Epstein-Barr virus-encoded LMP2A induces an epithelial-mesenchymal transition and increases the number of side population stem-like cancer cells in nasopharyngeal carcinoma.

It has been recently reported that a side population of cells in nasopharyngeal carcinoma (NPC) displayed characteristics of stem-like cancer cells. However, the molecular mechanisms underlying the modulation of such stem-like cell populations in NPC remain unclear. Epstein-Barr virus was the first identified human tumor virus to be associated with various malignancies, most notably NPC. LMP2A, the Epstein-Barr virus encoded latent protein, has been reported to play roles in oncogenic processes. We report by immunostaining in our current study that LMP2A is overexpressed in 57.6% of the nasopharyngeal carcinoma tumors sampled and is mainly localized at the tumor invasive front. We found also in NPC cells that the exogenous expression of LMP2A greatly increases their invasive/migratory ability, induces epithelial-mesenchymal transition (EMT)-like cellular marker alterations, and stimulates stem cell side populations and the expression of stem cell markers. In addition, LMP2A enhances the transforming ability of cancer cells in both colony formation and soft agar assays, as well as the self-renewal ability of stem-like cancer cells in a spherical culture assay. Additionally, LMP2A increases the number of cancer initiating cells in a xenograft tumor formation assay. More importantly, the endogenous expression of LMP2A positively correlates with the expression of ABCG2 in NPC samples. Finally, we demonstrate that Akt inhibitor (V) greatly decreases the size of the stem cell side populations in LMP2A-expressing cells. Taken together, our data indicate that LMP2A induces EMT and stem-like cell self-renewal in NPC, suggesting a novel mechanism by which Epstein-Barr virus induces the initiation, metastasis and recurrence of NPC.

Prognostic relevance of Bmi-1 expression and autoantibodies in esophageal squamous cell carcinoma.

BACKGROUND: Overexpression of Bmi-1 has been observed in a variety of cancers, and it has been suggested to be an independent prognostic marker for the patients. The objective of this study was to determine the level of Bmi-1 expression or its autoantibodies in human esophageal squamous cell carcinoma (ESCC) and to correlate it with clinicopathologic data. METHODS: We first examined Bmi-1 expression in ESCC cell lines and tumor samples by RT-PCR and Western blot analysis. We then analyzed Bmi-1 protein expression in 171 clinicopathologically characterized ESCC cases by immunohistochemistry. In addition, we detected its autoantibodies in sera of patients with ESCC by ELISA. RESULTS: We found that Bmi-1 expression was higher in the immortalized cells, cancer cell lines and most cancer tissue than in non-tumorous control tissue at both mRNA and protein level. In addition, Bmi-1 expression was observed in 64.3% (110 of 171) archive ESCC specimen by immunohistochemistry analysis, and the location of Bmi-1 in ESCC was in the nuclei instead of cytoplasm of tumor cells. There was a significant difference of Bmi-1 expression in patients categorized according to stage (P = 0.003) and pN classification (P = 0.047). Multivariate analysis suggested that Bmi-1 expression was an independent prognostic marker for ESCC patients. A prognostic significance of Bmi-1 was also found in the subgroup of T3~T4 and N1 tumor classification. Bmi-1 autoantibodies were detected in sera of 39.0% (62 of 159) ESCC patients. The correlations between anti-Bmi-1 antibodies and tumor stage (P = 0.040), or lymph node status (P < 0.001) were significant. CONCLUSIONS: Our results suggest that Bmi-1 protein is a valuable marker of ESCC progression. The presence of Bmi-1 autoantibodies in sera from patients with ESCC may have clinical utility in esophageal cancer diagnosis.

Sphingosine kinase 1 is associated with gastric cancer progression and poor survival of patients.

PURPOSE: The present study was to investigate the clinical significance of sphingosine kinase 1 (SPHK1), an oncoenzyme, in the development and progression of gastric cancer. EXPERIMENTAL DESIGN: mRNA and protein levels of SPHK1 expression in normal gastric epithelial cells, gastric cancer cell lines, and paired gastric cancer lesions and the adjacent noncancerous tissues were examined using reverse transcription-PCR and Western blotting. Immunohistochemistry was employed to analyze SPHK1 expression in 175 clinicopathologically characterized gastric cancer cases. Statistical analyses were applied to derive prognostic and diagnostic associations. RESULTS: Levels of SPHK1 mRNA and protein were higher in gastric cancer cell lines than in normal gastric epithelial cells. SPHK1 protein level was up-regulated in gastric cancer lesions compared with that in the paired adjacent noncancerous tissues. Gastric cancer tissues from 115 of 175 (65.7%) patients revealed high level of SPHK1 protein expression in contrast to the undetectable or marginally detectable expression of SPHK1 in the adjacent noncancerous gastric tissues. Significantly different expression levels of SPHK1 were found in patients at different clinical stages (P=0.003), T classification (P=0.035), and M classification (P=0.020). Patients with higher SPHK1 expression had shorter overall survival time, whereas those with lower SPHK1 expression survived longer. Further multivariate analysis suggested that SPHK1 up-regulation was an independent prognostic indicator for the disease. CONCLUSIONS: SPHK1 protein could be a useful marker for the prognosis of gastric cancer. Further study on the potential use of SPHK1 as a therapeutic target is also warranted.

Downregulation of BMI-1 enhances 5-fluorouracil-induced apoptosis in nasopharyngeal carcinoma cells.

5-Fluorouracil (5-FU) is an important chemotherapeutic agent for nasopharyngeal carcinoma (NPC). However, drug resistance may occur after several cycles of 5-FU-based chemotherapy. The oncogene B-cell-specific Moloney murine leukemia virus insertion site 1 (BMI-1) has been shown to be involved in the protection of cancer cells from apoptosis. In this study, 5-FU treatment could increase the percentage of apoptotic NPC cells among BMI-1/RNAi-transfected cells than that among cells transfected with the empty vector. The 50% inhibitory concentration (IC(50)) values of 5-FU were significantly decreased to a greater extent in the cells transfected with BMI-1/RNAi. Most importantly, the expression of phospho-AKT and the anti-apoptotic protein BCL-2 were downregulated in the cells in which BMI-1 expression was inhibited, whereas the apoptosis-inducer BAX was observed to be upregulated. Abrogation of AKT pathway by a PI3K inhibitor could not further increase the sensitivity to 5-FU in the cells with reduced BMI-1 expression. Taken together, BMI-1 depletion enhanced the chemosensitivity of NPC cells by inducing apoptosis; which is associated with inhibition of the PI3K/AKT pathway.

BPI700-Fc gamma1(700) chimeric gene expression and its protective effect in a mice model of the lethal E. coli infection.

BACKGROUND: Infections caused by gram-negative bacteria (GNB) often lead to high mortality in common clinical settings. The effect of traditional antibiotic therapy is hindered by drug-resistant bacteria and unneutralizable endotoxin. Few effective methods can protect high risk patients from bacterial infection. This study explored the protection of adeno-associated virus 2 (AAV2)-bacteriacidal permeability increasing protein 700 (BPI(700))-fragment crystallizable gamma one 700 (Fc gamma1(700)) chimeric gene transferred mice against the minimal lethal dose (MLD) of E. coli and application of gene therapy for bacterial infection. METHODS: After AAV2-BPI(700)-Fc gamma1(700) virus transfection, dot blotting and Western blotting were used to detect the target gene products in Chinese hamster ovary-K1 cells (CHO-K1cells). Reverse transcription-polymerase chain reaction and immunohistochemical assay were carried out to show the target gene expression in mice. Modified BPI-enzyme linked immunosorbent assay was used to identify the target gene products in murine serum. The protection of BPI(700)-Fc gamma1(700) gene transferred mice was examined by survival rate after MLD E. coli challenge. Colony forming unit (CFU) count, limulus amebocyte lysate kit and cytokine kit were used to quantify the bacteria, the level of endotoxin, and proinflammatory cytokine. RESULTS: BPI(1-199)-Fc gamma1 protein was identified in the CHO-K1 cell culture supernatant, injected muscles and serum of the gene transferred mice. After MLD E. coli challenge, the survival rate of AAV2-BPI(700)-Fc gamma1(700) gene transferred mice (36.7%) was significantly higher than that of AAV2-enhanced green fluorescent protein (AAV2-EGFP) gene transferred mice (3.3%) and PBS control mice (5.6%). The survival rate of AAV2-BPI(700)-Fc gamma1(700) gene transferred mice treated with cefuroxime sodium was 65.0%. The bacterium number in main viscera, the levels of endotoxin and proinflammatory cytokine (tumor necrosis factor-alpha and interleukin-1beta) in serum of the AAV2-BPI(700)-Fc gamma1(700) gene transferred mice were markedly lower than that of PBS control mice (P < 0.01). CONCLUSIONS: AAV2-BPI(700)-Fc gamma1(700) gene transferred mice can resist MLD E. coli infection through expressing BPI(1-199)-Fc gamma1 protein. Our findings suggested that AAV2 mediated BPI(700)-Fc gamma1(700) gene delivery could be used for protection and treatment of clinical GNB infection in high-risk individuals.

[Expression of macrophage colony stimulating factor in brains of PDAPPV717I transgenic mice].

OBJECTIVE: To identify the expression and distribution of macrophage colony stimulating factor (M-CSF) in brains of PDAPPV717I transgenic mice. METHODS: We detected the expression and distribution of M-CSF mRNA in brains of PDAPPV717I transgenic mice by using hybridization in situ and immunohistochemical staining. RESULTS: Expression of M-CSF mRNA was significantly higher in brains of PDAPPV717I transgenic mice than that in non-transgenic mice, and M-CSF mRNA in brain was mainly produced by reactive astrocytes. CONCLUSION: The results indicate that astrocytes play an important role in the onset/development of neuropathology of Alzheimer's disease.

[Serum levels of macrophage colony stimulating factor in the patients with Alzheimer's disease].

OBJECTIVE: To investigate the availability of serum level of macrophage clony stimulating factor (M-CSF) as a marker for early diagnosis of Alzheimer's disease (AD). METHODS: The serum levels of M-CSF in 70 patients with AD, 52 healthy controls, 22 patients with VAD (vascular dementia) were measured and the serum levels of IL-1 beta, IL-6, TNF-alpha in 32 patients with AD and 20 controls were measured as well. RESULTS: Serum levels of M-CSF were significantly elevated in patients with AD when compared with healthy controls (P < 0.01) and VAD controls (P < 0.05) respectively. At the early stage of mild dementia and middle dementia, serum levels of M-CSF were significantly elevated, but at the later stage of severe dementia, they returned to normal level. Serum levels of IL-1 beta were significantly elevated in AD patients compared with controls (P < 0.05), and serum levels of TNF-alpha and IL-6 were within the normal range in patients with AD. CONCLUSIONS: The results suggest that serum M-CSF level may provide a convenient and sensitive means for the early diagnosis of Alzheimer's disease.

[SPR detection of affinity of antibacterial peptides in bactericidal/permeability-increasing protein domain for endotoxin].

AIM: To study the affinity of endotoxin for three antibacterial peptides derived from N-terminal domain of bactericidal/permeability-increasing protein(BPI). METHODS: To design and synthesize three peptides from N-terminal domain of BPI, BPI22-36, BPI85-99 and BPI147-161.Surface plasmon resonance(SPR) was used to monitor the interaction between LPS/lipid A and the peptides and polymyxin B(PMB) immobilized on CM5 sensor chip. RESULTS: In three peptides, BPI147-161 was proved to have a best binding capacity with LPS/lipid A, followed by BPI85-99, but BPI22-36 could not interact with LPS/lipid A.The affinity constant K(A) of BPI147-161 with lipid A was 1.12 x 106 L/mol. In contrast, the K(A) of PMB was 5.58 x 106 L/mol. CONCLUSION: The results suggest that the peptide BPI147-161 from BPI effectively neutralize endotoxin and probably provide a novel treatment method for septic shock.

Pre-malignant nasopharyngeal epithelial cell models.

Experimental models that allow investigation of nasopharyngeal carcinoma(NPC) progression could provide valuable insights of the molecular mechanism of nasopharyngeal carcinogenesis as well as potential clinical intervention. Because Epstein-Barr virus only infects humans and a few species of monkeys, representative NPC animal models have not been available so far. Attempts to provide cell models for early nasopharyngeal carcinogenesis have involved in the studies of in vitro transformation of normal finite lifespan human nasopharyngeal epithelial cells (NPEC) to immortality. The first two immortalized NPECs were established by introduction of ectopic SV40T or HPV E6/E7. In order to avoid the unrelated molecular alterations caused by the viral oncogenes, we established and characterized two immortalized NPECs by introduction of Bmi-1, an oncogene which has been demonstrated to be overexpressed in NPC cells and specimens. In addition, human telomerase reverse transcriptase (hTERT) immortalized NPECs have been established in both Tsao's and our laboratory. Unlike the immortalized cells induced by viral oncogenes, these immortal NPECs maintain a normal p53 checkpoint, and are unlikely to have other undefined genetic lesions except presenting some molecular alterations which have been observed in NPC. Thus, the establishment of the immortalized NPECs can be used to further study the mechanism of NPC development using defined genetic elements, particularly in elucidating the role of EBV infection in NPC development.

Centromere protein H is a novel prognostic marker for human nonsmall cell lung cancer progression and overall patient survival.

BACKGROUND: Lung cancer is 1 of the leading causes of cancer death worldwide, and the high mortality from this disease is caused mainly by the lack of efficient diagnostic strategies for early-stage lung cancer. The objective of the current study was to investigate the expression pattern and clinicopathologic significance of centromere protein H (CENP-H) in patients with nonsmall cell lung cancer (NSCLC). METHODS: The expression profile of CENP-H in normal lung epithelial cells, NSCLC cell lines, NSCLC tissues, and adjacent noncancerous lung tissues were detected by reverse transcription-polymerase chain reaction (RT-PCR), real-time RT-PCR, and Western blot analysis. The expression level of CENP-H in 223 NSCLC tissues was measured by immunohistochemistry staining. Statistical analysis was performed to evaluate the clinicopathologic significance of CENP-H. RESULTS: The expression level of CENP-H was much higher in cancer cell lines and lung cancer tissues than that in normal cells and adjacent noncancerous lung tissues, respectively. Immunohistochemical analysis revealed positive CENP-H expression in 118 of 223 NSCLC tissues (52.9%). Statistical analysis revealed that CENP-H expression was correlated strongly with clinical stage (P=.018), tumor classification (P=.03), and Ki-67 expression (P < .001). Patients with lower CENP-H expression had better overall survival than patients with higher CENP-H expression. Further analysis suggested that CENP-H could predict prognosis only in patients with early-stage disease. Multivariate analysis suggested that CENP-H expression was an independent prognostic marker for survival in patients with NSCLC. CONCLUSIONS: The current results demonstrated that high CENP-H protein expression was related to poor outcome in patients with NSCLC. CENP-H may be used as a prognostic biomarker for patients lung patients, especially those with early-stage NSCLC.

The polycomb group protein Bmi-1 represses the tumor suppressor PTEN and induces epithelial-mesenchymal transition in human nasopharyngeal epithelial cells.

The polycomb group protein B lymphoma Mo-MLV insertion region 1 homolog (Bmi-1) is dysregulated in various cancers, and its upregulation strongly correlates with an invasive phenotype and poor prognosis in patients with nasopharyngeal carcinomas. However, the underlying mechanism of Bmi-1-mediated invasiveness remains unknown. In the current study, we found that upregulation of Bmi-1 induced epithelial-mesenchymal transition (EMT) and enhanced the motility and invasiveness of human nasopharyngeal epithelial cells, whereas silencing endogenous Bmi-1 expression reversed EMT and reduced motility. Furthermore, upregulation of Bmi-1 led to the stabilization of Snail, a transcriptional repressor associated with EMT, via modulation of PI3K/Akt/GSK-3beta signaling. Chromatin immunoprecipitation assays revealed that Bmi-1 transcriptionally downregulated expression of the tumor suppressor PTEN in tumor cells through direct association with the PTEN locus. This in vitro analysis was consistent with the statistical inverse correlation detected between Bmi-1 and PTEN expression in a cohort of human nasopharyngeal carcinoma biopsies. Moreover, ablation of PTEN expression partially rescued the migratory/invasive phenotype of Bmi-1-silenced cells, indicating that PTEN might be a major mediator of Bmi-1-induced EMT. Our results provide functional and mechanistic links between the oncoprotein Bmi-1 and the tumor suppressor PTEN in the development and progression of cancer.