Xueqiang Ding

Manganese superoxide dismutase induces migration and invasion of tongue squamous cell carcinoma via H2O2-dependent Snail signaling

Our previous studies had revealed that the dysregulation of manganese superoxide dismutase (SOD2) expression was a frequent event in tongue squamous cell carcinoma (TSCC) and may be associated with enhanced metastatic potential. To further evaluate the mechanism of SOD2-mediated metastasis in TSCC, TSCC cell lines with different metastatic potentials (i.e., the highly metastatic UM1 line and the UM2 line, which displays fewer metastases) were used. Compared to UM2 cells, UM1 cells exhibited significantly higher SOD2 activity and intracellular H(2)O(2); higher protein levels of Snail, MMP1, and pERK1/2; lower protein levels of E-cadherin; and no difference in catalase activity. Upon knockdown of SOD2 by RNA interference, UM1 cells displayed significantly reduced migration and invasion abilities; reduced activities of SOD2; lower intracellular H(2)O(2); decreased protein levels of Snail, MMP1, and pERK1/2; and increased protein levels of E-cadherin. The migration and invasion abilities of UM2 and SOD2 shRNA-transfected UM1 cells were enhanced by H(2)O(2) treatment and accompanied by increased protein levels of Snail, MMP1, and pERK1/2 and decreased protein levels of E-cadherin. Moreover the migration and invasion abilities of UM1 cells were decreased after catalase treatment. Thus, we conclude that the SOD2-dependent production of H(2)O(2) contributes to both the migration and the invasion of TSCC via the Snail signaling pathway, through increased Snail, MMP1, and pERK1/2 protein levels and the repression of the E-cadherin protein.

Down-regulation of tumor suppressor MTUS1/ATIP is associated with enhanced proliferation, poor differentiation and poor prognosis in oral tongue squamous cell carcinoma

Microtubule‐associated tumor suppressor gene (MTUS1, also known as mitochondrial tumor suppressor) is a recently identified tumor suppressor gene that has been implicated in several cancer types. The expression of MTUS1 gene leads to 5 known transcript variants and codes for 5 isoforms of Angiotensin II AT2 receptor interacting protein (ATIP). In this study, we first confirmed that the down‐regulation of MTUS1/ATIP was a frequent event in oral tongue squamous cell carcinoma (OTSCC) and the premalignant lesion (leukoplakia). We further demonstrated that the down‐regulation of MTUS1/ATIP was correlated with poor differentiation and enhanced proliferation (Ki67 proliferation index). Statistical analysis suggests that the down‐regulation of MTUS1/ATIP was associated with reduced overall survival. Isoform specific quantitative RT‐PCR assays revealed that ATIP1, ATIP3a and ATIP3b were the major isoforms of the MTUS1 gene products in oral tongue epithelial cells. Significant down‐regulations were observed for all 3 ATIP isoforms in OTSCC as compared to matching normal tissues. In vitro functional study showed that the restoration of ATIP1 expression led to G1 arrest, apoptosis and reduction of cell proliferation in OTSCC cell lines. These ATIP1‐induced cellular changes were accompanied by reduced phosphorylation of ERK1/2 and up‐regulation of p53. Taken together, these data suggest that MTUS1 plays major roles in the progression of OTSCC, and may serve as a biomarker or therapeutic target for patients with OTSCC.

Combretastatin A4 phosphate induces programmed cell death in vascular endothelial cells.

Combretastatin A4 phosphate (CA4P) is currently undergoing clinical trials as a tumor vascular-targeting agent. Here, we defined the antivascular effect and programmed cell death (PCD) induced by CA4P in vascular endothelial cells. CA4P induced time- and dose-dependent antiproliferative activities against human umbilical vein endothelial cells (HUVECs), and caused G2/M arrest accompanied with DNA fragmentation. The in vitro wound assay and tube formation assay indicated that CA4P potently inhibited migration of endothelial cells and tube formation. The apoptosis and autophagy marker microtubule-associated protein light chain 3 (LC3)-II was induced in CA4P-treated HUVECs. The current study demonstrates that CA4P is a promising antivascular agent with potent PCD-inducing activities. CA4P may be useful in the treatment of cancer and hemangioma.

SOD2 is a C-myc target gene that promotes the migration and invasion of tongue squamous cell carcinoma involving cancer stem-like cells.

Our previous studies revealed that manganese superoxide dismutase (SOD2) contributes to the migration and invasion of tongue squamous cell carcinoma (TSCC). The purpose of the current study was to further clarify the mechanisms of SOD2 in the migration and invasion of TSCC. Side population (SP) cells were used as cancer stem-like cells and further assessed by sphere and colony formation assays, and the expression of stem cell markers (Bmi1, Nanog and ABCG2). We found that UM1 cells (TSCC cells with increased SOD2 expression, migration and invasion abilities) possessed a higher proportion of SP cells, sphere and colony formation, and expressed a higher level of stem cell markers compared to UM2 cells (reduced SOD2 expression, migration and invasion abilities). SOD2 expression as well as migration and invasion abilities were enhanced in SP cells compared to non-SP cells. Knockdown of SOD2 in UM1 cells or SP cells inhibited the migration and invasion abilities, reduced sphere and colony formation, and the expression of stem cell markers. Direct binding of the C-myc protein to the SOD2 promoter was demonstrated by chromatin immunoprecipitation and luciferase assays. Knockdown of C-myc in UM1 cells inhibited SOD2 expression as well as migration and invasion abilities. Our results indicate that cancer stem-like cells play an important role in the migration and invasion of TSCC. SOD2 is a direct target gene of C-myc and C-myc-SOD2-mediated migration and invasion of TSCC involve cancer stem-like cells.

Folic acid rescue of ATRA-induced cleft palate by restoring the TGF-β signal and inhibiting apoptosis

BACKGROUND Cleft palate is a frequent congenital malformation with a heterogeneous etiology, for which folic acid (FA) supplementation has a protective effect. To gain more insight into the molecular pathways affected by FA, TGF-β signaling and apoptosis in mouse embryonic palatal mesenchymal (MEPM) cells of all-trans retinoic acid (ATRA)-induced cleft palate in organ culture were tested. METHODS C57BL/6J mice embryonic palates were explanted on embryonic day 14 and cultured in DMEM/F12 medium with or without ATRA or FA for 72 h. The palatal fusion was examined by light microscopy. Immunohistochemistry was used to detect TGFβ3/TGF receptor II and caspase 9 in MEPM cells. TUNEL was used to detect apoptosis. RESULTS Similar to development in vivo, palatal development and fusion were normal in control medium. ATRA inhibited palatal development and induced cleft palate, which can be rescued by FA. A higher apoptosis rate and caspase-9 in MEPM cells were detected in the ATRA group than in the control or the ATRA+FA group. Compared with the control or the ATRA+FA group, ATRA had little effect on TGF-β3 in MEPM cells but significantly inhibited TGF-β receptor II. CONCLUSIONS Folic acid can rescue the cultured palates to continue developing and fusing that were inhibited and resulted in cleft palate by ATRA. Apoptosis and TGFβ signaling in MEPM cells were involved in folic acid rescued ATRA-induced cleft palate.

Downregulation of miR-221 Inhibits Cell Migration and Invasion through Targeting Methyl-CpG Binding Domain Protein 2 in Human Oral Squamous Cell Carcinoma Cells

Oral squamous cell carcinoma (OSCC), the most frequent of all oral cancers, is a type of highly malignant tumors with a high capacity to invade locally and form distant metastases. An increasing number of studies have shown that microRNAs (miRNAs) play an important role in regulating cancer metastasis and invasion. In the present study, we detected the expression of miR-221 in two highly metastatic OSCC cell lines and two OSCC cell lines that are less metastatic using quantitative real-time PCR analysis (qRT-PCR). The qRT-PCR results indicate that miR-221 is upregulated in highly metastatic OSCC cell lines. Then, miR-221 expression was knocked down by transfection with miR-221 inhibitor, and UM1 cell migration and invasion were assessed using transwell migration and invasion assays. The results indicate that inhibition of miR-221 suppressed migration and invasion of UM1 cells. Furthermore, methyl-CpG binding domain protein 2 (MBD2) was identified as a direct target gene of miR-221. Additionally, MBD2 silencing could partly reverse the effect of miR-221 on cell migration and invasion. In conclusion, downregulation of miR-221 inhibits cell migration and invasion at least partially through targeting MBD2 in the human OSCC cell line UM1.

Downregulation of miR-221 enhances the sensitivity of human oral squamous cell carcinoma cells to Adriamycin through upregulation of TIMP3 expression.

Aberrantly expressed microRNAs (miRNAs) are involved in oral tumorigenesis since they can alter the expression of proteins involved in cancer progression. It remains unclear whether miRNA-221 influences the resistance of human oral squamous cell carcinoma cells to Adriamycin. We therefore investigated the role of miR-221 in the sensitivity of oral squamous cell carcinoma cells to chemotherapy. Tca8113 and UM2 cells were treated with different concentrations of Adriamycin. Quantitative real-time PCR (qRT-PCR) revealed miR-221 upregulation after Adriamycin treatment of Tca8113 and UM2 cells. By using miR-221 inhibitor mimics, we found that depleting cells of miR-221 increases the sensitivity of the cells to Adriamycin. The expression of tissue inhibitor of metalloproteinase-3 (TIMP3), a target of miR-221, was decreased in cells treated with Adriamycin. TIMP3 depletion reversed the effect of a miR-221 inhibitor mimics on cell survival rates and apoptosis. Together, these results reveal that silencing of miR-221 enhances the sensitivity of human oral squamous cell carcinoma cells to Adriamycin through upregulation of TIMP3 expression.

MTUS1/ATIP3a down-regulation is associated with enhanced migration, invasion and poor prognosis in salivary adenoid cystic carcinoma

BackgroundMicrotubule-associated tumor suppressor gene (MTUS1) has been identified as tumor suppressor gene in many malignant tumors. In this study, we investigated the role of MTUS1 in the development of salivary adenoid cystic carcinoma (SACC) and its functional effect on the migration and invasion of SACC.MethodsArchival clinical samples including 49 primary SACC were examined for MTUS1 expression by immunohistochemistry. Statistical analyses were performed to evaluate the correlation between MTUS1 with histopathological features and survival. The expression of MTUS1/ATIP (AT2 receptor-interacting protein) isoforms was determined in SACC tissue samples and cell lines using quantitative RT-PCR assays. Then we investigated whether the migration and invasion of SACC were mediated by MTUS1/ATIP3a using in vitro cell migration and invasion assay.ResultsWe confirmed that the down-regulation of MTUS1 was a frequent event in SACC, and was correlated with distant metastasis and associated with reduced overall survival and disease free survival. Isoform specific quantitative RT-PCR assays revealed that ATIP1, ATIP3a and ATIP3b were the major isoforms of the MTUS1 gene products in SACC, and were significant down-regulation in SACC as compared to matching normal tissues. For functional analyses, we found that SACC-LM cells (SACC cell line with higher migration and invasion ability) possessed a lower expression level of ATIP3a compared to SACC-83 cells (lower migration and invasion ability). Restoration of ATIP3a expression in SACC-LM cells induced anti-proliferative activity and inhibited the migration and invasion ability. Knockdown of ATIP3a promoted the proliferation, migration and invasion ability of SACC-83 cells. Restoration of ATIP3a inhibited the phosphorylation of ERK (extracellular-regulated kinase) 1/2, the expression of Slug and Vimentin in SACC-LM cells, while knockdown of ATIP3a increased the phosphorylation of ERK1/2, the expression of Slug and Vimentin in SACC-83 cells.ConclusionsOur studies confirm that MTUS1 plays an important role in the progression of SACC, and may serve as a biomarker or therapeutic target for patients with SACC. MTUS1/ATIP3a down-regulation contributes to the proliferation, migration and the invasion abilities of SACC.

Dysregulation of AKT1, a miR-138 target gene, is involved in the migration and invasion of tongue squamous cell carcinoma.

BACKGROUND AKT1, also known as PKBα, is abnormally expressed in various malignancies. In this study, we aimed to evaluate the role of AKT1 in the tongue squamous cell carcinoma (TSCC) and further clarify the mechanisms of AKT1 in the migration and invasion of TSCC. METHODS At first, immunohistochemistry (IHC) was conducted to detect the expression of AKT1 in TSCC. Then, we determined the role of AKT1 in the migration and invasion of TSCC and further investigated whether AKT1 was the target gene of miR-138 using dual luciferase reporter assays and Western blot. RESULTS Immunohistochemistry results suggested that AKT1 dysregulation was a frequent event in TSCC, and upregulation of AKT1 was correlated with lymph node metastasis and associated with reduced overall survival. UM1 cells with higher migratory and invasive abilities had more robust AKT1 protein expression than UM2 cells with lower migratory and invasive abilities. The migration and invasion abilities were inhibited in UM1 cells upon AKT1 knockdown, meanwhile resulted in a decline of metastasis-related proteins (vimentin, slug, and pERK1/2), and upregulation of E-cadherin. Luciferase assays revealed that AKT1 was directly targeted by miR-138, and ectopic transfection of miR-138 reduced the expression of AKT1 protein. CONCLUSIONS Our results confirm that upregulation of AKT1, a miR-138 target gene, is a frequent event in TSCC and contributes to the aggressive behaviors and poor prognosis of TSCC.

In vitro effect of iASPP on cell growth of oral tongue squamous cell carcinoma

iASPP is an inhibitory member of the apoptosis-stimulating proteins of P53 (ASPP) family. iASPP is over expressed in several malignant tumors and potentially affects cancer progression. However, the expression and potential role of iASPP in oral tongue squamous cell carcinoma (OTSCC) have not been addressed. In our study, we detected iASPP expression in OTSCC by immunohistochemistry. iASPP expression is up-regulated in OTSCC tissues. Moreover, in clinical pathology specimens, we found that increased iASPP expression correlates with poor differentiation and lymph node metastasis. Using multicellular tumor spheroids (MTS) and flow cytometry, we demonstrated that iASPP down-regulation arrests OTSCC cells at the G0/G1 phase, induces OTSCC cell apoptosis and inhibits OTSCC cell proliferation. These results indicate that iASPP plays a significant role in the progression of OTSCC and may serve as a biomarker or therapeutic target for OTSCC patients.