Antigen retrieval was performed by microwave at 95℃ for 15min with buffer solution of citrate salts (pH 6.0) (Maixin Bio., Beijing, China). For the detection of 8-oxo-dGsn in mitochondria, 5mg/ml RNase A (10mg/ml, TakaRa Biotechnology Co., Ltd, Dalian, China) treated sections were directly subjected to IHC with the 15A3 mAb. While to detect the 8-oxo-Gsn in RNA, the sections were treated with 3N HCl at room temperature (RT) for 30min, thus denaturing the nuclear and mitochondrion DNA, then the sections were treated with 50mM Tris-Base for 5 min, furthermore, the sections were treated with DNase I (1000U/ml, TakaRa Biotechnology Co., Ltd, Dalian, China) to remove nuclear or mitochondira DNA, then subjected to IHC with the 15A3 mAb. Besides, the MTH1 staining was performed according to the instructions provided by manufacturer.

Statistical analyses
Data analyses were performed using SPSS software, version 20.0 (SPSS Inc., Chicago, IL, USA). The χ 2 test was used to evaluate the MTH1 and 8-oxo-dGsn/ Gsn expression differences between gastric cancer and para-cancer tissues. The association between MTH1 or 8-oxo-dGsn/Gsn expression and other clinicopathological parameters, including gender, age, clinical stage, histological grade, lymph node metastasis, tumor size and depth of invasion were also analyzed. P values of less than 0.05 were considered statistically significant.

Patient characteristics
The mean age of the 51 patients studied was 63.27 years (range 42-87), including 34 male and 17 female. No significant difference was found in age and gender (P=0.43). Within the total sample, 11 gastric cancer are determined as pathological grade I, 13 as grade II, 5 as grade II-III, 13 as grade III, and 8 grade IV.

Assessment of MTH1 and 8-oxo-dGsn/Gsn expression
Of the 51 gastric cancer cases, 24 (47.1%) exhibited positive MTH1 expression (mainly in the cytoplasm) and 27 (52.9%) exhibited negative MTH1 expression. In the corresponding para-cancer tissue samples, only 6 (11.7%) exhibited positive MTH1 expression. The difference in MTH1 expression between gastric cancer and para-cancer tissues was found to be statistically significant (P<0.05).

Association between MTH1, 8-oxo-dG/G expression with other clinicopathological features
There was no significant association between MTH1 or 8-oxo-dGsn/Gsn expression and gender, age, tumor size, tumor location, tumor invasion depth, clinical stage, as well as lymph node metastasis (P>0.05). However, both MTH1 and 8-oxo-Gsn expression exhibited a significant positive correlation with pathological grade (P<0.05). The results are presented in Table 2.

Discussion
Accumulation of ROS is a double-edged sword. On one hand, ROS has been proved to be an endogenous class of carcinogens in the past decades (Guyton et al., 1993;Cerutti et al., 1994;Feig et al., 1994), on another, excessive ROS might induce cancer cells apoptosis, necrosis, or autophagy (Green et al., 1998;Golstein et al., 2007;Orrenius et al., 2007;Levine et al., 2008), leading to a low survival of cancer cells.
8-Oxo-dGTP is a product of dGTP oxidation and can be inserted into opposite dA or dC residues of template DNA at almost identical efficiencies, so is the same as 8-oxo-GTP. As a result, G:C to T:A or T:A to G:C transversion mutations occur (Shibutani et al., 1991, Maki et al., 1992Cai et al., 1997). As to mtDNA, which is more susceptible to mutations or damage induced by high level of ROS production in mithchondria of cancer cells, due to limited repair mechanisms compared to nuclear DNA (Zhu et al., 2005;Trachootham et al., 2006;Clay Montier et al., 2009). And now it is clear that mtDNA mutation influences OXPHOS function which associated with tumorigenesis (Woo et al., 2012;Yadav et al., 2013). In our study, 52.9% (27/51) gastric cancer samples show positive expression of 8-oxo-dGsn in mtDNA, besides,   no significant difference was found with other parameters, which means that oxidated mtDNA is prevalent among the whole gastric cancer, meanwhile, it is also a metabolic dysfunction signal of cancer cells, or even for normal cells.
The MTH1 protein sanitizes both oxidized dNTP and NTP pools, for example, by converting 8-oxoGTP to monophosphate, thereby preventing incorporation of 8-oxo-7, 8-dihydroguanine into RNA or 8-hydoxy-2'deoxyguanosine into DNA. Recently, Helge et al. (2014) and Huber et al. (2014) reported on Nature that, MTH1 is required for cancer survival, suggesting anticancer strategy by inhibiting MTH1 protein, thus, resulting in excessive oxidative damage of cancer necleic acid, causing a considerable decrease of cancer survival. Our study revealed the high expression of MTH1 protein, which just accorded with the reports of Helge Gad and Huber, moreover, we found MTH1 selectively high expressed in well differentiated gastric cancer.
Therefore, We speculated that the increased ROS level of gastric cancer mainly originated from its dysfunctional mitochondria. The level of ROS might dramatically relevant with mitochondrial capacity (quantity, size and maturity level of mitochondria), and oxidized RNA might produce abnormal protein that, with the dysfunctional mitochondria together, influence cancer differentiation and cancer behavior. However, MTH1 is a momentous protein for cancer cells defending oxidative damage. Thus, a novel anticancer strategy could be actualized through enhancing cancer oxidative stress and inhibiting MTH1 protein, simultaneously.