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 confirmatory of the reports from the literature13,14,15,16,17,18,19. 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 rates17,19. In fact, the vascular and perineural invasion of the tumor are fundamental mechanisms for metastatization and recurrence of tumors and constitute important prognostic factors14,15,16. Regarding the demographic relationships, our results are also in agreement with the previous reports of older age being a factor for better prognosis20,21. The classical relation between alcohol consumption and worse prognosis22,23,24,25 was also documented here. Unexpectedly, in contrast with previous reports26,27,28 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 first to investigate the presence of metals in oral cancer by µ-XRF analysis6. 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 fifteen 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 data29,30,31. Our finding 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. The evolution of techniques that can be used in the clinical laboratory to determine chromium and chloride in OSCC tumor tissues may offer an interesting tool to routinely evaluate the prognosis of those patients as based on the presence of those elements. These consistent relationships of the two metals and prognosis found here can also make grounds for noval therapeutic targets.
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.32,33. 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 finding 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, specifically 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 development6,34,35.
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 high expression of OGG1/2, there was less occurrence of disease relapse.
The protective action against disease relapse offered by high 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 tumor36. In OGG1/2 KO mice, ROS accumulation occurred, together with non-repair of oxidative damage generated in DNA by suppressing the Nrf2 pathway37. 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 repair37. Thus, our finding that disease relapse was less frequent in patients with high 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 high relation with the size of the tumors. In this respect, high nuclear expression of Ref-1 determined sixfold larger tumors, while cytoplasmic high expression of SOD-1 and Trx were related to four and threefold larger tumors, respectively. The association between tumor size and the high 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 early-response protein-1 (Egr-1), NF-κB, p53, HIF1α (AP-1), which are involved in various cellular processes, including cell survival and growth38,39,40,41. Trx-1 expression has been related to cancer development and spread42. Trx-1 has redox activity and is related to activation of different transcription factors of inflammation regulation, including NF-kB and activator protein-1 (AP-1)12. In addition, its action may increase expression of HIF1α, a hypoxia transcription factor43, possibly by inhibiting the degradation of HIF1α44. Trx-1 binds and inhibits pro-apoptotic proteins, including apoptosis signal by regulating kinase-1 (Ask-1)45. 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 inflammatory factors, or by activation of angiogenesis via HIF-VEGF.
The results show that detectable amounts chromium and chlorine in the tumor tissue, as well as the occurrence of high protein expression of OGG1/2, SOD1, Ref-1 and Trx-1 may influence the tumor growth and predict the clinical evolution of OSCC. These findings, obtained by an innovative methodological approach, may lead to the establishment of useful tools not only for disease prognosis but eventually for acquisition of new therapeutic targets.
The acquisition of practical techniques that can be used in the clinical laboratory to determine chromium and chloride in OSCC tumoral tissues may bring the current results to the routine Oncology practice.
