Search

Supplementary Figure S1.

Purpose: To investigate expression, regulation, potential role and targets of miR-195 and miR-497 in breast cancer.Experimental Design: The expression patterns of miR-195 and miR-497 were initially examined in breast cancer tissues and cell lines by Northern blotting and quantitative real-time PCR. Combined bisulfite restriction analysis and bisulfite sequencing were carried out to study the DNA methylation status of miR-195 and miR-497 genes. Breast cancer cells stably expressing miR-195 and miR-497 were established to study their role and targets. Finally, normal, fibroadenoma and breast cancer tissues were employed to analyze the correlation between miR-195/497 levels and malignant stages of breast tumor tissues.Results: MiR-195 and miR-497 were significantly downregulated in breast cancer. The methylation state of CpG islands upstream of the miR-195/497 gene was found to be responsible for the downregulation of both miRNAs. Forced expression of miR-195 or miR-497 suppressed breast cancer cell proliferation and invasion. Raf-1 and Ccnd1 were identified as novel direct targets of miR-195 and miR-497. miR-195/497 expression levels in clinical specimens were found to be correlated inversely with malignancy of breast cancer.Conclusions: Our data imply that both miR-195 and miR-497 play important inhibitory roles in breast cancer malignancy and may be the potential therapeutic and diagnostic targets. Clin Cancer Res; 17(7); 1722–30. ©2011 AACR.

Background: The therapeutic resistance to ionising radiation (IR) and anti-angiogenesis mainly impair the prognosis of patients with glioblastoma. The primary and secondary MET aberrant activation is one crucial factor for these resistances. The kringle 1 domain of hepatocyte growth factor (HGFK1), an angiogenic inhibitor, contains a high-affinity binding domain of MET; however, its effects on glioblastoma remain elusive. Methods: We formed the nanoparticles consisting of a folate receptor-targeted nanoparticle-mediated HGFK1 gene (H1/pHGFK1) and studied its anti-tumoural and radiosensitive activities in both subcutaneous and orthotopic human glioma cell-xenografted mouse models. We then elucidated its molecular mechanisms in human glioblastoma cell lines in vitro. Results: We demonstrated for the first time that peritumoural injection of H1/pHGFK1 nanoparticles significantly inhibited tumour growth and prolonged survival in tumour-bearing mice, as well as enhanced the anti-tumoural efficacies of IR in vivo by reducing Ki-67 expression, enhancing TUNEL staining-indicated apoptotic indexes, reducing microvascular intensity and reversing IR-induced MET overexpression in tumour tissues. Furthermore, we showed that HGFK1 suppressed the proliferation and induced cell apoptosis and enhanced sensitivity to IR in glioblastoma cell lines, mainly by suppressing the activities of MET receptor, down-regulating ATM-Chk2 axis but up-regulating Chk1. Conclusions: H1/pHGFK1 exerts anti-tumoural and radiosensitive activities mainly through the inhibition and reversal of IR-induced MET and ATM–Chk2 axis activities in glioblastoma. H1/pHGFK1 nanoparticles are a potential radiosensitiser and angiogenic inhibitor for glioblastoma treatment.

Breast cancer is the second highest cause of carcinoma-related death caused by distant metastasis in women. Estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2) and progesterone receptor (PR) are three classified makers of breast cancer, which are defined as ER + , HER2 + , and the most serious ER − PR − HER2− (triple-negative). It is well known that ErbB2 (V-Erb-B2 avian erythroblastic leukemia viral oncogene homolog 2) plays an important part in breast cancer. However, the molecular mechanisms underlying ErbB2 action needs to be well studied. In this report, we discovered that the decreased expression levels of miR-34a were inversely correlated with the increased ErbB2 levels in breast cancer. A luciferase reporter assay was done to understand the potential correlation between ErbB2 and miR-34a. Over-expression of miR-34a reduces ErbB2 expression and suppresses breast cancer cell invasion and growth in vitro. What's more, reduced expression of ErbB2 inhibits breast Cancer cell proliferation and re-expression of ErbB2 reversed miR-34a-dependent tumor suppression. Meanwhile, miR-34a levels were correlated inversely with breast cancer malignancy. Our study demonstrates that miR-34a, like ErbB2, might be a diagnostic target in breast cancer.

Interferon-induced transmembrane protein 1 (IFITM1) is one of the interferon-induced transmembrane protein family members. In this study, we reported that the elevated IFITM1 expression in human colorectal cancer (CRC) significantly correlated with CRC lymph node and distance metastasis as well as a more advanced clinical stage. Importantly, elevated IFITM1 expression is an independent prognostic factor for poor survival. To investigate the molecular mechanisms, we showed that over-expression of IFITM1 in CRC cells promoted, whereas knockdown of IFITM1 expression inhibited, cell migration/invasion and tumorigenicity in vitro. Furthermore, we identified Caveolin-1 (CAV1) as a downstream target of IFITM1-induced cell invasion, as knockdown of CAV1 abrogated siIFITM1 mediated inhibition of cell invasion in CRC cells. In addition, in a CRC cohort of 229 patients, the expression of IFITM1 inversely correlated with the expression of CAV1. These results suggested that IFITM1 promotes the aggressiveness of CRC cells, and it is a potential prognostic marker and therapeutic target for CRC.

SNPs in human AFP promoter are associated with serum AFP levels in hepatocellular carcinoma (HCC), suggesting that AFP promoter variants may generate better transcriptional activities while retaining high specificity to AFP-producing cells. We sequenced human AFP promoters, cloned 15 different genotype promoters and tested their reporter activities in AFP-producing and non-producing cells. Among various AFP variant fragments tested, EA4D exhibited the highest reporter activity and thus was selected for the further study. EA4D was fused with tBid and coupled with nano-particle vector (H1) to form pGL3-EA4D-tBid/H1. pGL3-EA4D-tBid/H1 could express a high level of tBid while retain the specificity to AFP-producing cells. In a HCC tumor model, application of pGL3-EA4D-tBid/H1 significantly inhibited the growth of AFP-producing-implanted tumors with minimal side-effects, but had no effect on non-AFP-producing tumors. Furthermore, pGL3-EA4D-tBid/H1 could significantly sensitize HCC cells to sorafenib, an approved anti-HCC agent. Collectively, pGL3-EA4D-tBid/H1, a construct with the AFP promoter EA4D and the novel H1 delivery system, can specifically target and effectively suppress the AFP-producing HCC. This new therapeutic tool shows little toxicity in vitro and in vivo and it should thus be safe for further clinical tests.

MicroRNAs (miRNAs) are small, non-coding RNAs which regulate gene expression at the post-transcriptional level. Abnormal expression of miRNAs occurs frequently in human tumors. Despite the fact that reduced expression of miR-128 has been observed in glioma tissues and cells, the role of miR-128 in tumors has not been fully characterized. In the present study, cell adhesion assays indicated that overexpression of miR-128 can promote cell-cell adhesion. Target site prediction algorithms indicated that miR-128 binds the 3'-untranslated regions of erythropoietin-producing hepatocellular receptor (Eph)B1 and EphB2 mRNAs. Luciferase reporter assays confirmed that miR-128 binds and regulates EphB1 and EphB2 mRNAs. Overexpression of EphB2 reduced the ability of miR-128 to promote cell-cell adhesion. The wound-healing assay indicated that miR-128 significantly inhibited cell migration via EphB2. This study revealed the novel functions of miR-128 in cell-cell adhesion and cell migration in glioma cells through the regulation of EphB2, and identified EphB1 and EphB2 as novel miR-128 targets.

Breast cancer is the second highest cause of carcinoma-related death caused by distant metastasis in women. Estrogen receptor (ER), human epidermal growth factor receptor 2, (HER2) and progesterone receptor (PR) are three classified makers of breast cancer, which are defined as ER+, HER2+, and the most serious ER-PR-HER2- (triple-negative). It is well known that ErbB2 (V-Erb-B2 avian erythroblastic leukemia viral oncogene homolog 2) plays an important part in breast cancer. However, the molecular mechanisms underlying ErbB2 action needs to be well studied. In this report, we discovered that the decreased expression levels of miR-34a were inversely correlated with the increased ErbB2 levels in breast cancer. A luciferase reporter assay was done to understand the potential correlation between ErbB2 and miR-34a. Over-expression of miR-34a reduces ErbB2 expression and suppresses breast cancer cell invasion and growth in vitro. What's more, reduced expression of ErbB2 inhibits breast Cancer cell proliferation and re-expression of ErbB2 reversed miR-34a-dependent tumor suppression. Meanwhile, miR-34a levels were correlated inversely with breast cancer malignancy. Our study demonstrates that miR-34a, like ErbB2, might be a diagnostic target in breast cancer.

Angiogenesis has been found as an attractive target for drug therapy as it is necessary for tumor growth. Accumulating evidences show that microRNAs (miRNAs), which are a group of highly conserved, single-stranded, short non-coding RNAs, play important roles through directly targeting angiogenic factors and protein kinases. The purpose of this study is to investigate the role of miR-195 in breast cancer development and angiogenesis through targeting IRS1. We show that miR-195 is inversely related with Insulin receptor substrate 1 (IRS1) in both breast cancer cells and breast cancer tissues. Induction of miR-195 could suppress IRS1 protein expression through binding to its 3'UTR regions either by transfection with miR-195 oligo or by infection with lentivirus encoding miR-195 gene. Moreover, re-expression of IRS1 reverses miR-195-mediated repression of tumor cell growth and miR-195 inhibits tumor angiogenesis through suppressing IRS1-VEGF axis. These data suggest that miR-195 mimics are potential therapeutic agents for breast cancer diagnose.

The recent outbreak of enterovirus 71 (EV71) infected millions of children and caused over 1,000 deaths. To date, neither an effective vaccine nor antiviral treatment is available for EV71 infection. Interferons (IFNs) have been successfully applied to treat patients with hepatitis B and C viral infections for decades but have failed to treat EV71 infections. Here, we provide the evidence that EV71 antagonizes type I IFN signaling by reducing the level of interferon receptor 1 (IFNAR1). We show that the host cells could sense EV71 infection and stimulate IFN-β production. However, the induction of downstream IFN-stimulated genes is inhibited by EV71. Also, only a slight interferon response and antiviral effects could be detected in cells treated with recombinant type I IFNs after EV71 infection. Further studies reveal that EV71 blocks the IFN-mediated phosphorylation of STAT1, STAT2, Jak1, and Tyk2 by reducing IFNAR1. Finally, we identified the 2A protease encoded by EV71 as an antagonist of IFNs and show that the protease activity is required for reducing IFNAR1 levels. Taken together, our study for the first time uncovers a mechanism used by EV71 to antagonize type I IFN signaling and provides new targets for future antiviral strategies.

miR-124 is a brain-enriched microRNA that plays a crucial role in neural development and has been shown to be down-regulated in glioma and medulloblastoma, suggesting its possible involvement in brain tumor progression. Here, we show that miR-124 is down-regulated in a panel of different grades of glioma tissues and in all of the human glioma cell lines we examined. By integrated bioinformatics analysis and experimental confirmation, we identified SNAI2, which is often up-regulated in glioma, as a direct functional target of miR-124. Because SNAI2 has been shown to regulate stem cell functions, we examined the roles of miR-124 and SNAI2 in glioma cell stem-like traits. The results showed that overexpression of miR-124 and knockdown of SNAI2 reduced neurosphere formation, CD133 + cell subpopulation, and stem cell marker (BMI1, Nanog, and Nestin) expression, and these effects could be rescued by re-expression of SNAI2. Furthermore, enhanced miR-124 expression significantly inhibited glioma cell invasion in vitro. Finally, stable overexpression of miR-124 and knockdown of SNAI2 inhibited the tumorigenicity and invasion of glioma cells in vivo. These findings reveal, for the first time, that the tumor suppressor activity of miR-124 could be partly due to its inhibitory effects on glioma stem-like traits and invasiveness through SNAI2. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc., link_to_subscribed_fulltext

Anti-angiogenesis-related therapies have become the standard care for patients with advanced hepatocellular carcinoma (HCC), as HCC is a highly vascularized solid tumor. Unfortunately, only modest and limited efficacies are observed. Emerging evidence have attributed to the limited efficacy to the presence of cancer stem cells (CSCs) in the tumor. CSCs predominantly drives angiogenesis via releasing proangiogenic factors and exosomes. They have the ability to resistant intratumoral hypoxia via autophagy or by directly forming the tubular structure to obtain blood. On the other hand, the vascular niche in tumor microenvironment also releases growth factors via juxtacrine and paracrine mechanisms to support the growth of CSCs and maintain its stemness features. This positive feedback loop between angiogenesis and CSCs exists in liver tumor microenvironment that is responsible for the development and poor prognosis of HCC. In this review, we summarize recent advances in our understanding of the crosstalks between angiogenesis and CSCs, and their interactions in liver tumor microenvironment and their purpose that an effective anti-angiogenic therapy should also target CSCs for HCC treatment.

The authors have previously isolated a putative oncogene, eukaryotic initiation factor 5A2 (EIF5A2) from 3q26. In this study, EIF5A2 was characterised for its role in colorectal carcinoma (CRC) aggressiveness and underlying molecular mechanisms.The expression dynamics of EIF5A2 were examined by immunohistochemistry in a cohort of carcinomatous and non-neoplastic colorectal tissues and cells. A series of in-vivo and in-vitro assays was performed to elucidate the function of EIF5A2 in CRC and its underlying mechanisms.The overexpression of EIF5A2 was examined by immunohistochemistry in 102/229 (44.5%) CRC patients, and it was significantly correlated with tumour metastasis and determined to be an independent predictor of shortened survival (p0.05). Ectopic overexpression of EIF5A2 in CRC cells enhanced cell motility and invasion in vitro and tumour metastasis in vivo, and induced epithelial-mesenchymal transition (EMT). The depletion of EIF5A2 expression prevented CRC cell invasiveness and inhibited EMT. Importantly, the metastasis-associated protein 1 (MTA1) gene was identified as a potential downstream target of EIF5A2 in CRC cells, and knockdown of MTA1 eliminated the augmentation of carcinoma cell migration, invasion and EMT by ectopic EIF5A2. The overexpression of EIF5A2 in CRC cells substantially enhanced the enrichment of c-myc on the promoter of MTA1, and MTA1 upregulation by EIF5A2 was partly dependent on c-myc.The data suggest that EIF5A2 plays an important oncogenic role in CRC aggressiveness by the upregulation of MTA1 to induce EMT, and EIF5A2 could be employed as a novel prognostic marker and/or effective therapeutic target for CRC.

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. It is more prevalent in men than women. Related to this, recent genetic studies have revealed a causal role for androgen receptor (AR) in hepatocarcinogenesis, but the underlying molecular mechanism remains unclear. Here, we used genome-wide location and functional analyses to identify a critical mediator of AR signaling - cell cycle-related kinase (CCRK) - that drives hepatocarcinogenesis via a signaling pathway dependent on β-catenin and T cell factor (TCF). Ligand-bound AR activated CCRK transcription and protein expression via direct binding to the androgen- responsive element of the CCRK promoter in human HCC cell lines. In vitro analyses showed that CCRK was critical in human cell lines for AR-induced cell cycle progression, hepatocellular proliferation, and malignant transformation. Ectopic expression of CCRK in immortalized human liver cells activated β-catenin/TCF signaling to stimulate cell cycle progression and to induce tumor formation, as shown in both xenograft and orthotopic models. Conversely, knockdown of CCRK decreased HCC cell growth, and this could be rescued by constitutively active β-catenin or TCF. In primary human HCC tissue samples, AR, CCRK, and β-catenin were concordantly overexpressed in the tumor cells. Furthermore, CCRK overexpression correlated with the tumor staging and poor overall survival of patients. Our results reveal a direct AR transcriptional target, CCRK, that promotes hepatocarcinogenesis through the upregulation of β-catenin/TCF signaling., published_or_final_version

It has been suggested that trimethylation of lysine 27 on histone H3 (H3K27me3) is a crucial epigenetic process in tumorigenesis. However, the expression dynamics of H3K27me3 and its clinicopathological/prognostic significance in hepatocellular carcinoma (HCC) are unclear. In this study, immunohistochemical analysis (IHC) was used to examine protein expression of H3K27me3 in HCC tissues from two independent cohorts and corresponding nontumorous hepatocellular tissues by tissue microarray. The optimal cutpoint of H3K27me3 expression was assessed by the X-tile program. Our results showed that the cutpoint for high expression of H3K27me3 in HCCs was determined when more than 70% of the tumor cells showed positive staining. High expression of H3K27me3 was observed in 134 of 212 (63.2%) and 76 of 126 (60.4%) of HCCs in the testing and validation cohorts, respectively. Correlation analysis demonstrated that high expression of H3K27me3 in HCCs was significantly correlated with large tumor size, multiplicity, poor differentiation, advanced clinical stage and vascular invasion (P < 0.05). In addition, high expression of H3K27me3 in HCC patients was associated closely with shortened survival time, independent of serum α-fetoprotein levels, tumor size and multiplicity, clinical stage, vascular invasion and relapse as evidenced by univariate and multivariate analysis in both cohorts (P < 0.05). In different subsets of HCC patients, H3K27me3 expression was also a prognostic indicator in patients with stage II tumors (P < 0.05). Thus, these findings provide evidence that a high expression of H3K27me3, as detected by IHC, correlates closely with vascular invasion of HCCs and is an independent molecular marker for poor prognosis in patients with HCC. ©2011 The Feinstein Institute for Medical Research., link_to_OA_fulltext

The present study aimed to construct a 1.5X hepatitis B virus (HBV) replication system in vitro that could generate high level of HBV viruses. This system would help compare the replication capacity among the virus strains associated with high and low risk of hepatocellular carcinoma (HCC). Four strains of HBV were isolated from two HCC patients and two HBV carriers. After molecular cloning, four corresponding constructs named as HBV-1.5Xs were generated. Each of them has one and a half copies of HBV 3.2 kb genome, a 5′-end redundant sequence of 1.1 kb to nt715 and a 3′-end redundant sequence of 500 bp to nt2325 that situated after the poly (A) sequence. The HepG2 cells were transfected with the HBV-1.5Xs, and the levels of HBsAg, HBeAg and viral DNA were then detected in both the supernatant and the cells. After 24 h and 48 h of transfection, a high OD value of HBsAg of 3.5 was observed in the supernatant and also in some of the diluted cell lysate samples. The HBeAg level was relatively low in all strain samples of HBV. The log 10 values of viral loads were also determined with the cell lysate having a higher value (10–11 per ml) than that of the supernatant (6–7 per ml). The results showed that the novel HBV-1.5X system was capable to generate high level of HBV in a consistent manner. However, no significant difference was found among the replication capacities among these strains in vitro . The HBV-1.5X system may be a useful platform that assists the establishment of stable cell lines and transgenic mice for the investigation of viral pathogenesis, particularly for the various strains of HBV.

Combined treatment using nonviral agent-mediated enzyme/prodrug therapy and immunotherapy had been proposed as a powerful alternative method of cancer therapy. The present study was aimed to evaluate the cytotoxicity in vitro and the therapeutic efficacy in vivo when the cytosine deaminase/5-fluorocytosine (CD/5-FC) and TNF-related apoptosis-inducing ligand (TRAIL) genes were jointly used against rat C6 glioma cells. The potency of the FA-PEG-PEI used as a nonviral vector was tested in the FR-expressed C6 glioma cells and Wistar rats. The C6 glioma cells and animal model were treated by the combined application of FA-PEG-PEI/pCD/5-FC and FA-PEG-PEI/pTRAIL. The antitumor effect was evaluated by survival assays and tumor volume. This study revealed a significant increase of cytotoxicity in vitro following the combined application of FA-PEG-PEI/pCD/5-FC and FA-PEG-PEI/pTRAIL treatments in C6 glioma cells. Animal studies showed a significant growth inhibition of the C6 glioma xenografts using the combined treatment. These results demonstrated that the combined treatment generated additive cytotoxic effect in C6 glioma cells in both in vitro and in vivo conditions, and indicated that such treatment method using both enzyme/prodrug therapy and TRAIL immunotherapy might be a promising therapeutic strategy in treating glioma.

MicroRNAs (miRNAs) are a class of endogenous, small non-protein coding single-stranded RNA molecules, which are crucial post-transcriptional regulators of gene expression. Previous studies have shown that miRNAs participate in a wide range of biological functions and play important roles in various human diseases including glioma. However, the role of miRNAs in mediating glioblastoma cell migration and invasion has not been elucidated. Using miRNA microarray, we identified miR-146b as one of the miRNAs that is significantly dysregulated in human glioblastoma tissue. We showed that miR-146b overexpression by transfection with the precursor miR-146b, or knock-down by Locked Nucleic Acid (LNA)-modified anti-miR-146b, has no effect on the growth of human glioblastoma U373 cells. However, precursor miR-146b transfection significantly reduced the migration and invasion of U373 cells, while LNA-anti-miR-146b transfection generated the opposite result. Furthermore, we discovered that a matrix metalloproteinase gene, MMP16, is one of the downstream targets of miR-146b. Taken together, our findings suggest that miR-146b is involved in glioma cell migration and invasion by targeting MMPs, and implicate miR-146b as a metastasis-inhibiting miRNA in glioma.

MicroRNAs (miRNAs) are non-protein-coding RNAs that function as post-transcriptional gene regulators. Recent evidence has shown that miRNA plays a pivotal role in the development of many cancers including glioma, a lethal brain cancer. We have recently compared the miRNA expression profiles between normal brain and glioma tissues from Chinese patients by miRNA microarray and identified a panel of differentially expressed miRNAs. Here, we studied the function of one miRNA, miR-15b, in glioma carcinogenesis and elucidated its downstream targets. Over-expression of miR-15b resulted in cell cycle arrest at G0/G1 phase while suppression of miR-15b expression resulted in a decrease of cell populations in G0/G1 and a corresponding increase of cell populations in S phase. We further showed that CCNE1 (encoding cyclin E1) is one of the downstream targets of miR-15b. Taken together, our findings indicate that miR-15b regulates cell cycle progression in glioma cells by targeting cell cycle-related molecules.

Hepatocellular carcinoma (HCC) is an aggressive liver cancer but clinically validated biomarkers that can predict natural history of malignant progression are lacking. The present study explored the proteome-wide patterns of HCC to identify biomarker signature that could distinguish cancerous and non-malignant liver tissues. A retrospective cohort of 80 HBV-associated HCC was included and both the tumor and adjacent non-tumor tissues were subjected to proteome-wide expression profiling by 2-DE method. The subjects were randomly divided into the training (n=55) and validation (n=25) subsets, and the data analyzed by classification-and-regression tree algorithm. Protein markers were characterized by MALDI-ToF/MS and confirmed by immunohistochemistry, western blotting and qPCR assays. Proteomic expression signature composed of six biomarkers (haptoglobin, cytochrome b5, progesterone receptor membrane component 1, heat shock 27 kDa protein 1, lysosomal proteinase cathepsin B, keratin I) was developed as a classifier model for predicting HCC. We further evaluated the model using both leave-one-out procedure and independent validation, and the overall sensitivity and specificity for HCC both are 92.5% respectively. Clinical correlation analysis revealed that these biomarkers were significantly associated with serum AFP, total protein levels and the Ishak’s score. The described model using biomarker signatures could accurately distinguish HCC from non-malignant tissues, which may also provide hints and guidance on how normal hepatocytes are transformed to malignant state during tumor progression.

We evaluated the antidiabetic effects of a mixed vegetable powder-formula I (MVP-FI), which is a dry powder mixture of over 65 kinds of vegetables and fruits, using the db/db type 2 diabetes mouse model. The db/db mice at 8-10 weeks of age were randomly divided into three groups: vehicle treatment, 1.575 g/kg MVP-FI treatment, and 3.15 g/kg MVP-FI treatment. During 12 days of treatment, we measured food intake and body weight changes, fasting blood glucose levels, and plasma lipid levels. Our results showed that the food intake and the body weight of MVP-FI-treated group were decreased gradually. Moreover, the fasting blood glucose level of the treated group was significantly dropped to a normal level comparable to that of the lean mice. Furthermore, we also found that the plasma triglyceride level in the treated group was dropped, whereas the high-density lipoprotein (HDL) level was increased and total cholesterol/HDL-cholesterol ratio was decreased. Taken together, these results suggest that the diabetic conditions of the db/db mice have been improved after 12 days treatment with MVP-FI. The antihyperglycemic and antiobese activities of the MVP-FI, as demonstrated in the present study, may have important clinical implications for improving the management of type 2 diabetic patients. © 2008 Yeung et al, publisher and licensee Dove Medical Press Ltd., published_or_final_version

Insulin inhibits the transcription of the microsomal triglyceride transfer protein (MTTP), which plays a pivotal role in lipoprotein assembly and secretion. Here, we provide evidence that a hepatocyte nuclear factor 1 binding element (HNF1A element) within the MTTP promoter serves as a novel negative insulin-responsive element. Deletion/mutation mapping of the MTTP gene promoter identified a modified HNF1A element that is crucial to the negative insulin effect. Chimeric promoter containing this HNF1A element and minimal TEAD1 promoter also responded negatively toward insulin treatment. Gel shift assay demonstrated that HNF1A but not HNF1B binds to this element. Enforced expression of HNF1A was sufficient to reconstitute the negative insulin responsiveness of MTTP promoter in TM4SF1 myocytes that are HNF1A negative. Furthermore, replacing this element with consensus HNF1A element preserved the negative insulin response, suggesting that negative insulin responsiveness is a generic characteristic of HNF1A element. Given that many genes implicated in diabetes contain HNF1A element, the potential regulation of these genes by insulin via HNF1A element may provide important clues for the manifestation and treatment of diabetic metabolic syndromes.

Chemoresistance and therapeutic selectivity are major obstacles to successful chemotherapy of ovarian cancer. Manganese superoxide disumutase (MnSOD) is an important antioxidant enzyme responsible for the elimination of superoxide radicals. We reported here that MnSOD was significantly elevated in ovarian cancer cells and its overexpression was one of the mechanisms that increased resistance to apoptosis in cancer cells. Knockdown of MnSOD by small-interfering RNA (siRNA) led to an increase in superoxide generation and sensitisation of ovarian cancer cells to the two front-line anti-cancer agents doxorubicin and paclitaxel whose action involved free-radical generation. This synergistic effect was not observed in non-transformed ovarian surface epithelial cells. Furthermore, our results revealed that this combination at the cellular level augmented activation of caspase-3 and caspase-9, but not caspase-8, suggesting involvement of an intrinsic apoptotic pathway. Evaluation of signalling pathways showed that MnSOD siRNA enhanced doxorubicin- and paclitaxel-induced phosphorylation of extracellular signal-regulated kinase 1/2. Akt activation was not affected. These results identify a novel chemoresistance mechanism in ovarian cancer, and show that combination of drugs capable of suppressing MnSOD with conventional chemotherapeutic agents may provide a novel strategy with a superior therapeutic index and advantage for the treatment of refractory ovarian cancer.

Background Interferon-α2 (IFNα2) is routinely used for anti-hepatitis B virus (HBV) treatment. However, the therapeutic efficiency is unsatisfactory, particularly in East Asia. Such inefficiency might be a result of the short half-life, relatively low local concentration and strong side-effects of interferons. Frequent and repeated injection is also a big burden for patients. In the present study, a single dose of vector-delivered IFNα1 was tested for its anti-HBV effects. Methods Adeno-associated viral vector (AAV-IFNα1) was generated to deliver the IFNα1 gene into hepatocytes. IFNα1, hepatitis B surface (HBsAg) and e (HBeAg) antigens were measured by enzyme-linked immunosorbent assay and/or western blotting. The level of viral DNA was measured by quantitative real-time polymerase chain reaction. Results AAV-IFNα1 effectively transduced HBV-producing cells (HepAD38) and mouse hepatocytes, where IFNα1 was expressed in a stable manner. Both intracellular and extracellular HBsAg and HBeAg were significantly reduced in vitro. In the HBV-producing mice, the concentration of IFNα1 in the liver was eight-fold higher than that in plasma. Compared with control groups, HBeAg/HBsAg antigen levels were reduced by more than ten-fold from day 1–5, and dropped to an undetectable level on day 9 in the AAV-IFNα1 group. Concurrently, the level of viral DNA decreased over 30-fold for several weeks. Conclusions A single dose administration of AAV-IFNα1 viral vector displayed prolonged transgene expression and superior antiviral effects both in vitro and in vivo. Therefore, the use of AAV-IFNα1 might be a potential alternative strategy for anti-HBV therapy. Copyright © 2008 John Wiley & Sons, Ltd.

Background High incidence of stroke at interchange period of autumn and winter was demonstrated by epidemiological survey, and the specific causes should be further investigated. Objective To investigate the influence of artificial cold exposure on the incidence of stroke in renovascular hypertensive rats (RHR), and analyze the association with blood pressure and cold-inducible RNA binding protein (CIRP) mRNA expression in brain tissue. Design A completely randomized grouping design, a randomized control animal trial. Settings Lab of Neurology, the First Affiliated Hospital of Sun Yat-sen University; Department of Chemistry, Open laboratory of Chemical Biology, Institute of Molecular Technology for Drug Discovery and Synthesis, University of Hong Kong. Materials Male SD rats ( n = 460), weighing 80–100 g were obtained from Guangdong Province Health Animal Unit. A modified RXZ-300A intelligent artificial climate cabinet (Ningbo Jiangnan Instrument Co., Ltd., China). Methods The experiment were processed in the Lab of Neurology, the First Affiliated Hospital of Sun Yat-sen University and the Open Laboratory of Chemical Biology, Institute of Molecular Technology for Drug Discovery and Synthesis, University of Hong Kong from October 2004 to November 2005. Rats ( n = 400) were operated to establish 2-kidney 2-clip RHR model as described previously. The sham-operated rats ( n = 60) served as normotensive controls. Eight weeks later, 300 of RHR were randomly selected according to their systolic blood pressure (SBP) and divided into 3 sub-groups ( n = 100 per group): mild hypertensive group (SBP of 160–200 mm Hg), moderate hypertensive group (SBP of 200–220 mm Hg) and severe hypertensive group (SBP > 220 mm Hg). Each group was further divided into two groups ( n = 50) under ACE and non-ACE. Normal sham-operated SD rats ( n = 60), SBP n = 30) under ACE and non-ACE. To establish the ACE and non-ACE treatment, rats were housed individually in artificial climate cabinet, and ACE was designed as three cycles of 12-hour light of 22 °C (7:00–19:00) and 12-hour dark of 4 °C(19:00–7:00). The non-ACE group was kept at 22 °C throughout the experiment. Main outcome measures Blood Pressure changes were measured and stroke symptom were observed; Expression of the CIRP were examined by reverse transcription-polymerase chain reaction. Results Finally 360 rats were involved in the analysis of results. Incidence of stroke: The incidence of stroke in 2k2c RHR was significantly higher after a three-day intermittent (12-hour) ACE (29.3%) as compared with that in non-ACE (17.3%) ( P 220 mm Hg) was found to have much higher incidence of stroke (66%, 33/50) than the mild (8%, 4/50) and moderate (18%) hypertensive 2k2c RHR. CIRP mRNA in brain tissue: ACE treatment stimulated the mRNA expression of CIRP in non-stroke 2k2c RHR but not in stroke 2k2c RHR ( P Conclusion High blood pressure and low expression of CIRP are associated with ACE induced stroke.

BACKGROUND Median survival for patients with glioblastoma multiforme, the most aggressive glioma, is only 12-15 months, despite multimodal treatment that includes surgery, chemotherapy, and radiotherapy. Thus, identification of genes that control the progression of glioblastoma multiforme is crucial for devising new therapies. We investigated the involvement of cell cycle-related kinase (CCRK), a novel protein kinase that is homologous to cyclin-dependent kinase 7, in glioblastoma multiforme carcinogenesis. METHODS We analyzed the expression levels of CCRK in 26 glioma patient samples (19 high-grade and seven low-grade) and normal brain by semiquantitative reverse transcription-polymerase chain reaction assays. CCRK expression was knocked down in human glioma U-373 MG and U-87 MG cells with small-interfering RNAs and short hairpin RNAs (siCCRK and shCCRK, respectively), and cell proliferation, cell cycle distribution, and cyclin-dependent kinase 2 (CDK2) phosphorylation were examined. A subcutaneous nude mouse xenograft model (n = 4 mice per group) was used to study the effect of CCRK knockdown and overexpression on tumorigenicity and growth of glioblastoma multiforme cells in vivo. All statistical tests were two-sided. RESULTS CCRK mRNA was elevated at least 1.5-fold and as much as 3.7-fold in 14 (74%) of 19 high-grade glioblastoma multiforme patient samples and in four (80%) of five glioma cell lines examined compared with normal brain tissue. Suppression of CCRK by siCCRK inhibited the proliferation of U-373 MG and U-87 MG glioblastoma cells in a time- and dose-dependent manner. The growth-inhibiting effect of siCCRK was mediated via G1- to S-phase cell cycle arrest and reduced CDK2 phosphorylation. CCRK knockdown statistically significantly suppressed glioma cell growth in vivo as indicated by the mean tumor volumes at week 6 after tumor cell injection (U-373-control = 1352 mm3, U-373-shCCRK = 294 mm3, difference = 1058 mm3, 95% confidence interval [CI] = 677 to 1439 mm3, P

This paper aims to understand the situation and epidemiology trend of neural tube defects in Guizhou province, China from 1996 to 2004. Pregnant women from 17 hospitals in Guizhou province were chosen for investigation of perinatal infants from January 1996 to December 2004. Of 1,208 birth defect cases studied in this 9-year period, a total of 122 cases were identified as neural tube defects (NTD), making up a 10.10% of the total birth defects. The average prevalence rate of NTD was 12.21 per 10,000 births; however, there is no significant difference between the genders. In this study, the age of mothers during pregnancy was shown to be one of the major factors affecting the prevalence rate of NTD and the differences between infants with or without NTD and the usage of folic acid as a supplement during pregnancy were found to be statistical significant. NTD was found to be the most common and serious form of human birth defect in Guizhou province. For prevention, folic acid supplement is thought to be efficacious in inhibiting NTD, and thus, it is essential for people in socially deprived areas to effectively reduce the prevalence of NTD in China.

Enhancer of zeste homolog 2 (EZH2) has been shown to be overexpressed in hepatocellular (HCC). We investigated the potential role of EZH2 in HCC tumorigenesis and examined the usefulness of RNA interference (RNAi) targeting EZH2 as a form of HCC treatment. Lentivirus-mediated RNAi was employed to knock-down EZH2 expression in human hepatoma cells to study the function of EZH2 in tumorigenesis and evaluate the treatment efficacy. Lentivirus-mediated RNAi effectively reduced EZH2 expression. Suppression of EZH2 in HCC cells significantly reduced their growth rate in vitro and markedly diminished their tumorigenicity in vivo. Moreover, in a mice model of established large-sized HCC, we showed that intratumor injection of lentiviral (Lenti)-shRNA (short hairpin RNA) or siRNA (small interfering RNA) targeting EZH2 produced significant tumor regression. To understand its molecular mechanism of action, we employed proteomic profiling technique and found that stathmin 1 is the downstream target of EZH2, as Lenti-shEZH2 treatment decreased stathmin protein expression, and ectopic overexpression of stathmin prevented Lenti-shEZH2 mediated tumor growth inhibition. Conclusion: Results from our study suggested for the first time that EZH2 plays a key role in HCC tumorigenesis, and is a novel therapeutic target for HCC. (HEPATOLOGY 2007;46:200–208.)

SARS-associated coronavirus was identified as the etiological agent of severe acute respiratory syndrome and a large virus pool was identified in wild animals. Virus generates drug resistance through fast mutagenesis and escapes antiviral treatment. siRNAs targeting different genes would be an alternative for overcoming drug resistance. Here, we report effective siRNAs targeting structural genes (i.e., spike, envelope, membrane, and nucleocapsid) and their antiviral kinetics. We also showed the synergistic effects of two siRNAs targeting different functional genes at a very low dose. Our findings may pave a way to develop cost effective siRNA agents for antiviral therapy in the future.

Alcohol is the most common teratogen in humans. Excessive alcohol consumption in pregnant women may lead to fetal alcohol syndrome (FAS), which is characterized by microcephaly, growth retardation, facial dysmorphology, and abnormalities in the central nervous system. Despite extensive studies, the molecular mechanisms underlying FAS remain unclear. To this end, we established a Xenopus laevis embryo model to study the effects of alcohol on fetal development. We will discuss the relationship between alcohol-induced oxidative and nitrosative stresses and their differential and critical roles in the downregulation of key developmental genes and birth defects in the X. laevis embryo model.

We have previously shown that gene therapy using adeno-associated virus (AAV) carrying bone morphogenetic proteins (BMPs) is a promising strategy for new bone formation in vivo in SD rats. However, it had a relatively low transduction efficiency. We investigate here whether enhanced osteogenic activity can be achieved without eliciting a severe immune response, using a cocktail of AAV-BMP2 and adenovirus (Ad)-BMP2 as a vector system. The muscles of SD rats were injected with either AAV-BMP2, Ad-BMP2, or an AAV-BMP2/Ad-BMP2 cocktail, and the in vivo bone formation was determined at eight weeks post-injection. Radiographic examination demonstrated that the addition of a low level of Ad-BMP2 to AAV-BMP2 produced significantly higher new bone formation than the use of AAV-BMP2 alone. Histological and immunohistological analysis revealed an enlarged bone-forming area and a long-term BMP2 expression, without pronounced infiltration of lymphocytes. Our results provide the first evidence that the introduction of a low level of adenovirus in vivo in immunocompetent subjects can greatly enhance AAV-mediated gene transfer, without inducing severe immune responses. This cocktail vector system may offer an attractive way of improving the efficiency of AAV-based gene delivery.

Nasopharyngeal carcinoma (NPC) is a highly metastatic tumor characterized by close association with EBV. Of the EBV-encoded products, latent membrane protein-1 (LMP-1) is thought to be the only oncoprotein playing an essential role in cell transformation as well as tumor metastasis. In this study, we tested the effect of suppressing LMP-1 by RNA interference (RNAi) on the proliferative and metastatic potentials of an EBV-positive NPC cell line, C666. We showed that stably suppressing LMP-1 by short hairpin RNA (shRNA) plasmid significantly altered cell motility, substratum adhesion, and transmembrane invasion ability. However, it has little effect on the rate of cell growth and cell cycle control. These results demonstrated the effectiveness of RNAi in suppressing LMP-1, supporting an important role of LMP-1 in NPC metastasis, and suggested a potential application of RNAi-mediated therapeutic strategy for EBV-related NPC.

The putative NTPase/helicase protein from severe acute respiratory syndrome coronavirus (SARS-CoV) is postulated to play a number of crucial roles in the viral life cycle, making it an attractive target for anti-SARS therapy. We have cloned, expressed, and purified this protein as an N-terminal hexahistidine fusion in Escherichia coli and have characterized its helicase and NTPase activities. The enzyme unwinds double-stranded DNA, dependent on the presence of a 5′ single-stranded overhang, indicating a 5′o 3′ polarity of activity, a distinct characteristic of coronaviridae helicases. We provide the first quantitative analysis of the polynucleic acid binding and NTPase activities of a Nidovirus helicase, using a high throughput phosphate release assay that will be readily adaptable to the future testing of helicase inhibitors. All eight common NTPs and dNTPs were hydrolyzed by the SARS helicase in a magnesium-dependent reaction, stimulated by the presence of either single-stranded DNA or RNA. The enzyme exhibited a preference for ATP, dATP, and dCTP over the other NTP/dNTP substrates. Homopolynucleotides significantly stimulated the ATPase activity (15–25-fold) with the notable exception of poly(G) and poly(dG), which were non-stimulatory. We found a large variation in the apparent strength of binding of different homopolynucleotides, with dT24 binding over 10 times more strongly than dA24 as observed by the apparent Km .

Hepatitis B virus (HBV), a serious infectious and widespread human pathogen, represents a major health problem worldwide. Chronic HBV infection has a very high risk of evolving into hepatocellular carcinoma. Although considerable progress was made during the recent past, the pathogenesis of HBV infection is still elusive and a definite diagnosis of HBV infected liver information still relies on biopsy histological test. In this report, we used proteomics technology to globally examine HBV infected serum samples aiming at searching for disease-associated proteins that can be used as serological biomarkers for diagnosis and/or target proteins for pathogenetic study. By comparing with normal and HBV negative serum samples, we found that at least seven proteins were significantly changed in HBV infected sera. These greatly altered proteins were identified to be haptoglobin beta and alpha2 chain, apolipoprotein A-I and A-IV, alpha1-antitrypsin, transthyretin and DNA topoisomerase IIbeta. The alteration of these proteins is displayed not only in quantity but also in patterns (or specificity), which can be correlated with necroinflammatory scores. In particular, apolipoprotein A-I presents heterogeneous change in expression level with different isoforms and alpha1-antitrypsin produces evidently different fragments implying diverse cleavage pathways. These unique phenomena appear specific to HBV infection. A combination simultaneously considering the quantities and isoforms of these proteins could be a useful serum biomarker (or index) for HBV diagnosis and therapy.

Microsomal triglyceride transfer protein (MTP) is rate limiting for the assembly and secretion of apolipoprotein B-containing lipoproteins. Elevated hepatic MTP mRNA level, presumably as a result of impaired insulin signaling, has been implicated in the pathophysiology of dyslipidemia associated with insulin resistance/type 2 diabetes. In this study, we showed that insulin decreases MTP mRNA level mainly through transcriptional regulation in HepG2 cells. We further characterized the corresponding signal transduction pathway, using chemical inhibitors and constitutively active and dominant negative forms of regulatory enzymes. We demonstrated that insulin inhibits MTP gene transcription through MAPKerk cascade but not through the PI 3-kinase pathway. Activation of ras through farnesylation is not a prerequisite for the inhibition. In addition, cellular MAPKerk and MAPKp38 activities play a counterbalancing role in regulating the MTP gene transcription. These complex regulations may represent a means to fine-tuning MTP gene transcription in response to a diverse set of environmental stimuli and may have important implications for the onset and development of diabetes-associated dyslipidemia.

T box (Tbx) genes are a large family of transcription regulators that play critical roles in invertebrate and vertebrate development. Mutations in Tbx5 gene have been found to cause Holt-Oram syndrome (HOS) in humans. Partial dysfunction of TBX5 in mouse also causes HOS phenotype. Little is known about its molecular and cellular mechanism. Here, we report that ectopic expression of TBX5 inhibited colony formation, induced apoptosis, and decreased the growth rate of cells. The two point mutations in T domain and a truncated mutation in C-terminal found in human HOS patients produced TBX5 mutant proteins with a significantly reduction of colony suppression activity. Deletion of the DNA-binding domain, however, nearly completely abrogated its ability to suppress colony formation. These results reveal TBX5 as a new regulator of apoptosis and cell growth, suggesting a possible mechanism for Holt-Oram syndrome, and a potential reagent for controlling tumor growth.

Apoptosis is an active process critical for the homeostasis of organisms. Enzymes of the caspase family are responsible for executing this process. We have previously shown that peroxynitrite (ONOO−), a biological product generated from the interaction of nitric oxide and superoxide, induces apoptosis of HL-60 cells. The aim of this study was to elucidate the mechanisms involved in the execution process of peroxynitrite-induced apoptosis. Proteolytic cleavage of poly(ADP-ribose) polymerase, an indication of caspase-3 family protease activation and an early biochemical event accompanying apoptosis, was observed in a time-dependent manner during peroxynitrite-induced apoptosis of HL-60 cells. Activation of caspase-3 during peroxynitrite-induced apoptosis was substantiated by monitoring proteolysis of the caspase-3 proenzyme and by measuring caspase-3 activity with a fluorogenic substrate. Furthermore, pretreatment of HL-60 cells with N-acetyl-Asp-Glu-Val-Asp-aldehyde, a specific inhibitor of caspase-3, but not N-acetyl-Tyr-Val-Ala-Asp-aldehyde, a specific inhibitor of caspase-1, decreased peroxynitrite-induced apoptosis. These results suggest that the activation of a caspase-3 family protease is essential for initiating the execution process of peroxynitrite-induced apoptosis of HL-60 cells.

The liver plays a central role in lipoprotein metabolism. In particular, very-low density lipoprotein (VLDL) is assembled in the hepatocytes and secreted into the blood circulation. The VLDL is then catabolized to low-density lipoprotein by lipoprotein lipase and hepatic triglyceride lipase. Obese subjects, especially those with visceral fat accumulation, are frequently associated with hyperlipidemia, non-insulin-dependent diabetes mellitus (NIDDM), and hypertension. The mechanism of hyperlipidemia in visceral fat obesity has not yet been elucidated. Otsuka Long-Evans Tokushima Fatty (OLETF) rat is an animal model of NIDDM, characterized by obesity with visceral fat accumulation, hyperlipidemia, and late-onset insulin resistance. To elucidate the mechanism of hyperlipidemia observed in OLETF rats, we focused on the production of VLDL by the liver and investigated hepatic messenger RNA (mRNA) levels of microsomal triglyceride transfer protein (MTP), acyl-coenzyme A synthetase (ACS), and apolipoprotein B (apo B), which play important roles in VLDL synthesis and secretion. In 6-week-old OLETF rats, in which insulin resistance had not been manifested, visceral fat weight was already higher and portal free fatty acid (FFA) and VLDL-triglyceride levels were elevated compared with the control rats. Hepatic ACS activity and mRNA levels, and MTP mRNA levels were also increased in OLETF rats, whereas apo B mRNA levels were similar; these results suggest that the enhanced expression of both ACS and MTP genes associated with visceral fat accumulation before developing insulin resistance may be involved in the pathogenesis of hyperlipidemia in obese animal models with NIDDM.

Gliomas are the most common and aggressive primary tumors in the central nervous system. Recently, Max interactor-1 (MXI1), an antagonist of c-Myc that is involved in brain tumor progression, has been reported to be deregulated in a variety of tumors including glioma. However, the mechanism of MXI1 deregulation in gliomas remains unclear. In this study, we show that the relative expression level of MXI1 is markedly down-regulated in glioma cell lines. Using integrated bioinformatic analysis and experimental confirmation, we identified several miRNAs by screening a panel of predicted miRNAs that may regulate the MXI1 3'UTR. The strongest inhibitory miRNA, miR-155, can attenuate the activity of a luciferase reporter gene that is fused with the MXI1 3'UTR and decrease the expression levels of MXI1 mRNA and protein in U87 glioma cells. The potential role of miR-155 in promoting glioma cell proliferation by targeting MXI1 was confirmed in various glioma cell lines by rescue experiments using MTT assays, EdU incorporation assay, and cell counting experiments. In addition, we determined that the level of MXI1 mRNA was inversely correlated with the expression of miR-155 in 18 sets of glioblastoma multiforme specimens. These findings reveal for the first time that the targeting of MXI1 by miR-155 may result in a reduction in MXI1 expression and promote glioma cell proliferation; this result suggests a novel function of miR-155 in targeting MXI1 in glioma-genesis.

Diallyl sulfone (DASO2) is a metabolite of diallyl sulfide, a compound derived from garlic. The present study investigated the effect of DASO2 as a protective agent against acetaminophen (APAP)-induced hepatotoxicity in mice. Oral administration of DASO2 protected mice against the APAP-induced hepatotoxicity in a dose- and time-dependent manner. When administrated 1 hour prior to, immediately after, or 20 minutes after a toxic dose of APAP, DASO2 at a dose of 25 mg/kg completely protected mice from development of hepatotoxicity, as indicated by liver histopathology and serum lactate dehydrogenase levels. Protective effect was observed when DASO2 at a dose as low as 5 mg/kg was given to mice 1 hour prior to APAP administration. Oral administration of DASO2 to mice 1 hour prior to a toxic dose of APAP significantly inhibited the APAP-induced glutathione depletion in the liver. DASO2 treatment also decreased the levels of oxidative APAP metabolites in the plasma without affecting the concentrations of nonoxidative APAP metabolites. In liver microsomes, 0.1 mM of DASO2 caused a 60% decrease in the rate of APAP oxidation to N-acetyl-p-benzoquinone imine, which was determined as glutathione conjugate. This inhibitory effect is mainly due to its inhibition of cytochrome P450 2E1 activity; with an IC50 value equal to 0.11 mM. DASO2 also slightly inhibited the activities of P450s 3A and 1A, with IC50 values >5 mM. Furthermore, a single oral dose of DASO2 inactivated P450 2E1- and P450 1A-dependent activities in liver microsomes. The results suggest that the protective effect of DASO2 against APAP-induced hepatotoxicity is due to its ability to block acetaminophen bioactivation mainly by the inactivation and inhibition of P450 2E1. © 1996 John Wiley & Sons, Inc.

The recombinant adenovirus is evolving as a promising gene delivery vector for gene therapy due to its efficiency in transducing different genes into most types of cells. However, the host-immune response elicited by primary inoculation of an adenovirus can cause rapid clearance of the vector, impairing the efficacy of the adenovirus and hence obstructing its clinical application. We have previously synthesized a biodegradable co-polymer consisting of a low molecular weight PEI (MW 600 Da), cross-linked with β-cyclodextrin, and conjugated with folic acid (PEI-CyD-FA, named H1). Here we report that coating the adenovirus vector (Adv) with H1 (H1/rAdv) could significantly improve both the efficacy and biosafety of Adv. Enhanced transfection efficiency as well as prolonged duration of gene expression were clearly demonstrated either by intratumoral or systemic injection of a single dose of H1/rAdv in immunocompetent mice. Importantly, repeated injections of H1/rAdv did not reduce the transfection efficiency in immunocompetent mice. Furthermore, H1 transformed the surface charge of the adenovirus capsomers from negative to positive in physiological solution, suggesting that H1 coated the capsid protein of the adenovirus. This could shelter the epitopes of capsid proteins of the adenovirus, resulting in a reduced host-immune response and enhanced transfection efficiency. Taken together, these findings suggest that H1/rAdv is an effective gene delivery system superior to the adenovirus alone and that it could be considered as a preferred vehicle for gene therapy.

Chemoradiotherapy (CRT) is a standard treatment for oesophageal squamous cell carcinoma (ESCC) in its advanced stages. The telomerase/telomere interacting protein PinX1 contributes to telomere maintenance, tumourigenicity, and influences the DNA damage agent-induced apoptotic response in telomerase-positive cancer cells. However, the clinical and biological significance of PinX1 in human ESCCs remains unclear. We examined the expression dynamics of PinX1 by immunohistochemistry in a learning cohort (n = 98) and a validation cohort (n = 59) of ESCC patients treated with definite chemoradiotherapy (CRT). A series of in vivo and in vitro assays were performed to elucidate the effect of PinX1 on ESCC cells' CRT response and underlying mechanisms. Knockdown of PinX1 did not affect ESCC cells' chemosensitivities to 5-fluorouracil and cisplatin, but substantially increased ESCC cells' therapeutic efficacy of radiation both in vitro and in vivo. Ectopic overexpression of PinX1 dramatically enhanced ESCC cells' resistance to radiotherapy. Furthermore, we demonstrated that PinX1 resistance to radiotherapy (RT) was attributed to PinX1 maintaining telomere stability, reducing ESCC cell death by RT-induced mitosis catastrophe (MC). High expression of Pinx1 correlated positively with ESCC's resistance to CRT, and was a strong and independent predictor for short disease-specific survival (DSS) of ESCC patients. Our data suggest that PinX1 could serve as a novel predictor for a CRT response to ESCC patients, and the pathway of PinX1-mediated telomere stability might represent a new target to improve the RT effect of ESCC.

MiR-145 is known as a tumor suppressor in numerous human cancers. However, its role in tumor angiogenesis remains poorly defined. In this study, we found that miR-145 was significantly downregulated in breast cancer tissues by using 106 cases of normal and cancer tissues as well as in breast cancer cells. MiR-145 exhibited inhibitory role in tumor angiogenesis, cell growth and invasion and tumor growth through the post-transcriptional regulation of the novel targets N-RAS and VEGF-A. In addition, we provide evidence that the expression levels of miR-145 correlate inversely with malignancy stages of breast tumors, although there is no association between miR-145 levels and hormone receptor levels in breast cancer. Taken together, these results demonstrate that miR-145 plays important inhibitory role in breast cancer malignancy by targeting N-RAS and VEGF-A, which may be potential therapeutic and diagnostic targets.

Nasopharyngeal carcinoma (NPC) is a highly malignant cancer with local invasion and early distant metastasis. NPC is highly prevalent in the Southern China and South-eastern Asia. The genetic susceptibility, endemic environment factors, and Epstein-Barr virus (EBV) infection are believed to be the major etiologic factors of NPC. Once metastasis occurs, the prognosis is very poor. It is urgently needed to develop biomarkers for early clinical diagnosis/prognosis, and novel effective therapies for nasopharyngeal carcinoma. In this paper, we systematically reviewed the current progress of miRNA studies in NPC. It has been shown that both host encoded miRNAs and EBV encoded miRNAs play key roles in almost all the steps of epithelia cell carcinogenesis, including epithelial-mesenchymal to stem-like transition, cell growth, migration, invasion, and tumorigenesis. More importantly, some miRNAs could be secreted out and play a role in the microenvironments. The level of sera miRNAs is correlated with the copy numbers of host miRNAs in tumor biopsies. Promising results of gene therapy have been also achieved by lentiviral delivered miRNAs. Taken together, cell free miRNAs would be potential biomarkers of early clinical diagnosis/prognosis; while some miRNAs could be further developed into therapeutic agents in the future.

In this study we describe a novel polymer, mPPS-FA, synthesized as a potential gene transfer vector. To complete mPPS-FA, folic acid was conjugated to a backbone (named mPPS) consisting of a copolymer of methyl PEG-2000, PEI-600, and sebacoyl chloride. (1)H NMR, FT-IR, and UV spectroscopy were used to characterize the structure of mPPS-FA. It was revealed that mPPS-FA holds the ability to bind plasmid DNA yielding positively charged particles (polyplexes). Dynamic light scattering (DLS) and TEM techniques were used to study the size and morphology of the formed mPPS-FA/DNA nanocomplexes. The mPPS-FA/DNA nanoparticles exhibited low cytotoxicity as transfection of B16-F0, U87MG, CHO-1, and Ho-8910 cells produced >80% viability indicating low cytotoxicity of the polymer. The ability of mPPS-FA to deliver EGFP plasmid to melanoma B16-F0, U87, CHO-1, Ho-8910, and A549 cells was investigated in vitro as compared to the lipid-based transfection agent Lipofectamine2000 and Linear PEI 22 kDa (L-PEI 22 kDa). We found that mPPS-FA/DNA complexes yielded the highest GFP transfection efficiency in B16-F0, U87, CHO-1, and Ho-8910 cells, which all highly express folate receptors (FR), at an mPPS-FA/DNA ratio (w/w) of 15. Furthermore, the transfection of mPPS-FA/DNA complexes in CHO-1 cells could be competitively blocked by free folic acid molecules. In contrast, in low FR expressing A549 cells, mPPS-FA showed similar low transfection efficiency as mPPS. Taken together, mPPS-FA showed the highest efficiency in vitro and the potential to be developed as a nonviral gene carrier.

Interleukin-2 (IL-2) has been shown to possess antitumor activity in numerous preclinical and clinical studies. However, the short half-life of recombinant IL-2 protein in serum requires repeated high-dose injections, resulting in severe side effects. Although adenovirus-mediated IL-2 gene therapy has shown antitumor efficacy, the host antibody response to adenoviral particles and potential biosafety concerns still obstruct its clinical applications. Here we report a novel nanopolymer for IL-2 delivery, consisting of low molecular weight polyethylenimine (600Da) linked by β-cyclodextrin and conjugated with folate (named H1). H1 was mixed with IL-2 plasmid to form H1/pIL-2 polyplexes of around 100 nm in diameter. Peritumoral injection of these polyplexes suppressed the tumor growth and prolonged the survival of C57/BL6 mice bearing B16–F1 melanoma grafts. Importantly, the antitumor effects of H1/pIL-2 (50 μg DNA) were similar to those of recombinant adenoviruses expressing IL-2 (rAdv-IL-2; 2 × 108 pfu). Furthermore, we showed that H1/pIL-2 stimulated the activation and proliferation of CD8+, CD4+ T cell, and natural killer cells in peripheral blood and increased the infiltration of CD8+, CD4+ Tcells, and natural killer cells into the tumor environment. In conclusion, these results show that H1/pIL-2 is an effective and safe melanoma therapeutic with an efficacy comparable to that of rAdv-IL-2. This treatment represents an alternative gene therapy strategy for melanoma. Mol Cancer Ther; 10(6); 1082–92. ©2011 AACR.