Min Ruan

Growth inhibition and induction of apoptosis in human oral squamous cell carcinoma Tca‐8113 cell lines by shikonin was partly through the inactivation of NF‐κB pathway

Shikonin, a naphthoquinone pigment isolated from the Chinese herbal therapeutic, Zicao, has been shown to exhibit antioxidant and anticancer effects. In this study, its ability to induce apoptosis in cultured Tca-8113 oral cancer cells was studied. Treatment of the Tca-8113 cells with a variety of concentrations of Shikonin (10-40 microm) resulted in dose- and time-dependent sequences of events marked by apoptosis, as shown by the loss of cell viability, chromatin condensation, internucleosomal DNA fragmentation and sub-G1 phase accumulation. Furthermore, apoptosis in the Tca-8113 cells was accompanied by the activation of protease caspase-8, -9, -3 and low expression of Bcl-2 protein. Interestingly, inactivation of the NF-kappaB pathway was found in shikonin-induced apoptosis in Tca-8113 cells. These results raise the possibility that the anti-tumor effects of Shikonin in Tca-8113 cells are at least partly through the inactivation of the NF-kappaB pathway and subsequent activation of protease caspase family. Pharmacological inhibition of the NF-kappaB activity by Shikonin might be a powerful treatment option for OSCC in which activation of NF-kappaB plays a critical role in tumor growth and progression.

Increased expression of Toll-like receptor-9 has close relation with tumour cell proliferation in oral squamous cell carcinoma

OBJECTIVE Toll-like receptor-9 (TLR-9), a new member of the interleukin-1 receptor superfamily, was recently found to have a high level of expression in many carcinoma specimens. The objective of this study was to examine the TLR-9 expression and its role in tumour cell proliferation in oral squamous cell carcinoma. MATERIALS AND METHODS Western blot and immunohistochemistry were used to detect TLR-9 protein in oral squamous cell carcinoma (OSCC) Tca-8113 cell lines and clinical specimens (n=60). The relationship between TLR-9 expression and clinicopathologic features was analysed. Cell proliferation and inflammatory chemokines secretion were tested by MTT and ELISA methods respectively. RESULTS Results showed that TLR-9 expression level was higher in OSCC tissues than in paired adjacent normal tissues (P<0.01), and the expression level of TLR-9 was significantly associated with tumour size (P=0.001), tumour clinical stage (P=0.003) and Ki-67 expression (P<0.01). In vitro results also suggested that stimulation of Tca-8113 cells with TLR-9 agonist CpG-ODN could significantly increase tumour cell proliferation as well as subsequent IL-1α and IL-6 secretions (P<0.01), which could be partially inhibited by usage of anti-TLR-9 protein. CONCLUSIONS It was therefore hypothesized that increased expression of TLR-9 may be of great value in assessing the development of OSCC, and could be used as a new target for OSCC prevention and therapy in future.

Activation of Toll-like receptor-9 promotes cellular migration via up-regulating MMP-2 expression in oral squamous cell carcinoma.

Purpose Activation of Toll like receptors (TLRs) signaling has been implicated in promoting malignant cell invasion and metastatic potential. Previously we demonstrated that increased TLR-9 expression predicted poor survival in oral cancer patients. The objective of this study is to further investigate the roles and potential molecular mechanisms of TLR-9 signaling in human oral cancer cell invasion. Methods Cell migration, invasion and protein expression were detected by wound healing assay, Transwell chambers model and western blot. The secretion and activity levels of metalloproteinases-2/9 were quantified by ELISA and Gelatin zymography. EMSA and ChIP assays were employed to detect the activity of AP-1signal pathway. TLR-9 siRNA transfection was used to regulate the expression and activity of TLR-9 in oral cancer cell line HB cells. Result The results of both wound healing assay and in vitro Transwell assay revealed that activation of TLR-9 induced dose- and time- dependent migration and invasion of HB cells. An increased expression, secretion and activity of MMP-2 were observed upon the treatment of CpG-ODN. The TLR-9 signaling-mediated MMP-2 expression appeared to be a consequence of AP-1 activation, because that their DNA binding activity was enhanced by CpG-ODN treatment. All these influences were efficiently repressed by the knockdown of TLR-9 through siRNA or pretreatment of an AP-1 inhibitor. Conclusion Activation of TLR-9 signaling could promote human oral cancer HB cells invasion with the induction of MMP-2 presentation by attenuating AP-1 binding activity, suggesting a novel anti-metastatic application for TLR-9 targeted therapy in oral cancer in the future.

Prognostic factors in primary salivary gland mucoepidermoid carcinoma: an analysis of 376 cases in an Eastern Chinese population

Mucoepidermoid carcinoma (MEC) is an infrequent malignant neoplasm that originates most commonly in the salivary glands. The present study aimed to provide new information on prognostic factors in patients with salivary gland MEC. A retrospective analysis of the medical records of patients diagnosed with primary salivary gland MEC between 2003 and 2010 was conducted. The incidence of MEC in the minor salivary glands (62.2%) was almost twice that in the major salivary glands (37.8%). The most frequently affected sites were the parotid gland and palate. Lymph node metastasis was reported more frequently in male than female patients (P = 0.02), in high-grade than low/intermediate grade lesions (P < 0.001), and in lesions involving the submandibular gland (P < 0.001). The disease-free survival (DFS) at 5 years was 80.47%, with rates of 98.0%, 86.5%, and 38.5% for low-, intermediate-, and high-grade tumours, respectively. Among various clinicopathological factors, the only independent prognostic factor was histological grade (P < 0.001). Primary tumour site and histological grade are two important factors affecting cervical lymph node metastasis. Histological grade is the only independent factor affecting survival beyond tumor lymph node metastasis (TNM) staging in salivary gland MEC. Further advances in therapy are needed to improve the outcomes for patients with high-grade lesions.

Increased expression of MUC1 predicts poor survival in salivary gland mucoepidermoid carcinoma.

BACKGROUND Mucoepidermoid carcinoma (MEC) is a malignant neoplasm that originates most commonly in the major and minor salivary glands. The aim of this study is to determine the relationship between mucin-1 (MUC1) expression and patient outcome based on a large number of cases. PATIENTS AND METHODS Surgical specimens from 357 patients with primary salivary gland MEC and 10 patients with normal salivary gland tissue were examined by immunohistochemistry. The relationship between MUC1 expression and the clinicopathological data and patient survival was analyzed. RESULTS Results showed that MUC1 expression level was higher in MEC tissues than in paired normal tissues (P = 0.001), and the expression level of MUC1 was significantly associated with gender (P = 0.02), location (P = 0.001), grade (P = 0.001), stage (P = 0.0018) and lymph node metastasis (P = 0.001). In addition, increased expression of MUC1 was confirmed as a strong predictor of poor survival in salivary gland MEC (HR 2.175 [95% CI 1.263, 3.745]; P = 0.0051). CONCLUSION The findings indicate that an increased expression of MUC1 may be of great value in assessing the development and prognosis of salivary gland MEC, and could be used as a new molecule target to improve outcomes for these patients in the future.

The secretion of IL-6 by CpG-ODN-treated cancer cells promotes T-cell immune responses partly through the TLR-9/AP-1 pathway in oral squamous cell carcinoma

Increasing evidence suggests that communication between tumor and immune cells can alter the tumor microenvironment in ways that promote tumor development. The purpose of this study was to characterize the immune response elicited by TLR-9-activated OSCC cells, to identify the cytokines involved in the signaling pathway and to elucidate the molecular mechanism of this pathway in OSCC cells. MTS, flow cytometry and ELISA assay were used to evaluate T-cell immune responses, cancer cell proliferation and pro-inflammatory cytokine secretion, respectively. Western blot analysis, EMSA and ChIP assay were employed to detect the activity of the NF-κB and AP-1 signaling pathways. A marked response was observed when T-cells were co-cultured with supernatants from CpG-ODN-treated OSCC cells. This response was characterized by increased CD4+ and CD8+ T-cell proliferation and an increase in IFN-γ production by the CD4+ T-cell population. Treatment of OSCC cells with CpG-ODN resulted in an increase in IL-6 secretion as well as an increase in AP-1 binding activity to the IL-6 promoter. Moreover, blockage of the TLR-9/AP-1 pathway significantly decreased IL-6 expression and T-cell immune response. In human OSCC, the TLR-9 pathway, when stimulated by CpG-ODNs, promotes a T-cell immune response mediated by AP-1-activated IL-6 secretion. Although the complete molecular mechanism has yet to be understood, these findings provide evidence linking tumor cell activities to immune system responses. In addition, the TLR-9/AP-1/IL-6 pathway provides new therapeutic targets for the prevention and treatment of OSCC.

Increased tumor-infiltrating plasmacytoid dendritic cells predicts poor prognosis in oral squamous cell carcinoma

OBJECTIVE Accumulating evidence suggests that plasmacytoid dendritic cells (pDC) have a dual role not only in initiating anti-tumor immune responses but also in inducing immune tolerance to facilitate cancer development. The aim of this study was to investigate the distribution and function of tumor-infiltrating pDCs in primary oral squamous cell carcinoma (OSCC) and their relation to patient outcome. METHODS The distribution of pDCs in 10 normal oral mucosa and 60 OSCC tissues was detected by immunohistochemistry. The population of pDCs in six OSCC patients and six healthy donors was evaluated by flow cytometry. The relationship between tumor-infiltrating pDCs and clinicopathological data and patient outcome was analyzed accordingly. The capacity of pDCs to produce cytokines, such as IFN-α, IL-6, IL-8 and TNF-α in response to TLR-9 ligands (CpG-ODN) was measured by ELISA. RESULT PDCs were detected at high levels in 38.3% of the OSCC tissues, primarily in the stroma, but were absent in normal oral mucosa. The frequency of pDCs in OSCC tissue was significantly higher than that observed in normal oral mucosa. However, the distribution and population of circulating pDCs was similar between healthy donors and OSCC patients. Kaplan-Meier analysis revealed a significant association of increasing number of tumor-infiltrating pDCs with lymph node metastasis and overall survival. Multivariate analysis confirmed that high levels of tumor-infiltrating pDCs was an independent prognostic factor. Further cytokine analysis revealed a decreased secretion of IFN-α, IL-6 and TNF-α, which indicated an impaired function of tumor-infiltrating pDCs. CONCLUSIONS The increased number of tumor-infiltrating pDCs correlates with an adverse outcome in primary OSCC patients. This finding is not only suggestive of the contribution of pDCs in the progression of oral cancer but also presents an opportunity and a new target for OSCC immune therapy in oral cancer management.

Comprehensive and in-depth analysis of microRNA and mRNA expression profile in salivary adenoid cystic carcinoma

OBJECTIVES To conduct an integrated analysis of microRNA and mRNA expression profile and further discover vital molecules to uncover novel pathogenic mechanisms in salivary adenoid cystic carcinoma (SACC). MATERIALS AND METHODS MicroRNA and mRNA expression profiles were obtained from six paired primary SACC tumors and corresponding adjacent normal glands using high-throughput next-generation sequencing technology followed by an overall integrated bioinformatics analysis and subsequently molecular biology techniques validation. RESULTS Compared with adjacent noncancerous normal gland, 2107 significant differentially expressed mRNA were determined in SACC. Gene ontology and KEGG pathway analysis suggested that the differentially expressed genes were relevant to many significant biological implications. Venn diagram analysis of differentially expressed genes in different group identified 29 differentially expressed overlapping mRNA. 40 differentially expressed microRNAs were also identified in SACC. Furthermore, integrated analysis of microRNA and mRNA expression profiles recognized a core microRNA-mRNA regulatory network and unmasked many novel genes including SCUBE3, CA6, hsa-miR-885-5p and other molecules which may play an essential role in the carcinogenesis of SACC. Also, Q-PCR and immunohistochemistry results reveal the high expression and distribution of SCUBE3 in SACC and dual luciferase reporter assay also preliminarily validated that SCUBE3 was a target of hsa-miR-885-5p. CONCLUSION Contemporary microRNA/mRNA analysis have uncovered many mRNAs and microRNAs worthy further exploration in SACC. These are bound to help us shed light on the overall genetic background of SACC and further elucidate the potential molecular mechanism of SACC.

Culture supernatants of oral cancer cells induce impaired IFN-α production of pDCs partly through the down-regulation of TLR-9 expression

OBJECTIVES The aim of the present study was to investigate whether tumor-derived supernatants down-regulate the immune function of plasmacytoid dendritic cells (pDCs) in oral cancer and the potential molecular mechanisms of this effect. MATERIALS AND METHODS Immunohistochemistry (IHC) and flow cytometry were used to detect tumor-infiltrating and peripheral blood pDCs. MTS and flow cytometry were employed to evaluate the immune response of CD4+ T cells. Real-time PCR and ELISA assays were used to identify TLR-7 and TLR-9 expression, IFN-α production and tumor-secreted soluble cytokines. RESULTS The proportion of pDCs (0.121%±0.043%) was significantly higher in Oral squamous cell carcinoma (OSCC) samples than in normal tissue (0.023%±0.016%) (P = 0.021). TLR9 mRNA was significantly lower in tumor-infiltrating pDCs and positively correlated to low IFN-α production (r = 0.956; P<0.01). The supernatant of oral cancer cells negatively regulated TLR9 mRNA expression and the subsequent IFN-α production of pDCs, which inhibited the immune response of CD4+ T cells. The neutralizing antibodies blocking assay showed that the specific inhibitory effect of pDC functionality was associated with the soluble fraction of the oral cancer environment, which is mainly mediated by IL-10 and TGF-β cooperation. CONCLUSION Tumor-derived supernatants may impair the function of tumor-infiltrating pDCs, which subsequently decreases the immune response of CD4+ T cells in human oral cancer through TGF-β- and IL-10- dependent mechanisms. Careful manipulation of these impaired pDCs may help develop an important alternative immunotherapy for the treatment of oral cancer.

OP148: Salivary gland adenoid cystic carcinoma with cervical lymph node metastasis: A preliminary study of 62 cases

Adenoid cystic carcinoma is an infrequent malignant neoplasm that originates most commonly in the major and minor salivary glands. The purpose of this study is to provide new information on salivary glands adenoid cystic carcinoma with cervical lymph node metastasis. Based on 616 patients who underwent primary tumor resection from 1995 to 2008, 62 cases with cervical lymph node metastasis were chosen and analyzed accordingly. General incidence of cervical lymph node metastasis was approximately 10%.The base of tongue, mobile tongue and mouth floor were the most three frequent sites reporting lymph nodes metastasis, with incidences of 19.2%, 17.6% and 15.3% respectively. Majority of cases occurred via a classic “tunnel-style” metastasis and the level Ib and II regions were the most frequently involved regions. Primary site and lymphovascular invasion were found to be significantly associated with lymph nodes metastasis. High patient mortality was also significantly correlated with lymph node positive cases. Tongue-mouth floor complex have a high propensity for lymph node metastasis, which occurs through a classic “tunnel-style” metastasis. Peritumoral lymphovascular invasion could be taken as strong predictor for lymph node metastasis, which ultimately leads to poor prognosis of ACC patient. Selective neck dissection should be performed in such cases.