Search

Abstract The development of resistance to anticancer drugs is believed to cause chemotherapy failure in pancreatic cancer (PC). The efflux of anticancer drugs mediated by ATP-binding cassette (ABC) transporters is a widely accepted mechanism for chemoresistance, but for ABCA subfamily members, which are characterized by their ability to transport lipids and cholesterol, its role in chemoresistance remains unknown. Here we found that the expression of ABCA8, a member of ABCA subfamily transporters, was significantly increased in human PC cells after gemcitabine (GEM) treatment, as well as in established GEM-resistant (Gem-R) PC cells. Importantly, ABCA8 knockdown reversed the chemoresistance phenotype of Gem-R cells, whereas ABCA8 overexpression significantly decreased the sensitivity of human PC cells to GEM, both in vitro and in vivo, demonstrating an important role of ABCA8 in regulating chemosensitivity. Moreover, our results showed that treatment with taurocholic acid (TCA), an endogenous substrate of ABCA8, also induced GEM insensitivity in PC cells. We further demonstrated that ABCA8 mediates the efflux of TCA out of PC cells, and that extracellular TCA activates extracellular signal-regulated kinase (ERK) signaling via the sphingosine 1-phosphate receptor 2 (S1PR2), which is responsible for ABCA8-induced GEM ineffectiveness. Together, these findings reveal a novel TCA-related mechanism of ABCA subfamily transporter-mediated chemoresistance that goes beyond the role of a drug pump and suggest ABCA8 or the TCA-S1RP2-ERK pathway as potential targets for improving the effectiveness of and overcoming the resistance to chemotherapy in PC.

Abstract Background Collagens are the most abundant proteins in extra cellular matrix and important components of tumor microenvironment. Recent studies have showed that aberrant expression of collagens can influence tumor cell behaviors. However, their roles in hepatocellular carcinoma (HCC) are poorly understood. Methods In this study, we screened all 44 collagen members in HCC using whole transcriptome sequencing data from the public datasets, and collagen type IV alpha1 chain (COL4A1) was identified as most significantly differential expressed gene. Expression of COL4A1 was detected in HCC samples by quantitative real-time polymerase chain reaction (qRT-PCR), western blot and immunohistochemistry (IHC). Finally, functions and potential mechanisms of COL4A1 were explored in HCC progression. Results COL4A1 is the most significantly overexpressed collagen gene in HCC. Upregulation of COL4A1 facilitates the proliferation, migration and invasion of HCC cells through FAK-Src signaling. Expression of COL4A1 is upregulated by RUNX1 in HCC. HCC cells with high COL4A1 expression are sensitive to the treatment with FAK or Src inhibitor. Conclusion COL4A1 facilitates growth and metastasis in HCC via activation of FAK-Src signaling. High level of COL4A1 may be a potential biomarker for diagnosis and treatment with FAK or Src inhibitor for HCC.

Background: Hepatic fibrosis is caused by chronic liver injury and may progress toward liver cirrhosis, and even hepatocellular carcinoma. However, current treatment is not satisfactory. Therefore, there is a mandate to find novel therapeutic targets to improve therapy, and biomarkers to monitor therapeutic response. Methods: Liver fibrosis was induced by carbon tetrachloride (CCl4) or thioacetamide (TAA) in wild type (WT) or CTHRC1−/− mice, followed by immunofluorescence and immunohistochemical analyses. CTHRC1 monoclonal antibody (mAb) was used to abrogate the effect of CTHRC1 in vitro and in vivo. Results: Here, we reported that collagen triple helix repeat containing 1 (CTHRC1), a secreted protein derived from hepatic stellate cells (HSCs), was significantly up-regulated in fibrotic liver tissues. CTHRC1 promoted HSCs transformation from a quiescent to an activated state, and enhanced migratory or contractile capacities of HSCs by activating TGF-β signaling. Meanwhile, CTHRC1 competitively bound to Wnt noncononical receptor and promoted the contractility but not activation of HSCs. CCl4 or TAA-induced liver fibrosis was attenuated in CTHRC−/− mice compared with littermate control, while a monoclonal antibody of CTHRC1 suppressed liver fibrosis in WT mice treated with CCl4 or TAA. Interpretation: We demonstrated that CTHRC1 is a new regulator of liver fibrosis by modulating TGF-β signaling. Targeting CTHRC1 could be a promising therapeutic approach, which can suppress TGF-β signaling and avoid the side effects caused by directly targeting TGF-β. CTHRC1 could also be a potential biomarker for monitoring response to anti-fibrotic therapy. Fund: This study was supported by the National Natural Science Foundation of China (ID 81672358, 81871923, 81872242, 81802890), the Shanghai Municipal Education Commission—Gaofeng Clinical Medicine Grant Support (ID 20181708), the Natural Science Foundation of Shanghai (ID 17ZR1428300, 18ZR1436900), and Shanghai Municipal Health Bureau (ID 2018BR32). The funders did not play a role in manuscript design, data collection, data analysis, interpretation nor writing of the manuscript. Keywords: Liver fibrosis, CTHRC1, HSCs, TGF-β signaling

Background/Aims: Epidermal growth factor receptor variant III (EGFRvIII), the most frequent EGFR variant, is constitutively activated without binding to EGF and is correlated with a poor prognosis. CH12, a human-mouse chimeric monoclonal antibody, has been developed in our laboratory and selectively binds to overexpressed EGFR and EGFRvIII. A previous study had reported that EGFR could influence autophagic activity, and autophagy is closely related to tumor development and the response to drug therapy. In this study, we aimed to elucidate the effect of CH12 on autophagy and efficacy of combining CH12 with an autophagy inhibitor against EGFRvIII-positive tumors. Methods: EGFRvIII was overexpressed in liver cancer, glioblastoma and breast cancer, and the change in the autophagy-relevant protein levels was analyzed by western blot assays, LC3 punctate aggregation was analyzed by immunofluorescence. The interaction of Beclin-1 and Rubicon was assessed by co-immunoprecipitation (Co-IP) after CH12 treatment. The efficacy of ATG7 or Beclin-1 siRNA in combination with CH12 in Huh-7-EGFRvIII cells was assessed by CCK-8 assays. The autophagy and apoptosis signaling events in Huh-7-EGFRvIII cells upon treatment with control, CH12, siRNA or combination for 48 h were assessed by western blot assays. Results: Our results showed that, in cancer cell lines overexpressing EGFRvIII, only the liver cancer cell lines Huh-7 and PLC/PRF/5 suggested autophagy activation. We then investigated the mechanism of autophagy activation after EGFRvIII overexpression. The results showed that EGFRvIII interacted with Rubicon, an autophagy inhibition protein, and released Beclin-1 to form the inducer complex, thus contributing to autophagy. In addition, CH12, via inhibiting the phosphorylation of EGFRvIII, promoted the interaction of EGFRvIII with Rubicon, further inducing autophagy. In vitro assays suggested that knocking down the expression of the key proteins ATG7 or Beclin-1 in the autophagy pathway with siRNA inhibits tumor cell proliferation. Combining autophagy-related proteins 7 (ATG7) or Beclin-1 siRNA with CH12 in Huh-7-EGFRvIII cells showed better inhibition of cell proliferation. Conclusion: EGFRvIII could induce autophagy, and CH12 treatment could improve autophagy activity in EGFRvIII-positive liver cancer cells. The combination of CH12 with an autophagy inhibitor or siRNA against key proteins in the autophagy pathway displayed more significant efficacy on EGFRvIII-positive tumor cells than monotherapy, and induced cell apoptosis.

Circular RNAs are formed from exon back-splicing, the significance of these endogenous RNAs is beginning to be unraveled. Here, the authors identify thousands of circular RNAs differentially expressed between normal and cancer tissues and show that an abundant circular RNA generated from HIPK3regulates cell growth.

Small cell lung cancer (SCLC) is an aggressive disease with poor survival. A few sequencing studies performed on limited number of samples have revealed potential disease-driving genes in SCLC, however, much still remains unknown, particularly in the Asian patient population. Here we conducted whole exome sequencing (WES) and transcriptomic sequencing of primary tumors from 99 Chinese SCLC patients. Dysregulation of tumor suppressor genes TP53 and RB1 was observed in 82% and 62% of SCLC patients, respectively, and more than half of the SCLC patients (62%) harbored TP53 and RB1 mutation and/or copy number loss. Additionally, Serine/Arginine Splicing Factor 1 (SRSF1) DNA copy number gain and mRNA over-expression was strongly associated with poor survival using both discovery and validation patient cohorts. Functional studies in vitro and in vivo demonstrate that SRSF1 is important for tumorigenicity of SCLC and may play a key role in DNA repair and chemo-sensitivity. These results strongly support SRSF1 as a prognostic biomarker in SCLC and provide a rationale for personalized therapy in SCLC.

Several types of epidermal growth factor receptor (EGFR) gene alternations have been observed in human tumors. Here we present a novel EGFR variant with aberrant splicing of exon 4 (named as de4 EGFR). Variant-specific polymerase chain reaction showed that de4 EGFR was expressed in some glioma (4/40), prostate cancer (3/11), and ovarian cancer (3/9) tissues but not in tissues adjacent to tumors or normal tissues. de4 EGFR displayed an enhanced transformation and a higher metastasis-promoting capacity in comparison to wild-type EGFR. With minimal EGF-binding activity, de4 EGFR underwent ligand-independent autophosphorylation and self-dimerization. Moreover, in serum-starved condition, de4 EGFR expression in U87 MG cells significantly upregulated the extracellular signal-regulated kinase and AKT phosphorylation and expression of JUN and Src. Importantly, E-cadherin expression was barely detectable in the U87 MG cells expressing de4 EGFR and restored expression of E-cadherin in these cells inhibited their metastatic behaviors. Taken together, we identified a novel EGFR variant with increased metastasis-promoting activity that may become a promising new target for cancer therapy.

We present herein a novel method of pIII-based antibody phage display using Hpd3cells—bacterial cells bearing the genome of a gene-III-lacking helper phage (VCSM13d3). A high level of single-chain variable fragments (scFvs) was displayed in the consequent phagemid particles using Hpd3cells to rescue the phagemid encoding scFv-pIII. Hpd3cells considerably improved the specific enrichment factor when used for constructing an immunized antibody library. In addition, using Hpd3cells could overcome pIII resistance and can contribute to the efficient enrichment of specific binding antibodies from a phage display library, thereby increasing the chance of obtaining more diverse antibodies specific for target antigens.

Ligand or peptide-targeted phagemid particles are being pursued as vehicles for receptor-mediated gene delivery. Here we describe a helper phage in which the protein III (pIII) protein is modified by the addition of a ligand peptide sequence at the amino terminus. Phagemid particles can be prepared with the help of this modified helper phage and should display the ligand peptide in most of the pIII proteins on the phagemid surface. Using such a method, it is not necessary for the phagemid to encode the pIII protein, which leaves a larger space for cloning genes of interest. In addition, the technique should allow for the rapid testing of peptide ligands selected from phage display libraries using phagemids encoding various reporter genes (e.g., green fluorescent protein, luciferase, β-galactosidase) and therapeutic genes.

Gastrointestinal stromal tumors (GISTs) are the major gastrointestinal mesenchymal tumors with a variable malignancy ranging from a curable disorder to highly malignant sarcomas. Metastasis and recurrence are the main causes of death in GIST patients. To further explore the mechanism of metastasis and to more accurately estimate the recurrence risk of GISTs after surgery, the clinical significance and functional role of collagen triple helix repeat containing-1 (CTHRC1) in GIST were investigated. We found that CTHRC1 expression was gradually elevated as the risk grade of NIH classification increased, and was closely correlated with disease-free survival and overall survival in 412 GIST patients. In vitro experiments showed that recombinant CTHRC1 protein promoted the migration and invasion capacities of primary GIST cells. A luciferase reporter assay and pull down assay demonstrated that recombinant CTHRC1 protein activated noncanonical Wnt/PCP-Rho signaling but inhibited canonical Wnt signaling. The pro-motility effect of CTHRC1 on GIST cells was reversed by using a Wnt5a neutralizing antibody and inhibitors of Rac1 or ROCK. Taken together, these data indicate that CTHRC1 may serve as a new predictor of recurrence risk and prognosis in post-operative GIST patients and may play an important role in facilitating GIST progression. Furthermore, CTHRC1 promotes GIST cell migration and invasion by activating Wnt/PCP-Rho signaling, suggesting that the CTHRC1-Wnt/PCP-Rho axis may be a new therapeutic target for interventions against GIST invasion and metastasis.

MicroRNAs (miRNAs) are small, single-stranded, non-coding RNAs that play pivotal roles in human cancer development and progression, such as tumor metastasis. Here, we identified the miRNAs that regulate hepatocellular carcinoma (HCC) cell migration by a high-throughput screening method using the classical wound-healing assay with time-lapse video microscopy and validation with a transwell migration assay. Eleven miRNAs (miR-134, -146b-3p, -188-3p, -525-3p, -661, -767-5p, -891a, -891b, -1244, -1247 and miR-1471) were found to promote or inhibit HCC cell migration. Further investigation revealed that miR-134 suppressed the invasion and metastasis of HCC cells in vitro and in vivo, and integrin beta 1 (ITGB1) was a direct and functional target gene of miR-134. Moreover, miR-134 inhibited the phosphorylation of focal adhesion kinase (FAK) and the activation of RhoA downstream of the ITGB1 pathway, thereby decreasing stress fiber formation and cell adhesion in HCC cells. In conclusion, we demonstrated that miR-134 is a novel metastasis suppressor in HCC and could be a potential therapeutic target for the treatment of HCC.

Hepatitis B Virus (HBV) mutations play a role in the development of hepatocellular carcinoma (HCC). However, the association between HBV polymerase gene mutations and HCC has not been reported. In this study, we conducted a multi-stage study to identify HCC-related mutations in the reverse transcriptase (RT) domain of the HBV polymerase gene.A total of 231 HCCs and 237 non-HCC controls from Qidong, China, were included in this study. The entire sequence of HBV RT was first compared between 29 HCC and 35 non-HCC cases, and candidate mutations were then evaluated in two independent validation sets.There were 15 candidate mutations identified from the discovery set, with A799G and T1055A being consistently associated with HCC across all studies. A pooled analysis of samples revealed that A799G, A987G, and T1055A were independent risk factors for HCC, with adjusted odds ratios of 5.53 [95% confidence interval (CI), 1.69-18.10], 4.20 (95%CI, 1.15-15.35), and 3.78 (95%CI, 1.45-9.86), respectively. A longitudinal study showed that these mutations were detectable 4-5 years prior to HCC diagnosis.Our study provides evidence the first that HBV RT contains naturally occurring mutations that can be used as predictive markers for HCC.

Liver cancer is one of leading causes of cancer-related deaths. A deeper mechanistic understanding of liver cancer could lead to the development of more effective therapeutic strategies. In our previous work, we screened 646 miRNAs and identified 11 that regulate liver cancer cell migration. The current study shows that miR-525-3p is frequently up-regulated in liver cancer tissues, and enhanced expression of miR-525-3p can promote liver cancer cell migration and invasion. Zinc finger protein 395 (ZNF395) is the direct functional target gene for miR-525-3p, and it is frequently down-regulated in liver cancer tissues. High expression of ZNF395 can significantly inhibit while knockdown of ZNF395 expression can markedly enhance the migration and invasion of liver cancer cells, suggesting that ZNF395 suppresses metastasis in liver cancer. Down-regulation of ZNF395 can mediate miR-525-3p induced liver cancer cell migration and invasion. In conclusion, miR-525-3p promotes liver cancer cell migration and invasion by directly targeting ZNF395, and the fact that miR-525-3p and ZNF395 both play important roles in liver cancer progression makes them potential therapeutic targets.

Hepatocellular carcinoma (HCC) is one of the most fatal cancers. In almost all populations, males have a higher HCC rate than females. Here we sought to explore the roles and mechanisms of acetylcholine (Ach) and androgen receptor (AR) on regulating the fate determinations of HCC. Ach activated AR and promoted its expression in HCC cells. Ach enhanced HCC cell migration and invasion but inhibited their apoptosis. Ach had no obvious effects on the migration, invasion, or apoptosis in AR-negative HCC cells. Elevation of migration and invasion induced by Ach was eliminated in AR-knockdown HCC cells. In contrast, Ach stimulated the migration and invasion but suppressed apoptosis in AR over-expressed HCC cells. Additionally, AR agonist R1881 promoted the migration and invasion but reduced the apoptosis of SNU-449 cells, whereas AR antagonist casodex inhibited the migration and invasion but stimulated the apoptosis of SNU-449 cells. STAT3 and AKT phosphorylation was activated by Ach in HCC cells. Collectively, these data suggest that Ach activates STAT3 and AKT pathways and acts on AR to promote the migration and invasion but inhibit the apoptosis of HCC cells. This study thus provides novel insights into carcinogenesis of liver cancer by local interaction between neurotransmitter Ach and hormone receptor AR in HCC.

A novel human gene, FN1BP1 (fibronectin 1 binding protein 1), was identified using the human placenta cDNA library. Northern blotting showed a transcript of ∼2.8 kb in human placenta, liver, and skeletal muscle tissues. This mRNA transcript length was similar to the full FN1BP1 sequence obtained previously. We established a conditionally induced stable cell line of Hep3B-Tet-on-FN1BP1 to investigate the preliminary function and mechanism of the secretory FN1BP1 protein. Cell-proliferation and colony-conformation assays demonstrated that FN1BP1 protein suppressed Hep3B cell growth and colonization in vitro. Analysis of Atlas human cDNA expression indicated that after FN1BP1 Dox-inducing expression for 24 h, 19 genes were up-regulated and 22 genes were down-regulated more than two-fold. Most of these gene changes were related to cell-cycle-arrest proteins (p21cip1, p15, and cyclin E1), transcription factors (general transcription factors, zinc finger proteins, transcriptional enhancer factors), SWI/SNF (SWItch/Sucrose NonFermentable) complex units, early-response proteins, and nerve growth or neurotrophic factors. Down-regulated genes were subject to colony-stimulating factors (e.g., GMSFs), and many repair genes were involved in DNA damage (RAD, ERCC, DNA topoisomerase, polymerase, and ligase). Some interesting genes (p21cip1, ID2, GMSF, ERCC5, and RPA1), which changed in the cDNA microarray analysis, were confirmed by semi-qRT-PCR, and similar changes in expression were observed. FCM cell-cycle analysis indicated that FN1BP1 over-expression could result in G1 phase arrest. FN1BP1 might inhibit cell growth and/or colony conformation through G1 phase arrest of the Hep3B cell cycle. These results indicate the potential role of FN1BP1 as a treatment target for hepatocellular carcinoma.

The mammalian target of the rapamycin (mTOR) pathway, which drives cell proliferation, is frequently hyperactivated in a variety of malignancies. Therefore, the inhibition of the mTOR pathway has been considered as an appropriate approach for cancer therapy. In this study, we examined the roles of mTOR in the maintenance and differentiation of cancer stem-like cells (CSCs), the conversion of conventional cancer cells to CSCs and continuous tumor growth in vivo. In H-Ras-transformed mouse liver tumor cells, we found that pharmacological inhibition of mTOR with rapamycin greatly increased not only the CD133+ populations both in vitro and in vivo but also the expression of stem cell-like genes. Enhancing mTOR activity by over-expressing Rheb significantly decreased CD133 expression, whereas knockdown of the mTOR yielded an opposite effect. In addition, mTOR inhibition severely blocked the differentiation of CD133+ to CD133- liver tumor cells. Strikingly, single-cell culture experiments revealed that CD133- liver tumor cells were capable of converting to CD133+ cells and the inhibition of mTOR signaling substantially promoted this conversion. In serial implantation of tumor xenografts in nude BALB/c mice, the residual tumor cells that were exposed to rapamycin in vivo displayed higher CD133 expression and had increased secondary tumorigenicity compared with the control group. Moreover, rapamycin treatment also enhanced the level of stem cell-associated genes and CD133 expression in certain human liver tumor cell lines, such as Huh7, PLC/PRC/7 and Hep3B. The mTOR pathway is significantly involved in the generation and the differentiation of tumorigenic liver CSCs. These results may be valuable for the design of more rational strategies to control clinical malignant HCC using mTOR inhibitors.

Background The transformation of healthy gastric tissue into intestinal metaplasia (IM) is thought to be a critical premalignant step in the development of intestinal-type gastric adenocarcinoma (GA). How such premalignancies contribute to the development of GA is, however, poorly understood. Methods In this study, the extent and clonal complexity in IM tissue from patients without gastric cancer were analysed by measuring variations of multiple microsatellite (MS) markers. Results Even though these tissues are generally regarded as clinically benign, we found extensive MS length heterogeneity between and within individual IM glands, indicating that complex genome diversity is already pervasive in these tissues. Based on a clonal relationship analysis, we found that there exist multiple clones within individual IM glands and that MS alterations can accumulate in these clones. Moreover, we found spatially distant IM glands with the same MS phenotype, suggesting that these MS alterations were progressed by gland fission. Conclusions These results provide evidence that genetic instability is an early event, present within metaplastic tissues of otherwise non-cancer patients, and such frequent genetic alterations can be part of the pathophysiological rationale for the requirement of this phase during gastric carcinogenesis. [ABSTRACT FROM AUTHOR]

Background and Aims: Orthotopic liver transplantation (OLT) can be an effective treatment option for certain patients with early stage hepatocellular carcinoma (HCC) meeting Milan, UCSF, or Hangzhou criteria. However, HCC recurrence rates post-OLT range from 20 to 40 %, with limited follow-up options. Elucidating genetic drivers common to primary and post-OLT recurrent tumors may further our understanding and help identify predictive biomarkers of recurrence-both to ultimately help manage clinical decisions for patients undergoing OLT.Methods: Whole exome and RNA sequencing in matched primary and recurrent tumors, normal adjacent tissues, and blood from four Chinese HCC patients was conducted. SiRNA knockdown and both qRT-PCR and Western assays were performed on PLCPRF5, SNU449 and HEPG2 cell lines; immunohistochemistry and RNA Sequencing were conducted on the primary tumors of Chinese HCC patients who experienced tumor recurrence post-OLT (n = 9) or did not experience tumor recurrence (n = 12).Results: In three independent HCC studies of patients undergoing transplantation (n = 21) or surgical resection (n = 242, n = 44) of primary tumors (total n = 307), HERC5 mRNA under-expression correlated with shorter: time to tumor recurrence (p = 0.007 and 0.02) and overall survival (p = 0.0063 and 0.023), even after adjustment for relevant clinical variables. HERC5 loss drives CCL20 mRNA and protein over-expression and associates with regulatory T cell infiltration as measured by FOXP3 expression. Further, matched primary and recurrent tumors from the 4 HCC patients indicated clonal selection advantage of Wnt signaling activation and CDKN2A inactivation.Conclusions: HERC5 plays a crucial role in HCC immune evasion and has clinical relevance as a reproducible prognostic marker for risk of tumor recurrence and survival in patients. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is one of the leading causes for cancer related mortality worldwide. Poor prognosis of HCC patients is mainly due to frequent metastasis and recurrence. Deregulation of metastasis suppressors in malignant cells plays critical roles during cancer metastasis. Thus, novel metastasis suppressors are urgently needed to be uncovered to shed new light on molecular mechanisms driving HCC metastasis. In the present study, decreased expression of leukemia inhibitory factor receptor (LIFR) was demonstrated in HCC, and its expression levels were even lower in HCC with metastasis. Downregulated LIFR expression predicted poor prognosis in HCC patients. LIFR was an independent and significant risk factor for their recurrence and survival. Silencing LIFR resulted in forced metastasis of HCC cells, whereas ectopic overexpression of LIFR attenuated migration and invasion of HCC cells in vitro and in vivo. Moreover, LIFR knockdown could activate phosphoinositide 3-kinase/V-akt Murine Thymoma Viral Oncogene Homolog (PI3K/AKT) signaling through enhancing phosphorylation of Janus kinase 1 (JAK1), which successively promoted matrix metalloproteinase 13 (MMP13) expression and HCC metastasis. Combination of LIFR and p-AKT or MMP13 was a more powerful predictor of poor prognosis for HCC patients. Together, these findings conclude that LIFR functions as a novel metastasis suppressor in HCC and may serve as a prognostic biomarker for HCC patients. [ABSTRACT FROM AUTHOR]

The article discusses research which identified new viral variations associated with hepatocellular carcinoma (HCC). A comparative study on the complete sequence of hepatitis B virus isolated from 20 HCC and 35 non-HCC patients in Qidong, China was carried out to verify the relationships of four selected mutations in the HBV core (C) gene to HCC. Results implied that A2189C and G2203W are new predictive markers for HCC. The study also highlights the influence of genetic variants in the HBV C gene on HCC progression.

Background: Compared with viral vectors, nonviral vectors are less immunogenic, more stable, safer and easier to replication for application in cancer gene therapy. However, nonviral gene delivery system has not been extensively used because of the low transfection efficiency and the short transgene expression, especially in vivo. It is desirable to develop a nonviral gene delivery system that can support stable genomic integration and persistent gene expression in vivo. Here, we used a composite nonviral gene delivery system consisting of the piggyBac (PB) transposon and polyethylenimine (PEI) for long-term transgene expression in mouse ovarian tumors. Methods: A recombinant plasmid PB [Act-RFP, HSV-tk] encoding both the herpes simplex thymidine kinase (HSV-tk) and the monomeric red fluorescent protein (mRFP1) under PB transposon elements was constructed. This plasmid and the PBase plasmid were injected into ovarian cancer tumor xenografts in mice by in vivo PEI system. The antitumor effects of HSV-tk/ ganciclovir (GCV) system were observed after intraperitoneal injection of GCV. Histological analysis and TUNEL assay were performed on the cryostat sections of the tumor tissue. Results: Plasmid construction was confirmed by PCR analysis combined with restrictive enzyme digestion. mRFP1 expression could be visualized three weeks after the last transfection of pPB/TK under fluorescence microscopy. After GCV admission, the tumor volume of PB/TK group was significantly reduced and the tumor inhibitory rate was 81.96% contrasted against the 43.07% in the TK group. Histological analysis showed that there were extensive necrosis and lymphocytes infiltration in the tumor tissue of the PB/TK group but limited in the tissue of control group. TUNEL assays suggested that the transfected cells were undergoing apoptosis after GCV admission in vivo. Conclusion: Our results show that the nonviral gene delivery system coupling PB transposon with PEI can be used as an efficient tool for gene therapy in ovarian cancer. [ABSTRACT FROM AUTHOR]

Nontoxic and biodegradable poly(ε‐caprolactone)‐b‐poly(ethylene glycol)‐b‐poly(ε‐caprolactone) triblock copolymers were synthesized by the solution polymerization of ε‐caprolactone in the presence of poly(ethylene glycol). The chemical structure of the resulting triblock copolymer was characterized with 1H NMR and gel permeation chromatography. In aqueous solutions of the triblock copolymers, the micellization and sol–gel‐transition behaviors were investigated. The experimental results showed that the unimer‐to‐micelle transition did occur. In a sol–gel‐transition phase diagram obtained by the vial‐tilting method, the boundary curve shifted to the left, and the gel regions expanded with the increasing molecular weight of the poly(ε‐caprolactone) block. In addition, the hydrodynamic diameters of the micelles were almost independent of the investigated temperature (25–55 °C). The atomic force microscopy results showed that spherical micelles formed at the copolymer concentration of 2.5 × 10−4 g/mL, whereas necklace‐like and worm‐like shapes were adopted when the concentration was 0.25 g/mL, which was high enough to form a gel. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 45: 605–613, 2007 [ABSTRACT FROM AUTHOR]

In recent years, capsid-modified filamentous bacteriophage has become a potential vector for gene delivery into mammalian cells. However, little was known about how the carried gene in the single-stranded genome expressed in mammalian cells. To explore whether the orientation of the carried gene affects its expression in the cells, we prepared EGF-displayed phagemid particles whose genome carried the GFP gene or luciferase gene. The phagemid carried reporter genes either in the same orientation (called (+)gene) or in the contrary orientation (called (−)gene) to filamentous origin. Using these phagemid particles to infect H1299 cells, we found that the phages with (−) reporter genes had about 2-fold transduction efficiency as those with (+) reporter genes. These results indicated that phagemid carrying (−)gene of interest presented a better procedure in phage-mediated gene therapy. Furthermore, camptothecin (CPT) treatment was also applied and found to enhance both kinds of phagemid particles, and (−)gene still produced about 1.5- to 2-fold transduction efficiency compared to those with (+)gene. Thus, it is imperative that we clone the genes of interest in the reverse orientation to filamentous origin to enhance their expressions when preparing phagemid gene delivery vectors. Also, the results suggested that CPT could enhance both the replication of single-stranded DNA and its transcription. [ABSTRACT FROM AUTHOR]

Background: Our previous cDNA array results indicated KIAA0101 as one of the differentially expressed genes in human hepatocellular carcinoma (HCC) as compared with non-cancerous liver. However, it is necessary to study its expression at protein level in HCC and its biological function for HCC cell growth. Method: Western blot and tissue array were performed to compare KIAA0101 protein expression level in paired human HCC and non-cancerous liver tissues from the same patients. Investigation of its subcellular localization was done by using dual fluorescence image examination and enriched mitochondrial protein Western blot analysis. The in vitro cell growth curve was used for examing the effect of over-expression of KIAA0101 in HCC cells. FACS was used to analyze the cell cycle pattern in KIAA0101 expression positive (+) and negative (-) cell populations isolated by the pMACSKKII system after KIAA0101 cDNA transfection. Results: Western blot showed KIAA0101 protein expression was down-regulated in HCC tissues as compared with their counterpart non-cancerous liver tissues in 25 out of 30 cases. Tissue array also demonstrated the same pattern in 161 paired samples. KIAA0101 was predominantly localized in mitochondria and partially in nuclei. KIAA0101 cDNA transfection could inhibit the HCC cell growth in vitro. In cell cycle analysis, it could arrest cells at the G1 to S phase transition. Conclusion: KIAA0101 protein expression was down-regulated in HCC. This gene could inhibit the HCC cell growth in vitro and presumably by its blocking effect on cell cycle. [ABSTRACT FROM AUTHOR]

Epidermal growth factor receptor (ErbB1, EGFR) is overexpressed in a variety of human cancer cells. It has been considered as a rational target for drug delivery. To identify novel ligands with specific binding capabilities to EGFR, we screened a phage display peptide library and found an enriched phage clone encoding the amino acid sequence YHWYGYT-PQNVI (designated as GE11). Competitive binding assay and Scatchard analysis revealed that GE11 peptide bound specifically and efficiently to EGFR with a dissociation constant of ∼22 nM, but with much lower mitogenic activity than with EGF. We showed that the peptides were internalized preferentially into EGFR highly expressing cells, and they accumulated in EGFR overexpressing tumor xenografts after i.v. delivery in vivo. In gene delivery studies, GE11-conjugated polyethylenimine (PEI) vectors were less mitogenic, but still quite efficient at transfecting genes into EGFR highly expressing cells and tumor xenografts. Taken together, GE11 is a potentially safe and efficient targeting moiety for selective drug delivery systems mediated through EGFR. [ABSTRACT FROM AUTHOR]

The gene HCAP1 (HCC-associated Protein 1), one variant of GEMIN4, has been mapped in a minimum LOH region on chromosome 17p13.3 and encodes a 1047-amino acid protein. Function predictions based on the amino acid sequence of protein HCAP1 revealed it to contain one helix-loop-helix motif and one leucine zipper domain. Using yeast two-hybrid screening, five zinc-finger proteins were identified as HCAP1-interacting proteins. Among them, NDP52 (nuclear dot protein 52) appeared most frequently in positive clones and was the most strongly interacting protein. Then, the interaction between HCAP1 and NDP52 was confirmed by GST pull-down assay and a coimmunoprecipitation experiment. Moreover, an immunofluorescent staining assay indicated that NDP52 colocalizes with HCAP1 in the cytoplasm. By deletion analysis, the leucine zipper domain of HCAP1 and the zinc finger domain of NDP52 were identified as important regions responsible for the interaction. [ABSTRACT FROM PUBLISHER]

Background: Hepatocellular carcinoma (HCC) presents one of the major health threats in China today. A better understanding of the molecular genetics underlying malignant transformation of hepatocytes is critical to success in the battle against this disease. The methylation state of C5 of the cytosine in the CpG di-nucleotide that is enriched within or near the promoter region of over 50 % of the polymerase II genes has a drastic effect on transcription of these genes. Changes in the methylation profile of the promoters represent an alternative to genetic lesions as causative factors for the tumor-specific aberrant expression of the genes. Methods: We have used the methylation specific PCR method in conjunction with DNA sequencing to assess the methylation state of the promoter CpG islands of twenty genes. Aberrant expression of these genes have been attributed to the abnormal methylation profile of the corresponding promoter CpG islands in human tumors. Results: While the following sixteen genes remained the unmethylated in all tumor and normal tissues: CDH1, APAF1, hMLH1, BRCA1, hTERC, VHL, RARβ, TIMP3, DAPK1, SURVIVIN, p14ARF, RB1, p15INK4b, APC, RASSF1c and PTEN, varying degrees of tumor specific hypermethylation were associated with the p16INK4a, RASSF1a, CASP8 and CDH13 genes. For instance, the p16INK4a was highly methylated in HCC (17/29, 58.6%) and less significantly methylated in non-cancerous tissue (4/29. 13.79%). The RASSF1a was fully methylated in all tumor tissues (29/29, 100%), and less frequently methylated in corresponding noncancerous tissue (24/29, 82.75%). Conclusions: Furthermore, co-existence of methylated with unmethylated DNA in some cases suggested that both genetic and epigenetic (CpG methylation) mechanisms may act in concert to inactivate the p16INK4a and RASSF1a in HCC. Finally, we found a significant association of cirrhosis with hypermethylation of the p16INK4a and hypomethylation of the CDH13 genes. For the first time, the survey was carried out on such an extent that it would not only provide new insights into the molecular mechanisms underscoring the aberrant expression of the genes in this study in HCC, but also offer essential information required for a good methylation-based diagnosis of HCC. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented about the examination of hTERT mRNA in the serum of patients with liver diseases.

Active drug efflux by the adenosine triphosphate-binding cassette (ABC) transporter ABCG2 is one of the common mechanisms causing multiple drug resistance in various human cancers. In the intrinsic drug resistance of hepatocellular carcinoma (HCC), the role of ABCG2 is closely associated with ‘side population (SP)’, a minor subset of cancer stem-like cells with unique capacity to extrude lipophilic dye Hoechst 33342 and many chemotherapeutic agents. In this study, we showed that ABCG2 was intrinsically expressed in a subgroup of HCC tissues and its expression pattern significantly influenced the levels of drug efflux from HCC cell lines. In MHCC-97L HCC cell line with intrinsic ABCG2 expression, we confirmed the importance of SP cells to the drug efflux-related chemotherapy resistance and found that the SP analysis provided an efficient method to evaluate the functional activity of ABCG2 transporter. In this cell line, we discovered that the SP proportion was modulated by the treatments of Akt signaling inhibitors and serum supplement, which led to the finding that Akt signaling was able to regulate the SP cells’ efflux activity via altering the subcellular localization of ABCG2 transporter. We further demonstrated that the Akt signaling inhibition attenuated the doxorubicin efflux from MHCC-97L cells and increased the drug efficacy. Our results indicate the protective role of intrinsic ABCG2 expression in HCC cells and suggest that suppressing Akt signaling could help overcome the drug efflux by ABCG2 transporter. [ABSTRACT FROM AUTHOR]

Human hepatocellular carcinoma (HCC) is considered difficult to cure because it is resistant to radio- and chemotherapy and has a high recurrence rate after curative liver resection. Epidermal growth factor receptor variant III (EGFRvIII) has been reported to express in HCC tissues and cell lines. This article describes the efficacy of an anti-EGFRvIII monoclonal antibody (mAb CH12) in the treatment of HCC xenografts with EGFRvIII expression and the underlying mechanism of EGFRvIII as an oncogene in HCC. The results demonstrated that CH12 bound preferentially to EGFRvIII with a dissociation constant (Kd) of 1.346 nm/liter. In addition, CH12 induces strong antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity in Huh7-EGFRvIII (with exogenous expression of EGFRvIII) and SMMC-7721 (with endogenous expression of EGFRvIII) cells. Notably, CH12 significantly inhibited the growth of Huh7-EGFRvIII and SMMC-7721 xenografts in vivo with a growth inhibition ratio much higher than C225, a U. S. Food and Drug Administration-approved anti-EGFR antibody. Treatment of the two HCC xenografts with CH12 significantly suppressed tumor proliferation and angiogenesis. Mechanistically, in vivo treatment with CH12 reduced the phosphorylation of constitutively active EGFRvIII, Akt, and ERK. Down-regulation of the apoptotic protectors Bcl-xL, Bcl-2, and the cell cycle regulator cyclin D1, as well as up-regulation of the cell-cycle inhibitor p27, were also observed after in vivo CH12 treatment. Collectively, these results indicate that the monoclonal antibody CH12 is a promising therapeutic agent for HCC with EGFRvIII expression. [ABSTRACT FROM AUTHOR]

Liquid chromatography/mass spectrometry (LC/MS) followed by multivariate statistical analysis has been successfully applied to the plasma phospholipids metabolic profiling in type 2 diabetes mellitus (DM-2). Principal components analysis and partial least-squares discriminant analysis (PLS-DA) models were tested and compared in class separation between the DM2 and control. The application of an orthogonal signal correction filtered model highly improved the class distinction and predictive power of PLS-DA models. Additionally, unit variance scaling was also tested. With this methodology, it was possible not only to differentiate the DM2 from the control but also to discover and identify the potential biomarkers with LC/MS/MS. The proposed method shows that LC/MS combining with multivariate statistical analysis is a complement or an alternative to NMR for metabonomics applications. [ABSTRACT FROM AUTHOR]