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Abstract Background Epigenetic silencing of tumor suppressor genes play important roles in NPC tumorgenesis. Tissue factor pathway inhibitor-2 (TFPI-2), is a protease inhibitor. Recently, TFPI-2 was suggested to be a tumor suppressor gene involved in tumorigenesis and metastasis in some cancers. In this study, we investigated whether TFPI-2 was inactivated epigenetically in nasopharyngeal carcinoma (NPC). Methods Transcriptional expression levels of TFPI-2 was evaluated by RT-PCR. Methylation status were investigated by methylation specific PCR and bisulfate genomic sequencing. The role of TFPI-2 as a tumor suppressor gene in NPC was addressed by re-introducing TFPI-2 expression into the NPC cell line CNE2. Results TFPI-2 mRNA transcription was inactivated in NPC cell lines. TFPI-2 was aberrantly methylated in 66.7% (4/6) NPC cell lines and 88.6% (62/70) of NPC primary tumors, but not in normal nasopharyngeal epithelia. TFPI-2 expression could be restored in NPC cells after demethylation treatment. Ectopic expression of TFPI-2 in NPC cells induced apoptosis and inhibited cell proliferation, colony formation and cell migration. Conclusions Epigenetic inactivation of TFPI-2 by promoter hypermethylation is a frequent and tumor specific event in NPC. TFPI-2 might be considering as a putative tumor suppressor gene in NPC.

Background: Epigenetic changes, including DNA methylation, disrupt normal cell function, thus contributing to multiple steps of carcinogenesis. Nasopharyngeal carcinoma (NPC) is endemic in southern China and is highly associated with Epstein-Barr virus (EBV) infection. Significant changes of the host cell methylome are observed in EBV-associated NPC with cancer development. Epigenetic marks for NPC diagnosis are urgently needed. In order to explore DNA methylation marks, we investigated DNA methylation of candidate genes in EBV-associated nasopharyngeal carcinoma.Methods: We first employed methyl-capture sequencing and cDNA microarrays to compare the genome-wide methylation profiles of seven NPC tissues and five non-cancer nasopharyngeal epithelium (NNE) tissues. We found 150 hypermethylated CpG islands spanning promoter regions and down-regulated genes. Furthermore, we quantified the methylation rates of seven candidate genes using bisulfite amplicon sequencing for nine NPC and nine NNE tissues.Results: All seven candidate genes showed significantly higher methylation rates in NPC than in NNE tissues, and the ratios (NPC/NNE) were in descending order as follows: ITGA4 > RERG > ZNF671 > SHISA3 > ZNF549 > CR2 > RRAD. In particular, methylation levels of ITGA4, RERG, and ZNF671 could distinguish NPC patients from NNE subjects.Conclusions: We identified the DNA methylation rates of previously unidentified NPC candidate genes. The combination of genome-wide and targeted methylation profiling by next-generation sequencers should provide useful information regarding cancer-specific aberrant methylation. [ABSTRACT FROM AUTHOR]

Reactive nitrogen species are considered to participate in inflammation-related carcinogenesis through DNA damage. 8-Nitroguanine is a specific marker of inflammation-related carcinogenesis. Epstein-Barr virus infection-related nasopharyngeal carcinoma (NPC) is one of the most prevalent malignant tumors in southern China and Southeast Asia, and its prognosis has been poor for decades. Previously, we demonstrated that nitrative DNA damage, such as 8-nitroguanine formation, occurs in cancer cells of NPC patients (Ma et al. Int. J. Cancer 2008). In the present study, to investigate the involvement of stem cells in NPC, we performed immunohistochemical analyses to examine nitrative DNA lesions (8-nitroguanine) and several cancer stem / progenitor cell markers (CD44v6, CD24 and ALDH1A1) in nasopharyngeal tissues obtained from chronic nasopharyngitis and NPC patients. The staining intensity of 8-nitroguanine was significantly higher in cancer cells and inflammatory cells in stroma of NPC than in chronic nasopharyngitis tissues. Expression levels of CD44v6 and ALDH1A1 were significantly increased in cancer cells of primary NPC specimens in comparison to chronic nasopharyngitis tissues. 8-Nitroguanine was detected in CD44v6- or ALDH1A1-positive stem cells in NPC tissues. In the case of CD24 staining, there was no significant difference between NPC and chronic nasopharyngitis tissues. Intensive staining of CD44v6 and ALDH1A1 were also detected in NPC cell line HK1 compared to immortalized nasopharyngeal epithelial cells NP460 by a double immunofluorescence study and western blotting assay. In conclusion, CD44v6 and ALDH1A1 could be candidates of cancer stem cell markers for NPC. The present results indicate the possible mechanism by which inflammation causes NPC by inducing inflammatory processes and formation of 8-nitroguanine in CD44v6- and / or ALDH1A1-positive stem cells, and mutant stem cells participate in NPC development. [ABSTRACT FROM AUTHOR]

Background: The potential effect of alcohol or tea intake on the risk of nasopharyngeal carcinoma (NPC) remains controversial., Methods: In a population-based case-control study in southern China, we assessed alcohol or tea intake from 2,441 histopathologically confirmed NPC cases and 2,546 controls. We calculated mean daily ethanol (g/day) and tea intake (mL/day). Fully adjusted ORs with 95% confidence intervals (CI) were estimated using logistic regression; potential dose-response trends were evaluated using restricted cubic spline analysis., Results: Compared with nondrinkers, no significantly increased NPC risk in men was observed among current alcohol drinkers overall (OR, 1.08; 95% CI, 0.93-1.25), nor among current heavy drinkers (OR for ≥90 g/day ethanol vs. none, 1.32; 95% CI, 0.95-1.84) or former alcohol drinkers. Current tea drinking was associated with a decreased NPC risk (OR, 0.73; 95% CI, 0.64-0.84). Compared with never drinkers, those with the low first three quintiles of mean daily current intake of tea were at significantly lower NPC risk (OR, 0.53, 0.68, and 0.65, respectively), but not significant for the next two quintiles. Current daily tea intake had a significant nonlinear dose-response relation with NPC risk., Conclusions: Our study suggests no significant association between alcohol and NPC risk. Tea drinking may moderately reduce NPC risk, but the lack of a monotonic dose-response association complicates causal inference., Impact: Tea drinking might be a healthy habit for preventing NPC. More studies on biological mechanisms that may link tea with NPC risk are needed., (©2020 American Association for Cancer Research.)

Collagen (COL) genes participate in tumor extracellular matrix (ECM)-receptor interactions and focal adhesion pathways, which play a crucial role in tumor invasion and metastasis. The prognostic value of COL genes has been shown for several malignancies. In the present study, we analyzed multiple microarray datasets using the Oncomine database to identify alterations of COL genes in gastric cancer (GC). Gene expression levels were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) in GC tissues and matched adjacent tissues. The prognostic value of differentially expressed COL genes in GC was evaluated by Kaplan-Meier survival analysis based on the complete mRNA transcriptomics data from The Cancer Genome Atlas (TCGA). We found that seven COL genes (COL1A2, COL4A1, COL4A2, COL6A1, COL6A2, COL6A3, and COL11A1) were elevated in GC. Among them, stepwise multivariate Cox regression was applied, and it was determined that COL4A1 and COL4A2 were signature and independent prognostic biomarkers in GC patients with obviously different overall survival (OS). High expression of COL4A1, COL4A2, COL6A1, COL6A2, and COL6A3 was correlated with poorer prognosis of GC patients treated by surgery only, while higher expression of COL4A1 and COL11A1 correlated with poorer survival of patients treated by 5-fluorouracil-based adjuvant therapy. Our results indicate that overexpression of COL genes might be utilized as novel prognostic markers for GC and assist with therapy selection., Competing Interests: Declaration of Competing Interest We declare that we have no conflicts of interest., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Epstein-Barr virus (EBV)-encoded latent membrane protein 2A (LMP2A) promotes the motility of nasopharyngeal carcinoma (NPC) cells. Previously, we have shown that the localization of integrin β4 (ITGβ4) is regulated by LMP2A, with ITGβ4 concentrated at the cellular protrusions in LMP2A-expressing NPC cells. In the present study, we aim to further investigate mechanisms involved in this process and its contribution to cell motility. We show that expression of LMP2A was correlated with increased epidermal growth factor receptor (EGFR) activation, elevated levels of intracellular Ca 2+ , calpain activation and accelerated cleavage of ITGβ4. Activation of EGFR and calpain activity was responsible for a redistribution of ITGβ4 from the basal layer of NPC cells to peripheral membrane structures, which correlated with an increased migratory capacity of NPC cells. Furthermore, we demonstrated that the calpain inhibitor calpastatin was downregulated in NPC primary tumors. In conclusion, our results point to LMP2A-mediated targeting of the EGFR/Ca 2+ /calpain/ITGβ4 signaling system as a mechanism underlying the increased motility of NPC cells. We suggest that calpain-facilitated cleavage of ITGβ4 contributes to the malignant phenotype of NPC cells., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Objective: To discover the relationship of transcriptional levels and promoter methylation status of CHFR gene in human nasopharyngeal carcinoma,to discuss the significance and epigenetic mechanism of CHFR inactivation in NPC, and to evaluate the feasibility of detecting methylated CHFR in nasopharyngeal swab as a means for diagnosis of NPC., Method: Transcriptional levels of CHFR was evaluated by RT-PCR. Methylation specific PCR was used to detect the methylation status of CHFR in NPC cells, normal nasopharyngeal epithelia, primary tumors and their paired nasopharyngeal swabs. Detailed methylation status was confirmed by bisulfite sequencing. NPC cells were treated by the methyltransferase inhibitor 5-aza-dC and the reactivation of CHFR was evaluated by RT-PCR., Result: CHFR transcription was inactivated in NPC. The methylation frequency in NPC primary tumors and their paired swabs were 65.5% and 63.8%, respectively, with a 86.2% concordance. Bisulfite sequencing revealed a dense methylation in NPC cells and primary tumors, but all the normal nasopharyngeal epithelia were unmethylated. CHFR expression were restored after 5-aza-dC treatment., Conclusion: CHFR is epigenetically inactivated by promoter methylation in NPC. Detecting methylated CHFR can be served as a useful non-invasive means for diagnosis of NPC.

Unlabelled: Gastric carcinoma is frequent, particularly in China, and therapy is often inefficient. Because cancer cells are partly or mainly dependent on glycolysis to generate adenosine triphosphate ATP (Warburg effect) and/or to produce precursors (of lipid, nucleotides, etc.) for building new cells, any inhibition of glycolysis may slow down the cell proliferation and/or may kill cells. The antitumor effect of citrate, an anti-glycolytic agent inhibiting phosphofructokinase (PFK) was tested on two human gastric carcinoma cell lines., Materials and Methods: Cell viability and morphology were assessed after 24-72 h exposure to citrate (5, 10, 220 mM). Apoptosis was assessed by annexin V-FITC/PI staining and Western immunobloting., Results: A 3-day continuous exposure to citrate led to near destruction of the cell population in both cell lines, apoptotic cell death occurred through the mitochondrial pathway in a dose- and time-dependent manner, associated with the reduction of the anti-apoptotic Mcl-1 protein in both lines., Conclusion: Citrate demonstrates strong cytotoxic activity against two gastric cancer lines, leading to an early diminution of expression of Mcl-1 and to massive apoptotic cell death involving the mitochondrial pathway.

Objective: To investigate the effects of situation of misdiagnosis of nasopharyngeal carcinoma (NPC) on distant metastasis., Method: The history of diagnosis and treatment of 85 newly diagnosed cases with nasopharyngeal carcinoma were studied by using itemized questionnaire purposely; 433 patients with different prognosis were analyzed retrospectively for the misdiagnoses and mistreatment, including surgical biopsy in the neck., Result: (1) The rate of misdiagnosis of 85 patients was 72.64%, and the percentage decreased as the level of the hospitals increased; the majority of the patients (77.36%) were diagnoses within 1 month after the first symptom had appeared; the number of diseases misdiagnosed was 20, most common of which were lymphnoditis, tuberculosis of lymph node and secretory tympanitis; (2) Our data showed that among 433 patients analysed retrospectively, 60 cases had undergone surgical biopsy in the neck, 75% of whom had never received nasopharyngeal biopsy; 43 cases had underwent nasopharyngeal biopsy after the pathological diagnosis as metastatic carcinoma of neck biopsy (71.67%) and the rest (20.0%) received radiotherapy directly or after negative nasopharyngeal biopsy for merely 1 to 4 times; of those 43 cases who were diagnosed as NPC by nasopharyngeal biopsy, 79.17% got positive results at first sampling. (3) Rate of misdiagnosis and mistreatment including surgical biopsy in the neck of patients who had been tumor-free for 5 years or above was significantly lower than that of those who experienced distant metastasis after or before treatment (P < 0.05)., Conclusions: Misdiagnosis and mistreatment including biopsy by surgery of neck is common even in high-grade hospitals; it is doctor that is responsible for this situation; the high occurrence rate of misdiagnosis and mistreatment, biopsy by neck surgery, especially the delayed treatment after the neck biopsy are the factors that contribute to distant metastasis of NPC.

Objective: To investigate the pathogenesis of unknown nosebleed patients., Method: The ELISA test were used to detected plasma Urokinase-type plasminogen activator (uPA) and Urokinase-type plasminogen activator receptor (uPAR) level in 19 cases unknown factor nosebleed patients and 36 health persons., Result: The results showed uPAR and uPA level in nosebleed group (before treatment) uPAR (0.14 +/- 0.04) microg/L, uPA (0.24 +/- 0.09) microg/L; (after treatment) uPAR (0.08 +/- 0.02) microg/L, uPA (0.18 +/- 0.07) microg/L. And normal group uPAR (0.07 +/- 0.03) microg/L, uPA (0.17 +/- 0.05) microg/L. The uPAR and uPA level in nosebleed group before treatment is higher than that in normal group (P <0.05). There is no significant difference between nosebleed group after treatment and normal group (P>0.05)., Conclusion: The reasons of uPAR and uPA level high in unknown factor nosebleed patients were not clear, maybe relation to vascular endothelial cell, smooth muscle cell and neutrophil-monocytic release more uPAR and uPA. So uPAR and uPA density of nostril accumulation is more high in its microenvironment, that fibrinolytic system activated increase and result in its hyperactivity, and happened nosebleed when blood be in hypocoagulable state.

Objective: This study was to explore the expression and clinicopathologic features of Tyrosine kinase receptors B (TrkB) and its ligand brain-derived neurotrophic factor (BDNF) in nasopharyngeal carcinoma (NPC)., Method: Immunohistochemistry was adopted to detect the expression level of TrkB and BDNF in NPC patients., Result: Both TrkB and BDNF were expressed in NPC as well as in chronic inflammation. The active expression rate of TrkB in NPC was 82.5% (47/57) and BDNF was 52.6% (30/57), both of which were higher than those in chronic inflammation (P < 0.05). The degree of TrkB expression was more marked in T3 + T4, III + IV stage NPC than that in T1 +T2, I + II stage NPC (P < 0.05). TrkB abnormal expression rate of nasopharyngeal carcinoma with lymph node metastasis was higher than that of NPC without lymph node metastasis (P < 0.05). No statistical significance for degrees of TrkB expression in pathologic type grades was found (P > 0.05). There were no statistical significance for degrees of BDNF expression in T stage, clinical stage and lymph node metastasis in NPC (P > 0.05). The expression of TrkB was unrelated to the expression of BDNF (r = 0.049, P > 0.05)., Conclusion: The high expression rate of TrkB and BDNF maybe plays an important role in development of NPC. It is suggested that TrkB and its ligand BDNF may act as an important index for forecasting the development and metastasis of NPC.

Objective: To investigate the feasibility and reliability of detection of Epstein-Barr virus (EBV) latent membrane protein-1 (LMP1) gene by nasopharyngeal swab in the diagnosis of nasopharyngeal carcinoma (NPC). To investigate the distribution of 30 bp deletion variant of LMP-1 gene in the local population., Method: Nasopharyngeal cells were collected by nasopharyngeal swab, and then DNA was extracted, which was subsequently confirmed by amplification of sequence of beta-thalassemia gene by polymerase chain reaction (PCR). And sequence of LMP1 was amplified with specific primer to verify the significance of LMP1 in the diagnosis of NPC., Result: DNA was obtained from 96.4% nasopharyngeal swab samples, LMP1 was detected in 33 of 36 samples, while 2 of 45 samples from normal control, with sensitivity 91.7%, and specialty 95.6%. 30 bp deletion of LMP1 gene was found in 80.6% of NPC samples, and wild type 11.1%., Conclusion: Our study suggests that nasopharyngeal swab could be effective method for gene-detection. As a parameter in diagnosis of NPC, LMP1 gene is maybe superior to EBVCA-IgA. 30 bp deletion of LMP1 oncogene is widespread in NPC patients.

Objective: To study the relation between Mta1 gene (metastasis associated gene, meta gene) and lymph node metastasis of laryngeal carcinomas., Method: Reverse transcription polymerase chain reaction(RT-PCR) technique was used to detect the expression of Mta1 mRNA in 30 carcinoma of larynx tissue cases, 12 lymph nodes and 10 normal larynx tissue cases., Result: The frequency of MTA1 overexpression was 100% (8/8) in primary carcinomas of larynx with metastasis but only 22.7% (5/22) in toes without metastasis (P<0.01). Expression of MTA1 was 87.5%(7/8) in lymph nodes with metastasis but only 0(0/4) in toes without metastasis(P<0.05). The frequency of Mta1 overexpression was 43.3% (13/30) in primary carcinomas of larynx., Conclusion: Our data suggest that the overexpression of the MTA1 gene correlates with lymph node metastasis of carcinomas of larynx. Mta1 gene will probably become a valuable gene for judging metastasis of laryngeal carcinoma.

Objective: To study the differences of morphology, phenotype and function of dendritic cell (DC) derived from the healthy individuals and the patients with nasopharyngeal carcinoma (NPC)., Method: DC were isolated and developed from peripheral blood monocytes from 12 NPC patients and 9 healthy volunteers in vitro, and were studied in terms of shape and phenotype of CD83, CD1a,CD86, HLA-DR by flow cytometry then. MTT method was used to measured the capability of DC in mixed lymphocyte reactions (MLR)., Result: The shape of DC derived from monocyte in the healthy volunteers' peripheral blood was more typical than that of NPC patients'. The expression rate of CD83, CD1a on DC from healthy volunteers was significantly higher than that from NPC patients (P < 0.05). The DC from healthy volunteers had the higher expression level of CD1a and CD83 than that from NPC patients, but not significantly( P > 0.05). DC in healthy volunteer's group could trigger proliferation of allogenous lymphocyte (P < 0.05) but it could not significantly do that in NPC patients group (P > 0.05)., Conclusion: These results reveal that the yield of DC derived from NPC patient's monocyte is lower than that from healthy individual's in peripheral blood,and the DC from NPC patient may be in an immature state and could not trigger the proliferation of lymphocyte.

Objective: To study the relation between T lymphoma invasion/metastasis1 (Tiam1) gene and nasopharyngeal carcinoma progression., Method: Using of reverse transcription polymerase chain (RT-PCR) technique on 41 nasopharyngeal carcinoma tissue and 20 normal nasopharyngeal tissue to proceed the test of mRNA overexpression., Result: Average expression of the Tiam1 gene in nasopharyngeal carcinoma tissue (1.183 +/- 0.73) is higher than in normal nasopharyngeal tissue (0.87 +/- 0.45) (P < 0.01). The positive relationship between the frequency of Tiam1 overexpression and clinical staging and T staging was very significant (P < 0.05) and increased gradually; The frequency of Tiam1 overexpression was 59.3% (16/27) in patients with lymph node metastasis, 21.4% (3/ 14) in patients without lymph node metastasis, overexpression between them was very significant (P < 0.05)., Conclusion: Average expression of the Tiam1 gene in nasopharyngeal carcinoma tissue is higher than in normal nasopharyngeal tissue. Our data suggest that the overexpression of the Tiam1 gene correlates invasion and metastasis of nasopharyngeal carcinoma.