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Find video protocols related to scientific articles indexed in Pubmed.
Role of ATM in the Formation of Replication Compartment During Lytic Replication of EBV in Nasopharyngeal Epithelial Cells.
J. Virol.
PUBLISHED: 10-31-2014
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Epstein-Barr virus (EBV), a type of oncogenic herpesvirus, is associated with human malignancies. Previous studies have shown that lytic reactivation of EBV in latently-infected cells induces ATM-dependent DNA damage response (DDR). The involvement of ATM activation has been implicated in inducing viral lytic genes transcription to promote lytic reactivation. Its contribution to the formation of replication compartment during lytic reactivation of EBV remains poorly defined. In this study, the role of ATM in viral DNA replication was investigated in EBV-infected nasopharyngeal epithelial cells. We observed that induction of lytic infection of EBV triggers ATM activation and localization of DDR proteins at the viral replication compartments. Suppression of ATM activity using siRNA approach or specific chemical inhibitor profoundly suppressed replication of EBV DNA and production of infectious virion in EBV-infected cells induced to undergo lytic reactivation. We further showed that phosphorylation of Sp1 at Serine-101 residue is essential in promoting the accretion of EBV replication proteins at the replication compartment which is crucial for replication of viral DNA. Knockdown of Sp1 expression by siRNA effectively suppressed replication of viral DNA and localization of EBV replication proteins to the replication compartments. Our study supports an important role of ATM activation in lytic reactivation of EBV in epithelial cells and phosphorylation of Sp1 is an essential process downstream of ATM activation involved in the formation of viral replication compartments. Our study revealed an essential role of ATM-dependent DDR pathway in lytic reactivation of EBV suggesting a potential anti-viral replication strategy using specific DDR inhibitors.
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Suppression of esophageal tumor growth and chemoresistance by directly targeting the PI3K/AKT pathway.
Oncotarget
PUBLISHED: 10-27-2014
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Esophageal cancer is the sixth most common cause of cancer-related deaths worldwide. Novel therapeutic intervention is urgently needed for this deadly disease. The functional role of PI3K/AKT pathway in esophageal cancer is little known. In this study, our results from 49 pairs of human esophageal tumor and normal specimens demonstrated that AKT was constitutively active in the majority (75.5%) of esophageal tumors compared with corresponding normal tissues. Inhibition of the PI3K/AKT pathway with specific inhibitors, wortmannin and LY294002, significantly reduced Bcl-xL expression, induced caspase-3-dependent apoptosis, and repressed cell proliferation and tumor growth in vitro and in vivo without obvious toxic effects. Moreover, significantly higher expression level of p-AKT was observed in fluorouracil (5-FU)-resistant esophageal cancer cells. Inactivation of PI3K/AKT pathway markedly increased the sensitivity and even reversed acquired resistance of esophageal cancer cells to chemotherapeutic drugs in vitro. More importantly, the resistance of tumor xenografts derived from esophageal cancer cells with acquired 5-FU resistance to chemotherapeutic drugs was significantly abrogated by wortmannin treatment in animals. In summary, our data support PI3K/AKT as a valid therapeutic target and strongly suggest that PI3K/AKT inhibitors used in conjunction with conventional chemotherapy may be a potentially useful therapeutic strategy in treating esophageal cancer patients.
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Epstein-Barr virus infection and persistence in nasopharyngeal epithelial cells.
Chin J Cancer
PUBLISHED: 09-16-2014
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Epstein-Barr virus (EBV) infection is closely associated with undifferentiated nasopharyngeal carcinoma (NPC), strongly implicating a role for EBV in NPC pathogenesis; conversely, EBV infection is rarely detected in normal nasopharyngeal epithelial tissues. In general, EBV does not show a strong tropism for infecting human epithelial cells, and EBV infection in oropharyngeal epithelial cells is believed to be lytic in nature. To establish life-long infection in humans, EBV has evolved efficient strategies to infect B cells and hijack their cellular machinery for latent infection. Lytic EBV infection in oropharyngeal epithelial cells, though an infrequent event, is believed to be a major source of infectious EBV particles for salivary transmission. The biological events associated with nasopharyngeal epithelial cells are only beginning to be understood with the advancement of EBV infection methods and the availability of nasopharyngeal epithelial cell models for EBV infection studies. EBV infection in human epithelial cells is a highly inefficient process compared to that in B cells, which express the complement receptor type 2 (CR2) to mediate EBV infection. Although receptor(s) on the epithelial cell surface for EBV infection remain(s) to be identified, EBV infection in epithelial cells could be achieved via the interaction of glycoproteins on the viral envelope with surface integrins on epithelial cells, which might trigger membrane fusion to internalize EBV in cells. Normal nasopharyngeal epithelial cells are not permissive for latent EBV infection, and EBV infection in normal nasopharyngeal epithelial cells usually results in growth arrest. However, genetic alterations in premalignant nasopharyngeal epithelial cells, including p16 deletion and cyclin D1 overexpression, could override the growth inhibitory effect of EBV infection to support stable and latent EBV infection in nasopharyngeal epithelial cells. The EBV episome in NPC is clonal in nature, suggesting that NPC develops from a single EBV-infected nasopharyngeal epithelial cell, and the establishment of persistent and latent EBV infection in premalignant nasopharyngeal epithelium may represent an early and critical event for NPC development.
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p70 S6 kinase drives ovarian cancer metastasis through multicellular spheroid-peritoneum interaction and P-cadherin/b1 integrin signaling activation.
Oncotarget
PUBLISHED: 09-07-2014
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Peritoneal dissemination as a manifestation of ovarian cancer is an adverse prognostic factor associated with poor clinical outcome, and is thus a potentially promising target for improved treatment. Sphere forming cells (multicellular spheroids) present in malignant ascites of patients with ovarian cancer represent a major impediment to effective treatment. p70 S6 kinase (p70(S6K)), which is a downstream effector of mammalian target of rapamycin, is frequently hyperactivated in human ovarian cancer. Here, we identified p70(S6K) as an important regulator for the seeding and successful colonization of ovarian cancer spheroids on the peritoneum. Furthermore, we provided evidence for the existence of a novel crosstalk between P-cadherin and ?1 integrin, which was crucial for the high degree of specificity in cell adhesion. In particular, we demonstrated that the upregulation of mature ?1 integrin occurred as a consequence of P-cadherin expression through the induction of the Golgi glycosyltransferase, ST6Gal-I, which mediated ?1 integrin hypersialylation. Loss of p70(S6K) or targeting the P-cadherin/?1-integrin interplay could significantly attenuate the metastatic spread onto the peritoneum in vivo. These findings establish a new role for p70(S6K) in tumor spheroid-mesothelium communication in ovarian cancer and provide a preclinical rationale for targeting p70(S6K) as a new avenue for microenvironment-based therapeutic strategy.
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Effect of a qigong intervention program on telomerase activity and psychological stress in abused Chinese women: a randomized, wait-list controlled trial.
BMC Complement Altern Med
PUBLISHED: 08-15-2014
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Abused women, who suffer from chronic psychological stress, have been shown to have shorter telomeres than never abused women. Telomere shortening is associated with increased risk of cell death, and it is believed that adopting health-promoting behaviors can help to increase the activity of telomerase, an enzyme that counters telomere shortening. Qigong is an ancient Chinese mind-body integration, health-oriented practice designed to enhance the function of qi, an energy that sustains well-being. Therefore, an assessor-blind, randomized, wait-list controlled trial was developed to evaluate the effect of a qigong intervention on telomerase activity (primary objective) and proinflammatory cytokines, perceived stress, perceived coping, and depressive symptoms (secondary objectives) in abused Chinese women.
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The role of Epstein-Barr virus in epithelial malignancies.
J. Pathol.
PUBLISHED: 08-07-2014
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The close association of Epstein-Barr virus (EBV) infection with non-keratinizing nasopharyngeal carcinoma and a subset of gastric carcinomas suggests that EBV infection is a crucial event in these cancers. The difficulties encountered in infecting and transforming primary epithelial cells in experimental systems suggest that the role of EBV in epithelial malignancies is complex and multi-factorial in nature. Genetic alterations in the premalignant epithelium may support the establishment of latent EBV infection, which is believed to be an initiation event. Oncogenic properties have been reported in multiple EBV latent genes. The BamH1 A rightwards transcripts (BARTs) and the BART encoded microRNAs (miR-BARTs) are highly expressed in EBV-associated epithelial malignancies and may induce malignant transformation. However, enhanced proliferation may not be the crucial function of EBV infection in epithelial malignancies, at least in the early stages of cancer development. EBV-encoded gene products may confer anti-apoptotic properties and promote the survival of infected premalignant epithelial cells harbouring genetic alterations. Multiple EBV-encoded microRNAs have been reported to have immune evasion functions. Genetic alterations in host cells as well as inflammatory stroma could modulate expression of EBV gene expression and alter the growth properties of infected premalignant epithelial cells encouraging their selection during carcinogenesis.
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p21/Cyclin E pathway modulates anticlastogenic function of Bmi-1 in cancer cells.
Int. J. Cancer
PUBLISHED: 08-01-2014
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Apart from regulating stem cell self-renewal, embryonic development and proliferation, Bmi-1 has been recently reported to be critical in the maintenance of genome integrity. In searching for novel mechanisms underlying the anticlastogenic function of Bmi-1, we observed, for the first time, that Bmi-1 positively regulates p21 expression. We extended the finding that Bmi-1 deficiency induced chromosome breaks in multiple cancer cell models. Interestingly, we further demonstrated that knockdown of cyclin E or ectopic overexpression of p21 rescued Bmi-1 deficiency-induced chromosome breaks. We therefore conclude that p21/cyclin E pathway is crucial in modulating the anticlastogenic function of Bmi-1. As it is well established that the overexpression of cyclin E potently induces genome instability and p21 suppresses the function of cyclin E, the novel and important implication from our findings is that Bmi-1 plays an important role in limiting genomic instability in cylin E-overexpressing cancer cells by positive regulation of p21.
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Circulating Epstein-Barr virus microRNAs miR-BART7 and miR-BART13 as biomarkers for nasopharyngeal carcinoma diagnosis and treatment.
Int. J. Cancer
PUBLISHED: 07-09-2014
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More than 75% of nasopharyngeal carcinoma (NPC) patients have already developed local or regional spread at diagnosis, which hampers effective treatment and results in a poor prognosis. It is essential to characterize more sensitive and specific biomarkers for screening of high risk individuals and assessment of NPC treatment effectiveness. NPC is an Epstein-Barr virus (EBV) associated tumor in which only a few viral proteins but more than 20 BamHI A rightward transcripts (BART) microRNAs are detected, at abundant levels. We hypothesized that these BART microRNAs may be novel biomarkers for NPC. Systematic analysis of EBV BART microRNA expression profiles in EBV latently infected Mutu I and Mutu III cell lines, EBV-harboring NPC and noncancerous NP cells found that miR-BART3, miR-BART7 and miR-BART13 microRNAs are highly expressed and regularly secreted into the extracellular environment of NPC cells. These BART microRNAs were evaluated for used as potential NPC biomarkers. Analysis of plasma specimens obtained from NPC patients (n?=?89), and healthy (n?=?28) and non-NPC tumor patient controls (n?=?18) found levels of both miR-BART7 and miR-BART13, but not miR-BART3, to be distinctly presence among NPC patients, with elevated levels being particularly apparent among patients with advanced disease. Receiver operating characteristic curve analysis combining miR-BART7 and miR-BART13 levels produces a 90% predictive value for the presence of NPC. Analysis of 41 NPC patients before and after radiotherapy showed that miR-BART7 and miR-BART13, but not miR-BART3, were diminished after treatment. These results indicate that EBV microRNAs, miR-BART7 and miR-BART13, may constitute useful new serological biomarkers for diagnosis of NPC and prediction of treatment efficacy.
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F-box only protein 31 (FBXO31) negatively regulates p38 mitogen-activated protein kinase (MAPK) signaling by mediating lysine 48-linked ubiquitination and degradation of mitogen-activated protein kinase kinase 6 (MKK6).
J. Biol. Chem.
PUBLISHED: 06-16-2014
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The p38 MAPK signal transduction pathway plays an important role in inflammatory and stress responses. MAPKK6 (MKK6), a dual specificity protein kinase, is a p38 activator. Activation of the MKK6-p38 pathway is kept in check by multiple layers of regulations, including autoinhibition, dimerization, scaffold proteins, and Lys-63-linked polyubiquitination. However, the mechanisms underlying deactivation of MKK6-p38, which is crucial for maintaining the magnitude and duration of signal transduction, are not well understood. Lys-48-linked ubiquitination, which marks substrates for proteasomal degradation, is an important negative posttranslational regulatory machinery for signal pathway transduction. Here we report that the accumulation of F-box only protein 31 (FBXO31), a component of Skp1 · Cul1 · F-box protein E3 ligase, negatively regulated p38 activation in cancer cells upon genotoxic stresses. Our results show that FBXO31 binds to MKK6 and mediates its Lys-48-linked polyubiquitination and degradation, thereby functioning as a negative regulator of MKK6-p38 signaling and protecting cells from stress-induced cell apoptosis. Taken together, our findings uncover a new mechanism of deactivation of MKK6-p38 and substantiate a novel regulatory role of FBXO31 in stress response.
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miR-31 is consistently inactivated in EBV-associated nasopharyngeal carcinoma and contributes to its tumorigenesis.
Mol. Cancer
PUBLISHED: 05-24-2014
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As a distinctive type of head and neck cancers, nasopharyngeal carcinoma (NPC) is genesis from the clonal Epstein-Barr virus (EBV)-infected nasopharyngeal epithelial cells accumulated with multiple genetic lesions. Among the recurrent genetic alterations defined, loss of 9p21.3 is the most frequent early event in the tumorigenesis of EBV-associated NPC. In addition to the reported CDKN2A/p16, herein, we elucidated the role of a miRNA, miR-31 within this 9p21.3 region as NPC-associated tumor suppressor.
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EBNA1-specific luminescent small molecules for the imaging and inhibition of latent EBV-infected tumor cells.
Chem. Commun. (Camb.)
PUBLISHED: 05-13-2014
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An EBNA1-specific small molecule (JLP2) has been synthesised. As a strong binder and dimerization inhibitor of EBNA1 in vitro, JLP2 may be used as a selective luminescent agent for the imaging and inhibition of latent EBV-infected cancer cells.
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Down-regulation of LPA receptor 5 contributes to aberrant LPA signalling in EBV-associated nasopharyngeal carcinoma.
J. Pathol.
PUBLISHED: 03-25-2014
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Undifferentiated nasopharyngeal carcinoma (NPC) is a highly metastatic disease that is consistently associated with the Epstein-Barr virus (EBV) infection. In this study, we have investigated the contribution of lysophosphatidic acid (LPA) signalling to the pathogenesis of NPC. Here we demonstrate two distinct functional roles for LPA in NPC. First, we show that LPA enhances the migration of NPC cells and secondly that it can inhibit the activity of EBV-specific cytotoxic T cells. Focussing on the first of these phenotypes, we show that one of the LPA receptors, LPA receptor 5 (LPAR5), is down-regulated in primary NPC tissues and that this down-regulation promotes the LPA-induced migration of NPC cell lines. Furthermore, we found that EBV infection or ectopic expression of the EBV-encoded LMP2A gene was sufficient to down-regulate LPAR5 in NPC cell lines. Our data point to a central role for EBV in mediating the oncogenic effects of LPA in NPC and identify LPA signalling as a potential therapeutic target in this disease.
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Id1-induced IGF-II and its autocrine/endocrine promotion of esophageal cancer progression and chemoresistance--implications for IGF-II and IGF-IR-targeted therapy.
Clin. Cancer Res.
PUBLISHED: 03-05-2014
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To investigate the autocrine/endocrine role of Id1-induced insulin-like growth factor-II (IGF-II) in esophageal cancer, and evaluate the potential of IGF-II- and IGF-type I receptor (IGF-IR)-targeted therapies.
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Targeted Activation of Human V?9V?2-T Cells Controls Epstein-Barr Virus-Induced B Cell Lymphoproliferative Disease.
Cancer Cell
PUBLISHED: 02-14-2014
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Epstein-Barr virus-induced lymphoproliferative disease (EBV-LPD) after transplantation remains a serious and life-threatening complication. Herein we showed that the aminobisphosphonate pamidronate-expanded human V?9V?2-T cells efficiently killed EBV-transformed autologous lymphoblastoid B cell lines (EBV-LCL) through ?/?-TCR and NKG2D receptor triggering and Fas and TRAIL engagement. By inoculation of EBV-LCL in Rag2(-/-)?c(-/-) mice and humanized mice, we established lethal EBV-LPD with characteristics close to those of the human disease. Adoptive transfer of pamidronate-expanded V?9V?2-T cells alone effectively prevented EBV-LPD in Rag2(-/-)?c(-/-) mice and induced EBV-LPD regression in EBV(+) tumor-bearing Rag2(-/-)?c(-/-) mice. Pamidronate treatment inhibited EBV-LPD development in humanized mice through selective activation and expansion of V?9V?2-T cells. This study provides proof-of-principle for a therapeutic approach using pamidronate to control EBV-LPD through V?9V?2-T cell targeting.
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miR-135a leads to cervical cancer cell transformation through regulation of ?-catenin via a SIAH1-dependent ubiquitin proteosomal pathway.
Carcinogenesis
PUBLISHED: 02-06-2014
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Human papillomaviruses (HPVs) is the principal etiological agent of cervical cancer (CC). However, exposure to the high-risk type HPV alone is insufficient for tumor formation, and additional factors are required for the HPV-infected cells to become tumorigenic. Dysregulated microRNAs (miRNAs) expression is frequently observed in cancer but their roles in the formation of CC have not been fully revealed. In this study, we compared the expression of miR-135a in laser capture microdissected cervical specimens and confirmed overexpression of the miRNA in malignant cervical squamous cell carcinoma compared with precancerous lesions. Transient force-expression of miR-135a induced growth in low-density culture, anchorage-independent growth, proliferation and invasion of a HPV-16 E6/E7-immortalized cervical epithelial cell line, NC104-E6/E7. The observed effects were due to the inhibitory action of miR-135a on its direct target seven in absentia homolog 1 (SIAH1) leading to upregulation of ?-catenin/T cell factor signaling. miR-135a force-expression enhanced the growth of HeLa- and NC104-E6/E7-derived tumor in vivo. The effect of miR-135a could be partially nullified by SIAH1 force-expression. More importantly, the expression of SIAH1 and ?-catenin correlated with that of miR-135a in precancerous and cancerous lesions of cervical biopsies. By comparing the tumorigenic activities of miR-135a in E6/E7 positive/negative cell lines and in NC104-E6/E7 with or without E6/E7 knockdown, we demonstrated that HPV E6/E7 proteins are prerequisite for miR-135a as an oncomiR. Taken together, miR-135a/SIAH1/?-catenin signaling is important in the transformation and progression of cervical carcinoma.
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Etiological factors of nasopharyngeal carcinoma.
Oral Oncol.
PUBLISHED: 01-29-2014
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Nasopharyngeal carcinoma (NPC) is a common disease among southern Chinese. The major etiological factors proposed for NPC pathogenesis include genetic susceptibility, environment factors and EBV infection. In the high risk population, genetic susceptibility to NPC has been mapped to the HLA loci and adjacent genes in MHC region on chromosome 6p21. Consumption of preserved food including salted fish has been implicated in its etiology in earlier studies. Its contribution to pathogenesis of NPC remains to be determined. A decreasing trend of NPC incidence was observed in Hong Kong, Taiwan and Singapore in recent years which may be accounted by a change of dietary habits. A comprehensive epidemiological study will help to elucidate the relative importance of various risk factors in the pathogenesis of NPC. Despite the close association of EBV infection with NPC, the etiological role of EBV in NPC pathogenesis remains enigmatic. EBV infection in primary nasopharyngeal epithelial cells is uncommon and difficult to achieve. EBV does not transform primary nasopharyngeal epithelial cells into proliferative clones, which contrasts greatly with the well-documented ability of EBV to transform and immortalize primary B cells. Genetic alterations identified in premalignant nasopharyngeal epithelium may play crucial roles to support stable EBV infection. Subsequently, latent and lytic EBV gene products may drive clonal expansion and transformation of premalignant nasopharyngeal epithelial cells into cancer cells. Stromal inflammation in nasopharyngeal mucosa is believed to play an important role in modulating the growth and possibly drive the malignant transformation of EBV-infected nasopharyngeal epithelial cells. Furthermore, there are increasing evidences supporting a role of EBV infection to evade host immune surveillance. EBV-infected cells may have selective growth advantages in vivo by acquiring a stress-resistance phenotype. Understanding the etiological factors and pathogenesis of NPC will contribute effectively to the prevention and treatment of this disease.
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Berberine-induced tumor suppressor p53 up-regulation gets involved in the regulatory network of MIR-23a in hepatocellular carcinoma.
Biochim. Biophys. Acta
PUBLISHED: 01-21-2014
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To investigate the involvement of p53 in the regulatory network of microRNA-23a (miR-23a) in berberine-treated hepatocellular carcinoma (HCC) cells.
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Integrated mRNA and microRNA transcriptome sequencing characterizes sequence variants and mRNA-microRNA regulatory network in nasopharyngeal carcinoma model systems.
FEBS Open Bio
PUBLISHED: 01-01-2014
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Nasopharyngeal carcinoma (NPC) is a prevalent malignancy in Southeast Asia among the Chinese population. Aberrant regulation of transcripts has been implicated in many types of cancers including NPC. Herein, we characterized mRNA and miRNA transcriptomes by RNA sequencing (RNASeq) of NPC model systems. Matched total mRNA and small RNA of undifferentiated Epstein-Barr virus (EBV)-positive NPC xenograft X666 and its derived cell line C666, well-differentiated NPC cell line HK1, and the immortalized nasopharyngeal epithelial cell line NP460 were sequenced by Solexa technology. We found 2812 genes and 149 miRNAs (human and EBV) to be differentially expressed in NP460, HK1, C666 and X666 with RNASeq; 533 miRNA-mRNA target pairs were inversely regulated in the three NPC cell lines compared to NP460. Integrated mRNA/miRNA expression profiling and pathway analysis show extracellular matrix organization, Beta-1 integrin cell surface interactions, and the PI3K/AKT, EGFR, ErbB, and Wnt pathways were potentially deregulated in NPC. Real-time quantitative PCR was performed on selected mRNA/miRNAs in order to validate their expression. Transcript sequence variants such as short insertions and deletions (INDEL), single nucleotide variant (SNV), and isomiRs were characterized in the NPC model systems. A novel TP53 transcript variant was identified in NP460, HK1, and C666. Detection of three previously reported novel EBV-encoded BART miRNAs and their isomiRs were also observed. Meta-analysis of a model system to a clinical system aids the choice of different cell lines in NPC studies. This comprehensive characterization of mRNA and miRNA transcriptomes in NPC cell lines and the xenograft provides insights on miRNA regulation of mRNA and valuable resources on transcript variation and regulation in NPC, which are potentially useful for mechanistic and preclinical studies.
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Suppression of Vascular Endothelial Growth Factor via Inactivation of Eukaryotic Elongation Factor 2 by Alkaloids in Coptidis rhizoma in Hepatocellular Carcinoma.
Integr Cancer Ther
PUBLISHED: 12-19-2013
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To investigate the inhibitory effect of COPTIDIS RHIZOM: aqueous extract (CRAE) on vascular endothelial growth factor (VEGF) expression and tumor angiogenesis in hepatocellular carcinoma (HCC).
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Superior Antitumor Activity of a Novel Bispecific Antibody Cotargeting Human Epidermal Growth Factor Receptor 2 and Type I Insulin-like Growth Factor Receptor.
Mol. Cancer Ther.
PUBLISHED: 11-13-2013
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The humanized anti-HER2 monoclonal antibody (mAb) trastuzumab (Herceptin; Genentech) effectively inhibits human epidermal growth factor receptor 2 (HER2)-positive breast tumors. However, many patients responding to treatment often develop resistance. Cross-talk between type I insulin-like growth factor receptor (IGF-IR) and HER2 and elevated IGF-IR signaling have been implicated in tumor cell resistance to trastuzumab therapy. Previously, we reported that the anti-IGF-IR mAb m590 inhibits proliferation and migration of breast cancer MCF-7 cells in vitro. Here, we generated a "knobs-into-holes" bispecific antibody (Bi-Ab) against HER2 and IGF-IR by engineering trastuzumab and m590. We compared the effects of Bi-Ab treatment in vitro and in SKOV-3 HER2- and IGF-IR-overexpressing cancer xenograft mouse model with those of m590 and trastuzumab treatment alone or in combination. Bi-Ab effectively inhibited proliferation of HER2- and IGF-IR-overexpressing ovarian cancer SKOV-3 cells in vitro by ablating receptor phosphorylation and downstream PI3K/Akt and mitogen-activated protein kinase signaling. Bi-Ab more effectively inhibited cancer growth in SKOV-3 HER2- and IGF-IR-overexpressing cancer xenograft mouse model than m590 and trastuzumab alone or in combination. Mice bearing SKOV-3 HER2- and IGF-IR-overexpressing xenografts showed extensive and sustainable tumor regression when treated with Bi-Ab. Our results suggest that Bi-Ab has superior antitumor activity compared with monospecific antibodies, and cotargeting HER2 and IGF-IR may be clinically beneficial in minimizing the acquired resistance to trastuzumab therapy. Mol Cancer Ther; 13(1); 1-11. ©2013 AACR.
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The metalloprotease ADAMTS8 displays anti-tumor properties through antagonizing EGFR-MEK-ERK signaling and is silenced in carcinomas by CpG methylation.
Mol. Cancer Res.
PUBLISHED: 11-01-2013
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Disintegrins and metalloproteinases with thrombospondin motifs (ADAMTSs) have been reported dysregulated in various cancers. Through refining a loss of heterozygosity locus at 11q25 by array-CGH, we identified ADAMTS8 as a novel candidate tumor suppressor gene. Although ADAMTS8 downregulation has been reported in several tumors, its biological function and underlying mechanism remain largely unknown. Here, we found that ADAMTS8 is broadly expressed in normal tissues but frequently downregulated or silenced by promoter methylation in common carcinoma cell lines, including nasopharyngeal, esophageal squamous cell, gastric and colorectal carcinomas. Pharmacological or genetic demethylation restored ADAMTS8 expression, indicating that promoter methylation mediates its silencing. Aberrant methylation of ADAMTS8 was also detected in several types of primary tumors but rarely in normal tissues. Further functional studies showed that restoring ADAMTS8 expression suppressed tumor cell clonogenicity through inducing apoptosis. ADAMTS8 as a secreted protease inhibited EGFR signaling along with decreased levels of phosphorylated MEK and ERK. We further found that ADAMTS8 disrupted actin stress fiber organization and inhibited tumor cell motility. Thus, our data demonstrate that ADAMTS8 metalloprotease acts as a functional tumor suppressor through antagonizing EGFR-MEK-ERK signaling, in addition to its previously reported anti-angiogenesis function, and is frequently methylated in common tumors. Implications: This study uncovers the tumor suppressive funciton of ADAMTS8, one of the ADAMTS8 family memebers,and its frequent methylation in certain tumors could be developed as a potential biomarker.
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MiR-23a-mediated inhibition of topoisomerase 1 expression potentiates cell response to etoposide in human hepatocellular carcinoma.
Mol. Cancer
PUBLISHED: 10-02-2013
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microRNAs have been shown to regulate the chemosensitivity of cancer cells. The aim of this study is to investigate the role and mechanism of mir-23a in enhancing the anti-tumor effect of topoisomerase 2A (TOP2A) poison etoposide in human hepatocellular carcinoma (HCC).
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Perturbation of biogenesis and targeting of Epstein-Barr virus-encoded miR-BART3 microRNA by adenosine-to-inosine editing.
J. Gen. Virol.
PUBLISHED: 09-17-2013
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Epstein-Barr virus (EBV) encodes at least 44 mature microRNAs (miRNAs), some of which are abundantly expressed in nasopharyngeal carcinoma cells. EBV-encoded miR-BART6 miRNA is known to undergo adenosine-to-inosine (A-to-I) RNA editing, which impacts on processing and function. Whether additional EBV miRNAs might be A-to-I edited remains to be determined. In this study, we have reported on A-to-I editing of EBV miR-BART3. The A-to-I editing enzyme was expressed abundantly in EBV-infected epithelial carcinoma cells. pri-miR-BART3 was found to be edited at four sites in these cells and in nasopharyngeal carcinoma samples. Whereas editing of the second site located within the seed region prevented the targeting of DICE1 mRNA, editing of the third site effectively crippled the biogenesis of mature miR-BART3. Thus, A-to-I editing perturbs biogenesis and targeting of miR-BART3 and may contribute to its differential expression and function in EBV-infected epithelial cells.
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PARP1 is overexpressed in nasopharyngeal carcinoma and its inhibition enhances radiotherapy.
Mol. Cancer Ther.
PUBLISHED: 08-26-2013
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Nasopharyngeal carcinoma is a rare but highly invasive cancer. As options of agents for effective combination chemoradiotherapy for advanced nasopharyngeal carcinoma are limited, novel therapeutic approaches are desperately needed. The ubiquitin ligase CHFR is known to target PARP1 for degradation and is epigenetically inactivated in nasopharyngeal carcinoma. We present evidence that PARP1 protein is indeed overexpressed in nasopharyngeal carcinoma cells in comparison with immortalized normal nasopharyngeal epithelial cells. Tissue microarray analysis also indicated that PARP1 protein is significantly elevated in primary nasopharyngeal carcinoma tissues, with strong correlation with all stages of nasopharyngeal carcinoma development. We found that the PARP inhibitor AZD2281 (olaparib) increased DNA damage, cell-cycle arrest, and apoptosis in nasopharyngeal carcinoma cells challenged with ionizing radiation or temozolomide. Isobologram analysis confirmed that the cytotoxicity triggered by AZD2281 and DNA-damaging agents was synergistic. Finally, AZD2281 also enhanced the tumor-inhibitory effects of ionizing radiation in animal xenograft models. These observations implicate that PARP1 overexpression is an early event in nasopharyngeal carcinoma development and provide a molecular basis of using PARP inhibitors to potentiate treatment of nasopharyngeal carcinoma with radio- and chemotherapy.
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Polo-like kinase inhibitor Ro5203280 has potent antitumor activity in nasopharyngeal carcinoma.
Mol. Cancer Ther.
PUBLISHED: 05-17-2013
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Nasopharyngeal carcinoma is a cancer with its highest prevalence among the southern Chinese and is rare elsewhere in the world. The main treatment modalities include chemotherapy and radiotherapy. However, tumor chemoresistance often limits the efficacy of nasopharyngeal carcinoma treatment and reduces survival rates. Thus, identifying new selective chemotherapeutic drugs for nasopharyngeal carcinoma treatment is needed. In this current study, the antitumor efficacy of a polo-like kinase inhibitor, Ro5203280, was investigated. Ro5203280 induces tumor suppression both in vitro and in vivo. An inhibitory effect was observed with the highly proliferating cancer cell lines tested, but not with the nontumorigenic cell line. Real-time cell proliferation and fluorescence-activated cell sorting (FACS) analysis, together with immunohistochemical (IHC), immunofluorescence, and Annexin V staining assays, were used to evaluate the impact of drug treatment on cell cycle and apoptosis. Ro5203280 induces G2-M cell-cycle arrest and apoptosis. Western blotting shows it inhibits PLK1 phosphorylation and downregulates the downstream signaling molecule, Cdc25c, and upregulates two important mitosis regulators, Wee1 and Securin, as well as the DNA damage-related factor Chk2 in vitro and in vivo. In vivo tumorigenicity assays with Ro5203280 intravenous injection showed its potent ability to inhibit tumor growth in mice, with no observable signs of toxicity. These findings suggest the potential usefulness of Ro5203280 as a chemotherapeutic targeting drug for nasopharyngeal carcinoma treatment.
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FEZF2, a novel 3p14 tumor suppressor gene, represses oncogene EZH2 and MDM2 expression and is frequently methylated in nasopharyngeal carcinoma.
Carcinogenesis
PUBLISHED: 05-14-2013
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Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus-associated tumor prevalent in southern China and southeast Asia, with the 3p14-p12 locus reported as a critical tumor suppressor gene (TSG) region during its pathogenesis. We identified a novel 3p14.2 TSG, FEZF2 (FEZ family zinc finger 2), for NPC. FEZF2 is readily expressed in normal tissues including upper respiratory epithelium, testis, brain and ovary tissues, as well as in immortalized nasopharyngeal epithelial cell line NP69, but it is completely silenced in NPC cell lines due to CpG methylation of its promoter, although no homozygous deletion of FEZF2 was detected. 5-Aza-2-deoxycytidine treatment restored FEZF2 expression in NPC cell lines along with its promoter demethylation. FEZF2 was frequently downregulated in NPC tumors, with promoter methylation detected in 75.5% of tumors, but only in 7.1% of normal nasopharyngeal tissues. Restored FEZF2 expression suppressed NPC cell clonogenicity through inducing G2/M cell cycle arrest and apoptosis and also inhibited NPC cell migration and stemness. FEZF2 acted as a histone deacetylase-associated repressor downregulating multiple oncogenes including EZH2 and MDM2, through direct binding to their promoters. Concomitantly, overexpression of EZH2 was frequently detected in NPC tumors. Thus, we have identified FEZF2 as a novel 3p14.2 TSG frequently inactivated by promoter methylation in NPC, which functions as a repressor downregulating multiple oncogene expression.
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Identification of a recurrent transforming UBR5-ZNF423 fusion gene in EBV-associated nasopharyngeal carcinoma.
J. Pathol.
PUBLISHED: 05-08-2013
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Nasopharyngeal carcinoma (NPC) is a distinct type of head and neck cancer which is prevalent in southern China, south-east Asia and northern Africa. The development and stepwise progression of NPC involves accumulation of multiple gross genetic changes during the clonal expansion of Epstein-Barr virus (EBV)-infected nasopharyngeal epithelial cell population. Here, using paired-end whole-transcriptome sequencing, we discovered a number of chimeric fusion transcripts in a panel of EBV-positive tumour lines. Among these transcripts, a novel fusion of ubiquitin protein ligase E3 component n-recognin 5 (UBR5) on 8q22.3 and zinc finger protein 423 (ZNF423) on 16q12.1, identified from the NPC cell line C666-1, was recurrently detected in 12/144 (8.3%) of primary tumours. The fusion gene contains exon 1 of UBR5 and exons 7-9 of ZNF423 and produces a 94 amino acid chimeric protein including the original C-terminal EBF binding domain (ZF29-30) of ZNF423. Notably, the growth of NPC cells with UBR5-ZNF423 rearrangement is dependent on expression of this fusion protein. Knock-down of UBR5-ZNF423 by fusion-specific siRNA significantly inhibited the cell proliferation and colony-forming ability of C666-1 cells. The transforming ability of UBR5-ZNF423 fusion was also confirmed in NIH3T3 fibroblasts. Constitutive expression of UBR5-ZNF423 in NIH3T3 fibroblasts significantly enhanced its anchorage-independent growth in soft agar and induced tumour formation in a nude mouse model. These findings suggest that expression of UBR5-ZNF423 protein might contribute to the transformation of a subset of NPCs, possibly by altering the activity of EBFs (early B cell factors). Identification of the oncogenic UBR5-ZNF423 provides new potential opportunities for therapeutic intervention in NPC.
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A novel Hsp90 inhibitor AT13387 induces senescence in EBV-positive nasopharyngeal carcinoma cells and suppresses tumor formation.
Mol. Cancer
PUBLISHED: 04-16-2013
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Nasopharyngeal carcinoma (NPC) is an epithelial malignancy strongly associated with Epstein-Barr virus (EBV). AT13387 is a novel heat shock protein 90 (Hsp90) inhibitor, which inhibits the chaperone function of Hsp90 and reduces expression of Hsp90-dependent client oncoproteins. This study aimed to evaluate both the in vitro and in vivo antitumor effects of AT13387 in the EBV-positive NPC cell line C666-1.
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WITHDRAWN: Loss of heterozygosity analyses of esophageal squamous cell carcinoma and precursor lesions from a high incidence area in China.
Cancer Lett.
PUBLISHED: 04-10-2013
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This article has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.
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Bortezomib and SAHA synergistically induce ROS-driven caspase-dependent apoptosis of nasopharyngeal carcinoma and block replication of Epstein-Barr virus.
Mol. Cancer Ther.
PUBLISHED: 03-08-2013
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A novel drug combination of a proteasome inhibitor, bortezomib, and a histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), was tested in nasopharyngeal carcinoma (NPC), both in vitro and in vivo. Dose-response of different concentrations of bortezomib and SAHA on inhibition of cell proliferation of NPC was determined. Mechanisms of apoptosis and effects on lytic cycle activation of Epstein-Barr virus (EBV) were investigated. Combination of bortezomib and SAHA (bortezomib/SAHA) synergistically induced killing of a panel of NPC cell lines. Pronounced increase in sub-G1, Annexin V-positive, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cell populations were detected after treatment with bortezomib/SAHA when compared with either drug alone. Concomitantly, markedly augmented proteolytic cleavage of PARP, caspase-3, -7, -8, and -9, reactive oxygen species (ROS) generation, and caspase-8-dependent histone acetylation were observed. ROS scavenger, N-acetyl cysteine, diminished the apoptotic effects of bortezomib/SAHA, whereas caspase inhibitor Z-VAD-FMK significantly suppressed the apoptosis without decreasing the generation of ROS. Bortezomib inhibited SAHAs induction of EBV replication and abrogated production of infectious viral particles in NPC cells. Furthermore, bortezomib/SAHA potently induced apoptosis and suppressed the growth of NPC xenografts in nude mice. In conclusion, the novel drug combination of bortezomib and SAHA is highly synergistic in the killing of NPC cells in vitro and in vivo. The major mechanism of cell death is ROS-driven caspase-dependent apoptosis. Bortezomib antagonizes SAHAs activation of EBV lytic cycle in NPC cells. This study provides a strong basis for clinical testing of the combination drug regimen in patients with NPC.
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Targeting of DICE1 tumor suppressor by Epstein-Barr virus-encoded miR-BART3* microRNA in nasopharyngeal carcinoma.
Int. J. Cancer
PUBLISHED: 02-12-2013
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Latent infection with Epstein-Barr virus (EBV) is associated with several types of malignancies including nasopharyngeal carcinoma (NPC), which is particularly more prevalent in Southern China. EBV expresses at least 44 mature microRNAs (miRNAs) to modulate the activity of viral and cellular RNAs, but the targets of these EBV-encoded miRNAs in NPC are not well understood. In this report, we characterized DICE1 tumor suppressor to be a cellular target of EBV miR-BART3* miRNA. miR-BART3* was abundantly expressed in NPC cells. The target site of miR-BART3* located in the 3-untranslated region of DICE1 transcript was identified and characterized. Enforced expression of miR-BART3* or its precursor pre-miR-BART3 led to down-regulation of endogenous DICE1 expression. Inhibition of endogenous miR-BART3* in NPC cells with anti-miR-BART3* oligonucleotide inhibitor resulted in increased expression of DICE1 protein. On the contrary, expression of miR-BART3* overcame the growth suppressive activity of DICE1 and stimulated cell proliferation. Consistent with its tumor suppressive function, DICE1 was underexpressed in EBV-expressing NPC tumor tissues. Taken together, our findings suggest that EBV encoded miR-BART3* miRNA targets DICE1 tumor suppressor to promote cellular growth and transformation in NPC.
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A CD90(+) tumor-initiating cell population with an aggressive signature and metastatic capacity in esophageal cancer.
Cancer Res.
PUBLISHED: 02-04-2013
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Tumor-initiating cells (TIC), also known as cancer stem cells, are regarded widely as a specific subpopulation of cells needed for cancer initiation and progression. TICs have yet to be identified in esophageal tumors that have an increasing incidence in developed countries. Here, we report a CD90(+) cell population found in esophageal squamous cell carcinoma (ESCC), which is endowed with stem cell-like properties and high tumorigenic and metastatic potential. mRNA profiling of these cells suggested pathways through which they drive tumor growth and metastasis, with deregulation of an Ets-1/MMP signaling pathway and epithelial-mesenchymal transition figuring prominently. These cells possessed higher self-renewal activity and were sufficient for tumor growth, differentiation, metastasis, and chemotherapeutic resistance. CD90(+) TICs were isolated and characterized from ESCC clinical specimens as well as ESCC cell lines. In freshly resected clinical specimens, they represented a rare cell population, the levels of which correlated with strong family histories and lymph node metastasis. Our results prompt further study of this CD90(+) population of esophageal TICs as potential therapeutic targets.
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Role of MIF/CXCL8/CXCR2 signaling in the growth of nasopharyngeal carcinoma tumor spheres.
Cancer Lett.
PUBLISHED: 01-30-2013
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Macrophage migration inhibitory factor (MIF) and CXCL8 (also named IL-8) are strongly expressed in the tissues of nasopharyngeal carcinoma (NPC). However, their role in the growth of NPC has not been fully examined. This study aims to evaluate the functions of MIF and CXCL8 on the growth of NPC tumor spheres. The elevated expression of CXCL8 in tumor over normal tissues was confirmed in 37 pairs of biopsies from NPC patients. In the in vitro study, all the poorly differentiated NPC cell lines, including the EBV-positive C666-1, and the EBV-negative CNE-1, CNE-2, SUNE-1, HNE-1 and HONE-1 cells, were found to express CXCL8 and MIF. Therefore, the EBV-positive C666-1 cell was selected to examine for the role of MIF and CXCL8 in the growth of the NPC tumor spheres. Functional study showed that the growth of C666-1 tumor spheres, under the nutrient poor or growth factor supplemented culture conditions, could be inhibited by the CXCL8 specific peptide inhibitor. The growth of the tumor spheres could also be reduced by the CXCR2 specific inhibitor SB225002 or the PI3K/AKT inhibitor LY294002, indicating that the endogenously produced CXCL8 plays an autocrine role in the growth of the tumor spheres. Further mechanistic studies revealed that the gene expression of CXCL8 could be reduced by the MIF specific small interfering RNA (siRNA) or NF-?B inhibitor parthenolide, and the growth of tumor spheres was also reduced after MIF siRNA transfection. Taken together, the present study highlights the role of MIF/CXCL8/CXCR2 axis in the growth of NPC tumor spheres. Chemotherapeutic interference of this signaling pathway may help to control the growth of the NPC tumor.
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Efficient immortalization of primary nasopharyngeal epithelial cells for EBV infection study.
PLoS ONE
PUBLISHED: 01-01-2013
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Nasopharyngeal carcinoma (NPC) is common among southern Chinese including the ethnic Cantonese population living in Hong Kong. Epstein-Barr virus (EBV) infection is detected in all undifferentiated type of NPC in this endemic region. Establishment of stable and latent EBV infection in premalignant nasopharyngeal epithelial cells is an early event in NPC development and may contribute to its pathogenesis. Immortalized primary nasopharyngeal epithelial cells represent an important tool for investigation of EBV infection and its tumorigenic potential in this special type of epithelial cells. However, the limited availability and small sizes of nasopharyngeal biopsies have seriously restricted the establishment of primary nasopharyngeal epithelial cells for immortalization. A reliable and effective method to immortalize primary nasopharyngeal epithelial cells will provide unrestricted materials for EBV infection studies. An earlier study has reported that Bmi-1 expression could immortalize primary nasopharyngeal epithelial cells. However, its efficiency and actions in immortalization have not been fully characterized. Our studies showed that Bmi-1 expression alone has limited ability to immortalize primary nasopharyngeal epithelial cells and additional events are often required for its immortalization action. We have identified some of the key events associated with the immortalization of primary nasopharyngeal epithelial cells. Efficient immortalization of nasopharyngeal epithelial cells could be reproducibly and efficiently achieved by the combined actions of Bmi-1 expression, activation of telomerase and silencing of p16 gene. Activation of MAPK signaling and gene expression downstream of Bmi-1 were detected in the immortalized nasopharyngeal epithelial cells and may play a role in immortalization. Furthermore, these newly immortalized nasopharyngeal epithelial cells are susceptible to EBV infection and supported a type II latent EBV infection program characteristic of EBV-infected nasopharyngeal carcinoma. The establishment of an efficient method to immortalize primary nasopharyngeal epithelial cells will facilitate the investigation into the role of EBV infection in pathogenesis of nasopharyngeal carcinoma.
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Epigenetic regulation of pluripotent genes mediates stem cell features in human hepatocellular carcinoma and cancer cell lines.
PLoS ONE
PUBLISHED: 01-01-2013
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Activation of the stem cell transcriptional circuitry is an important event in cancer development. Although cancer cells demonstrate a stem cell-like gene expression signature, the epigenetic regulation of pluripotency-associated genes in cancers remains poorly understood. In this study, we characterized the epigenetic regulation of the pluripotency-associated genes NANOG, OCT4, c-MYC, KLF4, and SOX2 in a variety of cancer cell lines and in primary tumor samples, and investigated the re-activation of pluripotency regulatory circuits in cancer progression. Differential patterns of DNA methylation, histone modifications, and gene expression of pluripotent genes were demonstrated in different types of cancers, which may reflect their tissue origins. NANOG promoter hypomethylation and gene upregulation were found in metastatic human liver cancer cells and human hepatocellular carcinoma (HCC) primary tumor tissues. The upregulation of NANOG, together with p53 depletion, was significantly associated with clinical late stage of HCC. A pro-metastatic role of NANOG in colon cancer cells was also demonstrated, using a NANOG-overexpressing orthotopic tumor implantation mouse model. Demethylation of NANOG promoter was observed in CD133+(high) cancer cells. In accordance, overexpression of NANOG resulted in an increase in the population of CD133+(high) cells. In addition, we demonstrated a cross-regulation between OCT4 and NANOG in cancer cells via reprogramming of promoter methylation. Taken together, epigenetic reprogramming of NANOG can lead to the acquisition of stem cell-like properties. These results underscore the restoration of pluripotency circuits in cancer cells as a potential mechanism for cancer progression.
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Enhanced IL-6/IL-6R signaling promotes growth and malignant properties in EBV-infected premalignant and cancerous nasopharyngeal epithelial cells.
PLoS ONE
PUBLISHED: 01-01-2013
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Nasopharyngeal carcinoma (NPC) is etiologically associated with Epstein-Barr virus (EBV) infection. However, the exact role of EBV in NPC pathogenesis remains elusive. Activation of signal transducer and activator of transcription 3 (STAT3) is common in human cancers including NPC and plays an important role in the pathogenesis and progression of human cancers. Interleukin-6 (IL-6), a major inflammatory cytokine, is a potent activator of STAT3. In this study, we report that EBV-infected immortalized nasopharyngeal epithelial (NPE) cells often acquire an enhanced response to IL-6-induced STAT3 activation to promote their growth and invasive properties. Interestingly, this enhanced IL-6/STAT3 response was mediated by overexpression of IL-6 receptor (IL-6R). Furthermore, IL-6R overexpression enhanced IL-6-induced STAT3 activation in uninfected immortalized NPE cells in vitro, and promoted growth and tumorigenicity of EBV-positive NPC cell line (C666-1) in vivo. Moreover, it is shown for the first time that IL-6R was overexpressed in clinical specimens of NPC. IL-6 expression could also be strongly detected in the stromal cells of NPC and a higher circulating level of IL-6 was found in the sera of advance-staged NPC patients compared to the control subjects. Therefore, IL-6R overexpression, coupled with enhanced IL-6/STAT3 signaling may facilitate the malignant transformation of EBV-infected premalignant NPE cells into cancer cells, and enhance malignant properties of NPC cells.
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Lipopolysaccharide and hypoxia-induced HIF-1 activation in human gingival fibroblasts.
J. Periodontol.
PUBLISHED: 11-16-2011
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We previously reported that chronic periodontal inflammation causes the accumulation of the transcriptional activator hypoxia-inducible factor-1? (HIF-1?) in human gingival fibroblasts (HGFs) in vivo. Here, evidence is provided that bacterial lipopolysaccharides (LPS) and cellular hypoxia, both associated with periodontitis, can individually, or in combination, lead to the accumulation and activation of HIF-1 in HGF in vitro.
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Prognostic significance of phosphorylated RON in esophageal squamous cell carcinoma.
Med. Oncol.
PUBLISHED: 10-07-2011
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Esophageal squamous cell carcinoma (ESCC) is the most common type of esophageal cancer. RON is a transmembrane receptor overexpressed in various cancers; however, the clinical significance of its phosphorylated form (pRON) is not fully deciphered. This report is the first to investigate the expression and clinical significance of pRON in human ESCC. Quantitative polymerase chain reaction revealed an up-regulation of RON mRNA in 70% (7/10) of ESCC tissues when compared to the adjacent nontumor tissues. An overexpression of pRON protein was found in most of the ESCC cell lines studied (4/5) when compared to two non-neoplastic esophageal epithelial cells using immunoblot. In 64 ESCC tissues, pRON was localized at the cell membrane, cytoplasm and nucleus in 15 (23.4%), 63 (98.4%) and 61 (95.3%) cases using immunohistochemistry. Patients having high expression of cytoplasmic pRON significantly associated with shorter median survival when compared to those with low expression (25.41 months vs. 14.43 months), suggesting cytoplasmic pRON as a potential marker for poor prognosis in ESCC patients.
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Differential regulation of RNF8-mediated Lys48- and Lys63-based poly-ubiquitylation.
Nucleic Acids Res.
PUBLISHED: 09-12-2011
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Pairing of a given E3 ubiquitin ligase with different E2s allows synthesis of ubiquitin conjugates of different topologies. While this phenomenon contributes to functional diversity, it remains largely unknown how a single E3 ubiquitin ligase recognizes multiple E2s, and whether identical structural requirements determine their respective interactions. The E3 ubiquitin ligase RNF8 that plays a critically important role in transducing DNA damage signals, interacts with E2s UBCH8 and UBC13, and catalyzes both K48- and K63-linked ubiquitin chains. Interestingly, we report here that a single-point mutation (I405A) on the RNF8 polypeptide uncouples its ability in catalyzing K48- and K63-linked ubiquitin chain formation. Accordingly, while RNF8 interacted with E2s UBCH8 and UBC13, its I405A mutation selectively disrupted its functional interaction with UBCH8, and impaired K48-based poly-ubiquitylation reactions. In contrast, RNF8 I405A preserved its interaction with UBC13, synthesized K63-linked ubiquitin chains, and assembled BRCA1 and 53BP1 at sites of DNA breaks. Together, our data suggest that RNF8 regulates K48- and K63-linked poly-ubiquitylation via differential RING-dependent interactions with its E2s UBCH8 and UBC13, respectively.
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The LIM domain protein, CRIP2, promotes apoptosis in esophageal squamous cell carcinoma.
Cancer Lett.
PUBLISHED: 06-01-2011
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The group 2 LIM domain protein, Cysteine-rich intestinal protein 2 (CRIP2) was found to play an important role in esophageal squamous cell carcinoma (ESCC) tumorigenesis. Subcellular fractionation studies show that CRIP2 is expressed in the nucleus. Real-time quantitative PCR shows CRIP2 expression is down-regulated in ESCC tissues and cell lines. Functional studies reveal that CRIP2 reduces colony formation, growth, and invasion abilities. Furthermore, over-expression of CRIP2 induces apoptosis through induction of active caspases 3 and 9 proteins. In conclusion, this study shows CRIP2 plays an important role in the development of ESCC.
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Id1 interacts and stabilizes the Epstein-Barr virus latent membrane protein 1 (LMP1) in nasopharyngeal epithelial cells.
PLoS ONE
PUBLISHED: 05-22-2011
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The EBV-encoded latent membrane protein 1 (LMP1) functions as a constitutive active form of tumor necrosis factor receptor (TNFR) and activates multiple downstream signaling pathways similar to CD40 signaling in a ligand-independent manner. LMP1 expression in EBV-infected cells has been postulated to play an important role in pathogenesis of nasopharyngeal carcinoma. However, variable levels of LMP1 expression were detected in nasopharyngeal carcinoma. At present, the regulation of LMP1 levels in nasopharyngeal carcinoma is poorly understood. Here we show that LMP1 mRNAs are transcribed in an EBV-positive nasopharyngeal carcinoma (NPC) cell line (C666-1) and other EBV-negative nasopharyngeal carcinoma cells stably re-infected with EBV. The protein levels of LMP1 could readily be detected after incubation with proteasome inhibitor, MG132 suggesting that LMP1 protein is rapidly degraded via proteasome-mediated proteolysis. Interestingly, we observed that Id1 overexpression could stabilize LMP1 protein in EBV-infected cells. In contrary, Id1 knockdown significantly reduced LMP1 levels in cells. Co-immunoprecipitation studies revealed that Id1 interacts with LMP1 by binding to the CTAR1 domain of LMP1. N-terminal region of Id1 is required for the interaction with LMP1. Furthermore, binding of Id1 to LMP1 suppressed polyubiquitination of LMP1 and may be involved in stabilization of LMP1 in EBV-infected nasopharyngeal epithelial cells.
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Cysteine-rich intestinal protein 2 (CRIP2) acts as a repressor of NF-kappaB-mediated proangiogenic cytokine transcription to suppress tumorigenesis and angiogenesis.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 05-03-2011
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Chromosome 14 was transferred into tumorigenic nasopharyngeal carcinoma and esophageal carcinoma cell lines by a microcell-mediated chromosome transfer approach. Functional complementation of defects present in the cancer cells suppressed tumor formation. A candidate tumor-suppressor gene, cysteine-rich intestinal protein 2 (CRIP2), located in the hot spot for chromosomal loss at 14q32.3, was identified as an important candidate gene capable of functionally suppressing tumor formation. Previous studies have shown that CRIP2 is associated with development. To date, no report has provided functional evidence supporting a role for CRIP2 in tumor development. The present study provides unequivocal evidence that CRIP2 can functionally suppress tumorigenesis. CRIP2 is significantly down-regulated in nasopharyngeal carcinoma cell lines and tumors. CRIP2 reexpression functionally suppresses in vivo tumorigenesis and angiogenesis; these effects are induced by its transcription-repressor capability. It interacts with the NF-?B/p65 to inhibit its DNA-binding ability to the promoter regions of the major proangiogenesis cytokines critical for tumor progression, including IL6, IL8, and VEGF. In conclusion, we provide compelling evidence that CRIP2 acts as a transcription repressor of the NF-?B-mediated proangiogenic cytokine expression and thus functionally inhibits tumor formation and angiogenesis.
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Tumor suppressor dual-specificity phosphatase 6 (DUSP6) impairs cell invasion and epithelial-mesenchymal transition (EMT)-associated phenotype.
Int. J. Cancer
PUBLISHED: 04-20-2011
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Suppressive effects of DUSP6 in tumorigenesis and EMT-associated properties were observed. Dual-specificity phosphatase (DUSP6) is a MAP kinase phosphatase (MKP) negatively regulating the activity of ERK, one of the major molecular switches in the MAPK signaling cascade propagating the signaling responses during malignancies. The impact of DUSP6 in EMT and its contribution to tumor dissemination has not yet been characterized. Due to differences in tumor microenvironments affecting cell signaling during cancer progression, DUSP6 may play varying roles in tumor development. We sought to examine the potential role of DUSP6-mediated tumorigenesis and EMT-associated properties in two aerodigestive tract cancers, namely, esophageal squamous cell carcinoma (ESCC) and nasopharyngeal carcinoma (NPC). Significant loss of DUSP6 was observed in 100% of 11 ESCC cell lines and 71% of seven NPC cell lines. DUSP6 expression was down-regulated in 40% of 30 ESCC tumor tissues and 75% of 20 NPC tumor tissues compared to their respective normal counterparts. Suppressive effects of DUSP6 in tumor formation and cancer cell mobility are seen in in vivo tumorigenicity assay and in vitro colony formation, three-dimensional Matrigel culture, cell migration and invasion chamber tests. Notably, overexpression of DUSP6 impairs EMT-associated properties. Furthermore, tissue microarray analysis reveals a clinical association of DUSP6 expression with better patient survival. Taken together, our study provides a novel insight into understanding the functional impact of DUSP6 in tumorigenesis and metastasis of ESCC and NPC.
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Catalytic activity of Matrix metalloproteinase-19 is essential for tumor suppressor and anti-angiogenic activities in nasopharyngeal carcinoma.
Int. J. Cancer
PUBLISHED: 04-01-2011
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The association of Matrix metalloproteinase-19 (MMP19) in the development of nasopharyngeal carcinoma (NPC) was identified from differential gene profiling, which showed MMP19 was one of the candidate genes down-regulated in the NPC cell lines. In this study, quantitative RT-PCR and Western blot analysis showed MMP19 was down-regulated in all seven NPC cell lines. By tissue microarray immunohistochemical staining, MMP19 appears down-regulated in 69.7% of primary NPC specimens. Allelic deletion and promoter hypermethylation contribute to MMP19 down-regulation. We also clearly demonstrate that the catalytic activity of MMP19 plays an important role in antitumor and antiangiogenesis activities in comparative studies of the wild-type and the catalytically inactive mutant MMP19. In the in vivo tumorigenicity assay, only the wild-type (WT), but not mutant, MMP19 transfectants suppress tumor formation in nude mice. In the in vitro colony formation assay, WT MMP19 dramatically reduces colony-forming ability of NPC cell lines, when compared to the inactive mutant. In the tube formation assay of human umbilical vein endothelial cells and human microvascular endothelial cells (HMEC-1), secreted WT MMP19, but not mutant MMP19, induces reduction of tube-forming ability in endothelial cells with decreased vascular endothelial growth factor (VEGF) in conditioned media detected by enzyme-linked immunosorbent assay (ELISA). The anti-angiogenic activity of WT MMP19 is correlated with suppression of tumor formation. These results now clearly show that catalytic activity of MMP19 is essential for its tumor suppressive and anti-angiogenic functions in NPC.
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Molecular changes during arsenic-induced cell transformation.
J. Cell. Physiol.
PUBLISHED: 02-24-2011
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Arsenic and its derivatives are naturally occurring metalloid compounds widely distributed in the environment. Arsenics are known to cause cancers of the skin, liver, lung, kidney, and bladder. Although numerous carcinogenic pathways have been proposed, the exact molecular mechanisms remain to be delineated. To further characterize the role of oxidative stress in arsenite-induced cell transformation via the reactive oxygen species (ROS)-mediated Ras/Erk pathway, here we demonstrated arsenite-induced rat lung epithelial cell (LEC) transformation, epithelial-mesenchymal transition, stimulation of the extracellular signal-regulated kinase signaling pathway, and enhancement of cell proliferation. However, there was no evidence of activation of the phosphoinositide 3-kinase/protein kinase B pathway in arsenite-induced transformed LECs. Since ROS is involved in arsenite-induced LEC cell transformation, Redox-status regulatory proteins (Cu/Zn SOD and thioredoxin) and arsenite-induced LEC cell transformation were significantly inhibited by concurrent treatment with the antioxidants. Our experimental results clearly demonstrated that induction of p-ERK and cell proliferation by arsenite is mediated via oxidative stress, since antioxidants can inhibit arsenite-induced cell transformation.
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Inhibition of NOTCH3 signalling significantly enhances sensitivity to cisplatin in EBV-associated nasopharyngeal carcinoma.
J. Pathol.
PUBLISHED: 02-16-2011
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Nasopharyngeal carcinoma (NPC) is an EBV-associated epithelial malignancy which is prevalent in south-east Asia and southern China. Despite the multiple genetic and epigenetic changes reported, the contribution of dysregulated signalling pathways to this distinct type of head and neck cancer is not well understood. Here we demonstrate the up-regulation of NOTCH ligands (JAG1 or DLL4) and effector (HEY1) in the majority of EBV-positive tumour lines and primary tumours. Among the NOTCH receptors, NOTCH3 was over-expressed in all EBV-positive tumour lines and 92.5% of primary tumours. Aberrant activation of NOTCH3 signalling was consistently detected in all these samples. These findings imply that NOTCH3 may play an crucial role in the development of NPC. By NOTCH3 specific siRNA, NOTCH3 signalling was suppressed and thereby significant growth inhibition and apoptosis induction occurred in NPC cells. Down-regulation of a number of targets involved in cell proliferation, eg CCND1, C-MYC,NFKB1, and survival, eg BCL2, BCL-XL, SURVIVIN, was confirmed in the NOTCH3 knockdown NPC cells. Importantly, NOTCH3 knockdown highly enhanced the sensitivity of NPC cells to cisplatin treatment. Furthermore, we revealed that the ability of NPC cells to form spheroids in vitro and tumours in nude mice was also significantly decreased after knockdown of NICD3 expression. These findings indicate that activation of NOTCH3 pathway is a critical oncogenic event in NPC tumourigenesis. Targeting NOTCH3 signalling may serve as a potential therapeutic approach for treating patients suffering from EBV-associated NPC.
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The epigenetic modifier PRDM5 functions as a tumor suppressor through modulating WNT/?-catenin signaling and is frequently silenced in multiple tumors.
PLoS ONE
PUBLISHED: 02-08-2011
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PRDM (PRDI-BF1 and RIZ domain containing) proteins are zinc finger proteins involved in multiple cellular regulations by acting as epigenetic modifiers. We studied a recently identified PRDM member PRDM5 for its epigenetic abnormality and tumor suppressive functions in multiple tumorigeneses.
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Loss of ?Np63? promotes mitotic exit in epithelial cells.
FEBS Lett.
PUBLISHED: 01-16-2011
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Protein p63 is a key regulator in cell proliferation and cell differentiation in stratified squamous epithelium. ?Np63? is the most commonly expressed p63 isoform, which is often overexpressed in human tumor. In the present work we report the potential involvement of ?Np63? in cell cycle regulation. ?Np63? accumulated in mitotic cells but its expression decreased during mitotic exit. Moreover, ?Np63? knockdown promoted mitotic exit. ?Np63? shares a conserved destruction box (D-box) motif with other potential targets of the Anaphase-Promoting Complex/Cyclosome (APC/C). Overexpression of APC/C coactivator Cdh1 destabilized ?Np63?. Our results suggest that ?Np63? level is cell cycle-regulated and may play a role in the regulation of mitotic exit.
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p21-activated kinase 4 regulates ovarian cancer cell proliferation, migration, and invasion and contributes to poor prognosis in patients.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 10-06-2010
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Ovarian cancer is a lethal gynecological malignancy, and to improve survival, it is important to identify novel prognostic and therapeutic targets. In this study, we present a role for p21-activated kinase 4 (Pak4) in ovarian cancer progression. We show a significant association between increased expression of Pak4 and its activated form, phosphorylated (p)-Pak4 Ser(474), with metastasis of ovarian cancers, shorter overall and disease-free survival, advanced stage and high-grade cancers, serous/clear cell histological subtypes, and reduced chemosensitivity. Pak4 overexpression was also observed in ovarian cancer cell lines. Pak4 and p-Pak4 expression were detected both in the nucleus and cytoplasm of ovarian cancer cells, in vitro as well as in vivo. Stable knockdown of Pak4 in ovarian cancer cell lines led to reduced cell migration, invasion, and proliferation, along with reduced c-Src, ERK1/2, and epidermal growth factor receptor (EGFR) activation and decreased matrix metalloproteinase 2 (MMP2) expression. Conversely, Pak4 overexpression promoted ovarian cancer cell migration and invasion in a c-Src, MEK-1, MMP2, and kinase-dependent manner, and induced cell proliferation through the Pak4/c-Src/EGFR pathway that controls cyclin D1 and CDC25A expression. Stable knockdown of Pak4 also impeded tumor growth and dissemination in nude mice. This report reveals the association between Pak4 and important clinicopathologic parameters, suggesting Pak4 to be a significant prognostic marker and potential therapeutic molecular target in ovarian cancer. The implied possible cross-talk between Pak4 and EGFR suggests the potential of dual targeting of EGFR and Pak4 as a unique therapeutic approach for cancer therapy.
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Berberine induces autophagic cell death and mitochondrial apoptosis in liver cancer cells: the cellular mechanism.
J. Cell. Biochem.
PUBLISHED: 09-11-2010
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Extensive studies have revealed that berberine, a small molecule derived from Coptidis rhizoma (Huanglian in Chinese) and many other plants, has strong anti-tumor properties. To better understand berberine-induced cell death and its underlying mechanisms in cancer, we examined autophagy and apoptosis in the human hepatic carcinoma cell lines HepG2 and MHCC97-L. The results of this study indicate that berberine can induce both autophagy and apoptosis in hepatocellular carcinoma cells. Berberine-induced cell death in human hepatic carcinoma cells was diminished in the presence of the cell death inhibitor 3-methyladenine, or following interference with the essential autophagy gene Atg5. Mechanistic studies showed that berberine may activate mitochondrial apoptosis in HepG2 and MHCC97-L cells by increasing Bax expression, the formation of permeable transition pores, cytochrome C release to cytosol, and subsequent activation of the caspases 3 and 9 execution pathway. Berberine may also induce autophagic cell death in HepG2 and MHCC97-L cells through activation of Beclin-1 and inhibition of the mTOR-signaling pathway by suppressing the activity of Akt and up-regulating P38 MAPK signaling. This is the first study to describe the role of Beclin-1 activation and mTOR inhibition in berberine-induced autophagic cell death. These results further demonstrate the potential of berberine as a therapeutic agent in the emerging list of cancer therapies with novel mechanisms.
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Expression of Epstein-Barr virus-encoded LMP1 and hTERT extends the life span and immortalizes primary cultures of nasopharyngeal epithelial cells.
J. Med. Virol.
PUBLISHED: 09-10-2010
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Cell immortalization is regarded as an early and pre-requisite step in tumor development. Defining the specific genetic events involved in cell immortalization may provide insights into the early events of carcinogenesis. Nasopharyngeal carcinoma is common among the Southern Chinese population. Epstein-Barr virus (EBV) infection is associated closely with nasopharyngeal carcinoma. The involvement of LMP1 (an EBV-encoded oncogene) has been implicated in the pathogenesis of nasopharyngeal carcinoma. In this study, LMP1 expression, in combination with ectopic expression of hTERT (catalytic unit of human telomerase), was shown to extend the life span of primary cultures of nasopharyngeal epithelial cells and facilitate the immortalization of one of the cell lines (NP446). This is the first report on the successful immortalization of nasopharyngeal epithelial cells involving LMP1. The events associated with the immortalization of nasopharyngeal epithelial cells by LMP1/hTERT were characterized. Expression of c-Myc, Bmi-1, and Id-1 were upregulated at an early stage of immortalization. At a later stage of immortalization, downregulation of p21 and p16 expression were observed. Upregulation of EGFR expression and activation of MAPK signaling pathway were observed in LMP1/hTERT-immortalized nasopharyngeal epithelial cells. The LMP1/hTERT-immortalized NP446 cells were non-tumorigenic in immunosuppressed nude mice and retained anchorage-dependent growth, suggesting that additional events are required for tumorigenic transformation. The ability of the EBV-encoded LMP1, in the presence of hTERT expression, to extend the life span and immortalize primary cultures of nasopharyngeal epithelial cells supports the involvement of EBV infection and its viral products in the early stage of pathogenesis of nasopharyngeal carcinoma.
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Up-regulation of microRNAs, miR21 and miR23a in human liver cancer cells treated with Coptidis rhizoma aqueous extract.
Exp Ther Med
PUBLISHED: 08-20-2010
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Coptidis rhizoma (CR; Huanglian in Chinese) has been used for the treatment of cancer in Chinese medicine, and recent studies have supported its use in cancer therapy. MicroRNAs (miRNAs) play an important role in the pathophysiology of human cancers. We examined alterations in the miRNA profile of hepatocellular carcinoma (HCC) cells after treatment with Coptidis rhizoma aqueous extract (CRAE). An on-chip microarray method was used to detect alterations in the expression profile of miRNAs in human HCC MHCC97-L cells after exposure to 175 ?g/ml CRAE. Altered expression of several miRNAs was detected in the MHCC97-L cells after treatment with 175 ?g/ml CRAE. The microarray results were validated by quantitative real-time PCR (qRT-PCR). Consistent results were obtained; qRT-PCR confirmed that both miR-21 and miR-23a were significantly up-regulated. TargetScan and PicTar microRNA databases were used to predict the possible target genes of the altered miRNAs. The results showed that the altered miRNAs after CRAE treatment may serve as markers for the therapy of liver cancer. To the best of our knowledge, this is the first report on the up-regulation of miRNAs, miR21 and miR23a in human liver cancer cells treated with CRAE. Our results suggest that CRAE targets miR-21 and miR-23a in liver cancer cells supporting the potential application of CRAE in the treatment of HCC.
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Curcumin alters the migratory phenotype of nasopharyngeal carcinoma cells through up-regulation of E-cadherin.
Anticancer Res.
PUBLISHED: 08-05-2010
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Curcumin is a natural polyphenol. It is a potent suppressor of nuclear factor kappa B (NF-kappaB). High NF-kappaB levels have suppressive effect on E-cadherin (molecule related to cell-cell adhesion) in nasopharyngeal carcinoma (NPC) cells. We hypothesized that suppressing NF-kappaB by curcumin could up-regulate E-cadherin expression in NPC cells.
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Extracellular protease ADAMTS9 suppresses esophageal and nasopharyngeal carcinoma tumor formation by inhibiting angiogenesis.
Cancer Res.
PUBLISHED: 06-15-2010
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ADAMTS metalloprotease family member ADAMTS9 maps to 3p14.2 and shows significant associations with the aerodigestive tract cancers esophageal squamous cell carcinoma (ESCC) and nasopharyngeal carcinoma (NPC). However, the functional impact of ADAMTS9 on cancer development has not been explored. In this study, we evaluated the hypothesized antiangiogenic and tumor-suppressive functions of ADAMTS9 in ESCC and NPC, in stringent tumorigenicity and Matrigel plug angiogenesis assays. ADAMTS9 activation suppressed tumor formation in nude mice. Conversely, knockdown of ADAMTS9 resulted in clones reverting to the tumorigenic phenotype of parental cells. In vivo angiogenesis assays revealed a reduction in microvessel numbers in gel plugs injected with tumor-suppressive cell transfectants. Similarly, conditioned medium from cell transfectants dramatically reduced the tube-forming capacity of human umbilical vein endothelial cells. These activities were associated with a reduction in expression levels of the proangiogenic factors MMP9 and VEGFA, which were consistently reduced in ADAMTS9 transfectants derived from both cancers. Taken together, our results indicate that ADAMTS9 contributes an important function in the tumor microenvironment that acts to inhibit angiogenesis and tumor growth in both ESCC and NPC.
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The ECM protein LTBP-2 is a suppressor of esophageal squamous cell carcinoma tumor formation but higher tumor expression associates with poor patient outcome.
Int. J. Cancer
PUBLISHED: 05-31-2010
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Our previous studies of chromosome 14 transfer into tumorigenic esophageal squamous cell carcinoma (ESCC) cell line, SLMT, suggested the existence of tumor suppressor genes on chromosome 14. Gene expression profiling of microcell hybrids and the tumor segregants identified an interesting gene, LTBP-2 (latent transforming growth factor ? binding protein 2), which has been analyzed here for its role in ESCC. LTBP-2 maps to 14q24 and encodes a secreted protein, which is a component of the extracellular matrix microfibrils. LTBP-2 expression was downregulated in ESCC cell lines and tumor tissues. Promoter hypermethylation was found to be involved in LTBP-2 inactivation. Functional studies indicated its tumor-suppressive roles in ESCC. In the in vitro colony formation and Matrigel three-dimensional culture assays, LTBP-2 decreased the colony-forming abilities of ESCC cell lines. LTBP-2 expression was associated with reduction of cell migrating and invasive abilities. LTBP-2 could also reduce the tube-forming ability of endothelial cells. Moreover, LTBP-2 induced tumor suppression in in vivo nude mouse assays. Tissue microarray immunohistochemical staining analysis indicated that LTBP-2 expression is reduced in tumor tissues when compared to normal tissues, and LTBP-2 expression correlated significantly with the survival of ESCC patients. Thus, LTBP-2 appears to play an important role in ESCC.
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K252a induces anoikis-sensitization with suppression of cellular migration in Epstein-Barr virus (EBV)--associated nasopharyngeal carcinoma cells.
Invest New Drugs
PUBLISHED: 05-07-2010
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Recent studies revealed an unexpected role of the neurotrophin receptor pathway, BDNF/TrkB signaling, in cancer metastasis and anoikis (i.e. detachment-induced cell death). Survival of cancer cells in detached state (known as anoikis-resistance) is known to be pre-requisite for metastasis. Nasopharyngeal carcinoma (NPC), an endemic head and neck cancer in Southeast Asia, is highly invasive, metastatic, and etiologically associated with Epstein-Barr virus (EBV, an oncovirus) infection. Mechanistic studies on the invasive/metastatic nature of NPC can facilitate the development of anti-metastatic therapy in NPC. Thus far, the role of BDNF/TrkB signaling in virus-associated human cancer is unclear. Here, using multiple cell line models of NPC with EBV-association (HONE-1-EBV, HK1-LMP1 and C666-1), we investigated the potential involvement of BDNF/TrkB signaling in cellular migration and anoikis-resistant characteristics of NPC. We found that all three EBV-associated NPC cell lines tested were intrinsically anoikis-resistant (i.e. survived in detached state) and expressed both BDNF and TrkB. BDNF stimulation induced cellular migration, but not proliferation of these cells. Further, we examined if pharmacologic targeting of anoikis-resistance of NPC cells can be achievable by a proof-of-concept Trk inhibitor, K252a, in these EBV-associated NPC models. Our results demonstrated that K252a, was able to attenuate BDNF-induced migration and proliferation of NPC cells. More importantly, we demonstrated for the first time that K252a harbored potent anoikis-sensitization activity (i.e. sensitizing cancer cells to detachment-induced cell death) against EBV-associated human cancer cells, namely NPC cells. This proof-of-concept study demonstrated that K252a, a Trk inhibitor, can potentially be used as an anoikis-sensitizing agent in NPC.
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Bauhinia variegata var. variegata trypsin inhibitor: from isolation to potential medicinal applications.
Biochem. Biophys. Res. Commun.
PUBLISHED: 04-13-2010
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Here we report for the first time of a new Kunitz-type trypsin inhibitor (termed BvvTI) from seeds of the Camels foot tree, Bauhinia variegata var. variegata. BvvTI shares the same reactive site residues (Arg, Ser) and exhibits a homology of N-terminal amino acid sequence to other Bauhinia protease inhibitors. The trypsin inhibitory activity (K(i), 0.1 x 10(-9)M) of BvvTI ranks the highest among them. Besides anti-HIV-1 reverse transcriptase activity, BvvTI could significantly inhibit the proliferation of nasopharyngeal cancer CNE-1 cells in a selective way. This may partially be contributed by its induction of cytokines and apoptotic bodies. These results unveil potential medicinal applications of BvvTI.
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Anti-invasion, anti-proliferation and anoikis-sensitization activities of lapatinib in nasopharyngeal carcinoma cells.
Invest New Drugs
PUBLISHED: 03-30-2010
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Nasopharyngeal cancer (NPC) is a highly prevalent and invasive head and neck cancer in Asia. Disease recurrence and distant metastasis account for major NPC deaths. Therefore, more effective therapy is needed. Lapatinib, a dual tyrosine kinase inhibitor (TKI) against both EGFR and HER-2, has been known to exert potent antitumor activity against several cancer models. Given that both EGFR and HER-2 are co-expressed in NPC, we hypothesized that dual targeting of EGFR and HER-2 by this small molecule EGFR/HER-2 TKI would elicit anti-tumor activity in NPC. Using in vitro models of NPC, we demonstrated that lapatinib was able to efficiently inhibit the phosphorylation of both EGFR and HER-2. This was accompanied by significant growth inhibition of NPC cells (with maximal growth inhibition >90%). For the most lapatinib-sensitive cell line (HK1-LMP1, with IC(50) ? 600 nM), which harbored the highest levels of both EGFR and HER-2, inhibition of cell growth was associated G(0)/G(1) cell cycle arrest, marked PARP cleavage, caspase-3 cleavage, as well as significant downregulation of several important survival proteins (e.g. survivin, Mcl-1 and cyclin D1). NPC cells are intrinsically invasive. We found that lapatinib was able to inhibit cellular invasion of both HK1-LMP1 and HONE-1 cells. Furthermore, our data demonstrated for the first time that lapatinib harbored potent anoikis-sensitization activity (i.e. sensitizing cancer cells to detachment-induced apoptosis) in human cancer cells overexpressing both EGFR and HER-2 (HK1-LMP1 and HK1). Taken together, our findings suggest that lapatinib is a promising anti-cancer agent for NPC with anti-invasion and anoikis-sensitization activities.
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Regulation of chromatin architecture by the PWWP domain-containing DNA damage-responsive factor EXPAND1/MUM1.
Mol. Cell
PUBLISHED: 03-30-2010
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Dynamic changes of chromatin structure facilitate diverse biological events, including DNA replication, repair, recombination, and gene transcription. Recent evidence revealed that DNA damage elicits alterations to the chromatin to facilitate proper checkpoint activation and DNA repair. Here we report the identification of the PWWP domain-containing protein EXPAND1/MUM1 as an architectural component of the chromatin, which in response to DNA damage serves as an accessory factor to promote cell survival. Depletion of EXPAND1/MUM1 or inactivation of its PWWP domain resulted in chromatin compaction. Upon DNA damage, EXPAND1/MUM1 rapidly concentrates at the vicinity of DNA damage sites via its direct interaction with 53BP1. Ablation of this interaction impaired damage-induced chromatin decondensation, which is accompanied by sustained DNA damage and hypersensitivity to genotoxic stress. Collectively, our study uncovers a chromatin-bound factor that serves an accessory role in coupling damage signaling with chromatin changes in response to DNA damage.
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Poly(I:C) induces intense expression of c-IAP2 and cooperates with an IAP inhibitor in induction of apoptosis in cancer cells.
BMC Cancer
PUBLISHED: 03-26-2010
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There is increasing evidence that the toll-like receptor 3 (TLR3) is an interesting target for anti-cancer therapy. Unfortunately, most laboratory investigations about the impact of TLR3 stimulation on human malignant cells have been performed with very high concentrations--5 to 100 microg/ml--of the prototype TLR3 ligand, poly(I:C). In a previous study focused on a specific type of human carcinoma - nasopharyngeal carcinoma - we have shown that concentrations of poly(I:C) as low as 100 ng/ml are sufficient to induce apoptosis of malignant cells when combined to a pharmacological antagonist of the IAP family based on Smac mimicry.
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MicroRNA let-7 suppresses nasopharyngeal carcinoma cells proliferation through downregulating c-Myc expression.
J. Cancer Res. Clin. Oncol.
PUBLISHED: 02-12-2010
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This study aimed at evaluating the potential anti-proliferative effects of the microRNA let-7 family in nasopharyngeal carcinoma (NPC) cells. In addition, the association between let-7 suppression and DNA hypermethylation is examined.
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An RNA-directed nucleoside anti-metabolite, 1-(3-C-ethynyl-beta-d-ribo-pentofuranosyl)cytosine (ECyd), elicits antitumor effect via TP53-induced Glycolysis and Apoptosis Regulator (TIGAR) downregulation.
Biochem. Pharmacol.
PUBLISHED: 02-09-2010
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1-(3-C-ethynyl-beta-d-ribo-pentofuranosyl)cytosine (ECyd) is a ribose-modified nucleoside analog of cytidine with potent anticancer activity in several cancers. The main antitumor mechanism of this promising RNA-directed nucleoside anti-metabolite is efficient blockade of RNA synthesis in cancer cells. Here, we examined the therapeutic potential of this RNA-directed anti-metabolite in in vitro models of nasopharyngeal cancer (NPC). In a panel of 6 NPC cell lines, ECyd effectively inhibited cellular proliferation at nM concentrations (IC(50): approximately 13-44nM). Moreover, cisplatin-resistant NPC cells were highly sensitive to ECyd (at nM concentration). The ECyd-mediated growth inhibition was associated with G(2)/M cell cycle arrest, PARP cleavage (a hallmark of apoptosis) and Bcl-2 downregulation, indicating induction of apoptosis by ECyd in NPC cells. Unexpectedly, ECyd-induced significant downregulation of TIGAR, a newly described dual regulator of apoptosis and glycolysis. More importantly, this novel action of ECyd on TIGAR was accompanied by marked depletion of NADPH, the major reducing power critically required for cell proliferation and survival. We hypothesized that ECyd-induced TIGAR downregulation was crucially involved in the antitumor activity of ECyd. Indeed, overexpression of TIGAR was able to rescue NPC cells from ECyd-induced growth inhibition, demonstrating a novel mechanistic action of ECyd on TIGAR. We demonstrated for the first time that an RNA-directed nucleoside analog, ECyd, exerts its antitumor activity via downregulation of a novel regulator of apoptosis, TIGAR. Moreover, ECyd may represent a novel therapy for NPC.
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A lectin with anti-HIV-1 reverse transcriptase, antitumor, and nitric oxide inducing activities from seeds of Phaseolus vulgaris cv. extralong autumn purple bean.
J. Agric. Food Chem.
PUBLISHED: 01-26-2010
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Lectins/hemagglutinins are a class of sugar-binding proteins which agglutinate cells and/or precipitate glycoconjugates. They occur widely in plants but manifest significant differences in activities, which means only a few of them own exploitable potentials. The objective of this study was to find and characterize a multifunctional plant lectin with high potential values in food chemistry and medicine. A 60-kDa lectin from Phaseolus vulgaris L. cv. Extralong Autumn Purple Bean (EAPL) was purified by liquid chromatography, and the sequence of its first 20 N-terminal amino acids was ANEIYFSFQRFNETNLILQR. It was galactose-specific and manifested hemagglutinating activity toward erythrocytes of rabbit, rat, mouse, and human ABO blood types. EAPL manifested anti-HIV-1-RT activity, and it could inhibit the proliferation of human tumor cells by inducing the production of apoptotic bodies. The nitric oxide-inducing activity of EAPL may find application in tumor therapy.
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Epstein-Barr virus infection in immortalized nasopharyngeal epithelial cells: regulation of infection and phenotypic characterization.
Int. J. Cancer
PUBLISHED: 01-22-2010
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Epstein-Barr virus (EBV) infection has been postulated to be an early event involved in the pathogenesis of nasopharyngeal carcinomas (NPC). The lack of representative premalignant nasopharyngeal epithelial cell system for EBV infection has hampered research investigation into the regulation and involvement of EBV infection in NPC pathogenesis. We have compared the efficiency of EBV infection in nasopharyngeal epithelial cells with different biological properties including immortalized, primary and cancerous nasopharyngeal epithelial cells. EBV infection could be achieved in all the nasopharyngeal epithelial cells examined with variable infection rate. TGF-beta effectively enhanced EBV infection into nasopharyngeal epithelial cells both in the immortalized and primary nasopharyngeal epithelial cells. Stable infection of EBV was achieved in a telomerase-immortalized nasopharyngeal epithelial cell line, NP460hTert. The expression pattern of EBV-encoded genes and biological properties of this EBV infected cell line on long-term propagation were monitored. The EBV-infected nasopharyngeal epithelial cells acquired anchorage-independent growth and exhibited invasive growth properties on prolonged propagation. A distinguished feature of this EBV-infected nasopharyngeal epithelial cell model was its enhanced ability to survive under growth factor and nutrient starvation. This was evidenced by the suppressed activation of apoptotic markers and sustained activation of pAkt of EBV-infected cells compared to control cells under nutrient starvation. Examination of cytokine profiles of EBV-infected NP460hTert cells to nutrient and growth factor deprivation revealed upregulation of expression of MCP-1 and GRO-alpha. The establishment of a stable EBV infection model of premalignant nasopharyngeal epithelial cells will facilitate research investigation into the pathogenic role of EBV in NPC development.
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Genetic alterations in a telomerase-immortalized human esophageal epithelial cell line: implications for carcinogenesis.
Cancer Lett.
PUBLISHED: 01-21-2010
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Ectopic expression of viral oncoproteins disrupts cellular functions and limits the value of many existing immortalization models as models for carcinogenesis, especially for cancers without definitive viral etiology. Our newly established telomerase-immortalized human esophageal epithelial cell line, NE2-hTERT, retained nearly-diploid and non-tumorigenic characteristics, but exhibited genetic and genomic alterations commonly found in esophageal cancer, including progressive loss of the p16(INK4a) alleles, upregulation of anti-apoptotic proteins, epithelial-mesenchymal transition, whole-chromosome 7 gain and duplicated 5q arm. Our data also revealed a novel positive regulation of p16(INK4a) on cyclin D1. These findings probably represent early crucial events and mechanisms in esophageal carcinogenesis.
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Cucurbitacin I elicits anoikis sensitization, inhibits cellular invasion and in vivo tumor formation ability of nasopharyngeal carcinoma cells.
Carcinogenesis
PUBLISHED: 10-20-2009
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Nasopharyngeal carcinoma (NPC) is an Asian-prevalent head and neck cancer with high invasiveness. Although several important risk factors for NPC development have been identified, there is currently no preventive strategy for NPC, even in endemic regions. Signal transducer and activator of transcription 3 (STAT3) has been implicated in NPC carcinogenesis, which may serve as a potential target for cancer prevention. Here, we examined the chemopreventive potential of Cucurbitacin I, a natural-occurring selective inhibitor of JAK/STAT3, in NPC models. We hypothesized that Cucurbitacin I would prevent NPC invasion and tumor formation. Our data demonstrated that brief exposure of NPC cells to Cucurbitacin I was sufficient to significantly reduce the in vitro clonogenicity and in vivo tumorigenicity of NPC cells. The chemopreventive potential of Cucurbitacin I was further demonstrated by pre-dosing of the animals with Cucurbitacin I prior to tumor inoculation, which was found to be able to suppress tumor growth up to 7 days post-inoculation. The anti-proliferation activity of Cucurbitacin I was accompanied by downregulation of phospho-STAT3 and STAT3 target gene expression (e.g. cyclin D1 and Mcl-1). Cucurbitacin I also reduced the invasiveness of invasive NPC cell lines with elevated STAT3 activation. Furthermore, our data demonstrated for the first time that Cucurbitacin I harbored potent anoikis-sensitization activity (i.e. sensitizing cancer cells to detachment-induced cell death) against human cancer. Taken together, our results suggested that Cucurbitacin I may be a potent chemopreventive agent for NPC with anti-invasion and anoikis-sensitizing activities.
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Hypoxia-targeting by tirapazamine (TPZ) induces preferential growth inhibition of nasopharyngeal carcinoma cells with Chk1/2 activation.
Invest New Drugs
PUBLISHED: 09-23-2009
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Hypoxia is commonly developed in solid tumors, which contributes to metastasis as well as radio- and chemo-resistance. Nasopharyngeal carcinoma (NPC) is a highly invasive and metastatic head and neck cancer prevalent in Southeast Asia with a high incidence rate of 15-30/100,000 persons/year (comparable to that of pancreatic cancer in the US). Previous clinical studies in NPC showed that hypoxia is detected in almost 100% of primary tumors and overexpression of hypoxia markers correlated with poor clinical outcome. Tirapazamine (TPZ) is a synthetic hypoxia-activated prodrug, which preferentially forms cytotoxic and DNA-damaging free radicals under hypoxia, thus selectively eradicate hypoxic cells. Here, we hypothesized that specific hypoxia-targeting by this clinical trial agent may be therapeutic for NPC. Our findings demonstrated that under hypoxia, TPZ was able to induce preferential growth inhibition of NPC cells, which was associated with marked cell cycle arrest at S-phase and PARP cleavage (a hallmark of apoptosis). Examination of S-phase checkpoint regulators revealed that Chk1 and Chk2 were selectively activated by TPZ in NPC cells under hypoxia. Hypoxia-selectivity of TPZ was also demonstrated by preferential downregulation of several important hypoxia-induced markers (HIF-1?, CA IX and VEGF) under hypoxia. Furthermore, we demonstrated that TPZ was equally effective and hypoxia-selective even in the presence of the EBV oncoprotein, LMP1 or the EBV genome. In summary, encouraging results from this proof-of-concept study implicate the therapeutic potential of hypoxia-targeting approaches for the treatment of NPC.
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Two-photon plasma membrane imaging in live cells by an amphiphilic, water-soluble cyctometalated platinum(II) complex.
Inorg Chem
PUBLISHED: 09-03-2009
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An amphiphilic, water-soluble cyclometalated Pt(II) complex with two-photon emission properties has been developed as a molecular marker specific for in vitro plasma membrane staining.
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Targeting NF-kappaB signaling pathway suppresses tumor growth, angiogenesis, and metastasis of human esophageal cancer.
Mol. Cancer Ther.
PUBLISHED: 09-01-2009
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Esophageal cancer is the eighth most common malignancy, and one of the leading causes of cancer-related deaths worldwide. The overall 5-year survival rate of patients with esophageal cancer remains low at 10% to 40% due to late diagnosis, metastasis, and resistance of the tumor to radiotherapy and chemotherapy. NF-kappaB is involved in the regulation of cell growth, survival, and motility, but little is known about the role of this signaling pathway in the tumorigenesis of human esophageal squamous cell carcinoma (ESCC), the most common form of esophageal cancer. This study aims to explore the functions of NF-kappaB in human ESCC progression and to determine whether targeting the NF-kappaB signaling pathway might be of therapeutic value against ESCC. Our results from human ESCC cell lines and ESCC tissue indicated that NF-kappaB is constitutively active in ESCC. Exposure of ESCC cells to two NF-kappaB inhibitors, Bay11-7082 and sulfasalazine, not only reduced cancer cell proliferation, but also induced apoptosis and enhanced sensitivity to chemotherapeutic drugs, 5-fluorouracil, and cisplatin. In addition, Bay11-7082 and sulfasalazine suppressed the migration and invasive potential of ESCC cells. More importantly, the results from tumor xenograft and experimental metastasis models showed that Bay11-7082 had significant antitumor effects on ESCC xenografts in nude mice by promoting apoptosis, and inhibiting proliferation and angiogenesis, as well as reduced the metastasis of ESCC cells to the lungs without significant toxic effects. In summary, our data suggest that NF-kappaB inhibitors may be potentially useful as therapeutic agents for patients with esophageal cancer.
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