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Background: Foxp3 + regulatory T (Treg) cells and M2 macrophages are associated with increased tumour progression. However, the interaction between Treg cells and M2 macrophages remains unclear., Methods: The expression of FoxP3 and CD163 was detected by immunohistochemistry in 65 cases of laryngeal squamous cell carcinoma (LSCC). In vitro, the generation of activated Treg (aTreg) cells and M2 macrophages by interactions with their precursor cells were analysed by flow cytometry and ELISA. In vivo, the antitumour effects were assessed by combined targeting aTreg cells and M2 macrophages, and intratumoural immunocytes were analysed by flow cytometry., Results: In LSCC tissue, accumulation of aTreg cells and M2 macrophages predicted a poor prognosis and were positively associated with each other. In vitro, aTreg cells were induced from CD4 + CD25 - T cells by cancer cell-activated M2-like macrophages. Consequently, these aTreg cells skewed the differentiation of monocytes towards an M2-like phenotype, thereby forming a positive-feedback loop. Combined targeting aTreg cells and M2 macrophages led to potent antitumour immunity in vivo., Conclusions: The positive-feedback loop between aTreg cells and M2 macrophages is essential to maintain or promote immunosuppression in the tumour microenvironment and may be a potential therapeutic target to inhibit tumour progression.

The present study investigated the expression profile and the function of RP11-169D4.1 and explored its potential mechanisms in laryngeal squamous cell carcinoma. The biological function of RP11-169D4.1 was examined using the MTT assay, flow cytometric analysis, wound healing and transwell assays. The relationship between RP11-169D4.1 and miR-205-5p was discovered by Argonaute 2 protein immunoprecipitation. The target gene of RP11-169D4.1 was CDH1 which was assessed by Pearson's correlation analysis, RT-PCR and western blot assay. We demonstrated that RP11-169D4.1 expression was markedly decreased in LSCC tissues and cell lines. The overexpression of RP11-169D4.1 inhibited the proliferation, migration and invasion of LSCC cell lines as well as promoted apoptosis. We further verified that miR-205-5p had binding sites with RP11‑169D4.1 and that RP11-169D4.1 could regulate the expression of CDH1. Ectopic transfection of RP11-169D4.1 led to a significant reduction in the downstream signaling molecule AKT in LSCC cells. The long non-coding RNA RP11-169D4.1 may serve as a tumor suppressor and a promising therapeutic target in laryngeal cancer, which could inhibit the process of EMT by regulating CDH1.

MicroRNA controls cancer invasion by governing the expression of gene regulating migration and invasion. Here, we reported a novel regulatory pathway controlled by miR-744-3p, which enhanced expression of matrix metallopeptidase 9 (MMP-9) in laryngeal squamous cell carcinoma (LSCC). We profiled the differential micoRNA expression pattern in LSCC cell lines and normal epithelial cultures derived from the head and neck mucosa using microRNA microarray. MiR-7-1-3p, miR-196a/b and miR-744-3p were expressed differentially in the LSCC cell lines. Subsequent validation using real-time PCR revealed that high miR-744-3p level was positively correlated with regional lymph node metastasis of LSCC. Real-time cellular kinetic analysis showed that suppressing miR-744-3p could inhibit migration and invasion of LSCC cell lines and reduce the number of lung metastatic nodules in nude mice modules. In silico analysis revealed that miR-744-3p targeted 2 distinct signaling cascades which eventually upregulated MMP-9 expression in LSCC. First, miR-744-3p could suppress programmed cell death 4 (PDCD4), a direct suppressor of NF-κB (p65). PDCD4 could also prevent AKT activation and suppress MMP-9 expression. Further, suppressing miR-744-3p expression could restore phosphatase and tensin homolog (PTEN) expression. PTEN could inhibit AKT activation and inhibit MMP-9 expression in LSCC cells. The results revealed that suppressing miR-744-3p was effective to inhibit LSCC metastasis by inactivating AKT/mTOR and NF-κB (p65) signaling cascade. Targeting miR-744-3p could be a valuable therapeutic intervention to suppress the aggressiveness of LSCC., Competing Interests: No conflicts of interest were declared.

FoxP3+ regulatory T (Treg) cells have diverse functions in the suppression of antitumor immunity. We show that FoxP3hiCD45RA-CD4+ Treg cells [activated Treg (aTreg) cells] are the predominant cell population among tumor-infiltrating FoxP3+ T cells, and that high aTreg cell-infiltrating content is associated with reduced survival in patients with head and neck squamous cell carcinoma (HNSCC). In vitro studies have demonstrated that aTreg cells can suppress tumor-associated antigen (TAA) effector T cell immune responses in HNSCC. Moreover, C-C chemokine receptor 4 (CCR4) was specifically expressed by aTreg cells in the peripheral blood of HNSCC patients. Using a RayBiotech human chemokine antibody array, we showed that monocyte chemoattractant protein-1 (MCP-1), an endogenous CCR4-binding ligand, was specifically upregulated in the HNSCC microenvironment compared to the other four CCR4-binding ligands. Blocking MCP-1/CCR4 signaling-induced aTreg cell recruitment using a CCR4 antagonist evoked antitumor immunity in mice, and lead to inhibition of tumor growth and prolonged survival. Therefore, blocking aTreg cell trafficking in tumors using CCR4-binding agents may be an effective immunotherapy for HNSCC., Competing Interests: The authors declare that there are no conflicts of interest.

DJ-1, a novel mitogen-dependent oncogene, has an important role in the progression of human malignancies, whereas tumor suppressor phosphatase and tensin homolog (PTEN) is known to control a variety of processes associated with cell survival, proliferation and invasion. DJ-1 overexpression was reported to be negatively correlated with PTEN expression in tumor tissues of patients with laryngeal squamous cell carcinoma (LSCC). In the present study, the effect of DJ-1 on PTEN in laryngeal cancer cells was investigated by transfecting DJ-1-specific small interfering (si)RNA into Hep-2 and SNU-899 cells. Cell survival and cell proliferative and invasive capacity were then evaluated. The results showed that siRNA targeting of DJ-1 effectively upregulated PTEN expression, resulting in enhanced cell death as well as decreased proliferation and invasion of Hep-2 and SNU-899 cells. The results of the present study indicated, for the first time, to the best of our knowledge, that DJ-1-induced PTEN downregulation is associated with proliferative and invasive activity of laryngeal cancer cells. The DJ-1 gene may have an important role in the tumorigenesis of LSCC.

Background: The clinical efficiency and functional outcomes of supracricoid partial laryngectomy (SCPL) with cricothyroidopexy (CTP) were compared with those of the traditional SCPL with cricohyoidoepiglottopexy (CHEP) in treating laryngeal squamous carcinoma involving anterior vocal commissure (AVC)., Methods: From January 2000 to June 2009, 50 patients diagnosed with early- or intermediate-stage (T1b-T3 classification) glottic cancer involving AVC were treated with SCPL-CHEP or SCPL-CTP. Postoperative complications, local recurrence, survival rate, and speech performance were compared between these 2 surgical procedures., Results: Patients undergoing SCPL-CHEP or SCPL-CTP manifested similar levels of postoperative complications, tumor recurrence, and survival rates. However, the SCPL-CTP group showed significantly lower Voice Handicap Index (VHI) scores, higher maximum phonation time, and improved glottic reconstruction and closure than the SCPL-CHEP group., Conclusion: The SCPL-CTP procedure better preserves postoperative speech performance than the SCPL-CHEP procedure, underscoring the moderate effectiveness of SCPL-CTP as a treatment for laryngeal squamous carcinoma involving AVC., (Copyright © 2012 Wiley Periodicals, Inc.)

Objective: The therapeutic effect of middle frontal horizontal partial laryngectomy (MFHPL) in treating stage T1b squamous cell carcinoma of the glottic larynx involving anterior vocal commissure (AVC) was compared with that of the anterior frontolateral vertical partial laryngectomy (AFVPL). The feasibility and practical significance of MFHPL in clinical application was discussed in the present study., Methods: From January 1996 to January 2010, a total of 65 patients diagnosed with stage T1bN0M0 glottic laryngeal cancer were treated with MFHPL or AFVPL. The postoperative complications, glottic reconstruction, recurrence rate, voice quality and survival rates were evaluated and compared between two treatments., Results: AFVPL and MFHPL were performed in 34 and 31 patients, respectively. Flexible fiberoptic laryngoscopy revealed that in the MFHPL-treated patients the reconstructed glottis was spacious and symmetric. In contrast, AFVPL treatment resulted in irregular glottic area with poor symmetry and tubular glottis. The incidence of postoperative laryngeal stenosis significantly differed between the MFHPL- and AFVPL-treated groups (P = 0.025). No significant difference was detected in the 3- and 5-year overall- or tumor-free survival rates between two treatments. The Voice Handicap Index (VHI) and maximum phonation time (MPT) after surgery were 51.0±12.99 and 12.42±3.44 sec in the AFVPL-treated group; while in the MFHPL-treated patients they were 31.81±7.48 and 7.65±1.98 sec, respectively. Both differences in VHI (P = 0.012) and MPT (P = 0.024) were significant between two treatments., Conclusions: MFHPL was comparable to AFVPL with respect to postoperative complications, recurrence rate and survival rates, but possessed advantages over AFVPL in terms of the incidence of laryngeal stenosis and voice quality. Our study indicated that MFHPL has a potential value in clinical practice of treating stage T1b squamous cell carcinoma of the glottic larynx involving AVC.

Background: DJ-1 can induce the tumor cell proliferation and invasion via down-regulating PTEN in many malignant tumors, and correlated to prognostic significance. However, the tumorigenesis role and clinical significance of DJ-1 in supraglottic squamous cell carcinoma (SSCC) is unclear. We aimed to evaluate the DJ-1 the relationship between DJ-1 and clinicopathological data including patient survival., Methods: The expression of DJ-1 and PTEN in SSCCs (52) and adjacent non-cancerous tissues (42) was assessed by immunohistochemistry (IHC), and the relationship between DJ-1 and clinicopathological data was analyzed., Results: DJ-1 was detected mainly in SSCCs (88.5%) and less frequently in adjacent non-cancerous tissues (21.0%). PTEN expression was detected in 46.2% of SSCCs and in 90.5% of adjacent non-cancerous tissues. DJ-1 expression was linked to nodal status (P = 0.009), a highly significant association of DJ-1 expression with shortened patient overall survival (5-year survival rate 88.0% versus 53.9%; P = 0.007; log rank test) was demonstrated., Conclusions: Our data suggested that DJ-1 over-expression was linked to nodal status, and might be an independent prognostic marker for patients with SSCC.

Objective: To compare the real-time diagnostic accuracy of conventional white-light imaging (WLI) endoscopy with that of narrow-band imaging (NBI) endoscopy in patients at high risk for nasopharyngeal carcinoma (NPC)., Design: Prospective study., Setting: A university tertiary care center., Patients: From July 28 through October 27, 2009, a total of 211 consecutive patients at high risk for NPC were enrolled. A high-performance endoscopic system equipped with WLI and NBI modes was used for a detailed examination of the nasopharynx during the same endoscopy., Main Outcome Measures: Diagnostic efficacies of WLI and NBI were compared with pathologic findings. Lesions were classified according to the detailed morphologic epithelial microvessel observations during NBI., Results: A total of 285 lesions were detected, including 66 cancerous lesions. The sensitivity and negative predictive values of NBI in NPC screening were significantly higher than those of WLI (93.9% vs 71.2%, P = .001; and 98.1% vs 91.7%, P = .003; respectively); specificity and positive predictive value were not significantly different. During NBI, the presence of superficial, distorted, irregularly shaped microvessels indicated malignant lesions; 53 of 55 lesions (96.4%) with type IV intrapapillary capillary loops were confirmed on histologic testing as malignant. The false-negative and false-positive rates for NBI were 4.5% and 3.6%, respectively., Conclusions: Narrow-band imaging endoscopy is a promising tool to differentiate nonmalignant from malignant nasopharyngeal lesions on the basis of the morphologic findings of mucosal capillary vessels in vivo. In addition, NBI may increase the diagnostic value of endoscopy in populations at high risk for NPC.

Objective: To detect the expression of DJ-1 in laryngeal squamous cell carcinoma (LSCC) and to study the relationship between DJ-1 expression and clinical indexes of LSCC., Methods: The expressions of DJ-1 protein in 71 LSCC samples and 9 cases control samples from laryngeal mucosa tissues of non-LSCC patients were detected using streptavidin peroxidase immunohistochemistry staining and the relationships between DJ-1 protein expression and clinicopathologic characteristics were analyzed., Results: (1) The positive expression rate of DJ-1 protein in LSCC was 85.9%(61/71), which was significantly higher than the rate (55.5%, 5/9) in control laryngeal mucosa tissues (P < 0.05). (2) DJ-1 expression was related to tumor recurrence (P < 0.05), but not to sex, age, primary cancer position, T stage, clinical stage, lymph node metastasis and tumor differentiation. Tumor recurrence rate (53.3%) in the patients with higher expression of DJ-1 protein was higher than the rate (26.8%) in the patients with lower expression of DJ-1 protein (χ(2) = 5.164, P < 0.05). (3) With Kaplan-Meier curves and Cox regression analysis, the cumulative 5-year survival rates were correlated with DJ-1 expression levels in laryngeal cancer tissues or cervical lymph node metastasis (all P < 0.05), but not to sex, age, primary cancer position, T stage, clinical stage and tumor differentiation., Conclusions: The expression of DJ-1 protein in LSCC is higher than that in control laryngeal mucous tissues. Overexpression of DJ-1 is associated with poor overall survival in LSCC patients.

DJ-1 is frequently overexpressed in a large variety of solid tumors, but the DJ-1 expression in laryngeal squamous cell cancer and its clinical/prognostic significance is unclear. We aimed to evaluate DJ-1 protein expression in glottic squamous cell carcinoma (GSCC) and to correlate this with clinicopathological data including patient survival. The expression of DJ-1 in GSCCs (60) and adjacent normal tissue (44) was assessed by immunohistochemistry and western blot analysis. In addition, the role of DJ-1 was investigated in tumorigenesis by transfecting DJ1-specific siRNA into laryngeal squamous cell carcinoma (LSCC) Hep-2 cells. Our data showed that positive expression of DJ-1 was found in 85% of GSCCs. In univariate survival analysis of the GSCC cohorts, a highly significant association between DJ-1 expression with shortened patient overall survival (5-year survival rate 92.9%vs 66.6%; P = 0.001; log rank test) was demonstrated. In multivariate analyses, DJ-1, tumor grading, and pT status were significant prognostic parameters for shortened patient overall survival. Furthermore, siRNA targeting DJ-1 can effectively inhibit DJ-1 expression, resulting in enhanced apoptosis and less proliferation of Hep-2 cells. We concluded that DJ-1 overexpression might be a novel independent molecular marker for poor prognosis (shortened overall survival) of patients with GSCC.

Objective: To evaluate the effect of combined use of rapamycin and cisplatin in the chemotherapy of Hep-2 cells in vitro., Methods: The inhibitory effects of rapamycin and cisplatin, used alone or in combination, on the proliferation of Hep-2 cells were measured with MTT assay and median-effect plot analysis. The cell cycle changes after the treatment were analyzed using flow cytometry and Hoechst 33258 immunofluorescence staining., Results: The IC50 of rapamycin and cisplatin for inducing growth arrest of Hep-2 cells was 11.03 nmol/L and 8.81 micromol/L, respectively. Rapamycin alone caused cell cycle arrest of the Hep-2 cells in G1 phase. Rapamycin and cisplatin showed synergistic effects in the chemotherapy of Hep-2 cells (q > 1.15, King's Formula), causing significantly increased apoptosis ratio and growth inhibition rate of Hep-2 cells., Conclusion: Combined use of rapamycin and cisplatin significantly improves the chemotherapeutic effect against Hep-2 cells.

Objective: To detect the presence of ion channel protein and its role in cell growth and proliferation in human head and neck squamous carcinoma cells (SCC)., Methods: Human head and neck squamous carcinoma SCC-25 cell line was tested with transient receptor potential melastatin 7 (TRPM7) antibody using the method of immunocytochemistry. The role of TRPM7 in cell growth and proliferation was evaluated through its blockade by ion channel blockers and specific siRNA using lactate dehydrogenase (LDH) assay technique., Results: Clear immunoreactivity against TRPM7 was detected in almost all SCC-25 cells tested, whereas no immunoreactivity was observed in negative control. The inhibitory effect of Gd3+, a non-specific ion channel blocker, on cell growth and proliferation was potent. Addition of 10 micromol/L Gd3+ (n = 16) and 100 micromol/L Gd3+ (n = 16) in the culture medium significantly inhibited the growth of SCC-25 cells, as compared with control cells growing in normal medium (t was 4.1414 and 6.2661, P was 0.0256 and 0.0082 respectively). However, the effect of 2-APB was striking. Cell proliferation was almost totally suppressed in the presence of 100 micromol/L 2-APB (t = 13.4493, P = 0.0008, n = 16) compared with cells growing in normal medium. Suppression of TRPM7 expression by siRNA also significantly inhibited the growth and proliferation of these cells (t = 4.3446, P = 0.0002, n = 32, compared with nontransfected cells),whereas cells transfected with negative control siRNA showed no difference in cell proliferation compared with nontransfected cells., Conclusions: All of those results strongly suggest the existence of TRPM7 channel in human head and neck squamous carcinoma cells. Ion channel blockers serve as a potent inhibitor of SCC-25 cell proliferation. The striking inhibitory effect of 2-APB on cell growth and proliferation may promise clinical workers an inspiring remedy for fighting against carcinoma.