Lunquan Sun

STAT3 activation induced by Epstein-Barr virus latent membrane protein1 causes vascular endothelial growth factor expression and cellular invasiveness via JAK3 And ERK signaling

The principal Epstein-Barr virus (EBV) oncoprotein, latent membrane protein 1 (LMP1), has been suggested to contribute to the highly invasive nature of nasopharyngeal carcinoma (NPC). Signal transducer and activator of transcription 3 (STAT3) is a master transcriptional regulator in proliferation and apoptosis and is newly implicated in angiogenesis and invasiveness, which, in turn, are likely to contribute to the highly invasive character of NPC. The fundamental molecular mechanisms of LMP1-regulated STAT3 activation in NPC cell invasion have not been completely explored. Here, we showed that LMP1 signals the Janus kinase 3 (JAK3) and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways upon the activation of STAT3 as well as STAT transactivation activity. LMP1 induces vascular endothelial growth factor (VEGF) expression via the JAK/STAT and mitogen-activated protein kinase (MAPK)/ERK signalling pathways. Induction of STAT3 by the human viral oncoprotein LMP1 may contribute to the invasion of NPC.

Targeting autophagy to sensitive glioma to temozolomide treatment.

Temozolomide (TMZ), an alkylating agent, is widely used for treating primary and recurrent high-grade gliomas. However, the efficacy of TMZ is often limited by the development of resistance. Recently, studies have found that TMZ treatment could induce autophagy, which contributes to therapy resistance in glioma. To enhance the benefit of TMZ in the treatment of glioblastomas, effective combination strategies are needed to sensitize glioblastoma cells to TMZ. In this regard, as autophagy could promote cell survival or autophagic cell death, modulating autophagy using a pharmacological inhibitor, such as chloroquine, or an inducer, such as rapamycin, has received considerably more attention. To understand the effectiveness of regulating autophagy in glioblastoma treatment, this review summarizes reports on glioblastoma treatments with TMZ and autophagic modulators from in vitro and in vivo studies, as well as clinical trials. Additionally, we discuss the possibility of using autophagy regulatory compounds that can sensitive TMZ treatment as a chemotherapy for glioma treatment.

Down-Regulation of EBV-LMP1 Radio-Sensitizes Nasal Pharyngeal Carcinoma Cells via NF-κB Regulated ATM Expression

Background The latent membrane protein 1 (LMP1) encoded by EBV is expressed in the majority of EBV-associated human malignancies and has been suggested to be one of the major oncogenic factors in EBV-mediated carcinogenesis. In previous studies we experimentally demonstrated that down-regulation of LMP1 expression by DNAzymes could increase radiosensitivity both in cells and in a xenograft NPC model in mice. Results In this study we explored the molecular mechanisms underlying the radiosensitization caused by the down-regulation of LMP1 in nasopharyngeal carcinoma. It was confirmed that LMP1 could up-regulate ATM expression in NPCs. Bioinformatic analysis of the ATM ptomoter region revealed three tentative binding sites for NF-κB. By using a specific inhibitor of NF-κB signaling and the dominant negative mutant of IkappaB, it was shown that the ATM expression in CNE1-LMP1 cells could be efficiently suppressed. Inhibition of LMP1 expression by the DNAzyme led to attenuation of the NF-κB DNA binding activity. We further showed that the silence of ATM expression by ATM-targeted siRNA could enhance the radiosensitivity in LMP1 positive NPC cells. Conclusions Together, our results indicate that ATM expression can be regulated by LMP1 via the NF-κB pathways through direct promoter binding, which resulted in the change of radiosensitivity in NPCs.

Therapeutic Evaluation of Epstein-Barr Virus-encoded Latent Membrane Protein-1 Targeted DNAzyme for Treating of Nasopharyngeal Carcinomas

The ability of the 10–23 DNAzyme to specifically cleave RNA with high efficiency has fuelled expectation that this agent may have useful applications for targeted therapy. Here, we, for the first time, investigated the antitumor and radiosensitizing effects of a DNAzyme (DZ1) targeted to the Epstein-Barr virus (EBV)-LMP1 mRNA of nasopharyngeal carcinoma (NPC) in patients. Preclinical studies indicated that the DNAzyme was safe and well tolerated. A randomized and double-blind clinical study was conducted in 40 NPC patients who received DZ1 or saline intratumorally, in conjunction with radiation therapy. Tumor regression, patient survival, EBV DNA copy number and tumor microvascular permeability were assessed in a 3-month follow-up. The mean tumor regression rate at week 12 was significantly higher in DZ1 treated group than in the saline control group. Molecular imaging analysis showed that DZ1 impacted on tumor microvascular permeability as evidenced by a faster decline of the Ktrans in DZ1-treated patients. The percentage of the samples with undetectable level of EBV DNA copy in the DZ1 group was significantly higher than that in the control group. No adverse events that could be attributed to the DZ1 injection were observed in patients.

EBV encoded miR-BHRF1-1 potentiates viral lytic replication by downregulating host p53 in nasopharyngeal carcinoma

miRNAs (microRNAs) are a class of non-coding small RNAs. The Epstein-Barr-virus (EBV) encoded miR-BHRF1-1 is barely expressed in most nasopharyngeal carcinoma (NPC) cells with EBV latent infection. Here, we used a strategy of overexpression and inhibition of miR-BHRF1-1 and showed that miR-BHRF1-1 is involved in TPA-induced accumulation of EBV lytic proteins and viral copies in late lytic cycle. The data further suggested that the miR-BHRF1-1-potentiated induction of EBV lytic replication was accompanied by inhibiting p53 expression. Our results demonstrated that the EBV original pathogen miR-BHRF1-1 is involved in the control of EBV late lytic replication by directly targeting the host p53 gene.

FOXC2 promotes chemoresistance in nasopharyngeal carcinomas via induction of epithelial mesenchymal transition

Paclitaxel (Taxol) is currently used as the front-line chemotherapeutic drug for many types of human cancers. However, the emergence of drug resistance has been a major obstacle to the effective treatment of cancers in clinical settings. The transcription factor Forkhead box protein C2 (FOXC2) was recently demonstrated to activate the epithelial-mesenchymal transition (EMT). In this article, we present a novel role of FOXC2 in regulating chemoresistance of nasopharyngeal carcinoma (NPC) through the EMT. Using an EMT PCR array based on the screening of 84 genes, the expression of FOXC2 was notably upregulated in paclitaxel-resistant NPC cells (CNE2/t). We observed that the paclitaxel-resistant cells exhibited characteristic EMT phenotypes. The silencing of FOXC2 expression in the resistant cells can reverse the EMT molecular markers and chemoresistant phenotypes, such as cellular morphology, proliferation and anoikis. In an NPC xenograft mouse model, the downregulation of FOXC2 expression in the resistant NPC cells increased their sensitivity to paclitaxel treatment, resulting in reduced tumor growth. Taken together, our results suggest that FOXC2-mediated EMT may be an alternative mechanism through which cancer cells can initiate and maintain drug resistance. Thus, targeting FOXC2 may provide a novel strategy for overcoming chemoresistance in NPC therapy.

Association of Follicle-Stimulating Hormone Receptor Polymorphisms with Ovarian Response in Chinese Women: A Prospective Clinical Study

Background Follicular stimulating hormone (FSH) is a glycoprotein and widely used for the treatment of infertility; FSH action is mediated by FSH receptor (FSHR), SNPs of which determine the ovarian response. Two polymorphisms of the FSHR gene were identified, which caused a change of threonine (T) to alanine (A) at position 307 and asparagine (N) to serine(S) at position 680. Both polymorphic sites give rise to three discrete variants of the FSHR: TT, TA, and AA for position 307; NN, NS, and SS for position 680. Methodology/Principal Findings 450 Chinese women were recruited in an assisted reproductive technology program from October 2011 to March 2012. FSHR polymorphisms at the positions 307 and 680 were examined by PCR-RFLP. Serum FSH and estradiol level, FSH amount, ovarian response and pregnancy rate were recorded during treatment. The basal FSH levels were higher in AA [7.38 ± 2.07 vs 6.34 ± 1.75, 6.63 ± 1.94, P<0.05, 95% CI (6.75, 8.01)] and SS [7.51 ± 2.01 vs 6.31 ± 1.75, 6.66 ± 1.96, P<0.05, 95% CI (6.88, 8.15)] compared to other genotypes respectively; the days for ovulation induction was slightly longer in AA and SS. Women with AA and SS have higher rates of poor response compared to carriers of other genotypes (P<0.05). Furthermore, there is a nearly complete linkage between these two polymorphisms in Chinese women (D’=0.95, r2=0.84). Conclusions/Significance In Chinese women receiving ART, the subjects with AA and SS genotypes have higher basal FSH levels, and these genotypes are associated with an increased risk of poor response. Our data suggested that the personalized FSH therapy may be applied according to patient’s genetic background in clinical settings. The linkage suggested that the polymorphisms of Thr307Ala and Asn680Ser may be used as TAG-SNP markers for analysis of potential genotyping in ART.

Knockdown of ANLN by lentivirus inhibits cell growth and migration in human breast cancer

Anillin (ANLN), an actin-binding protein, is required for cytokinesis. Recently, ANLN has been identified as a biomarker in diverse human cancers; however, the precise role of ANLN in breast cancer remains unclear. In this study, we firstly detected the expression of ANLN in 71 patients with breast cancer by immunohistochemistry, and found ANLN was highly expressed in breast cancer tissues. To evaluate the function of ANLN in breast cancer cells, we employed lentivirus-mediated RNA interference to knock down ANLN expression in two human breast cancer cell lines, MDA-MB-231, and ZR-75-30. Knockdown of ANLN remarkably inhibited the proliferation rate and colony formation ability of both breast cancer cell lines. Moreover, flow cytometry analysis showed that depletion of ANLN in MDA-MB-231 cells blocked the cell cycle progression, with more cells delayed at G2/M phase, due to phosphorylation of Cdc2 and suppression of Cyclin D1. Furthermore, knockdown of ANLN strongly suppressed the migration of breast cancer cells, strengthening the evidence that ANLN could be involved in breast cancer progression. Our results may suggest ANLN as a potential target candidate in breast cancer.

Delivery system for DNAzymes using arginine-modified hydroxyapatite nanoparticles for therapeutic application in a nasopharyngeal carcinoma model.

DNAzymes are synthetic, single-stranded, catalytic nucleic acids that bind and cleave target mRNA in a sequence-specific manner, and have been explored for genotherapeutics. One bottleneck restricting their application is the lack of an efficient delivery system. As an inorganic nanomaterial with potentially wide application, nano-hydroxyapatite particles (nHAP) have attracted increasing attention as new candidates for nonviral vectors. In this study, we developed an nHAP-based delivery system and explored its cellular uptake mechanisms, intracellular localization, and biological effects. Absorption of arginine-modified nanohydroxyapatite particles (Arg-nHAP) and DZ1 (latent membrane protein 1 [LMP1]-targeted) reached nearly 100% efficiency under in vitro conditions. Using specific inhibitors, cellular uptake of the Arg-nHAP/DZ1 complex was shown to be mediated by the energy-dependent endocytosis pathway. Further, effective intracellular delivery and nuclear localization of the complex was confirmed by confocal microscopy. Biologically, the complex successfully downregulated the expression of LMP1 in nasopharyngeal carcinoma cells. In a mouse tumor xenograft model, the complex was shown to be delivered efficiently to tumor tissue, downregulating expression of LMP1 and suppressing tumor growth. These results suggest that Arg-nHAP may be an efficient vector for nucleic acid-based drugs with potential clinical application.

Targeting EBV-LMP1 DNAzyme enhances radiosensitivity of nasopharyngeal carcinoma cells by inhibiting telomerase activity

The latent membrane protein 1 (LMP1), which is encoded by the Epstein–Barr virus (EBV), has been suggested to be one of the major oncogenic factors in nasopharyngeal carcinoma (NPC). In previous studies, we experimentally demonstrated that downregulation of LMP1 expression by targeting EBV-LMP1 DNAzyme (Dz1) could increase the radiosensitivity of NPC. However, how Dz1 contributes to the radiosensitivity in NPC is still not clear. In the present study, we confirmed that Dz1 decreases LMP1 expression and downregulates the expression of the catalytic subunit of telomerase (hTERT), both at the protein and mRNA levels (P < 0.01), and therefore, consequently inhibits telomerase activity (P < 0.05) in LMP1-positive NPC cells. We also observed that LMP1 mediated Akt phosphorylation is involved in the regulation of hTERT expression and phosphorylation. After LMP1 and hTERT expression was silenced by Dz1 and hTERT-targeted siRNA, respectively, the radiosensitivity increased in CNE1-LMP1 cells (P < 0.05). The inhibition was more significant after Dz1 treatment was combined with siRNA (P < 0.01). We concluded that hTERT expression and phosphorylation could be regulated by LMP1 through the Akt pathway, and Dz1 enhances radiosensitivity of LMP1-positive NPC cells by inhibiting telomerase activity.