Pathway In Human Hepatoma Cells Research Articles

Heat Shock Protein Family A Member 1 Promotes Intracellular Amplification of Hepatitis B Virus Covalently Closed Circular DNA.

Hepatitis B virus (HBV) contains a partially double-stranded relaxed circular DNA (rcDNA) genome that is converted into a covalently closed circular DNA (cccDNA) in the nucleus of the infected hepatocyte by cellular DNA repair machinery. cccDNA associates with nucleosomes to form a minichromosome that transcribes RNA to support the expression of viral proteins and reverse transcriptional replication of viral DNA. In addition to the de novo synthesis from incoming virion rcDNA, cccDNA can also be synthesized from rcDNA in the progeny nucleocapsids within the cytoplasm of infected hepatocytes via the intracellular amplification pathway. In our efforts to identify cellular DNA repair proteins required for cccDNA synthesis using a chemogenetic screen, we found that B02, a small-molecule inhibitor of DNA homologous recombination repair protein RAD51, significantly enhanced the synthesis of cccDNA via the intracellular amplification pathway in human hepatoma cells. Ironically, neither small interfering RNA (siRNA) knockdown of RAD51 expression nor treatment with another structurally distinct RAD51 inhibitor or activator altered cccDNA amplification. Instead, it was found that B02 treatment significantly elevated the levels of multiple heat shock protein mRNA, and siRNA knockdown of HSPA1 expression or treatment with HSPA1 inhibitors significantly attenuated B02 enhancement of cccDNA amplification. Moreover, B02-enhanced cccDNA amplification was efficiently inhibited by compounds that selectively inhibit DNA polymerase α or topoisomerase II, the enzymes required for cccDNA intracellular amplification. Our results thus indicate that B02 treatment induces a heat shock protein-mediated cellular response that positively regulates the conversion of rcDNA into cccDNA via the authentic intracellular amplification pathway. IMPORTANCE Elimination or functional inactivation of cccDNA minichromosomes in HBV-infected hepatocytes is essential for the cure of chronic hepatitis B virus (HBV) infection. However, lack of knowledge of the molecular mechanisms of cccDNA metabolism and regulation hampers the development of antiviral drugs to achieve this therapeutic goal. Our findings reported here imply that enhanced cccDNA amplification may occur under selected pathobiological conditions, such as cellular stress, to subvert the dilution or elimination of cccDNA and maintain the persistence of HBV infection. Therapeutic inhibition of HSPA1-enhanced cccDNA amplification under these pathobiological conditions should facilitate the elimination of cccDNA and cure of chronic hepatitis B.

Cryptolepine inhibits hepatocellular carcinoma growth through inhibiting interleukin-6/STAT3 signalling

BackgroundDiverse signalling pathways are involved in carcinogenesis and one of such pathways implicated in many cancers is the interleukin 6/signal transducer and activator of transcription 3 (IL-6/STAT3) signalling pathway. Therefore, inhibition of this pathway is targeted as an anti-cancer intervention. This study aimed to establish the effect of cryptolepine, which is the main bioactive alkaloid in the medicinal plant Cryptolepis sanguinolenta, on the IL-6/STAT3 signalling pathway.MethodsFirst, the effect of cryptolepine on the IL-6/STAT3 pathway in human hepatoma cells (HepG2 cells) was screened using the Cignal Finder Multi-Pathway Reporter Array. Next, to confirm the effect of cryptolepine on the IL-6/STAT3 signalling pathway, the pathway was activated using 200 ng/mL IL-6 in the presence of 0.5–2 μM cryptolepine. The levels of total STAT3, p-STAT3 and IL-23 were assessed by ELISA.ResultsCryptolepine downregulated 12 signalling pathways including the IL-6/STAT3 signalling pathway and upregulated 17 signalling pathways. Cryptolepine, in the presence of IL-6, decreased the levels of p-STAT3 and IL-23 in a dose-dependent fashion.ConclusionOur results demonstrated that cryptolepine inhibits the IL-6/STAT3 signalling pathway, and therefore cryptolepine-based remedies such as Cryptolepis sanguinolenta could potentially be used as an effective immunotherapeutic agent for hepatocellular carcinoma and other cancers.

Nitric Oxide Mediated Degradation of CYP2A6 via the Ubiquitin-Proteasome Pathway in Human Hepatoma Cells.

Several cytochrome P450 enzymes are known to be down-regulated by nitric oxide (NO). CYP2A6 is responsible for the metabolism of nicotine and several other xenobiotics, but its susceptibility to down-regulation by NO has not been reported. To address this question, we used Huh7 human hepatoma cell lines to express CYP2A6 with a C-terminal V5 tag (CYP2A6V5). NO donor treatment [dipropylenetriamine NONOate (DPTA)] down-regulated CYP2A6 protein to approximately 40% of control levels in 4 hours. An NO scavenging agent protected CYP2A6 from down-regulation by DPTA in a concentration-dependent manner, demonstrating that the down-regulation is NO-dependent. Experiments with the protein synthesis inhibitor cycloheximide showed that CYP2A6 protein down-regulation occurs posttranslationally. In the presence of proteasome inhibitors MG132 or bortezomib, NO-treated cells showed an accumulation of a high molecular mass signal, whereas autophagy inhibitors chloroquine and 3-methyladenine and the lysosomal and calpain inhibitor E64d had no effect. Immunoprecipitation of CYP2A6 followed by Western blotting with an antiubiquitin antibody showed that the high molecular mass species contain polyubiquitinated CYP2A6 protein. This suggests that NO led to the degradation of protein via the ubiquitin-proteasome pathway. The down-regulation by NO was blocked by the reversible CYP2A6 inhibitor pilocarpine but not by the suicide inhibitor methoxsalen, demonstrating that down-regulation requires NO access to the active site but does not require catalytic activity of the enzyme. These findings provide novel insights toward the regulation of CYP2A6 in a human cell line and can influence our understanding of CYP2A6-related drug metabolism. SIGNIFICANCE STATEMENT: This study demonstrates that the nicotine metabolizing enzyme CYP2A6 is down-regulated by nitric oxide, a molecule produced in large amounts in the context of inflammation and that is also inhaled from cigarette smoke. This occurs via ubiquitination and proteasomal degradation, and does not require catalytic activity of the enzyme. This work adds to the growing knowledge of the selective effect and mechanism of action of nitric oxide (NO) on cytochrome P450 enzymes and suggests a possible novel mode of interaction between nicotine and NO in cigarette smokers.

TPP-related mitochondrial targeting copper (II) complex induces p53-dependent apoptosis in hepatoma cells through ROS-mediated activation of Drp1

BackgroundIn recent years, copper complexes have gradually become the focus of potential anticancer drugs due to their available redox properties and low toxicity. In this study, a novel mitochondrion-targeting copper (II) complex, [Cu (ttpy-tpp)Br2] Br (simplified as CTB), is first synthesized by our group. CTB with tri-phenyl-phosphine (TPP), a targeting and lipophilic group, can cross the cytoplasmic and mitochondrial membranes of tumor cells. The present study aims to investigate how CTB affects mitochondrial functions and exerts its anti-tumor activity in hepatoma cells.MethodsMultiple molecular experiments including Flow cytometry, Western blot, Immunofluorescence, Tracker staining, Transmission Electron Microscopy and Molecular docking simulation were used to elucidate the underlying mechanisms. Human hepatoma cells were subcutaneously injected into right armpit of male nude mice for evaluating the effects of CTB in vivo.ResultsCTB induced apoptosis via collapse of mitochondrial membrane potential (MMP), ROS production, Bax mitochondrial aggregation as well as cytochrome c release, indicating that CTB-induced apoptosis was associated with mitochondrial pathway in human hepatoma cells. Mechanistic study revealed that ROS-related mitochondrial translocation of p53 was involved in CTB-mediated apoptosis. Simultaneously, elevated mitochondrial Drp1 levels were also observed, and interruption of Drp1 activation played critical role in p53-dependent apoptosis. CTB also strongly suppressed the growth of liver cancer xenografts in vivo.ConclusionIn human hepatoma cells, CTB primarily induces mitochondrial dysfunction and promotes accumulation of ROS, leading to activation of Drp1. These stimulation signals accelerate mitochondrial accumulation of p53 and lead to the eventual apoptosis. Our research shows that CTB merits further evaluation as a chemotherapeutic agent for the treatment of Hepatocellular carcinoma (HCC).

Celastrol inhibits hepatitis C virus replication by upregulating heme oxygenase-1 via the JNK MAPK/Nrf2 pathway in human hepatoma cells

background and purposeCelastrol, a quinone methide triterpene isolated from the root extracts of Tripterygium wilfordii, can greatly induce the gene expression activity of heme oxygenase-1 (HO-1) to achieve disease prevention and control. HO-1 induction was recently shown to result in anti-HCV activity by inducing type I interferon and inhibiting hepatitis C virus (HCV) NS3/4A protease activity. The aim of the present study is to evaluate the anti-HCV activity of celastrol and characterize its mechanism of inhibition. MethodsThe anti-HCV activity of celastrol was evaluated using the HCV subgenomic replicon and HCVcc infection systems. The anti-HCV mechanism of celastrol targeting HO-1 expression was clarified using specific inhibitors against several signaling pathways. The transcriptional regulation of celastrol on target gene expression was determined using promoter-based reporter activity assay. The synergistic effect of celastrol and a numbers of clinically used anti-HCV drugs was determined via a drug combination assay. ResultsCelastrol inhibited HCV replication in both the HCV subgenomic and HCVcc infection systems with EC50 values of 0.37 ± 0.022 and 0.43 ± 0.019 μM, respectively. Celastrol-induced heme oxygenase 1 (HO-1) expression promoted antiviral interferon responses and inhibition of NS3/4A protease activity, thereby blocking HCV replication. These antiviral effects were abrogated by treatment with the HO-1-specific inhibitor SnMP or silencing of HO-1 expression by transfection of shRNA, which indicates that HO-1 induction contributes to the anti-HCV activity of celastrol. JNK mitogen-activated protein kinase and nuclear factor erythroid 2-related factor 2 (Nrf2) were confirmed to be involved in the inductive effect of celastrol on HO-1 expression. Celastrol exhibited synergistic effects in combination with interferon-alpha, the NS5A inhibitor daclatasvir, and the NS5B inhibitor sofosbuvir. ConclusionCelastrol can serve as a potential supplement for blocking HCV replication. Targeting the JNK/Nrf2/HO-1 axis presents a promising strategy against HCV infection.

Synthesis and cytotoxic activities of novel 4-methoxy-substituted and 5-methyl-substituted (3'S,4'S)-(-)-cis-khellactone derivatives that induce apoptosis via the intrinsic pathway.

This study deals with the design and synthesis of a series of novel 4-methoxy-substituted and 5-methyl-substituted (3′S,4′S)-(−)-cis-khellactones. The newly synthesized compounds were characterized by 1H nuclear magnetic resonance (NMR), 13C-NMR, mass spectrometry, and elemental analysis. All the derivatives were subjected to in vitro cytotoxicity screening against HEPG-2 (human liver carcinoma), SGC-7901 (human gastric carcinoma), and LS174T (human colon carcinoma), by using the MTT assay. The results revealed that several of the 4-methoxy-substituted compounds exhibited potent cytotoxicity. Among these, compound 12e showed the highest activity against cancer cells which 50% inhibitory concentration (IC50) values were in the range of 6.1–9.2 μM with low toxicity on normal human hepatocyte. Preliminary investigation of possible mechanisms of action of compound 12e against HEPG-2 cells indicated possible induction of apoptosis, as determined by morphological observations and Annexin V/propidium iodide (PI) double staining, in addition to apparent dissipation of mitochondrial membrane potential (MMP), as measured by 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1) staining in combination with the activation of caspase-9 and caspase-3 by Western blot analysis. Overall, the data suggest that compound 12e may be a promising potential anti-cancer agent that could act primarily by inducing apoptosis through the mitochondria-mediated intrinsic pathway in human hepatoma cells.

The effects of urotensin II on migration and invasion are mediated by NADPH oxidase-derived reactive oxygen species through the c-Jun N-terminal kinase pathway in human hepatoma cells

AimsUrotensin II (UII) is a vasoactive neuropeptide involved in migration and invasion in various cell types. However, the effects of UII on human hepatoma cells still remain unclear. The aim of this study was to investigate the role and mechanism of UII on migration and invasion in human hepatoma cells. MethodsMigration was measured by wound healing assays and a Transwell® methodology, and invasion was analyzed using Matrigel® invasion chambers. Reactive oxygen species (ROS) levels were detected using a 2′, 7′-dichlorofluorescein diacetate probe, and flow cytometry, and protein expression levels were evaluated by western blotting. Cell proliferation and actin polymerization were examined using cell proliferation reagent WST-1 and F-actin immunohistochemistry staining. ResultsExposure to UII promoted migration and invasion in hepatoma cells compared with that in cells without UII. UII also increased matrix metalloproteinase-2 (MMP2) expression in a time-independent manner. Furthermore, UII markedly enhanced ROS generation and NADPH oxidase subunit expression, and consequently facilitated the phosphorylation of c-Jun N-terminal kinase (JNK). The UT antagonist urantide or the antioxidant/NADPH oxidase inhibitor apocynin decreased UII-induced ROS production. JNK phosphorylation, migration, invasion, and MMP9/2 expression were also reversed by pretreatment with apocynin. Urantide and JNK inhibitor SP600125 abrogated migration, invasion, or MMP9/2 expression in response to UII. UII induced actin polymerization and fascin protein expression, and could be reversed by apocynin and SP600125. ConclusionsExogenous UII induced migration and invasion in hepatoma cells that mainly involved NADPH oxidase-derived ROS through JNK activation. UT played an additional role in regulating hepatoma cells migration and invasion. Thus, our data suggested an important effect of UII in hepatocellular carcinoma metastasis.

Glabridin induces apoptosis and autophagy through JNK1/2 pathway in human hepatoma cells

BackgroundExtensive research results support the use of herbal medicine or natural food to augment therapy for various cancers. Studies have associated glabridin with numerous biological activities, such as regulating energy metabolism and estrogenic, neuroprotective, antiosteoporotic, and skin-whitening activities. Hypothesis/PurposeHowever, how glabridin affects tumor cell autophagy has not been clearly determined. MethodsAutophagy is a lysosomal degradation pathway essential for cell survival and tissue homeostasis. In this study, the roles of autophagy and related signaling pathways during glabridin-induced autophagy in human liver cancer cells were investigated. Additionally, the molecular mechanism of the anticancer effects of glabridin in human hepatoma cells was investigated. ResultsThe results revealed that glabridin significantly inhibited cell proliferation in human hepatoma cells. Glabridin induced apoptosis dose-dependently in Huh7 cells through caspase-3, -8, and -9 activation and PARP cleavage. Furthermore, autophagy was detected as early as 12h after exposure to a low dose of glabridin, as indicated by the up-regulated expression of LC3-II and beclin-1 proteins. The inhibition of JNK1/2 and p38 MAPK by specific inhibitors significantly reduced glabridin-induced activation of caspases-3, -8, and -9. Blocking autophagy sensitize the Huh7 cells to apoptosis. ConclusionThis study demonstrated for the first time that autophagy occurs earlier than apoptosis does during glabridin-induced apoptosis in human liver cancer cell lines. Glabridin induces Huh7 cell death through apoptosis through the p38 MAPK and JNK1/2 pathways and is a potential chemopreventive agent against human hepatoma.

The p53-inducible gene 3 involved in flavonoid-induced cytotoxicity through the reactive oxygen species-mediated mitochondrial apoptotic pathway in human hepatoma cells.

Flavonoids have been reported to exhibit prooxidant cytotoxicity against cancer cells, but the underlying mechanism is still poorly understood. Here we investigated the potential mechanism that p53-inducible gene 3 (PIG3), a NADPH:quinone oxidoreductase, mediated the prooxidant cytotoxicity of flavonoids on human hepatoma HepG2 cells. The results showed that flavonoids (apigenin, luteolin, kaempferol, and quercetin) inhibited the growth of HepG2 cells in a dosage- and time-dependent manner, and induced the morphological changes characteristic of apoptosis in HepG2 cells. We also found that expression of PIG3 was increased markedly in HepG2 cells treated with flavonoids at both mRNA and protein levels, which was accompanied by increased intracellular ROS production and a decreased mitochondrial membrane potential (ΔΨm). All these effects were largely reversed through knockdown of the PIG3 gene in HepG2 cells. Western blotting indicated that flavonoids increased cytochrome c release, upregulated the ratio of Bax/Bcl-2, and activated the caspases-9 and -3. Moreover, knockdown of PIG3 could reverse the changes of these apoptotic-related proteins. These results suggest that PIG3 plays an important role in regulating the prooxidant activity and apoptosis-inducing action of flavonoids on HepG2 cells though the ROS-triggered mitochondrial apoptotic pathway.

Carnosic acid induces autophagic cell death through inhibition of the Akt/mTOR pathway in human hepatoma cells.

The therapeutic goal of cancer treatment is now geared towards triggering tumour-selective cell death with autophagic cell death being required for the chemotherapy of apoptosis-resistant cancer. In this study, Carnosic acid (CA), a polyphenolic diterpene isolated from Rosemary (Rosemarinus officinalis), significantly induced autophagic cell death in HepG2 cells. Ca treatment caused the formation of autophagic vacuoles produced an increasing ratio of LC3-II to LC3-I in a time- and dose-dependent manner but had no effect on the levels of autophagy-related protein ATG6 and ATG13 expression. Autophagy inhibitors, 3-methyladenine (3-MA), chloroquine and bafilomycin A1, or ATG genes silencing in HepG2 cells significantly inhibited CA-induced autophagic cell death. The CA treatment decreased the levels of phosphorylated Akt and mTOR without any effects on PI3K or PTEN. Most importantly, overexpression of Akt and knockdown of PTEN attenuated autophagy induction in CA-treated cells. Taken together, our results indicated that CA induced autophagic cell death through inhibition of the Akt/mTOR pathway in human hepatoma cells. These findings suggest that CA has a great potential for the treatment of hepatoma via autophagic induction.

Andrographolide exerts anti‐hepatitis C virus activity by up‐regulating haeme oxygenase‐1 via the p38 MAPK/Nrf2 pathway in human hepatoma cells

This study aimed to evaluate the anti-hepatitis C virus (HCV) activity of andrographolide, a diterpenoid lactone extracted from Andrographis paniculata, and to identify the signalling pathway involved in its antiviral action. Using HCV replicon and HCVcc infectious systems, we identified anti-HCV activity of andrographolide by measuring protein and RNA levels. A reporter activity assay was used to determine transcriptional regulation of anti-HCV agents. A specific inhibitor and short hairpin RNAs were used to investigate the mechanism responsible for the effect of andrographolide on HCV replication. In HCV replicon and HCVcc infectious systems, andrographolide time- and dose-dependently suppressed HCV replication. When combined with IFN-α, an inhibitor targeting HCV NS3/4A protease (telaprevir), or NS5B polymerase (PSI-7977), andrographolide exhibited a significant synergistic effect. Andrographolide up-regulated the expression of haeme oxygenase-1 (HO-1), leading to increased amounts of its metabolite biliverdin, which was found to suppress HCV replication by promoting the antiviral IFN responses and inhibiting NS3/4A protease activity. Significantly, these antiviral effects were attenuated by an HO-1-specific inhibitor or HO-1 gene knockdown, indicating that HO-1 contributed to the anti-HCV activity of andrographolide. Andrographolide activated p38 MAPK phosphorylation, which stimulated nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated HO-1 expression, and this was found to be associated with its anti-HCV activity. Our results demonstrate that andrographolide has the potential to control HCV replication and suggest that targeting the Nrf2-HO-1 signalling pathway might be a promising strategy for drug development.

Bufalin induces G2/M phase arrest and triggers autophagy via the TNF, JNK, BECN-1 and ATG8 pathway in human hepatoma cells

Liver cancer is the fifth most common cause of cancer death worldwide. The study of more effective anti-hepatoma drugs is urgently required. Bufalin has been isolated from a traditional Chinese medicine and possesses less toxicity to normal cells. However, it has been found to inhibit growth of cancer cells. In this study, we aimed to investigate the efficacy and mechanism of bufalin in Huh7, Hep3B and HA22T human hepatoma cells. The three cell lines were treated with bufalin, the proliferation was detected by WST-1 assay and cell cycle was detected by flow cytometry analysis. The results showed that bufalin inhibited the proliferation of hepatoma cells and regulated the hepatoma cell death program in a dose- and time-dependent manner without typical features of apoptosis. RT-PCR arrays were used to investigate the autophagy transcriptional response triggered by bufalin and 13 genes were altered and further confirmed by real-time PCR. The translation levels of selected genes were examined by western blot analysis to reveal the bufalin-induced autophagy cascade. Bufalin synergized with the JNK pathway to induce autophagy of hepatoma cells and is closely associated with the upregulation of TNF, BECN-1, MAPK and ATG8, together with the downregulation of Bcl-2 and Bid. Our study provided a multi-angle evaluation system for anti-hepatoma pharmacology for pre-clinical drug investigation. In this case, bufalin was capable of inducing hepatoma cell autophagy, suggesting a potential regimen for single or combined chemotherapy to overcome hepatoma in clinical practice.

Sinomenine hydrochloride enhancement of the inhibitory effects of anti-transferrin receptor antibody-dependent on the COX-2 pathway in human hepatoma cells.

Transferrin receptor (TfR) has been used as a target for the antibody-based therapy of cancer due to its higher expression in tumors relative to normal tissues. Great potential has been shown by anti-TfR antibodies combined with chemotherapeutic drugs as a possible cancer therapeutic strategy. In our study, we investigated the anti-tumor effects of anti-TfR monoclonal antibody (mAb) alone or in combination with sinomenine hydrochloride in vitro. Results suggested that anti-TfR mAb or sinomenine hydrochloride could induce apoptosis, inhibit proliferation, and affect the cell cycle. A synergistic effect was found in relation to tumor growth inhibition and the induction of apoptosis when anti-TfR mAb and sinomenine hydrochloride were used simultaneously. The expression of COX-2 and VEGF protein in HepG2 cells treated with anti-TfR mAb alone was increased in line with increasing dosage of the agent. In contrast, COX-2 expression was dramatically decreased in HepG2 cells treated with sinomenine hydrochloride alone. Furthermore, we demonstrated that the inhibitory effects of sinomenine hydrochloride and anti-TfR mAb administered in combination were more prominent than when the agents were administered singly. To sum up, these results showed that the combined use of sinomenine hydrochloride and anti-TfR mAb may exert synergistic inhibitory effects on human hepatoma HepG2 cells in a COX-2-dependent manner. This finding provides new insight into how tumor cells overcome the interference of iron intake to survive and forms the basis of a new therapeutic strategy involving the development of anti-TfR mAb combined with sinomenine hydrochloride for liver cancer.

Anti-proliferative effects of cinnamaldehyde on human hepatoma cell lines

Abstract Cinnamaldehyde has been shown to exert diverse health benefits on humans. We investigated the molecular effects of cinnamaldehyde on the growth regulation mechanism and the Janus kinase–signal transducers and activators of the transcription (JAK–STAT) signalling pathway in human hepatoma cells. We found that cinnamaldehyde caused inhibition of cellular mitogenesis, partly by promoting apoptosis in two human hepatoma cancer cell lines – HepG2 and Hep3B. Amplified changes in caspase 3 activity, Bcl-2 protein expression, and mitochondrial cytochrome c release were displayed in cinnamaldehyde treatments in these cells. The ability of cinnamaldehyde to induce growth arrest was also verified by the observation that it significantly decreased the protein levels of cyclin D1 and proliferative cell nuclear antigen (PCNA) but increased the protein levels of p27Kip1 and p21Waf1/Cip1. Especially, the JAK2–STAT3/STAT5 signalling pathway was markedly blocked by cinnamaldehyde in human hepatoma cells. Suppressor of cytokine signalling 1 (SOCS1) and SOCS3 expressions were slightly activated by cinnamaldehyde. The specific JAK2 inhibitor, AG490, significantly prevented the phosphorylation of STAT3 and STAT5 proteins and enhanced the effect of cinnamaldehyde-inhibited cellular mitogenesis. Hence, these results suggest that cinnamaldehyde has a potent inhibitory effect against human hepatoma cell growth, and the JAK2–STAT3/STAT5 and that the apoptotic pathways may be important targets of cinnamaldehyde.

VI-16, a newly synthesized flavonoid, induces apoptosis through the mitochondrial pathway in human hepatoma cells

VI-16, a newly synthesized flavonoid, has a hydroxy substitution at C5 position, a methoxyl substitution at C5 position, and a piperazine substitution at C7 position. Here, we firstly investigated the potential antitumor effect of VI-16 in HepG2 human hepatocarcinoma cells. The MTT assay showed that VI-16 inhibited HepG2 cell growth in a concentration- and time-dependent manner. To further investigate whether apoptosis induction contributed to the antitumor effects of VI-16, DAPI staining and Annexin-V/PI double staining were performed in our tests. The data showed that VI-16 could induce apoptotic cell death in HepG2 cells. Moreover, mechanistic studies revealed that VI-16-induced apoptosis was a caspase-dependent process by decreasing the expression of pro-caspase-3. The changes in the expression of caspase-8, capsase-9, Bax and bcl-2 after VI-16 treatment suggested that the mitochondrial pathway was involved in the apoptosis induced by VI-16. Furthermore, VI-16 could significantly increase the loss of mitochondrial membrane potential and the expression of p53. Taken together, these results demonstrated that apoptosis induced by VI-16 might be one of the mechanisms by which VI-16 acts as a preventive antitumor drug against human hepatoma.

Q39, a quinoxaline 1,4-Di-N-oxide derivative, inhibits hypoxia-inducible factor-1α expression and the Akt/mTOR/4E-BP1 signaling pathway in human hepatoma cells

Cumulative evidence has established that hypoxia-inducible factor-1α (HIF-1α) and its downstream target, vascular endothelial growth factor (VEGF), play a critical role in hepatocellular carcinoma angiogenesis, invasiveness and metastasis. 3-(4-bromophenyl)-2-(ethylsulfonyl)-6-methylquinoxaline 1,4-dioxide (Q39) has recently shown great antiproliferative activity in extensive cell lines in normoxia and hypoxia. In this study, Q39 exhibited high antiproliferative activity against hepatoma both in vitro and in vivo, mainly by inducing apoptosis. In addition, suppression of HIF-1α by Q39 resulted in a drastic decrease in VEGF expression. These results indicate that Q39 is an effective inhibitor of HIF-1α and provide new perspectives into the mechanism of its anticancer activity. Interestingly, neither the HIF-1α degradation rate nor the HIF-1α steady-state mRNA level was affected by Q39. Instead, suppression of HIF-1α accumulation by Q39 correlated with prominent dephosphorylation of mTOR and 4E-BP1, a pathway known to regulate protein expression at the translational level.

Alpha fetoprotein is a novel protein‐binding partner for caspase‐3 and blocks the apoptotic signaling pathway in human hepatoma cells

Although there is increasing evidence that alpha fetoprotein (AFP) may function as regulatory factor in the growth of tumor cells, the precise mechanism is still unclear. In the current study, we investigated the role of the cytoplasmic AFP in caspase-3-mediated signaling of apoptosis. Our results showed that low doses of TNF-related apoptosis-inducing ligand (TRAIL) elevated the activity of caspase-8, but not caspase-3. Caspase-3 colocalized and interacted with AFP in the cytoplasm of Bel 7402 cells, and translocated into nuclei in association with the occurrence of apoptosis while cells were under cotreatment with all-trans retinoic acid (ATRA) or TRAIL. AFP was able to form complexes with caspase-3 and block onward transmission of signaling from caspase-8. Knockdown of AFP increased the sensitivity of Bel 7402 cells to TRAIL, and thereby, triggered caspase-3 signaling. No intermolecule interaction occurred between AFP and caspase-8, nor was caspase-8 activity altered after AFP knockdown, demonstrating the selectivity of AFP in interfering with the apoptotic signaling pathway. The effect of AFP on caspase-3 was further confirmed by transfection of the AFP gene into HLE cells (AFP negative). We conclude that ATRA or TRAIL resistance in AFP producing hepatoma is at least, in part, attributable to the high level of the cytoplasmic AFP. Therefore, it is possible that the combination of AFP gene silencing together with ATRA/TRAIL cotreatment will benefit the enhancement of the chemotherapeutic efficiency of these agents on tumors.

Berberine induces apoptosis through a mitochondria/caspases pathway in human hepatoma cells

Berberine, a main component of Coptidis Rhizoma, is a plant alkaloid with a long history of medicinal use in Chinese medicine. Berberine has indicated significant antimicrobial activity against a variety of organisms including bacteria, viruses, fungi. The mechanism by which berberine initiates apoptosis remains poorly understood. In the present study, we demonstrated that berberine exhibited significant cytotoxicity in hepatoma HepG2 cells but is ineffective in Chang liver cells. Herein we investigated cytotoxicity mechanism of berberine in HepG2 cells. The results showed that HepG2 cells underwent internucleosomal DNA fragmentation after 24-h treatment with berberine (50 microM). Moreover, berberine induced the activation of caspase-8 and -3, and caused the cleavage of poly ADP-ribose polymerase (PARP) and the cytochrome c release, whereas the expression of Bid and anti-apoptosis factor Bcl-XL were decreased markedly. The loss of mitochondrial membrane potential (Delta psim) at 24 h and activation of Fas at 12 h were also seen in the berberine-treated HepG2 cells. These findings supported the fact that the inhibitors of caspases, DEVD-FMK, IETD-FMK and VAD-FMK, prevented apoptosis and restored the expression of Bcl-XL, Bcl-2 and Bid. These results indicated that the potential of anti-hepatoma activity of berberine may be mediated through a caspases-mitochondria-dependent pathway.

Emodin-induced apoptosis through p53-dependent pathway in human hepatoma cells

Most of the commonly used cytotoxic anticancer drugs have been shown to induce apoptosis in susceptible cells. However, the signaling pathway of their apoptotic effects remains undefined. In this study, the cytotoxic effect of emodin on various human hepatoma cell lines was investigated. Results demonstrated that emodin exhibited strongly suppressing effect on HepG2/C3A, PLC/PRF/5, and SK-HEP-1 cells, with the IC 50 value of 42.5, 46.6, and 53.1 μM, respectively. Furthermore, emodin induced apoptosis in HepG2/C3A cells was clearly verified by the appearance of DNA fragmentation and sub-G 1 accumulation. Besides, HepG2/C3A cells were found to be arrested in G 2/M phase after the cells were treated with 60 μM emodin for 48 h. Moreover, significant increase in the levels of apoptosis-related signals such as p53 (419.3 pg/ml), p21 (437.4 units/ml), Fas (6.6 units/ml), and caspase-3 (35.4 pmol/min) were observed in emodin treated HepG2/C3A cells. Taken together, emodin displays effective inhibitory effects on the growth of various human hepatoma cell lines and stimulates the expression of p53 and p21 that resulted in the cell cycle arrest of HepG2/C3A cells at G 2/M phase. Results also suggest that emodin-induced apoptosis in HepG2/C3A cells were mediated through the activation of p53, p21, Fas/APO-1, and caspase-3. It implies that emodin could be a useful chemotherapeutical agent for treatment of hepatocellular carcinoma (HCC).

Hepatitis B virus-X protein upregulates the expression of p21wafl/cipl and prolongs G1→S transition via a p53-independent pathway in human hepatoma cells

Progression through the cell cycle is controlled by the induction of cyclins and activation of cognate cyclin-dependent kinases. The human hepatitis B virus-X (HBV-X) protein functions in gene expression alterations, in the sensitization of cells to apoptotic killing and deregulates cell growth arrest in certain cancer cell types. We have pursued the mechanism of growth arrest in Hep3B cells, a p53-mutant human hepatocellular carcinoma (HCC) cell line. In stable or transient HBV-X transformed Hep3B cells, HBV-X increased protein and mRNA levels of the cyclin-dependent kinase inhibitor (CDKI) p21(waf1/cip1) increased binding of p21(waf1/cip1) with cyclin-dependent kinase 2 (CDK2), markedly inhibited cyclin E and CDK2 associated phosphorylation of histone H1 and induced the activation of a p21 promoter reporter construct. By using p21 promoter deletion constructs, the HBV-X responsive element was mapped to a region between -1185 and -1482, relative to the transcription start site. Promoter mutation analysis indicated that the HBV-X responsive site coincides with the ets factor binding sites. These data indicate that in human hepatocellular carcinoma cells, HBV-X can circumvent the loss of p53 functions and induces critical downstream regulatory events leading to transcriptional activation of p21(waf1/cip1). As a consequence, there is an increased chance of acquisition of mutations which can enhance the genesis of hepatomas. Our results also emphasize the chemotherapeutic potential of p21(waf1/cip1) inhibitors, particularly in the HBV-X infected hepatoma which lacks functional p53.