Chlorine and Chromium Elements, Proteins of Oxidative Stress and Dna Repair Pathways Are Related to Tumor Aggressiveness and Prognosis of Patients With Oral Cancer

Squamous cell (OCSCC), the the extremely aggressive, the response to treatment is poor and markers for prognosis of disease are scarce. The comparison of chemical and histopathological data obtained from the analysis of excised tumor fragments with the demographic and clinical evolution data is an effective strategy scarcely explored in OCSCC studies. The aim was to analyse OCSCC tissues for trace elements and protein expression of enzymes related with oxidative stress and DNA repair that can be candidates as markers of tumor aggressiveness and prognosis. and DNA purinic/apyrimidinic endonuclease/redox and performed by immunohistochemistry. Sociodemographic, clinical and hystopathological data were obtained from 4-year follow-up records.


Abstract
Background Squamous cell carcinoma (OCSCC), the most frequent cancer of the oral cavity, is extremely aggressive, the response to treatment is poor and markers for prognosis of disease are scarce. The comparison of chemical and histopathological data obtained from the analysis of excised tumor fragments with the demographic and clinical evolution data is an effective strategy scarcely explored in OCSCC studies. The aim was to analyse OCSCC tissues for trace elements and protein expression of enzymes related with oxidative stress and DNA repair that can be candidates as markers of tumor aggressiveness and prognosis.

Results
Disease relapse was higher in patients with chlorine and chromium presence and in those with tumors with strong OGG1/2 expression. Strong expression of SOD-1, Trx and Ref-1 was determinant of larger tumor. As expected, perineural, vascular invasions and alcohol consumption were markers of greater worse prognosis.

Conclusion
Presence of trace elements can be markers of disease prognosis. Strong expression of enzymes related with oxidative stress or DNA repair can be either harmful by stimulating tumor growth or bene cial by diminishing relapse rates. Interference on these players may bring novel strategies for the therapeutic management of OCSCC patients.

Background
Oral cancer squamous cell carcinoma (OCSCC) is the most common type of oral cavity cancers. These cancers predominanly occur between the fth and the seventh decades of life and are the sixth most prevalent cancer type in the world population [1]. Despite the improvement in diagnostic and therapeutic procedures, tumor relapse after surgical removal is frequent and the overall 5-year survival rates are low [2,3,4]. Smoking is the chief risk factor for OCSCC, followed by alcoholic addiction. HPV infection and genetic susceptibility are other OCSCC risk factors [5].
It has now been well established that non-essential metals and metaloids can induce carcinogenicity by favoring the generation of reactive oxygen species (ROS). The formation of metal-mediated free radicals can cause several modi cations in DNA bases, increases lipid peroxidation and elicits alterations in the body homeostasis of calcium and sulfhydryl [6].
Unbalance between ROS production and degradation leads to oxidative stress that may participate in the induction of in ammation and carcinogenesis [7]. Enzymes involved in the protection against damage from oxidative stress, such as superoxide dismutase (SOD-1), thioredoxin (Trx), and in DNA repair, such as purinic/apyrimidinic endonuclease/redox factor-1 (Ref-1) and 8-oxoguanine glycosylase (OGG) are important natural defenses against cancer development. Those enzymes may eventually be used as targets for acquisition of prognostic markers and therapeutic strategies [8].
SOD-1 catalyzes the dismutation of superoxide radical into oxygen and hydrogen peroxide (H 2 O 2 ) [8]. In the intracellular medium, metals react with H 2 O 2 and generate hydroxyl radical (OH − ) capable of acting against direct damages to DNA. Damaged DNA can be repaired by the action of OGG1 glycosylase, which excises the modi ed base [9]. Ref-1 protects the DNA structure against enzymatic degradation while the speci c enzymes complete the repair [10]. The activity of Ref-1 is regulated by Trx. [10,11].
Since OCSCC frequently relapses after surgery, it is of prime importance to determine the factors that contribute for the worse prognosis of those patients. In this study, we hypothesize whether differences in content in the tumoral tissue of metals and metaloids and in the protein expression of enzymes related with DNA repair and oxidative stress could be related to disease relapse. To this end, we analysed tumor fragments collected during the surgery of patients with OCSCC to match with the data of clinical evolution of those patients.

Study subjects
Tumor samples, demographic, hystopathological and clinical evolution data were obtained from 78 patients with hystopathological diagnosis of oral cavity squamous cell carcinoma (Table 1). They were selected at the Outpatient Clinic of the Arnaldo Vieira de Carvalho Cancer Institute (ICAVC) in São Paulo, Brazil, between January 2012 and May 2015. The follow-up period was 4 years. They were participants of an ongoing Genome Head and Neck Project (GENCAPO), involving a multi-institutional and multidisciplinary group that has been active since 2002. Tumor fragments inbedded in paraphin blocks were used for the analyses of protein and elementary characterization performed in this study.

Tissue microarrays
Tissue microarrays of the tumor fragments were made as previously described [12], with selection of two representative tumor areas, as evaluated by two experienced pathologists from tissue slides stained with hematoxylin/eosin, followed by extraction of two 1.5 mm diameter cylinders from each sample which were then added to the microarray receptor block using a tissue microarrayer (BEECHER INSTRUMENTS, Silver Spring, MD, USA). Sections were then removed from the TMA and mounted on microscopy slides. A pathologist checked the content of each spot. Spots bent or missing more than 70% of the tissue were excluded.

Qualitative Elementary Characterization
For the qualitative elemental characterization, tumor tissue samples (mean thickness of 450 µm; mean density of 0.54 g/cm 3 ) were removed from the TMA and subjected to dewaxing and rehydration processes with xylene, alcohol (the quality and integrity of these reagents were checked or purity in each batch used) and ultrapure water. They were then deposited in plastic support with Ultralene® lm (SPEX SAMPLEPREP, Metuchen, NJ, USA) and sent to the D09-XRF beamline equipment. For the elementary characterization, the synchrotron radiation-based µ-XRF technique was used to detect chemical elements from electron excitation energy absortion, which is speci c for each element. To obtain the spectra, a white beam with a power range of 4 to 24 keV and dimensions of 2 mm 2 was applied to the samples for 20 seconds and excited the eletons. Nine measurements in a 3x3 matrix were performed and later an averaging was performed to obtain the nal spectrum used in analyzes. For adjustment of characteristic X-ray spectra, determination of elements and their respective uorescent intensities, an analysis of a certi ed reference sample was carried out, Standard Reference Material® 1577b "Bovine Liver", produced by National Institute of Standards and Technology (Gaithersburg, MD, USA), under the same conditions as the test samples, PyMca 5.0.0 software program [13] was the basis for analyzes. Measurements of µXRF were performed in D09-X-Ray Fluorescence (D09-XRF) light line at the National Synchrotron Light Laboratory ( Fig. 1), Campinas, São Paulo, Brazil [14]. Spectra were obtained by the average of nine measured points and analysis was peformed in program PyMca 5.0.0. Each spectrum was veri ed, the characteristic peaks of the elements identi ed, and then identi cation values were assigned as (0) for absence and (1)  Cruz Biotechnology) were used in immunohistochemistry reaction with REVEAL Polymer-HRP (Spring Bioscience), according to the manufacturer's protocol. For each reaction negative controls (absence of primary and secondary antibody) were used. Protein expression was independently evaluated by two different observers, and con icting results were submitted to re-analysis. Protein analysis was semiquantitative so that samples were classi ed according to % of cells stained at: 0 (0% of labeled cells), 1 (< 10%); 2 (10 ≤ 50%) and 3 (> 50% of labeled cells); and by staining intensity in: 0 (negative), 1 (weak), 2 (moderate) and 3 (strong). Scores estimated from the % and intensity of staining were multiplied and their means calculated for each sample. On the basis of the nal score, each sample was cathegorized as negative (0), weak positive (1 ≤ 3) or strong positive (> 3), according to method used by studies that performed similar analyzes to the present study [15,16].

Statistical Analysis
For association tests, Chi-square test was used in bivariate analysis and, when necessary, Fisher's exact test, with a 5% margin of error and with Bonferroni correction. Multivariate logistic regression by modeling was used to adjust odds ratio (OR) and con dence interval (CI ≥ 95%). The variables that obtained a pvalue of less than 20% (p < 0.20) were inserted by the backward method in multivariate logistic regression model, with the signi cant variables remaining at the end of the model, at each stage (p < 0.05). For overall survival analysis, it was calculated the time interval (in months) between dates of surgery and death by disease of each patient or the last return in cases of survivors. The time interval for recurrencefree survival analysis was calculated using as end points the dates of global relapse, or the date of the last return in asymptomatic cases. The Kaplan-Meier model was used for survival analysis, using the Wilcoxon p-value and the Cox proportional hazards to adjust p-values, and to hazards ratio (HR) and CI (CI ≥ 95%). The values of OR and HR were adjusted for lymph node status (TNM). All analyses were performed using SPSS version 20 (IBM Corp., Armonk, NY, USA). Table 2 shows the results of the multivariate analysis between, on one hand, the data on demography, habits, histopathological features of the tumor and protein expression in the tumor cells and, on the other hand, of occurrence or not of relapse or death. Patients aged over 63 years were sevenfold less likely to relapse than those below this age cut-off (p < 0.05). In addition, we also observed that patients consuming alcoholic bevarages were eightfold more likely to relapse (p < 0.05) and sixfold more likely to die from the disease than those that did not consume alcohol (p < 0.01). Also, in Table 2, patients with tumors with vascular invasion were thirteenfold more likely to relapse (p < 0.01) and sixfold more likely to die (p < 0.05) as compared with those with tumors without vascular invasion. Perineural invasion was not related to disease relapse but death rates were increased fourfold in patients with tumors with this feature (p < 0.05).
Also shown in Table 2, all four proteins analysed here by immunohistochemistry were expressed in both nucleous and cytoplasm of the tumor cells (Fig. 2). In patients with tumors with strong expression of OGG1/2, the frequency of relapse was reduced by twenty-fourfold as compared to those with weak expression of this protein (p < 0.01). Regarding the other three proteins, namely Trx, SOD-1 and Ref-1, no correlations were found with relapse or death rates. In Table 3 it is shown that strong protein cytoplasmatic expressions of Trx, and SOD-1 and strong nuclear expression of Ref-1 were associated with larger tumor sizes. Strong cytoplasmic expression of Trx (p < 0.05) and SOD-1 (p < 0.05) were related to three and to vefold larger tumor sizes, respectively. The strong nuclear expression of Ref-1 was associated to sixfold larger tumor size (p < 0.05).
In Table 3, we also found by multivariate analysis that the lymph node vascular involvement was increased by fortysevenfold (p < 0.001) when tumors had vascular invasion. There was a trend for lymph node involvement in tumors of smokers that was not statistically signi cant (p = 0.09). The vascular invasion in tumors also increased the tumor aggressiveness by fteenfold (p < 0.05), whereas the presence of perineural invasion increased the vascular invasion by tenfold (p < 0.001). No association was found between tumor size and tumor vascular invasion (p = 0.211). In Table 4 it is shown that current alcohol consumption is a risk factor for shorter relapse-free survival and increased threefold the probability of disease relapsing (p < 0.05; Table 4). Current alcohol consumption also decreased the overall survival (p < 0.05). The four-year survival after surgery was only 42% in alcohol consumption and 74% in non-alcohol addicted (Fig. 3). The multivariate analysis also showed that alcohol consumption is a risk factor for shorter overall survival, with twofold increase in risk (p < 0.05; Table 4).
Patients with tumors with vascular invasion had lower probability of relapse-free survival (p < 0.01): within two years after surgery, 64% of patients with vascular invasion had disease relapse, as compared with 34% of those with tumors without vascular invasion. (Fig. 4). In the multivariate analysis, tumor vascular invasion appeared as vefold increased risk for shorter disease-free survival (HR = 5.108, CI = 1.536-16.989; Table 4).
Two years after surgery, 93% of patients with tumor vascular invasion died, as compared with 26% of those whose tumors did not have vascular invasion (Fig. 5). Multivariate analysis showed that tumor vascular invasion was a risk factor for shorter overall survival, increasing threefold the probability of death (HR = 2.954, CI = 1.164-7.499; Table 4).
In Table 4 it is also shown that the presence in the tumor of chlorine was associated with vefold decrease in the disease relapse rates (HR = 0.210, CI = 0.046-0.970). There was a trend not statiscally con rmed (p = 0.069) that presence of chromium would be related to shorter relapse-free survival.

Discussion
The results of this study respecting the relationships between the histopathological characteristics of OSCC tumors and the data of the clinical evolution of the patients were con rmatory of the reports from the literature [17][18][19][20][21][22][23]. In several previous studies, the presence of vascular and perineural invasion of the tumor has been related with worse prognosis, i.e., disease relapse, shorter survival and higher death rates [21,23]. In fact, the vascular and perineural invasion of the tumor are fundamental mechanisms for metastatization and recurrence of tumors and constitute important prognostic factors [18][19][20]. Regarding the demographic relationships, our results are also in agreement with the previous reports of older age being a factor for better prognosis [24,25]. The classical relation between alcohol consumption and worse prognosis [26][27][28][29] was also documented here. Unexpectedly, in contrast with previous reports [30][31][32] the smoking habit was not determinant of worse prognosis. It is possible that, in our study, the fact that among non-smokers few had never smoked, and many were ex-smokers, has concealed this rather classical relationship. Therefore, in general terms, the Brazilian population sample studied here had the typical prognostic features of those from other countries.
Our research group was the rst to investigate the presence of metals in oral cancer by µ-XRF analysis [33]. Using this simple and straightforward approach, it is possible to perform multielemental analysis without the disruptive preparation of the tissue samples. It is of note that among the fteen different metals analysed in the tumor fragments only two had relationship with the disease evolution data of the patients. The presence of two of those two metals, chlorine and chromium was associated to disease relapse.
In the patients studied here, the concentration in the serum of the metals was not determined. Nonetheless, in none of the previous studies with elevated serum chlorine was found in oral cancer patients, in contrast, high concentrations of Cu and Zn were reported in patients with this cancer type but with no relationship with the clinical evolution or demographic data [34][35][36]. Our nding that the presence of chromium in the tumor was determinant of worse clinical evolution and that of chloride of better evolution suggests that the analysis of the tumor metal content may be important to unravel new mechanisms underlying the course of the disease.
Some hints of mechanisms whereby the presence of chromium may adversely affect the clinical evolution, with increased relapse rates, can be suggested by the studies of Shi et al. [37,38]. Those authors postulate that reduction of hexavalent Cr to trivalent Cr generates oxygen radicals with activation of signaling pathways of apoptosis inhibition, via PI3K and AKT. The inhibition of apoptosis leads to the accumulation of mutations. This favors microenvironmental changes that stimulate tumor progression and relapse.
In respect to our nding of the relationship between presence of chlorine and decreased rate of tumor recurrence, it is possible that excess chlorine consequent to disturbances in the ionic channel function, speci cally the chloride intracellular chanell 1 e 4 (CLIC1 CLIC4), lowers the cytoplasmic pH thereby inducing the tumor cell apoptosis. In fact, it has been recently shown that CLIC1 is involved in the regulation of the cell cycle and of cell volume, as well as in the regulation of apoptosis. It is postulated that CLIC1 has an important role in tumor development [33,39,40].
Regarding the protein expression of the four different enzymes studied here, only OGG1/2 expression showed relation with disease prognosis. Nonetheless, the expression of the three other enzymes, SOD1, Ref-1 and Trx were related to the tumor size. In patients with tumors exihibiting strong expression of OGG, there was less occurrence of disease relapse.
The protective action against disease relapse offered by strong OGG1/2 expression in the tumors can be ascribed to the reduction of ROS and of mitochondrial DNA damage resulting from the action of this enzyme. OGG1/2 inhibits the activation of p-AKT and of HIF1 which leads to decrease in progression and metastatization of tumors, as observed in mice models of breast tumor [41]. In OGG1/2 KO mice, ROS accumulation occurred, together with non-repair of oxidative damage generated in DNA by suppressing the Nrf2 pathway [42]. Progression of hepatocellular adenocarcinoma induced by phenobarbital to hepatocellular carcinoma was increased in the KO animals, which highlights the importance of OGG1/2 as a key enzyme acting in DNA repair [42]. Thus, our nding that disease relapse was less frequent in patients with strong OGG1/2 expression was in line with those anti-neoplastic actions of this enzyme.
Noteworthy was the fact that the expression of the three other enzymes that were unrelated to disease progression had otherwise strong relation with the size of the tumors. In this respect, strong nuclear expression of Ref-1 determined sixfold larger tumors, while cytoplasmic strong expression of SOD-1 and Trx were related to four and threefold larger tumors, respectively. The association between tumor size and the strong expression of these enzymes can be accounted for the activities of the enzymes that favor tumor growth. SOD-1 increases the oxidative burden, Ref-1 activates transcription factors such as earlyresponse protein-1 (Egr-1), NF-κB, p53, HIF1α (AP-1), which are involved in various cellular processes, including cell survival and growth [43][44][45][46]. Trx-1 expression has been related to cancer development and spread [47]. Trx-1 has redox activity and is related to activation of different transcription factors of in ammation regulation, including NF-kB and activator protein-1 (AP-1) [11]. In addition, its action may increase expression of HIF1α, a hypoxia transcription factor [48], possibly by inhibiting the degradation of HIF1α [49]. Trx-1 binds and inhibits pro-apoptotic proteins, including apoptosis signal by regulating kinase-1 (Ask-1) [50]. Thus, increased protein expression of Trx-1 suggests that increase in the activity of this enzyme would favor tumor growth. This may occur by either altering in ammatory factors, or by activation of angiogenesis via HIF-VEGF.

Conclusion
The results show that detectable amounts chromium and chlorine in the tumor tissue, as well as the Availability of data and materials The data generated or analyzed during this study are included in this article and supplementary les.
Extra information is available with the corresponding author upon request.

Competing interests
The authors declare that no competing interests exist.   Recurrence-free survival plot. Kaplan-Meier curve is shown for recurrence-free survival in patients with squamous cell carcinoma of the oral cavity according to vascular invasion.