Yixiang Wang

Hydrogen sulfide accelerates cell cycle progression in oral squamous cell carcinoma cell lines

OBJECTIVE To investigate the cell cycle regulator role of the third gaseous transmitter hydrogen sulfide (H2 S) in three oral SCC cell lines by using NaHS, a donor of H2 S. METHODS The synchronized oral squamous cell carcinoma cell lines (Cal27, GNM, and WSU-HN6) were treated with different concentrations of NaHS and then subjected to cell proliferation, cell cycle, and Western blot analyses. RESULTS The CCK-8 assay results showed that the exogenously administered H2 S donor, NaHS, induced CAL-27, and GNM cell proliferation in a concentration-dependent manner, and the cell cycle analysis indicated that NaHS accelerated cell cycle progression of the synchronized CAL-27, GNM, and WSU-HN6 cells. Western blot analysis revealed that the cell cycle regulatory genes RPA70 and RB1 were significantly down-regulated and that proliferating cell nuclear antigen (PCNA) and CDK4 were markedly up-regulated by NaHS at specific time points in the cell cycle. In addition, our results indicated that the phosphorylation of Akt and Erk1/2 was involved in exogenous H2 S-induced oral SCC cell proliferation. CONCLUSIONS H2 S is a potential pro-proliferative factor of human oral SCC cells that accelerates the progression of the SCC cell cycle; thus, H2 S plays a deleterious role in oral SCC cancer development.

Molecular characteristics of homologous salivary adenoid cystic carcinoma cell lines with different lung metastasis ability

Although the homologous salivary adenoid cystic carcinoma (SACC) cell lines SACC-83 and SACC-LM have already been used as SACC models to investigate the underlying mechanisms of metastasis, the molecular features of these SACC cell lines remain unclear. We screened 136 genes related to metastasis in order to investigate the biological and molecular properties of these two cell lines by short tandem repeat (STR) profiling, immunostaining, transwell invasion assay, real-time PCR and western blotting. STR and immunostaining results showed that SACC-83 and SACC-LM are homologous cancer cell lines, derived from adenoepithelial cells and, to date, are not contaminated by each other or other cancer cell lines. Transwell invasion assay results showed that SACC-LM had increased invasion ability compared to SACC-83. 29 of the 136 differentially expressed genes including EREG, S100P, cyclooxygenase (COX)-2, phospho-Akt (p-Akt), matrix metalloproteinase (MMP)-1, MMP-2, MMP-3, MMP-9, MMP-13 and MMP-14 were found following gene screening in SACC-83 and SACC-LM cells. Compared with SACC-83, SACC-LM presents higher expression of COX-2, S100P and lower expression of MMP-2, p-Akt, which could be candidates for identifying the homologous pair cell lines.

High MMP-21 expression in metastatic lymph nodes predicts unfavorable overall survival for oral squamous cell carcinoma patients with lymphatic metastasis

The aim of the present study was to examine the clinical significance of lymph node metastatic (LNM) foci in predicting the overall survival of oral squamous cell carcinoma (OSCC) patients with LNM. MMP-21 was screened based on the LNM animal model of OSCC. Then four proteins, matrix metalloproteinase (MMP)-2, MMP-21, vascular endothelial growth factor (VEGF)-C and VEGF receptor (VEGFR)-3 were examined by immunohistochemistry in 63 OSCC specimens, including the primary tumors (PTs) and the corresponding LNM foci. The expression levels between the PTs and LNM foci were compared by Wilcoxon paired test. Relationships between expression of the four proteins and patient overall survival were assessed by Kaplan-Meier based on the median of the labeling index. The Cox proportional hazards model was used to assess the relative hazard factors. MMP-21 and VEGF-C expression levels were higher in the LNM foci than levels in the PTs. Results showed that MMP-2 and VEGF-C expression levels in the PTs and MMP-2, MMP-21 and VEGF-C expression in the LNM foci correlated with the overall survival of the OSCC patients with lymphatic metastasis. MMP-21 expression level in the LNM foci was the most reliable predictor among all the tested factors. These results suggest that high MMP-21 expression in LNM foci can be used to predict survival in OSCC patients with LNM. Characteristics of LNM foci may be more reliable than PT characteristics in predicting the overall survival of OSCC patients with lymphatic metastasis.

The prognostic value of glycerol-3-phosphate dehydrogenase 1-like expression in head and neck squamous cell carcinoma.

In this study, we sought to determine the prognostic significance of glycerol‐3‐phosphate dehydrogenase 1‐like (GPD1L) expression in head and neck squamous cell carcinoma (HNSCC).

Hydrogen sulfide promotes cell proliferation of oral cancer through activation of the COX2/AKT/ERK1/2 axis.

Hydrogen sulfide, the third gaseous transmitter, is one of the main causes of halitosis in the oral cavity. It is generally considered as playing a deleterious role in many oral diseases including oral cancer. However, the regulatory mechanisms involved in the effects of hydrogen sulfide on oral cancer growth remain largely unknown. In the present study, we investigated the underlying mechanisms through CCK-8 assay, EdU incorporation, real-time PCR, western blot and pathway blockade assays. Our results showed that hydrogen sulfide promoted oral cancer cell proliferation through activation of the COX2, AKT and ERK1/2 pathways in a dose-dependent manner. Blocking any of the three above pathways inhibited hydrogen sulfide-induced oral cancer cell proliferation. Meanwhile, blockade of COX2 by niflumic acid downregulated NaHS-induced p-ERK and p-AKT expression. Inactivation of the AKT pathway by GSK690693 significantly decreased NaHS‑induced p-ERK1/2 expression, and inhibition of the ERK1/2 pathway by U0126 markedly increased NaHS-induced p-AKT expression. Either the AKT or ERK1/2 inhibitor did not significantly alter the COX2 expression level. Our data revealed, for the first time, that hydrogen sulfide promotes oral cancer cell proliferation through activation of the COX2/AKT/ERK1/2 axis, suggesting new potential targets to eliminate the effect of hydrogen sulfide on the development of oral cancer.

Melanoma differentiation-associated gene-7/interleukin-24 as a potential prognostic biomarker and second primary malignancy indicator in head and neck squamous cell carcinoma patients

The significance of melanoma differentiation-associated gene-7/interleukin-24 (MDA-7/IL-24) expression in head and neck squamous cell carcinoma (HNSCC) remains unclear. This study was designed to investigate and evaluate the clinical significance of MDA-7/IL-24 expression in HNSCC by detecting expression by immunostaining in 131 HNSCC specimens. The function of MDA-7/IL-24 was investigated by real-time polymerase chain reaction (PCR) and Western blot in Ad5.mda-7-infected HNSCC cell lines. Our results showed that MDA-7/IL-24 was mainly expressed in the cytoplasm of HNSCC cells. MDA-7/IL-24 high patients presented with a favorable postoperative prognosis compared with MDA-7/IL-24 low patients, and high expression of MDA-7/IL-24 was significantly correlated with a lower incidence of second primary malignancies (SPMs) in the head and neck regions. In vitro assays showed that high expression of MDA-7/IL-24 could upregulate the expression of the epithelial terminal differentiation markers cytokeratin (KRT) 1, KRT4, KRT13, phosphorylated endoplasmic reticulum stress protein (p)-EIF2a, and the apoptosis-related protein cleaved caspase-3. It also downregulated the epithelial proliferative markers KRT5, KRT14, Integrin β4, and anti-apoptosis protein Bcl-2, which might be partially involved in the underlying mechanisms of Ad.mda-7-mediated HNSCC differentiation and apoptosis. Our results indicate that MDA-7/IL-24 can be a prognostic biomarker and an indicator of second primary malignancies (SPM) in HNSCC.

Interpretation of immunohistochemistry data of tumor should consider microenvironmental factors

The influence of tumor surrounding microenvironment is often neglected when immunohistochemistry is performed to investigate tumor properties and search biomarkers of cancer. This study was designed to evaluate whether the influence of tumor microenvironment on biological features of tumor cells should be taken into account for interpretation of the immunohistochemistry data of tumor specimens. In this study, we showed an example by using three tumor cell lines (HeLa, WSU-HN6, and Tca83) to establish tumor-caused bone destruction models in nude mice and then to investigate the influence of bone marrow microenvironment (BMM) on biological features of tumor cells. Immunohistochemistry results showed that, compared with tumor cells located outside of BMM, tumor cells located inside of BMM presented huge differences in the expression of inflammation-related proteins including tumor necrosis factor-α (TNF-α), TNF receptor-associated factor protein-6 (TRAF-6), phosphorylated-NF-κB p65 (p-p65), interleukin (IL)-6 and IL-11, matrix metalloproteinases including MMP-1, MMP-2, MMP-9, and MMP-13; and osteogenesis-related proteins including runt-related transcription factor 2 (RUNX2), bone sialoprotein (BSP), and osteocalcin (OCN) in all the models. However, when we compared the cell line pair derived from different sites (outside and inside of BMM, respectively) of the same HeLa tumor sample by real-time PCR, Western blot, and immunocytochemistry, the differences aforementioned in tumor tissues were not found. In addition, we verified that normal human bone marrow could not cause the above changes detected in vivo. Our results suggested that tumor-modified microenvironment could give the new biological features of the invaded tumor cells. Therefore, we should consider the influence of the surrounding microenvironment on tumor cells when we analyze tumor properties using immunohistochemistry.

Editing genomic DNA in cancer cells with high genetic variance: Benefit or risk?

The generation of stably-transfected cell lines is a common and very important technology in cancer science. Considerable knowledge in the field of life sciences has been gained through the modification of the genetic code. However, there is a risk in evaluating exogenous gene function through editing genomic DNA in a cancer cell with high genetic variance. In the present study, we showed that genomic DNA status should be considered when evaluating the exogenous gene function in a cancer cell line with high variant genome through stable transfection technology, immunostaining, wound healing assay, transwell invasion assay, real-time PCR, western blot and karyotyping analysis. Our results showed that the S100P expression level was not related to the migration and invasion abilities in these stably transfected cell lines derived from a human salivary adenoid cystic carcinoma cell line SACC-83. The MMP expression pattern was detected by western blot analysis which matched the biological behaviors in these cells. The genomic analysis showed that SACC-83 presented hypotetraploid karyotyping with high variance. Our data indicated that establishment of stable transgenic cancer cell lines should consider the status of genetic variance in a cancer cell to avoid any biased conclusion.