Expression And The Role Of Thioredoxin System Proteins In Patients With Squamous Cell Carcinoma Of The Tongue

BackgroundOral squamous cell carcinoma (OSCC) is one of the most common malignant tumors of the top ten at present, while the incidence of tongue squamous cell carcinoma (TSCC) ranks the first among the oral cancer in China. TSCC is known for its invasive nature, propensity for lymph node metastasis, and poor prognosis. Comprehensive treatment usually involves surgery, radiotherapy, chemotherapy etc., but the five-year survival of TSCC patients is still low. To explore positively effective preventive measures and drugs becomes very urgent and necessary.The thioredoxin (Trx) system, comprising Trx and Trx reductase (TrxR) plus a reduced form of NADPH, is a major antioxidant system integral to maintenance of the intracellular redox state. Trx protein is one of the most important regulators of redox balance and, thus, of redox-controlled cell functions. The oxidized form of Trx can only be reduced by TrxR within the cell. Several studies have investigated Trx system function or expression in a variety of human tumors and overexpression of Trx was related to poor prognosis and increased proliferation of tumor cells. Therefore, the Trx system protein may play an important role in proliferation and differentiation of tumor cells. Inhibition of the Trx system protein function may induce growth-inhibition of tumor cells. Therefore, the purpose of the present study was to investigate the expression levels of Trx and TrxR-1in human TSCC tissues and the relationships with clinicopathological features and clinical outcome, as well as its role in development of TSCC. The study was divided into three parts as follows:Part One:Overexpression of thioredoxin system proteins predicts poor prognosis in patients with squamous cell carcinoma of the tongueObjective To evaluate the expression of Trx and TrxR-1and the relationships with clinicopathological features and clinical outcome in TSCC. Materials and Methods Immunohistochemistry was employed to analyze the protein expression levels of Trx and TrxR-1in65TSCC tissue samples and10normal oral mucosa samples. The results were then evaluated semiquantitatively and compared to other clinicopathological variables. Statistical analysis was performed with the SPSS16.0. P <0.05was considered statistically significant. Results The expression of Trx and TrxR-1was predominantly manifested in the cytoplasm of tumour cells, with diffuse distribution in nuclei. The percentages of TSCC specimens with Trx and TrxR-1expression were100%(65/65) and95.4%(62/65), respectively, whereas the corresponding rates of expression in normal tissues were almost low, with Trx (9/10) and TrxR-1(10/10). Both Trx and TrxR-1expression levels were significantly higher in TSCC tissues as compared with the10normal oral mucous samples (P<0.01). A highly significant association between Trx and TrxR-1expression in TSCCs was revealed (P=0.001), and the expression of Trx was correlated with tumour cell differentiation (P=0.001). Moreover, Kaplan-Meier analysis revealed that Trx expression and TNM stage were significantly related with5-year survival rate (P=0.033,0.000), while TrxR-1expression was not associated with survival (P=0.092).Conclusion The results indicated that high expression of Trx and TrxR-1was associated with tumourigenesis in TSCC, and overexpression of Trx might predict poor prognosis. Much additional studies into these areas are required to fully elucidate the role of the Trx system in tumourigenesis and as a possible therapeutic target.Part Two:Induction of apoptosis of human tongue squamous cell carcinoma cells by myricetin targeting thioredoxin reductaseObjective To detect the proliferative and apoptotic effect on CAL-27cells when treated with myricetin. Materials and Methods Morphology changes of CAL-27cells treated or without treated by myricetin were observed by optical microscope; the changes of ultramicrostructure in the cells treated with myricetin at120μmol/L for12h were observed by transmission electron microscope; tumor cells were treated with different concentrations of myricetin for6,12, and24h and detected the growth inhibition by MTT assay; cancer cells were treated with Hoechst33258for12h and fluorescent microscopy was used to observe the morphological feature; cells were treated with0,40,80,120,160, and200μmol/L of myricetin for12h and stained with Annexin V and PI, and florescence intensity was measured by flow cytometry; SPSS16.0was used for all analysis and data was indicated by mean±standard deviation (χ±S). Results The morphology changes of CAL-27cells treated with myricetin was observed. When treated with certain myricetin, we observed the cancer cells developed morphological changes, including chromatin condensation, nuclear fragmentation, and apoptotic bodies by transmission electron microscope and fluorescent microscopy. To determine the proliferative activity of HOSCC cells, we found that cell viability was inhibited by myricetin in a time-, and dose-dependent manner. There were significant differences between different concentration-groups and time-groups (P<0.05). The percentage of growth inhibition of the cancer cells treated with200μmol/L myricetin for24h reached to94.21%. Flow cytometry showed that the apoptosis rate of CAL-27cells increased from (14.60%±2.44%) to (62.91%±3.54%) gradually, with the prolonging of myricetin role. There were significant differences (P<0.05). Conclusion Being treated with myricetin induced growth-inhibition and apoptosis of human tongue squamous cell carcinoma (HTSCC) CAL-27cells.Part Three:The apoptotic mechanisms of human tongue squamous cell carcinoma CAL-27cells treated with myricetinObjective To investigate the TrxR activity in CAL-27cells treated with certain myricetin and explain the possible molecular mechanism with special emphasis on the downstream regulator of Trx and caspase for induction of apoptosis of human tongue squamous cell carcinoma (HTSCC) CAL-27cells by myricetin. Materials and Methods CAL-27cell were treated with different concentrations of myricetin in serum-free medium for6,12, and24hours and the cells lysates were subjected to TrxR activity assay; the protein expression status of p-ASK1, ASK1, p-p38, p38, p-JNK and JNK in the CAL-27cells treated with myricetin at40,80,120,160, and200μmol/L for12h, or120μmol/L for30min,1,6, and12h was respectively detected by western blotting assay and the test results were investigated through semi-quantitative analysis; CAL-27cell were treated with different concentrations of myricetin for6,12, and24hours and the cells lysates were subjected to caspase3activity assay; SPSS16.0was used for all analysis and data was indicated by mean±standard deviation (χ±S).Results TrxR activity of CAL-27was decreased when treated with myricetin in a time-, and dose-dependent manner. When treated with120μmol/L myricetin for12h, the rate of inhibition exceeded90%;200μmol/L myricetin for24h, the TrxR activity was almost absent. Western blotting analysis demonstrated that myricetin treatment increased protein phosphorylation of ASK1, p38, and JNK in CAL-27cells with a time-, and dose-dependent manner. Caspase3activity was also increased when treated with myricetin in CAL-27cells with a time-, and dose-dependent manner. There was also statistical difference (P<0.05). Conclusion Myricetin-exerted apoptotic effects on human tongue squamous cell carcinoma CAL-27cells involve TrxR/Trx/ASK1/p38/JNK signaling pathway.