Search

Chemotherapies such as 5-fluorouracil (5-FU) and cisplatin (CDDP) have been widely used to treat laryngeal squamous cell carcinoma (LSCC), the second most common head and neck squamous cell carcinoma. However, chemoresistance seriously impairs chemotherapeutic efficacy. Our present study reveals that 5-FU and CDDP treatment increase the expression of histone deacetylase 1 (HDAC1) in LSCC cells. Consistently, increased levels of HDAC1 are observed in chemoresistant cells. Knockdown of HDAC1 significantly restores the sensitivity of LSCC cells, as HDAC1 increases the expression of interleukin-8 (IL-8), which is essential for LSCC chemoresistance. Mechanistically, HDAC1 directly initiates the transcription of IL-8 though binding to its promoter. Simultaneously, si-HDAC1 increases the levels of miR-93, which binds to the 3'UTR of IL-8 mRNA to trigger its degradation. In summary, the HDAC1/IL-8 axis can confer chemotherapeutic resistance to LSCC cells., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Although elevated serum gamma-glutamyltransferase (GGT) is a known indicator of increased risk of several cancers, the clinical value of repeated measurements of GGT has not been determined. Therefore, we aimed to investigate whether repeatedly elevated serum GGT levels are associated with the risk of respiratory cancer incidence., Methods: We included participants who had undergone the Korean Health screening four times during 2009-2012 and had previously undergone four consecutive examinations. Those who were diagnosed with respiratory cancer before the date of examination were excluded. The participants obtained one GGT point if their GGT levels were in the highest quartile (the quartile 4 group). We analyzed the association between GGT points and respiratory cancer incidence by Cox proportional hazard models., Results: During mean follow-up of 6.39 ± 1.2 years, 3,559,109 participants were enrolled. Of them, 8,944 (0.34%) men and 1,484 (0.14%) women were newly diagnosed with respiratory cancer. In multivariate analysis adjusted for confounding factors, male participants with 4 GGT points had a significantly higher hazards of developing respiratory cancer than those with 0 GGT points (hazard ratio [HR]: 1.39; 95% confidence interval [CI]: 1.31-1.48). Among female, participants with the highest points of GGT also had sixfold increased risk of developing laryngeal cancer. However, no significant association was observed between GGT points and lung cancer incidence among women (HR: 0.95; 95% CI: 0.81-1.11)., Conclusion: Repeatedly elevated serum levels of GGT were associated with a higher risk of respiratory cancer incidence, especially in men. This finding suggests that physicians can identify a person with a higher risk of respiratory cancer through a simple repeated measurement of GGT., (© 2021 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Abstract: To investigate whether laryngeal cancer cells express erythropoietin (Epo) and erythropoietin receptor (EpoR) and what is their possible relationship with clinical and pathological features of the tumor.We performed immunohistochemical analysis of Epo and EpoR expression on 78 tissue samples of invasive and in situ squamous cell laryngeal carcinoma.The statistical analysis showed a weak positive and statistically significant correlation of EpoHS and EpoR HS expression levels. Epo HS and EpoR HS levels did not correlate with patient sex or age, type of diagnosis, cancer stage, histological tumor grade, presence or absence of disease recurrence, type of oncologic cancer therapy provided, or results of selected laboratory blood work. The results show a statistically significant difference in Epo expression with respect to survival.We confirmed the presence of Epo an EpoR in malignant laryngeal tumors and demonstrated the correlation between Epo expression and survival. Further studies are needed to more precisely define the role of Epo and EpoR in treatment of patients with laryngeal cancer., Competing Interests: The authors declare that they have no conflicts of interest to the publication of this article., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

Objectives: The gastric H+/K+ ATPase proton pump has previously been shown to be expressed in the human larynx, however its contribution to laryngopharyngeal reflux (LPR) signs, symptoms and associated diseases such as laryngeal cancer is unknown. Proton pump expression in the larynx of patients with LPR and laryngeal cancer was investigated herein. A human hypopharyngeal cell line expressing the proton pump was generated to investigate its effects., Study Design: In-vitro translational., Methods: Laryngeal biopsies were obtained from three LPR and eight LSCC patients. ATP4A, ATP4B and HRPT1 were assayed via qPCR. Human hypopharyngeal FaDu cell lines stably expressing proton pump were created using lentiviral transduction and examined via transmission electron microscopy and qPCR for genes associated with inflammation or laryngeal cancer., Results: Expression of ATP4A and ATP4B was detected in 3/3 LPR, 4/8 LSCC-tumor and 3/8 LSCC-adjacent specimens. Expression of ATP4A and ATP4B in FaDu elicited mitochondrial damage and expression of IL1B, PTGS2, and TNFA (P < .0001); expression of ATP4B alone did not., Conclusions: Gastric proton pump subunits are expressed in the larynx of LPR and LSCC patients. Mitochondrial damage and changes in gene expression observed in cells expressing the full proton pump, absent in those expressing a single subunit, suggest that acid secretion by functional proton pumps expressed in upper airway mucosa may elicit local cell and molecular changes associated with inflammation and cancer., Level of Evidence: NA Laryngoscope, 131:130-135, 2021., (© 2020 The American Laryngological, Rhinological and Otological Society, Inc.)

Objectives: The current study was performed to determine the presence of pepsin in saliva and laryngeal tissue among participants with benign and malignant laryngeal neoplasms., Study Design: Case-control study included three groups of patients with: (1) benign laryngeal neoplasms, (2) malignant laryngeal neoplasms and (3) control subjects without symptoms or signs of laryngopharyngeal reflux (LPR)., Methods: Eighty-one voluntary participants were included into study. They were recruited from a group of patients with histologically proven benign and malignant laryngeal neoplasms and in case of control subjects among patients with nasal septum deformation without symptoms of LPR. Morning saliva samples were collected preoperatively. Tumor biopsies were collected by directoscopy of larynx and the control samples from interarytenoid unit of larynx. All samples were analyzed by Enzyme-Linked Immunosorbent Assay (ELISA) and Immunohistochemistry., Results: Pepsin was found in all samples of saliva and tissue biopsies in groups with malignant and benign neoplasms. The highest concentration of pepsin was found in a group of patients with malignant laryngeal neoplasms. Patients with benign laryngeal neoplasms had lower concentrations and the control subjects presented with the lowest concentration of pepsin measured from their saliva. Differences were not statistically significant. Immunohistochemical (IHC) analysis showed the largest number of high positive samples in the group of malignant lesions., Conclusion: These results suggest that pepsin and LPR can contribute to the development of benign and malignant laryngeal neoplasms. Further prospective studies, with far more patients, are necessary to prove the role of pepsin in multifactorial etiology of laryngeal neoplasms., (© 2020 The Author(s).)

Purpose: Abnormal DNA methylation plays an important role in clinical diagnosis and prognosis of various tumors. DNA methylation is catalyzed by DNA methyltransferase (DNMT). However, the methylation status of MAGE-A1 and MAGE-A3 promoter regions in LSCC is unclear. To investigate the methylation levels of MAGE-A1, -A3 in LSCC and corresponding normal tissues. The expression of DNMTs (DNMT1, DNMT3a and DNMT3b) in LSCC and the relationship between the methylation status of MAGE-A1, -A3 were analyzed., Materials and Methods: We examined methylation status of MAGE-A1, -A3 in LSCC by using MSP. Meanwhile, the expression level of DNMTs in LSCC was detected by immunohistochemistry. And further analysis the correlation between DNMTs expression level and methylation status of MAGE-A1 and MAGE-A3., Results: The unmethylation rate of MAGE-A1, -A3 were 39.62% and 46.23%. The expression of DNMTs was 33.02% to 37.74%. The level of demethylation of MAGE-A1 and MAGE-A3 were negative related to DNMTs protein. MAGE-A1 and MAGE-A3 unmethylation status and DNMT3a expression were independent prognostic factors for LSCC., Conclusions: The DNMTs may participate in the methylation process of MAGE-A1 and MAGE-A3, which may play an important role in the occurrence and development of LSCC., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Andrographolide is a natural diterpenoid from Andrographis paniculata that has been proposed as an anticancer agent as well as a chemosensitizer for use in combination with anticancer drugs. Carboplatin is the first-line chemotherapeutic agent for advanced laryngeal carcinoma. However, the clinical efficacy of carboplatin is limited by drug resistance and side effects. The aim of this study was to investigate whether andrographolide has a synergistic antitumor effect with carboplatin on human laryngeal cancer cells. Hep-2 cells were exposed to andrographolide with or without carboplatin. The effects of indicated therapies were examined using the Cell Counting Kit-8 assay, the colony-forming assay, the Hoechst 33342/PI double staining, and flow cytometry analysis. The molecular mechanism was assessed by reactive oxygen species (ROS) detection and western blot. At the sublethal concentration, andrographolide increased carboplatin sensitivity of Hep-2 cells by increasing carboplatin-induced apoptosis and inhibiting cell viability. Moreover, we found that andrographolide sensitized carboplatin mainly through the induction of ROS generation and apoptotic signaling. Taken together, these results indicate that andrographolide, along with carboplatin, synergistically inhibited cell proliferation and induced mitochondrial apoptosis of Hep-2 cells by increasing the intracellular ROS, regulating the mitogen-activated protein kinase and phosphatidylinositol 3-kinase (PI3K/AKT) pathways, altering the BCL2/BAX ratio, and ultimately activating the cleavage of Caspase-3 and PARP. These results suggest that andrographolide sensitizes human laryngeal cancer cells to carboplatin-induced apoptosis by increasing ROS levels.

Caspases (cysteine-aspartic proteases) represent a family of enzymes that modify several functions crucial for cell homeostasis such as inflammation and apoptosis. According to their implication in the apoptotic pathways, caspases are characterized as initiators and executioners, respectively. In the first group have been inserted caspase-2,-8,-9, and -10, whereas caspase-3,-6, and-7 belong to the second category. All of these normal actions of the caspase complex that induce apoptosis are altered in carcinoma progression and establishment. In cancer tissues, programmed cell death is inhibited due to a deregulation in expression of apo- and anti-apoptotic proteins. This genetic imbalance drives the cancer cell to immortalization which reflects the aberrant tissue proliferation. For this reason, caspases and the other apoptotic molecules are considered as important targets for specific targeted therapeutic strategies for enhancing the apoptotic levels inside the malignant tumor cells cores. In the current review we explored the role of caspases deregulation in laryngeal squamous cell carcinoma (LSCC). In malignancies -including LSCC- its deregulation leads the cells to immortalization due to apoptosis inhibition and telomerase overexpression. Caspase-dependent apoptotic rates are decreased in LSCC. For this reason, caspases are considered a very promising target for applying targeted therapeutic strategies in order to enhance apoptosis in the corresponding patients suffering of LSCC.

BACKGROUND Laryngeal cancer is one of the major malignancies of the neck and head and is responsible for considerable mortality across the globe. The treatments for laryngeal cancer mainly involve surgical interventions followed by chemotherapy. However, due to unsatisfactory results, constant relapses and the adverse effects associated with the currently used drugs, there is pressing need to develop effective drug options for treatment of laryngeal cancer. Therefore, this study was undertaken to investigate the anticancer effects of a plant-derived alkaloid, Mukonal, against human AMC-HN-8 laryngeal cancer cells. MATERIAL AND METHODS The WST-1 and clonogenic assays were employed to determine the cell viability. Apoptosis was detected by Hoechst and AO/EB staining. Cell migration and cell cycle analysis was performed by Transwell assay and flow cytometry, respectively. Protein expression was examined by Western blotting. RESULTS The results revealed that Mukonal reduced the viability of laryngeal cancer cells dose-dependently. The IC50 of Mukonal was found to be 10 µM. However, the effects of Mukonal on the normal HuLa-PC cells was found to be 140 µM. The decrease in the viability of the AMC-HN-8 laryngeal cancer cells was found to be due to the induction of apoptosis and G2/M cell cycle arrest. Mukonal also suppressed the cell migration and of the AMC-HN-8 laryngeal cancer cells. Mukonal could also inhibit the PI3K/AKT and MEK/ERK signalling pathways in a concentration-dependent manner. CONCLUSIONS Taken together, we conclude that Mukonal could prove a beneficial lead molecule for the treatment of laryngeal cancer.

Background: PTK6 is involved in cell proliferation, invasion and migration. Patients with lower PTK6 expression predicts poor prognosis of LSCC. However, the mechanism of PTK6 in LSCC progression remains unclear. We investigated the role of PTK6 in the pathogenesis of LSCC., Methods: Human LSCC tissues and paired adjacent non-tumor tissues were obtained to evaluate PTK6 expression. The biological function of PTK6 in LSCC was determined by overexpression of PTK6 in Hep-2 cells in vitro and in nude mice. The potential PTK6 target factors and signaling pathways were identified by Western blotting assay and immunohistochemical staining., Results: PTK6 was downregulated in tissues of human LSCC. Biological function investigation of PTK6 demonstrated that overexpression of PTK6 significantly decreased cell growth, clonogenicity, invasion and migration capacity in vitro and suppressed xenograft tumor growth as well as lung metastasis in vivo. PTK6 suppresses LSCC proliferation mainly by inhibiting c-myc and cyclinD1 expression. In addition, PTK6 promotes cell apoptosis in LSCC. Moreover, PTK6 mitigated LSCC invasion and migration through regulating EMT and MMP-9., Conclusion: PTK6 plays a tumor suppressor role in LSCC by regulating c-myc and cyclinD1 expression, cell apoptosis, EMT and MMP-9., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Squamous cell carcinomas of the head and neck (SCCHN) affect anatomical sites including the oral cavity, nasal cavity, pharynx, and larynx. Laryngeal cancers are characterized by high recurrence and poor overall survival, and currently lack robust molecular prognostic biomarkers for treatment stratification. Using an algorithm for integrative clustering that simultaneously assesses gene expression, somatic mutation, copy number variation, and methylation, we for the first time identify laryngeal cancer subtypes with distinct prognostic outcomes, and differing from the non-prognostic laryngeal subclasses reported by The Cancer Genome Atlas (TCGA). Although most common laryngeal gene mutations are found in both subclasses, better prognosis is strongly associated with damaging mutations of the methyltransferases NSD1 and NSD2, with findings confirmed in an independent validation cohort consisting of 63 laryngeal cancer patients. Intriguingly, NSD1/2 mutations are not prognostic for nonlaryngeal SCCHN. These results provide an immediately useful clinical metric for patient stratification and prognostication.

Background/aim: Phosphatase and tensin homolog (PTEN) (gene locus: 10q23.3) -a tumor suppressor gene- is deleted, mutated or epigenetically hyper-methylated in a variety of malignancies. PTEN acts as a negative regulator in PI3K/AKT/mTOR signaling transduction pathway. Our aim was to investigate PTEN protein expression patterns in laryngeal squamous cell carcinomas (LSCC)., Materials and Methods: Using tissue microarray technology, fifty (n=50) primary LSCCs were cored and re-embedded into one recipient block. Immunohistochemistry and digital image analysis were implemented for evaluating protein expression levels., Results: Abnormal protein expression (low to negative staining intensity values) was observed in 28/50 (56%) tissue cores. Overall PTEN expression was associated with the anatomical region of the malignancies (p=0.039), whereas a borderline correlation with the differentiation grade was also assessed (p=0.05)., Conclusion: Aberrant expression of PTEN tumor-suppressor gene in LSCCs seems to affect their biological behavior. Well-differentiated tumors express moderate to high protein levels, an evidence of normal gene function, whereas loss of its expression correlates with a progressive tumor dedifferentiation. Additionally, loss of its expression is detected more frequently in specific anatomical regions of the larynx (glottis, predominantly, and partially supraglottis)., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Purpose: Topoisomerases (types: I/IIa-b/IIIa-b) represent a super-family of nucleic enzymes involved in the DNA replication, transcription, recombination, and also chromosome topological formation. Topoisomerase's I (Topo I- gene location: 20q12) aberrant expression is a frequent genetic event in a variety of solid malignancies. Topo I inhibition promotes cell death due to DNA damage and for this reason it is a target for specific targeted chemotherapy (camptothecin, topotecan, irinotecan). Our aim was to investigate the role of abnormal Topo I protein expression in laryngeal squamous cell carcinomas (LSCC) in which there are very limited data regarding the influence of the marker., Methods: Using tissue microarray (TMA) technology, 50 formalin-fixed, paraffin-embedded primary laryngeal SCCs were cored and re-pembedded into one recipient block. Immunohistochemistry was performed using anti- Topo I antibody. Digital image analysis was also implemented for evaluating objectively the protein expression levels on the corresponding stained nuclei., Results: Topo I protein overexpression (moderate to high staining intensity values) was observed in 32/50 (64%) tissue cores, whereas low expression rates were detected in 18/50 (36%) cases. Topo I overall expression was strongly associated with the differentiation grade of the examined tumors (p=0.021). No other statistical correlations were identified., Conclusions: Topo I overexpression is observed in a significant subset of LSCCs affecting the level of differentiation in them. Additional molecular studies focused on the mechanism of Topo I gene/protein deregulation (i.e. amplification, abnormal epigenetic promoter methylation, mRNA aberrant expression) are necessary discriminating the eligible patients for applying specific chemotherapeutic strategies based on anti-Topo I agents.

MicroRNAs (miRNAs) play important roles in gene regulation during laryngocarcinoma. MiR-9 is a potential oncomiR, but its function in laryngocarcinoma is not known. The aim of this study is to investigate the roles of miR-9 in laryngocarcinoma. We found miR-9 expression was higher in laryngocarcinoma tissues compared with their normal controls, so did the laryngocarcinoma cells. Cellular function of miR-9 indicated that miR-9 restoration in laryngocarcinoma cells could promote cell proliferation and metastasis. Phosphatase and tensin homolog (PTEN) was predicted as a target gene of miR-9 and verified using luciferase reporter assay. PTEN expression was down-regulated in the laryngocarcinoma cells with miR-9 overexpression. We also found that miR-9 expression was negatively associated with PTEN expression in laryngocarcinoma tissues., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Photodynamic therapy (PDT) has been developed as a promising treatment modality for laryngeal cancer. 9-Hydroxypheophorbide α (9-HPbD), a novel chlorophyll-derived photosensitizer, has a longer absorption wavelength, which increases the penetration of light to malignant tissues. Carboplatin (CBDCA), a second-generation platinum derivative, also has gained more popularity for the treatment of laryngeal cancer. Our previous studies have elucidated that 9-HPbD-PDT could inhibit the migration and invasion of HEp-2 cells. The objective of this study is to investigate the change of migration and invasion of HEp-2 cells induced by a combined modality of CBDCA and 9-HPbD-PDT in vitro. A wound healing assay, cell migration assay and Matrigel invasion assay were used to evaluate the cellular migration and invasion. Reactive oxygen species (ROS) and Western blots for epithelial-mesenchymal transition (EMT) markers (E-cadherin, N-cadherin and vimentin), MMPs (MMP-2 and MMP-9) and MEK/ERK signalling pathway were performed to investigate the possible mechanisms that may be involved. We observed that CBDCA and 9-HPbD-PDT administration synergistically inhibited the migration and invasion of HEp-2 cells. Moreover, the combined modality cooperatively repressed the EMT process and down-regulated expressions of MMP-2 and MMP-9 via ROS-mediated inhibition of phosphorylation in the MEK/ERK signalling pathway. Our results suggested that the combination of CBDCA and 9-HPbD-PDT might be a promising therapeutic strategy for laryngeal cancer metastasis.

BACKGROUND The angiotensin-converting enzyme (ACE, CD143) gene plays a crucial role in the pathology of many cancers. Previous studies mostly focused on the gene polymorphism, but the other functions of ACE have rarely been reported. The purpose of this study was to investigate the expression of ACE and its biological function, as well as its prognostic value, in laryngeal cancer. MATERIAL AND METHODS The expression of ACE was detected by quantitative real-time polymerase chain reaction (qRT-PCR) analysis in 106 patients with laryngeal cancer and 85 healthy people. Then the cell proliferation was estimated after the cell lines Hep-2 were transfected with pGL3-ACE and empty vector, respectively. In addition, the relationship between ACE expression and clinicopathologic characteristics was analyzed. Finally, Kaplan-Meier analysis was used to evaluate the overall survival of patients with different ACE expression, while Cox regression analysis was conducted to reveal the prognostic value of ACE in laryngeal cancer. RESULTS Our results demonstrate that ACE is over-expressed in laryngeal cancer and thus promotes cell proliferation. The up-regulation of ACE was significantly influenced by tumor stage and lymph node metastasis. Patients with high ACE expression had a shorter overall survival compared with those with low ACE expression according to Kaplan-Meier analysis. The ACE gene was also found to be an important factor in the prognosis of laryngeal cancer. CONCLUSIONS Our study shows that the ACE gene was up-regulated, which promoted the cell proliferation, and it could be an independent prognostic marker in laryngeal cancer.

Objective: To study the association between human 8-oxoguanine giycosylase 1 (hOGG1) gene polymorphisms and the risk of laryngeal carcinoma. Methods: A total of 86 patients with laryngeal carcinoma treated in The Air Force General Hospital and the Bozhou People's Hospital of Anhui Province from Jan 2010 to Dec 2014 were included in experimental group, and 86 healthy adults were selected as control group. The hOGG1 gene polymorphisms of subjects in two group were detected with by polymerase chain reaction amplification and gene sequencing technology, the differences in hOGG1 gene polymorphisms between two groups were analyzed. Results: The frequencies of hOGG1 gene heterozygous type (Ser/Cys) and mutant type (Cys/Cys) in experimental group were higher than those in control group( P <0.05). Compared to individuals with hOGG1 gene Ser/Ser, individuals with hOGG1 gene Ser/Cys had a 2.97-fold increased risk of laryngeal carcinoma, and individuals with hOGG1 gene Cys/Cys had a 8.09-fold increased risk of laryngeal carcinoma. Stratified by smoking or not, it was found that the proportion of heterozygous type or mutant type in smokers was higher significantly than non-smokers ( P <0.05). Conclusion: Individuals with hOGG1 gene heterozygous type (Ser/Cys) or mutant type (Cys/Cys) have a higher risk of laryngeal carcinoma, with interaction between these gene types and smoking, and the gene types could be used as predictors for the occurrence of laryngeal carcinoma.

Background: Aldehyde dehydrogenase isoform 1 (ALDH1) has been shown to be a marker of cancer stem cells (CSCs). These stem cells may be responsible for tumour perpetuation as well as local and distant invasion. Several studies have shown that CSCs are more chemoradiotherapy (CRT)-resistant and may be responsible for tumour recurrence. Other studies, in contrast, have found ALDH1 expression to be indicative of a better prognosis., Methods: We retrospectively evaluated 84 patients diagnosed and treated for laryngeal cancer between 2006 and 2011. All patients underwent curative-intent radiotherapy or CRT at our institution. 57 of the 84 tumour samples contained sufficient material for ALDH1 assessment., Results: ALDH1 expression was detected in 17.5 % (10/57) of the tissue samples. None of the tumours from stage I patients tested positive for ALDH1. The relapse rate in ALDH1 + patients was 10 versus 51.2 % for ALDH1-. No differences in overall survival were observed between the groups; however, disease-free survival was 90 % for the ALDH1 + group versus 48.9 % for ALDH1- patients (p = 0.034)., Conclusion: The patients in this study with ALDH1 + tumours had better outcomes than their counterparts with ALDH1- tumours. This finding suggests that not all CSCs are resistant to conventional cancer treatments. It may also imply that new methods of correctly identifying these cells are needed.

Revival of dormant tumor cells may be an important tumor metastasis mechanism. We hypothesized that aurora kinase A (AURKA), a cell cycle control kinase, promotes the transition of laryngeal squamous cell carcinoma (LSCC) cells from G0 phase to active division. We therefore investigated whether AURKA could revive dormant tumor cells to promote metastasis. Western blotting revealed that AURKA expression was persistently low in dormant laryngeal cancer Hep2 (D-Hep2) cells and high in non-dormant (T-Hep2) cells. Decreasing AURKA expression in T-Hep2 cells induced dormancy and reduced FAK/PI3K/Akt pathway activity. Increasing AURKA expression in D-Hep2 cells increased FAK/PI3K/Akt pathway activity and enhanced cellular proliferation, migration, invasion and metastasis. In addition, FAK/PI3K/Akt pathway inhibition caused dormancy-like behavior and reduced cellular mobility, migration and invasion. We conclude that AURKA may revive dormant tumor cells via FAK/PI3K/Akt pathway activation, thereby promoting migration and invasion in laryngeal cancer. AURKA/FAK/PI3K/Akt inhibitors may thus represent potential targets for clinical LSCC treatment., Competing Interests: All authors declare no conflicts interest.

Objective: To investigate the role of oxidative stress in smoking-related laryngeal squamous carcinoma through detecting the expression of antioxidant enzymes in smoking patients. Method: A total of 138 cases with laryngeal squamous cell carcinoma enrolled in the first hospital affiliated the northern he bei college from 2012 to 2015 and forty five volunteers were conducted. All participants were divided into three groups according to smoking index: group A(heavy smoking, 88 cases of laryngeal cancer patients) and group B(no smoking 50 cases of laryngeal cancer patients) and C group(45 heavy smoking volunteers).Catalase(CAT), glutathione peroxidase(GSH-px) and malondialdehyde(MDA) and the expression of NRF2 in serum, tissue adjacent to carcinoma, and carcinoma tissues from each groups were measured, respectively. Result: ①the expression of the CAT and GSH-px in group A were significantly lower than that of group B( P <0.05), but higher than that of group C( P <0.05); ②the MDA level of group A is significantly higher than group B( P <0.05) and C group( P <0.01);③NRF2 was highly expressed in carcinoma tissues, and the expression level was negatively correlated with degree of carcinoma differentiation ( P <0.05). Conclusion: Compared with nonsmoking patients, heavy smoking patients with laryngeal cancer were under more severe oxidative stress. NRF2 expression level in patients with laryngeal squamous cell carcinomas was associated with pathological stage., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Clinical Otorhinolaryngology Head and Neck Surgery.)

Laryngeal squamous cell carcinoma (LSCC) is a multifaceted and genomically complex disease and cellular and preclinical studies have demystified wide ranging molecular mechanisms which underpin its development and progression and resistance against wide ranging molecular therapeutics. Oxidative stress is a widely studied molecular mechanism and reportedly involved in carcinogenesis. Increasingly it is being realized that accumulation of Reactive Oxygen Species (ROS) activates defensive mechanism to counteract oxidative stress induced damage. Manganese superoxide dismutase (MnSOD) and glutathione peroxidase (GPx) are important members of defensive machinery. We investigated whether the polymorphisms of MnSOD (Ala-9Val, rs4880) and GPx1 (Pro198Leu, rs1050450) are associated with LSCC and also evaluated possible interactions between these polymorphisms and various lifestyle factors or pathological features of patients. For this purpose, 67 LSCC patients and 73 healty controls were enrolled. Molecular assessment of MnSOD and GPx1 variants were determined with polymerase chain reaction-restriction fragment length polymorphism techniques. We found that the frequency of both heterozygous PL genotype and P allele was considerably higher in patients with advanced tumor stage (T3/T4) than in those with early tumor stage (T1/T2) (OR= 5.106; 95% CI=1.372-19.004; p<0.001, OR=5.787; 95% CI =1.564-21.414; p<0.001 respectively). Although the frequency of ValVal/LL combine genotype was significantly decreased (OR=0.204, 95% CI=0.055-0.760; p=0.021), the frequency of ValAla/PL combine genotypes was higher in patients with stage T3/T4 than in those patients with stage T1/T2 (p=0.027). Consequently, we have concluded that variants of GPx1 and MnSOD should not be considered as a risk factor of LSCC, only may be accepted as a prognostic markers. Use of new technologies such as metabolomics and deep DNA sequencing will prove to be helpful in developing a deeper knowledge related to how cancer cell metabolism adapts and provides a buffer against increased oxidative stress.

Background: The survival rate of patients with head and neck squamous cell carcinoma (HNSCC) stands at approximately 50% and this has not improved in decades. This study developed a novel sirtuin-3 (SIRT3) inhibitor (LC-0296) and examined its role in altering HNSCC tumorigenesis., Materials and Methods: The effect of the SIRT3 inhibitor, LC-0296, on cell survival, proliferation, and apoptosis, and reactive oxygen species levels in HNSCC cells were studied., Results: LC-0296 reduces cell proliferation and promotes apoptosis of HNSCC cells but not of normal human oral keratinocytes. This inhibitory effect is mediated, in part, via modulation of reactive oxygen species levels. Additionally, LC-0296 works synergistically to increase the sensitivity of HNSCC cells to radiation and cisplatin treatment., Conclusion: Development of novel SIRT3 inhibitors, such as LC-0296, might enable the development of new targeted therapies to treat and improve the survival rate of patients with head and neck cancer., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Nitric oxide synthase (eNOS/NOS3) is responsible for the endothelial synthesis of nitric oxide (NO(•)). G894T polymorphism leads to the amino acid substitution from Glu298Asp that causes lower NOS3 activity and basal NO(•) production in NOS3 894T (298Asp) allele carriers compared with the GG homozygotes. NO(•) acts as an antioxidant protecting against Fenton's reaction which generates highly reactive hydroxyl radicals. Allelic variation of NOS3 may influence an individual's risk of laryngeal cancer (LC). In the current study we have examined the possible relationship between NOS3 G894T genotypes and various systemic oxidative damage markers such as protein carbonyl, advanced oxidation protein products, Cu, Zn-superoxide dismutase, thiol group fractions, and lipid hydroperoxides in LC patients. Genotyping was carried out by PCR-RFLP. In LC patients with TT genotype, Cu, Zn-superoxide dismutase activities and nonprotein thiol levels were significantly higher than the controls. In patients with GT and GG genotype, high levels of lipid hydroperoxides showed statistical significance when compared to controls. Our results indicate a potential relationship among G894T polymorphism of NOS3, and impaired redox homeostasis. Further studies are required to determine the role of NOS3 gene polymorphism and impaired plasma redox homeostasis.

Objective: This study aimed to investigate correlation between full-length spleen tyrosine kinase [SYK (L)] expression and clinical characteristics of laryngeal squamous cell carcinoma (LSCC), and explore effects of SYK (L) on invasion and metastasis of LSCC., Methods: The human laryngeal cancer Hep-2 cells with low SYK (L) expression were transfected with pIRES2-EGFP-SYK (L) vector and empty vector pIRES2-EGFP to generate Hep-2-SYK (L) cells and Hep-2-neo cells. The cell invasion and migration abilities were determined., Results: The SYK (L) positive expression rate in LSCC tissues was significantly lower than in vocal cord dysplasia tissues and normal laryngeal tissues (P < 0.05). There was a significant correlation between SYK (L) expression and LSCC T stage, histopathological grade and lymph node metastasis (P < 0.05). mRNA expression of SYK (L) in Hep-2-SYK (L) cells was significantly higher than in Hep-2-neo cells and Hep-2 cells (P < 0.01). The protein expression of SYK (L) in Hep-2-SYK (L) cells was markedly higher than in Hep-2-neo cells and Hep-2 cells (P < 0.01). The number of invasive cells was significantly lower in Hep-2-SYK (L) group than in Hep-2-neo group and Hep-2 group (P < 0.01). The average number of migrating cells in Hep-2-SYK (L) group also markedly reduced as compared to Hep-2-neo group and Hep-2 group (P < 0.01)., Conclusion: The SYK (L) expression was down-regulated in LSCC, which was closely correlated with cancer growth and lymph node metastasis. SYK (L) up-regulation was able to inhibit the invasion and metastasis of LSCC, therefore suppressing tumor development. Thus, SYK (L) may be a potential target for the LSCC treatment.

Previous reports have demonstrated that a non-neuronal cholinergic system is expressed aberrantly in airways. A proliferative effect is exerted directly by cholinergic agonists through the activation of nicotinic and muscarinic receptors. In cancer, particularly those related with smoking, the mechanism through which tumour cells respond to aberrantly activated cholinergic signalling is a key question. Fifty paired pieces of larynx squamous cell carcinoma and adjacent non-cancerous tissue were compared in terms of their acetylcholinesterase activity (AChE). The AChE activity in non-cancerous tissues (0.248 ± 0.030 milliunits per milligram of wet tissue; mU/mg) demonstrates that upper respiratory tissues express sufficient AChE activity for controlling the level of acetylcholine (ACh). In larynx carcinomas, the AChE activity decreased to 0.157 ± 0.024 mU/mg (p=0.009). Larynx cancer patients exhibiting low ACh-degrading enzymatic activity had a significantly shorter overall survival (p=0.031). Differences in the mRNA levels of alternatively spliced AChE isoforms and molecular compositions were noted between glottic and supraglottic cancers. Our results suggest that the low AChE activity observed in larynx squamous cell carcinoma may be useful for predicting the outcome of patients., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Objective: Investigate the effect of oxidative stress on the occurrence and development of laryngeal squamous cell carcinoma associated with smoking, and the clinical diagnostic value of catalase on smoking related laryngeal squamous cell carcinoma., Method: Collecting 119 smokers(including the smoking related laryngeal cancer group 68 cases, the control group 51 cases), the indexes of catalase (CAT), malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH), nitric oxide (NO) in blood plasma and cancerous tissue in two groups were compared. The association between these oxidative stress indicators and the occurrence and severity of smoking related laryngeal squamous cell carcinoma was analysised by SPSS 17.0., Result: (1) Compared with control group, the smoke frequency and amount, CAT, MDA, GSH increased significantly in the smoking related laryngeal cancer group (P = 0.000; 0.000; 0.000; 0.000; 0.000); whereas SOD, NO decreased (P = 0.000; 0.000). (2) The lower the differentiation degree, the higher the serum CAT (P = 0.000) and the higher CAT, MDA, GSH of larynx tissue (P = 0.000; 0.000; 0.000), but the lower the serum NO (P = 0.000) and the lower SOD, NO of larynx tissue (P = 0.000; 0.000); The higher the clinical stage, the higher CAT of serum and larynx tissue and the higher GSH of larynx tissue (P = 0.000; 0.001), the lower NO of larynx tissue (P = 0.009). (3) The serum CAT, MDA were independent risk factors of smoking related laryngeal squamous cell carcinoma (OR = 1.060, 2.475; P < 0.01, P < 0.05)., Conclusion: Oxidative stress is the key factor of the occurrence of smoking related laryngeal squamous cell carcinoma, and the CAT can be used as the indicator of clinical diagnosis of smoking related laryngeal squamous cell carcinoma.

Background: Laryngeal squamous cell carcinoma (LSCC) is still a problem worldwide. In some publications interactions between the expression of matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9, and their tissue inhibitors (TIMPs) implicated during cancer progression were suggested., Methods: The immunohistochemical staining using primary antibody against MMP-2, MMP-9, TIMP-1, TIMP-2, and TIMP-3 were performed. The research group consists of primary N(0) LSCC (20 cases), primary N(+) LSCC (17 cases), and 18 cases of normal mucosa., Results: Studied MMPs and TIMPs were localized in tumor cells and tumor stroma compartment. MMP-2 expression was higher in stroma compared to tumor cells. MMP-9, TIMP-1, TIMP-2, and TIMP-3 expression was higher in tumor cells than in tumor stroma (P < 0.05). In tumor stroma MMP-2, MMP-9, TIMP-1, and TIMP-3 expression, in LSCC N(0) vs. LSCC N(+) was significantly higher (P < 0.05). The ratios between MMP-2 and TIMP-3 expression were statistically significant (N(0) vs. N(+); P = 0.012). The analyses using classification trees predicted the probability of metastases according to TIMP-3/MMP-14/MMP-2 and MMP-9/TIMP-1 expression levels., Conclusions: The presence of MMP-2, MMP-9, TIMP-1, TIMP-2, TIMP-3 expression in tumor cells and in tumor stroma, and additionally different expression according to lymph node involvement suggested of their impact during cancer progression. The significant correlation between TIMP-3 expression and the presence of lymph node metastases and MMP-2 expression might suggest the importance of TIMP-3 as a prognostic factor during tumor progression. The evaluation of molecular markers which participate in MMP-2 activation pathway have a major impact during metastasis., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Although targeting radioresistant tumor cells is essential for enhancing the efficacy of radiotherapy, the signals activated in resistant tumors are still unclear. This study shows that ERp57 contributes to radioresistance of laryngeal cancer by activating STAT3. Increased ERp57 was associated with the radioresistant phenotype of laryngeal cancer cells. Interestingly, increased interaction between ERp57 and STAT3 was observed in radioresistant cells, compared to the control cells. This physical complex is required for the activation of STAT3 in the radioresistant cells. Among STAT3-regulatory genes, Mcl-1 was predominantly regulated by ERp57. Inhibition of STAT3 activity with a chemical inhibitor or siRNA-mediated depletion of Mcl-1 sensitized radioresistant cells to irradiation, suggesting that the ERp57-STAT3-Mcl-1 axis regulates radioresistance of laryngeal cancer cells. Furthermore, we observed a positive correlation between ERp57 and phosphorylated STAT3 or Mcl-1 and in vivo interactions between ERp57 and STAT3 in human laryngeal cancer. Importantly, we also found that increased ERp57-STAT3 complex was associated with poor prognosis in human laryngeal cancer, indicating the prognostic role of ERp57-STAT3 regulation. Overall, our data suggest that ERp57-STAT3 regulation functions in radioresistance of laryngeal cancer, and targeting the ERp57-STAT3 pathway might be important for enhancing the efficacy of radiotherapy in human laryngeal cancer.

Rho-associated coiled-coil containing protein kinase (ROCK) over-expression has been implicated in the progression of many tumor types. The aim of this study was to explore the roles of ROCK1 and ROCK2 in human laryngeal squamous cell carcinoma (LSCC). ROCK1 and ROCK2 expression levels were examined in 50 cases of human LSCC samples by immunohistochemistry. Effects of ROCK1 and ROCK2 on LSCC cell proliferation and motility were investigated in the presence of the ROCK inhibitor Y-27632. The results showed that ROCK1 expression was positively correlated with tumor size and lymph node metastasis (P < 0.05); ROCK2 positively correlated with tumor size (P < 0.05). Inhibition of ROCK1 and ROCK2 by Y-27632 significantly inhibits proliferation, migration, and invasion of LSCC cells. Our data indicate that expression of ROCK1 and ROCK2 are closely associated with tumor growth and lymph node metastasis of LSCC. Thus, these two ROCK isoforms may be useful as molecular makers for LSCC diagnosis and may be useful therapeutic targets as well.

Aim: Overexpression of histone demethylase PHF8 has been reported to function as an oncoprotein in many cancers; however, the implications of PHF8 involvement in laryngeal and hypopharyngeal squamous cell carcinoma (LHSCC) remain unclear. This study aims to explore the expression of PHF8 and its clinical significance in LHSCC., Materials & Methods: Western blotting and immunohistochemistry were performed to evaluate PHF8 protein expression in fresh and archived LHSCC samples. Global expressions of H3K27 and H3K9 methylation were analyzed in a cell line with PHF8 siRNA treatment., Results & Conclusion: In our study, PHF8 was upregulated in fresh LHSCC tissues. Immunohistochemical staining revealed that the expression of PHF8 was positively associated with T classification, clinical stage, primary tumor position and tumor relapse. Survival analysis demonstrated that high PHF8 expression was significantly associated with shorter overall survival and disease-free survival. Moreover, PHF8 regulates the levels of H3K9me2 and H3K27me2 in LHSCC. Taken together, PHF8 might be a novel prognostic marker for this disease.

Objective: To study the expression of DNA-dependent protein kinase (DNA-PK) in human laryngeal squamous cell carcinoma (LSCC) and normal laryngeal mucosa (NLM), and to analysize the relationship between the expression and the clinicopathologic parameters of LSCC., Method: Immunohistochemical technique (Envision) was used to detect the expression of DNA-PK in 64 cases of LSCC and 15 cases of NLM. To investigate an investigation was conducted on the relationship between the expression and clinico-pathological features of LSCC., Result: DNA-PK was lowly expressed in NLM and highly expressed in LSCC,the positive rate of DNA-PK expression was 26.67% (4/15), 78.13% (50/64), respectively, and there was significant different difference between the two groups (P < 0.05). Its expression was correlated with the level of histodifferentiation (P < 0.05), but not with TNM stages and neck lymph node metastasis (P > 0.05)., Conclusion: DNA-PK may be involved in disease development of LSCC.

Glutathione S-transferase T1 (GSTT1) polymorphic variation has been implicated as a risk factor for various cancers. However, previous studies investigating the association between GSTT1 null genotype and laryngeal cancer risk in Asians reported conflicting outcomes. In the present study, the possible association of laryngeal cancer risk with GSTT1 null genotype was explored by a meta-analysis. Relevant studies were identified through a systemic search of PubMed and Chinese National Knowledge Infrastructure databases. Six studies with a total of 1,824 individuals were included in the meta-analysis. The pooled odds ratio (OR) with 95 % confidence interval (CI) was used to assess the association. Meta-analysis of all included studies showed that there was an obvious association between GSTT1 null genotype and laryngeal cancer risk in Asians (OR = 2.41, 95 % CI 1.27-4.57, P = 0.007, I (2) = 86 %). After adjusting for heterogeneity, there was still an obvious association between GSTT1 null genotype and laryngeal cancer risk in Asians (OR = 1.75, 95 % CI 1.36-2.24, P < 0.001, I (2) = 0 %). The findings from the meta-analysis suggest that GSTT1 null genotype is associated with laryngeal cancer risk in Asians.

Laryngeal carcinoma is a common malignant neoplasm occurring in the head and neck, threatening human health. Protein casein kinase 2-alpha (CK2α) has been indicated to participate in the pathogenesis of this cancer; however, the underlying mechanisms still need to be elucidated. In this study, short hairpin RNA (shRNA)-mediated RNA interference (RNAi) technology was utilized to inhibit the CK2α expression in HEp-2 laryngeal carcinoma cells. Results showed that both mRNA and protein expression levels of endogenous CK2α were markedly decreased in HEp-2 cells transfected with CK2α specific shRNA. Transwell assays revealed that stable knockdown of CK2α significantly inhibited the migration and invasion of HEp-2 cells. As compared with cells treated with negative control shRNA, epithelial cadherin (E-cadherin) expression was increased, but snail, slug and vimentin were decreased in cells transfected with CK2α shRNA, indicating that inhibition of CK2α expression may suppress the epithelial-mesenchymal transition (EMT) process of laryngeal carcinoma in vitro. Moreover, suppression of CK2α was found to enhance the apoptosis induced by cisplatin in laryngeal carcinoma cells, probably through inhibition of permeability glycoprotein (P-glycoprotein) and multidrug-resistance protein (MRP1). In conclusion, our study may provide a promising therapeutic strategy for human laryngeal carcinoma by targeting CK2α., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Background: 5-fluorouracil (5-FU) is an antifolate chemotherapeutic that has become established in many therapeutic regimes, but sensitivity variations and development of resistance are common problems that limit the efficiency of the treatments. Inter-individual variations to 5-FU outcome have been attributed to different expression profiles of genes related to folate metabolism., Methods: To elucidate the mechanisms of variations to 5-FU outcome, the authors investigated MTHFR, DHFR, TYMS and SLC19A1 folate genes expression for 5-FU response in laryngeal cancer cell line (Hep-2). Concentrations of 10, 50, and 100 ng/mL of 5-FU chemotherapeutic were added separately in Hep-2 cell line for 24 hours at 37 °C. Cell sensibility was evaluated with fluorescein isothiocyanate (FITC) label Bcl-2 by flow cytometry. The real-time quantitative PCR (qPCR) technique was performed for quantification of gene expression using TaqMan(®) Gene Expression Assay. ANOVA and Bonferroni's post hoc tests were utilized to statistical analysis., Results: The numbers of viable Hep-2 cells with 10, 50, and 100 ng/mL concentrations of 5-FU chemotherapy were 15.87, 28.3 and 68.9%, respectively. Statistical analysis showed significant association between control group and increased expression for TYMS gene in cells treated with 100 ng/mL/5-FU chemotherapy (P<0.05)., Conclusions: The authors found association between the highest 5-FU dose chemotherapy and increased expression levels for TYMS folate gene in laryngeal cancer cell line. Although these experiments were performed in vitro, the results suggest that genetic factors are thought to play an important role in drug metabolism and may be useful for predicting treatment outcomes., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

A family of small non-coding RNAs, ~22 nt in length, known as microRNAs (miRNAs), regulating ~30% of all human gene expression, have been reported to be involved in the pathogenesis of a number of types of cancers, including laryngeal squamous cell carcinoma (LSCC). In the current study, miR-34a and miR-34c were observed to be downregulated in human LSCC tissues. Ectopic expression of miR-34a and miR-34c in Hep-2 cells significantly induced the cell proliferation and migration ability in vitro. UDP-N-acetyl-α-D-galactosamine:polypeptide-N-acetylgalactosaminyltransferase 7 (GALNT7), whose expression is negatively regulated by miR-34a and miR-34c in Hep-2 cells, is confirmed to be a novel direct target gene of miR-34a and miR-34c. In conclusion, the current results suggest that miR-34a and miR-34c may function as tumor suppressors in LSCC through downregulation of GALNT7. The study of miR-34a, miR-34c and its novel target, GALNT7, may serve as novel potential makers for LSCC therapy.

Objective: S100A4 is a member of the S100 family of calcium-binding proteins, which possesses a wide range of biological functions, such as regulation of angiogenesis, cell survival, motility, and invasion. Here, we demonstrate for the first time a major role of S100A4 in the cell invasion properties of the human laryngeal squamous carcinoma cells (LSCC) and evaluated the mechanism., Materials and Methods: Cultured human LSCC cell line Hep-2 was overexpressed by transfection of pcDNA3.1-S100A4 plasmid. For this, cellular Hep-2 expression was quantified by Western blot analysis. Moreover, cell invasion and migration assays were performed. Furthermore, the impact of the S100A4 on NF-kB activity and MMP-9 expression was detected., Results: We found S100A4 potently promoted Hep-2 invasion, by increasing cell motility and matrix metalloproteinase-9 (MMP-9) production. The increase in MMP-9 production was mediated by activation of nuclear factor-kB transcriptional activity by S100A4. After MMP-9 and NF-kBp65 was inhibited by BB94 treatment and NF-kBp65 siRNA transfected, pcDNA3.1-S100A4 induced cell invasion and migration was decreased., Conclusions: Our findings thus establish S100A4 as a major factor in the invasive abilities of Hep-2 cells.

We investigated the expression of Src homology 2 domain-containing phosphatase (SHP-2) in laryngeal carcinoma and its clinical significance. Expression of SHP-2 was detected by immunohistochemical staining in normal mucosal tissues and various grades of laryngeal carcinoma. We looked for possible correlations between expression of SHP-2 in laryngeal carcinoma and clinical staging and lymph node metastasis. Immunochemical staining results revealed that the SHP-2 expression was significantly higher (88.24%) in laryngeal carcinoma than in normal mucosal tissue (25%). Additionally, the expression of SHP-2 was significantly correlated with lymph node metastasis, but not with clinical stage and gender of patients with laryngeal carcinoma. Therefore, SHP-2 may be useful as a prognostic marker for laryngeal carcinoma and as a therapeutic target in laryngeal carcinoma treatment.

Purpose: Matrix metalloproteinases (MMPs) is a superfamily of proteins involved in angiogenesis and metastatic tissue invasion in many cancers. Overexpression of MMP- 9 has been detected in significant proportions of laryngeal squamous cell carcinomas (LSCCs), but its prognostic impact remains unclear. In this study we performed a digital image analysis for analyzing MMP-9 protein expression in a series of LSCCs correlating them with clinicopathological factors., Methods: MMP-9 protein expression level was determined immunohistochemically in 30 tissue sections surgically derived from patients (21 male and 9 female) with LSCC. Using digital image analysis, we measured their corresponding protein expression levels (staining intensity/S.I. range values 0-255)., Results: Moderate and high MMP-9 protein expression levels (grouping as 2+/3+ overexpression) were detected in 19/30 (63.3%) cases. Statistical significance was observed correlating stage with SI (p=0.02), whereas a borderline association with differentiation grade of the examined tumors was also registered (p=0.05). Interestingly, high levels of MMP-9 expression were observed in cases that demonstrated a significant level of inflammatory (predominantly lymphocytic) infiltration., Conclusion: MMP-9 protein overactivation is a frequent and significant genetic event in LSCC, correlating with its biological behavior (increased TNM stage). MMP-9 seems to mediate an epithelial-stromal intra-reaction correlating also with induction of specific inflammation pathways.

Accelerated radiotherapy (AR) improves the poor prognosis associated with epidermal growth factor receptor (EGFR) overexpression frequently seen in head and neck carcinomas. Combining AR with carbogen and nicotinamide (ARCON) counteracts enhanced tumour cell proliferation- and hypoxia-related radioresistance. The purpose of this study was to investigate if EGFR expression levels are associated with response to ARCON in patients with carcinoma of the larynx. Patients (N=272) with advanced stage larynx carcinoma were randomised between AR alone and ARCON. Paraffin-embedded biopsies from these patients were processed for immunohistochemical staining of EGFR. EGFR fraction was quantitated by automated image analysis and related to clinical outcome. A large variation was observed in EGFR fraction between tumours with expression levels ranging from 0 to 0.93 (median fraction 0.4). No difference in 5-year locoregional control was found between low and high EGFR expressing tumours in the AR arm (69% versus 75%), which is in line with the established effect of AR in EGFR overexpressing tumours. There was, however, a significant association in the ARCON arm: patients with low EGFR levels had a better 5-year locoregional control (88% versus 72% p=0.02) and disease-specific survival (92% versus 77% p=0.01). ARCON improved locoregional control relative to AR only in patients with low EGFR expression (hazard ratio (HR) 0.34 p=0.009). In conclusion, only in tumours with a low EGFR fraction, adding hypoxia modification to AR has an additive beneficial effect on outcome. EGFR expression is a predictive biomarker for the selection of patients that will or will not respond to ARCON., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background and Purpose: In a phase III trial in patients with advanced stage laryngeal carcinoma comparing ARCON (accelerated radiotherapy with carbogen breathing and nicotinamide) to accelerated radiotherapy alone (AR) the prognostic and predictive value of CAIX, a hypoxia-associated protein, was investigated., Material and Methods: 261 Paraffin embedded tumor biopsies and 79 fresh frozen biopsies from patients entered in the trial were immunohistochemically stained for CAIX. CAIX-fraction and CAIX expression pattern were related to tumor control and patient survival., Results: Low CAIX-fraction was prognostic for worse regional control and overall survival in patients treated with AR. Patients with a low CAIX-fraction treated with ARCON had better regional control and metastasis-free survival compared to AR (RC 97% vs 71%, p < 0.01 and MFS 92% vs 69%, p = 0.06). Patients with a perinecrotic CAIX staining pattern had a significantly worse local control, metastasis-free and overall survival compared to patients with a diffuse pattern (65% vs 84%, p = 0.01, 70% vs 96%, p < 0.01 and 42% vs 71%, p < 0.01 respectively), and this could not be improved with ARCON. After multivariate analysis CAIX pattern and N-stage emerged as significant predictors for metastasis-free survival and overall survival., Conclusions: ARCON improves regional control and metastasis-free survival only in patients with low CAIX expression. The different patterns of CAIX expression suggest different mechanisms of upregulation and have important prognostic value., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Aim: The objective of this study was to investigate whether cyclooxygenase-2 (COX-2), 12-lipoxygenase (12-LOX), and inducible nitric oxide synthase (iNOS) have a role in carcinogenesis of head and neck squamous cell carcinoma (HNSCC)., Materials and Methods: Twenty-two patients with HNSCC were included in the study. Cancer tissues and adjacent normal mucosa were obtained from each patient. Real-time PCR was used to assess the expression of COX-2, 12-LOX, and iNOS., Results: COX-2 and 12-LOX mRNA expressions are significantly increased in HNSCC compared with adjacent normal mucosa. Expression of iNOS was not significantly elevated in overall head and neck cancer tissues compared with normal mucosa. However, iNOS expression was found to be significantly elevated in patients with laryngeal cancer., Conclusion: These data suggest that COX-2 and 12-LOX may play a role in carcinogenesis of head and neck cancer. iNOS as well as COX-2 and 12-LOX may play a role in carcinogenesis of laryngeal cancer.

The aim of this study was to investigate the effect of a B-cell-specific MLV integration site-1 (Bmi-1) RNA interference (RNAi) expression vector on the proliferation and invasiveness of laryngeal carcinoma. We constructed a lentiviral vector expressing Bmi-1-specific short hairpin RNA (shRNA), and transfected it into HEp-2 cells. Bmi-1 gene expression was detected by real-time RT-PCR and western blot analysis. We used flow cytometry and TUNEL assay to analyze the apoptosis of transfected cells, and examined cellular growth in vitro by MTT assay. We established an animal model and evaluated the therapeutic effects of small interfering RNA (siRNA) against Bmi-1. siRNA against Bmi-1 significantly knocked down Bmi-1 expression in HEp-2 cells, induced cell cycle arrest at the G1 phase, inhibited cell proliferation and promoted cell apoptosis. Lentiviral Bmi-1-shRNA vector transfection also significantly reduced cell migration. The formation and growth rate of xenograft tumors in mice transfected with siRNA against Bmi-1 was significantly reduced. The loss of mitochondrial membrane potential, the release of cytochrome c from the mitochondria into the cytosol, and the increased activity of caspase-3, -8 and -9 occurred concomitantly with the inhibition of Bmi-1. Our data indicate that siRNA against Bmi-1 significantly suppresses tumor growth and induces apoptosis in vitro and in vivo.

Members of the ADAMTS family of proteases degrade proteoglycans and thereby have the potential to alter tissue architecture and regulate cellular functions. Aggrecanases are the main enzymes responsible for aggrecan degradation, due to their specific cleavage pattern. In this study, the expression status, the macromolecular organization and localization of ADAMTS-1, ADAMTS-4/aggrecanase-1 and ADAMTS-5/aggrecanase-2 in human normal larynx and laryngeal squamous cell carcinoma (LSCC) were investigated. On mRNA level, the results showed that ADAMTS-4 was the highest expressed enzyme in normal larynx, whereas ADAMTS-5 was the main aggrecanase in LSCC presenting a stage-related increase up to stage III (8-fold higher expression compared to normal), and thereafter decreased in stage IV. Accordingly, immunohistochemical analysis showed that ADAMTS-5, but not ADAMTS-4, was highly expressed by carcinoma cells. Sequential extraction revealed an altered distribution and organization of multiple molecular forms (latent, activated and fragmented forms) of the enzymes within the cancerous and their corresponding macroscopically normal laryngeal tissues, compared to the normal ones. Importantly, these analyses indicated that critical macromolecular changes occurred from the earliest LSCC stages not only in malignant parts of the tissue but also in areas that were not in proximity to carcinoma cells and appeared otherwise normal. Overall, the results of the present study show that ADAMTS-5/aggrecanase-2 is the main aggrecanase present in laryngeal carcinoma suggesting a critical role for the enzyme in aggrecan degradation and laryngeal tissue destruction during tumor progression., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Cancer progression involves multiple proteolytic interactions, with metalloproteinases (MMPs) performing a crucial role. MMP-2, a major MMP, plays a key role in the degradation of basement membranes. Mechanisms underlying MMP-2 activation had to be investigated. Membrane-type matrix metalloproteinases are not only responsible for the regulation of extracellular matrix remodeling, but also involved in the activation of several inactive MMPs. The aim of this study was to evaluate the expression of pro-MMP2, MMP-14, and MMP-15 in tumor cells and tumor stroma. Immunohistochemical studies were performed on paraffin-embedded tissue sections including laryngeal squamous cell carcinoma (SCC). We found the expression of pro-MMP2 in 58% of cases, MMP-14 in 78%, and MMP-15 in 98% of cases of SCC. In all tumor cases, we revealed a higher expression of pro-MMP2 in tumor stoma than in tumor cells. The expression of MMP-14 and MMP-15 was higher in tumor cells than in the stroma. Moreover, we found a statistically significant difference between the expression of MMP-14 and MMP-15 in the tumor in comparison with the surrounding stroma (P < 0.05). An analysis of expression levels of MT-MMPs by classification trees showed that the probability of metastases was related to decreased expression of MMP-14 and increased expression of MMP-15. Our results may suggest that tumor cells with low MMP-14 expression invade tumor stroma and form metastases. Probably, in such cases, tumor progression is stimulated by MMP-15 in an MMP-14 independent pathway, a novel (alternative) mechanism., (© 2012 John Wiley & Sons A/S. All rights reserved.)

Aim of this study was to investigate the correlation of matrix metalloproteinase-9 (MMP-9) expression with histopathologic and clinical characteristics of laryngeal squamous cell carcinoma, and to assess the role of MMP-9 expression in patient survival. Study included 196 patients with squamous cell carcinoma of the larynx treated at ENT Department, Split University Hospital Centre, from January 1, 2000 till December 31, 2009. The level of MMP-9 expression showed a statistically significant correlation (p < 0.001) with the disease histopathologic grade, stage, metastatic potential, recurrence potential, and survival. Kaplan-Meier curve yielded a statistically significant survival difference (p < 0.001) among the three patient groups with different levels of MMP-9 expression. The survival curve clearly showed the MMP-9 expression as a predictor of survival to be significantly (p < 0.001) associated with survival. In this study, MMP-9 expression as a biological marker showed a potential predictive value in laryngeal squamous cell carcinoma.

The development of laryngeal squamous cell carcinomas (LSCC) is strongly influenced by the host immune system. Indoleamine 2,3-dioxygenase (IDO) can promote and maintain an immunosuppressive microenvironment which can impede the efficacy of anticancer responses. The purpose of the current study is to investigate the prognostic value of intratumoral IDO expression in LSCC. The expression of IDO protein was retrospectively assessed by immunohistochemistry in 187 LSCC patients. The potential association of tumor IDO expression with clinical parameters and tumor-infiltrating lymphocytes (TILs) was analyzed separately. Survival curves were estimated by the Kaplan-Meier method, and differences between groups were determined by log-rank test. Multivariate logistic regression analysis was performed to determine the independent factors associated with survival. Based on the evaluation score, 90 carcinomas (48.1 %) were identified with high IDO expression and 97 carcinomas (51.9 %) showed low expression. Tumor IDO expression was not associated with clinical stage, presence of metastases, and other clinicopathological parameters. Also, high IDO expression was not correlated with tumor-infiltrating CD3(+) and CD8(+) TILs. Instead it was positively related with the density of FOXP3(+) Tregs. Furthermore, multivariate analysis identified a significant association of overall survival and disease-free survival with tumor IDO status. IDO high expression represents a significant negative prognostic factor in patients with LSCC. Current results provide further support for using IDO as an immunotherapeutic target in LSCC. The precise role of tumoral IDO in human LSCC remains to be elucidated in the future.

Background: The diagnostic role of cyclooxygenase-2 (COX-2) expression in laryngeal atypical hyperplasia, vocal fold nodule, and laryngeal squamous cell carcinoma was examined., Methods: Specimens obtained from patients diagnosed with vocal fold nodule (n = 35), atypical hyperplasia (n = 35), laryngeal squamous cell carcinoma (n = 35), and clinical parameters were evaluated retrospectively., Results: Although no staining was observed in patients with vocal fold nodules, staining was noted in laryngeal atypical hyperplasia and squamous cell carcinoma. The percentage of COX-2 staining was the highest in the carcinoma group., Conclusion: It was determined that COX-2 staining was significantly associated with laryngeal squamous cell carcinoma. It should be noted that overexpression of COX-2, a potentially important factor in the evolution of carcinogenesis in precancerous lesions, might be an indicator of the development of carcinoma., (Copyright © 2012 Wiley Periodicals, Inc.)

Although tumor resistance remains a significant impediment to successful radiotherapy, associated regulatory markers and detailed molecular mechanisms underlying this phenomenon are not well defined. In this study, we identified inositol polyphosphate 4-phosphatase type II (INPP4B) as a novel marker of radioresistance by systematically analyzing Unigene libraries of laryngeal cancer. INPP4B was highly expressed in radioresistant laryngeal cancer cells and was induced by treatment with either radiation or anticancer drugs in various types of cancer cells. Ectopic INPP4B overexpression increased radioresistance and anticancer drug resistance by suppressing apoptosis in HEp-2 cells. Conversely, INPP4B depletion with small interfering RNA resensitized HEp-2 as well as A549 and H1299 cells to radiation- and anticancer drug-induced apoptosis. Furthermore, radiation-induced INPP4B expression was blocked by inhibition of extracellular signal-regulated kinase (ERK). INPP4B depletion significantly attenuated radiation-induced increases in Akt phosphorylation, indicating an association of INPP4B-mediated radioresistance with Akt survival signaling. Taken together, our data suggest that ERK-dependent induction of INPP4B triggers the development of a tumor-resistance phenotype via Akt signaling and identify INPP4B as a potentially important target molecule for resolving the radioresistance of cancer cells.

Objective: This study aimed to investigate the relationship between focal adhesion kinase expression and clinicopathological factors, cell apoptosis and proliferation, and overall survival, in laryngeal squamous cell carcinoma., Methods: Immunohistochemical staining was used to detect the expression of focal adhesion kinase and proliferating cell nuclear antigen in tissue samples. Apoptotic cells were assessed using the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labelling ('TUNEL') method., Results: The proportion of focal adhesion kinase expression was 73.26 per cent in tumour tissues, significantly greater than in normal tissues (p < 0.05), and was significantly related to laryngeal cancer clinical stage, lymph node metastasis and overall survival (p < 0.05). In the tumour cases assessed, up-regulation of focal adhesion kinase expression was significantly associated with decreased cell apoptosis and increased proliferation (p < 0.05)., Conclusion: These findings suggest that focal adhesion kinase may affect laryngeal squamous cell carcinoma progression through regulation of cell apoptosis and proliferation. Focal adhesion kinase expression is a major prognostic factor in laryngeal cancer patients.

Introduction: The laryngeal cancer is the most common malignancy of the head and neck squamous cell carcinoma. Recent studies have revealed the role of genetic variations in the risk for laryngeal cancer. Polymorphic genes of matrix metalloproteinases may affect an individual genetic predisposition to the occurrence and clinical implications of the disease. THE AIM OF THE PRESENT STUDY: was to evaluate the role of polymorphic variants of MMP-1 and MMP-2 genes in the development of laryngeal carcinoma., Material and Methods: DNA was extracted from 633 individuals including 261 patients with laryngeal carcinoma and 372 healthy volunteers. Genotyping was carried out using TaqMan(®) SNP Genotyping Assays (Applied Biosystems, USA). The distribution of polymorphisms of metalloproteinases were analysed: -1607 1G/2G MMP-1 and -1306 C/T MMP-2 gene., Results: The distribution of genotypes of -1607 1G/2G MMP-1 polymorphism was significantly different in controls vs patients (χ(2) 15.05, p<0.001). The 2G allele carriers of MMP-1 -1607 1G/2G polymorphism were at higher risk for laryngeal carcinoma development (p<0.001). In the present study, 2G/2G polymorphic variant was the independent factor of cancer development (p=0.0015)., Conclusion: Presented data suggest an implication of MMP-1 polymorphisms in the laryngeal carcinoma susceptibility. The presence of the MMP-1 2G allele seemed to be associated with increased risk for the disease. In summary, the current study have provided the evidence that an individual's risk for carcinoma of larynx is modulated by genetic factors., (Copyright © 2012 Polish Otolaryngology Society. Published by Elsevier Urban & Partner Sp. z.o.o. All rights reserved.)