GW501516-activated PPARβ/δ promotes liver fibrosis via p38-JNK MAPK-induced hepatic stellate cell proliferation

Background

After liver injury, the repair process comprises activation and proliferation of hepatic stellate cells (HSCs), which produce extracellular matrix (ECM) proteins. Peroxisome proliferator-activated receptor beta/delta (PPARβ/δ) is highly expressed in these cells, but its function in liver repair remains incompletely understood. This study investigated whether activation of PPARβ/δ with the ligand GW501516 influenced the fibrotic response to injury from chronic carbon tetrachloride (CCl₄) treatment in mice. Wild type and PPARβ/δ-null mice were treated with CCl₄ alone or CCl₄ co-administered with GW501516. To unveil mechanisms underlying the PPARβ/δ-dependent effects, we analyzed the proliferative response of human LX-2 HSCs to GW501516 in the presence or absence of PPARβ/δ.

Results

We found that GW501516 treatment enhanced the fibrotic response. Compared to the other experimental groups, CCl₄/GW501516-treated wild type mice exhibited increased expression of various profibrotic and pro-inflammatory genes, such as those involved in extracellular matrix deposition and macrophage recruitment. Importantly, compared to healthy liver, hepatic fibrotic tissues from alcoholic patients showed increased expression of several PPAR target genes, including phosphoinositide-dependent kinase-1, transforming growth factor beta-1, and monocyte chemoattractant protein-1. GW501516 stimulated HSC proliferation that caused enhanced fibrotic and inflammatory responses, by increasing the phosphorylation of p38 and c-Jun N-terminal kinases through the phosphoinositide-3 kinase/protein kinase-C alpha/beta mixed lineage kinase-3 pathway.

Conclusions

This study clarified the mechanism underlying GW501516-dependent promotion of hepatic repair by stimulating proliferation of HSCs via the p38 and JNK MAPK pathways.     

Proline-rich tyrosine kinase 2 (Pyk2) promotes cell motility of hepatocellular carcinoma through induction of epithelial to mesenchymal transition

Aims

Proline-rich tyrosine kinase 2 (Pyk2), a non-receptor tyrosine kinase of the focal adhesion kinase (FAK) family, is up-regulated in more than 60% of the tumors of hepatocellular carcinoma (HCC) patients. Forced overexpression of Pyk2 can promote the proliferation and invasion of HCC cells. In this study, we aimed to explore the underlying molecular mechanism of Pyk2-mediated cell migration of HCC cells.

Methodology/Principal Findings

We demonstrated that Pyk2 transformed the epithelial HCC cell line Hep3B into a mesenchymal phenotype via the induction of epithelial to mesenchymal transition (EMT), signified by the up-regulation of membrane ruffle formation, activation of Rac/Rho GTPases, down-regulation of epithelial genes E-cadherin and cytokeratin as well as promotion of cell motility in presence of lysophosphatidic acid (LPA). Suppression of Pyk2 by overexpression of dominant negative PRNK domain in the metastatic HCC cell line MHCC97L transformed its fibroblastoid phenotype to an epithelial phenotype with up-regulation of epithelial genes, down-regulation of mesenchymal genes N-cadherin and STAT5b, and reduction of LPA-induced membrane ruffle formation and cell motility. Moreover, overexpression of Pyk2 in Hep3B cells promoted the phosphorylation and localization of mesenchymal gene Hic-5 onto cell membrane while suppression of Pyk2 in MHCC97L cells attenuated its phosphorylation and localization.

Conclusion

These data provided new evidence of the underlying mechanism of Pyk2 in controlling cell motility of HCC cells through regulation of genes associated with EMT.     

Molecular network profiling of U373MG human glioblastoma cells following induction of apoptosis by novel marine-derived anti-cancer 1,2,3,4-tetrahydroisoquinoline alkaloids

Background

Glioblastoma is the most aggressive form of brain tumors showing resistance to treatment with various chemotherapeutic agents. The most effective way to eradicate glioblastoma requires the concurrent inhibition of multiple signaling pathways and target molecules involved in the progression of glioblastoma. Recently, we obtained a series of 1,2,3,4-tetrahydroisoquinoline alkaloids with potent anti-cancer activities, including ecteinascidin-770 (ET-770; the compound 1a) and renieramycin M (RM; the compound 2a) from Thai marine invertebrates, together with a 2’-N-4”-pyridinecarbonyl derivative of ET-770 (the compound 3). We attempted to characterize the molecular pathways responsible for cytotoxic effects of these compounds on a human glioblastoma cell line U373MG.

Methods

We studied the genome-wide gene expression profile on microarrays and molecular networks by using pathway analysis tools of bioinformatics.

Results

All of these compounds induced apoptosis of U373MG cells at nanomolar concentrations. The compound 3 reduced the expression of 417 genes and elevated the levels of 84 genes, while ET-770 downregulated 426 genes and upregulated 45 genes. RM decreased the expression of 274 genes and increased the expression of 9 genes. The set of 196 downregulated genes and 6 upregulated genes showed an overlap among all the compounds, suggesting an existence of the common pathways involved in induction of apoptosis. We identified the ErbB (EGFR) signaling pathway as one of the common pathways enriched in the set of downregulated genes, composed of PTK2, AKT3, and GSK3B serving as key molecules that regulate cell movement and the nervous system development. Furthermore, a GSK3B-specific inhibitor induced apoptosis of U373MG cells, supporting an anti-apoptotic role of GSK3B.

Conclusion

Molecular network analysis is a useful approach not only to characterize the glioma-relevant pathways but also to identify the network-based effective drug targets.     

LncRNA MAGI2-AS3 inhibits hepatocellular carcinoma cell proliferation and migration by targeting the miR-374b-5p/SMG1 signaling pathway

Long noncoding RNAs (lncRNAs) have been proven to play critical roles in cancer progression. Recently, lncRNA MAGI2-AS3 has been revealed to be a tumor suppressor and inhibit cell growth by targeting the Fas/FasL signalling pathway in breast cancer. However, the role and underlying mechanism of MAGI2-AS3 in hepatocellular carcinoma (HCC) remain largely unknown. In the current study, we found that MAGI2-AS3 expression is downregulated in HCC tissues and closely associated with some clinical characteristics (tumor size, lymph node metastasis, and TNM stage) and poor overall survival. Overexpression of MAGI2-AS3 inhibits HCC cell proliferation and migration in vitro, while impedes tumor growth in vivo accordantly. In addition, our data suggest that MAGI2-AS3 could function as an endogenous sponge of miR-374b-5p by directly binding to it and suppressing its expression. Furthermore, miR-374b-5p upregulation could restore the inhibitory effect of MAGI2-AS3 on HCC cells processes. Moreover, suppressor with morphogenetic effect on genitalia family member 1 (SMG1) is positively regulated by MAGI2-AS3 via absorbing miR-374b-5p in HCC cells. More important, SMG1 knockdown reverses the suppressive function of MAGI2-AS3 in HCC cell processes. Taken together, we reveal a functional MAGI2-AS3/miR-374b-5p/SMG1 axis that suppresses HCC progression, potently suggesting a new road for HCC treatment.     

The role of proline rich tyrosine kinase 2 (Pyk2) on cisplatin resistance in hepatocellular carcinoma

Aims

We previously demonstrated Proline rich tyrosine kinase 2 (Pyk2) plays important roles in regulating tumor progression, migration and invasion in hepatocellular carcinoma (HCC). In this study, we aimed to examine the role of proline rich tyrosine kinase 2 (Pyk2) on cisplatin resistance in HCC and to explore its underlying molecular mechanism.

Methodology/Principal Findings

Stable transfectants either overexpressing or suppressing Pyk2 were established in different HCC cell lines. MTT, colony formation and Annexin-V assays were employed to examine their in vitro responses to cisplatin. Xenograft ectopic and orthotopic nude mice models were generated to investigate the in vivo responses of them to cisplatin treatment. cDNA microarray was performed to identify Pyk2-induced genes which were further validated by quantitative real-time RT-PCR using clinical HCC samples. In vitro functional study demonstrated that Pyk2-overexpressing HCC transfectants exhibited relatively lower cytotoxicity, higher colony-forming ability and lower apoptosis to cisplatin compared with the control transfectants. Moreover, Pyk2 overexpressing HCC transfectants had a higher survival rate under cisplatin treatment by up-regulation of AKT phosphorylation. In vivo xenograft nude mice model demonstrated that Pyk2-overexpressing transfectants developed higher tolerance to cisplatin treatment together with less tumor necrosis and apoptosis. cDNA microarray analysis revealed that there were more than 4,000 genes differentially expressed upon overexpression of Pyk2. Several upregulated genes were found to be involved in drug resistance and invasion in cancers. Among them, the expression profiles of MDR1, GAGE1, STAT1 and MAP7 were significantly associated with the expression of Pyk2 in clinical HCC samples.

Conclusions

Our results may suggest a new evidence of Pyk2 on promoting cisplatin resistance of HCC cells through preventing cell apoptosis, activation of AKT pathway and upregulation of drug resistant genes.     

Recurrence-associated pathways in hepatitis B virus-positive hepatocellular carcinoma

Background

Despite the recent identification of several prognostic gene signatures, the lack of common genes among experimental cohorts has posed a considerable challenge in uncovering the molecular basis underlying hepatocellular carcinoma (HCC) recurrence for application in clinical purposes. To overcome the limitations of individual gene-based analysis, we applied a pathway-based approach for analysis of HCC recurrence.

Results

By implementing a permutation-based semi-supervised principal component analysis algorithm using the optimal principal component, we selected sixty-four pathways associated with hepatitis B virus (HBV)-positive HCC recurrence (p < 0.01), from our microarray dataset composed of 142 HBV-positive HCCs. In relation to the public HBV- and public hepatitis C virus (HCV)-positive HCC datasets, we detected 46 (71.9%) and 18 (28.1%) common recurrence-associated pathways, respectively. However, overlap of recurrence-associated genes between datasets was rare, further supporting the utility of the pathway-based approach for recurrence analysis between different HCC datasets. Non-supervised clustering of the 64 recurrence-associated pathways facilitated the classification of HCC patients into high- and low-risk subgroups, based on risk of recurrence (p < 0.0001). The pathways identified were additionally successfully applied to discriminate subgroups depending on recurrence risk within the public HCC datasets. Through multivariate analysis, these recurrence-associated pathways were identified as an independent prognostic factor (p < 0.0001) along with tumor number, tumor size and Edmondson’s grade. Moreover, the pathway-based approach had a clinical advantage in terms of discriminating the high-risk subgroup (N = 12) among patients (N = 26) with small HCC (<3 cm).

Conclusions

Using pathway-based analysis, we successfully identified the pathways involved in recurrence of HBV-positive HCC that may be effectively used as prognostic markers.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1472-x) contains supplementary material, which is available to authorized users.     

Gene expression profiling of subcutaneous adipose tissue in morbid obesity using a focused microarray: Distinct expression of cell-cycle- and differentiation-related genes

Background

Prevalence of obesity is increasing to pandemic proportions. However, obese subjects differ in insulin resistance, adipokine production and co-morbidities. Based on fasting plasma analysis, obese subjects were grouped as Low Acylation Stimulating protein (ASP) and Triglyceride (TG) (LAT) vs High ASP and TG (HAT). Subcutaneous (SC) and omental (OM) adipose tissues (n = 21) were analysed by microarray, and biologic pathways in lipid metabolism and inflammation were specifically examined.

Methods

LAT and HAT groups were matched in age, obesity, insulin, and glucose, and had similar expression of insulin-related genes (InsR, IRS-1). ASP related genes tended to be increased in the HAT group and were correlated (factor B, adipsin, complement C3, p < 0.01 each). Differences between LAT and HAT group were almost exclusively in SC tissue, with little difference in OM tissue. Increased C5L2 (p < 0.01), an ASP receptor, in HAT suggests a compensatory ASP pathway, associated with increased TG storage.

Results

HAT adipose tissue demonstrated increased lipid related genes for storage (CD36, DGAT1, DGAT2, SCD1, FASN, and LPL), lipolysis (HSL, CES1, perilipin), fatty acid binding proteins (FABP1, FABP3) and adipocyte differentiation markers (CEBPα, CEBPβ, PPARγ). By contrast, oxidation related genes were decreased (AMPK, UCP1, CPT1, FABP7). HAT subjects had increased anti-inflammatory genes TGFB1, TIMP1, TIMP3, and TIMP4 while proinflammatory PIG7 and MMP2 were also significantly increased; all genes, p < 0.025.

Conclusion

Taken together, the profile of C5L2 receptor, ASP gene expression and metabolic factors in adipose tissue from morbidly obese HAT subjects suggests a compensatory response associated with the increased plasma ASP and TG.     

Association Analysis of Genetic Variants in the Myosin IXB Gene in Acute Pancreatitis

Introduction

Impairment of the mucosal barrier plays an important role in the pathophysiology of acute pancreatitis. The myosin IXB (MYO9B) gene and the two tight-junction adaptor genes, PARD3 and MAGI2, have been linked to gastrointestinal permeability. Common variants of these genes are associated with celiac disease and inflammatory bowel disease, two other conditions in which intestinal permeability plays a role. We investigated genetic variation in MYO9B, PARD3 and MAGI2 for association with acute pancreatitis.

Methods

Five single nucleotide polymorphisms (SNPs) in MYO9B, two SNPs in PARD3, and three SNPs in MAGI2 were studied in a Dutch cohort of 387 patients with acute pancreatitis and over 800 controls, and in a German cohort of 235 patients and 250 controls.

Results

Association to MYO9B and PARD3 was observed in the Dutch cohort, but only one SNP in MYO9B and one in MAGI2 showed association in the German cohort (p < 0.05). Joint analysis of the combined cohorts showed that, after correcting for multiple testing, only two SNPs in MYO9B remained associated (rs7259292, p = 0.0031, odds ratio (OR) 1.94, 95% confidence interval (95% CI) 1.35-2.78; rs1545620, p = 0.0006, OR 1.33, 95% CI 1.16-1.53). SNP rs1545620 is a non-synonymous SNP previously suspected to impact on ulcerative colitis. None of the SNPs showed association to disease severity or etiology.

Conclusion

Variants in MYO9B may be involved in acute pancreatitis, but we found no evidence for involvement of PARD3 or MAGI2.     

Treatment with captopril abrogates the altered expression of alpha1 macroglobulin and alpha1 antiproteinase in sera of spontaneously hypertensive rats

Background

Proteins that are associated with hypertension may be identified by comparing the 2-dimensional gel electrophoresis (2-DE) profiles of the sera of spontaneously hypertensive rats (SHR) with those generated from normotensive Spraque-Dawley rats (SDR).

Results

Five proteins of high abundance were found to be significantly altered when the 2-DE serum profiles of the SHR were compared to those that were similarly generated from the SDR. Analysis by mass spectrometry and database search identified the proteins as retinol binding protein 4, complement C3, albumin (19.9 kDa fragment), alpha1 macroglobulin and alpha1 antiproteinase, which are all known to be associated with hypertension. The altered expression of the two latter proteins was found to be abrogated when similar analysis was performed on sera of the SHR that were treated with captopril.

Conclusion

Our data suggests that serum alpha1 macroglobulin and alpha1 antiproteinase are potentially useful complementary biomolecular indicators for monitoring of hypertension.     

WNT and NOTCH signaling pathways as activators for epidermal growth factor receptor in esophageal squamous cell carcinoma

Background

Esophageal squamous cell carcinoma (ESCC) is the most common histological type of esophageal cancer, with a poor prognosis. Deregulation of WNT and NOTCH signaling pathways is important in ESCC progression, which can be due to either malfunction of their components or crosstalk with other pathways. Therefore, identification of new crosstalk between such pathways may be effective to introduce new strategies for targeted therapy of cancer. A correlation study was performed to assess the probable interaction between growth factor receptors and WNT/NOTCH pathways via the epidermal growth factor receptor (EGFR) and Musashi1 (MSI1), respectively.

Methods

Levels of MSI1/EGFR mRNA expression in tumor tissues from 48 ESCC patients were compared to their corresponding normal tissues using real-time polymerase chain reaction.

Results

There was a significant correlation between EGFR and MSI1 expression (p?=?0.05). Moreover, there was a significant correlation between EGFR/MSI1 expression and grade of tumor differentiation (p?=?0.02).

Conclusion

This study confirms a direct correlation between MSI1 and EGFR and may support the important role of MSI1 in activation of EGFR through NOTCH/WNT pathways in ESCC.

     

Low-intensity pulsed ultrasound activates the phosphatidylinositol 3 kinase/Akt pathway and stimulates the growth of chondrocytes in three-dimensional cultures: a basic science study

Introduction

The effect of low-intensity pulsed ultrasound (LIPUS) on cell growth was examined in three-dimensional-cultured chondrocytes with a collagen sponge. To elucidate the mechanisms underlying the mechanical activation of chondrocytes, intracellular signaling pathways through the Ras/mitogen-activated protein kinase (MAPK) and the integrin/phosphatidylinositol 3 kinase (PI3K)/Akt pathways as well as proteins involved in proliferation of chondrocytes were examined in LIPUS-treated chondrocytes.

Methods

Articular cartilage tissue was obtained from the metatarso-phalangeal joints of freshly sacrificed pigs. Isolated chondrocytes mixed with collagen gel and culture medium composites were added to type-I collagen honeycomb sponges. Experimental cells were cultured with daily 20-minute exposures to LIPUS. The chondrocytes proliferated and a collagenous matrix was formed on the surface of the sponge. Cell counting, histological examinations, immunohistochemical analyses and western blotting analysis were performed.

Results

The rate of chondrocyte proliferation was slightly but significantly higher in the LIPUS group in comparison with the control group during the 2-week culture period. Western blot analysis showed intense staining of type-IX collagen, cyclin B₁ and cyclin D₁, phosphorylated focal adhesion kinase, and phosphorylated Akt in the LIPUS group in comparison with the control group. No differences were detected, however, in the MAPK, phosphorylated MAPK and type-II collagen levels.

Conclusion

LIPUS promoted the proliferation of cultured chondrocytes and the production of type-IX collagen in a three-dimensional culture using a collagen sponge. In addition, the anabolic LIPUS signal transduction to the nucleus via the integrin/phosphatidylinositol 3-OH kinase/Akt pathway rather than the integrin/MAPK pathway was generally associated with cell proliferation.     

Genetic variants associated with the progression of hepatocellular carcinoma in hepatitis C Egyptian patients

Background

Hepatocellular carcinoma (HCC) associated to infection with hepatitis C virus (HCV) has become the fastest-rising cause of cancer-related deaths. Genetic variations may play an important role in the development of HCC in HCV patients. Ghrelin exerts anti-inflammatory, antifibrotic and hepatoprotective effects on chronically injured hepatic tissues. Ghrelin gene shows several single nucleotide polymorphisms (SNPs) including − 604G/A, Arg51Gln, and Leu72Met. Hemochromatosis gene (HFE) mutations namely C282Y and H63D may cause hepatic iron overload, thus increasing the risk of HCC in HCV patients.

Aim

To investigate the association of progression of HCC with ghrelin and HFE gene polymorphisms in HCV Egyptian patients.

Methods

Seventy-nine chronic HCV patients (thirty-nine developed HCC and forty did not), and forty healthy control subjects were included in the study. The polymorphisms were evaluated by PCR/RFLP analysis, and related protein levels were measured by either ELISA or colorimetric assays.

Results

The three tested SNPs on ghrelin gene were detected in the studied groups, only one SNP (Arg51Gln) showed significantly higher GA, AA genotypes and A allele frequencies in hepatitis C patients who developed HCC than in hepatitis C patients without HCC and controls. Of the two mutations studied on HFE gene only H63D heterozygous allele was detected, and its frequency did not statistically differ among studied groups.

Conclusion

Our results suggest that A allele at position 346 of the ghrelin gene is associated with susceptibility to HCC in hepatitis C patients.     

Hypoxia promotes the migration and invasion of human hepatocarcinoma cells through the HIF-1α–IL-8–Akt axis

Background

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third most common cause of cancer-related death worldwide. The 5-year survival rate remains low despite considerable research into treatments of HCC, including surgery, radiotherapy and chemotherapy. Many mechanisms within HCC still require investigation, including the influence of hypoxia, which has a crucial role in many cancers and is associated with metastasis. Hypoxia inducible factor-1α (HIF-1α) is known to regulate the expression of many chemokines, including interleukin-8 (IL-8), which is associated with tumor metastasis. Although many studies have reported that HIF-1α is associated with HCC migration and invasion, the underlying mechanisms remain unknown.

Methods

The expression level of HIF-1α was determined in HCC cells. The correlation of IL-8 and HIF-1α expressions was assessed via knockdown of HIF-1α. HCC cells were also used to assess the influence of HIF-1α on HCC cell migration and invasion. LY294002, an inhibitor of the Akt pathway, was used to confirm the associated signaling pathways.

Results

We observed a significant attenuation of cell migration and invasion after silencing of HIF-1α. Exogenously expressing IL-8 restored migration and invasion. Akt was found to be involved in this process.

Conclusion

Hypoxia promotes HCC cell migration and invasion through the HIF-1α–IL-8–Akt axis.

     

Three steps to the immortality of cancer cells: senescence,polyploidy and self-renewal

Background

Hepatocellular carcinoma (HCC) exhibits strong intrinsic and acquired drug resistance which is the main obstacle to chemotherapy. Overexpression of ATP binding cassette (ABC) proteins correlates with activation of mitogen activated protein kinase (MAPK) pathway in HCC. Here, we systematically investigated the inhibition of MAPK pathway and its role in regulating HCC cell growth as well as ABC proteins MRP1 and MRP3 expression.

Methods

The Raf1 kinase inhibitor (GW5074) and different MEK inhibitors (U0126 and AZD6244) were used to treat HCC cells to identify their effects on HCC cell growth and ABC proteins expression in vitro. Cell viability tests were performed after the treatment of MAPK pathway inhibitors and in combination with gemcitabine or doxorubicin. Western blot was applied to assess the changes of MAPK pathway and protein expression of MRP1 and MRP3. Flow cytometry was used to measure intracellular doxorubicin accumulation after the treatment of MEK inhibitors.

Results

Both Raf1 inhibitor (GW5074) and MEK inhibitors (U0126 and AZD6244) suppressed HCC cell growth in a dose dependent manner. Pre-treatment of MEK inhibitor U0126 or AZD6244 sensitized HCC cells to gemcitabine or doxorubicin based chemotherapy. Raf1 inhibitor GW5074 had no effect on MRP1 and MRP3 protein expression. Treatment of gemcitabine or doxorubicin activated phosphorylated ERK and induced the upregulation of MRP1 and MRP3. MEK inhibitors U0126 and AZD6244 deactivated phosphorylated ERK, decreased endogenous MRP1 expression, reversed gemcitabine or doxorubicin induced MRP1 and MRP3 upregulation, and increased the intracellular doxorubicin accumulation.

Conclusion

This study provides evidence that MEK inhibitors sensitize HCC cells to chemotherapy by increasing intracellular chemodrug accumulation. MEK inhibirors U0126 and AZD6244 reduced MRP1 as well as MRP3 expression, and may contribute partially to the sensitization. The combination of MEK inhibitor and conventional chemotherapy may offer new therapeutic option for the treatment of resistant HCC.