Genexol inhibits primary tumour growth and metastases in gemcitabine-resistant pancreatic ductal adenocarcinoma

Introduction

Gemcitabine, the current standard of care for pancreatic ductal adenocarcinoma (PDA), has a less than 10% partial response rate. Genexol-PM, a modified form of paclitaxel, has been shown to have antitumour effects in clinical trials of metastatic breast and small-lung-cell carcinoma. The aim of the present study was to determine if Genexol would be a beneficial treatment for gemcitabine-resistant PDA.

Materials and methods

We measured the in vitro IC50s of gemcitabine and genexol in cell lines sensitive and resistant to gemcitabine. In vivo, animals with orthotopic pancreatic tumours, resistant to gemcitabine, were treated with phosphate-buffered saline (PBS), gemcitabine, Genexol or gemcitabine+Genexol. Tumour progression was monitored using red fluorescent protein imaging.

Results

We showed equivalent IC50s for gemcitabine-sensitive and gemcitabine-resistant cell lines when treated with genexol. In vivo treatment with genexol resulted in a greater per cent reduction in tumour size, less metastatic spread and longer survival compared with treatment with gemcitabine.

Discussion

Genexol proved to be an effective treatment for gemcitabine-resistant PDA. These data combined with the successful clinical use of genexol in Phase II trials of other malignancies suggests it maybe an effective treatment for pancreatic cancer, specifically for those patients resistant to gemcitabine.     

Dysregulation of MicroRNA-34a Expression in Colorectal Cancer Inhibits the Phosphorylation of FAK Via VEGF

Background

MicroRNAs (miRs) are small non-coding RNAs that play important roles in cancer development where they can act as oncogenes or as tumor-suppressors. MiR-34a is a tumor suppressor that is frequently downregulated in a number of tumor types. However, little is known about the role of miR-34a in colorectal cancer (CRC).

Aim

This study aims to show the dysregulation of miR-34a in CRC and to characterize the function and mechanism of miR-34a in CRC cell lines.

Methods

The expression of miR-34a was detected using real-time PCR on CRC tissues and adjacent non-tumorous tissues. The ELISA was used to assess vascular endothelial growth factor (VEGF). The focal adhesion kinase (FAK) and phosphorylated FAK Y397 (pY397FAK) were measured by Western blot. The functions of miR-34a in vivo were measured by migration, invasion, CCK-8 assay and flow cytometry.

Results

MiR-34a was significantly downregulated and pY397FAK was upregulated in CRC cancer tissues. It plays an important role in inhibiting migration and invasion and in increasing apoptosis of CRC cells. Bioinformatic analysis suggested that VEGF may be a target of miR-34a, and this hypothesis was proved by ELISA and RT-PCR. The level of pY397FAK that could be activated by VEGF was downregulated in miR-34a overexpression CRC cell lines. The phosphorylation of FAK at 397 sites in miR-34a-stable cell lines was completely rescued by extra VEGF treatment.

Conclusion

MiR-34a is frequently downregulated in CRC and modulates the phosphorylation of FAK by negatively regulating VEGF.     

Estrogen is increased in male cholangiocarcinoma patients’ serum and stimulates invasion in cholangiocarcinoma cell lines in vitro

Purpose

Cholangiocarcinoma is defined as a chronic liver disease with altered estrogen metabolism and could result in estrogen retention. Estrogenic response was known as a promoting factor in progression of some cancer. In this study, we determined the significant increase of estrogen level in cholangiocarcinoma patients’ sera.

Methods

The estrogen levels in cholangiocarcinoma patients’ sera were measured and correlated with clinical presentations. Estrogen receptor-α expressions in cholangiocarcinoma tissues were detected by immunohistochemistry method. KKU-100 and KKU-M213 cholangiocarcinoma cell lines were treated with 17β-estradiol and tested the proliferative and invasive effects.

Results

The estrogen levels showed positive correlations with serum bilirubin and alkaline phosphatase and a negative correlation with albumin. This study also showed an association with shorter survival times when patients with low and high serum estrogen levels were compared. In vitro studies demonstrated the effect of estrogen on cell proliferation and invasion in dose-dependent manners, which could be inhibited by tamoxifen, a clinical used estrogen antagonist. Invasion showed an association with the TFF1 gene expression and could be inhibited by small interfering RNA against TFF1 gene. Estrogen receptor-α was the main estrogen receptor that response to 17β-estradiol stimulation.

Conclusions

TFF1 trefoil protein could be one of the effectors for estrogen-induced invasion in cholangiocarcinoma via the estrogen receptor-α. These findings could lead to an understanding of the mechanism of cholangiocarcinoma progression.     

Protein–Protein Interaction Network Analysis in Chronic Obstructive Pulmonary Disease

Background

The aim of this study was to investigate the gene expression profile of chronic obstructive pulmonary disease (COPD) patients and non-COPD patients.

Methods

Microarray raw data (GSE29133) was downloaded from Gene Expression Omnibus, including three COPD samples and three normal controls. Gene expression profiling was performed using Affymetrix human genome u133 plus 2.0 GeneChip. Differentially expressed genes were identified by Student’s t test and genes with p < 0.05 were considered significantly changed. Up- and downregulated genes were submitted to the molecular signatures database (MSigDB) to search for a possible association with other previously published gene expression signatures. Furthermore, we constructed a COPD protein–protein interaction (PPI) network and used the connectivity map (cMap) to query for potential drugs for COPD.

Results

A total of 680 upregulated genes and 530 downregulated genes in COPD were identified. The MSigDB investigation found that upregulated genes were highly similar to gene signatures that respond to interferon and downregulated genes were similar to erythroid progenitor cells from fetal livers of E13.5 embryos with KLF1 knocked out. A PPI network consisting of 814 gene/proteins and 2,613 interactions was identified by Search Tool for the Retrieval of Interacting Genes. The cMap predicted helveticoside, disulfiram, and lanatoside C as the top three possible drugs that could perhaps treat COPD.

Conclusion

Comprehensive analysis of the gene expression profile for COPD versus control reveals helveticoside, disulfiram, and lanatoside C as potential molecular targets in COPD. This evidence provides a new breakthrough in the medical treatment of patients with COPD.     

miR-145 functions as tumor suppressor and targets two oncogenes,ANGPT2 and NEDD9, in renal cell carcinoma

Purpose

Abnormal expression of miRNAs is closely related to a variety of human cancers. The purpose of this study is to identify new tumor suppressor miRNA and elucidate its physiological function and mechanism in renal cell carcinoma (RCC).

Methods

The expression of miR-145 in 45 RCC and adjacent normal tissues was performed by quantitative RT-PCR. Cell proliferation, migration, invasion, apoptosis and cycle assays were carried out for functional analysis after miR-145 transfection. Two target genes of miR-145 were identified by luciferase reporter assay. The altered expression of 84 epithelial to mesenchymal transition (EMT)-related genes after miR-145 transfection was detected by RT² Profiler EMT PCR array.

Results

The expression of miR-145 was downregulated in RCC compared to their normal adjacent tissues. Restoring miR-145 expression in RCC cell lines dramatically suppressed cell proliferation, migration and invasion, and induced cell apoptosis and G2-phase arrest. We further validated those miR-145 targets two oncogenes, ANGPT2 and NEDD9 in RCC. In addition, miR-145 was found to regulate numerous genes involved in the EMT.

Conclusions

These findings demonstrate that miR-145 functions as tumor suppressor in RCC, suggesting that miR-145 may be a potential therapeutic target for RCC.     

Inhibition of silent information regulator 1 induces glucose metabolism disorders of hepatocytes and enhances hepatitis C virus replication

Background and objective

Glucose metabolism disorders including insulin resistance (IR) and type 2 diabetes are frequent and important cofactors of chronic hepatitis C (CHC). Silent information regulator 1 (SIRT1) plays a key role in the regulation of hepatic glucose metabolism. We investigated the possible effect of HCV replication on glucose metabolism of hepatocytes and expression of SIRT1 using Huh-7.5 cells harboring the HCV replicon.

Methods

The level of reactive oxygen species (ROS) and value of NAD⁺/NADH and ATP/ADP were detected. Glucose uptake by hepatocytes and glucose production were measured. The activity and expression levels of SIRT1 and expression of its downstream glucose-metabolism genes were measured.

Results

In replicon cells, the level of ROS increased and the value of nicotinamide adenine dinucleotide (NAD⁺)/NADH decreased, then the activity and expression level of mRNA and protein of SIRT1 decreased. Inhibition of SIRT1 not only increased insulin receptor substrate-1 phosphorylation and decreased Akt phosphorylation, inhibited cell surface expression of glucose transporter 2 and suppressed cellular glucose uptake, but it also decreased phosphorylation of forkhead box O1, then upregulated phosphoenolpyruvate carboxykinase and glucose 6-phosphatase genes and downregulated the glucokinase gene, thus promoting glucose production. Interferon treatment restored the aforementioned changes. SIRT1 activator improved glucose metabolism disorders by an increase in glucose uptake and a decrease in glucose production, and it inhibited HCV replication.

Conclusions

HCV replication decreasing the NAD⁺/NADH ratio may downregulate the activity and expression of SIRT1, then change the expression profile of glucose metabolism-related genes, thereby causing glucose metabolism disorders of hepatocytes and promoting HCV replication. Treatment with SIRT1 activator improves glucose metabolic disorders and inhibits HCV replication, suggesting that restoration of SIRT1 activity may be a promising new therapeutic approach for CHC patients with IR.     

Wilms’ tumor 1 gene modulates Fas-related death signals and anti-apoptotic functions in hepatocellular carcinoma

Background

The Wilms’ tumor 1 (WT1) gene is known to be overexpressed in hepatocellular carcinoma (HCC) and to upregulate tumor growth and oncogenic potential, although the detailed mechanisms remain to be elucidated.

Methods

We identified host genes involved in WT1 gene modulation of human liver cancer cell lines in vitro, and further characterized genes related to apoptosis. Moreover, we evaluated the alteration of genes by WT1 in 40 HCC and 58 non-HCC human liver samples collected at resection.

Results

Analysis of the effect of small interfering RNAs-mediated knock-down of WT1 on apoptosis using an annexin V labeling assay, and on modulation of the activity of caspases-3, -8 and -9, indicated that WT1 has an anti-apoptotic role. We identified three apoptosis-related genes that were modulated by WT1; the cellular FLICE-inhibitory proteins (cFLIP) gene was upregulated, and Fas-associated death domain (FADD) and nuclear factor kappa B (NF-κB) were downregulated. Interestingly, knock-down of FADD or NF-κB resulted in the upregulation of WT1, and the expression of cFLIP changed in parallel with WT1 expression. We further evaluated WT1-mediated alteration of genes in HCC and non-HCC human liver samples. Both HCC and non-HCC tissues that expressed relatively high levels of WT1 showed cFLIP overexpression.

Conclusions

WT1 modulates cFLIP, FADD and NF-κB, and has an anti-apoptotic role in HCC. This mechanism of action of WT1 could be related to the tumor growth and oncogenic potential of HCC.     

Expression and biological role of δ-catenin in human ovarian cancer

Objective

δ-Catenin is found to be involved in the progression of several human cancers. However, its expression pattern and biological roles in human ovarian cancers are not clear. In this study, we examined the expression pattern of δ-catenin in 149 ovarian cancer specimens. We also depleted and overexpressed δ-catenin expression in ovarian cancer cell lines and investigated its role in cell proliferation and invasion.

Methods

δ-Catenin expression was analyzed in 149 archived ovarian cancer specimens using immunohistochemistry. siRNA knockdown and plasmid transfection were performed in SKOV3, SW626, and OVCAR3 cell lines. MTT, colony formation assay, soft agar colony assay, and matrigel invasion assay were carried out to assess the role of δ-catenin in cell proliferation and invasion. We also performed cell cycle analysis in δ-catenin depleted and overexpressed cells. In addition, we examined the level of several cell cycle-related molecules using Western blot.

Results

Of the 149 patients in the study, 104 (69.7?%) showed δ-catenin overexpression. δ-catenin overexpression positively correlated with advanced FIGO stage. δ-Catenin depletion in ovarian cancer cell lines inhibited ovarian cancer cell proliferation and invasion. Depletion of δ-catenin also blocked cell cycle progression and downregulated cyclin D1 expression in ovarian cancer cells. Overexpression of δ-catenin enhanced cell proliferation, invasion, and upregulated cyclinD1 expression.

Conclusions

δ-Catenin is overexpressed in ovarian cancers and associated with advanced stage. Our data provide evidence that δ-catenin regulates the ovarian cancer cell proliferation, invasion, and cell cycle. δ-Catenin thus has potential as a therapeutic target.     

Susceptibility to cytotoxic T cell lysis of cancer stem cells derived from cervical and head and neck tumor cell lines

Purpose

To explore cancer stem cell susceptibility to a host’s cytotoxic T lymphocyte (CTL)-mediated immune response.

Methods

We compared the susceptibility of putative CSC generated from cancer cell lines to immunologic recognition and killing by alloantigen-specific CD8⁺ CTL. CSC-enriched spheroid culture-derived cells (SDC) exhibited higher expression of ALDH, ICAM1 and of stem/progenitor cell markers on all 3 tumor cell lines investigated and lower MHC class I on the cervical cancer cell line as compared to their monolayer-derived cells (MDC).

Results

The expression of ICAM1 and MHCI was upregulated by IFN-γ treatment. CSC populations were less sensitive to MHC class I-restricted alloantigen-specific CD8⁺ CTL lysis as compared to matched MDC. IFN-γ pretreatment resulted in over-proportionally enhanced lysis of SDC. Finally, the subset of ALDH^high expressing SDC presented more sensitivity toward CD8⁺ CTL killing than the ALDH^low SDC.

Conclusions

Tumor therapy resistance has been attributed to cancer stem cells (CSC). We show in vitro susceptibility of CSC to CTL-mediated lysis. Immunotherapy targeting of ALDH⁺ CSC may therefore be a promising approach. Our results and method may be helpful for the development and optimization of adjuvants, as here exemplified for INF-γ, for CSC-targeted vaccines, independent of the availability of CSC-specific antigens.     

Downregulating sCLU Enhances the Sensitivity of Hepatocellular Carcinoma Cells to Gemcitabine by Activating the Intrinsic Apoptosis Pathway

Purpose

The purpose of this study was to investigate whether the therapeutic activity of gemcitabine (GCB) in hepatocellular carcinoma (HCC) could be increased by the down-regulation of secretory clusterin (sCLU), a glycoprotein that is considered to play a cytoprotective role in the resistance to chemotherapy.

Methods

The expression of sCLU was detected in HCC tumor tissues and cell lines. A cell viability and apoptosis assay were performed in parental HCC cells or the same cells transfected with sCLU shRNA and treated with or without GCB. The potential downstream pathways were investigated using the Human Apoptosis RT² Profiler? PCR Array.

Results

The expression levels of sCLU in HCC tissues were significantly higher than in adjacent non-tumor liver tissues and were associated with the histological grade and transarterial chemoembolization. sCLU overexpression was also found in three HCC cell lines and hepatocytes. The depletion of sCLU synergistically increased GCB sensitivity in Bel7402 and SMMC7721 cells and induced cell apoptosis. Based on the PCR array analysis, sCLU depletion also resulted in the up-regulation of BNIP1, GADD45A, TNFRSF10A, and TRADD and down-regulation of AKT1 in Bel7402 and SMMC7721 cells compared with the parental controls. These results were further supported by a Western blot analysis, which showed increased GADD45a protein expression and the decreased expression of phosphorylated AKT. GADD45a overexpression also increased the sensitivity to GCB in the Bel7402 and SMMC7721 cells.

Conclusion

Targeting sCLU may be a useful method to enhance the cytotoxic effect of GCB in hepatocellular carcinoma.     

Aspirin May Prevent Cholangiocarcinoma: A Case–Control Study from the United Kingdom

Background

The proliferation of cholangiocarcinoma cells is suppressed in cell culture by nonsteroidal antiinflammatory drugs (NSAIDs) through the inhibition of cyclo-oxygenase-2 enzyme and also by statins which decrease the production of mediators of the cell cycle.

Aims

To investigate whether there is an inverse association between NSAIDs, including aspirin, and the development of cholangiocarcinoma and, for the first time in a Western population, between statin use and the development of cholangiocarcinoma.

Methods

This epidemiological study had a case–control design in which cases of cholangiocarcinoma diagnosed in Norwich between 2004 and 2010 and in Leicester in 2007 were identified from clinical databases. Controls were patients with basal cell carcinomas treated in the respective dermatology departments. The case notes of all subjects were reviewed to confirm diagnoses and obtain information on medication use. The data were analyzed using unconditional logistic regression to calculate odds ratios (OR) with 95 % confidence intervals (CI).

Results

In total, 81 cases of cholangiocarcinoma and 275 controls were identified. For all cases there was radiological evidence of cancer and 86 % of the cases involved the extrahepatic biliary system. Aspirin use was inversely associated with the development of cholangiocarcinoma (OR 0.45, 95 % CI 0.22–0.92), but there were no significant associations between the development of cholangiocarcinoma and NSAIDs (OR 0.39; 95 % CI 0.11–1.42) or statins (OR 0.58; 95 % CI 0.28–1.19).

Conclusions

The epidemiological data from this study support the biological evidence for aspirin having a protective effect against the development of cholangiocarcinoma. Aspirin use should be measured in future etiological studies and assessed as a chemoprevention agent in those at high risk of developing this type of cancer.     

Diet high in fat and sucrose induces rapid onset of obesity-related metabolic syndrome partly through rapid response of genes involved in lipogenesis, insulin signalling and inflammation in mice

Background

Frequent consumption of a diet high in fat and sucrose contributes to lifestyle-related diseases. However, limited information is available regarding the short-term effects of such a diet on the onset of obesity-associated metabolic abnormalities.

Methods

Male C57BL/6?J mice were divided into two groups and fed a standard chow diet (control group) or a high fat?Chigh sucrose diet containing 21% fat and 34% sucrose (HF?CHS diet group) for 2 or 4?weeks.

Results

The HF?CHS diet significantly induced body weight gain beginning at week 1 and similarly increased mesenteric white adipose tissue weight and plasma insulin levels at weeks 2 and 4. Plasma resistin levels were notably elevated after feeding with the HF?CHS diet for 4?weeks. Measurement of hepatic triglycerides and Oil Red O staining clearly indicated increased hepatic lipid accumulation in response to the HF?CHS diet as early as 2?weeks. Quantitative PCR analysis of liver and white adipose tissue indicated that, starting at week 2, the HF?CHS diet upregulated mRNA expression from genes involved in lipid metabolism and inflammation and downregulated genes involved in insulin signalling. Although plasma cholesterol levels were also rapidly increased by the HF?CHS diet, no differences were found between the control and HF?CHS diet?Cfed animals in the expression of key genes involved in cholesterol biosynthesis.

Conclusions

Our study demonstrates that the rapid onset of hepatosteatosis, adipose tissue hypertrophy and hyperinsulinemia by ingestion of a diet high in fat and sucrose may possibly be due to the rapid response of lipogenic, insulin signalling and inflammatory genes.     

Comparison of HCV-associated gene expression and cell signaling pathways in cells with or without HCV replicon and in replicon-cured cells

Background

Hepatitis C virus (HCV) replication is affected by several host factors. Here, we screened host genes and molecular pathways that are involved in HCV replication by comprehensive analyses using two genotypes of HCV replicon-expressing cells, their cured cells and naïve Huh7 cells.

Methods

Huh7 cell lines that stably expressed HCV genotype 1b or 2a replicon were used. The cured cells were established by treating HCV replicon cells with interferon-alpha. Expression of 54,675 cellular genes was analyzed by GeneChip DNA microarray. The data were analyzed by using the KEGG Pathway database.

Results

Hierarchical clustering analysis showed that the gene-expression profiles of each cell group constituted clear clusters of naïve, HCV replicon-expressed, and cured cell lines. The pathway process analysis between the replicon-expressing and the cured cell lines identified significantly altered pathways, including MAPK, steroid biosynthesis and TGF-beta signaling pathways, suggesting that these pathways were affected directly by HCV replication. Comparison of cured and naïve Huh7 cells identified pathways, including steroid biosynthesis and sphingolipid metabolism, suggesting that these pathways were required for efficient HCV replication. Cytoplasmic lipid droplets were obviously increased in replicon-expressing and cured cells as compared to naïve cells. HCV replication was significantly suppressed by peroxisome proliferator-activated receptor (PPAR)-alpha agonists but augmented by PPAR-gamma agonists.

Conclusion

Comprehensive gene expression and pathway analyses show that lipid biosynthesis pathways are crucial to support proficient virus replication. These metabolic pathways could constitute novel antiviral targets against HCV.     

WTAP regulates migration and invasion of cholangiocarcinoma cells

Background

Wilms’ tumor 1-associating protein (WTAP) is a nuclear protein that has been associated with the regulation of proliferation and apoptosis. Although its dynamic expression and physiological functions in vascular cells have been reported, its expression and roles in cholangiocarcinoma cells are poorly characterized.

Methods

To examine the expression of WTAP in patient tissues, we performed immunohistochemistry. To examine motility of cholangiocarcinoma cells, we employed Boyden chamber, wound healing and Matrigel invasion assays, and a liver xenograft model.

Results

Immunohistochemistry in patient tissues showed WTAP overexpression in cholangiocarcinoma tissues and correlation of WTAP expression with metastasis of cholangiocarcinoma cells. Overexpression or knockdown of WTAP significantly increased or decreased the motility of cholangiocarcinoma cells. Moreover, WTAP overexpression or knockdown significantly increased or decreased tumorigenicity of cholangiocarcinoma cells in an orthotopic xenograft model. Furthermore, microarray study showed that WTAP induce the expressions of MMP7, MMP28, cathepsin H and Muc1.

Conclusion

WTAP is overexpressed in cholangiocarcinoma and regulates motility of cholangiocarcinoma cells     

Identification of Disease-Associated DNA Methylation in B Cells from Crohn’s Disease and Ulcerative Colitis Patients

Background

Changes in the methylation status of inflammatory bowel disease (IBD)-associated genes could significantly alter levels of gene expression, thereby contributing to disease onset and progression. We previously identified seven disease-associated DNA methylation loci from intestinal tissues of IBD patients using the Illumina GoldenGate BeadArray assay.

Aims

In this study, we extended this approach to identify IBD-associated changes in DNA methylation in B cells from 18 IBD patients [9 Crohn??s disease (CD) and 9 ulcerative colitis (UC)]. B cell DNA methylation markers are particularly favorable for diagnosis due to the convenient access to peripheral blood.

Methods

We examined DNA methylation profiles of B cell lines using the Illumina GoldenGate BeadArray assay. Disease-associated CpGs/genes with changes in DNA methylation were identified by comparison of methylation profiles between B cell lines from IBD patients and their siblings without IBD. BeadArray data were validated using a bisulfite polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) method. To verify that observed changes in DNA methylation were not due to virus transformation, we compared specific CpG DNA methylation levels of GADD45A and POMC between B cell lines and matching peripheral blood B lymphocytes from five individuals.

Results

Using this approach with strict statistical analysis, we identified 11 IBD-associated CpG sites, 14 CD-specific CpG sites, and 24 UC-specific CpG sites with methylation changes in B cells.

Conclusions

IBD- and subtype-specific changes in DNA methylation were identified in B cells from IBD patients. Many of these genes have important immune and inflammatory response functions including several loci within the interleukin (IL)-12/IL-23 pathway.