Apoptosis In Hep3B Cells Research Articles

Peroxiredoxin 3 is resistant to oxidation-induced apoptosis of Hep-3b cells

Although peroxiredoxin 3 (PRX3) was reported to be overexpressed in liver cancer, the precise function of PRX3 in the development and/or progression of liver cancer remained to be obscure. The present study was conducted to investigate the response of PRX3 to oxidative stress in hepatocellular carcinoma (HCC) cells. After successful knockdown of PRX3 expression by small interfering RNA, we treated HCC cell lines Hep-3b and Hep-G2 with gradient concentrations of H2O2 and detected cell proliferation, apoptosis, and the level of reactive oxygen species (ROS) in the cells. After low-dose (5-20 μmol/l) H2O2 treatment, the ROS level was significantly higher in PRX3-knockdown Hep-3b cells than in controls. In addition, PRX3 down-regulation resulted in decreased proliferation, increased apoptosis, and increased caspase 3 activity of Hep-3b cells. We did not notice significant difference between PrxIII knockdown and control Hep-G2 cells in ROS level, cell viability or apoptosis. Our results suggest that PRX3 is an indispensable ROS scavenger that protects tumor cells against oxidative damage and subsequent apoptosis, which provides a clue that PRX3 may be involved in the chemotherapeutic resistance of liver cancer. The underlying mechanism for PRX3 function needs further investigation.

Tumor necrosis factor-α attenuates starvation-induced apoptosis through upregulation of ferritin heavy chain in hepatocellular carcinoma cells

BackgroundTumor microenviroment is characteristic of inflammation, ischemia and starvation of nutrient. TNF-α, which is an extraordinarily pleiotropic cytokine, could be an endogenous tumor promoter in some tumor types. The basic objective of this study was to investigate the effects of TNF-α on the cell viability and apoptosis of hepatocellular carcinoma cells under serum starvation, and to identify the molecular mechanisms involved.MethodsFor this purpose, five different concentrations of TNF-α and two different serum settings (serum-cultured and serum-deprived) were used to investigate the effects of TNF-α on the cell viability and apoptosis of Hep3B and SMMC-7721 cells.ResultsTNF-α (10 ng/ml) attenuated serum starvation-induced apoptosis of hepatocellular carcinoma cells, and autophagy conferred this process. BAY11-7082, a specific inhibitor of NF-κB, reversed the suppression of serum starvation-induced apoptosis by TNF-α. Moreover, TNF-α-induced NF-κB transactivation was suppressed by autophagy inhibitor 3-MA. In addition, TNF-α up-regulated Ferritin heavy chain (FHC) transiently by NF-κB activation and FHC levels were correlated with the TNF-α-induced protection against serum starvation-mediated apoptosis of hepatocellular carcinoma cells. Furthermore, FHC-mediated inhibition of apoptosis depended on suppressing ROS accumulation.ConclusionsOur findings suggested that autophagy conferred the TNF-α protection against serum starvation-mediated apoptosis of hepatocellular carcinoma cells, the mechanism involved with the activation of the TNF-α/ NF-κB /FHC signaling pathway.

Estrogen receptor α (ESR1) over-expression mediated apoptosis in Hep3B cells by binding with SP1 proteins

Previous studies have reported that estrogen receptors (ERs) are expressed in normal human liver, chronic hepatitis, and benign hepatic tumor tissues. However, decreased expression of ERs can be observed in hepatocellular carcinoma (HCC) and the role of ERs in HCC is not fully understood. Thus, the present study aimed to investigate the molecular mechanism induced by the overexpression of ERα (ERα (ESR1)) in Hep3B cells. We first detected the induction of apoptosis in ER-negative Hep3B cells using DNA fragmentation assay and flow cytometry. We found that ERα and ERα plus 17β-estradiol treatment increased apoptosis in Hep3B cells. Additionally, western blotting showed increased expression of active caspase 3 and tumor necrosis factor α (TNFα (TNF)) in ERα-transfected cells. To further understand the importance of SP1-binding sites in the TNFα promoter, ERα-negative Hep3B cells were co-transfected with ERα and a wild-type TNFα plasmid or TNFα with deleted SP1 regions. Deletion of both distant and primal SP1 sites abolished the activity of ERα, and similar results were observed by blocking the expression of SP1 protein using mithramycin (MA). This result indicates that SP1 protein is essential for ERα-activated TNFα promoter activity. Co-immunoprecipitation assay further confirmed the binding interaction between ERα and SP1 in a ligand-dependent manner. In general, we demonstrate that the overexpression of ERα mediates apoptosis in ERα-negative Hep3B cells by the binding of ERα to SP1 protein. Additionally, this ERα-SP1 complex binds to the proximal and distal sites of the TNFα gene promoter and further induces the expression of active caspase 3 in a ligand-dependent manner.

OP-11 Serum C-reactive protein (CRP) as biomarker for overall survival in patients with sorafenib therapy with hepatocellular carcinoma

treat several diseases. In particular, a major biological activity of G. frondosa is its antitumor activity, which has been a target for scientific and clinical research. G. frondosa is a mushroom indigenous to many Asian countries; it is an edible fungus with a large fruiting body characterized by overlapping caps. Liver cancer is a leading cause of cancer deaths, for which there is currently no successful chemotherapy. The aim of this study is to investigate the chemopreventive effects of G. frondosa in a liver cancer cell-line, Hep3B. Recent studies have revealed that G. frondosa inhibits tumor growth by inducing apoptosis. Methods: To investigate the inhibition of cell growth and ability by GFW treatment, we used a MTT assay, colony formation assay, soft agar assay and wound healing assay. Further, we also made use of phase contrast microscopy, flow cytometry and Western blot to detect autophagy and apoptosis. We next examined the ability of GFW to modulate tumor growth in xenograft mouse model. Results: This study examined the effect of G. frondosa on viability and apoptosis of human liver cancer cells Hep3B. We found that treatment with water extract of G. frondosa (GFW) (15.6 or 7.8 μg/mL) significantly reduced Hep3B cancer cells growth by 95% and 90% respectively, and induced cytoplasmic vacuolations. The autophagy-associated proteins LC3A, LC3B, ATG3, ATG5, ATG7, and Beclin-1 were also increased in Hep3B cancer cells treated with GFW. GFW inhibited the phosphorylation of PI3K (Tyr458/Tyr199) an important maker in autophagy signaling pathway, in addition, GFW increased the phosphorylation of JNK (Thr183/Tyr185), and down-regulation of Bcl-2. Moreover, prolonged exposure of Hep3B cells to GFW eventually caused apoptosis which was associated with suppression of phosphorylated Akt (Ser473) and ERK (Thr202/Tyr204), and activation of caspase-3 and -9, and concomitant degradation of PARP. We also demonstrated a GFW-induced therapeutic effect on Hep3B tumor development in nude mice. The feeding of GFW (50 mg kg–1 day–1) significantly inhibited tumor growth by 77.4%, as compared with the control group, in nude mice inoculated with Hep3B cancer cells. Conclusion: In conclusion, our results demonstrated that the PI3K signaling pathway promotes autophagy in GFW treated Hep3B cells. On the other hand, we also found that inhibition of ERK and of the PKC-delta signaling pathway promotes apoptosis in GFW-treated Hep3B cells. These findings implied that GFW may be used as a novel therapeutic reagent for the treatment of hepatocellular carcinoma.

PTHrP is a novel mediator for TGF-β-induced apoptosis

Parathyroid hormone-related protein (PTHrP) is a polyhormone secretory protein that plays fundamental roles in the development and function of various tissues. Transforming growth factor (TGF)-β is an important tumor suppressor that induces cell cycle arrest and apoptosis. Increased PTHrP expression has been implicated in TGF-β-induced growth inhibition in human hepatocellular carcinoma cells. However, whether PTHrP is involved in TGF-β-induced apoptosis remains unknown. Using Hep3B and HuH-7, two human hepatocellular carcinoma cell lines, the current study examined the hypothesis that TGF-β-induced apoptosis is mediated by the induction of PTHrP expression. We found that (1) TGF-β induces PTHrP mRNA expression, protein expression and secretion in a time-dependent fashion; (2) knockdown of PTHrP gene expression or neutralization of secreted PTHrP isoforms blocks TGF-β-induced apoptosis; and (3) TGF-β-induced PTHrP expression is Smad3-dependent. Thus, we have identified PTHrP as a novel mediator for TGF-β-induced apoptosis in Hep3B cells. Our findings provide further insights into the mechanisms through which TGF-β conveys tumor suppression activity.

Anticancer effects of O-desmethylangolensin are mediated through cell cycle arrest at the G2/M phase and mitochondrial-dependent apoptosis in Hep3B human hepatocellular carcinoma cells

In the present study, in order to investigate the anticancer effects of O-desmethylangolensin (O-DMA) on human hepatocellular carcinoma Hep3B cells, we first examined the antiproliferative effect of O-DMA. When Hep3B cells were treated with O-DMA at various concentrations (5-200µM) for 24, 48 or 72h, cell proliferation decreased significantly in a dose- and time-dependent manner. Moreover, O-DMA exposure at the IC50 concentration for 72h arrested cells at the G2/M phase, which was accompanied by a reduction in CDK1, and an increase in cyclinA andB. Under the same conditions, O-DMA significantly increased the number of sub-G1 phase cells. Additionally, an AnnexinV assay revealed that exposure to O-DMA affected the rate of cell apoptosis. O-DMA caused the downregulation of Bcl-2 and upregulation of Bax, which led to cytochromec release from the mitochondria and activation of caspase-3. Taken together, these data suggest that O-DMA exhibits anticancer activity by arresting the cell cycle at G2/M phase and causing mitochondrial-dependent apoptosis in Hep3B cells.

Reactive Oxygen Species Play a Central Role in Hexavalent Chromium-Induced Apoptosis in Hep3B Cells without the Functional Roles of p53 and Caspase-3

Background/Aims: Hexavalent chromium [Cr(VI)] and its compounds, which have the extensive application in diverse industries including metallurgy, textile and electroplating, are known to be genotoxic and mutagenic to humans. Although it is supported by a large body of literatures that p53 and caspase-3 played key roles in Cr(VI)-induced cytotoxicity, it is clear that Cr(VI) could induce apoptosis either without activating caspase, or in a p53-independent manner. Methods: In the present study, by using Z-VAD-fmk to inhibit caspase-3 in p53-deficient Hep3B cells, we explored the effect of Cr(VI) on apoptosis induction and the related mechanisms when the functions of p53 and caspase were simultaneously blocked. Results: We found that Cr(VI) still induced DNA damage, mitochondrial injury, oxidative stress and apoptosis in Hep3B cells without the functional roles of p53 and caspase-3, and the mechanism study revealed that this was in a ROS-dependent manner since NAC co-treatment showed the protective effect against Cr(VI)-induced apoptosis. Conclusion: Our research has disclosed the mechanism involved in Cr(VI)-induced cytotoxicity following the loss of p53 and caspase-3 functions and shed light on the importance of using antioxidants for primary and secondary prevention in Cr(VI) occupational exposure populations.

Smh-3 induces G2/M arrest and apoptosis through calcium-mediated endoplasmic reticulum stress and mitochondrial signaling in human hepatocellular carcinoma Hep3B cells

In the present study, we investigated the antitumor effects of Smh-3 on the viability, cell cycle and apoptotic cell death in human hepatocellular carcinoma Hep3B cells in vitro. We also investigated the molecular mechanisms involved in the effects of Smh-3 on human hepatoma Hep3B cells, including the effects on protein and mRNA levels which were determined by western blotting and DNA microarray methods, respectively. The results demonstrated that Smh-3 induced growth inhibition, cell morphological changes and induction of G(2)/M arrest and apoptosis in Hep3B cells. DNA microarray assay identified numerous differentially expressed genes related to angiogenesis, autophagy, calcium-mediated ER stress signaling, cell adhesion, cell cycle and mitosis, cell migration, cytoskeleton organization, DNA damage and repair, mitochondrial-mediated apoptosis and cell signaling pathways. Furthermore, Smh-3 inhibited CDK1 activity, mitochondrial membrane potential (ΔΨm) and increased the cytosolic Ca(2+) release and caspase-4, caspase-9 and caspase-3 activities in Hep3B cells. Western blot analysis demonstrated that Smh-3 increased the protein levels of caspase-4 and GADD153 that may lead to ER stress and consequently apoptosis in Hep3B cells. Taken together, Smh-3 acts against human hepatocellular carcinoma Hep3B cells in vitro through G(2)/M phase arrest and induction of calcium-mediated ER stress and mitochondrial-dependent apoptotic signaling pathways.

Inhibition of CIP2A determines erlotinib-induced apoptosis in hepatocellular carcinoma

Erlotinib is a small-molecular inhibitor of epidermal growth factor receptor (EGFR). Here, we identify that cancerous inhibitor of protein phosphatase 2A (CIP2A) is a major determinant mediating erlotinib-induced apoptosis in hepatocellular carcinoma (HCC). Erlotinib showed differential effects on apoptosis in 4 human HCC cell lines. Erlotinib induced significant apoptosis in Hep3B and PLC5 cell lines; however, Huh-7 and HA59T cell lines showed resistance to erlotinib-induced apoptosis at all tested doses. Down-regulation of CIP2A, a cellular inhibitor of protein phosphatase 2A (PP2A), mediated the apoptotic effect of erlotinib in HCC. Erlotinib inhibited CIP2A in a dose- and time-dependent manner in all sensitive HCC cells whereas no alterations in CIP2A were found in resistant cells. Overexpression of CIP2A upregulated phospho-Akt and protected Hep3B cells from erlotinib-induced apoptosis. In addition, silencing CIP2A by siRNA restored the effects of erlotinib in Huh-7 cells. Moreover, adding okadaic acid, a PP2A inhibitor, abolished the effects of erlotinib on apoptosis in Hep3B cells; and forskolin, a PP2A agonist enhanced the effect of erlotinib in resistant HA59T cells. Combining Akt inhibitor MK-2206 with erlotinib restored the sensitivity of HA59T cells to erlotinib. Furthermore, in vivo xenograft data showed that erlotinib inhibited the growth of PLC5 tumor but had no effect on Huh-7 tumor. Erlotinib downregulated CIP2A and upregulated PP2A activity in PLC5 tumors, but not in Huh-7 tumors. In conclusion, inhibition of CIP2A determines the effects of erlotinib on apoptosis in HCC. CIP2A may be useful as a therapeutic biomarker for predicting clinical response to erlotinib in HCC treatment.

Dieckol, isolated from Ecklonia stolonifera, induces apoptosis in human hepatocellular carcinoma Hep3B cells.

Phlorotannins have been reported to demonstrate several biological properties, including antioxidant activity, and activities useful in the treatment of diabetic complications and in chemoprevention of several vascular diseases. In this study, we focused on the apoptosis induced by dieckol, a marine algal phlorotannin isolated from Ecklonia stolonifera, on human hepatocellular carcinoma (HCC) Hep3B cells. Dieckol reduced the numbers of viable cells and increased the numbers of apoptotic cells in a dose-dependent manner. Immunoblotting analysis revealed that dieckol increased the expression levels of cleaved caspases-3, 7, 8, and 9, and cleaved poly(ADP-ribose) polymerase. Dieckol increased the permeability of mitochondrial membranes and the release of cytochrome c from mitochondria into the cytosol with apoptosis-inducing factor. In addition, dieckol induced increased expression of truncated Bid and Bim. The results indicate that dieckol induces apoptosis via the activation of both death receptor and mitochondrial-dependent pathways in HCC Hep3B cells.

Methyl methanesulfonate induces apoptosis in p53-deficient H1299 and Hep3B cells through a caspase 2- and mitochondria-associated pathway

Methyl methanesulfonate (MMS) has been shown to induce apoptosis in various cell types through p53-dependent pathways. Nevertheless, pharmacological and genetic blockade of p53 functions results in similar or delayed sensitivity to MMS treatment, suggesting the presence of p53-independent apoptotic mechanisms. To understand the p53-independent mechanisms that are engaged during MMS-induced apoptosis, we established MMS-induced apoptotic cell models using p53-deficient H1299 and Hep3B cells. Our results demonstrated that MMS at concentrations of 50, 100, 200, 400 and 800μM induced the formation of gammaH2AX foci, and that at higher concentrations, 400 and 800μM, MMS treatment led to apoptosis in the two cell lines. This apoptotic cell death was concurrent with the loss of mitochondrial membrane potential, nuclear-cytosolic translocation of active caspase 2, release of cytochrome c from mitochondria, and the cleavage of caspase 9, caspase 3 and PARP. However, MMS-induced DNA damage failed to stabilize the p53 family members TAp73 and DNp73. These results demonstrated a p53- and p73-independent mechanism for MMS-induced apoptosis that involves the nuclear-cytosolic translocation of active caspase 2 as well as the mitochondria-mediated pathway.

Etoposide induces a mixed type of programmed cell death and overcomes the resistance conferred by Bcl-2 in Hep3B hepatoma cells

The Bcl-2 protein is known to exert not only anti-apoptotic but also anti-autophagic activities. Numerous studies have demonstrated that etoposide, which is one of the most widely used cancer chemotherapy agents, induces apoptotic cell death. However, the exact molecular mechanism leading to cell death by etoposide remains to be resolved. This study aimed to dissect the mode of cell death induced by etoposide in Hep3B hepatoma cells. Furthermore, this study was conducted to examine whether etoposide overcomes the resistance conferred by Bcl-2 in Hep3B hepatoma cells. We observed that Hep3B cells treated with etoposide show not only apoptotic but autophagic phenotypes. Autophagy inhibition by 3-methyladenine (3MA) and caspase inhibition by zVAD-fmk effectively decreased autophagic and apoptotic phenotypes, respectively. However, either zVAD-fmk or 3MA only partially prevented cell death. These data indicate that etoposide concomitantly induces autophagic cell death and apoptosis in Hep3B cells. Importantly, etoposide can effectively induce cell death in Bcl-2-overexpressing Hep3B cells. Conversely, staurosporine, which exclusively induces apoptosis in Hep3B cells, did not efficiently induce cell death in Bcl‑2‑overexpressing Hep3B cells. Staurosporine-treated Hep3B cells also showed an autophagic phenotype. While autophagy is cell death-inducing in Hep3B cells treated with etoposide, it is cytoprotective in Hep3B cells treated with staurosporine. To this end, we observed that etoposide-induced mixed type of programmed cell death is associated with the dissociation of Bcl-2 from Beclin-1. Taken together, etoposide induces a mixed type of programmed cell death and overcomes the resistance conferred by Bcl-2 in Hep3B hepatoma cells.

ADAM10 overexpression confers resistance to doxorubicin-induced apoptosis in hepatocellular carcinoma

Chemoresistance represents a major obstacle to successful treatment of hepatocellular carcinoma (HCC). A disintegrin and metalloproteinase 10 (ADAM10) is known to be frequently upregulated in many cancers. We aimed to determine the biological function of ADAM10 in the chemoresistance of HCC cells. Overexpression of ADAM10 in three HCC cell lines (HepG2, Hep3B, and Huh7) conferred protection against doxorubicin-induced apoptosis, as determined by Annexin V staining. Western blot analysis revealed that ADAM10-overexpressing cells had a significantly lower amount of cleaved caspase-3 and an elevated expression of myeloid cell leukemia-1 (Mcl-1), a prosurvival member of the Bcl-2 family. Conversely, RNA interference-mediated silencing of endogenous ADAM10 potentiated doxorubicin-induced apoptosis in HepG2 and Hep3B cells, which was coupled with increased cleavage of caspase-3 and decreased expression of Mcl-1. Ectopic expression of ADAM10 resulted in a marked increase in the phosphorylation of phosphatidylinositol 3-kinase (PI3-K) and Akt. Most interestingly, the pretreatment with the PI3-K inhibitor LY294002 significantly enhanced doxorubicin-induced apoptosis and diminished the Mcl-1 expression in ADAM10-overexpressing Huh7 cells. Our data provide evidence that ADAM10 plays an important role in modulating the chemosensitivity of HCC cells, which, at least partially, involves the activation of the PI3-K/Akt pathway. ADAM10 may be a promising target for the improvement of chemotherapeutic efficacy in HCC.

Suppression of AMF/PGI‐mediated tumorigenic activities by ursolic acid in cultured hepatoma cells and in a mouse model

Our previous studies demonstrated that autocrine motility factor/phosphoglucose isomerase (AMF/PGI) possesses tumorigenic activities through the modulation of intracellular signaling. We then investigated the effects of ursolic acid (UA), oleanolic acid (OA), tangeretin, and nobiletin against AMF/PGI-mediated oncogenesis in cultured stable Huh7 and Hep3B cells expressing wild-type or mutated AMF/PGI and in a mouse model in this study. The working concentrations of the tested compounds were lower than their IC10 , which was determined by Brdu incorporation and colony formation assay. Only UA efficiently suppressed the AMF/PGI-induced Huh7 cell migration and MMP-3 secretion. Additionally, UA inhibited the AMF/PGI-mediated protection against TGF-β-induced apoptosis in Hep3B cells, whereas OA, tangeretin, and nobiletin had no effect. In Huh7 cells and tumor tissues, UA disrupted the Src/RhoA/PI 3-kinase signaling and complex formation induced by AMF/PGI. In the Hep3B system, UA dramatically suppressed AMF/PGI-induced anti-apoptotic signaling transmission, including Akt, p85, Bad, and Stat3 phosphorylation. AMF/PGI enhances tumor growth, angiogenesis, and pulmonary metastasis in mice, which is correlated with its enzymatic activity, and critically, UA intraperitoneal injection reduces the tumorigenesis in vivo, enhances apoptosis in tumor tissues and also prolongs mouse survival. Combination of sub-optimal dose of UA and cisplatin, a synergistic tumor cell-killing effects was found. Thus, UA modulates intracellular signaling and might serve as a functional natural compound for preventing or alleviating hepatocellular carcinoma.

MicroRNA‐122 suppresses cell proliferation and induces cell apoptosis in hepatocellular carcinoma by directly targeting Wnt/β‐catenin pathway

To validate whether the anti-cancer effect of microRNA-122 (miR-122) on hepatocellular carcinoma (HCC) is mediated through regulating Wnt/β-catenin signalling pathways. The expression levels of miR-122 in HCC tissues and varied hepatoma cells were quantified by real-time PCR. MiR-122 agomir was transfected into HepG2, Hep3B cells to over-express miR-122. The effect of over-expression miR-122 on proliferation and apoptosis of HepG2 and Hep3B cells was evaluated using CCK-8 kit and flow cytometer respectively. The 3'-UTR segments of Wnt1 containing the miR-122 binding sites were amplified by PCR and the luciferase activity in the transfected cells was assayed. Wnt1 mRNA level was quantified using RT-PCR. Protein levels of Wnt1, β-catenin and TCF-4 were detected using Western blotting. In comparison with the expression level of miR-122 in para-cancerous tissues or Chang liver cell, the expression level in HCC tissues or varied hepatoma cells was significantly decreased (P<0.05). Over-expression of miR-122 significantly inhibited the proliferation (P<0.05), and promoted the apoptosis of HepG2 and Hep3B cells. Over-expressed miR-122 down-regulated the protein levels of Wnt1, β-catenin and TCF-4 (P<0.05). MiR-122 suppressed the luciferase activity of the pmiR-Wnt1-wt by approximately 50% compared with the negative control, while mutation or removal of the miR-122 binding site using siRNA or mir-122 inhibitor blocked the suppressive effect (P<0.05). MiR-122 expression is down-regulated in human HCC. Over-expression of miR-122 inhibits HCC cell growth and promotes the cell apoptosis by affecting Wnt/β-catenin-TCF signalling pathway.

Hispolon Induces Apoptosis and Cell Cycle Arrest of Human Hepatocellular Carcinoma Hep3B Cells by Modulating ERK Phosphorylation

Hispolon is an active phenolic compound of Phellinus igniarius , a mushroom that has recently been shown to have antioxidant, anti-inflammatory, and anticancer activities. This study investigated the antiproliferative effect of hispolon on human hepatocellular carcinoma Hep3B cells by using the MTT assay, DNA fragmentation, DAPI (4,6-diamidino-2-phenylindole dihydrochloride) staining, and flow cytometric analyses. Hispolon inhibited cellular growth of Hep3B cells in a time-dependent and dose-dependent manner, through the induction of cell cycle arrest at S phase measured using flow cytometric analysis and apoptotic cell death, as demonstrated by DNA laddering. Hispolon-induced S-phase arrest was associated with a marked decrease in the protein expression of cyclins A and E and cyclin-dependent kinase (CDK) 2, with concomitant induction of p21waf1/Cip1 and p27Kip1. Exposure of Hep3B cells to hispolon resulted in apoptosis as evidenced by caspase activation, PARP cleavage, and DNA fragmentation. Hispolon treatment also activated JNK, p38 MAPK, and ERK expression. Inhibitors of ERK (PB98095), but not those of JNK (SP600125) and p38 MAPK (SB203580), suppressed hispolon-induced S-phase arrest and apoptosis in Hep3B cells. These findings establish a mechanistic link between the MAPK pathway and hispolon-induced cell cycle arrest and apoptosis in Hep3B cells.

Characterization of 9-Nitrocamptothecin Liposomes: Anticancer Properties and Mechanisms on Hepatocellular Carcinoma In Vitro and In Vivo

BackgroundHepatocellular carcinoma (HCC) is the third most common cause of cancer related mortality worldwide. 9-Nitrocamptothecin (9NC) is a potent topoisomerase-I inhibitor with strong anticancer effect. To increase the solubility and stability, we synthesized a novel 9NC loaded liposomes (9NC-LP) via incorporating 9NC into liposomes. In the present study, we determined the effects of 9NC and 9NC-LP on in vitro and in vivo, and the underlying mechanisms.Methodology/Principal FindingsWe first analyzed the characteristics of 9NC-LP. Then we compared the effects of 9NC and 9NC-LP on the proliferation and apoptosis of HepG2, Bel-7402, Hep3B and L02 cells in vitro. We also investigated their anticancer properties in nude mice bearing HCC xenograft in vivo. 9NC-LP has a uniform size (around 190 nm) and zeta potential (∼−11 mV), and exhibited a steady sustained-release pattern profile in vitro. Both 9NC and 9NC-LP could cause cell cycle arrest and apoptosis in a dose-dependent and p53-dependent manner. However, this effect was not ubiquitous in all cell lines. Exposure to 9NC-LP led to increased expression of p53, p21, p27, Bax, caspase-3, caspase-8, caspase-9 and apoptosis-inducing factor, mitochondrion-associated 1 and decreased expression of Bcl-2, cyclin E, cyclin A, Cdk2 and cyclin D1. Furthermore, 9NC-LP exhibited a more potent antiproliferative effect and less side effects in vivo. Western blot analysis of the xenograft tumors in nude mice showed similar changes in protein expression in vivo.Conclusions/SignificanceIn conclusion, 9NC and 9NC-LP can inhibit HCC growth via cell cycle arrest and induction of apoptosis. 9NC-LP has a more potent anti-tumor effect and fewer side effects in vivo, which means it is a promising reagent for cancer therapy via intravenous administration.

CF102 an A3 adenosine receptor agonist mediates anti‐tumor and anti‐inflammatory effects in the liver

The Gi protein-associated A(3) adenosine receptor (A(3) AR) is a member of the adenosine receptor family. Selective agonists at the A(3) AR, such as CF101 and CF102 were found to induce anti-inflammatory and anti-cancer effects. In this study, we examined the differential effect of CF102 in pathological conditions of the liver. The anti-inflammatory protective effect of CF101 was tested in a model of liver inflammation induced by Concanavalin A (Con. A) and the anti-cancer effect of CF102 was examined in vitro and in a xenograft animal model utilizing Hep-3B hepatocellular carcinoma (HCC) cells. The mechanism of action was explored by following the expression levels of key signaling proteins in the inflamed and tumor liver tissues, utilizing Western blot (WB) analysis. In the liver inflammation model, CF102 (100 µg/kg) markedly reduced the secretion of serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase in comparison to the vehicle-treated group. Mechanistically, CF102 treatment decreased the expression level of phosphorylated glycogen synthase kinase-3β, NF-κB, and TNF-α and prevented apoptosis in the liver. This was demonstrated by decreased expression levels of Fas receptor (FasR) and of the pro-apoptotic proteins Bax and Bad in liver tissues. In addition, CF102-induced apoptosis of Hep-3B cells both in vitro and in vivo via de-regulation of the PI3K-NF-κB signaling pathway, resulting in up-regulation of pro-apoptotic proteins. Taken together, CF102 acts as a protective agent in liver inflammation and inhibits HCC tumor growth. These results suggest that CF102 through its differential effect is a potential drug candidate to treat various pathological liver conditions.

Abstract 4097: Sorafenib sensitizes solid tumors to TRAIL or TRAIL receptor agonist antibodies

Abstract Solid carcinomas display considerable resistance to chemotherapeutic drugs and tumor cell apoptosis-inducing agents such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell death. A phase III clinical trial with the multi-kinase inhibitor sorafenib (Nexavar) in Hepatoceullular carcinoma (HCC) was completed leading to the first FDA-approved drug therapy for liver cancer in 2007. Phase I/II trials have been completed with a fully-human anti-TRAIL death receptor 1 (DR4) agonist antibody (mapatumumab) in non small-cell lung carcinoma and non-Hodgkin's lymphoma and phase I trials with an anti-TRAIL-R2 (DR5) monoclonal antibody (lexatumumab) in advanced cancers. We hypothesized that sorafenib and TRAIL or sorafenib and TRAIL receptor agonist antibodies (mapatumumab or lexatumumab) may overcome resistance to apoptosis in a panel of TRAIL-resistant solid carcinoma cell lines. We observed that sorafenib induces apoptosis of HepG2 and Hep3B cells in a dose-dependent manner. In addition, sorafenib is a potent sensitizer of HCC cell lines to TRAIL. We have also found that single agent lexatumumab causes cell death in HepG2, Hep3B and SNU449 cells whereas mapatumumab does not. The combination of sorafenib with TRAIL or either TRAIL receptor agonist antibody enhances apoptosis in HCC cell lines. We found that in other solid tumor types including prostate, breast and colon cancer there is a synergistic interaction between sorafenib and TRAIL or TRAIL receptor agonist antibodies. Furthermore, these combinations prevent the growth of tumors in vivo. Sorafenib downregulates the expression of Mcl-1 protein, a member of the Bcl-2 family that has been previously found to be overexpressed in about 50% of HCC patient samples. Sorafenib was also found to inhibit Stat3 activation by Jak2 kinases. These findings suggest a molecular rationale for combining these agents in therapy of solid tumors in clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4097. doi:10.1158/1538-7445.AM2011-4097

Differential effects of somatostatin on tnf receptors and apoptosis in hepatocellular carcinoma cell lines

IntroductionSomatostatin has antitumour activity in animal models of hepatocellular carcinoma. In human studies both favourable and unfavourable results have been reported. A 40% fraction of treated patients are benefited. Since...