[Show abstract][Hide abstract] ABSTRACT: Liver fibrosis represents the consequences of a sustained wound healing response to chronic liver injuries, and its progression toward cirrhosis is the major cause of liver-related morbidity and mortality worldwide. However, anti-fibrotic treatment remains an unconquered area for drug development. Accumulating evidence indicate that oxidative stress plays a critical role in liver fibrogenesis. In this study, we found that PQQ, a natural anti-oxidant present in a wide variety of human foods, exerted potent anti-fibrotic and ROS-scavenging activity in Balb/C mouse models of liver fibrosis. The antioxidant activity of PQQ was involved in the modulation of multiple steps during liver fibrogenesis, including chronic liver injury, hepatic inflammation, as well as activation of hepatic stellate cells and production of extracellular matrix. PQQ also suppressed the up-regulation of RACK1 in activated HSCs in vivo and in vitro. Our data suggest that PQQ suppresses oxidative stress and liver fibrogenesis in mice, and provide rationale for the clinical application of PQQ in the prevention and treatment of liver fibrosis.
[Show abstract][Hide abstract] ABSTRACT: Liver fibrosis represents the consequences of a sustained wound healing response to chronic liver injury, and activation of quiescent hepatic stellate cells (HSCs) into a myofibroblast-like phenotype is considered as the central event of liver fibrosis. RACK1, the receptor for activated C-kinase 1, is a classical scaffold protein implicated in numerous signaling pathways and cellular processes; however, the role of RACK1 in liver fibrosis is little defined. Herein, we report that RACK1 is up-regulated in activated HSCs in transforming growth factor beta 1 (TGF-β1)-dependent manner both in vitro and in vivo, and TGF-β1 stimulates the expression of RACK1 through NF-κB signaling. Moreover, RACK1 promotes TGF-β1 and platelet-derived growth factor (PDGF)-mediated activation of pro-fibrogenic pathways as well as the differentiation, proliferation and migration of HSCs. Depletion of RACK1 suppresses the progression of TAA-induced liver fibrosis in vivo. In addition, the expression of RACK1 in fibrogenic cells also positively correlates well with the stage of liver fibrosis in clinical cases. Our results suggest RACK1 as a downstream target gene of TGF-β1 involved in the modulation of liver fibrosis progression in vitro and in vivo, and propose a strategy to target RACK1 for liver fibrosis treatment.
[Show abstract][Hide abstract] ABSTRACT: Chronic neurodegeneration is in part caused by a vicious cycle of persistent microglial activation and progressive neuronal cell loss. However, the driving force behind this cycle remains poorly understood. In this study, we used medium conditioned by necrotic differentiated-PC12 cells to confirm that damaged neurons can release soluble injury signals, including heat shock protein 60 (HSP60), to efficiently promote the neurotoxic cycle involving microglia. Since lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) has previously been identified as a novel receptor for HSP60, we hypothesize that LOX-1 through binding to extracellular HSP60 promotes microglia-mediated neuroinflammation. In this study, we observed that LOX-1 expression is induced upon toxic microglial activation, and discovered that LOX-1 is necessary in microglia for sensing soluble neuronal injury signal(s) in the conditioned medium to induce generation of pro-inflammatory mediators (IL-1β, TNF-α, NO and ROS) that promote neurotoxicity. Employing a unique eukaryotic HSP60-overexpression method, we further demonstrated that extracellular HSP60 acts on microglial LOX-1 to boost the production of pro-inflammatory factors (IL-1β, NO and ROS) in microglia and to propagate neuronal damage. These results indicate that LOX-1 is essential in microglia for promoting an inflammatory response in the presence of soluble neuronal-injury signals such as extracellular HSP60, thereby linking neuroinflammation and neurotoxicity.
No preview · Article · Jul 2012 · Neurochemistry International
[Show abstract][Hide abstract] ABSTRACT: Prosaposin, a secreted protein, is a well-known pleiotropic growth factor. Although a previous report has indicated that prosaposin is overexpressed in breast cancer cell lines, the role of prosaposin in the development of breast cancer remains to be identified. Here, we first revealed that prosaposin upregulated estrogen receptor alpha expression, nuclear translocation and transcriptional activity by western blot, immunofluorescence assay and dual luciferase reporter gene assay, respectively. Furthermore, we demonstrated prosaposin upregulated estrogen receptor alpha expression through MAPK-signaling pathway using MAPK inhibitor. Proliferation assay and tumor xenograft experiments in nude mice (n = 6 per group) further confirmed prosaposin could promote breast cancer growth significantly in vitro and in vivo. These findings suggested that prosaposin might enhance estrogen receptor alpha-mediated signaling axis and play a role in breast cancer development and progression.
[Show abstract][Hide abstract] ABSTRACT: Coordinated translation initiation is coupled with cell cycle progression and cell growth, whereas excessive ribosome biogenesis and translation initiation often lead to tumor transformation and survival. Hepatocellular carcinoma (HCC) is among the most common and aggressive cancers worldwide and generally displays inherently high resistance to chemotherapeutic drugs. We found that RACK1, the receptor for activated C-kinase 1, was highly expressed in normal liver and frequently upregulated in HCC. Aberrant expression of RACK1 contributed to in vitro chemoresistance as well as in vivo tumor growth of HCC. These effects depended on ribosome localization of RACK1. Ribosomal RACK1 coupled with PKCβII to promote the phosphorylation of eukaryotic initiation factor 4E (eIF4E), which led to preferential translation of the potent factors involved in growth and survival. Inhibition of PKCβII or depletion of eIF4E abolished RACK1-mediated chemotherapy resistance of HCC in vitro. Our results imply that RACK1 may function as an internal factor involved in the growth and survival of HCC and suggest that targeting RACK1 may be an efficacious strategy for HCC treatment.
Full-text · Article · Jun 2012 · The Journal of clinical investigation
[Show abstract][Hide abstract] ABSTRACT: Hepatocellular carcinoma (HCC) is among the most common and aggressive cancers worldwide, and novel therapeutic strategies are urgently required to improve clinical outcome. Interferon-alpha (IFN-α) and sorafenib are widely used as anti-tumor agents against various malignancies. In this study, we investigated the combined effects of IFN-α and sorafenib against HCC. We demonstrated that the combination therapy synergistically suppressed HCC cellular viability, arrested cell cycle propagation and induced apoptosis in HCC cells. Further research revealed that IFN-α and sorafenib collaboratively regulated the expression levels of cell cycle-related proteins Cyclin A and Cyclin B as well as the pro-survival Bcl-2 family proteins Mcl-1, Bcl-2 and Bcl-X(L). Moreover, sorafenib inhibited IFN-α induced oncogenic signaling of STAT3, AKT and ERK but not the activation of the tumor suppressor STAT1. Xenograft experiments also confirmed the combined effects of IFN-α and sorafenib on tumor growth inhibition and apoptosis induction in vivo. In conclusion, these results provide rationale for the clinical application of IFN-α and sorafenib combination therapy in HCC treatment.
No preview · Article · May 2012 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: Patients with chronic hepatitis B virus (HBV) infection are at risk for metastatic hepatocellular carcinoma (HCC). Metastatic cancer cells develop resistance to anoikis. The serine/threonine p21-activated kinase (PAK) 1 regulates cytoskeletal dynamics and protects cells from anoikis; it also promotes virus replication. We investigated the effects of PAK1 on anoikis resistance in human hepatoma cells and in mice.
We transfected human hepatoma cells with pHBV1.3 (to mimic HBV replication) or plasmids encoding different HBV proteins; we performed colony formation and anoikis assays. We knocked down levels of PAK1 and Bcl2, or inhibited their activity, in hepatoma cells and quantified anoikis and growth of tumor xenografts in nude mice; we also measured anoikis of tumor cells isolated from ascites of the mice. We performed immunohistochemical analysis of PAK1 levels in HCC samples from patients.
Human hepatoma cells transfected with pHBV1.3 expressing hepatitis B virus X protein (HBx) underwent anchorage-independent proliferation, were resistant to anoikis, and had higher levels of Bcl2 than nontransfected cells. Expression of HBx increased mitochondrial levels of Bcl2 and PAK1, which interacted physically. Anoikis resistance of Huh7 and SK-Hep1 cells required PAK1 activity and Bcl2. Expression of HBx promoted growth of Huh7 xenograft tumors in mice; PAK1 knockdown reduced growth of these tumors in mice and anoikis of cells isolated from these tumors. In human HCC samples, increased levels of PAK1 correlated with poor prognosis, HBV infection, and portal vein tumor thrombosis.
The HBV protein HBx up-regulates PAK1, allows hepatoma cells to become resistant to anoikis, and promotes growth of aggressive xenograft tumors in mice. HBx induction of PAK1 might promote progression of HCC in patients with chronic HBV infection.
[Show abstract][Hide abstract] ABSTRACT: Of the three envelope glycoproteins encoded by hepatitis B virus (HBV) that are collectively referred to as HBV surface antigen (HBsAg), the large HBsAg (LHBs) glycoprotein is expressed preferentially in HBV-associated hepatocellular carcinoma. LHBs can act as an oncogene in transgenic mice, but how it contributes functionally to hepatocarcinogenesis remains unclear. In this study, we determined the molecular and functional roles of LHBs during HBV-associated hepatocarcinogenesis. LHBs increased tumor formation of hepatoma cells. Moreover, expression of LHBs but not other HBV envelope glycoproteins specifically promoted proliferation of hepatoma and hepatic cells in vitro. Mechanistic investigations revealed that these effects were caused by activation of the Src/PI3K/Akt pathway through proximal stimulation of PKCα/Raf1 signaling by LHBs. Proliferation induced by stable LHBs expression was associated with increased G(1)-S cell-cycle progression and apoptosis resistance mediated by Src kinase activation, as established in hepatocellular carcinoma clinical specimens. Importantly, LHBs-induced cellular proliferation and tumor formation were reversed by administration of the Src inhibitor saracatinib. Together, our findings suggest that LHBs promotes tumorigenesis of hepatoma cells by triggering a PKCα/Raf1 to Src/PI3K/Akt signaling pathway, revealing novel insights into the underlying mechanisms of HBV-associated hepatocarcinogenesis.
[Show abstract][Hide abstract] ABSTRACT: p53-Induced ring-H2 protein (Pirh2), a recently identified ubiquitin-protein ligase, interacts with p27(Kip1) to promote ubiquitination of p27(Kip1) independently of p53. High Pirh2 and low p27(Kip1) immunoreactivity are associated with a poor prognosis in several cancers, including resistant phenotypes. In the present study, we investigated the role of Pirh2 and p27(Kip1) in human hepatocellular carcinoma (HCC) progression. Immunohistochemical analysis was performed on formalin-fixed paraffin sections of 87 specimens. Statistical analysis showed that expression of Pirh2 was negatively related to p27(Kip1) expression (r = 0.787; P < .05), and Pirh2 expression correlated significantly with histologic grade (P < .001), venous invasion (P = .004), tumor size (P = .024), and the presence of multiple tumor-bearing lymph nodes (P = .017), whereas p27(Kip1) expression correlated significantly with histologic grade (P < .001), venous invasion (P = .048), and cirrhosis (P = .028). By Kaplan-Meier analysis, the survival curves of low versus high expressers of Pirh2 and p27(Kip1) showed significant separation (P < .01). Molecular interaction could be demonstrated between Pirh2 and p27(Kip1) in three HCC cell lines. In vitro, following release of two HCC cell lines from serum starvation, the expression of Pirh2 was upregulated, whereas p27(Kip1) was downregulated. Our results suggest that Pirh2 mediates the degradation of p27(Kip1) and participates in cell proliferation in human HCC. These findings provide a rational framework for further development of Pirh2 inhibitors as a novel class of anti-tumor agents.
[Show abstract][Hide abstract] ABSTRACT: Trihydrophobin 1 (TH1) is a member of the negative elongation factor complex, which is involved in transcriptional pausing. Although the negative elongation factor complex attenuates the estrogen receptor α-mediated transcription, little is known about the relationship between TH1 and tumor progression. Here, we report that the protein level of TH1 was negatively correlated with the aggressiveness of human breast cancer. Immunohistochemical analysis revealed that TH1 expression in clinical stage III-IV primary breast cancer tissues was statistically significantly lower than that in stage I-II breast tissues (P < 0.01), and especially inversely associated with lymph node metastasis (P < 0.001). Furthermore, we showed that overexpression of TH1 in MDA-MB-231 breast cancer cells inhibited, and knockdown of TH1 in MCF-7 cells enhanced, cell proliferation and migratory ability. Moreover, upregulation of TH1 in MDA-MB-231 cells resulted in the decrease of cyclin D1, β-catenin, and ERK activity, and the increase of p21. In contrast, knockdown of TH1 in MCF-7 cells enhanced the expression of cyclin D1 and β-catenin, increased the activity of ERK, and downregulated the expression of p21. Additionally, overexpression of TH1 in MDA-MB-231 cells prevented. However, knockdown of TH1 in MCF-7 cells induced a number of molecular and cellular alterations associated with epithelial-mesenchymal transition. Taken together, our results suggest that TH1 might play an important role in regulation of proliferation and invasion in human breast cancer, and could be a potential target for human breast cancer treatment.
[Show abstract][Hide abstract] ABSTRACT: The 70-kDa family of heat shock proteins (HSP70), in particular, plays a vital role in cellular protection and has been detected in various tissues subject to stress. HSPA12B is the newest member of the HSP70 family but is distinct from the HSP70 family. In this study, we elucidated the dynamic expression changes and localization of HSPA12B in lipopolysaccharide (LPS)-induced neuroinflammatory processes in adult rats. HSPA12B expression was strongly induced in active microglial cells in inflamed spinal cord. In vitro studies indicated that the up-regulation of HSPA12B may be involved in the subsequent microglia activation following LPS challenge. The elevated HSPA12B expression was regulated by activation of MAPK-p38 and ERK1/2 pathways, less contribution of the SAPK/JNK pathway in microglial cells. Collectively, these results suggested HSPA12B may be important for host defense in microglia-mediated immune response. Understanding the cell signal pathway may provide a novel strategy against inflammatory and immune reaction in neuroinflammtion in CNS.
No preview · Article · Jul 2010 · Journal of the neurological sciences
[Show abstract][Hide abstract] ABSTRACT: Human heat shock protein 60 (hsp60) is a mitochondrial protein that functions as a molecular chaperone. Recently, it has been observed that hsp60 can become exposed on the cell surface and released into the extracellular space. Extracellular hsp60 is thought to function as a danger signal that activates the immune response. However, concerns have been raised that the effects of recombinant hsp60 on cytokines might be the result of contamination with bacterial components, given that the recombinant hsp60 protein used in these studies was produced with a bacterial expression system. In the present study, recombinant hsp60 was produced using a eukaryotic expression system, and the resulting protein was purified. The results obtained demonstrated that recombinant hsp60 was secreted efficiently from cells when fused to the leader peptide of interleukin-2 and the secreted protein was modified by N-linked glycosylation. Furthermore, we successfully obtained unglycosylated recombinant protein that was capable of binding to macrophages.
No preview · Article · Mar 2010 · Protein Expression and Purification
[Show abstract][Hide abstract] ABSTRACT: Src-suppressed C kinase substrate (SSeCKS), a protein kinase C substrate, is a major lipopolysaccharide (LPS) response protein, regulating the inflammatory process. In the process of spinal inflammatory diseases by LPS intraspinal injection, expression of SSeCKS in the spinal cord was increased, mainly in active astrocytes and neurons. Induced SSeCKS was colabeled with terminal deoxynucleotidyl transferase-mediated biotinylated-dUTP nick-end labeling (an apoptosis maker) in the late inflammation processes. These results indicated that SSeCKS might correlate with the inflammatory reaction and late neurodegeneration after LPS injection. A cell type-specific action for SSeCKS was further studied within C6 cells and PC12 cells. Knockdown of SSeCKS by small-interfering RNAs (siRNAs) blocked the LPS-induced inducible nitric oxide synthase (iNOS) expression in C6 cells, while overexpression SSeCKS enhanced iNOS expression. SSeCKS is also participated in regulation of PC12 cell viability. Loss of SSeCKS rescued PC12 cell viability, and excessive SSeCKS exacerbated the cell death upon conditioned medium and tumor necrosis factor-alpha exposure. This study delineates that SSeCKS may be important for host defenses in spinal inflammation and suggests a valuable molecular mechanism by which astrocytes modify neuronal viability during pathological states.
[Show abstract][Hide abstract] ABSTRACT: Src-suppressed C kinase substrate (SSeCKS), is an in vivo and in vitro protein kinase C substrate that may have a role in both mitogenic regulation and cytoskeletal arrangement. In this study, we mainly investigated the mRNA and protein expression and cellular localization of SSeCKS during chronic constriction injury (CCI). Reverse transcriptase-mediated PCR and western blot analysis revealed that SSeCKS was present in normal whole spinal cord. It gradually increased, and reached a peak at 2 weeks for its mRNA level and 7 days for its protein level after CCI. The protein expression of SSeCKS was further analyzed by immunohistochemistry. The positively stained areas for SSeCKS changed with the similar pattern to that of protein expression detected by immunoblotting analysis. Double immunofluorescence staining showed SSeCKS immunoreactivity was mostly co-localized with neurons, partly with activated astrocytes and rarely with microglia in the superficial laminar of spinal dorsal horn. In cell culture, the expression of pro-inflammation cytokines, p-ERK, and SSeCKS was increased in the spinal astrocytes after stimulated by damaged sensory neurons. However, SSeCKS gene silencing by siRNA inhibited the up-regulation of p-ERK and the pro-inflammation cytokines. Taken together, activated astrocytes released cytokines and iNOS after neuropathic pain via SSeCKS-ERK signaling. SSeCKS might be critical for the activation of astrocytes in the neuropathic pain.
No preview · Article · Nov 2009 · Neuromolecular medicine
[Show abstract][Hide abstract] ABSTRACT: CLEC-2 is a C-type lectin-like receptor and plays an important role in platelet activation. Snake venom toxin rhodocytin and the endogenous sialoglycoprotein podoplanin are identified as ligands for CLEC-2 and function as stimulators in platelet activation. We also previously indentified two splice variants of murine CLEC-2 as well as a soluble fragment cleaved from the full-length form. However, little is known about the interacting partners with the cytoplasmic region of CLEC-2. In this study, we reported that RACK1, the receptor for activated C-kinase 1, associated with the cytoplasmic tail of CLEC-2. Moreover, overexpression of RACK1 decreased the stability of CLEC-2 through promoting its ubiquitin-proteasome degradation, without impairing surface expression and downstream signaling of CLEC-2. Taken together, these results suggest RACK1 as a novel modulator of CLEC-2 expression.
No preview · Article · Sep 2009 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: Src-suppressed protein kinase C substrate (SSeCKS) is a protein kinase C substrate protein, which plays an important role in mitogenic regulatory activity. In the early stage of nerve injury, expression of SSeCKS in the PNS increases, mainly in Schwann cells (SCs). However, the exact function of SSeCKS in the regulation of SC proliferation remains unclear. In this study, we found that tumor necrosis factor-alpha (TNF-alpha) induced both SSeCKS alpha isoform expression and SC growth arrest in a dose-dependent manner. By knocking down SSeCKS alpha isoform expression, TNF-alpha-induced growth arrest in SCs was partially rescued. Concurrently, the expression of cyclin D1 was reduced and the activity of extracellular signal-regulated kinase 1/2 was decreased. A luciferase activity assay showed that cyclin D1 expression was regulated by SSeCKS at the transcription level. In addition, the cell fragments assay and immunofluorescence revealed that TNF-alpha prevented the translocation of cyclin D1 into the nucleus, while knocking down SSeCKS alpha isoform expression prompted cyclin D1 redistribution to the nucleus. In summary, our data indicate that SSeCKS may play a critical role in TNF-alpha-induced SC growth arrest through inhibition of cyclin D1 expression thus preventing its nuclear translocation.
Full-text · Article · Sep 2009 · Journal of Neurochemistry
[Show abstract][Hide abstract] ABSTRACT: Gliomas are the most common type of primary intracranial tumor. Although tumor grade predicts the clinical course of most patients, molecular characteristics of individual tumors have emerged as important prognostic factors for patients with gliomas. Reduced expression of p27 protein is known as an independent prognostic marker in a large variety of cancers and is associated with an unfavorable prognosis. It is believed that phosphorylation of p27 on Ser10 has been shown to be required for the binding of CRM1, a carrier protein for nuclear export. This study assessed whether CRM1, Ser10-phosphorylated p27, and p27 correlated with each other, with glioma pathological stage, and with patient outcome.
Immunohistochemical and Western blot analysis were performed in 70 cases of human gliomas and normal brain tissues. Survival analyses were performed using the Kaplan-Meier method.
High CRM1 expression (80% of cancer cell nuclei stained) was observed in 70 specimens and was related to the grade of malignancy. A strong inverse correlation was evident between p27 levels and both Ser10-phosphorylated p27 (P < 0.001) and CRM1 level (P < 0.001). We also reviewed each grade of tumors separately and investigated whether CRM1 expression predicted patient survival within each subgroup. In brief, CRM1 overexpression was significantly associated with overall survival (P < 0.001).
The current results showed that CRM1 and p27 expression were associated with glioma grade and that high CRM1 protein expression might be related to poor outcome.
[Show abstract][Hide abstract] ABSTRACT: The early stage of inflammation involves the adhesion and transmigration of leukocytes across the blood-brain barrier (BBB) to the normally sequestered central nervous system (CNS). This process is regulated by the expression of a series of adhesion molecules. One of the most well-known components is intercellular adhesion molecule-1 (ICAM-1). It was described as a ligand of the membrane-bound integrin receptors lymphocyte function-associated antigen-1 (LFA-1) and monocyte adhesion molecules-1 (Mac-1) on leukocytes, and was involved in the adhesion and transmigration of leukocytes. Studies have demonstrated the upregulation of ICAM-1 in many tissues after lipopolysaccharide (LPS) stimulation, for example. In the CNS, recent studies just focus on the relatively acute effects in brain tissues, but neglected the possibly existed differences between the brain and the spinal cord following traumatic lesions. Our data demonstrated the upregulation of ICAM-1, LFA-1, and Mac-1 in the spinal cords of LPS intraspinal injected rats, and the location of ICAM-1 in microglia cells. These results suggested a possible role of this molecule in microglia-mediated immune response and antigen presenting in CNS immune diseases.
No preview · Article · Sep 2008 · Neuromolecular medicine
[Show abstract][Hide abstract] ABSTRACT: Lipopolysaccharide is a major constituent of the outer membrane of Gram-negative bacteria. It activates monocytes and macrophages to produce cytokines such as tumor necrosis factor-alpha and interleukins IL-1beta and IL-6. These cytokines appear to be responsible for the neurotoxicity observed in peripheral nervous system inflammatory disease. It has been reported that, in the central nervous system, the expression level of intercellular adhesion molecule-1 (ICAM-1) was dramatically upregulated in response to LPS, as well as many inflammatory cytokines. ICAM-1 contributes to multiple processes seen in central nervous system inflammatory disease, for example migration of leukocytes to inflammatory sites, and adhesion of polymorphonuclear cells and monocytes to central nervous system cells. In the present study, we found that lipopolysacharide evoked ICAM-1 mRNA and protein expression early at 1 h post-injection, and the most significant increase was seen at 4 h. Immunofluorescence double-labeling suggested that most of the ICAM-1-positive staining was located in Schwann cells. Using Schwann cell cultures, we demonstrated that ICAM-1 expression in Schwann cells is regulated by mitogen-activated protein kinases, especially the p38 and stress-activated protein kinase/c-Jun N-terminal kinase pathways. Thus, it is thought that upregulation of ICAM-1 expression in Schwann cells may be important for host defenses after peripheral nervous system injury, and reducing the biosynthesis of ICAM-1 and other cytokines by blocking the cell signal pathway might provide a new strategy against inflammatory and immune reaction after peripheral nerve injury.