Yinying Dong

Vascular endothelial cells facilitated HCC invasion and metastasis through the Akt and NF-κB pathways induced by paracrine cytokines

BackgroundIt is well documented that cancer cells secrete angiogenic factors to recruit and sustain tumor vascular networks. However, little is known about the effects of endothelial cells on the behavior of tumor cells. The study here was to determine the roles of endothelial cells in HCC cell growth, migration and invasion.MethodsA mixture of highly metastatic MHCC97H cells and HUVEC cells, as well as MHCC97H cells alone were subcutaneously injected into nude mice to observe the effects of HUVECs on HCC growth. The biological characteristics of MHCC97H cells respectively treated with conditioned medium (CM) derived from HUVECs and endothelial cell basal medium (EBM) in vitro, such as proliferation, migration and invasion, invasion/metastasis associated gene expression, were comparatively analyzed. Differential cytokines between CM and EBM were screened and identified using human cytokine array. Effects of the interested differential cytokine CCL2, IL-8 and CXCL16 and its related signaling pathways were further investigated in HCC cells.ResultsSubcutaneous tumorigenicity of MHCC97H cells in nude mice was promoted by HUVECs and its invasion/metastasis associated genes were significantly upregulated. The in vitro, proliferation, migration and invasion of HCC cells treated with CM were all significantly enhanced as compared to those with EBM stimulation. Simultaneously, PI3K/Akt and ERK1/2 pathway in HCC cells were activated by CM. Total of 25 differential cytokines were identified between CM and EBM such as angiopoietin-2, CCL2 (MCP-1), uPA, endostatin, CXCL16, IL-8, pentraxin 3 etc. The selected differential cytokines CCL2, IL-8 and CXCL16 all modulated the expressions of HCC invasion/metastasis genes, especially MMP2 and MMP9. In exposure to CCL2 or CXCL16 alone, upregulation in AKT phosphorylation but no change in ERK phosphorylation were found in MHCC97H cells, moreover the contents of nuclear transcription factor NF-κB were increased as compared to the control. However, no effects on the activation of Akt and ERK pathway in MHCC97H were found in exposure to IL-8.ConclusionThis study expands the contribution of endothelial cells to the progression of HCC. It unveils a new paradigm in which endothelial cells function as initiators of molecular crosstalks that enhance survival, migration and invasion of HCC cells.

Increasing matrix stiffness upregulates vascular endothelial growth factor expression in hepatocellular carcinoma cells mediated by integrin β1.

Matrix stiffness as a novel regulation factor involves in modulating the pathogenesis of hepatocellular carcinoma (HCC) invasion or metastasis. However, the mechanism by which matrix stiffness modulates HCC angiogenesis remains unknown. Here, using buffalo rat HCC models with different liver matrix stiffness backgrounds and an in vitro cell culture system of mechanically tunable Collagen1 (COL1)-coated polyacrylamide gel, we investigated the effects of different matrix stiffness levels on vascular endothelial growth factor (VEGF) expression in HCC cells and explored its regulatory mechanism for controlling HCC angiogenesis. Tissue microarray analysis showed that the expression levels of VEGF and CD31 were gradually upregulated in tumor tissues with increasing COL1 and lysyl oxidase (LOX) expression, indicating a positive correlation between tumor angiogenesis and matrix rigidity. The expression of VEGF and the phosphorylation levels of PI3K and Akt were all upregulated in HCC cells on high-stiffness gel than on low-stiffness gel. Meanwhile, alteration of intergrin β1 expression was found to be the most distinctive, implying that it might mediate the response of HCC cells to matrix stiffness simulation. After integrin β1 was blocked in HCC cells using specific monoclonal antibody, the expression of VEGF and the phosphorylation levels of PI3K and Akt at different culture times were accordingly suppressed and downregulated in the treatment group as compared with those in the control group. All data suggested that the extracellular matrix stiffness stimulation signal was transduced into HCC cells via integrin β1, and this signal activated the PI3K/Akt pathway and upregulated VEGF expression. This study unveils a new paradigm in which matrix stiffness as initiators to modulate HCC angiogenesis.

Dehydroepiandrosterone Sulphate: Action and Mechanism in the Brain

Dehydroepiandrosterone sulphate (DHEAS) is synthesised from dehydroepiandrosterone by the enzyme sulphotransferase. DHEAS is one of the most important neurosteroids in the brain. The concentration of DHEAS in the brain is sometimes higher than peripheral system. At the cellular level, DHEAS has been shown to modulate a variety of synaptic transmission, including cholinergic, GABAergic dopaminergic and glutamatergic synaptic transmission. In addition to the effect on the release of a number of neurotransmitters, DHEAS could also modulate the activity of postsynaptic receptors. DHEAS has been found to have multiple important effects on brain functions, such as memory enhancing, antidepressant and anxiolytic effects, and may have relationships with many brain diseases.

Dynamic expression patterns of differential proteins during early invasion of hepatocellular carcinoma.

Background Tumor cell invasion into the surrounding matrix has been well documented as an early event of metastasis occurrence. However, the dynamic expression patterns of proteins during early invasion of hepatocellular carcinoma (HCC) are largely unknown. Using a three-dimensional HCC invasion culture model established previously, we investigated the dynamic expression patterns of identified proteins during early invasion of HCC. Materials and Methods Highly metastatic MHCC97H cells and a liver tissue fragment were long-term co-cultured in a rotating wall vessel (RWV) bioreactor to simulate different pathological states of HCC invasion. The established spherical co-cultures were collected on days 0, 5, 10, and 15 for dynamic expression pattern analysis. Significantly different proteins among spheroids at different time points were screened and identified using quantitative proteomics of iTRAQ labeling coupled with LC–MS/MS. Dynamic expression patterns of differential proteins were further categorized by K-means clustering. The expression modes of several differentially expressed proteins were confirmed by Western blot and qRT–PCR. Results Time course analysis of invasion/metastasis gene expressions (MMP2, MMP7, MMP9, CD44, SPP1, CXCR4, CXCL12, and CDH1) showed remarkable, dynamic alterations during the invasion process of HCC. A total of 1,028 proteins were identified in spherical co-cultures collected at different time points by quantitative proteomics. Among these proteins, 529 common differential proteins related to HCC invasion were clustered into 25 types of expression patterns. Some proteins displayed significant dynamic alterations during the early invasion process of HCC, such as upregulation at the early invasion stage and downregulation at the late invasion stage (e.g., MAPRE1, PHB2, cathepsin D, etc.) or continuous upregulation during the entire invasion process (e.g., vitronectin, Met, clusterin, ICAM1, GSN, etc.). Conclusions Dynamic expression patterns of candidate proteins during the early invasion process of HCC facilitate the discovery of new molecular targets for early intervention to prevent HCC invasion and metastasis.

Microarray profiling of circular RNAs and the potential regulatory role of hsa_circ_0071410 in the activated human hepatic stellate cell induced by irradiation

Radiation-induced liver fibrosis (RILF) is considered as a major complication of radiation therapy for liver cancer. Circular RNA (circRNA) has been recently identified as a functional noncoding RNA involving in various biological processes. However, the expression pattern and regulatory capacity of circRNA in the irradiated hepatic stellate cell (HSC), the main fibrogenic cell type, still remain unclear. A circRNA microarray was used to identify circRNA expression profiles in irradiated and normal HSC. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to confirm the dysregulated circRNAs. Bioinformatic analyses including gene ontology (GO), KEGG pathway and circRNA/microRNA interaction network analysis were applied to predict the potential functions of circRNAs. Compared with the normal HSC, 179 circRNAs were found to be up-regulated and 630 circRNAs were down-regulated in irradiated HSC (fold change ≥2.0 and P<0.05). Six dysregulated circRNAs selected randomly were successfully verified by qRT-PCR. Bioinformatic analyses indicated that dysregulated circRNA might be involved in the cell response to irradiation and biological processes of hepatic fibrosis. Furthermore, inhibition of hsa_circ_0071410 increased the expression of miR-9-5p, resulting in the attenuation of irradiation induced HSC activation. In summary, this study revealed the expression profile and potential function of differentially expressed circRNAs in irradiated HSC, which provides novel clues for RILF study.

The role of inflammatory cytokines and ERK1/2 signaling in chronic prostatitis/chronic pelvic pain syndrome with related mental health disorders

Mental health disorders(MHD) in chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) have been widely studied. However, the underlying role of inflammatory cytokines and their associated signaling pathways have not been investigated. Here, we report the potential role of cytokines and associated signaling pathways in CP/CPPS patients with MHD and in a CP/CPPS animal model. CP/CPPS patients (nu2009=u2009810) and control subjects (nu2009=u2009992) were enrolled in this case-control multicenter study, and serum cytokine levels were measured. Male Sprague-Dawley rats received multiple intracutaneous injections of an immuno-agent along with a pertussis-diphtheria-tetanus triple vaccine for autoimmune CP/CPPS development. The results revealed that, in CP/CPPS patients with significant MHD, elevated IL-1α, IL-1β, IL-4, IL-13, and TNF-α serum levels were observed. The above five cytokines in CP/CPPS rats were significantly elevated in prostate tissue (pu2009<u20090.05), and IL-1β levels were elevated in serum and cerebrospinal fluid. In behavioral tests, CP/CPPS rats showed anxiety- and depression-like symptoms, and impaired spatial and associative memory performance (pu2009<u20090.05). In the CP/CPPS group, ERK1/2 phosphorylation levels were increased in the amygdala and nucleus accumbens, and decreased in the hippocampus, but not caudate nucleus. Thus, prostate-derived cytokines, especially IL-1β, cross the blood brain barrier and may lead to enhanced ERK1/2 signaling in several brain areas, possibly underlying induction of CP/CPPS-related MHD.

Matrix stiffness-mediated effects on stemness characteristics occurring in HCC cells

Matrix stiffness as an important physical attribute of extracellular matrix exerts significant impacts on biological behaviors of cancer cells such as growth, proliferation, motility, metabolism and invasion. However, its influence on cancer stemness still remains elusive. Here, we explore whether matrix stiffness-mediated effects on stemness characteristics occur in HCC cells. As the substrate stiffness increased, HCC cells exhibited high proportion of cells with CD133(+)/EpCAM(+), high expression levels of CD133, EpCAM, Nanog and SOX2, greater self-renewing ability and oxaliplatin resistance. Simultaneously, their phosphorylation levels of Akt and mTOR, as well as p-4E-BP and SOX2 expressions were also obviously upregulated. Conversely, knockdown of integrin β1 partially attenuated higher stiffness-mediated stemness characteristics in HCC cells, and reversed the phosphorylation levels of Akt and mTOR, and expressions of p-4E-BP and SOX2, suggesting that integrin β1 may deliver higher stiffness signal into HCC cells and activate mTOR signaling pathway. Additionally, mTOR inhibitor suppressed the mTOR phosphorylation level and expression levels of p-4E-BP and SOX2 in HCC cells grown on higher stiffness substrate, as well as depressed their stemness properties significantly, favoring a regulating role of mTOR signaling pathway in matrix stiffness-mediated effects on stemness. In summary, matrix stiffness may be involved in the process of stemness regulation via activating integrin β1/Akt/mTOR/SOX2 signaling pathway. To the best of our knowledge, this study first reveals a novel regulating pathway to direct the stemness characteristics in HCC cells.

MicroRNA-146a-5p Negatively Regulates Pro-Inflammatory Cytokine Secretion and Cell Activation in Lipopolysaccharide Stimulated Human Hepatic Stellate Cells through Inhibition of Toll-Like Receptor 4 Signaling Pathways.

Lipopolysaccharide (LPS)/toll-like receptor 4 (TLR4) signaling pathway is demonstrated to be involved in the hepatic fibrosis. MicroRNA (miR)-146a-5p is a key regulator of the innate immune response. The functional significance of miR-146a-5p during the LPS/TLR4 mediated hepatic fibrosis process remains unclear. In this study, we found that TLR4 and α-smooth muscle actin (α-SMA) were up-regulated and miR-146a-5p was down-regulated in human hepatic stellate cell (HSC) line LX2 after LPS stimulation. Overexpression of miR-146a-5p inhibited LPS induced pro-inflammatory cytokines secretion through down-regulating the expression levels of TLR-4, IL-1 receptor-associated kinase 1 (IRAK1), TNF receptor associated factor-6 (TRAF6) and phosphorylation of nuclear factor-kappa B (NF-κB). Knockdown of IRAK1 and TRAF6 also suppressed pro-inflammatory cytokine production by inhibiting NF-κB phosphorylation. In addition, miR-146a-5p mimic blocked LPS induced TRAF6 dependent c-Jun N-terminal kinase (JNK) and Smad2 activation as well as α-SMA production. Taken together, these results suggest that miR-146a-5p suppresses pro-inflammatory cytokine secretion and cell activation of HSC through inhibition of TLR4/NF-κB and TLR4/TRAF6/JNK pathway.

Activation of the JNK-c-Jun pathway in response to irradiation facilitates Fas ligand secretion in hepatoma cells and increases hepatocyte injury

BackgroundIt is well established that some irradiated liver non-parenchymal cells secrete pro-inflammatory cytokines to facilitate the development of radiation-induced liver disease. However, little is known on whether the irradiated hepatoma cells-mediated non-irradiated hepatocyte injury occurs in HCC patients. Here, we elucidated the roles of the irradiated hepatoma cells in driving non-irradiated hepatocyte injury and its underlying mechanism.MethodsSMMC7721 cells were cultured and divided into irradiated (4-Gy X-ray, R) and non-irradiated (NR) groups. At 24th hour after irradiation, conditioned medium (CM) from these cultures was mixed with normal culture medium in specific proportions, and termed as 7721-R-CM and 7721-NR-CM. Following incubation with these CM compound, the biological characteristics of L02 cells related to liver cell injury including viability, apoptosis and liver dysfunction indices were comparatively analyzed. Simultaneously, the levels of proliferation- and apoptosis-related cytokines in irradiated and non-irradiated SMMC7721 cells were also measured. FasL as a cytokine with significantly differential expression, was selected to clarify its effects on L02 apoptosis. Subsequently, FasL expression following irradiation was examined in SMMC7721 and other HCC cells with varying malignant potentials, as well as in HCC tissues, the related mechanism of higher expression of FasL in irradiated HCC cells was further investigated.ResultsApoptosis and liver dysfunction indices were all significantly enhanced in L02 cells treated with 7721-R-CM, whereas viability was suppressed, compared to those with 7721-NR-CM stimulation. FasL was identified as a leading differential cytokine in the irradiated SMMC7721 cells. Higher proportion of apoptosis was also found in L02 cells following FasL incubation. A recombinant Fas-Fc protein, which blocks Fas-FasL interaction, ameliorated 7721-R-CM-induced apoptosis in L02 cells. FasL was highly expressed in a dose-dependent manner, and peaked at the 24th hour post-irradiation in different HCC cells and their culture supernatant. Meanwhile, phosphorylation levels of JNK, ERK, Akt, and p38 were all upregulated significantly in irradiated HCC cells. But, only JNK inhibition was validated to block radiation-induced FasL expression in HCC cells. c-Jun, the target transcription factor of JNK, was also activated.ConclusionIn HCC cells, the JNK-c-Jun pathway plays an important role in mediating irradiation- induced FasL expression, which may be critical in determining non-irradiated hepatocyte injury.

Pro-inflammation NF-κB signaling triggers a positive feedback via enhancing cholesterol accumulation in liver cancer cells

BackgroundHepatocellular carcinoma (HCC) develops in a complex microenvironment characterized by chronic inflammation. In recent years, cholesterol metabolic abnormalities have been implicated the importance in cancer cell physiology. This study was designed to investigate the relationship between inflammation and cholesterol accumulation in HCC cells.MethodsHuman HCC cells HepG2 and Huh7 were cultured and stimulated with lipopolysaccharide (LPS) for 24xa0h. The changes of HCC cells related to cholesterol metabolism including intracellular cholesterol concentrations, cholesterol uptake, and the expression of cholesterol-related genes 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), LDL receptor (LDLR), sterol regulatory element-binding transcription factor 2 (SREBF2), and proprotein convertase subtilisin/kexin 9 (PCSK9) were comparatively analyzed. Simultaneously, the effects of nuclear factor-kappa B (NF-κB) signaling pathway on cholesterol metabolism were clarified by knocking-down of nuclear factor kappa-B kinase subunit alpha (IKKα) and TGF-beta-activated kinase 1 and MAP3K7-binding protein 3 (TAB3) via RNAi and microRNA (miR)-195. Subsequently, the roles of cholesterol accumulation in LPS induced pro-inflammatory effects were further investigated.ResultsPro-inflammatory factor LPS significantly increased intracellular cholesterol accumulation by upregulating the expression of HMGCR, LDLR, and SREBF2, while downregulating the expression of PCSK9. These effects were revealed to depend on NF-κB signaling pathway by knocking-down and overexpression of IKKα and TAB3. Additionally, miR-195, a regulator directly targeting IKKα and TAB3, blocked the effects of cholesterol accumulation, further supporting the critical role of pro-inflammation NF-κB signaling in regulating cholesterol accumulation. Intriguingly, the accumulation of cholesterol conversely exerted an augmented pro-inflammation effects by further activating NF-κB signaling pathway.ConclusionsThese results indicated that pro-inflammation effects of NF-κB signaling could be augmented by a positive feedback via enhancing the cholesterol accumulation in liver cancer cells.