leupeptins and Carcinoma--Hepatocellular

Various antibacterial compounds, antitumor compounds, enzyme inhibitors and recent signal transduction inhibitors have been discovered from microorganisms and plants. Therefore, it should be possible to find antimetastatic compounds from these sources, if a simple assay system is available. We isolated several enzyme inhibitors from nature to inhibit experimental metastasis. Leupeptin is an old protease inhibitor and inhibited blood-borne lung metastasis of hepatoma cells in rats. A leupeptin analogue inhibiting urokinase inhibited in vitro invasion of human fibrosarcoma cells. Alpha-glucosidase inhibitors such as epi-CPL and baicalein inhibited in vitro invasion and in vivo metastasis of mouse melanoma cells. A mannosidase inhibitor, mannostatin A, also inhibited in vitro invasion of mouse melanoma cells. Oncogene function inhibitors induce normal phenotypes in the oncogene-expressing cells. As expected, they inhibited tumor cell invasion in vitro.

Topics: Animals; Carcinoma, Hepatocellular; Collagenases; Enzyme Inhibitors; Glycoside Hydrolases; Leupeptins; Lung; Matrix Metalloproteinase Inhibitors; Mice; Neoplasm Metastasis; Neoplasms, Experimental; Protease Inhibitors; Rats

How Wnt signaling is orchestrated in liver regeneration and tumorigenesis remains elusive. Recently, we identified transmembrane protein 9 (TMEM9) as a Wnt signaling amplifier.. TMEM9 facilitates v-ATPase assembly for vesicular acidification and lysosomal protein degradation. TMEM9 is highly expressed in regenerating liver and hepatocellular carcinoma (HCC) cells. TMEM9 expression is enriched in the hepatocytes around the central vein and acutely induced by injury. In mice, Tmem9 knockout impairs hepatic regeneration with aberrantly increased adenomatosis polyposis coli (Apc) and reduced Wnt signaling. Mechanistically, TMEM9 down-regulates APC through lysosomal protein degradation through v-ATPase. In HCC, TMEM9 is overexpressed and necessary to maintain β-catenin hyperactivation. TMEM9-up-regulated APC binds to and inhibits nuclear translocation of β-catenin, independent of HCC-associated β-catenin mutations. Pharmacological blockade of TMEM9-v-ATPase or lysosomal degradation suppresses Wnt/β-catenin through APC stabilization and β-catenin cytosolic retention.. Our results reveal that TMEM9 hyperactivates Wnt signaling for liver regeneration and tumorigenesis through lysosomal degradation of APC.

Topics: Adenomatous Polyposis Coli Protein; Animals; beta Catenin; Carbon Tetrachloride; Carcinogenesis; Carcinoma, Hepatocellular; Cell Nucleus; Chemical and Drug Induced Liver Injury; Disease Models, Animal; Gene Knockout Techniques; HEK293 Cells; Hep G2 Cells; Humans; Leupeptins; Liver Neoplasms; Liver Regeneration; Lysosomes; Male; Membrane Proteins; Mice; Mice, Knockout; Proteolysis; Vacuolar Proton-Translocating ATPases; Wnt Signaling Pathway; Xenograft Model Antitumor Assays

Hepatocellular carcinoma (HCC) is one of the most lethal and prevalent cancers worldwide. CD147 (EMMPRIN or basigin) is a leading gene relating to hepatocarcinogenesis and metastasis, and is detected in transmembrane, exosome or circulating forms in HCC patients. The endosome recycling of CD147 further enhances the function of this oncoprotein from a dynamic perspective. However, previous studies about CD147 mainly focused on one separate form, and little attention has been paid to how the different forms of tumor-derived CD147 changes. Moreover, uncovering the roles of the residual C-terminal portion of CD147 after shedding is inevitable to fully understand CD147 promoting tumor progression. In this study, we discovered that under low-cholesterol condition, CD147 endocytosis is inhibited but its shedding mediated by ADAM10 is enhanced. Further procession of residual CD147 in the lysosome produces nuclear-localized CD147-ICD (intracellular domain of CD147), which contributes to autophagy through NF-κB-TRAIL-caspase8-ATG3 axis. As autophagy endows cancer cells with increased adaptability to chemotherapy, and HAb 18 (a specific antibody targeting CD147) inhibits CD147 shedding and sequential CD147-ICD enhances autophagy, we found the combination of HAb 18 and cisplatin exhibited marked antitumor efficiency.

Topics: ADAM10 Protein; ADP-Ribosylation Factor 6; ADP-Ribosylation Factors; Amyloid Precursor Protein Secretases; Animals; Antibodies, Monoclonal; Antineoplastic Agents; Autophagy; Basigin; Beclin-1; beta-Cyclodextrins; Carcinoma, Hepatocellular; Cell Line, Tumor; Cisplatin; Drug Resistance, Neoplasm; Female; Humans; Leupeptins; Liver Neoplasms; Membrane Proteins; Mice; Mice, Nude; Proteolysis; Simvastatin

Metastasis associated 1 protein (MTA1) is one of the prime facilitators of metastatic progression in all solid tumors including hepatocellular carcinoma (HCC). However, the underlying regulatory mechanism of MTA1 expression in HCC is not clear. In this study, we evaluated MTA1 transcript and protein expression in HCC and normal hepatic cell lines. The results revealed that MTA1 protein expression had a significantly increase in HCC cell line, HuH6, compared with that in normal hepatic cell line, THLE-2. Determination of protein half-life using cycloheximide (CHX) treatment did not reveal any statistically significant difference in protein turn-over rates between THLE-2 (3.3 ± 0.25 h) and HuH6 (3.6 ± 0.15 h) cell lines. MTA1 protein level was stabilized in THLE-2 cells after treatment with MG-132 to levels similar to those observed in HuH6 cells. Mass spectrometric analysis of FLAG immunoprecipitates of FLAG-MTA1 transfected THLE-2 cells after MG-132 treated revealed candidate ubiquitin ligases that were interacting with MTA1. RNAi-mediated silencing of each prospective ubiquitin ligase in THLE-2 cells indicated that knockdown of TRIM25 resulted in stabilization of MTA1 protein, indicating TRIM25 as a putative E3 ligase for MTA1. Coimmunoprecipitation of FLAG-tagged MTA1, but not IgG, in MG-132 treated and untreated THLE-2 cells cotransfected with either FLAG-MTA1 or Myc-TRIM25 revealed robust polyubiquitinated MTA1, confirming that the TRIM25 is the ubiquitin ligase for MTA1 degradation. Overexpression of TRIM25 in HuH6 and RNAi mediated silencing of TRIM25 in THLE-2 cells inhibited and increased the cell migration and invasion, respectively. Analysis of The Cancer Genome Atlas data for assessment of TRIM25 transcript level and MTA1 protein expression in 25 HCC patients confirmed an inverse correlation between the expression of TRIM25 and MTA1. Cumulatively, our data reveal a novel mechanism of post-translational to regulate MTA1 expression in normal hepatic cells, which is repressed in HCC. © 2017 IUBMB Life, 69(10):795-801, 2017.

Topics: Atlases as Topic; Carcinoma, Hepatocellular; Cell Line; Cell Line, Tumor; Cell Movement; Cycloheximide; Disease Progression; Gene Expression Regulation, Neoplastic; Half-Life; Hepatocytes; Histone Deacetylases; Humans; Leupeptins; Liver Neoplasms; Protein Binding; Protein Stability; Proteolysis; Repressor Proteins; RNA, Small Interfering; Signal Transduction; Trans-Activators; Transcription Factors; Tripartite Motif Proteins; Ubiquitin-Protein Ligases; Ubiquitination

Suppressor of cytokine signaling 1 (SOCS1) is considered as a tumor suppressor protein in hepatocellular carcinoma (HCC), but the underlying mechanisms remain unclear. Previously, we have shown that SOCS1-deficient hepatocytes displayed increased responsiveness to hepatocyte growth factor (HGF) due to enhanced signaling via the MET receptor tyrosine kinase. As aberrant MET activation occurs in many tumors including HCC, here we elucidated the mechanisms of SOCS1-mediated regulation. SOCS1 attenuated HGF-induced proliferation of human and mouse HCC cell lines and their growth as tumors in NOD.scid.gamma mice. Tumors formed by SOCS1 expressing HCC cells showed significantly reduced MET expression, indicating that SOCS1 not only attenuates MET signaling but also regulates MET expression. Mechanistically, SOCS1 interacted with MET via the Src homology 2 domain and this interaction was promoted by MET tyrosine kinase activity. The SOCS1-mediated reduction in MET expression does not require the juxtamembrane Y1003 residue implicated in Cbl-mediated downmodulation. Moreover, the proteasome inhibitor MG-132, but not the inhibitors of lysosomal degradation bafilomycin and chloroquine, reversed the SOCS1-mediated reduction in MET expression, indicating that this process is distinct from Cbl-mediated downmodulation. Accordingly, SOCS1 promoted polyubiquitination of MET via K48-dependent but not K63-mediated ubiquitin chain elongation. Furthermore, siRNA-mediated downmodulation of Cbl did not abolish SOCS1-mediated reduction in MET expression in HCC cells. SOCS1-dependent ubiquitination of endogenous MET receptor occurred rapidly following HGF stimulation in HCC cells, leading to proteasomal degradation of phosphorylated MET receptor. These findings indicate that SOCS1 mediates its tumor suppressor functions, at least partly, by binding to MET and interfering with downstream signaling pathways as well as by promoting the turnover of the activated MET receptor. We propose that loss of this control mechanism due to epigenetic repression of SOCS1 could contribute to oncogenic MET signaling in HCC and other cancers, and that MET inhibitors might be useful in treating these patients.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Chlorocebus aethiops; Chloroquine; COS Cells; Gene Expression Regulation, Neoplastic; HEK293 Cells; Hep G2 Cells; Humans; Leupeptins; Liver Neoplasms; Mice; Proto-Oncogene Proteins c-cbl; Proto-Oncogene Proteins c-met; Signal Transduction; Suppressor of Cytokine Signaling 1 Protein; Suppressor of Cytokine Signaling Proteins

Identification of substances with specific toxicity for carcinoma cells promises to facilitate the development of cancer chemotherapeutics that cause minimal side effects. Here, we show that knockdown of the farnesoid X receptor (FXR) effectively suppresses the proliferation of human hepatocellular carcinoma cell lines HepG2 and HLE accompanied by elevated expression of cyclin-dependent kinase (CDK) inhibitor p16/INK4a and p21/Cip1 proteins. On the other hand, the growth of the primary human hepatocyte-derived cell line Fa2N-4 is not affected by the treatment with FXR siRNA irrespective of marked increases in the mRNAs of p16/INK4a and p21/Cip1. Surprisingly, the expression levels of p16/INK and p21/Cip1 proteins are left unchanged in Fa2N-4 cells that are subjected to the FXR siRNA treatment. Since the expression levels of these CDK inhibitor proteins in FXR-knockdown Fa2N-4 cells were elevated in the presence of proteasomal inhibitor MG132, these CDK inhibitors may be subjected to the proteasomal degradation, thereby counteracting the increased expression of their cognate mRNAs, therefore similar levels of p16 and p21 proteins were observed in control and FXR-knockdown Fa2N-4 cells. These results suggest that FXR-knockdown is effective for inhibiting the proliferation of hepatocellular carcinoma cells, not interfering with the regulatory mechanism of normal hepatocyte growth.

Topics: Carcinoma, Hepatocellular; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Gene Expression; Gene Knockdown Techniques; Hep G2 Cells; Hepatocytes; Humans; Leupeptins; Liver Neoplasms; Proteasome Endopeptidase Complex; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; RNA, Small Interfering

Cellular senescence evasion caused by the inactivation of tumor suppressive programs is implicated in tumor initiation and therapeutic resistance. Our previous study has shown that the downregulation of growth arrest and DNA damage 45G (GADD45G) contributes to senescence bypass in hepatocellular carcinoma (HCC). Here, we report that the Smad-interacting protein-1 (SIP1) is transcriptionally activated and functions critically in the GADD45G-induced tumor cell senescence. Knockdown of SIP1 significantly abrogates the suppressive effects of GADD45G on the growth of xenografted liver tumor in vivo. The essential role of SIP1 in GADD45G activities is further validated in the model of the proteasome inhibitor MG132-induced cell senescence. We further show that JNK but not p38 MAPK activation is involved in the GADD45G-mediated SIP1 upregulation, and that JNK inhibition counteracts the GADD45G-induced cellular senescence. More importantly, we show that GADD45G and SIP1 expression are coincidently downregulated in primary human HCC tissues. Together, our results establish that the dowregulation of GADD45G-SIP1 axis may contribute to cellular senescence evasion and HCC development.

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Growth Processes; Cell Line, Tumor; Cellular Senescence; Cysteine Proteinase Inhibitors; GADD45 Proteins; HEK293 Cells; Homeodomain Proteins; Humans; Intracellular Signaling Peptides and Proteins; Leupeptins; Liver Neoplasms; MAP Kinase Kinase 4; Mice; Mice, Nude; Repressor Proteins; Xenograft Model Antitumor Assays; Zinc Finger E-box Binding Homeobox 2

Advanced hepatocellular carcinoma (HCC) responds poorly to conventional systemic therapies. Therefore, new effective therapy strategies are urgently needed. Molecular targeted therapies have entered the field of anti-neoplastic treatment and are being used on their own and in combination with other drugs. Sorafenib inhibits proliferation and angiogenesis of HCC by suppressing the Raf serine/threonine kinases and the receptor tyrosine kinases. The proteasome inhibitor bortezomib has shown activity in a variety of solid tumors, including HCC. However, the precise anti-proliferative mechanisms of these agents remain unclear.. We treated human hepatoma cell lines (Huh7 and Hep3B) and immortalized human hepatocyte (OUMS29) with sorafenib and/or proteasome inhibitors, including epoxomicin and acetyl-leucyl-leucyl-norleucinal. Cytotoxic effects were examined by morphometric analyses of apoptosis and necrosis. Apoptosis was also evaluated by Western blotting of keratin18, PARP and caspase3. The activity of Akt and stress-activated protein kinases was examined by Western blotting.. Both sorafenib and proteasome inhibitors induced apoptosis in Huh7 and OUMS29. However, sorafenib attenuated proteasome inhibitor-induced apoptosis. Sorafenib induced necrosis, especially in combination with proteasome inhibitors. Sorafenib induced down-regulation of Akt synergistically in combination with proteasome inhibitors in Huh7. Sorafenib inhibited both the JNK and p38 pathways in a time- and dose-dependent manner. In addition, sorafenib also inhibited proteasome inhibitor-mediated JNK and p38 activation in both Huh7 and OUMS29.. Sorafenib enhances the anti-proliferative effect of proteasome inhibitors in part by inactivating the Akt signaling pathway and modulating stress-activated protein kinases. The combination of these agents could be an ideal molecular targeted therapy for HCC.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Death; Cell Line, Tumor; Down-Regulation; Drug Synergism; Humans; Leupeptins; Liver Neoplasms; Molecular Targeted Therapy; Niacinamide; Oligopeptides; Phenylurea Compounds; Proteasome Inhibitors; Protein Kinase Inhibitors; Protein Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Sorafenib

Liver cancer in men is the fifth most frequently diagnosed cancer worldwide. Programmed cell death 5 (PDCD5) is an apoptosis related gene and plays an important role in the pathogenesis and development of cancer. In this study, we confirmed that the levels of PDCD5 mRNA and protein were lower in hepatocellular carcinoma (HCC) tissue compared to normal tissue. PDCD5 expression was significantly associated with HBV infection, tumor number, lymph node metastasis and the survival time of the patients with HCC. In addition, the serum levels of PDCD5 and AFP in the patients were significantly positively correlated. We also confirmed that upregulation of PDCD5 was able to inhibit cell proliferation and mobility, induce apoptosis and G1 arrest. Interestingly, PDCD5 also inhibited proteasome activity similarly to the proteosome inhibitor MG132. PDCD5 could be considered as a reliable marker of favorable prognosis of HCC patients.

Topics: Aged; alpha-Fetoproteins; Apoptosis; Apoptosis Regulatory Proteins; Biomarkers, Tumor; Carcinoma, Hepatocellular; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Leupeptins; Liver Neoplasms; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Proteins; Prognosis; Treatment Outcome

Despite the antitumour effect of ursolic acid observed in several cancers, the underlying mechanism remains unclear. Thus, in the present study, the roles of AMP-activated protein kinase (AMPK) and glycogen synthase kinase 3 beta (GSK3β) were examined in ursolic acid induced apoptosis in HepG2 hepatocellular carcinoma cells. Ursolic acid significantly exerted cytotoxicity, increased the sub-G1 population and the number of ethidium homodimer and terminal deoxynucleotidyl transferase(TdT) mediated dUTP nick end labeling positive cells in HepG2 cells. Also, ursolic acid enhanced the cleavages of poly-ADP-ribose polymerase (PARP) and caspase3, attenuated the expression of astrocyte elevated gene (AEG1) and survivin in HepG2 cells. Interestingly, ursolic acid increased the phosphorylation of AMPK and coenzyme A carboxylase and also enhanced phosphorylation of GSK3β at inactive form serine 9, whereas ursolic acid attenuated the phosphorylation of AKT and mTOR in HepG2 cells. Conversely, AMPK inhibitor compound C or GSK3β inhibitor SB216763 blocked the cleavages of PARP and caspase 3 induced by ursolic acid in HepG2 cells. Furthermore, proteosomal inhibitor MG132 suppressed AMPK activation, GSK3β phosphorylation, cleaved PARP and deceased AEG-1 induced by ursolic acid in HepG2 cells. Overall, our findings suggest that ursolic acid induced apoptosis in HepG2 cells via AMPK activation and GSK3β phosphorylation as a potent chemopreventive agent.

Topics: AMP-Activated Protein Kinases; Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Cell Adhesion Molecules; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Hep G2 Cells; Humans; Indoles; Leupeptins; Liver Neoplasms; Maleimides; Membrane Proteins; Phosphorylation; Poly(ADP-ribose) Polymerases; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; RNA-Binding Proteins; TOR Serine-Threonine Kinases; Triterpenes; Ubiquitination; Ursolic Acid

The activating receptor natural killer group 2, member D (NKG2D) and its ligands play a crucial role in immune response to tumors. NKG2D ligand expression in tumors has been shown to be associated with tumor eradication and superior patient survival, but the involvement of NKG2D ligands in the immune response against hepatocellular carcinoma (HCC) still remains to be elucidated.. We investigated the expression of NKG2D ligands in HCC tissues collected from 54 patients and HCC cell lines. We also examined the proteasome expression and the effect of inhibition of proteasome activity on NKG2D ligand expression in HCC tissues and cell lines.. In dysplastic nodules (DN), well-differentiated (well-HCC), and moderately-differentiated HCCs (mod-HCC), UL16-binding protein (ULBP) 1 was expressed predominantly in tumor cells, but not in poorly-differentiated HCCs (poor-HCC). Remarkably, recurrence-free survival of patients with ULBP1-negative HCC was significantly shorter than that of patients with ULBP1-positive HCC (p=0.006). Cox regression analysis revealed that loss of ULBP1 expression was an independent predictor of early recurrence (p=0.008). We confirmed that ULBP1 was expressed in the well- and mod-HCC cell lines, but not in the poor-HCC cell line KYN-2. However, inhibition of proteasome activity resulted in significant up-regulation of ULBP1 expression in KYN-2. Moreover, we found that 20S proteasome expression was more abundant in KYN-2 than that in the well- and mod-HCC cell lines.. ULBP1 is prevalently expressed in DN to mod-HCC, but loss of its expression correlates with tumor progression and early recurrence.

Topics: Aged; Carcinoma, Hepatocellular; Cell Differentiation; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Disease-Free Survival; Female; Gene Expression; GPI-Linked Proteins; Humans; Intracellular Signaling Peptides and Proteins; Killer Cells, Natural; Leupeptins; Ligands; Liver Neoplasms; Male; Middle Aged; Neoplasm Recurrence, Local; NK Cell Lectin-Like Receptor Subfamily K; Proteasome Endopeptidase Complex; RNA, Messenger; RNA, Neoplasm

The ubiquitin-proteasome system and autophagy-lysosome system are 2 major protein degradation pathways in eukaryotic cells, which are tightly linked to cancer. Proteasome inhibitors have been approved in clinical use against hematologic malignancies, but their application in solid tumors is uncertain. Moreover, the role of autophagy after proteasome inhibition is controversial.. Two proteasome inhibitors, 2 autophagy inhibitors, and 3 hepatocellular carcinoma (HCC) cell lines were investigated in the current study. In vitro, cell proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cell apoptosis was evaluated by flow cytometry analysis of annexin-V/propidium iodide staining, and autophagy was evaluated by green fluorescent protein-light chain 3 (GFP-LC3) redistribution and LC3 Western blot analysis. In vivo, Ki-67 staining was used to detect cell proliferation, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining was used to detect apoptosis, and electron microscopy and p62 immunohistochemical staining were used to detect autophagy.. Proteasome inhibitors suppressed proliferation, induced apoptosis, and activated autophagy in HCC cell lines in vitro, and autophagy exerted a protective role after proteasome inhibition. In vivo, anticancer effects of bortezomib on the MHCC-97H orthotopic model (human HCC cells) were different from the effects observed on the Huh-7 subcutaneous model (human HCC cells). The autophagy inhibitor chloroquine interacted synergistically with bortezomib to suppress proliferation and induce apoptosis in both tumor models.. The current results indicated that simultaneous targeting of the proteasome and autophagy pathways may represent a promising method for HCC treatment.

Topics: Adenine; Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Boronic Acids; Bortezomib; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Chloroquine; Cysteine Proteinase Inhibitors; Humans; Leupeptins; Membrane Proteins; Microtubule-Associated Proteins; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Pyrazines

Abstract Raf-1 kinase inhibitor protein (RKIP) is a tumor and metastasis suppressor that promotes drug-induced apoptosis in cancer cells. It is frequently downregulated, both at the mRNA and protein level, in hepatocellular carcinoma (HCC), but the mechanisms leading to this reduction are obscure. We sequenced the whole RKIP gene in three human HCC cell lines (HA22T/VGH, HepG2, and Hep3B), and in five clinical HCC samples, but could not find any gene variant that might account for their low RKIP levels. We also examined whether gene methylation may be responsible for the altered RKIP expression. No methylation of the RKIP gene was found in the tumor samples, while among the cell lines only Hep3B showed methylation of the gene, which was reduced by treatment with 5-aza-2'-deoxycytidine (5-AZA). The same treatment caused upregulation of RKIP at the mRNA, but not at the protein level, indicating that gene methylation is not a principal mechanism of the decrease in RKIP in the Hep3B cells. Furthermore, different elements consistently suggested that RKIP may be a target repressed by miR-224, a miRNA that is frequently and specifically upregulated in HCC, but our results excluded that this occurs, at least in the HCC cell lines. Factors like Snail, EZH2, and HDAC, have been implicated in the RKIP downregulation present in breast and prostate tumors, though some of our results from the cell lines do not support that they play such a role in HCC; however, this aspect is worthy of further study. However, recent results of ours and others suggest a significant involvement of proteosomal degradation and of its pharmacological inhibition. In conclusion, the causes of RKIP downregulation in HCC remain incompletely understood. However, we think that the present observations will be useful to generate further research, with the ultimate possible goal of devising specific approaches to restore the relevant antitumor function of the factor.

Topics: Antineoplastic Agents; Azacitidine; Carcinoma, Hepatocellular; Cell Line, Tumor; DNA Methylation; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Leupeptins; Liver Neoplasms; MicroRNAs; Mutation; Phosphatidylethanolamine Binding Protein; Promoter Regions, Genetic; RNA Interference

The hepatoma upregulated protein (HURP) represents a putative oncogene that is overexpressed in many human cancers, especially hepatocellular carcinoma (HCC). HURP plays an important role during mitotic spindle formation, a process that is targeted by various anti-cancer drugs like taxol. However, the role of HURP during the establishment of taxol chemoresistance in HCC remains unclear. In this study, we observed that high HURP protein level correlates with taxol resistance in HCC cells. Following HURP knockdown, HCC cells show a more sensitive response to taxol treatment. Notably, sorafenib, a tyrosine kinase inhibitor approved for the treatment of HCC, inhibits HURP expression primarily at the transcriptional level and sensitizes HCC cells to sub-lethal doses of taxol. By using real-time PCR and chromatin immunoprecipitation assays, we observed that the NF-κB family member c-Rel represents a putative transcription factor that activates HURP gene expression. In addition, the inhibitory effect of sorafenib on HURP expression was attributed to a reduced translation and nuclear translocation of c-Rel. Accordingly, downregulation of c-Rel using short-hairpin RNA was shown to reduce HURP protein level and enhance taxol-induced cell death. Taken together, our results indicate that HURP acts as a novel survival protein that protects HCC cells against taxol-induced cell death. In addition, the regulation of HURP gene expression by NF-κB signaling appears to be critical for the response of HCC cells to taxol.

Topics: Apoptosis; Benzenesulfonates; Carcinoma, Hepatocellular; Cell Line, Tumor; Eukaryotic Initiation Factor-4E; Humans; Leupeptins; Liver Neoplasms; Neoplasm Proteins; NF-kappa B; Niacinamide; Paclitaxel; Phenylurea Compounds; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-rel; Pyridines; Sorafenib

Nuclear factor-kappa B (NF-kappaB) regulates the expression of various genes, several genes involved in inflammation and tumorigenesis, including those of the liver. A role for NF-kappaB has been implicated in the pathogenesis of hepatocellular carcinoma. This transcription factor can regulate hTERT gene transcription. Expression of hTERT was found to be at high levels in hepatocellular carcinoma. However, positive effects of NF-kappaB on hTERT protein synthesis in HepG(2) cells are unknown. In this study, we show that LPS (specific binding to TLR4 to activate NF-kappaB) was positive for NF-kappaB p65 mRNA expression and activation, and also up-regulated hTERT mRNA and protein expressions at 36h in a dose-dependent manner. In contrast, MG-132 (blocking the activity of 26S proteasome and thereby preventing nuclear translocation of NF-kappaB) significantly inhibited activation of NF-kappaB and mRNA expression. And also reduced the expression of hTERT at both mRNA and protein levels at 36h in a dose-dependent manner. Furthermore, dexamethasone inhibited LPS-induced activation of NF-kappaB and expression of the hTERT in HepG(2) cells. These findings suggest that NF-kappaB may modulate hTERT mRNA level, importantly, in protein level in HepG(2) cells and dexamethasone inhibits LPS-induced hTERT via blocking NF-kappaB.

Topics: Carcinoma, Hepatocellular; Cell Survival; Dexamethasone; Dose-Response Relationship, Drug; Hep G2 Cells; Humans; Leupeptins; Lipopolysaccharides; Molecular Targeted Therapy; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Biosynthesis; RNA, Messenger; Telomerase; Time Factors; Transcription Factor RelA; Transcription, Genetic; Up-Regulation

Melanoma antigen (MAGE)-A4 is processed to generate a C-terminal fragment with proapoptotic activity. Here we demonstrate that Adriamycin promotes generation of the processed MAGE-A4 by activating the proteasome. The proteasome is known to prevent accumulation of toxic proteins to maintain cellular homeostasis.. Treatment of hepatoma cells expressing MAGE-A4 with a sublethal dose of Adriamycin increased the MAGE-A4 processing and sensitized the cells to Adriamycin-induced apoptosis. The processing of MAGE-A4 was inhibited by the proteasome inhibitors MG115, MG132, lactacystin and epoxamicin. MAGE-A4 was coimmunoprecipitated with the S6 proteasomal ATPase, and present in the fractions containing the proteasome during glycerol gradient centrifugation. Consistent with the notion that the proteasome cleaves MAGE-A4, the 26S proteasome, ubiquitin, and cell lysates were necessary for efficient in vitrocleavage of MAGE-A4.. The present study suggests that a low dose of Adriamycin increases the proteasome activity, which either maintains cellular homeostasis or leads to apoptosis depending, at least under the present conditions, on the expression of MAGE-A4.

Topics: Acetylcysteine; Animals; Antigens, Neoplasm; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Chlorocebus aethiops; COS Cells; Doxorubicin; HEK293 Cells; Humans; Leupeptins; Liver Neoplasms; Neoplasm Proteins; Proteasome Endopeptidase Complex; Ubiquitin

The nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) plays a critical role in HCV replication and is an attractive target for the therapy of HCV infection. So far, little is known about the posttranslational regulation of NS5A protein and its precise role in HCV RNA replication. Our objectives were to elucidate the down-regulation of NS5A protein and HCV RNA replication by zinc mesoporphyrin (ZnMP) and the mechanism by which this process occurs.. Human hepatoma cells expressing HCV proteins were used to investigate the posttranslational regulation of ZnMP on NS5A protein by Western blots and immunoprecipitation. Real-time quantitative reverse transcription polymerase chain reaction was used to determine the effects of ZnMP on HCV RNA replication.. ZnMP selectively and markedly down-regulated NS5A protein levels by increasing degradation of NS5A protein (half-life fell from 18.7 hours to 2.7 hours). The proteasome inhibitors epoxomicin and MG132 significantly abrogated degradation of NS5A protein by ZnMP without affecting levels of NS5A in the absence of ZnMP. Analysis of immunoprecipitates with an antiubiquitin antibody revealed polyubiquitination of NS5A, suggesting that ZnMP induces ubiquitination of NS5A protein. In addition, 10 micromol/L of ZnMP reduced HCV replication by approximately 63% in the Con1 replicon cells, approximately 70% in J6/Japanese fulminant hepatitis 1 HCV-transfected cells, and approximately 90% in J6/Japanese fulminant hepatitis 1 HCV-infected cells without affecting cell viability.. ZnMP produces a rapid and profound down-regulation of the NS5A protein by enhancing its polyubiquitination and proteasome-dependent catabolism. ZnMP may hold promise as a novel agent to treat HCV infection.

Topics: Antiviral Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Down-Regulation; Half-Life; Hepacivirus; Humans; Leupeptins; Liver Neoplasms; Metalloporphyrins; Oligopeptides; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Protein Stability; RNA, Viral; Time Factors; Transfection; Ubiquitination; Viral Nonstructural Proteins; Virus Replication

Pigment epithelium-derived factor (PEDF) has several biological actions on tumor cells, but its effects are cell-type dependent. The aim of this study was to examine the pathophysiological role of PEDF in hepatocellular carcinoma (HCC). PEDF expression was examined in various hepatoma cell lines and human HCC tissues, and was seen in various hepatoma cell lines including HepG2 cells. In human HCC tissues, PEDF expression was higher than in adjacent non-HCC tissues. In addition, serum PEDF levels were higher in HCC patients than in non-HCC patients, and curative treatment of HCC caused significant reductions in serum PEDF levels compared with pretreatment levels. In vitro experiments, camptothecin (CPT) was used to induce apoptosis and the effect of PEDF was investigated by knockdown of the PEDF gene in CPT-treated HepG2 cells. Knockdown of the PEDF gene enhanced CPT-induced apoptosis, simultaneously down-regulating Bcl-xL expression in HepG2 cells. Expression of apoptosis-related molecules and effects of bafilomycin A1 on CPT-induced apoptosis were also examined in PEDF gene knockdown HepG2 cells. Treatment with bafilomycin A1 suppressed CPT-induced decreases in Bcl-xL expression and increases in apoptosis in PEDF gene knockdown HepG2 cells. PEDF may, therefore, exert anti-apoptotic effects through inhibition of lysosomal degradation of Bcl-xL in CPT-treated HepG2 cells.

Topics: Apoptosis; bcl-X Protein; Camptothecin; Carcinoma, Hepatocellular; Cell Cycle; Cyclophilins; Densitometry; Eye Proteins; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Hep G2 Cells; Humans; Leupeptins; Liver; Liver Neoplasms; Lysosomes; Macrolides; Nerve Growth Factors; Protein Processing, Post-Translational; Recombinant Proteins; RNA, Messenger; Serpins

To test whether nuclear factor kappa B plays an important role in the apoptosis-inhibitory effect of hepatitis B virus (HBV) P22(e) protein.. HepG2 cells were transfected with recombination plasmid pEGFP-HBVP22(e). The Act-D and TNF alpha were used to induce apoptosis. NF-kappa B inhibitor ALLN were used to inhibit the signaling pathway. The activation of NF-kappa B was EMSA, and the nulear translocation of NF-kappa B was determined by immuno-staining.. Laser scanning confocal microscopy and EMSA indicated that HBV P22(e) protein enhanced the nuclear translocation of NF-kappa B after apoptosis induction. ALLN treatment inhibited the nuclear translocation of NF-kappa B, and blocked the apoptosis-inhibiting effect of HBV P22(e) protein.. This study indicates that HBV P22(e) protein inhibits apoptosis of hepatocyte via the NF-kappa B signaling pathway.

Topics: Apoptosis; Carcinoma, Hepatocellular; Hep G2 Cells; Hepatitis B Core Antigens; Hepatitis B virus; Humans; Leupeptins; Liver Neoplasms; NF-kappa B; Plasmids; Signal Transduction; Transfection; Viral Core Proteins

Hypoxia-inducible factor-1alpha (HIF-1alpha) is destabilized via the ubiquitin-proteasome system. Thus HIF-1alpha expression is robustly upregulated by proteasome inhibition, but paradoxically its activity is reduced. In the present study, we investigated the mechanism underlying the paradoxical response of HIF-1alpha to proteasome inhibition. In both Hep3B and HEK293 cells, a proteasome inhibitor MG132 noticeably attenuated hypoxic induction of erythropoietin and VEGF mRNAs. MG132 inactivated HIF-1alpha C-terminal transactivation domain (CAD), independently of factor inhibiting HIF-1 (FIH) and inhibited p300 recruitment by HIF-1alpha. We next tested the possibility that CITED2 is involved in the HIF-1 inactivation. CITED2 was found to be degraded via the ubiquitin-proteasome system and thus was stabilized by proteasome inhibition. Both the activity and the p300 binding of HIF-1alpha were inhibited by CITED2 expression and recovered by CITED2 siRNA in the presence of MG132. These results suggest that CITED2 is stabilized by proteasome inhibition and inactivates HIF-1 by interfering with the HIF-1alpha-p300 interaction. This may be an important mode-of-action for proteasome inhibition-based cancer therapy.

Topics: Carcinoma, Hepatocellular; Cell Hypoxia; Cells, Cultured; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; E1A-Associated p300 Protein; Erythropoietin; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Immunoprecipitation; Kidney; Leupeptins; Liver Neoplasms; Mixed Function Oxygenases; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Repressor Proteins; RNA, Messenger; Trans-Activators; Transcription Factors; Ubiquitin; Vascular Endothelial Growth Factor A

Down-regulation of interleukin (IL)-6-type cytokine signaling has been shown to occur, among other mechanisms, via induction of the feedback inhibitor SOCS3 (suppressor of cytokine signaling 3). Binding of SOCS3 to the phosphorylated Tyr(759) in the cytoplasmic region of gp130, the common signal transducing receptor chain of all IL-6-type cytokines, is necessary for inhibition of Janus kinase-mediated signaling. In the present study, we analyzed the effect of SOCS3 on signal transduction by the proinflammatory cytokine oncostatin M (OSM), which signals through a receptor complex of gp130 and the OSM receptor (OSMR). OSM leads to a much stronger and prolonged induction of SOCS3 in HepG2 hepatoma cells and murine embryonal fibroblasts (MEF) compared with IL-6. A negative effect of SOCS3 on OSM signaling was confirmed using MEF cells lacking SOCS3. We can show that the OSMR-mediated signaling is inhibited by SOCS3 to a similar extent as previously described for gp130. However, the inhibition occurs independent of tyrosine motifs within the OSMR. Instead, SOCS3 interacts directly with JAK1 in a stimulation-dependent manner, a mechanism so far only known for SOCS1.

Topics: Animals; Antineoplastic Agents; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Transformed; Cell Line, Tumor; Cell Transformation, Viral; Cytokine Receptor gp130; Cytokines; Fibroblasts; Gene Expression Regulation; Genes, Reporter; Humans; Interleukin-6; Janus Kinase 1; Leupeptins; Liver Neoplasms; Luciferases; Mice; Oncostatin M; Precipitin Tests; Protein Binding; Protein-Tyrosine Kinases; Receptors, Amino Acid; Receptors, Cytokine; Receptors, Oncostatin M; Signal Transduction; Suppressor of Cytokine Signaling 3 Protein; Suppressor of Cytokine Signaling Proteins

The ubiquitin-proteasome pathway is responsible for regulating cell cycle proteins, tumor-suppressor molecules, oncogenes, transcription factors, and pro- and anti-apoptotic proteins. The aim of this study is to evaluate the effects of proteasome inhibitors on human hepatocellular carcinoma (HCC) cells. HCC cells SK-Hep1, HLE and HepG2 were treated with the proteasome inhibitors MG132 and MG115. Our data showed that both inhibitors induce apoptosis in the three cell types tested in a dose-dependent manner. Moreover, subtoxic levels of MG132 and MG115 sensitized HCC cells to TRAIL-induced apoptosis. To investigate the mechanism of increased TRAIL sensitivity in HCC cells, we first examined surface expression of TRAIL and its receptors. MG132 upregulated TRAIL and its receptors (TRAIL-R1 and -R2) in SK-Hep1 and HLE, whereas MG115 upregulated them in SK-Hep1. MG132 downregulated expression of X-linked inhibitor of apoptosis protein (XIAP) in SK-Hep1 and HLE, and of survivin in all three cell-types. MG115 downregulated expression of XIAP in SK-Hep1, and survivin in SK-Hep1 and HepG2. Furthermore, MG132 downregulated phospho-AKT and its downstream target phospho-BAD, indicating that MG132 activated the mitochondrial apoptosis pathway by inhibiting phosphorylation of AKT and BAD. In conclusion, proteasome inhibitors induced apoptosis and augmented TRAIL sensitivity via both the IAP family and AKT pathways. Thus, combining proteasome inhibitors with a TRAIL agonist may provide a new therapeutic strategy for HCC.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Caspase Inhibitors; Caspases; Down-Regulation; Drug Synergism; Humans; Leupeptins; Mice; Oncogene Protein v-akt; Phosphorylation; Protease Inhibitors; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proto-Oncogene Proteins c-akt; Receptors, TNF-Related Apoptosis-Inducing Ligand; Receptors, Tumor Necrosis Factor; TNF-Related Apoptosis-Inducing Ligand

Lipid esters stored in cytoplasmic lipid droplets (CLDs) of hepatocytes are used to synthesize very low-density lipoproteins (VLDLs), into which apolipoprotein B (ApoB) is integrated cotranslationally. In the present study, by using Huh7 cells, derived from human hepatoma and competent for VLDL secretion, we found that ApoB is highly concentrated around CLDs to make "ApoB-crescents." ApoB-crescents were seen in <10% of Huh7 cells under normal conditions, but the ratio increased to nearly 50% after 12 h of proteasomal inhibition by N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal. Electron microscopy showed ApoB to be localized to a cluster of electron-lucent particles 50-100 nm in diameter adhering to CLDs. ApoB, proteasome subunits, and ubiquitinated proteins were detected in the CLD fraction, and this ApoB was ubiquitinated. Interestingly, proteasome inhibition also caused increases in autophagic vacuoles and ApoB in lysosomes. ApoB-crescents began to decrease after 12-24 h of proteasomal inhibition, but the decrease was blocked by an autophagy inhibitor, 3-methyladenine. Inhibition of autophagy alone caused an increase in ApoB-crescents. These observations indicate that both proteasomal and autophagy/lysosomal degradation of ApoB occur around CLDs and that the CLD surface functions as a unique platform for convergence of the two pathways.

Topics: Apolipoproteins B; Autophagy; Carcinoma, Hepatocellular; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Cytoplasm; Humans; Leupeptins; Lipids; Lipoproteins, VLDL; Liver Neoplasms; Proteasome Endopeptidase Complex

High NaCl activates the transcription factor tonicity-responsive enhancer/osmotic response element binding protein (TonEBP/OREBP) by increasing its abundance and transactivation, the latter signaled by a variety of protein kinases. In addition, high NaCl causes TonEBP/OREBP to translocate into the nucleus, but little is known about the signals directing this translocation. The result is increased transcription of protective genes, including those involved in accumulation of organic osmolytes. High NaCl also damages DNA, and DNA damage activates ataxia telangiectasia-mutated (ATM) kinase through autophosphorylation on serine 1981. We previously found that ATM is involved in the high NaCl-induced increase in TonEBP/OREBP transactivation. The purpose of the present studies was to test whether ATM is also involved in high NaCl-induced TonEBP/OREBP nuclear translocation. We quantified TonEBP/OREBP in nuclear and cytoplasmic extracts from cultured cells by Western blot analysis. In COS-7 cells, wortmannin, an inhibitor of ATM, reduces high NaCl-induced nuclear translocation of TonEBP/OREBP. We used AT cells (in which ATM is inactive) to test the specificity of this effect. Nuclear translocation of native TonEBP/OREBP and of its recombinant NH2-terminal rel homology domain, which contains the nuclear localization signal, is reduced in AT cells and is restored when the cells are reconstituted with functional ATM. In conclusion, activation of ATM contributes to high NaCl-induced nuclear translocation of TonEBP/OREBP.

Topics: Androstadienes; Animals; Ataxia Telangiectasia Mutated Proteins; Carcinoma, Hepatocellular; Cell Cycle Proteins; Cell Line, Tumor; Cell Nucleus; Chlorocebus aethiops; COS Cells; Cysteine Proteinase Inhibitors; DNA Damage; DNA-Binding Proteins; Humans; Leupeptins; Liver Neoplasms; NFATC Transcription Factors; Osmotic Pressure; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Recombinant Proteins; Signal Transduction; Sodium Chloride; Transcription Factors; Transfection; Tumor Suppressor Proteins; Water-Electrolyte Balance; Wortmannin

We recently demonstrated that heavy metals, Hg2+, Pb2+, and Cu2+ induced Cyp1a1 gene expression, yet the mechanisms involved remain unknown. To explore the molecular mechanisms involved in the modulation of Cyp1a1 by heavy metals, Hepa 1c1c7 cells were treated with the metals in the presence and absence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent Cyp1a1 inducer. Time-dependent effect study showed that all metals significantly induced the basal Cyp1a1 mRNA. This was apparent 3 h after treatment, and levels remained elevated for at least 24 h. At the inducible level, Hg2+ and Pb2+ further increased, while Cu2+ decreased, the TCDD-mediated induction of Cyp1a1 mRNA. The RNA synthesis inhibitor, actinomycin D, completely blocked the Cyp1a1 induction by heavy metals. The protein synthesis inhibitor, cycloheximide, and 26S proteasome inhibitor, carbobenzoxy-L-leucyl-L-leucyl-leucinal (MG-132), super-induced the metal-mediated induction of Cyp1a1 mRNA. In addition, all three metals induced aryl hydrocarbon receptor/xenobiotic-responsive element (AhR/XRE) binding, suggesting an AhR-dependent mechanism. Cyp1a1 mRNA and protein decay experiments showed that the three metals did not significantly affect the half-life of mRNA; however, they significantly decreased the degradation rate of its protein, implying a posttranslational regulation of the Cyp1a1 by the heavy metals. A significant decrease in TCDD-mediated induction of Cyp1a1 activity associated with an increase in HO-1 mRNA and a decrease in cellular heme content was observed after all metals treatment. This suggests that heme degradation plays a role in reducing Cyp1a1 activity. This is the first demonstration that heavy metals can directly induce Cyp1a1 gene expression in an AhR-dependent manner through transcriptional and posttranslational mechanisms.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cycloheximide; Cytochrome P-450 CYP1A1; Dactinomycin; Drug Interactions; Enzyme Induction; Gene Expression Regulation, Neoplastic; Hepatocytes; Leupeptins; Metals, Heavy; Mice; Polychlorinated Dibenzodioxins; Protein Processing, Post-Translational; Protein Synthesis Inhibitors; Receptors, Aryl Hydrocarbon; Receptors, Cytoplasmic and Nuclear; Response Elements; RNA, Messenger

Host genes involved in lipid metabolism are differentially regulated during the early stages of hepatitis C virus (HCV) infection. The majority of lipids synthesized in the liver are exported to other tissues in the form of lipoproteins. The formation of these lipoproteins is dependent upon the association of triglycerides with apolipoprotein B100. Using the HCV subgenomic replicon expression system, we show that secretion of apoB100 is significantly reduced. Inhibition of apoB100 degradation by ALLN did not improve secretion. Triglyceride levels as well as microsomal triglyceride transfer protein mRNA and activity levels were reduced in replicon-expressing cells, indicating potential reasons for the observed decrease. Further evidence is presented for the interaction between the HCV nonstructural protein 5A and apoB100. These results provide further insight into the alteration of lipid metabolism by HCV.

Topics: Apolipoprotein B-100; Apolipoproteins B; Carcinoma, Hepatocellular; Carrier Proteins; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Genes, Reporter; Genome, Viral; Hepacivirus; Humans; Immunoprecipitation; Leupeptins; Lipids; Liver; Liver Neoplasms; Oleic Acid; Plasmids; Promoter Regions, Genetic; Protein Binding; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Transcription, Genetic; Transfection; Triglycerides; Viral Nonstructural Proteins

Proteasome inhibitors, like MG132, can exert cell growth inhibitory and apoptotic effects in different tumor types. The apoptotic mechanism of these compounds involves the activation of the effector caspases. beta-catenin, also an oncogene, represents one of the substrates of these proteases, but the consequences of its cleavage are poorly understood. We investigated its function during apoptosis induced by MG132 in three hepatocellular carcinoma (HCC) cell lines, endowed (HepG2 and HuH-6) or not (HA22T/VGH) with activating mutations of beta-catenin. Induction of apoptosis was associated with cell growth inhibition, accumulation of the cells at the G(2)/M phases of the cell cycle, as well as with fragmentation of beta-catenin (but not of alpha- or gamma-catenin) in all the cell lines. The cleavage of beta-catenin was inhibited by the caspase inhibitors Z-VAD-fmk and Z-DEVD-fmk. Fragmented beta-catenin was found in the nuclei of the treated cells. Analyses through the reporter plasmid pTOPflash showed that MG132 significantly reduces Tcf transcriptional activity in the cells. This was associated with a decrease in the mRNA expression of survivin and c-myc, which are target genes of the APC/beta-catenin/Tcf signaling. Nevertheless, Z-VAD-fmk or Z-DEVD-fmk did not reverse the MG132 effects on Tcf transcriptional activity, suggesting that the compound may affect this activity also by other mechanisms. Overall, the present study supports the therapeutic potential of the proteasome inhibitors in HCC.

Topics: alpha Catenin; Amino Acid Chloromethyl Ketones; Apoptosis; beta Catenin; Carcinoma, Hepatocellular; Caspase Inhibitors; Caspases; Cell Division; Cell Line, Tumor; Cell Nucleus; Cysteine Proteinase Inhibitors; Cytoskeletal Proteins; Cytosol; Desmoplakins; DNA-Binding Proteins; G2 Phase; gamma Catenin; Humans; Inhibitor of Apoptosis Proteins; Leupeptins; Lymphoid Enhancer-Binding Factor 1; Microtubule-Associated Proteins; Neoplasm Proteins; Oligopeptides; Peptide Fragments; Proto-Oncogene Proteins c-myc; RNA, Messenger; Survivin; Trans-Activators; Transcription Factors; Transcriptional Activation

Chronic ethanol consumption causes increased oxidative damage in the liver. Induction of CYP2E1 is one pathway involved in how ethanol produces oxidative stress. Ethanol can cause protein accumulation, decreased proteolysis, and decreased proteasome activity. The objective of this study was to investigate the effect of inhibition of the proteasome activity on CYP2E1-dependent toxicity. HepG2 cells over-expressing CYP2E1 (E47 cells) were treated with arachidonic acid (AA) plus iron, agents important in development of alcoholic liver injury and which are toxic to E47 cells by a mechanism dependent on CYP2E1, oxidative stress, and lipid peroxidation. Addition of various proteasome inhibitors was associated with significant potentiation of the loss of cell viability caused by AA plus iron. Potentiation of toxicity was associated with increased oxidative damage as reflected by an increase in lipid peroxidation and accumulation of oxidized and nitrated proteins in E47 cells and an enhanced decline in mitochondrial membrane potential. Antioxidants prevented the loss of viability and the potentiation of this loss of viability by proteasome inhibition. CYP2E1 levels were elevated about 3-fold by the proteasome inhibitors. Inhibition of proteasome activity also potentiated toxicity of AA alone and toxicity after treatment to remove glutathione (GSH). Similar results were found in hepatocytes from pyrazole-treated rats with high levels of CYP2E1. In conclusion, proteasome activity plays an important role in modulating CYP2E1-mediated toxicity in HepG2 cells by regulating CYP2E1 levels and by removal of oxidized proteins. Such interactions may be important in CYP2E1-catalyzed toxicity of hepatotoxins and in alcohol-induced liver injury.

Topics: Antioxidants; Arachidonic Acid; Buthionine Sulfoximine; Carcinoma, Hepatocellular; Cysteine Endopeptidases; Cytochrome P-450 CYP2E1; Drug Synergism; Endopeptidases; Humans; Iron; Leupeptins; Lipid Peroxides; Liver Neoplasms; Lysosomes; Membrane Potentials; Mitochondria, Liver; Multienzyme Complexes; Proteasome Endopeptidase Complex; Tumor Cells, Cultured; Tyrosine

Studies in hepatocyte cultures indicate that apolipoprotein (apo) B-100 production is regulated largely by intracellular degradation and the proteasome pathway is a major mechanism for the degradation. In the present study, we have examined the detailed itinerary of apoB degradation through its secretory pathway in HepG2 cells. We found that ubiquitin-dependent proteasomal degradation of apoB largely occurred on the cytosolic surface of rough and smooth endoplasmic reticulum (ER) and that a small proportion of apoB was dislodged from the secretory organelles into the cytosolic compartment where it underwent ubiquitination for proteasomal degradation. The transmembrane conformation of apoB persisted as the protein was transported through the Golgi apparatus. We further demonstrated that proteasomal degradation of apoB was associated the Golgi apparatus but Golgi-associated apoB was not ubiquitinated, indicating an ubiquitin-independent proteasomal degradation of apoB is associated with this organelle. We conclude that apoB undergoes proteasomal degradation while going through different compartments of the secretory pathway; further, ER-associated proteasomal degradation of apoB in the ER is ubiquitin-dependent whereas that occurring in the Golgi is ubiquitin-independent.

Topics: Apolipoprotein B-100; Apolipoproteins B; Biological Transport; Calpain; Carcinoma, Hepatocellular; Cell Compartmentation; Cysteine Endopeptidases; Cytosol; Endoplasmic Reticulum; Enzyme Inhibitors; Fluorescent Antibody Technique; Golgi Apparatus; Humans; Leupeptins; Microsomes, Liver; Multienzyme Complexes; Proteasome Endopeptidase Complex; Tumor Cells, Cultured; Ubiquitin

Because retention of mutant alpha(1)-antitrypsin (alpha(1)-AT) Z in the endoplasmic reticulum (ER) is associated with liver disease in alpha(1)-AT-deficient individuals, the mechanism by which this aggregated glycoprotein is degraded has received considerable attention. In previous studies using stable transfected human fibroblast cell lines and a cell-free microsomal translocation system, we found evidence for involvement of the proteasome in degradation of alpha(1)-ATZ (Qu, D., Teckman, J. H., Omura, S., and Perlmutter, D. H. (1996) J. Biol. Chem. 271, 22791-22795). In more recent studies, Cabral et al. (Cabral, C. M., Choudhury, P., Liu, Y., and Sifers, R. N. (2000) J. Biol. Chem. 275, 25015-25022) found that degradation of alpha(1)-ATZ in a stable transfected murine hepatoma cell line was inhibited by tyrosine phosphatase inhibitors, but not by the proteasomal inhibitor lactacystin and concluded that the proteasome was only involved in ER degradation of alpha(1)-ATZ in nonhepatocytic cell types or in cell types with levels of alpha(1)-AT expression that are substantial lower than that which occurs in hepatocytes. To examine this important issue in further detail, in this study we established rat and murine hepatoma cell lines with constitutive and inducible expression of alpha(1)-ATZ. In each of these cell lines degradation of alpha(1)-ATZ was inhibited by lactacystin, MG132, epoxomicin, and clasto-lactacystin beta-lactone. Using the inducible expression system to regulate the relative level of alpha(1)-ATZ expression, we found that lactacystin had a similar inhibitory effect on degradation of alpha(1)-ATZ at high and low levels of alpha(1)-AT expression. Although there is substantial evidence that other mechanisms contribute to ER degradation of alpha(1)-ATZ, the data reported here indicate that the proteasome plays an important role in many cell types including hepatocytes.

Topics: Acetylcysteine; alpha 1-Antitrypsin; Animals; Antibiotics, Antineoplastic; Carcinoma, Hepatocellular; Cell Line; Cells, Cultured; Cysteine Endopeptidases; Electrophoresis, Polyacrylamide Gel; Endoplasmic Reticulum; Fibroblasts; HeLa Cells; Hepatocytes; Humans; Lactones; Leupeptins; Liver; Mice; Multienzyme Complexes; Mutation; Oligopeptides; Precipitin Tests; Proteasome Endopeptidase Complex; Protein Binding; Rats; Time Factors; Transfection; Tumor Cells, Cultured

Large, free polymannose oligosaccharides generated during glycoprotein biosynthesis rapidly appear in the cytosol of HepG2 cells where they undergo processing by a cytosolic endo H-like enzyme and a mannosidase to yield the linear isomer of Man5GlcNAc (Man[alpha 1-2]Man[alpha 1-2]Man[alpha 1-3][Man alpha 1-6]Man[beta 1-4] GlcNAc). Here we have examined the fate of these partially trimmed oligosaccharides in intact HepG2 cells. Subsequent to pulse-chase incubations with D-[2-3H]mannose followed by permeabilization of cells with streptolysin O free oligosaccharides were isolated from the resulting cytosolic and membrane-bound compartments. Control pulse-chase experiments revealed that total cellular free oligosaccharides are lost from HepG2 cells with a half-life of 3-4 h. In contrast use of the vacuolar H+/ATPase inhibitor, concanamycin A, stabilized total cellular free oligosaccharides and enabled us to demonstrate a translocation of partially trimmed oligosaccharides from the cytosol into a membrane-bound compartment. This translocation process was unaffected by inhibitors of autophagy but inhibited if cells were treated with either 100 microM swainsonine, which provokes a cytosolic accumulation of large free oligosaccharides bearing 8-9 residues of mannose, or agents known to reduce cellular ATP levels which lead to the accumulation of the linear isomer of Man5GlcNAc in the cytosol. Subcellular fractionation studies on Percoll density gradients revealed that the cytosol-generated linear isomer of Man5GlcNAc is degraded in a membrane-bound compartment that cosediments with lysosomes.

Topics: Anti-Bacterial Agents; Bacterial Proteins; Biological Transport; Carbohydrate Sequence; Carcinoma, Hepatocellular; Cell Fractionation; Cell Membrane Permeability; Cytosol; Enzyme Inhibitors; Golgi Apparatus; Humans; Leupeptins; Liver Neoplasms; Lysosomes; Macrolides; Mannans; Mannose; Molecular Sequence Data; Oligosaccharides; Proton-Translocating ATPases; Streptolysins; Swainsonine; Tumor Cells, Cultured

The linkage between the conformation of apolipoprotein B100 (apoB) and the intracellular assembly and degradation of apoB-containing lipoproteins was investigated in the present study. Disruption of disulfide bond formation in newly synthesized apoB molecules through the use of the reducing agent DTT resulted in a decrease in the secretion of apoB-containing lipoproteins from HepG2 cells compared with control cells. The synthesis of total apoB (apoB100 plus nascent chains), as well as a number of control proteins, such as albumin and alpha 1-antitrypsin, was decreased significantly in DTT-treated cells. However, the intracellular accumulation of full-length apoB100 molecules was not inhibited in the presence of DTT. Subcellular fractionation indicated that apoB molecules isolated from the microsomes of DTT-treated cells had an increased association with the microsomal membrane compared with apoB isolated from untreated cells. Analysis of the distribution of apoB-containing lipoproteins from the lumen of isolated microsomes demonstrated that in the presence of DTT, there was a shift in the distribution, such that there was a decrease in the formation of HDL-sized (lipid-poor) apoB-containing lipoproteins and a decrease in the formation of LDL/VLDL apoB particles. Alterations in apoB conformation and their impact on degradation were also investigated by using DTT and by inhibiting N-linked glycosylation with tunicamycin. DTT appeared to change the rate and pattern of apoB degradation. Degradation was accelerated in both intact and permeabilized HepG2 cells. ApoB degradation occurred in DTT-treated permeabilized cells without the usual generation of the 70-kD and 335-kD fragments and was largely N-acetyl-leucyl-leucyl-norleucinal (ALLN) insensitive. In tunicamycin-treated cells, DTT further accelerated the degradation of unglycosylated apoB. Overall, the data suggest that the misfolding of apoB may prevent the proper association of apoB with lipids, resulting in impairment of the assembly of mature apoB-containing lipoproteins. Alteration in the conformation of apoB also appears to alter the degradation pathway of apoB, such that the protein is degraded through a pathway that is at least in part ALLN insensitive.

Topics: Animals; Apolipoprotein B-100; Apolipoproteins B; Carcinoma, Hepatocellular; Cystine; Dithiothreitol; Glycosylation; Humans; Leupeptins; Lipoproteins; Lipoproteins, HDL; Lipoproteins, LDL; Lipoproteins, VLDL; Liver; Liver Neoplasms; Microsomes, Liver; Molecular Weight; Neoplasm Proteins; Protease Inhibitors; Protein Conformation; Protein Denaturation; Protein Folding; Protein Processing, Post-Translational; Sulfhydryl Reagents; Tumor Cells, Cultured; Tunicamycin

It has been proposed that inefficient translocation across the endoplasmic reticulum (ER) membrane gives rise to transmembrane forms of apolipoprotein B-100 (apoB). However, we previously demonstrated that the amino-terminal 50% of apoB (apoB-50) was efficiently translocated across the ER membrane in the nonhepatic cell line COS-1. To determine whether liver-specific factors modulate apoB membrane translocation or topology, hybrid proteins containing 300 amino acid overlapping segments of apoB-48 were transiently expressed in HepG2 cells and their protease sensitivities were examined in membrane vesicles. The hybrid proteins demonstrated the same range of protection from exogenously added protease (75-100%) as a transfected secretory control protein. When endogenous apoB was examined, its protection from trypsin in intact membranes was -80%, a value similar to that of two endogenous secretory control proteins, transferrin and alpha 2-macroglobulin. No discretely sized fragments of apoB were generated by trypsin digestion of membranes unless they were first permeabilized with detergent. In contrast to the behavior of apoB and other control proteins, albumin predominantly resisted degradation by trypsin in both intact and detergent permeabilized membranes. HepG2 cells were treated with ALLN, a protease inhibitor that has been proposed to inhibit the turnover of partially translocated forms of apoB. Although an -6-fold increase in intracellular apoB was observed in ALLN-treated cells, no corresponding increase in protease sensitivity was observed. These results indicate that the efficient translocation of apoB across the ER membrane occurs independently of its ability to undergo assembly into a secretion competent lipoprotein.

Topics: Apolipoprotein B-100; Apolipoprotein B-48; Apolipoproteins B; Biological Transport; Carcinoma, Hepatocellular; Endoplasmic Reticulum; Humans; Immunoblotting; Intracellular Membranes; Leupeptins; Lipoproteins; Liver; Liver Neoplasms; Protease Inhibitors; Trypsin; Tumor Cells, Cultured

Receptor-mediated endocytosis of tissue-type plasminogen activator (t-PA)-plasminogen activator inhibitor type 1 (PAI-1) complexes results in their clearance by Hep G2 cells. After complexes are internalized, the t-PA component is degraded. However, neither the locus of intracellular catabolism nor the fate of PAI-1 has been elucidated. To characterize these aspects of t-PA-PAI-1 catabolism, the subcellular distribution of a prebound cohort of ligand molecules was delineated after internalization at 37 degrees C. 125I-t-PA.PAI-1 and t-PA.125I-PAI-1 were compared in separate experiments. After ligand uptake, intracellular vesicles were separated on density gradients. Internalized 125I-t-PA.PAI-1 concentrated initially in endosomes. After 20 minutes of uptake, the complex began to appear in lysosomes. Subsequently, low molecular weight labeled ligand fragments were detected in culture media. A panel of lysosomotropic agents, including primaquine, chloroquine, ammonium chloride, and a combination of leupeptin and pepstatin A, inhibited degradation. When t-PA.125I-PAI-1 rather than 125I-t-PA.PAI-1 was internalized, strikingly different results were observed. Although the kinetics of internalization and the intracellular itinerary were indistinguishable for the differently labeled complexes, the 125I-PAI-1 component of t-PA.125I-PAI-1 resisted rapid degradation. After a rapid loss of t-PA, the 125I-PAI-1 moiety persisted in lysosomes for up to 180 minutes. Thus, internalized t-PA.PAI-1 is targeted to lysosomes in which PAI-1 is relatively more stable than t-PA.

Topics: Ammonium Chloride; beta-N-Acetylhexosaminidases; Carcinoma, Hepatocellular; Chloroquine; Endocytosis; Humans; Kinetics; Leupeptins; Liver Neoplasms; Lysosomes; Pepstatins; Plasminogen Activator Inhibitor 1; Primaquine; Recombinant Proteins; Subcellular Fractions; Tissue Plasminogen Activator; Tumor Cells, Cultured

A study of insulin-receptor internalization and recycling was undertaken in primary cultures of rat hepatocytes and a human hepatoma cell line (HepG2). Receptors were quantitated by measuring 125I-insulin binding to partially purified soluble receptor preparations from untreated cells (total receptors) and trypsinized cells (intracellular receptors). In resting HepG2 cells, exposure to insulin results in internalization of insulin receptors, the rate and extent of which is dependent on the insulin concentration. However, receptors do not accumulate inside the cell in proportion to the higher rates of internalization at high concentrations of insulin. This lack of accumulation is explained by much higher recycling rates after exposure to high concentrations of insulin. Similar results were noted for primary cultures of rat hepatocytes. These results imply qualitatively different fates for receptors internalized after exposure to different concentrations of insulin. To further investigate the possibility of different pathways for insulin-receptor internalization and processing, cells in low (1 ng/ml) or high (100 ng/ml) concentrations of insulin were exposed to drugs or treatments known to affect receptor metabolism. Hypotonic shock and hypokalemia, which arrest coated-pit formation, blocked internalization of insulin and insulin receptors at low concentrations of insulin but allowed internalization in response to high concentrations of insulin. The lysosomotropic drugs monensin and chloroquine caused intracellular accumulation of insulin and its receptors internalized at low concentrations of insulin but had a relatively smaller effect on receptors internalized at high concentrations of insulin. All internalization is blocked by 2,4-dinitrophenol. We conclude that high doses of insulin lead to insulin-receptor internalization and recycling through a pathway that is functionally distinct from the pathway taken by receptors internalized by low (physiologic) concentrations of insulin. The pharmacologic experiments raise the possibility that the high-dose pathway, unlike the low-dose pathway, may proceed independently of coated pits and endosomal acidification.

Topics: 2,4-Dinitrophenol; Animals; Biological Transport; Biotransformation; Carcinoma, Hepatocellular; Cells, Cultured; Chloroquine; Coated Pits, Cell-Membrane; Dinitrophenols; Humans; Insulin; Leupeptins; Liver; Liver Neoplasms; Monensin; Osmolar Concentration; Potassium; Rats; Receptor, Insulin; Tumor Cells, Cultured

Basic fibroblast growth factor (FGF) was purified by heparin-Sepharose chromatography from two sources, brain and hepatoma cells. Brain cell-derived basic FGF (brFGF) and hepatoma cell-derived basic FGF (heFGF) were found to exist in multiple forms whose molecular weights depended on whether they were extracted from their respective tissue or cells at neutral or acid pH. When extracted at pH 7.0 brFGF and heFGF comigrated on NaDodSO4/PAGE with a Mr of approximately 18,400. When extracted at pHs 3.5-4.5, acid proteinases cleaved brFGF and heFGF to lower molecular weight forms but to different extents. brFGF was cleaved to a Mr 18,000 form at acid pH by a brain-derived acid proteinase that could be inhibited by pepstatin. heFGF was cleaved mostly to a Mr 16,500 form at acid pH by a hepatoma cell-derived acid proteinase that was inhibited by leupeptin. Electrophoretic transfer blot analysis using site-specific anti-FGF antibodies suggested that the cleavages occurred at the amino-terminal ends of brFGF and heFGF. Cleavage to lower molecular weight forms of brFGF and heFGF did not affect growth factor activity or chromatographic behavior on heparin-Sepharose columns.

Topics: Amino Acid Sequence; Animals; Aspartic Acid Endopeptidases; Brain; Carcinoma, Hepatocellular; Cattle; Cell Line; Endopeptidases; Fibroblast Growth Factors; Humans; Leupeptins; Liver Neoplasms; Molecular Weight; Protease Inhibitors

Studies were made on the effect of protease inhibitors (pepstatin, leupeptin, and antipain) on the induction of tumors in mice by the alpha-isomer of 1,2,3,4,5,6-hexachlorocyclohexane (alpha-BHC) and by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). Animals were given 0.05% alpha-BHC in the diet or 0.05% BBN in their drinking water and then fed on a powder diet supplemented with 0.1% protease inhibitors. The incidence of liver nodular hyperplasias was significantly higher in the group treated with alpha-BHC and leupeptin than in the group treated with alpha-BIC alone, but pepstatin and antipain did not affect induction of liver nodular hyperplasias by alpha-BHC. None of the three protease inhibitors influenced the induction of bladder tumors by BBN.

Topics: Animals; Butylhydroxybutylnitrosamine; Carcinoma, Hepatocellular; Hexachlorocyclohexane; Hyperplasia; Leupeptins; Liver; Liver Neoplasms; Mice; Nitrosamines; Pepstatins; Protease Inhibitors; Urinary Bladder Neoplasms

1. Rates of degradation of normal and abnormal protein were measured in hepatoma cells after labelling first for 16h with [14C]leucine plus L-arginine and then for 3h with [3H]-leucine plus the arginine analogue, L-canavanine. 2. Over the first 2h of the degradation period, canavanine-containing proteins were degraded at approximately 5 times the average degradation rate of normal proteins. 3. Degradation of normal proteins was inhibited by about 30% by insulin, cycloheximide, puromycin, leupeptin, antipain and foetal calf serum, whereas these agents had a negligible effect on the breakdown of canavanine-containing proteins. 4. Other compounds inhibited degradation of both classes of protein to equal extents. 5. Combination experiments showed no additional inhibitory effects on the degradation of normal proteins over degradation measured in the presence of a single selective inhibitor. 6. In contrast with the results with a 16 h labelling period, the degradation of normal proteins labelled for only 3 h was not inhibited by insulin. 7. These results are explained by a model with two distinct pathways of protein turnover. The first of these pathways involves the formation of autophagic vacuoles and would be completely inhibited by each of the selective inhibitors. Normal and canavanine-containing proteins would be catabolized by this pathway at equal rates. We propose that degradation by a second pathway is not regulated by the agents tested, but by the inherent stability of each protein.

Topics: Amino Acids; Arginine; Canavanine; Carcinoma, Hepatocellular; Cells, Cultured; Cycloheximide; Fetal Blood; Insulin; Leucine; Leupeptins; Liver Neoplasms; Neoplasm Proteins; Neoplasms, Experimental; Pepstatins; Phenylmethylsulfonyl Fluoride; Phosphoenolpyruvate Carboxykinase (GTP); Puromycin