calpain and Liver-Neoplasms

Rapid progression is the major cause of the poor prognosis of hepatocellular carcinoma (HCC); however, the underlying mechanism remained unclear. Here, we found Calpain-2 (CAPN2), a well-established protease that accelerates tumor progression in several malignancies, is overexpressed in HCC and acts as an independent predictor for poor outcomes. Furthermore, CAPN2 promoted the proliferation and invasion of HCC, and showed a positive correlation with the levels of invasion-related markers. Mechanistically, a novel CAPN2-SRC positive regulatory loop was identified upstream of β-catenin to prevent its ubiquitination and degradation, and subsequently promoted HCC progression: CAPN2 could proteolyze PTP1B to form a truncation of approximately 42 kDa with increased phosphatase activity, resulting in reduced SRC Y530 phosphorylation and increased SRC kinase activity; meanwhile, CAPN2 itself was a bone fide substrate of SRC that was primarily phosphorylated at Y625 by SRC and exhibited increased proteolysis activity upon phosphorylation. Interestingly, the CAPN2-SRC loop could not only restrain most of cytoplasmic β-catenin degradation by inhibiting GSK3β pathway, but also prevented TRIM33-induced nuclear β-catenin degradation even in β-catenin-mutant cells. Present study identified a CAPN2-SRC positive loop responsible for intracellular β-catenin accumulation and signaling activation, and targeting CAPN2 protease activity might be a promising approach for preventing HCC progression.

Topics: beta Catenin; Calpain; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Humans; Liver Neoplasms; src-Family Kinases; Transcription Factors; Wnt Signaling Pathway

Interleukin-1α (IL-1α) plays an important role in inflammation and hematopoiesis. Many tumors have increased IL-1α expression. However, the immune regulatory role of secreted IL-1α in tumor development and whether it can be targeted for cancer therapy are still unclear. Here, we found that tumoral-secreted IL-1α significantly promoted hepatocellular carcinoma (HCC) development

Topics: Calpain; Carcinoma, Hepatocellular; CD8-Positive T-Lymphocytes; Humans; Interleukin-1alpha; Liver Neoplasms; Tumor Microenvironment

Hepatocellular carcinoma (HCC) is the second prominent cause of cancer-associated death worldwide. Usually, HCC is diagnosed in advanced stages, wherein sorafenib, a multiple target tyrosine kinase inhibitor, is used as the first line of treatment. Unfortunately, resistance to sorafenib is usually encountered within six months of treatment. Therefore, there is a critical need to identify the underlying reasons for drug resistance. In the present study, we investigated the proteomic and metabolomics alterations accompanying sorafenib resistance in hepatocellular carcinoma Hep3B cells by employing ultra-high-performance liquid chromatography quadrupole time of flight mass spectrometry (UHPLC-QTOF-MS). The Bruker Human Metabolome Database (HMDB) library was used to identify the differentially abundant metabolites through MetaboScape 4.0 software (Bruker). For protein annotation and identification, the Uniprot proteome for Homo sapiens (Human) database was utilized through MaxQuant. The results revealed that 27 metabolites and 18 proteins were significantly dysregulated due to sorafenib resistance in Hep3B cells compared to the parental phenotype. D-alanine, L-proline, o-tyrosine, succinic acid and phosphatidylcholine (PC, 16:0/16:0) were among the significantly altered metabolites. Ubiquitin carboxyl-terminal hydrolase isozyme L1, mitochondrial superoxide dismutase, UDP-glucose-6-dehydrogenase, sorbitol dehydrogenase and calpain small subunit 1 were among the significantly altered proteins. The findings revealed that resistant Hep3B cells demonstrated significant alterations in amino acid and nucleotide metabolic pathways, energy production pathways and other pathways related to cancer aggressiveness, such as migration, proliferation and drug-resistance. Joint pathway enrichment analysis unveiled unique pathways, including the antifolate resistance pathway and other important pathways that maintain cancer cells' survival, growth, and proliferation. Collectively, the results identified potential biomarkers for sorafenib-resistant HCC and gave insights into their role in chemotherapeutic drug resistance, cancer initiation, progression and aggressiveness, which may contribute to better prognosis and chemotherapeutic outcomes.

Topics: Alanine; Amino Acids; Antineoplastic Agents; Biomarkers; Calpain; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Folic Acid Antagonists; Glucose; Humans; L-Iditol 2-Dehydrogenase; Liver Neoplasms; Metabolic Networks and Pathways; Nucleotides; Phosphatidylcholines; Proline; Protein Kinase Inhibitors; Proteome; Proteomics; Sorafenib; Succinic Acid; Superoxide Dismutase; Tyrosine; Ubiquitin Thiolesterase; Uridine Diphosphate

Hepsin is a transmembrane serine protease implicated in many biological processes, including hepatocyte growth, urinary protein secretion, auditory nerve development, and cancer metastasis. Zymogen activation is critical for hepsin function. To date, how hepsin is activated and regulated in cells remains an enigma. In this study, we conducted site-directed mutagenesis, cell expression, plasma membrane protein labeling, trypsin digestion, Western blotting, and flow cytometry experiments in human hepatoma HepG2 cells, where hepsin was originally discovered, and SMMC-7721 cells. Our results show that hepsin is activated by autocatalysis on the cell surface but not intracellularly. Moreover, we show that hepsin undergoes ectodomain shedding. In the conditioned medium from HepG2 and SMMC-7721 cells, we detected a soluble fragment comprising nearly the entire extracellular region of hepsin. By testing protease inhibitors, gene knockdown, and site-directed mutagenesis, we identified calpain-1 as a primary protease that acted extracellularly to cleave Tyr52 in the juxtamembrane space of hepsin. These results provide new insights into the biochemical and cellular mechanisms that regulate hepsin expression and activity.

Topics: Calpain; Carcinoma, Hepatocellular; Cell Membrane; Enzyme Activation; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Neoplasm Proteins; Protein Domains; Serine Endopeptidases

Tumor necrosis factor-α has been proven an effective anticancer agent in preclinical studies. However, the translation of TNFα from research to clinic has been blocked by significant systemic toxicity and limited efficacy at maximal tolerated dose, which need urgently to be solved.. The level of cytosolic Ca. Our study provides the evidence supporting a novel mechanism by which TNFα induces extracellular Ca

Topics: Animals; Apoptosis; Biomarkers; Calcium; Calpain; Carcinoma, Hepatocellular; Cell Line, Tumor; Disease Models, Animal; Extracellular Space; Humans; Liver Neoplasms; Membrane Potential, Mitochondrial; Mice; Mitochondria; Models, Biological; Receptors, Tumor Necrosis Factor; RNA, Small Interfering; Transient Receptor Potential Channels; Tumor Necrosis Factor-alpha; Xenograft Model Antitumor Assays

Tumor necrosis factor-related apoptosis-induced ligand (TRAIL) induces apoptosis selectively in cancer cells without affecting the majority of normal human cells. However, hepatocellular carcinoma (HCC) cells often display resistance to TRAIL-induced apoptosis. Ibulocydine (IB) is an isobutyrate ester pro-drug of a novel synthetic Cdk inhibitor that targets Cdk7 and Cdk9. In this study, we show that treatment with subtoxic doses of IB in combination with TRAIL displays potent cytotoxicity in TRAIL-resistant human HCC cells. Combination of IB and TRAIL was found to synergistically induce apoptosis through activation of caspases, which was blocked by a pan-caspase inhibitor (zVAD). Although the expression of Mcl-1 and survivin were reduced by IB plus TRAIL, overexpression of Mcl-1 and survivin did not block the cell death induced by co-treatment. Moreover, overexpression of Bcl-xL did not significantly interfere with the cell death induced by co-treatment of IB and TRAIL. Interestingly, the combination treatment induced cleavage of Bax, which was translocated to mitochondria upon induction of apoptosis. Furthermore, down-regulation of Bax by small interfering RNA effectively reduced the cell death and loss of mitochondrial membrane potential (MMP) caused by co-treatment with IB and TRAIL. Finally, pre-treatment of HCC cells with a calpain inhibitor effectively blocked IB plus TRAIL-induced cleavage of Bax and apoptosis. Collectively, our results demonstrate that IB increases the sensitivity of human HCC cells to TRAIL via mitochondria signaling pathway mediated by calpain-induced cleavage of Bax, suggesting that combined treatment with IB and TRAIL may offer an effective therapeutic strategy for human HCC.

Topics: Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Calpain; Carcinoma, Hepatocellular; Caspases; Cell Line, Tumor; Drug Synergism; Humans; Liver Neoplasms; Mitochondria; Prodrugs; Pyrimidine Nucleosides; RNA, Small Interfering; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand; Up-Regulation

Liver resection is still the most commonly used therapeutic treatment for hepatocellular carcinoma (HCC), and liver regeneration promotes HCC growth in the regenerating liver. The high recurrence/metastasis of HCC is the main cause of death for HCC patients after liver resection. However, how the augmented growth and metastasis of residual HCC induced by the promoted liver regeneration following liver resection can be abolished remains unclear. In this study, a rat model with liver cirrhosis and diffused HCC was established by administration of diethylnitrosamine. Recombinant miR-203 adenovirus was administered to induce hepatic miR-203 overexpression and 30% partial hepatectomy (PH) followed. The effect of miR-203 on the proliferation, invasion and metastasis of the residual HCC in the remnant cirrhotic liver with promoted regeneration was investigated. We found that the basic spontaneous regeneration of the non-tumorous liver by 30% PH promoted proliferation, invasion and lung metastasis of the hepatic residual HCC. miR-203 overexpression further promoted the regeneration of the non-tumorous liver by upregulating Ki67 expression and enhancing IL-6/SOCS3/STAT3 pro-proliferative signals. Importantly, miR-203 overexpression markedly inhibited the proliferation, invasion and metastasis of hepatic residual HCC through suppressing expression of Ki67, CAPNS1 and lung metastasis. Moreover, it was found that miR-203 overexpression reversed the epithelial-mesenchymal transition induced by hepatectomy through targeting IL-1β, Snail1 and Twist1. In conclusion, our results suggested that miR-203 overexpression inhibited the augmented proliferation and lung metastasis of the residual HCC induced by the promoted liver regeneration following PH partly by regulating epithelial-mesenchymal transition.

Topics: Animals; Calpain; Carcinoma, Hepatocellular; Cell Movement; Cell Proliferation; Diethylnitrosamine; Epithelial-Mesenchymal Transition; Hepatectomy; Interleukin-1beta; Interleukin-6; Ki-67 Antigen; Liver; Liver Neoplasms; Male; MicroRNAs; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Recurrence, Local; Rats; Rats, Wistar; Snail Family Transcription Factors; STAT3 Transcription Factor; Suppressor of Cytokine Signaling 3 Protein; Twist-Related Protein 1

The antiepileptic drug phenobarbital (PB) exerts hepatic effects related to cell proliferation and tumorigenesis which are closely linked to the Wnt/β-catenin signaling pathway. This pathway is, amongst others, regulated by calpain proteases. We now identified PB as an inhibitor of Wnt/β-catenin signaling in mouse hepatoma cells. Further analyses revealed that PB inhibits calpain activity, an effect which is at least in parts mediated by a transcriptional regulation of calpain mRNA levels and which is furthermore independent of the constitutive androstane receptor, the known mediator of most effects of PB in liver cells.

Topics: Animals; Calpain; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Liver Neoplasms; Mice; Phenobarbital; Structure-Activity Relationship

Our previous study shows that Calpain 6 (CAPN6) expression is regulated by PI3K-Akt in liver cancer through POU2F1 and CAPN6 which promote cell proliferation and inhibit apoptosis of liver cancer cells. microRNAs (miRNAs) plays important roles in regulation of gene expression. However, whether miRNAs regulates CAPN6 expression and its cellular function is still unknown. This study aims to investigate how miRNAs regulate liver cancer apoptosis through POU2F1-CAPN6. It was verified that POU2F1 could promote cell proliferation and inhibit apoptosis through CAPN6. Using methods of bioinformatics, miR-449a was predicted as a potential regulator of both CAPN6 and POU2F1. It was verified that CAPN6 and POU2F1 were the target genes of miR-449a by luciferase assay. CAPN6 and POU2F1 protein and mRNA levels decreased in liver cancer cells with miR-449a overexpression using western blot and real time RT-PCR assays. miR-449a expression was lower in liver cancer tissues compared with their normal ones, so did the cells. Overexpression of miR-449a inhibited cell proliferation, induced G1 phase arrest and cell apoptosis in liver cancer. Further research demonstrated that miR-449a inhibited cancer cell proliferation and induced apoptosis via suppressing both POU2F1 and CAPN6. The study indicated that miR-449a functions as a tumor inhibitor in liver cancer by decreasing POU2F1 and CAPN6 expression in liver cancer.

Topics: Apoptosis; Biomarkers, Tumor; Calpain; Carcinoma, Hepatocellular; Cell Cycle; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Male; MicroRNAs; Microtubule-Associated Proteins; Middle Aged; Neoplasm Grading; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Staging; Octamer Transcription Factor-1; Prognosis; Signal Transduction; Survival Rate; Tumor Cells, Cultured

Tumor metastasis is the main reason of death for hepatocellular carcinoma (HCC) patients. Cell migration and invasion are two prerequisites for tumor metastasis, in which TRPM7 and MMPs play an important role. In our study, we found that bradykinin (BK) could upregulate the expression of TRPM7 and dynamically regulate the phosphorylation of non-muscle myosin IIA heavy chain (NMHC-IIA) Ser-1943 in HepG2 cells. The influx of Ca

Topics: Bradykinin; Calcium; Calpain; Carcinoma, Hepatocellular; Cell Movement; Exocytosis; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Matrix Metalloproteinase 2; Molecular Motor Proteins; Myosin Heavy Chains; Neoplasm Invasiveness; Phosphorylation; Phosphoserine; Podosomes; Protein Serine-Threonine Kinases; Receptor, Bradykinin B2; RNA Interference; RNA, Messenger; Signal Transduction; src-Family Kinases; TRPM Cation Channels; Up-Regulation

SerpinB3 has been recently described as an early marker of liver carcinogenesis, but the potential mechanistic role of this serpin in tumor development is still poorly understood. Overexpression of Myc often correlates with more aggressive tumour forms, supporting its involvement in carcinogenesis. Yes-associated protein (Yap), the main effector of the Hippo pathway, is a central regulator of proliferation and it has been found up-regulated in hepatocellular carcinomas. The study has been designed to investigate and characterize the interplay and functional modulation of Myc by SerpinB3 in liver cancer. Results from this study indicate that Myc was up-regulated by SerpinB3 through calpain and Hippo-dependent molecular mechanisms in transgenic mice and hepatoma cells overexpressing human SerpinB3, and also in human hepatocellular carcinomas. Human recombinant SerpinB3 was capable to inhibit the activity of Calpain in vitro, likely reducing its ability to cleave Myc in its non oncogenic Myc-nick cytoplasmic form. SerpinB3 indirectly increased the transcription of Myc through the induction of Yap pathway. These findings provide for the first time evidence that SerpinB3 can improve the production of Myc through direct and indirect mechanisms that include the inhibition of generation of its cytoplasmic form and the activation of Yap pathway.

Topics: Adaptor Proteins, Signal Transducing; Animals; Antigens, Neoplasm; Calpain; Carcinogenesis; Carcinoma, Hepatocellular; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Mice; Mice, Transgenic; Phosphoproteins; Proto-Oncogene Proteins c-myc; Serpins; Transcription Factors; Transcription, Genetic; YAP-Signaling Proteins

Calpain small subunit 1 (Capn4) has been identified as a major gene that promotes metastasis of hepatocellular carcinoma (HCC). However, the mechanism by which Capn4 promotes progression of HCC is not understood. In this study, we found that Capn4 expression was increased in highly metastatic HCC cell lines and in tumour tissue from HCC patients compared to healthy patient tissue. Over-expression of Capn4 in HCC cells enhanced tumour cell growth in vitro and increased invasiveness, tumourigenicity and lung metastasis in vivo. Protein microarray analyses showed that expression of multiple proteins was regulated by Capn4. Interestingly, Capn4 was found to physically associate with FAK and promoted hyperactivity of the FAK-Src signalling pathway via increased phosphorylation of specific tyrosine residues of FAK, Src and p130Cas. Knock-down of Capn4 expression suppressed the malignant behaviour of HCC cells and inhibited the FAK-Src signalling pathway. Furthermore, Capn4-mediated invasion and metastasis of HCC cells required up-regulation of matrix metalloproteinase-2 (MMP2) through activation of this signalling pathway. Our clinical data revealed that Capn4 expression correlated well with the levels of phospho-FAK, and over-expression of both Capn4 and phospho-FAK correlates with the poorest survival outcomes in HCC. In conclusion, our data showed that Capn4 can contribute to HCC growth and metastasis via activation of the FAK-Src signalling pathway and MMP2.

Topics: Aged; Animals; Blotting, Western; Calpain; Carcinoma, Hepatocellular; Female; Fluorescent Antibody Technique; Focal Adhesion Kinase 1; Heterografts; Humans; Immunoprecipitation; Kaplan-Meier Estimate; Liver Neoplasms; Male; Mice; Mice, Nude; Microscopy, Confocal; Middle Aged; Neoplasm Invasiveness; Proportional Hazards Models; Signal Transduction; src-Family Kinases; Tissue Array Analysis; Transfection

Rosin, the traditional Chinese medicine, is reported to be able to inhibit skin cancer cell lines. In this report, we investigate the inhibitory effect against HCC cells of QC2, the derivative of rosin's main components dehydroabietic acid.. MTT assay was used to determine the cytotoxicity of QC2. Morphological changes were observed by time-lapse microscopy and transmission electron microscopy and the cytoskeleton changes were observed by laser-scanning confocal microscopy. Cytomembrane integrity and organelles damage were confirmed by detection of the reactive oxygen (ROS), lactate dehydrogenase (LDH), and mitochondrial membrane potential (Δψm). The underlying mechanism was manifested by Western blotting. The oncotic cell death was further confirmed by detection of oncosis related protein calpain.. Swelling cell type and destroyed cytoskeleton were observed in QC2-treated HCC cells. Organelle damage was visualized by transmission electron microscopy. The detection of ROS accumulation, increased LDH release, and decreased ATP and Δψm confirmed the cell death. The oncotic related protein calpain was found to increase time-dependently in QC2-treated HCC cells, while its inhibitor PD150606 attenuated the cytotoxicity.. Dehydroabietic acid derivative QC2 activated oncosis related protein calpain to induce the damage of cytomembrane and organelles which finally lead to oncosis in HCC cells.

Topics: Abietanes; Adenosine Triphosphate; Amino Acid Chloromethyl Ketones; Calpain; Carcinoma, Hepatocellular; Caspase 3; Caspase Inhibitors; Cell Death; Cell Line, Tumor; Cell Membrane; Cytoskeleton; Hepatocytes; Humans; Imidazoles; Indoles; Liver Neoplasms; Membrane Potential, Mitochondrial; Reactive Oxygen Species

Calpains are a conserved family of calcium-dependent cysteine proteinases involved in various cellular functions. Two ubiquitous isoforms, µ- and m-calpain, are key members of the calpain family that play essential roles in regulating cell migration and invasion. However, it remains unclear whether they are involved in the progression of hepatocellular carcinoma (HCC). Here, we investigated the functions of µ- and m-calpain in the invasive and metastatic processes of human hepatoma cells. Our results indicated that the expression levels of calpains were elevated in HCC cells compared with those in normal hepatic cells. Our results indicated that small interfering RNA (siRNA)-mediated silencing of µ- and m-calpain expressions significantly suppressed the adhesive, migrative and invasive potentials of human hepatoma cells. The matrix metalloproteinases (MMPs) are key regulators of malignant tumour invasion and metastasis. siRNA-mediated down-regulation of µ- and m-calpain expressions also significantly attenuated MMP-2 and MMP-9 secretion. Thus µ- and m-calpain may play important roles in the invasion and metastasis of human hepatoma cells, and calpains may be drug targets for preventing HCC metastasis.

Topics: Calpain; Carcinoma, Hepatocellular; Cell Adhesion; Cell Line; Cell Movement; Down-Regulation; Hep G2 Cells; Humans; Liver Neoplasms; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; RNA Interference; RNA, Small Interfering

Osteopontin (OPN) is a secreted phosphoprotein, originally characterized in malignant-transformed epithelial cells. OPN is associated with tumor metastasis of several tumors and is overexpressed in hepatocellular carcinoma (HCC) tissue involving HCC invasion and metastasis. Importantly, OPN is significantly up-regulated in liver injury, inflammation, and hepatitis C virus (HCV)-associated HCC. However, the underlying mechanisms of OPN activation and its role in HCV-mediated liver disease pathogenesis are not known. In this study, we investigated the mechanism of OPN activation in HCV-infected cells. We demonstrate that HCV-mediated Ca(2+) signaling, elevation of reactive oxygen species, and activation of cellular kinases such as p38 MAPK, JNK, PI3K, and MEK1/2 are involved in OPN activation. Incubation of HCV-infected cells with the inhibitors of AP-1 and Sp1 and site-directed mutagenesis of AP-1- and Sp1-binding sites on the OPN promoter suggest the critical role of AP-1 and Sp1 in OPN promoter activation. In addition, we show the in vivo interactions of AP-1 and Sp1 with the OPN promoter using chromatin immunoprecipitation assay. We also show the calpain-mediated processing of precursor OPN (∼75 kDa) into ∼55-, ∼42-, and ∼36-kDa forms of OPN in HCV-infected cells. Furthermore, we demonstrate the critical role of HCV-induced OPN in increased phosphorylation of Akt and GSK-3β followed by the activation of β-catenin, which can lead to EMT of hepatocytes. Taken together, these studies provide an insight into the mechanisms of OPN activation that is relevant to the metastasis of HCV-associated HCC.

Topics: beta Catenin; Calcium Signaling; Calpain; Carcinoma, Hepatocellular; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Extracellular Signal-Regulated MAP Kinases; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Hepacivirus; Hepatitis C; Hepatocytes; Humans; Liver Neoplasms; Neoplasm Metastasis; Osteopontin; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt; Sp1 Transcription Factor; Transcription Factor AP-1

Calcium (Ca(2+)) signals are involved in important checkpoints in cell death pathways and promote both apoptosis and autophagy. However, the relationship between autophagy and apoptosis in response to Ca(2+) level elevation is poorly understood. Here, we provided evidence that the influx of extracellular Ca(2+) triggered by Trichokonin VI (TK VI), an antimicrobial peptide, induced calpain-dependent apoptosis and autophagy in hepatocellular carcinoma (HCC) cells. Remarkably, TK VI preferentially induced apoptosis that was associated with calpain-mediated Bax and Atg5 cleavage, which resulted in the collapse of the mitochondrial membrane potential and cytochrome c release. Interestingly, truncated, but not full-length Atg5, associated with Bcl-xL and promoted the intrinsic pathway. Moreover, TK VI treatment induced reactive oxygen species (ROS) accumulation, an effect in which Bak might play a major role. This accumulation of ROS resulted in the subsequent disposal of damaged mitochondria within autophagosomes via Atg5-mediated and mitochondria-selective autophagy. Both the inhibition of calpain activity and Bax deficiency activated a switch that promoted an enhancement of autophagy. The inhibition of both apoptosis and autophagy significantly attenuated the TK VI cytotoxicity, indicating that the two processes had stimulatory effects during TK VI-meditated cell death. These results suggested that calpain, Bak and Atg5 were molecular links between autophagy and apoptosis and revealed novel aspects of the crosstalk between these two processes. The potential of TK VI is proposed as a promising anticancer agent for its well-characterized activity of Ca(2+) agonist and as a possible novel therapeutic strategy that acts on cancer cell mitochondria.

Topics: Alamethicin; Antimicrobial Cationic Peptides; Apoptosis; Autophagy; Autophagy-Related Protein 5; bcl-2 Homologous Antagonist-Killer Protein; bcl-X Protein; Calcium; Calcium Signaling; Calpain; Carcinoma, Hepatocellular; Cell Line, Tumor; Humans; Liver Neoplasms; Membrane Potential, Mitochondrial; Microtubule-Associated Proteins; Mitochondria; Oxidative Stress; Reactive Oxygen Species; RNA Interference

Calpain small subunit 1 (Capn4) has been shown to correlate with the metastasis/invasion of hepatocellular carcinoma. This study aimed to investigate the role of Capn4 in intrahepatic cholangiocarcinoma (ICC).. Capn4 expression was measured in 33 ICC tissues by quantitative real-time polymerase chain reaction and western blot. The role of Capn4 in the migration, invasion and proliferation of ICC cells and matrix metalloproteinase 2 (MMP2) expression were assessed after Capn4 depletion by specific small interfering RNA. Capn4 expression was further examined by immunohistochemistry in a tissue microarray consisting of 140 ICC patients and 13 normal liver tissues, and the prognostic role of Capn4 in ICC was evaluated by Kaplan-Meier and Cox regression analyses.. Capn4 expression was significantly higher in the ICC tissues compared to the peritumor tissues. Capn4 down-regulation impaired the migration/invasion ability of HCCC-9810 and QBC939 cells in vitro and decreased MMP2 expression. Capn4 overexpression significantly correlated with the presence of lymphatic metastasis of ICC (p = 0.026) and the tumor-node-metastasis (TNM) stage (p = 0.009). The postoperative 2- and 5-year overall survivals in patients with Capn4(low) were higher than those in the Capn4(high) group. The cumulative recurrence rate in patients with Capn4(low) was much lower than in the Capn4(high) group. Multivariate analysis showed that Capn4 overexpression was an independent prognostic marker in ICC.. Capn4 overexpression was implicated in ICC metastasis/invasion, and Capn4 overexpression may be used as a molecular therapeutic target for ICC.

Topics: Adult; Aged; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Calpain; Cell Line, Tumor; Cell Proliferation; Cholangiocarcinoma; Female; Gene Expression Regulation, Neoplastic; Humans; Kaplan-Meier Estimate; Liver Neoplasms; Lymphatic Metastasis; Male; Matrix Metalloproteinase 2; Middle Aged; Neoplasm Invasiveness; Neoplasm Staging; Prognosis; Proportional Hazards Models

Natural flavonoids are associated with anti-proliferation of cancer growth. However, the antioxidant and anti-proliferation effects of AE (aloe-emodin) have not been well studied. We have investigated how AE affects the proliferation of hepatic hepatocellular carcinoma cells and exerts an anti-cancer effect. The cytotoxic effect of AE was demonstrated using an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay and Huh-7 cells were inhibited by AE treatment in both dose- and time-dependent manners. The IC(50) level of AE was ∼75 μM. AE also has anti-proliferative effects via induction of DNA damage and apoptosis. 2-DE (two-dimensional electrophoresis) revealed that several proteins were related to the anti-cancer effects of AE. CAPN2 (calpain-2) and UBE3A (ubiquitin-protein ligase E3A), which are associated with the apoptosis signalling pathway, were verified by Western blotting. AE exhibited potent anti-proliferative effects on Huh-7 cells via down-regulation of CAPN2 and UBE3A. The findings support the possibility of AE being a chemopreventative agent.

Topics: Aloe; Antineoplastic Agents, Phytogenic; Apoptosis; Calpain; Carcinoma, Hepatocellular; Cell Line, Tumor; DNA Damage; Down-Regulation; Emodin; Humans; Liver Neoplasms; Ubiquitin-Protein Ligases

Ground glass hepatocytes harboring hepatitis B virus (HBV) pre-S2 mutants have been recognized as pre-neoplastic lesions of hepatocellular carcinoma (HCC). The pre-S2 mutants accumulated in endoplasmic reticulum (ER) can induce ER stress, upregulate cyclin A and promote hepatocyte proliferation. Notably, cyclin A was aberrantly detected in the cytoplasm, instead of nucleus, of pre-S2 mutant-transgenic mice livers, thereby raising the potential role of cytoplasmic cyclin A in HBV hepatocarcinogenesis. In this study, we confirmed that cyclin A was detected in the cytoplasm in the majority of HBV-related HCC tissues. In vitro, the pre-S2 mutant-initiated ER stress could induce cytoplasmic cyclin A mediated via cleavage by the calcium-dependent protease μ-calpain, resulting in an N-terminal truncated product which was preferentially located in the cytoplasm. The aberrant cyclin A expression subsequently induced centrosome overduplication, and this effect was abolished by calpain-specific inhibitors or RNA interference targeting to cyclin A. Overall, our data indicate that HBV pre-S2 mutant may elicit aberrant cyclin A expression and centrosome overduplication through ER stress induction and thereby represent a potential mechanism for the chromosome instability in HBV hepatocarcinogenesis.

Topics: Animals; Calpain; Carcinoma, Hepatocellular; Cell Growth Processes; Cell Transformation, Neoplastic; Centrosome; CHO Cells; Chromosomal Instability; Cricetinae; Cyclin A; Endoplasmic Reticulum; Hepatitis B Surface Antigens; Hepatitis B virus; Hepatocytes; Humans; Liver; Liver Neoplasms; Mice; Mice, Transgenic; Mutation; Protein Precursors; RNA Interference; Tumor Cells, Cultured

Hepatitis B virus X protein (HBx) plays an important role in the development of hepatocellular carcinoma (HCC). However, the mechanism remains unclear. Recently, we have reported that HBx promotes hepatoma cell migration through the upregulation of calpain small subunit 1 (Capn4). In addition, several reports have revealed that osteopontin (OPN) plays important roles in tumor cell migration. In this study, we investigated the signaling pathways involving the promotion of cell migration mediated by HBx. We report that HBx stimulates several factors in a network manner to promote hepatoma cell migration. We showed that HBx was able to upregulate the expression of osteopontin (OPN) through 5-lipoxygenase (5-LOX) in HepG2-X/H7402-X (stable HBx-transfected cells) cells. Furthermore, we identified that HBx could increase the expression of 5-LOX through nuclear factor-κB (NF-κB). We also found that OPN could upregulate Capn4 through NF-κB. Interestingly, we showed that Capn4 was able to upregulate OPN through NF-κB in a positive feedback manner, suggesting that the OPN and Capn4 proteins involving cell migration affect each other in a network through NF-κB. Importantly, NF-κB plays a crucial role in the regulation of 5-LOX, OPN and Capn4. Thus, we conclude that HBx drives multiple cross-talk cascade loops involving NF-κB, 5-LOX, OPN and Capn4 to promote cell migration. This finding provides new insight into the mechanism involving the promotion of cell migration by HBx.

Topics: Arachidonate 5-Lipoxygenase; Blotting, Western; Calpain; Carcinoma, Hepatocellular; Cell Movement; Enzyme-Linked Immunosorbent Assay; Feedback, Physiological; Humans; Liver Neoplasms; Luciferases; Osteopontin; Real-Time Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Trans-Activators; Tumor Cells, Cultured; Viral Regulatory and Accessory Proteins

Proteolysis affects every protein at some point in its life cycle. Many biomarkers of disease or cancer are stable proteolytic fragments in biological fluids. There is great interest and a challenge in proteolytically modified protein study to identify physiologic protease-substrate relationships and find potential biomarkers. In this study, two human hepatocellular carcinoma (HCC) cell lines with different metastasis potential, MHCC97L, and HCCLM6, were researched with a high-throughput and sensitive PROTOMAP platform. In total 391 proteins were found to be proteolytically processed and many of them were cleaved into persistent fragments instead of completely degraded. Fragments related to 161 proteins had different expressions in these two cell lines. Through analyzing these significantly changed fragments with bio-informatic tools, several bio-functions such as tumor cell migration and anti-apoptosis were enriched. A proteolysis network was also built up, of which the CAPN2 centered subnetwork, including SPTBN1, ATP5B, and VIM, was more active in highly metastatic HCC cell line. Interestingly, proteolytic modifications of CD44 and FN1 were found to affect their secretion. This work suggests that proteolysis plays an important role in human HCC metastasis.

Topics: Biomarkers, Tumor; Calpain; Carcinoma, Hepatocellular; Cell Line, Tumor; Humans; Liver Neoplasms; Mass Spectrometry; Peptide Fragments; Protein Interaction Maps; Proteolysis; Proteome; Proteomics; Reproducibility of Results

Hepatocellular carcinoma (HCC) is one of the most common cancers in the world which is highly chemoresistant to currently available chemotherapeutic agents. Thus, novel therapeutic targets are needed to be sought for the successful treatment of HCC. Peptaibols, a family of peptides synthesized non-ribosomally by the Trichoderma species and other fungi, exhibit antibiotic activities against bacteria and fungi. Few studies recently showed that peptaibols exerted cytotoxicity toward human lung epithelial and breast carcinoma cells. However, the mechanism involved in peptaibol-induced cell death remains poorly understood.. Here, we showed that Trichokonin VI (TK VI), a peptaibol from Trichoderma pseudokoningii SMF2, induced growth inhibition of HCC cells in a dose-dependent manner. It did not obviously impair the viability of normal liver cells at lower concentration. Moreover, the suppression of cell viability resulted from the programmed cell death (PCD) with characteristics of apoptosis and autophagy. An influx of Ca2+ triggered the activation of mu-calpain and proceeded to the translocation of Bax to mitochondria and subsequent promotion of apoptosis. On the other hand, typically morphological characteristics consistent with autophagy were also observed by punctate distribution of MDC staining and the induction of LC3-II, including extensive autophagic vacuolization and enclosure of cell organelles by these autophagosomes. More significantly, specific depletion of Bak expression by small RNA interfering (siRNA) could partly attenuate TK VI-induced autophagy. However, siRNA against Bax led to increased autophagy.. Taken together, these findings showed for the first time that peptaibols were novel regulators involved in both apoptosis and autophagy, suggesting that the class of peptaibols might serve as potential suppressors of tumor cells.

Topics: Alamethicin; Anti-Infective Agents; Apoptosis; Autophagy; bcl-2 Homologous Antagonist-Killer Protein; bcl-X Protein; Calcium; Calpain; Carcinoma, Hepatocellular; Cell Line, Tumor; Dose-Response Relationship, Drug; Down-Regulation; Drug Screening Assays, Antitumor; Humans; Liver Neoplasms; Models, Biological; Peptaibols; Protein Transport; Time Factors

Liver transplantation (LT) is one of the best therapeutic options for nonresectable hepatocellular carcinoma (HCC). Unfortunately, some HCC patients succumb to the disease after LT, which reduces long- and medium-term survival. To identify the proteins associated with HCC invasion and metastasis, HCC patients undergoing LT with complete follow-up data were included in this study and were categorized into recurrence and nonrecurrence groups. We extracted the total protein from the acquired homogeneous tumor cells and applied a cleavable isotope-coded affinity tag technology to quantitate relative changes in protein levels between the two groups. We identified a total of 149 proteins with two-dimensional liquid chromatography coupled with tandem mass spectrometry, including 52 differentially expressed proteins by at least two-fold. Among them, calpain small subunit 1 (Capn4), a protein with relevant interactions with many migration-invasion-related proteins, has attracted more attention. First, Capn4 overexpression in the recurrence group was confirmed via real-time polymerase chain reaction and western blotting in another cohort of 40 HCC patients undergoing LT. Second, Capn4 was associated with enhanced invasiveness in vitro. The small interfering RNA-mediated knockdown expression of Capn4 in HCC cell lines significantly inhibited its mobile and invasive ability. Tissue microarray in a further 192 cases revealed that Capn4 significantly correlated with invasive phenotype of HCC, and univariate and multivariate analyses indicated that Capn4 is an independent prognostic factor for recurrence and survival of HCC patients.. Our study revealed that Capn4 overexpression underlies invasion and metastasis after LT for HCC and might be a candidate biomarker for future diagnosis and a target for therapy.

Topics: Calpain; Carcinoma, Hepatocellular; China; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Liver Transplantation; Neoplasm Invasiveness; Neoplasm Metastasis; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction; Prognosis; Proteome; RNA, Neoplasm; RNA, Small Interfering

Topics: Biomarkers, Tumor; Calpain; Carcinoma, Hepatocellular; Humans; Liver Neoplasms; Liver Transplantation; Neoplasm Recurrence, Local; Predictive Value of Tests; Risk Factors; X-Linked Inhibitor of Apoptosis Protein

An unresolved question regarding the physiopathology of hepatitis C virus (HCV) infection is the remarkable efficiency with which host defenses are neutralized to establish chronic infection. Modulation of an apoptotic response is one strategy used by viruses to escape immune surveillance. We previously showed that HCV proteins down-regulate expression of BH3-only Bcl2 interacting domain (Bid) in hepatocytes of HCV transgenic mice. As a consequence, cells acquire resistance to Fas-mediated apoptosis, which in turn leads to increased persistence of experimental viral infections in vivo. This mechanism might participate in the establishment of chronic infections and the resulting pathologies, including hepatocellular carcinoma. We now report that Bid is also down-regulated in patients in the context of noncirrhotic HCV-linked tumorigenesis and in the HCV RNA replicon system. We show that the nonstructural HCV viral protein NS5A is sufficient to activate a calpain cysteine protease, leading to degradation of Bid. Moreover, pharmacological inhibitors of calpains restore both the physiological levels of Bid and the sensitivity of cells toward a death receptor-mediated apoptotic signal. Finally, human HCV-related tumors and hepatocytes from HCV transgenic mice that display low Bid expression contain activated calpains.. Calpains activated by HCV proteins degrade Bid and thus dampen apoptotic signaling. These results suggest that inhibiting calpains could lead to an improved efficiency of immune-mediated elimination of HCV-infected cells.

Topics: Adult; Aged; Animals; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Calpain; Carcinoma, Hepatocellular; Cells, Cultured; Disease Models, Animal; Female; Hepacivirus; Hepatitis C, Chronic; Humans; Liver; Liver Neoplasms; Male; Mice; Mice, Transgenic; Middle Aged; Replicon; Signal Transduction; Viral Nonstructural Proteins; Viral Proteins

Antrodia cinnamomea is well known in Taiwan as a traditional medicine for treating cancer and inflammation. The purpose of this study was to evaluate the apoptotic effects of ethylacetate extract from A. cinnamomea (EAC) fruiting bodies in Hep 3B, a liver cancer cell line. EAC decreased cell proliferation of Hep 3B cells by inducing apoptotic cell death. EAC treatment increased the level of calcium (Ca2+) in the cytoplasm and triggered the subsequent activation of calpain and caspase-12. EAC also initiated the mitochondrial apoptotic pathway through regulation of Bcl-2 family proteins expression, release of cytochrome c, and activation of caspase-9 in Hep 3B cells. Furthermore, the mitochondrial apoptotic pathway amplified the calpain pathway by Bid and Bax interaction and Ca2+ translocation. We have therefore concluded that the molecular mechanisms during EAC-mediated proliferation inhibition in Hep 3B cells were due to: (1) apoptosis induction, (2) triggering of Ca2+/calpain pathway, (3) disruption of mitochondrial function, and (4) apoptotic signaling being amplified by cross-talk between the calpain/Bid/Bax and Ca2+/mitochondrial apoptotic pathways.

Topics: Apoptosis; BH3 Interacting Domain Death Agonist Protein; Calcium; Calpain; Carcinoma, Hepatocellular; Caspase 12; Caspases; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Drugs, Chinese Herbal; Endoplasmic Reticulum; Enzyme Activation; Enzyme Inhibitors; Flow Cytometry; Fruit; Humans; Liver Neoplasms; Membrane Potentials; Mitochondrial Membranes; Polyporales; Proto-Oncogene Proteins c-bcl-2

Integrins mediate many fundamental cellular processes by binding to components of the extracellular matrix. We showed previously that integrin beta(1A) could inhibit cell proliferation. Integrin beta(1A) stimulated the promoter activity of p21(cip1) and enhanced its transcription in SMMC-7721 cells. In this study, we demonstrated that integrin beta(1A) upregulated p27(kip1) at the post-translational level in SMMC-7721 cells. Our results showed that integrin beta(1A) increased the p27 protein amount, both in cytoplasm and nucleus, but did not affect the p27 mRNA amount. Cycloheximide treatment experiment revealed that the half-life of p27 protein was prolonged in integrin beta1A overexpressing cells, indicating that integrin beta(1A) inhibited the degradation of p27 protein. Our data also provided evidence that both the proteasome and calpain were involved in the degradation of p27 protein in SMMC-7721 cells. Integrin beta(1A) decreased the Skp2 expression and repressed the activity of calpain during G1 phase in SMMC-7721 cells. Taken together, these results indicated that integrin beta(1A) might upregulate the protein amount of p27 through repressing Skp2-dependent proteasome degradation and calpain-mediated proteolysis in SMMC-7721 cells.

Topics: Calpain; Carcinoma, Hepatocellular; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p27; G1 Phase; Half-Life; Humans; Integrin beta1; Liver Neoplasms; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; RNA, Messenger; S-Phase Kinase-Associated Proteins; Up-Regulation

Endoplasmic reticulum (ER) was recently suggested as a third subcellular compartment in apoptotic execution. Survivin is a member of inhibitors of apoptosis and ursodeoxycholic acid (UDCA) prevents apoptosis from various apoptotic stimuli. To assess the activity of survivin and the effect of UDCA on the survivin in ER stress-mediated apoptosis, we treated hepatoma cell lines with thapsigargin (TG). TG-induced apoptosis was assessed by morphological changes, DNA fragmentation, cleavages of poly(ADP-ribose)polymerase (PARP), and activation of calpain and caspase-12. The level of survivin was decreased after TG treatment in hepatoma cell lines indicating that survivin play an important role in ER stress-mediated apoptosis. UDCA prevented decrease in survivin levels and inhibited TG-induced apoptosis and caspase-12 activation suggesting an anti-apoptotic effect of UDCA.

Topics: Apoptosis; Calpain; Carcinoma, Hepatocellular; Caspase 12; Caspases; DNA Fragmentation; Endoplasmic Reticulum; Enzyme Activation; Genes, bcl-2; Humans; Inhibitor of Apoptosis Proteins; Liver Neoplasms; Microtubule-Associated Proteins; Neoplasm Proteins; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Stress, Physiological; Survivin; Thapsigargin; Tumor Cells, Cultured; Ursodeoxycholic Acid

Calpain, a cytosolic Ca(2+)-dependent proteinase, plays a pivotal role in cell injury. In this study, we investigated the effect of calpain-mu antisense oligonucleotide on oxidative stress induced-hepatocyte injury.. Hemagglutinating virus of Japan liposome complex with one of three types of antisense oligonucleotide (AS-1, AS-2, AS-3) or scramble oligonucleotide was added to the culture medium of HuH7 cells and incubated for 6 days. The expression of calpain-mu protein was examined by Western blotting. After the addition of tert-butyl hydroperoxide, bleb formation was examined by phase contrast microscopy, and cell viability was assessed by the release of lactate dehydrogenase.. Incubation of HuH7 cells with AS-2 resulted in a decrease in the amount of calpain on day 4 and a further decrease to almost undetectable levels on day 6, whereas scramble oligonucleotide had no effect. Bleb formation was observed 120 min after the addition of tert-butyl hydroperoxide in scramble oligonucleotide-treated cells as in untreated cells. In contrast, it was rarely observed in AS-2-treated cells. Lactate dehydrogenase release was significantly suppressed in AS-2-treated cells, compared with that in scramble oligonucleotide treated-cells.. Our findings suggest that calpain activation is involved in the pathogenesis of oxidative stress injury and that transfection of calpain antisense may potentially protect against ischemia/reperfusion liver injury.

Topics: Calpain; Cell Survival; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Gene Expression; Hepatocytes; Humans; L-Lactate Dehydrogenase; Liver Neoplasms; Oligonucleotides, Antisense; Oxidative Stress; tert-Butylhydroperoxide; Transfection; Tumor Cells, Cultured

CYP2E1-dependent mitochondrial damage, in the presence or absence of extracellular calcium, was investigated. HepG2 cells expressing CYP2E1 (E47 cells) were preloaded with arachidonic acid (AA), washed, and incubated with iron-nitrilotriacetate 1:3 complex (Fe-NTA) in minimum essential medium (MEM) (1.8mM Ca(2+)) or Ca(2+)-free MEM (SMEM). Toxicity in SMEM was CYP2E1-dependent, necrotic, and lipid peroxidation-dependent. Intracellular calcium did not significantly change during the incubation in SMEM. Mitochondrial damage preceded the loss of plasma membrane integrity and was significant at 12h of incubation, in coincidence with the toxicity. E47 cells treated with AA+Fe in MEM also showed a decline of mitochondrial membrane potential (Delta(Psi)(m)) that preceded the loss of plasma membrane integrity, but starting at earlier times, e.g., 3h than in SMEM. The decline in Delta(Psi)(m) and the toxicity in both MEM and SMEM were inhibited by alpha-tocopherol and cyclosporin A, while the calpain inhibitor calpeptin was only effective in MEM. In conclusion, oxidative damage to mitochondria and the permeability transition plays a role in the CYP2E1-dependent toxicity of Fe+AA in HepG2 cells, both in MEM and SMEM. Ca(2+) mobilization and activation of calpain contributes to the more rapid onset of mitochondrial damage in MEM, while oxidative damage and lipid peroxidation are involved in the Ca(2+)-independent later onset of mitochondrial damage.

Topics: alpha-Tocopherol; Arachidonic Acid; Calcium; Calpain; Carcinoma, Hepatocellular; Culture Media; Cyclosporine; Cysteine Proteinase Inhibitors; Cytochrome P-450 CYP2E1; Cytosol; Dipeptides; Humans; Intracellular Membranes; Iron; Lipid Peroxidation; Liver Neoplasms; Mitochondria, Liver; Oxidative Stress; Permeability; Tumor Cells, Cultured

The ability of ricin, a type II ribosome-inactivating protein, to induce hepatoma cell (BEL7404) to apoptosis in vitro was examined by fluorescence microscopy, flow cytometry, and DNA fragmentation assay. As a Bcl-2 lacking model, BEL7404 bore unique advantage to study the effect of over-expressing Bcl-2 on the apoptosis induced by the inhibitor of protein synthesis. By establishing a Bcl-2 over-expressing cell line (BEL7404/ Bcl-2), we found that Bcl-2 could promote the survival of the hepatoma cell against ricin insult. The ricin-induced apoptosis of BEL7404 was accompanied by increased expression of Bak and decreased levels of Bcl-xl and Bax. Caspases and PARP cleavage activity were found to be implicated in the death process. Through the inhibitor tests, our results excluded the participation of calcium-dependent proteases or protein kinase C in the apoptotic process induced by ricin, though an elevation of intracellular calcium did occur as an immediate response to ricin treatment. Cycloheximide, another protein synthesis inhibitor, did synergistically enhance rather than inhibit the cytotoxicity of ricin to hepatoma cell BEL7404. Actually, cycloheximide alone was able to induce hepatoma cell BEL7404 to death that could also be inhibited by over-expressing Bcl-2. The elevation of apoptotic protein Bak was discussed to challenge the notion that ricin exerted its cytotoxicity through nonspecific inhibition of all the de novo protein synthesis.

Topics: Apoptosis; Calpain; Carcinoma, Hepatocellular; Caspases; Cells, Cultured; Cycloheximide; Drug Synergism; Humans; Liver Neoplasms; Protein Kinase C; Proteins; Proto-Oncogene Proteins c-bcl-2; Ricin

Ethanol is known to induce apoptosis in hepatocytes. However, intracellular signaling events of ethanol-induced death are still only partially understood. We studied such processes in ethanol-induced apoptosis in HepG2 cells as a model system for human liver cells. We determined the incidence of apoptosis by DNA fragmentation and tested the effects of various known inhibitors. Ethanol induces apoptosis in HepG2 cells in a dose- and time-dependent manner as well as in rat primary hepatocytes. This effect was not mediated through the death receptor CD95 and the tumor necrosis factor receptors. It was efficiently inhibited by the caspase inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (zVAD-fmk), the Ca(2+) chelator EGTA, and the serine protease inhibitor N-p-tosyl-l-lysine chloromethyl ketone (TLCK). Upon ethanol treatment, the intracellular calcium ion concentration was increased and cytochrome c was released from the mitochondria, and caspases were activated. EGTA and TLCK could inhibit cytochrome c release from the mitochondria. Furthermore, overexpression of Bcl-x(L) saved cells from ethanol-induced apoptosis. These data suggest that ethanol-induced apoptosis in liver cells is initiated by the intracellular Ca(2+) elevation in the cytoplasm and activation of TLCK-sensitive serine proteases. Our data provide new insight into ethanol-induced apoptosis in liver cells and may lead to therapeutic strategies to prevent liver damage.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; bcl-X Protein; Blotting, Western; Calcium; Calmodulin; Calpain; Carcinoma, Hepatocellular; Caspase 3; Caspase 9; Caspases; Cell Separation; Cytochrome c Group; Densitometry; Dose-Response Relationship, Drug; Egtazic Acid; Endopeptidases; Enzyme Activation; Ethanol; Fas Ligand Protein; fas Receptor; Flow Cytometry; Humans; Ions; Liver Neoplasms; Membrane Glycoproteins; Membrane Potentials; Mitochondria; Models, Biological; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Tosyllysine Chloromethyl Ketone; Transfection; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The activity of a novel Ca2+-dependent protease in preneoplastic and neoplastic tissues of male Fisher 344 rat liver was examined during experimental liver carcinogenesis. This protease activity is significantly high in hyperplastic nodules and hepatocellular carcinomas which were induced by the method of Solt and Farber. Taken together with our previous report on the promoter-specific induction of this protease, the results suggest the possible importance of this protease in the promotion stage of liver carcinogenesis.

Topics: Animals; Calpain; Liver Neoplasms; Liver Neoplasms, Experimental; Male; Precancerous Conditions; Rats; Rats, Inbred F344