OBJECTIVE: To investigate the effect and mechanism of tea polyphenol (TP) on the proliferation, apoptosis, migration and invasion of nasopharyngeal carcinoma(NPC) cell line HONEl. METHOD: After treated with different concentration of tea polyphenol, CCK-8 assay, fluorescent staining, cell scratching assay and transwell assay were applied to detect the effect of tea polyphenol on the HONE1 cells. Furthermore, the expression of protein VEGF was investigated by flow cytometry assay. RESULT: It was found that tea polyphenol could inhibit NPC cell proliferation significantly in a dose-dependent manner, however, little impact was observed in normal nasopharyngeal epithelial cell line NP69. Furthermore, it was demonstrated by fluorescent staining assay that tea polyphenol could induce NPC cell apoptosis, and cell scratching assay and transwell assay showed that tea polyphenol could inhibit cell migration and invasion. CONCLUSION Tea polyphenol can significantly inhibit cell proliferation, induce cell apoptosis and decreased the migration and invasion ability of NPC cells in vitro. Tea polyphenol might be a tumor suppressor of NPC cells.
Apoptosis ; drug effects ; Carcinoma ; Cell Line, Tumor ; drug effects ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Humans ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; pathology ; Polyphenols ; pharmacology ; Tea ; chemistry

OBJECTIVE: To explore the effects of laminaria japonica polysaccharides(LJP) and tea polyphenols (TP) on nasopharyngeal carcinoma(NPC) cells HONE1 and CNE2, and further to explore the underlying mechanism of antitumor activity of LJP on NPC cell in vivo. METHOD: To identify the logarithmic growth phase of NPC cells HONE1 and CNE2 through cell growth curve and doubling time by means of MTT, then inhibition of the cells proliferation were detected with LJP and TP separately and combined. With LJP treatment, cell apoptosis of HONE1 was examined by double staining assay. A tumor model,established by subcutaneously inoculation of NPC cell HONE1 into nude mice,was used to evaluate the inhibitory effect of LJP in vivo. RESULT: Both LJP and TP had inhibition effect on two groups of cell proliferation, and LJP and TP combined effect of inhibition were higher than the two separate on two sets of experimental cell proliferation, whose effect was concentration-dependent. LJP could induce apoptosis of HONE1. With the increasing concentration of LJP, apoptosis rate increased. The apoptosis rate was(49.51 +/- 1. 89) % (P<0. 01) when treated with 320 mg/L LJP. The inhibition rate was between 50% to 60% at 72 h after treatment with 320 mg/L LJP. Compared to control group, the growth of xenografts in nude mice was significantly suppressed after administration of LJP at a dose-dependent manner. The inhibition rates were 33. 7%(P<0. 05)and 47. 0%(P<0. 01) when treated with 25.0 mg/kg and 50. O mg/kg respectively. Whereas the inhibition rate of 12.5 mg/kg group was only 16. 4%(P>0. 05). CONCLUSION LJP and TP can inhibit the proliferation of NPC cells HONE1 and CNE2 respectively,and combined use has a significant effect. LJP can inhibit the growth of NPC probably by inducing apoptosis of NPC cells in vitro and in vivo.
Animals ; Carcinoma ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Laminaria ; chemistry ; Mice ; Mice, Nude ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; pathology ; Polyphenols ; pharmacology ; Polysaccharides ; pharmacology ; Tea ; chemistry ; Xenograft Model Antitumor Assays

OBJECTIVE: To explore the proliferation inhibition and apoptosis of polysaccharides extracts from polysaccharides extracts from Hedyotic diffusa (PEHD) on Human Nasopharyngeal Carcinoma (NPC)cell line CNE2 cells in vitro. METHOD: CNE2 cells treated with various concentrations of PEHD were detected by MTT assay at 24 h, 48 h, and 72 h. The apoptotic cells were analyzed by flow cytometry with Annexin V/PI staining. The expression levels of Bax, Bcl-2 and caspase-3 protein were examined by Western blotting method. RESULT: The growth of CNE2 cells were suppressed after treatment with PEHD (P < 0.05), MTT assay showed that the highest cell inhibition rate reached to 76.5%, the inhibition in the doses from 2 to 6 mg/ml showed dose-and-time-dependent. The percent of apoptosis in 4 and 6 mg/ml PEHD treatment groups for 48 h were 31.32%, 46.28%, respectively, and significantly higher than that in control groups, 4.86% (P < 0.01). After the cells being treated with PEHD for 48 h, the expression of Bax and caspase-3 protein increased, and the expression of Bcl-2 protein decreased gradually. CONCLUSION PEHD could inhibited the growth of CNE2 cells and was dose-and-time-dependent, the mechanism may involve induction of cell apoptosis, which was associated with the activation of Bax and caspase-3 protein and the down-regulation of Bcl-2 protein expression.
Apoptosis ; Carcinoma ; Caspase 3 ; metabolism ; Cell Line, Tumor ; drug effects ; Cell Proliferation ; Down-Regulation ; Hedyotis ; chemistry ; Humans ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; pathology ; Plant Extracts ; pharmacology ; Polysaccharides ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo evaluate the inhibitory effect of deuterium-depleted water (DDW) on the proliferation of nasopharyngeal carcinoma (NPC) cells in vitro and explore the possible mechanism.

METHODSThe growth inhibition of NPC cells and preosteoblast MC3T3-E1 cells following DDW treatment was measured by MTT assay and plate colony formation assay. The changes in migration and invasion of NPC cells were evaluated using Transwell and boyden chamber assays. The protein expression of proliferating cell nuclear antigen (PCNA) was determined using Western blotting. Flow cytometry was employed to evaluate the changes in cell cycle distribution after DDW treatment.

RESULTSDDW with deuterium concentrations of 100, 75 and 50 ppm significantly suppressed the cell proliferation (P<0.05) and lowered colony formation capacity and invasiveness of the NPC cells (P<0.01). Western blotting demonstrated a down-regulated expression of PCNA in the cells by DDW. DDW also caused obvious cell cycle arrest in the NPC cells with reduced cells in S phase and significantly increased cells in G(1) phase (P<0.05). Rather than causing growth inhibition, DDW promoted the growth of normal control MC3T3-E1 cells.

CONCLUSIONDDW possesses selective biological effects to inhibit the proliferation of NPC cells in vitro, suggesting the potential of DDW as a novel nontoxic adjuvant therapeutic agent in antitumor therapy.

Carcinoma ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Deuterium ; administration & dosage ; pharmacology ; Humans ; Nasopharyngeal Neoplasms ; pathology ; Proliferating Cell Nuclear Antigen ; metabolism ; Water ; chemistry

OBJECTIVE: To express the recombinant SPLUNC1 protein in HNE1 cells and to study its function of bactericidal and binding to lipopolysaccharide (LPS). METHODS: Full length of SPLUNC1 gene was cloned into pCMV-tag4A vector and stably transfected into HNE1 cell lines, the supernatant of cell cultures was collected. After being treated with the supernatant, the Pseudomonas aeruginosa was seeded to LB soft agar plate, and the bacteria clones were counted and analyzed. For in vitro LPS binding assay, LPS was coated to 96-well plates. We incubated in the plate with SPLUNC1 protein, and detected the binded SPLUNC1 protein by ELISA. Incubating the FITC-LPS with the SPLUNC1 stably transfected or control cells, the intracellular intensity of fluorescence was observed under the fluorescence microscope. RESULTS: SPLUNC1 inhibited the bacteria clone formation obviously. Although the binding efficiency of LPS and SPLUNC1 in vitro was very low, more FITC-LPS entered into the SPLUNC1 stably transfected cells. CONCLUSION SPLUNC1 can inhibit the growth of Pseudomonas aeruginosa and bind LPS, and play an important defensive role in innate immunity of the upper airway.
Cell Line, Tumor ; Glycoproteins ; isolation & purification ; pharmacology ; Humans ; Membrane Proteins ; chemistry ; Nasopharyngeal Neoplasms ; genetics ; pathology ; Phosphoproteins ; isolation & purification ; pharmacology ; Pseudomonas aeruginosa ; drug effects ; Respiratory Mucosa ; chemistry ; immunology ; Respiratory System ; chemistry ; immunology ; Transfection

This study reviewed 65 cases of polymorphic reticulosis (PR) with respect to clinical and histopathologic bases, and immunohistochemical studies were done using MT1 and UCHL as T-cell markers, MB2 as a B-cell marker and alpha-1-antichymotrypsin as a histiocytic marker. The results obtained were as follows: 1. The male to female ratio was 2.4:1 and the mean age of patients was 44.5 years. The sites involved primarily were the nasal cavity, tonsil and pharynx and about one-fourth of the total cases showed extensive involvement of two anatomical sites at initial presentation. 2. Almost all cases showed characteristic histologic features similar to those of peripheral T-cell lymphoma and showed positive reaction to the T-cell marker. The above immunohistochemical findings suggest strongly that quite a significant portion of PR is in fact T-cell lymphoma.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Antigens, Differentiation, T-Lymphocyte/*analysis ; Antigens, Neoplasm/analysis ; Blood Sedimentation ; Comparative Study ; Female ; Head and Neck Neoplasms/chemistry/*pathology ; Human ; Lymphoma, T-Cell/*pathology ; Male ; Middle Age ; Nasopharyngeal Neoplasms/chemistry/pathology ; Neoplasm Proteins/analysis ; Reticuloendotheliosis/metabolism/*pathology ; Support, Non-U.S. Gov't ; Tumor Markers, Biological/analysis

BACKGROUNDThis study was designed to detect methylation of E-cadherin gene promoter and gene mutation of beta-catenin in exon 3 and their expression of protein and mRNA in primary tumor and lymph node metastatic tumor of nasopharyngeal carcinoma (NPC), and investigate the mechanism of invasion and metastasis of neoplastic cells in NPC.

METHODSFourty-two fresh biopsy samples were taken from untreated NPC patients at the Affiliated Hospital of Sun Yat-sen Medical College, Sun Yat-sen University, Guangzhou, China during the period of 1999-2002. Among them 21 were taken from primary tumors and the other 21 from lymph node metastatic tumors. The gene promoter methylation of E-cadherin was detected by methylation-specific PCR (MSP). The mutation in exon 3 of beta-catenin was detected by direct sequencing analysis. RT-PCR, Western blot and immunohistochemical staining were used to detect the mRNA and protein expression patterns in both primary and metastatic tumors of NPC.

RESULTSDown-regulated expression of E-cadherin in metastatic tumor was compared with that in primary tumor. Reduced expression of E-cadherin was found to be correlated with lymph node metastatic tumor of NPC (P = 0.004); but there was no obvious correlation between primary and metastatic tumors in the expression of beta-catenin (P = 0.698). The mRNA expression level of E-cadherin in metastatic tumors decreased significantly compared with that in primary tumors. However, little change was observed in the mRNA level of beta-catenin in different tumor tissues. Only 4 samples (19.1%) displayed gene promoter methylation of E-cadherin in primary tumor and 10 samples (47.6%) showed methylated form of E-cadherin. The gene promoter methylation of E-cadherin was more common in metastatic tumor than in primary tumor of NPC (P = 0.024). Only 2 (4.76%) of the 42 samples showed mutations in exon 3 of beta-catenin at 41 (T41A, ACC-->GCC) and codon 47 (S47T, AGT-->ACT). The cytoplasmic and nuclear expression of beta-catenin in tumor was not found in any samples of NPC.

CONCLUSIONSThe results suggest that the downregulation of E-cadherin results from the gene promoter aberrant methylation of E-cadherin and that the methylation of E-cadherin plays an important role in invasion and metastasis of tumor cells in NPC. However, beta-catenin mutation is an infrequent event in NPC, and beta-catenin is not a critical factor influencing the invasion and metastasis of tumor cells in NPC.

Adult ; Aged ; Blotting, Western ; Cadherins ; analysis ; genetics ; Cytoskeletal Proteins ; analysis ; genetics ; DNA Methylation ; Female ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Mutation ; Nasopharyngeal Neoplasms ; chemistry ; genetics ; pathology ; Neoplasm Metastasis ; Promoter Regions, Genetic ; Trans-Activators ; analysis ; genetics ; beta Catenin

OBJECTIVETo clarify if Epstein-Barr virus encoded LMP1 induces matrix metalloproteinase 9 expression via NF-kappa B or AP-1 signaling pathway, which gives evidence to the elucidation of the mechanism of LMP1- mediated carcinogenesis.

METHODSTo determine whether LMP1 or its mutants contribute to MMP9 production via NF-kappa B or AP-1 transcription factor, MMP9-chloramphenicol acetyl transferase (CAT), NF-kappa B mut 9-CAT, AP-1 mut MMP9-CAT were transfected into human nasopharyngeal carcinoma cells stably expressing LMP1 (HNE2-LMP1) or its mutants, [HNE2-LMP1 (1-185), HNE2-LMP1 (1-231), HNE2-LMP1 delta 187-351] by electroporation technic. The difference of MMP9 reporter activity among those cell lines was detected by CAT assay and expression of MMP9 was determined in nasopharyngeal carcinoma cells stably expressing LMP1 or its mutants by zymographic analysis. In the meantime, efforts were made to demonstrate if LMP1 regulates NF-kappa B or AP-1 activation using reporter gene analysis.

RESULTSIn contrast with vector-transfected cells, MMP9 CAT activity in HNE2-LMP1, HNE2-LMP1 (1-185), HNE2-LMP1(1-231), HNE2-LMP1 delta 187-351 increased 7.2, 1.3, 3.3, 4.0 times respectively. Zymographic analysis demonstrated that the 92 kDa MMP9 expression was induced in HNE2-LMP1, HNE2-LMP1(1-231) and HNE2-LMP1 delta 187-351 cells, whereas it was negative in HNE2-pSG5 and HNE2-LMP1 (1-185) cells. As compared to the HNE2 cells, NF-kappa B or AP-1 reporter activity in HNE2-LMP1 cells were increased 13.8, 8.4 fold respectively. Moreover, In contrast with MMP9 CAT-transfected cells, MMP9 CAT activity in NF-kappa B mut MMP9-CAT or AP-1 mut MMP9-CAT transfected HNE2-LMP1, HNE2-LMP1 (1-185), HNE2-LMP1(1-231) and HNE2-LMP1 delta 187-351 cells were significantly decreased by 18.1% or 16.3%, 35.0% or 33.3%, 29.1% or 26.1% from the original level. However, there was no difference in NF-kappa B mut MMP9-CAT or AP-1 mut MMP9-CAT transfected HNE2-pSG5, HNE2-LMP1 (1-185) cells.

CONCLUSIONIn nasophargyngeal carcinoma, Epstein-Barr virus-encoded LMP1 induces MMP9 transcription and enzymatic activity via an NF-kappa B or AP-1 signaling pathway, which may contribute to invasiveness and metastasis.

Gene Expression ; drug effects ; Herpesvirus 4, Human ; chemistry ; Humans ; Matrix Metalloproteinase 9 ; biosynthesis ; NF-kappa B ; metabolism ; Nasopharyngeal Neoplasms ; pathology ; Signal Transduction ; Transcription Factor AP-1 ; metabolism ; Tumor Cells, Cultured ; Viral Matrix Proteins ; pharmacology

OBJECTIVETo study whether macrophage migration inhibitory factor (MIF) can increase the ability of invasion of nasopharyngeal carcinoma cell lines in vitro, and to investigate the mechanism of invasion and metastasis of tumor cells during the early stage of nasopharyngeal carcinoma (NPC).

METHODSThe invasion and migration of NPC cell lines, CNE-1 and CNE-2, were evaluated by micron-migration assay in a chamber with 8- micro m porosity polycarbonate filter membrane. Flow cytometry and western blotting were adopted respectively to evaluate the protein expression level of matrix metalloproteinase 2 and 9 (MMP2, MMP9) in MIF treated or non-treated tumor cell lines. The concentrations of interleukin 8 (IL-8) secreted into the culture supernatant by the cells were measured by using Enzyme-linked immunoabsorbent assay (ELISA).

RESULTS(1) After treatment with MIF for 24 hours, the number of cells passing through the 8- micro m filter membrane were increased in CNE-1 (113.7 +/- 20.9) and CNE-2 (311.3 +/- 48.9), as compared with that of non-MIF treated NPC cells. A significant statistic difference (P = 0.005, P = 0.001) was obtained in both CNE-1 and CNE-2 cells. (2) After treatment with MIF, the number of MMP9-positive cells increased in both CNE-1 (from 28.5% +/- 2.45% to 82.4% +/- 3.49%, P = 0.001) and CNE-2 (from 32.8% +/- 3.48% to 86.1% +/- 1.62%, P = 0.002) cell lines. In addition, an enhanced MMP9 protein expression up to 3-fold was observed in both cell lines. However, the expression level of MMP2 did not changed significantly between treated and non-treated cell lines (P > 0.05). (3) The concentration of IL-8 in the culture supernatant of CNE-2 was 1201.8 +/- 593.3 pg/ml after treatment with MIF for 24 h, remarkably higher than that without MIF treatment (32.7 +/- 20.1 pg/ml, P = 0.026). A similar change was not detected in CNE-1 (P = 0.581) cells.

CONCLUSIONS(1) MIF can increase cell migration of CNE-1 and CNE-2 NPC cell lines in vitro. (2) A higher expression level of MMP9 and an up-regulated IL-8 by MIF may play a very important role in the progress of NPC, such as invasion and metastasis.

Cell Line, Tumor ; Cell Movement ; drug effects ; Enzyme-Linked Immunosorbent Assay ; Humans ; Interleukin-8 ; analysis ; Macrophage Migration-Inhibitory Factors ; pharmacology ; Matrix Metalloproteinase 2 ; analysis ; Matrix Metalloproteinase 9 ; analysis ; Nasopharyngeal Neoplasms ; chemistry ; pathology ; Neoplasm Invasiveness

OBJECTIVETo evaluate the roles of p27(kip1), p16 gene protein and proliferating cell nuclear antigen expression in nasopharyngeal carcinoma (NPC).

METHODSThe EnVision immunohistochemical method was used to detect the expression of p27(kip1), p16 gene protein and PCNA in 66 cases of non-keratinized carcinoma (NKC) and 25 cases of non-tumor nasopharyngeal tissue.

RESULTS(1) The positive expression rates of p27(kip1), p16 gene protein were 65%, 68% in NKC respectively. There were significant differences between NKC and non-tumor group (P < 0.05). (2) The expression of p27(kip1), p16 protein correlated with cranial nerve encroaching and the 5-year survival rates of the patients (P < 0.05), but had no significant correlation to lymph node metastases and clinical staging (P > 0.05). The expression of PCNA was related to clinical staging and to the patient's 5-year survival rates (P < 0.05), but not to lymph node metastases and cranial nerve encroaching (P > 0.05). (3) The positive expression of p27(kip1), p16 gene protein and PCNA were correlated.

CONCLUSIONThe results suggest that immunological labeling of p27(kip1), p16 gene protein and PCNA might be used to determine the prognosis of NKC.

Adult ; Aged ; Cell Cycle Proteins ; analysis ; Cyclin-Dependent Kinase Inhibitor p16 ; analysis ; Cyclin-Dependent Kinase Inhibitor p27 ; Female ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Nasopharyngeal Neoplasms ; chemistry ; mortality ; pathology ; Prognosis ; Proliferating Cell Nuclear Antigen ; analysis ; Tumor Suppressor Proteins ; analysis