Conditioned culture medium of bone marrow mesenchymal stem cells promotes phenotypic transformation of microglia by regulating mitochondrial autophagy.

Objective: To study the mechanism by which conditioned medium of bone marrow mesenchymal stem cells (BMSCs-CM) facilitates the transition of pro-inflammatory polarized microglia to an anti-inflammatory phenotype. Methods: BV2 cells, a mouse microglia cell line, were transformed into a pro-inflammatory phenotype using lipopolysaccharide. The expression of phenotypic genes in BV2 cells was detected using real-time quantitative PCR (RT-qPCR). Enzyme-linked immunosorbent assay was used to measure inflammatory cytokine levels in BV2 cells co-cultured with BMSCs-CM. The expressions of mitophagy-associated proteins were determined using western blot. The mitochondrial membrane potential and ATP levels in BV2 cells were measured using JC-1 staining and an ATP assay kit, respectively. Additionally, we examined the proliferation, apoptosis, and migration of C8-D1A cells, a mouse astrocyte cell line, co-cultured with BV2 cells. Results: After co- culture with BMSCs -CM, the mRNA expression of tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase significantly decreased in pro-inflammatory BV2 cells, whereas the expression of CD206 and arginase-1 significantly increased. Moreover, TNF-α and interleukin-6 levels significantly decreased, whereas transforming growth factor-ß and interleukin-10 levels significantly increased. Furthermore, co-culture with BMSCs-CM increased mitophagy-associated protein expression, ATP levels, mitochondrial and lysosomal co-localization in these cells and decreased reactive oxygen species levels. Importantly, BMSCs-CM reversed the decrease in the proliferation and migration of C8-D1A cells co-cultured with pro-inflammatory BV2 cells and inhibited the apoptosis of C8-D1A cells. Conclusion: BMSCs-CM may promote the transition of polarized microglia from a pro-inflammatory to an anti-inflammatory phenotype by regulating mitophagy and influences the functional state of astrocytes.

Erratum: Involvement of miR-619-5p in resistance to cisplatin by regulating ATXN3 in oral squamous cell carcinoma: Erratum.

[This corrects the article DOI: 10.7150/ijbs.54014.].

Cytoplasmic eIF6 promotes OSCC malignant behavior through AKT pathway.

BACKGROUND: Eukaryotic translation initiation factor 6 (eIF6), also known as integrin ß4 binding protein, is involved in ribosome formation and mRNA translation, acting as an anti-association factor. It is also essential for the growth and reproduction of cells, including tumor cells. Yet, its role in oral squamous cell carcinoma (OSCC) remains unclear. METHODS: The expression characteristics of eIF6 in 233 samples were comprehensively analyzed by immunohistochemical staining (IHC). Effects of eIF6 over-expression and knockdown on cell proliferation, migration and invasion were determined by CCK-8, wound healing and Transwell assays. Western blot, immunofluorescence (IF) and co-immunoprecipitation (co-IP) were performed for mechanical verification. RESULTS: We found that cytoplasmic eIF6 was abnormally highly expressed in OSCC tissues, and its expression was associated with tumor size and the clinical grade. Amplification of eIF6 promoted the growth, migration and invasion capabilities of OSCC cell lines in vitro and tumor growth in vivo. Through Western blot analysis, we further discovered that eIF6 significantly promotes epithelial-mesenchymal transformation (EMT) in OSCC cells, while depletion of eIF6 can reverse this process. Mechanistically, eIF6 promoted tumor progression by activating the AKT signaling pathway. By performing co-immunoprecipitation, we discovered a direct interaction between endogenous eIF6 and AKT protein in the cytoplasm. CONCLUSION: These results demonstrated that eIF6 could be a new therapeutic target in OSCC, thus providing a new basis for the prognosis of OSCC patients in the future. Video abstract.

Involvement of miR-619-5p in resistance to cisplatin by regulating ATXN3 in oral squamous cell carcinoma.

MicroRNAs are major post-transcriptional regulators responsible for the development of human cancers, including OSCC. The specific role of miR-619-5p in OSCC, however, is rarely reported. Cisplatin is one of the mostly applied chemotherapy drugs of OSCC. Nevertheless, drug resistance of cisplatin following the initial chemotherapy largely restricts its clinical benefits, and the mechanism of cisplatin resistance is unclear. This study intends to explore the biological function of miR-619-5p in the development of cisplatin resistance in OSCC cell lines and a xenograft model, as well as the potential molecular mechanism. Our results showed that miR-619-5p was down-regulated in OSCC samples and cisplatin-resistant OSCC cells. Ectopically expressed miR-619-5p inhibited proliferative, migratory and invasive abilities of OSCC cisplatin-resistant cells. The putative target gene ATXN3 was predicted by bioinformatic analysis and confirmed by dual-luciferase reporter assay. Importantly, ATXN3 was responsible for the regulatory effects of miR-619-5p on biological behaviors of cisplatin-resistant OSCC cells. Moreover, miR-619-5p mimics and ATXN3-siRNA significantly enhanced ATXN3 knockdown in both HN6/CDDPR and CAL27/CDDPR cells and inhibited expression of PI3K and AKT. In vivo evidences demonstrated that intratumoral injection of miR-619-5p agomir remarkably slowed down the growth of OSCC in xenograft mice. Collectively, microRNA-619-5p was the vital regulator for regulating cisplatin resistance of OSCC, which may be served as a potential therapeutic target.

Expression and oncogenic properties of membranous Notch1 in oral leukoplakia and oral squamous cell carcinoma.

Notch1 signaling is essential for tissue development and tumor progression. This signaling pathway has also been implicated in oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC). However, the role of Notch1 expression in OL and its malignant transformation is unknown. This study aimed to examine the Notch1 expression patterns by immunohistochemistry (IHC) in a cohort of 78 Chinese patients with OL and to analyze the relationship between the patterns and progression of OL to OSCC. Strong Notch1 staining was observed in 10 (13%) of the 78 OL patients, but it was not associated with any of the clinicopathological parameters. However, we observed membranous Notch1 expression in 24 (31%) of the OL samples. Membranous Notch1 expression was significantly associated with the severity of dysplasia (P<0.001) and development of OSCC (P=0.003). By multivariate analysis, membranous Notch1 expression was found to be the only independent factor for OSCC development in the patient population (P=0.019). Among the 24 patients with membranous Notch1 expression, 11 (46%) developed OSCC compared to 8 (15%) of the 54 patients without such expression (P=0.001, determined by log­rank test). Furthermore, we established a 4­nitroquinoline­1­oxide (4NQO)­induced murine OSCC model and studied the Notch1 expression patterns in different stages of carcinogenesis. We observed that the extent of expression of membranous Notch1 increased during carcinogenesis. These data indicated a relationship between membranous Notch1 expression and OSCC risk in patients with OL and suggested that membranous Notch1 served as a biomarker for assessing OSCC risk.

Comparison between Thermal Desorption Tubes and Stainless Steel Canisters Used for Measuring Volatile Organic Compounds in Petrochemical Factories.

OBJECTIVE: The purpose of this study was to compare thermal desorption tubes and stainless steel canisters for measuring volatile organic compounds (VOCs) emitted from petrochemical factories. METHODS: Twelve petrochemical factories in the Mailiao Industrial Complex were recruited for conducting the measurements of VOCs. Thermal desorption tubes and 6-l specially prepared stainless steel canisters were used to simultaneously perform active sampling of environmental air samples. The sampling time of the environmental air samples was set up on 6 h close to a full work shift of the workers. A total of 94 pairwise air samples were collected by using the thermal adsorption tubes and stainless steel canisters in these 12 factories in the petrochemical industrial complex. To maximize the number of comparative data points, all the measurements from all the factories in different sampling times were lumped together to perform a linear regression analysis for each selected VOC. Pearson product-moment correlation coefficient was used to examine the correlation between the pairwise measurements of these two sampling methods. A paired t-test was also performed to examine whether the difference in the concentrations of each selected VOC measured by the two methods was statistically significant. RESULTS: The correlation coefficients of seven compounds, including acetone, n-hexane, benzene, toluene, 1,2-dichloroethane, 1,3-butadiene, and styrene were >0.80 indicating the two sampling methods for these VOCs' measurements had high consistency. The paired t-tests for the measurements of n-hexane, benzene, m/p-xylene, o-xylene, 1,2-dichloroethane, and 1,3-butadiene showed statistically significant difference (P-value < 0.05). This indicated that the two sampling methods had various degrees of systematic errors. Looking at the results of six chemicals and these systematic errors probably resulted from the differences of the detection limits in the two sampling methods for these VOCs. CONCLUSIONS: The comparison between the concentrations of each of the 10 selected VOCs measured by the two sampling methods indicted that the thermal desorption tubes provided high accuracy and precision measurements for acetone, benzene, and 1,3-butadiene. The accuracy and precision of using the thermal desorption tubes for measuring the VOCs can be improved due to new developments in sorbent materials, multi-sorbent designs, and thermal desorption instrumentation. More applications of thermal desorption tubes for measuring occupational and environmental hazardous agents can be anticipated.

[Expression of full-length spleen tyrosine kinase in the oral squamous cell carcinoma and its relationship with tumor invasion and metastasis].

OBJECTIVE: To investigate the expression of full-length spleen tyrosine kinase [SYK (L)] mRNA and protein in human oral squamous cell carcinoma (OSCC) as well as its possible effects on the invasion and metastasis of OSCC. METHODS: The expression of SYK (L) was detected in 27 cases of OSCC tissues and its matched adjacent non-cancerous tissues by real-time quantitative polymerase chain reaction (RT-qPCR), Western blot, and immunohistochemistry. Fourteen cases of normal oral gingival tissues were also analyzed as a normal control. RESULTS: Reduced mRNA and protein expression of SYK (L) in OSCC tissues was observed compared with that in normal oral gingival tissues (P<0.01) and adjacent non-cancerous tissues (P<0.05). SYK(L) expression was significantly associated with lymph-node metastasis (P<0.05). CONCLUSION: SYK(L) is a candidate tumor suppressor for OSCC tissues, and has an inhibitive effect on the initiation, proliferation, and lymph-node metastasis of human OSCC.

Clinicopathological Characteristics and Outcome Predictors in Squamous Cell Carcinoma of the Maxillary Gingiva and Hard Palate.

PURPOSE: Squamous cell carcinoma (SCC) located in the maxillary gingiva and hard palate is relatively rare. There are few published guidelines for the treatment of SCC of the maxilla. The aim of the present study was to characterize the clinicopathologic features of SCC of the maxillary gingiva and hard palate and determine factors that predict outcome and lead to a strategic treatment plan. MATERIALS AND METHODS: A retrospective cohort study of patients with SCC of the maxillary gingiva and hard palate was conducted from 2003 to 2012 at the Department of Oral and Maxillofacial Surgery, Affiliated Hospital of Stomatology, Nanjing Medical University. Clinicopathologic characteristics, treatments, outcome predictors, and 3- and 5-year overall survival rates were analyzed. The Kaplan-Meier method was used to evaluate 3- and 5-year overall survival rates. Univariate and multivariate Cox regression analyses were used to identify predictors of survival. A P value less than .05 was considered statistically significant. RESULTS: The 3- and 5-year survival rates of the 62 participants were 66.6 and 57.3%, respectively. Univariate analyses showed statistically significant (P < .05) associations between patient survival rate and tumor differentiation grade, T classification, marginal status, cervical lymphatics, and local recurrence. Occult lymph node metastases of maxillary SCC in tumor stages T2 to T4 occurred in 20 to 40% of patients. Patients who presented with lesions located after the first premolar plane area and received postoperative radiotherapy had a better prognosis. CONCLUSION: Elective neck dissection is recommended for management of T2 to T4 SCCs in the maxillary gingiva and hard palate. Postoperative radiotherapy can improve the prognosis and decrease the recurrence of SCC after the first premolar plane area.

Neuropilin-1 promotes epithelial-to-mesenchymal transition by stimulating nuclear factor-kappa B and is associated with poor prognosis in human oral squamous cell carcinoma.

BACKGROUND: The epithelial-to-mesenchymal transition (EMT) is a key process in carcinogenesis, invasion, and metastasis of oral squamous cell carcinoma (OSCC). In our previous studies, we found that neuropilin-1 (NRP1) is overexpressed in tongue squamous cell carcinoma and that this overexpression is associated with cell migration and invasion. Nuclear factor-kappa B (NF-κB) plays an essential role both in the induction and the maintenance of EMT and tumor metastasis. Therefore, we hypothesized that NRP1 induces EMT, and that NRP1-induced migration and invasion may be an important mechanism for promoting invasion and metastasis of OSCC through NF-κB activation. METHODS/RESULTS: The variations in gene and protein expression and the changes in the biological behavior of OSCC cell lines transfected with a vector encoding NRP1, or the corresponding vector control, were evaluated. NRP1 overexpression promoted EMT and was associated with enhanced invasive and metastatic properties. Furthermore, the induction of EMT promoted the acquisition of some cancer stem cell (CSC)-like characteristics in OSCC cells. We addressed whether selective inhibition of NF-κB suppresses the NRP1-mediated EMT by treating cells with pyrrolidinedithiocarbamate ammonium (PDTC), an inhibitor of NF-κB. Immunohistochemical analysis of NRP1 in OSCC tissue samples further supported a key mediator role for NRP1 in tumor progression, lymph node metastasis, and indicated that NRP1 is a predictor for poor prognosis in OSCC patients. CONCLUSION: Our results indicate that NRP1 may regulate the EMT process in OSCC cell lines through NF-κB activation, and that higher NRP1 expression levels are associated with lymph node metastasis and poor prognosis in OSCC patients. Further investigation of the role of NRP1 in tumorigenesis may help identify novel targets for the prevention and therapy of oral cancers.