metallothionein and Nasopharyngeal-Neoplasms

To investigate anticancer effect and potential mechanism of tanshinone II(A) (Tan II(A)) on human nasopharyngeal carcinoma cell line CNE cells.. Antiproliferative effect of Tan II(A) on CNE cells was evaluated by morphological examination, cell growth curves, colonial assay and MTT assay. Apoptosis detection was carried out using Hoechest 33258 and PI double-dyeing method. Intracellular Ca2+ concentration and mitochondria membrane potential were detected by fluorospectrophotometer. Bad and MT-1A transcript analysis in CNE cells was analyzed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR).. Tan II(A) could inhibit CNE cells proliferation in dose- and time-dependent manner. 50% inhibiting concentration of Tan II(A) on CNE cells in 24, 48, 72 h was 45.7, 24.8, 3.3 mg x L(-2), respectively. Typical apoptotic morphology such as chromatin aggregation was observed in CNE cells with Tan II(A) treated for 24 h, and the apoptotic inducing effect was in a dose-dependent manner. After treated with Tan II(A), intracellular Ca2+ concentration of CNE cells was increased, mitochondria membrane potential of the cells was decreased, relative mRNA level of Bad and MT-1A was up-regulated.. Tan II(A) had anticancer effect on CNE cells through apoptosis via calcineurin-dependent pathway and MT-1A downregulation.

Topics: Abietanes; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-Associated Death Protein; Calcium; Carcinoma; Cell Line, Tumor; Cell Proliferation; Drugs, Chinese Herbal; Gene Expression Regulation, Neoplastic; Humans; Membrane Potential, Mitochondrial; Metallothionein; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Signal Transduction

Photoactivation of hypericin is known to generate reactive oxygen species and induce phototoxic effects. However, modulation of the cellular antioxidant defense would influence the extent and severity of the photodynamic effects. We have previously shown that hypericin-mediated photodynamic therapy (PDT) induced a significant reduction of Glutathione S-transferase activity. In this study, we investigated the phototoxic effects of hypericin-mediated PDT in nasopharyngeal cancer (NPC) in vitro and analyzed the expression of metallothionein (MT), a family of potential free radical scavengers. HK1 NPC cells were subjected to PDT treatment in vitro, and the effects on cell death were analyzed by flow cytometry (using propidium iodide and Annexin V staining) and transmission electron microscopy. The expression profile of MT-1E and MT-2A isoforms (the only functional MT isoforms expressed in HK1 NPC cells) were determined by quantitative real-time RT-PCR. The results showed that hypericin PDT induced necrotic cell death as evidenced by the absence of a subdiploid peak and decreased Annexin-V fluorescence. Ultrastructural examination verified the presence of cell necrosis. There was a significant up-regulation of MT-1E and MT-2A isoforms six hours following PDT, with an approximately 50-fold rise in the expression level of MT-1E and a 15-fold increase of MT-2A. Hence, despite the up-regulation of MT, cells still succumbed to PDT-induced necrosis. It appears that the oxidative stress induced by PDT overwhelmed the antioxidant defense mechanism such as the alteration of MT levels in tumor cells.

Topics: Anthracenes; Flow Cytometry; Humans; In Vitro Techniques; Metallothionein; Nasopharyngeal Neoplasms; Necrosis; Perylene; Photochemotherapy; Photosensitizing Agents; Protein Isoforms; Reverse Transcriptase Polymerase Chain Reaction; Tumor Cells, Cultured; Up-Regulation

Biological functions of metallothionein (MT) proteins which are encoded by 10 functional MT isoforms, include cell proliferation, differentiation and apoptosis. The aim of this study was to compare the relative expression levels of functional MT mRNA isoforms in three nasopharyngeal cancer (NPC) cell lines with laryngeal carcinoma and embryonic lung cell lines by quantitative real-time RT-PCR. All the NPC lines exhibited expression of the MT-2A transcript, whereas the MT-1E isoform was expressed in well differentiated HK1 and moderately differentiated TW01 but not in poorly differentiated CNE2 cells. Interestingly, TW01 and HEp-2 laryngeal cancer cells exhibited similar expression profiles with both MT-1E and MT-2A isoforms being detected at levels below those of MRC-5 embryonic lung fibroblasts. Functional studies of the MT-2A isoform by down-regulating expression of this gene with MT-2A antisense oligonucleotide in CNE2 cells, showed a reduction in cell viability and proliferation. These findings may provide valuable information in the search for novel therapeutic strategies against NPC.

Topics: Antineoplastic Agents; Cell Proliferation; Cell Survival; Down-Regulation; Growth Inhibitors; Humans; Metallothionein; Nasopharyngeal Neoplasms; Oligodeoxyribonucleotides, Antisense; Protein Isoforms; RNA, Messenger; Tumor Cells, Cultured

The superoxide anion and the hydroxyl radical (OH.) play an important role in the radiotherapy of tumors. The superoxide dismutase (SOD) and the metallothionien (MT) are the main enzymes to clear the superoxide anion and OH. Up to now, there are few reports on the relationship between the nasopharyngeal carcinoma (NPC) and SOD or MT. This study was conducted to observe the dynamic changes of the activity of total superoxide dismutase (T-SOD) in the serum and the expression of MT in the tissue from the NPC patients treated with radiotherapy.. From December 2000 to January 2002, 46 patients with NPC were selected randomly to test the activity of T-SOD in serum using xanthine oxidase method and the expression of MT in tissue using immunohistochemistry before, during, and after radiotherapy. And 26 persons without cancer were enrolled as normal control.. Thirty-two patients were performed 3 times of examinations of T-SOD activity. The activity of T-SOD before radiotherapy (83.9+/-19.6 U(N)/ml) was lower than normal control (96.8+/-23.6 U(N)/ml) (P< 0.05). The activities of T-SOD in the serum samples before, during, and after the radiotherapy were 77.6+/-19.1, 87.1+/-18.6, and 96.3+/-31.6 in the NPC patients at stage I(P >0.05); 80.1+/-15.0, 78.0+/-35.4, and 110.6+/-72.0 in the NPC patients at stage IV(P >0.05); 79.8+/-18.2, 87.2+/-31.7, and 94.8+/-36.3 in the patients with complete response to radiotherapy (P >0.05); 98.5+/-18.6, 62.9+/-35.3, and 79.2+/-27.3 in the patients with part response to radiotherapy (P >0.05). Among the 32 patients, 27 patients were performed 3 times of tests of MT expression. The expression rate of MT in NPC tissues was 39.8+/-37.8% before radiotherapy, and markedly higher than the normal control 12.1+/-22.4% (P< 0.05). The expression rates of MT before, during, and after radiotherapy were 33.0+/-42.2%, 21.3+/-36.1%, and 5.0+/-10.0% in the tissues from the patients at stage I(P< 0.05); 62.2+/-40.5%, 9.2+/-12.8%, and 9.2+/-15.0% in the patients at stage IV( P< 0.05); 42.5+/-38.6%, 19.8+/-27.5%, and 10.0+/-13.9% in the patients with complete response to radiotherapy (P< 0.05); 32.3+/-37.2%, 1.43+/-2.43%, and 5.4+/-9.1% in the patients with part response to radiotherapy (P< 0.05).. The test of T-SOD in serum may be helpful for the diagnosis of initial NPC. But the dynamic change of the activity of total T-SOD in radiotherapy was not obvious. The expression rates of MT in NPC tissues increased markedly before radiotherapy, and decreased obviously after radiotherapy.

Topics: Adolescent; Adult; Aged; Female; Humans; Male; Metallothionein; Middle Aged; Nasopharyngeal Neoplasms; Superoxide Dismutase

The expression of metallothionein (MT), an intracellular ubiquitous low molecular weight protein thiol with antioxidant properties, was studied in nasopharyngeal cancer (NPC) and correlated with the apoptotic index. Immunohistochemical staining of randomly selected, formalin-fixed and paraffin-embedded normal and malignant nasopharyngeal tissues were analysed for the expression of MT using the commercially available E9 antibody directed against MT I and MT II isoforms. The corresponding apoptosis labelling indices were evaluated by the TUNEL method. Localization of MT at the ultrastructural level was studied by immunogold labelling. All the tumour sections (17 specimens) showed MT-immunopositivity. A direct correlation between the percentage of MT-positive cells and the staining intensity was noted (P < 0.001; Pearson's r = 0.95). There was absence of cytoplasmic staining and only nuclear staining (with localization in the nucleoplasm) was demonstrated in the tumour cells. In normal epithelium of the nasopharynx, the basal layer was stained. An inverse relationship was observed between the level of MT expression and the apoptotic index in the NPC tissues (P = 0.0059; Pearson's r = -0.6380). The results suggest that overexpression of MT in NPC may protect the tumour cells from entering into the apoptotic process and thereby contribute to tumour expansion. Preferential localization of MT in the nuclei of NPC cells may possibly enhance radioresistance since radiotherapy is known to eradicate tumour cells by free radical-induced apoptosis.

Topics: Adult; Aged; Aged, 80 and over; Apoptosis; Carcinoma; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Male; Metallothionein; Middle Aged; Nasopharyngeal Neoplasms; Radiation Tolerance; Regression Analysis

Metallothionein (MT) is a metal-binding protein with functional roles in cell growth, repair and differentiation. MT is reported to be differentially expressed in lymphocytes of malignant gastrointestinal lesions. The level of MT protein was examined by immunohistochemical analysis at light microscopic and ultrastructural level in infiltrating lymphocytes from 20 cases of undifferentiated nasopharyngeal carcinoma (NPC). MT expression was found to be absent in the infiltrating lymphocytes of NPC and in reactive lymphocytes of lymphoid hyperplasia in nasopharyngeal tissues. Ultrastructural examination confirmed the absence of MT immunoreactivity in the lymphoid infiltrate of NPC. On the other hand, malignant lymphoblasts of diffuse large cell lymphoma, showed MT-immunopositivity by immunoelectron microscopy. This study demonstrates a lack of MT expression in the lymphoid stroma of undifferentiated NPC, a further characteristic of its non-neoplastic nature.

Topics: Adult; Aged; Carcinoma; Female; Humans; Immunohistochemistry; Lymphocytes, Tumor-Infiltrating; Lymphoma, B-Cell; Male; Metallothionein; Microscopy, Electron; Microscopy, Immunoelectron; Middle Aged; Nasopharyngeal Neoplasms

Metallothionein (MT) is a cysteine-rich protein with pleiotropic functions and a high binding affinity for heavy metals. The present study was designed to examine the relationship between MT expression and tissue zinc levels in conjunction with cell proliferation in nasopharyngeal cancer (NPC). Proliferative activity in NPC was quantified by Ki67 immunolabelling and MT expression was determined by immunohistochemistry. Total zinc and subcellular zinc fractions were analysed by flame atomic absorption spectrometry. MT immunostaining was observed in the nuclei of NPC cells, with the percentage MT immunopositivity ranging from 3.0 to 59.7%. Thirteen tumours displayed weak MT staining and the remaining 11 showed moderate to strong immunostaining. There was a significant positive correlation between MT and Ki67 positivity (P = 0.0127). Tissue zinc levels were higher in NPC as compared with benign nasopharyngeal tissues (4.800 +/- 0.4610 versus 2.889 +/- 0.4045 microgram/g dry wt tissue, respectively; P = 0.0122). Nuclear zinc levels in NPC were significantly higher than levels in membrane and cytosolic fractions (mean zinc levels 1.4840 +/- 0.1489, 0.6286 +/- 0.0789 and 0.3014 +/- 0.0250 microgram/mg protein, respectively). A linear relationship was also observed between nuclear zinc levels and MT immunostaining (P = 0.0024) as well as with Ki67 immunopositivity (P = 0.0123). Our results show that MT and zinc are correlated with proliferative activity in NPC, providing further insights into the biology of this enigmatic and aggressive tumour.

Topics: Carcinoma; Cell Division; Cell Nucleus; Humans; Immunohistochemistry; Ki-67 Antigen; Metallothionein; Nasopharyngeal Neoplasms; Zinc