Cytolytic T lymphocytes (CTL) and natural killer (NK) cells have been implicated as important cells in antitumor responses. Our previous research has shown that high mobility group nucleosomal-binding domain 2 (HMGN2) could be released by IL-2 and PHA stimulated peripheral blood mononuclear cells (PBMCs) and also induced tumor cells apoptosis at low doses. In this study, we isolated and cultured PBMCs and CD8⁺ T cells to analyze the expression and antitumor effects of HMGN2. PBMCs from healthy donors were isolated using Human Lymphocyte Separation tube. CD8⁺ T cells were separated from the PBMCs using MoFlo XDP high-speed flow cytometry sorter. Activation of PBMCs and CD8⁺ T cells were achieved by stimulating with Phytohemagglutinin (PHA) or tumor antigen. In addition, the methods of ELISA, intracellular staining, and fluorescence-labeling assays were used. PHA induced PBMCs to release high levels of HMGN2, and CD8⁺ T cells was the major cell population in PBMCs that release HMGN2 after PHA activation. Tumor antigen-activated CD8⁺ T cells also released high levels of HMGN2. Supernatants of tumor antigen-activated CD8⁺ T cells were able to kill tumor cells in a dose-dependent manner. This antitumor effect could be significantly blocked by using an anti-HMGN2 antibody. Fluorescence-labeling assays showed that the supernatant proteins of activated CD8⁺ T cells could be transported into tumor cells, and the transport visibly decreased after HMGN2 was depleted by anti-HMGN2 antibody. These results suggest that HMGN2 is an anti-tumor effector molecule of CD8⁺ T cells.

Oral squamous cell carcinoma (OSCC) is capable of constructing a favorable immune escape environment through interactions of cells with cells and of cells with the environment. Programmed death ligand‑1 (PD‑L1) is a well‑recognized inhibitor of anti‑tumor immunity that plays an important role in tumor immune escape. However, the molecular mechanisms regulating PD‑L1 expression are not yet fully understood. In this study, to investigate the role of protein kinase D3 (PKD3) in the regulation of PD‑L1 expression, the expression and correlation of PKD3 and PD‑L1 were first analyzed by the immunostaining of human OSCC tissue sections, cell experiments and TCGA gene expression databases. The expression levels of PKD3 and PD‑L1 were found to be significantly higher in OSCC cells than in normal tissues or cells. In addition, the expression levels of PKD3 and PD‑L1 were found to be significantly positively correlated. Subsequently, it was found that the levsel of PD‑L1 expression decreased following the silencing of PKD3 and that the ability of interferon (IFN)‑γ to induce PD‑L1 expression was also decreased in OSCC. The opposite phenomenon occurred following the overexpression of PKD3. It was also found that the phosphorylation of signal transducer and activator of transcription (STAT)1/STAT3 was reduced by the knockdown of PKD3 in OSCC. Moreover, the expression level of PD‑L1 was decreased after the use of siRNA to knockdown STAT1 or STAT3. On the whole, the findings of this study confirm that PKD3 regulates the expression of PD‑L1 induced by IFN‑γ by regulating the phosphorylation of STAT1/STAT3. These findings broaden the understanding of the biological function of PKD3, suggesting that PKD is a potential therapeutic target for OSCC.

BACKGROUND: Oral squamous cell carcinoma (OSCC) usually originates from oral potentially malignant disorders (OPMD), such as oral leukoplakia (OLK) and oral lichen planus (OLP). Identifying biomarkers for the early diagnosis and evaluation of malignant transformation in OPMD could improve the survival rate of OSCC patients. OBJECTIVE: The present study aimed to screen for potential salivary biomarkers for evaluating the malignant transformation of OPMD. METHODS: Salivary proteases from OLK and OSCC patients or healthy donors and proteases in cultural medium from DOK and Cal-27 cells were detected with a human protease array kit. The concentrations of the salivary Kallikrein 5 (KLK5) and urokinase-type plasminogen activator (uPA) proteases were measured by ELISA. Receiver operating characteristics (ROC) to determine the potential value of these proteases in clinical diagnosis were calculated using SPSS software. Immunohistochemistry was used to detect the KLK5 and uPA expression in the oral organizations. RESULTS: The salivary protease spectrum was different among patients with OLK and OSCC and healthy donors. KLK5 and uPA levels in saliva tended to increase as the disease progressed (healthy < OPMD [OLK and OLP] < OSCC). ROC curves showed the optimum diagnostic cutoffs for KLK5 as a biomarker for OLK, OLP, and OSCC were 5.97, 6.03, and 9.45 pg/mL, respectively, while the cutoffs for uPA were 17.19, 17.26, and 20.96 pg/mL. Their combined analysis showed a higher sensitivity for the differential diagnosis of disease. Furthermore, higher levels of KLK5 and uPA were observed in OSCC tissues than in OLK and OLP. CONCLUSIONS: Salivary KLK5 and uPA are potential biomarkers for evaluating OLK and OLP malignant transformation and early diagnosis of OSCC.

A major obstacle of successful chemotherapy is the development of multidrug resistance (MDR) in the cancer cells, which is difficult to reverse. Metabolomic analysis, an emerging approach that has been increasingly applied in various fields, is able to reflect the unique chemical fingerprints of specific cellular processes in an organism. The assessment of such metabolite changes can be used to identify novel therapeutic biomarkers. In the present study, 1H nuclear magnetic resonance (NMR) spectroscopy was used to analyze the extracellular metabolomic spectrum of the Tca8113 oral squamous carcinoma cell line, in which MDR was induced using the carboplatin (CBP) and pingyangmycin (PYM) chemotherapy drugs in vitro. The data were analyzed using the principal component analysis (PCA) and partial least squares discriminant analysis (PLS‑DA) methods. The results demonstrated that the extracellular metabolomic spectrum of metabolites such as glutamate, glycerophosphoethanol amine, α‑Glucose and β‑Glucose for the drug‑induced Tca8113 cells was significantly different from the parental Tca8113 cell line. A number of biochemicals were also significantly different between the groups based on their NMR spectra, with drug‑resistant cells presenting relatively higher levels of acetate and lower levels of lactate. In addition, a significantly higher peak was observed at δ 3.35 ppm in the spectrum of the PYM‑induced Tca8113 cells. Therefore, 1H NMR‑based metabolomic analysis has a high potential for monitoring the formation of MDR during clinical tumor chemotherapy in the future.

Protein kinase D1 (PKD1), one of the protein kinase D (PKD) family members, plays a prominent role in multiple bio-behaviors of cancer cells. Low pH and hypoxia are unique characteristics of the tumor microenvironment. The aim of this study was to investigate the role and mechanism of PKD1 in regulating metabolism in the human tongue squamous cell carcinoma (TSCC) cell line SCC25 under a hypoxic condition, as well as growth and apoptosis. Here, we found that hypoxia not only induced the expression of HIF-1, but also induced the expression and activation of PKD1. Moreover, we inhibited the expression of PKD1 by shRNA interference, and the growth of SCC25 cells under hypoxia was significantly decreased, as well as the expression of HIF-1, while the percentage of apoptotic SCC25 cells was increased. Furthermore, stable silencing of PKD1 in SCC25 cells under a hypoxic condition decreased glucose uptake, lactate production and glycolytic enzyme (GLUT-1 and LDHA) expression, as well as reduced the phosphorylation of p38 MAPK. The results revealed that following inhibition of the expression of PKD1 under a hypoxic condition, the growth and metabolism of the SCC25 cells were significantly suppressed. In contrast, when PKD1 was overexpressed in SCC25 cells, the results were completely reversed, except for growth and apoptosis. Taken together, our results demonstrated that PKD1 not only regulates the hypoxic glycolytic metabolism of cancer cells via regulation of the expression of HIF-1 and glycolytic enzymes, but is also involved in the remodeling of the acidic tumor microenvironment. This study suggests that PKD1 may be a potential target for microenvironment-directed tumor biotherapy.