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Hepatocellular carcinoma (HCC) is a major subtype of primary liver cancer with a high mortality rate. Pyroptosis and autophagy are crucial processes in the pathophysiology of HCC. Searching for efficient drugs targeting pyroptosis and autophagy with lower toxicity is useful for HCC treatment. Mallotucin D (MLD), a clerodane diterpenoid from Croton crassifolius, has not been previously reported for its anticancer effects in HCC. This study aims to evaluate the inhibitory effects of MLD in HCC and explore the underlying mechanism. We found that the cell proliferation, DNA synthesis, and colony formation of HepG2 cells and the angiogenesis of HUVECs were all greatly inhibited by MLD. MLD caused mitochondrial damage and decreased the TOM20 expression and mitochondrial membrane potential, inducing ROS overproduction. Moreover, MLD promoted the cytochrome C from mitochondria into cytoplasm, leading to cleavage of caspase-9 and caspase-3 inducing GSDMD-related pyroptosis. In addition, we revealed that MLD activated mitophagy by inhibiting the PI3K/AKT/mTOR pathway. Using the ROS-scavenging reagent NAC, the activation effects of MLD on pyroptosis- and autophagy-related pathways were all inhibited. In the HepG2 xenograft model, MLD effectively inhibited tumor growth without detectable toxicities in normal tissue. In conclusion, MLD could be developed as a candidate drug for HCC treatment by inducing mitophagy and pyroptosis via promoting mitochondrial-related ROS production.

Mitochondria are involved in the regulation of apoptosis, making them a promising target for the development of new anticancer drugs. Doxorubicin (DOX), a chemotherapeutic drug, can induce reactive oxygen species (ROS)-mediated apoptosis, improving its anticancer effects. Herein, Rhein, an active ingredient in rhubarb, with the capability of self-assembly and mitochondrial targeting, was used in conjunction with DOX to form efficient nanomaterials (Rhein-DOX nanogel) capable of sustained drug release. It was self-assembled with a hydrogen bond, π-π stacking interactions, and hydrophobic interactions as the main driving force, and its loading efficiency was up to 100%. Based on its self-assembly characteristics, we evaluated the mechanism of this material to target mitochondria, induce ROS production, and promote apoptosis. The IC 50 of the Rhein-DOX nanogel (3.74 μM) was only 46.3% of that of DOX (11.89 μM), and the tumor inhibition rate of the Rhein-DOX nanogel was 79.4% in vivo, 2.3 times that of DOX. This study not only addresses the disadvantages of high toxicity of DOX and low bioavailability of Rhein, when DOX and Rhein are combined for the treatment of hepatoma, but it also significantly improved the synergistic antihepatoma efficacy of Rhein and DOX, which provides a new idea for the development of long-term antihepatoma agents with low toxicity.

Prunus spinosa L. (blackthorn) is used in traditional medicine as a remedy for various diseases. To establish its anticancer properties, we exposed human liver cancer cells (Hep G2) to a range of blackthorn flower extract concentrations (10-200 µg/mL) and determined cytotoxic activity with the neutral red and kenacid blue methods after 24, 48, and 72 h of incubation. Statistically significant inhibitory effects on Hep G2 cellular proliferation were observed at concentrations above 50 µg/mL (p<0.001-0.05). Cell viability was lower when determined with neutral red than kenacid blue method. In addition, we evaluated antioxidant/prooxidant effects of the blackthorn flower extract by measuring reactive oxygen species (ROS), and the results confirmed its prooxidant behaviour within the applied concentration range. Flow cytometry determined primarily necrotic and apoptotic cell death, which provides additional evidence of its cytotoxic effect on liver carcinoma.

Purpose: Sorafenib has been the only first systemic drug that improves survival of patients with advanced hepatocellular carcinoma (HCC). However, because the response rate of sorafenib is relatively low, novel therapeutic strategies are needed to improve survival in patients with HCC. This study investigated the effect of CMG002 alone and in combination with sorafenib on HCC in vitro and vivo., Methods: The effect of a newly developed dual PI3K/mTOR inhibitor, CMG002, on the proliferation of Huh-7 and HepG2 HCC cells was investigated using the MTT assay. Western blotting was performed to assess phosphorylation of the key enzymes in the Ras/Raf/MAPK and PI3K/AKT/mTOR pathways. HepG2 cells were inoculated into mice, which were treated with vehicle, sorafenib, CMG002, and their combinations. Tumor cell proliferation and tumor angiogenesis were evaluated by immunohistochemical analysis of Ki-67 and CD31, respectively. Tumor cell apoptosis was detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Levels of key enzymes in the Ras/Raf/MAPK and PI3K/AKT/mTOR pathways were evaluated by western blot analysis., Results: The combination of sorafenib and CMG002 additively inhibited Huh-7 and HepG2 cell proliferation compared to single-agent treatment. Sorafenib and CMG002 as single agents differentially inhibited or activated key enzymes in the Ras/Raf/MAPK and PI3K/AKT/mTOR pathways. The combination of sorafenib and CMG002 inhibited all key enzymes in the two pathways. Treatment with CMG002 for 4 weeks alone and in combination with sorafenib strongly inhibited tumor growth. CMG002 inhibited HCC cell proliferation, induced apoptosis, and decreased tumor angiogenesis. Furthermore, these effects were enhanced when CMG002 was combined with sorafenib., Conclusions: The combination of CMG002 and sorafenib significantly inhibited HCC cell proliferation and tumorigenesis by inhibiting the Ras/Raf/MAPK and PI3K/AKT/mTOR pathways. These findings suggest that CMG002 to be a potential novel candidate treatment for HCC.

The effects of epigallocatechin-3-gallate (EGCG) and metformin single treatment have been tested against hepatocellular carcinoma (HCC). This study aimed to assess the combination effects of EGCG and metformin on proliferation and apoptosis of HepG2cells and identified new potential molecular targets. The effect of EGCG and metformin against cell proliferation in HepG2 was determined using MTT assay. Reverse transcription polymerase chain reaction was applied to examine the gene expression of cyclin D1, lncRNA-AF085935, caspase-3, survivin and VEGF. The level of protein expression of glypican-3 was assessed by western blot. In HepG2 cells, EGCG and metformin combination treatment exhibited high significant effect against tumor proliferation. It significantly reduced cyclin D1, lncRNA-AF085935, glypican-3 and promoted apoptosis through increasing caspase3 and decreasing survivin compared to control cells. Moreover, EGCG and metformin treated cells showed decreased expression levels of VEGF. Our study provided new insights of the anticarcinogenic effects of EGCG and metformin on HCC through their effects on glypican-3 and lncRNA-AF085935.

YB-1 nuclear translocation/accumulation caused by anticancer agents leads to malignant transformation. Nuclear import of YB-1 requires a nuclear localization signal (YB-NLS). Previously, we identified five nucleocytoplasmic-shuttling proteins as YB-NLS binding proteins, and showed that they co-accumulate in the nucleus with YB-1 in response to treatment with actinomycin D. In addition, another group reported that transportin-1 is the molecule responsible for YB-1 nuclear translocation, binding to a region (PY-NLS) consistent with the YB-NLS. Recently, we found that indirubin 3'-oxime inhibits the nuclear localization of YB-1 in HepG2 cells and increases their sensitivity to actinomycin D. Here, we found that YB-1 nuclear translocation is dependent on the cellular mRNA level and that indirubin 3'-oxime inhibits the interaction between YB-1 and transportin-1. Interestingly, in cells showing inhibition of actinomycin D-induced YB-1 nuclear translocation by the compound, the YB-NLS-binding proteins as well as transportin-1 and its cargos were imported to the nucleus. Furthermore, the compound inhibited nuclear localization of the GFP-conjugated full-length YB-1 but not that of GFP-conjugated YB-NLS. These results indicate that indirubin 3'-oxime is a specific inhibitor of anticancer agent-induced YB-1 nuclear translocation, interacting with YB-1 itself in a region other than the YB-NLS/PY-NLS. This compound would increase the efficacy of cancer therapy., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The current study was designed and performed to investigate the effect of mefloquine on the proliferation and tumor formation potential of liver cancer stem cells. CD133 + HepG2 cells were identified using MACS and showed markedly higher tumor formation potential compared to the parental cells. The secondary tumors formed by CD133 + cells were markedly large in size and more in number compared to the parental cells. Mefloquine treatment of CD133 + HepG2 cells inhibited the proliferation selectively in concentration based manner. The rate of proliferation was inhibited to 82 and 12% in parental and CD133 + sphere forming cells, respectively on treatment with 10 μM concentration of mefloquine. The number of secondary tumors formed by primary tumors was decreased significantly on treatment with 10 μM mefloquine concentration. Treatment of the liver cancer stem cells with mefloquine markedly decreased the potential to undergo self-renewal at 10 μM concentration after 48 h. The results from western blot analysis showed significantly higher expression of cancer stem cell molecules β-catenin and cyclin D1 in LCSCs. Treatment of the LCSCs with various concentrations of mefloquine reduced the expression levels of β-catenin and cyclin D1. Administration of the CD133 + cell tumor xenografts in the mice led to the formation of large sized tumors in the control group. However, the tumor growth was inhibited significantly in the mice on treatment with 10 mg/kg doses of mefloquine after day 21. The tumor weight was significantly lower in the animals of mefloquine treatment group compared to the control group. Thus, mefloquine treatment inhibits self-renewal and proliferation potential of cells through targeting β-catenin pathway., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

BACKGROUND Hepatocellular carcinoma (HCC) is the most important cause of cancer-related deaths worldwide. Pirfenidone is an orally available small molecule with therapeutic potential for fibrotic diseases. MATERIAL AND METHODS In this study, we analyzed the effects of different pirfenidone concentrations on the proliferation of HepG2 HCC cells using Cell Counting Kit-8 (CCK-8) and colony formation assays. Flow cytometry was performed to measure the apoptotic effects of pirfenidone on HepG2 cells. Western blot analysis was performed to detect the expression of β-catenin and p-β-catenin. RESULTS Pirfenidone inhibited proliferation and promoted HepG2 cell apoptosis. In addition, Western blot results indicated that pirfenidone suppressed b-catenin expression in HepG2 cells. To assess the mechanism, we treated HepG2 cells with pirfenidone, and pirfenidone plus the β-catenin activator, SB-216763. The results revealed that SB-216763 accelerated proliferation and inhibited apoptosis in HepG2 cells treated with pirfenidone. Western blot results showed that SB-216763 upregulated β-catenin expression in HepG2 cells treated with pirfenidone. CONCLUSIONS In conclusions, pirfenidone may be a potential drug for HCC treatment.

A reductase-cleavable and thermo-responsive star-shaped polymer nanogel was prepared via an "arm-first" atom transfer radical polymerization approach. The nanogel consists of a thermo- and redox-sensitive core and a zwitterionic copolymer block. The dual sensitive core is composed of poly(N-isopropylacrylamide) that is formed by disulfide crosslinking of N-isopropylacrylamide. The zwitterionic copolymer block contains a poly(sulfobetaine methacrylate) component, a known anti-adsorptive moiety that extends blood circulation time, and a lactose motif of poly(2-lactobionamidoethyl methacrylamide) that specifically targets the asialoglycoprotein receptors (ASGP-Rs) of hepatoma. Doxorubicin (DOX) was encapsulated into the cross-linked nanogels via solvent extraction/evaporation method and dialysis; average diameter of both blank and DOX-loaded nanogels was ~120 nm. The multi-responsiveness of nanogel drug release in different temperatures and redox conditions was assessed. After 24 h, DOX release was only ~20% at 30°C with 0 mM glutathione (GSH), whereas over 90% DOX release was observed at 40°C and 10 mM GSH, evidence of dual responsiveness to temperature and reductase GSH. The IC 50 value of DOX-loaded nanogels was much lower in human hepatoma (HepG2) cells compared to non-hepatic HeLa cells. Remarkably, DOX uptake of HepG2 cells differed substantially in the presence and absence of galactose (0.31 vs 1.42 µg/mL after 48 h of incubation). The difference was non-detectable in HeLa cells (1.21 vs 1.57 µg/mL after 48 h of incubation), indicating that the overexpression of ASGP-Rs leads to the DOX-loaded lactosylated nanogels actively targeting hepatoma. Our data indicate that the lactose-decorated star-shaped nanogels are dual responsive and hepatoma targeted, and could be employed as hepatoma-specific anti-cancer drug delivery vehicle for cancer chemotherapy., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Monastrol and its analog oxomonastrol differ by replacement of the sulfur atom present in monastrol to an oxygen atom in oxomonastrol. Monastrol inhibits the mitotic kinesin family member 11 (EG5), which has been studied for its potential use in cancer therapy. The aim of this study was to investigate the effect of monastrol and oxomonastrol on HepG2/C3A cells. Our results showed that monastrol induced DNA damage, reduced cell proliferation, and up-regulated the cytochrome P450 family 1 subfamily A member 1 (CYP1A1) mRNA levels. However, oxomonastrol was cytotoxic only at the highest concentrations used, without reducing cell proliferation and viability. Moreover, no genotoxic damage or alteration of levels of mRNA were found. Our results suggest that monastrol has greater antiproliferative activity compared to oxomonastrol, and this effect is probably related to the DNA damage induced by monastrol and its possible bioactivation demonstrated by the increase in CYP1A1 mRNA expression. Moreover, these effects appear to be related to the presence of the sulfur atom in its structure., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: Acetylshikonin is one of the biologically active compounds derived from the root of Lithospermum erythrorhizon, a medicinal plant with anti-cancer and anti-inflammation activity. Although there have been a few previous reports demonstrating that acetylshikonin exerts anti-cancer activity in vitro and in vivo, it is still not clear what is the exact molecular target protein of acetylshikonin in cancer cells., Purpose: The purpose of this study is to evaluate the inhibitory effect of acetylshikonin against CYP2J2 enzyme which is predominantly expressed in human tumor tissues and carcinoma cell lines., Study Design: The inhibitory effect of acetylshikonin on the activities of CYP2J2-mediated metabolism were investigated using human liver microsomes (HLMs), and its cytotoxicity against human hepatoma HepG2 cells was also evaluated., Method: Astemizole, a representative CYP2J2 probe substrate, was incubated in HLMs in the presence or absence of acetylshikonin. After incubation, the samples were analyzed by liquid chromatography and triple quadrupole mass spectrometry. The anti-cancer activity of acetylshikonin was evaluated on human hepatocellular carcinoma HepG2 cells. WST-1, cell counting, and colony formation assays were further adopted for the estimation of the growth rate of HepG2 cells treated with acetylshikonin., Results: Acetylshikonin inhibited CYP2J2-mediated astemizole O-demethylation activity (K i = 2.1µM) in a noncompetitive manner. The noncompetitive inhibitory effect of acetylshikonin on CYP2J2 enzyme was also demonstrated using this 3D structure, which showed different binding location of astemizole and acetylshikonin in CYP2J2 model. It showed cytotoxic effects against human hepatoma HepG2 cells (IC 50 = 2μM). In addition, acetylshikonin treatment inhibited growth of human hepatocellular carcinoma HepG2 cells leading to apoptosis accompanied with p53, bax, and caspase3 activation as well as bcl2 down-regulation., Conclusion: Taken together, our present study elucidates acetylshikonin displays the inhibitory effects against CYP2J2 in HLMs and anti-cancer activity in human hepatocellular carcinoma HepG2 cells., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Oxaloacetate (OA) is one of the intermediates of the Krebs cycle. In addition to its role in energy production, OA may have other effects on the cell. We report here that OA could have a cell type dependent cytotoxic effect on the human hepatic carcinoma cell line HepG2 through induction of apoptosis and reactive oxygen species (ROS) accumulation. In our study, OA decreased the viability and colony formation of HepG2 cells and induced cell death. Caspase-3 activity was increased, the pro-apoptotic protein Bax was up-regulated, and the anti-apoptotic protein Bcl-2 was down-regulated in OA-treated HepG2 cells indicating that apoptosis through the intrinsic pathway was involved in the cell death. The ROS level in OA-treated HepG2 cells was increased. The anti-oxidant N-acetylcysteine (NAC) and glutathione (GSH) prevented the OA-induced decrease in cell but did not alter the enhanced apoptotic Bax/Bcl-2 mRNA ratio. These results suggest that the OA-induced apoptosis of HepG2 cell is not driven by oxidative damage and at least two distinct mechanisms, one mediated by ROS and one involving apoptosis, result in the cytotoxic effects of OA on HepG2 cells. These studies expand the biological functional repertoire of OA and provide a mechanism by which hepatocellular carcinoma may be targeted by OA.

5,6-Dihydroxy-3,7,4'-trimethoxyflavonol (AH5), 5,6,3'-trihydroxy-3,7,4'-trimethoxyflavonol (AH22), artemetin, and oroxylin A are four flavonoids with the same 2-phenyl-chromone skeleton isolated from the Chinese herb Aster himalaicus. The aim of this study was to evaluate the structure-activity relationship of these four analogs and the mediation of AH5 cytotoxicity via G2/M arrest and apoptosis in human hepatocellular carcinoma (HCC) cells. 3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay indicated AH5 showed the better potency to inhibit proliferation in human HCC cells, which suggested hydroxyl binding to C6 is necessary to anticancer properties, whereas binding to C3' attenuated the activities and increased toxicity in tested cells. Flow cytometry analysis revealed that AH5-induced G2/M arrest and significantly apoptosis in these cell lines. HepG-2 cells were used to further evaluate the antitumor effects and mechanisms of AH5. AH5-induced apoptosis was further confirmed by 4',6-diamidino-2-phenylindole (DAPI) staining and the increased ratio of Bax/Bcl-2. Moreover, AH5 induced the release of cytochrome C and the activation of caspase-9 and caspase-3, thus suggesting mitochondria activation might be involved. Western blot showed that AH5 induced the phosphorylation of Cdc2 and decreased the level of Cyclin B1. These results demonstrated that AH5 could be a proapoptotic leading compound for developing novel anticancer drugs.

Human hepatocellular carcinoma (HCC) has a high rate of tumor recurrence and metastasis, resulting in shortened survival time. The function of alpha-fetoprotein (AFP) as a regulatory factor in the growth of HCC cells has been well defined. The aim of this study was to investigate the use of a novel AFP-specific single-chain variable fragment that blocked AFP and inhibited HCC cell growth. The results indicated that the anti-AFP single-chain variable fragment (scFv) induced growth inhibition of AFP-expressing HCC cell lines in vitro through induction of G1 cell cycle arrest and apoptosis. The mechanism of apoptosis probably involved with blocking AFP internalization and regulation of the PTEN/PI3K/Akt signaling network. Moreover, the anti-AFP-scFv also effectively sensitized the HepG2 cells to paclitaxel (PTX) at a lower concentration. The combination effect of PTX and anti-AFP-scFv displayed a synergistic effect on HepG2 cells both in vitro and in vivo. Our results demonstrated that targeting AFP by specific antibodies has potential immunotherapeutic efficacy in human HCC.

Background: Brown macroalgae have attracted attention because they display a wide range of biological activities, including antitumoral properties. Inthis study we isolated isololiolide from Cystoseira tamariscifolia for the first time., Purpose: To examine the therapeutical potential of isololiolide against tumor cell lines., Methods/study Design: The structure of the compound was established and confirmed by 1D and 2D NMR as well as HRMS spectral analysis. The in vitro cytotoxicity was analyzed by colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in tumoral as well as in non-tumoral cell lines. Cell cycle arrest and induction of apoptosis were assessed by flow cytometry. Alteration of expression levels in proteins important in the apoptotic cascade was analyzed by western blotting., Results: Isololiolidewas isolated for the first time from the brown macroalga C.tamariscifolia. Isololiolide exhibited significant cytotoxic activity against three human tumoral cell lines, namely hepatocarcinoma HepG2 cells, whereas no cytotoxicity was found in non-malignant MRC-5 and HFF-1 human fibroblasts. Isololiolide completely disrupted the HepG2 normal cell cycle and induced significant apoptosis. Moreover, western blot analysis showed that isololiolide altered the expression of proteins that are important in the apoptotic cascade, increasing PARP cleavage and p53 expression while decreasing procaspase-3 and Bcl-2 levels., Conclusion: Isololiolide isolated from C. tamariscifolia is able to exert a selective cytotoxic activity on hepatocarcinoma HepG2 cells as well as induce apoptosis through the modulation of apoptosis-related proteins., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Citrus seeds are full of phenolic compounds, such as flavonoids. The aims of this study were to identify the types of flavonoids in Citrus seed extracts, the cytotoxic effect, mode of cell death, and signaling pathway in human hepatic cancer HepG2 cells. The flavonoids contain anticancer, free radical scavenging, and antioxidant activities. Neohesperidin, hesperidin, and naringin, active flavanone glycosides, were identified in Citrus seed extract. The cytotoxic effect of three compounds was in a dose-dependent manner, and IC50 levels were determined. The sensitivity of human HepG2 cells was as follows: hesperidin > naringin > neohesperidin > naringenin. Hesperidin induced HepG2 cells to undergo apoptosis in a dose-dependent manner as evidenced by the externalization of phosphatidylserine and determined by annexin V-fluorescein isothiocyanate and propidium iodide staining using flow cytometry. Hesperidin did not induce the generation of reactive oxygen species, which was determined by using 2',7'-dichlorohydrofluorescein diacetate and flow cytometry method. The number of hesperidin-treated HepG2 cells with the loss of mitochondrial transmembrane potential increased concentration dependently, using 3,3'-dihexyloxacarbocyanine iodide employing flow cytometry. Caspase-9, -8, and -3 activities were activated and increased in hesperidin-treated HepG2 cells. Bcl-xL protein was downregulated whereas Bax, Bak, and tBid protein levels were upregulated after treatment with hesperidin in a dose-dependent manner. In conclusion, the bioflavanone from Citrus seeds, hesperidin, induced human HepG2 cell apoptosis via mitochondrial pathway and death receptor pathway. Citrus seed flavonoids are beneficial and can be developed as anticancer drug or food supplement, which still needs further in vivo investigation in animals and human beings.

Hepatocellular carcinoma (HCC) is the most common primary liver tumor (PLT), with cholangiocarcinoma (CC) being the second most frequent. Glucose transporter 1 (GLUT-1) expression is increased in PLTs and therefore it is suggested as a therapeutic target. Flavonoids, like quercetin, are GLUT-1 competitive inhibitors and may be considered as potential therapeutic agents for PLTs. The objective of this study was evaluation of quercetin anticancer activity in three human HCC cell lines (HepG2, HuH7, and Hep3B2.1-7) and in a human CC cell line (TFK-1). The possible synergistic effect between quercetin and sorafenib, a nonspecific multikinase inhibitor used in clinical practice in patients with advanced HCC, was also evaluated. It was found that in all the cell lines, quercetin induced inhibition of the metabolic activity and cell death by apoptosis, followed by increase in BAX/BCL-2 ratio. Treatment with quercetin caused DNA damage in HepG2, Hep3B2.1-7, and TFK-1 cell lines. The effect of quercetin appears to be independent of P53. Incubation with quercetin induced an increase in GLUT-1 membrane expression and a consequent reduction in the cytoplasmic fraction, observed as a decrease in (18)F-FDG uptake, indicating a GLUT-1 competitive inhibition. The occurrence of synergy when sorafenib and quercetin were added simultaneously to HCC cell lines was noticed. Thus, the use of quercetin seems to be a promising approach for PLTs through GLUT-1 competitive inhibition.

Acquisition of cisplatin resistance is the common and critical limitation for hepatocellular carcinoma (HCC) therapy. Our study was aimed to determine whether there were conditions under which the addition of imperatorin would reverse the resistance of HCC cells to cisplatin-based therapy. In this study, we found that addition of imperatorin significantly enhanced the cytotoxicity of cisplatin to HCC cells. Since the Mcl-1 was overexpressed in HCC cell lines (HepG2, HepG3B, PLC, Huh7) compared with normal liver cell line (L-O2), we found that the Mcl-1 expression was downregulated by imperatorin but not influenced by cisplatin in HCC cells. In addition, our results showed the combination of imperatorin and cisplatin induced apoptosis and ∆Ψm collapse more significantly compared with treatment of imperatorin or cisplatin alone. Furthermore, the imperatorin-induced sensitization for cisplatin-cytotoxicity to HCC cells was abolished by the transfection of Mcl-1 expression plasmid. Finally, we found that the addition of imperatorin significantly reversed the resistance to cisplatin in cisplatin-resistant HCC cells, which was Mcl-1 dependent. In summary, our study revealed that combination with imperatorin could enhance the antitumor activity of cisplatin via targeting Mcl-1 and reverse the resistance to cisplatin in HCC.

Anticancer activity of a novel curcumin analog (E)-2-(4-hydroxy-3-methoxybenzylidene)-5-((E)-3-(4-hydroxy-3-methoxyphenyl)acryloyl)cyclopentanone (CUR3d) was studied using a human hepatocellular carcinoma cell line (HepG2). The results showed that CUR3d completely inhibits the tumor cell proliferation in a dose- and time-dependent manner. CUR3d at 100 μmol/L activated the pro-apoptotic caspase-3 along with downregulation of anti-apoptotic BIRC5 and Bcl2. CUR3d treatment controlled the cancer cell growth by downregulating the expression of PI3K/Akt (Akt1, Akt2) pathway along with NF-κB. CUR3d down-regulated the members of epidermal growth receptor family (EGFR, ERBB3, ERBB2) and insulin like growth receptors (IGF1, IGF-1R, IGF2). This correlated with the downregulation of G-protein (RHOA, RHOB) and RAS (ATF2, HRAS, KRAS, NRAS) pathway signaling. CUR3d also arrested cell cycle via inhibition of CDK2, CDK4, CDK5, CDK9, MDM2, MDM4 and TERT genes. Cell cycle essential aurora kinases (AURKα, AURKβ) and polo-like kinases (PLK1, PLK2, PLK3) were also modulated by CUR3d. Topoisomerases (TOP2α, TOP2β), important factors in cancer cell immortality, as well as HIF-1α were downregulated following CUR3d treatment. The expression of protein kinase-C family (PRKC-A, PRKC-D, PRKC-E) was also attenuated by CUR3d. The downregulation of histone deacetylases (Class I, II, IV) and PARP I further strengthened the anticancer efficacy of CUR3d. Downregulation of carcinogenic cathepsins (CTSB, CTSD) and heat shock proteins exhibited CUR3d's potency as a potential immunological adjuvant. Finally, the non-toxic manifestation of CUR3d in healthy liver and lung cells along with downregulation of drug resistant gene ABCC1 further warrant need for advance investigations., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

A lactoferrin-containing PEGylated liposome system (Lf-PLS) was developed and tested in vitro as a hepatoma-targeting drug delivery system. PEGylated liposomes (PLS) were successfully prepared using the thin film hydration method with peglipid post insertion. Lf was covalently conjugated onto the carboxyl terminal of DSPE-PEG2000-COOH on liposomes. Coumarin-6 was used to trace Lf-PLS with fluorescence. The cellular uptake of this system was carried out in asialoglycoprotein receptor (ASGPR) positive HepG2 cells via confocal microscopy and flow cytometry. The Lf-PLS liposome was observed as spherical or oval vesicles with the particle size around 130 nm, zeta potential about -30 mV and encapsulation efficiency more than 80%. The confocal microscopy images and flow cytometry data demonstrated that Lf-PLS resulted in significantly higher cell association by ASGPR positive HepG2 cells compared to PLS. The association between Lf-PLS and cells were dependent on the concentration, time and temperature, which was inhibited by pre-incubation with excessive free Lf. The results suggest that Lf-PLS has a good targeting effect on HepG2 cells in vitro. The targeting mechanism may be related to the specific binding of Lf and ASGPR on HepG2 cells, which guides Lf-PLS to the cell surface to induce an active endocytosis process. All these results demonstrated that Lf-PLS might be a potential drug delivery system in targeting hepatocellular carcinoma, which deserves more research on its targeting ability, antitumor efficiency, and metabolism in vivo for treatment of hepatomacellular carcinoma.

iRGD is a derivative of the integrin-binding peptide RGD, which selectively increases the penetrability of tumor tissue to various coadministered substances in several preclinical models. In this study, we investigated the ability of iRGD to improve the delivery of sorafenib and doxorubicin therapy in hepatocellular carcinoma (HCC) using established mouse models of the disease. A contrast-enhanced MRI method was developed in parallel to assess the in vivo effects of iRGD in this setting. We found that iRGD improved the delivery of marker substances to the tumors of HCC-bearing mice about three-fold without a parallel increase in normal tissues. Control peptides lacking the critical CendR motif had no effect. Similarly, iRGD also selectively increased the signal intensity from tumors in Gd-DTPA-enhanced MRI. In terms of antitumor efficacy, iRGD coadministration significantly augmented the individual inhibitory effects of sorafenib and doxorubicin without increasing systemic toxicity. Overall, our results offered a preclinical proof of concept for the use of iRGD coadministration as a strategy to widen the therapeutic window for HCC chemotherapy, as monitored by Gd-DTPA-enhanced MRI as a noninvasive, clinically applicable method to identify iRGD-reactive tumors., (©2015 American Association for Cancer Research.)

Baicalein (BA), isolated from the Chinese medicinal herb Scutellariae radix (Huangqin in Chinese), is a flavonoid with various pharmacological effects. Herein, we found that BA only slightly reduced the cell viability on HepG2 cells after 24-h treatment as determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. However, BA (50 μM) effectively blocked the colony formation. Meanwhile, BA remarkably induced the formation of autophagosomes after 24-h treatment as determined by immunofluorescence with monodansylcadaverine staining as well as transmission electron microscopy, respectively. Moreover, BA obviously up-regulated the expression of microtubule-associated protein 1A/1B-light chain 3-II in concentration-dependent and time-dependent manners in HepG2 cells. When combined with the autophagy inhibitor chloroquine and BA, the cell viability and colony formation were significantly decreased, indicating that BA triggered protective autophagy, which prevented cell death. Further study showed that BA concentration-dependently and time-dependently decreased the expression of p-AKT (S473), p-ULK1 (S757) and p-4EBP1 (T37 and S65), suggesting the involvement of protein kinase B (AKT)/mammalian target of rapamycin (mTOR) in BA-triggered autophagy., (Copyright © 2015 John Wiley & Sons, Ltd.)

Emerging evidence demonstrates that platelet-derived growth factor-D (PDGF-D) plays a critical role in epithelial-mesenchymal transition (EMT) and drug resistance in hepatocellular carcinoma (HCC) cells. However, the underlying mechanism has not been fully elucidated. The objective is to explore the molecular mechanism of PDGF-D-mediated EMT in drug resistance HCC cells. To achieve our goal, we used multiple approaches including Western blotting, real-time RT-PCR, wound healing assay, invasion assay, luciferase activity assay, transfection, and immunohistochemistry. We found that PDGF-D is highly expressed in gemcitabine-resistant (GR) HCC cells. Moreover, PDGF-D markedly inhibited miR-106a expression and subsequently upregulated Twist1 expression. Notably, PDGF-D expression was associated with miR-106a and Twist1 in HCC patients. Our findings provide a possible molecular mechanism for understanding GR chemoresistance in HCC cells. Therefore, inactivation of PDGF-D/Twist or activation of miR-106a could be a novel strategy for the treatment of HCC.

Objective: To study the effect and mechanism of 4-amino-1, 8-naphthalimide (4-AN) on the sensitive effect of arsenic trioxide (ATO) in hepatocellular carcinoma cells., Methods: Hepatocellular carcinoma HepG2 cells were divided into two groups according to whether they were treated with 4-AN or not. Cell viability was evaluated by MTT assay, population doubling experiment and colony formation assay; genic mechanism was explored by 8-OH-dG assay, single cell gel electrophoresis (comet assay) and microriucleus test., Results: At 2-10 micromol/L concentration of ATO, the cell viability and colony formation efficiency of the combinatio group (4-AN+ATO) were significantly lower than that of the ATO group (P<0.05); moreover, the tail-length (L-Tail) and olive tail moment (OTM) in comet assay were notablely higher than that of the ATO group (P<0.05). At 2-20 micromol/L concentration of ATO, the population doubling time and 8-OH-dG in combination group were significantly higher than that of ATO group (P<0.05). Results from DNA damage repair assay showed that the efficiency of DNA damage repair in combination group was remarkably lower than that of ATO group (P<0.05). At 5-20 micromol/L concentration of ATO, the frequency of micronucleated cells in combination group was significantly higher than that of ATO group (P<0.05)., Conclusion: 4-AN can significantly increase the sensitivity of ATO in treatment with hepatocellular carcinoma cells and prevent DNA damage repair may be a primary mechanism for this effect.

Objective: To investigate the effect of Biejiajian Pills on the expressions of the signal molecules and target genes of Wnt signal pathway in HepG2 cells and explore the mechanisms by which Biejiajian pills suppress the invasiveness of hepatocellular carcinoma., Methods: HepG2 cells were cultured for 48 h in the presence of serum collected from rats fed with Biejiajian Pills. The expressions of β-catenin, GSK-3β and P-GSK-3β in the cultured cells were assessed by Western blotting and the expressions of CD44v6 and VEGF were detected using immunohistochemistry., Results: HepG2 cells cultured with the serum of rats fed with Biejiajian Pills showed lowered expressions of β-catenin protein both in the cytoplasm and the nuclei with also inhibition of phosphorylation of GSK-3β and reduced expression of CD44v6 and VEGF., Conclusion: Biejiajian Pills can significantly reduce the expression of β-catenin by decreasing the phosphorylation of GSK-3β and blocking the Wnt/β-catenin signaling pathway to cause down-regulation of the target genes CD44v6 and VEGF, which may be one of the molecular mechanisms by which Biejiajian Pills suppress the proliferation and invasiveness of hepatocellular carcinoma.

Accelerated senescence is important because this process is involved in tumor suppression and has been induced by many chemotherapeutic agents. The platinum-based chemotherapeutic agent cisplatin displays a wide range of antitumor activities. However, the molecular mechanism of cisplatin-induced accelerated senescence in hepatocellular carcinoma (HCC) remains unclear. In the present study, the growth inhibitory effect of cisplatin on HepG2 and SMMC-7721 cells was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cellular senescence was then assessed by β-galactosidase assay. Senescence-related factors, including p53, p21, and p16, were evaluated by quantitative reverse transcription-polymerase chain reaction. Reactive oxygen species (ROS) was analyzed by flow cytometry. Our results revealed that cisplatin reduced the proliferation of HepG2 and SMMC-7721 cells in a dose- and time-dependent manner. Senescent phenotype observed in cisplatintreated hepatoma cells was dependent on p53 and p21 activation but not on p16 activation. Furthermore, cisplatininduced accelerated senescence depended on intracellular ROS generation. The ROS scavenger N-acetyl-L-cysteine also significantly suppressed the cisplatin-induced senescence of HepG2 and SMMC-7721 cells. In conclusion, our results revealed a functional link between intracellular ROS generation and cisplatin-induced accelerated senescence, and this link may be used as a potential target of HCC.

Natural flavonoids from plants have been demonstrated to possess promising chemopreventive activities against various diseases. 7-{4-[Bis-(2-hydroxy-ethyl)-amino]-butoxy}-5-hydroxy-8-methoxy-2-phenyl-chromen-4-one (V8), a newly synthesized derivative of wogonin may have antioxidant, antiviral, anti-inflammatory and anti-tumor potentials as wogonin. Based on the recent findings of V8, the anti-tumor activities and fundamental mechanisms by which V8 inhibits growth of hepatocellular carcinoma were further investigated in this study. After the treatment of V8, a significant inhibition of HepG2 cell proliferation was observed in a dose-dependent manner with the IC50 value of 23 μM using MTT assay. The exposure to V8 also resulted in apoptosis induction and an accumulation of ROS and Ca(2+). Meanwhile, a release of cytochrome c (Cyt-c), activation of BH-3 only proteins and Bax, decrease in mitochondrial membrane potential ΔΨ, as well as a suppression of Bcl-2, pro-caspase9 and pro-caspase3 expression were shown. Moreover, knocking down CHOP partly decreased the effect of V8-mediated apoptosis and activation of GRP78, p-PERK, p-eIF2α, ATF4 and CHOP modulated ER stress triggered by V8. In vivo, V8 inhibited the transplanted mice H22 liver carcinomas in a dose-dependent manner. Compared with wogonin, V8 exhibited stronger anti-proliferative effects both in vitro and in vivo. The underlying mechanism of activating PERK-eIF2α-ATF4 pathway by which V8 induces apoptosis was verified once again in vivo. The apoptosis induction via the mitochondrial pathway by modulating the ROS-mediated ER signaling pathway might serve to provide support for further studies of V8 as a possible anticancer drug in the clinical treatment of cancer.

Salvia chinensia Benth (S. chinensia) is a medical plant that has been traditionally applied for centuries in the treatment of malignant diseases including hepatocellular carcinoma (HCC). However, the scientific basis underlying its anti-HCC activity has not been fully established. In this study, the chemical profiles of total flavonoids from S. chinensia (TFSC) were explored. Thirteen compounds which constituted the major components of TFSC were separated and identified. Flow cytometry analysis and caspase activity assays showed that TFSC dose-dependently induced HepG2 and Huh-7 HCC cell apoptosis. TFSC was also shown to substantially suppress NF-κB activity in HCC cells. Moreover, TFSC significantly repressed transplanted murine H22 ascitic hepatic cancer cell growth in vivo. Further studies revealed that TFSC induced HCC cell apoptosis and inhibited expressional levels of NF-κB responsive genes in transplanted tumor tissues. In addition, the toxic impact of TFSC on tumor-bearing mice was undetectable. These results indicate that TFSC induces HCC cell apoptosis both in vitro and in vivo. The suppression of cellular NF-κB activity is implicated in the TFSC-mediated HCC cell apoptosis., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Prolonged endoplasmic reticulum (ER) stress may activate apoptotic pathways in cancer cells. It is suggested that ER stress has the potential of enhancing tumor death in cancer therapy. Galangin, a flavonol derived from Alpinia officinarum Hance, has been shown to suppress the proliferation of hepatocellular carcinoma cells (HCC). The aim of this study was to determine whether galangin was able to induce ER stress in HepG2, Hep3B and PLC/PRF/5 cells. The proliferation of HCC was tested by MTT method. Intracellular Ca(2+) levels were measured with Fluo3-AM.The proteins levels of GRP94, GRP78 and CHOP were detected by Western blot. To further understand the anti-HCC mechanism of galangin, mitogen-activated protein kinases (MAPKs) were detected. The results showed that galangin treatment induced ER stress was evidenced by increased protein levels of GRP94, GRP78 and CHOP, as well as increased free cytosolic Ca(2+) concentration. ER stress inhibitor 4-PBA and CHOP siRNA blocked significantly galangin-induced ER in all three cell lines. Further experiments showed that MAPKs involved in ER stress induced by galangin. In summary, galangin is identified as a stimulator of ER stress to suppress the proliferation of HCC, and may be used as a potential anti-cancer agent., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Toll-like receptors (TLRs) play a pivotal role in innate immunity, controlling inflammatory responses, and further development of adaptive immunity. Hepatitis virus can establish chronic infection, and the associated inflammatory responses are important determinants of virus-associated liver damage. However, the contributions of the host immune system to chronic presence of virus are not clear in patients with hepatitis virus infection. Chronic inflammatory conditions caused by persistent hepatitis virus infections and interferon (IFN)-γ-related immunopathology are known to be related to carcinogenesis. To gain insight into the role of immune modulation in the pathogenesis of hepatocellular carcinoma (HCC), we studied the expression of TLR7 in cancerous and non-cancerous liver tissue from 87 patients with HCC. Our results showed that TLR7 is significantly down-regulated in neoplastic hepatocytes (P < .001), especially in the patients with hepatitis B (n = 52) or C (n = 24) virus infection. We confirmed this decreased TLR7 expression by quantitative analysis of mRNA using real-time reverse transcription-polymerase chain reaction in 26 liver specimens of HCC patients. Using serial deletion analysis of the TLR7 promoter, a hepatocyte-specific regulatory region was found at nucleotides -156 to -98 in the TLR7 promoter. Furthermore, the effects of IFN-γ on TLR7 expression in a hepatoma cell line (HepG2) were investigated in vitro. We demonstrated that IFN-γ significantly decreased TLR7 promoter activity and expression in a dose-dependent manner. We thus propose that hepatitis virus induces down-regulation of TLR7 gene expression through IFN-γ, thereby modulating inflammatory signaling in hepatoma cells., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Hepatocellular carcinoma (HCC) is the most common sort of primary liver malignancy with poor prognosis. This study aimed at examining the effects of silibinin (a putative antimetastatic agent) on some transcriptional markers mechanistically related to HCC recurrence and metastasis in HepG-2 [hepatitis B virus (HBV)-negative and P53 intact) and PLC/PRF/5 (HBV-positive and P53 mutated) cells. The expression of 27 genes in response to silibinin was evaluated by real-time RT-PCR. The MMP gelatinolytic assay and microculture tetrazolium test (MTT) were tested. Silibinin was capable of suppressing the transcriptional levels of ANGPT2, ATP6L, CAP2, CCR6, CCR7, CLDN-10, cortactin, CXCR4, GLI2, HK2, ID1, KIAA0101, mortalin, PAK1, RHOA, SPINK1, and STMN1 as well as the enzymatic activity of MMP-2 but promoted the transcripts of CREB3L3, DDX3X, and PROX1 in both cells. Some significant differences between the cells in response to silibinin were detected that might be related to the differences of the cells in terms of HBV infection and/or P53 mutation, suggesting the possible influence of silibinin on HCC through biological functions of these 2 prognostic factors. In conclusion, our findings suggest that silibinin could potentially function as a multitargeting antimetastatic agent and might provide new insights for HCC therapy particularly for HBV-related and/or P53-mutated HCCs.

Aims: MicroRNA-1 (miR-1) has been demonstrated as a tumor-suppressive miRNA, which shows a down-regulated pattern in several human malignancies including hepatocellular carcinoma (HCC). However, the pathophysiologic roles of miR-1 and their mechanisms in HCC tumorigenesis are still not totally elucidated., Main Methods: Pre-miR-1 was cloned into pSuper plasmid to overexpress the miR-1 in hepatoma cells. Real-time PCR and Western blot were applied to detect miR-1, ET-1 mRNA and protein levels respectively. Dual luciferase reporter assay was conducted to investigate the binding site of miR-1 on 3'UTR of ET-1 mRNA. Proliferation of hepatoma cells was evaluated by MTT assay., Key Findings: We observed that over-expression of miR-1 by miRNA-expressing plasmid transfection in HepG2 and Hep3B cells significantly reduced the proliferation of these cells. To explore the mechanism, we examined the potential target genes of miR-1 by bioinformatics. A potent mitogen, Endothelin-1 (ET-1), attracted our attention. Elevated expression of ET-1 but reduced miR-1 level was detected both in human liver cancer tissues and in hepatoma cell lines using Western Blot and miRNA real-time PCR respectively. By the over-expression and inhibition of miR-1 in HepG2 and Hep3B, we confirmed that miR-1 negatively regulated ET-1 expression in hepatoma cells. A luciferase reporter assay showed that miR-1 regulation was established by pairing to a complementary binding site within the ET-1 3'UTR. Finally, attenuated proliferation of hepatoma cells by over-expression of miR-1 could be partially restored by exogenous ET-1 treatment., Significance: Our findings demonstrate that miR-1 could inhibit ET-1 expression to attenuate the proliferation of hepatoma cells., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The mechanisms underlying the antiproliferative and antitumor activities of aromatic turmerone (ar-turmerone), a volatile turmeric oil isolated from Curcuma longa Linn., have been largely unknown. In this study, 86% pure ar-turmerone was extracted by supercritical carbon dioxide and liquid-solid chromatography and its potential effects and molecular mechanisms on cell proliferation studied in human hepatocellular carcinoma cell lines. Ar-turmerone exhibited significant antiproliferative activity, with 50% inhibitory concentrations of 64.8 ± 7.1, 102.5 ± 11.5, and 122.2 ± 7.6 μg/mL against HepG2, Huh-7, and Hep3B cells, respectively. Ar-turmerone-induced apoptosis, confirmed by increased annexin V binding and DNA fragmentation, was accompanied by reactive oxygen species (ROS) production, mitochondrial membrane potential dissipation, increased Bax and p53 up-regulated modulator of apoptosis (PUMA) levels, Bax mitochondrial translocation, cytochrome c release, Fas and death receptor 4 (DR4) augmentation, and caspase-3, -8, and -9 activation. Exposure to caspase inhibitors, Fas-antagonistic antibody, DR4 antagonist, and furosemide (a blocker of Bax translocation) effectively abolished ar-turmerone-triggered apoptosis. Moreover, ar-turmerone stimulated c-Jun N-terminal kinase (JNK) and extracellular signal-related kinase (ERK) phosphorylation and activation; treatment with JNK and ERK inhibitors markedly reduced PUMA, Bax, Fas, and DR4 levels and reduced apoptosis but not ROS generation. Furthermore, antioxidants attenuated ar-turmerone-mediated ROS production; mitochondrial dysfunction; JNK and ERK activation; PUMA, Bax, Fas, and DR4 expression; and apoptosis. Taken together, these results suggest that ar-turmerone-induced apoptosis in HepG2 cells is through ROS-mediated activation of ERK and JNK kinases and triggers both intrinsic and extrinsic caspase activation, leading to apoptosis. On the basis of these observations, ar-turmerone deserves further investigation as a natural anticancer and cancer-preventive agent.

Objective: To investigate the anticancer activity of Polyalthia evecta (P. evecta) (Pierre) Finet & Gagnep against human hepatoma cell line (HepG2)., Methods: The anticancer activity was based on (a) the cytotoxicity against human hepatoma cells (HepG2) assessed using a neutral red assay and (b) apoptosis induction determined by evaluation of nuclei morphological changes after DAPI staining. Preliminary phytochemical analysis of the crude extract was assessed by HPLC analysis., Results: The 50% ethanol-water crude leaf extract of P. evecta (EW-L) showed greater potential anticancer activity with high cytotoxicity [IC50 = (62.8 ± 7.3)µg/mL] and higher selectivity in HepG2 cells than normal Vero cells [selective index (SI) = 7.9]. The SI of EW-L was higher than the positive control, melphalan (SI = 1.6) and the apoptotic cells (46.4 ± 2.6) % induced by EW-L was higher than the melphalan (41.6 ± 2.1)% (P<0.05). The HPLC chromatogram of the EW-L revealed the presence of various kinds of polyphenolics and flavonoids in it., Conclusions: P. evecta is a potential plant with anticancer activity. The isolation of pure compounds and determination of the bioactivity of individual compounds will be further performed.

Hep88 mAbs, a novel monoclonal antibodies against hepatocellular carcinoma cell line from Thai patient, has been proved earlier for its tumoricidal effect on HepG2 cell line. In the present study, we investigated not only Hep88 mAb's targeted proteins from HepG2 cell line by western blot analysis but also its inhibitory activity on those cells by MTT assay. Moreover the ultrastructural alteration induced by Hep88 mAb of HepG2 cell line compare with Chang liver cell line was also examined. The results demonstrated that Hep88 mAb had cytotoxic effect on HepG2 cell line but not Chang liver cell line. Additionally, recognizing proteins against Hep88 mAb have been found on both cell lines. The ultrastructural alteration detected from transmission electron microscopy included the appearing of intracellular vacuolization as well as the dilatation of endoplasmic reticulum and mitochondria have been observed. These findings are suggested that the death of HepG2 cell line after treatment with Hep88 mAb might be involved by an apoptosis-like program cell death (PCD) pathway. From all of these remarks, it is possible that Hep88 mAb can injure HCC cells by binding with its membrane-bound antigen and activated downstream intracellular signals which is finally leading cell to be death via apoptosis-like PCD.

Aims and Background: Previous studies showed that dihydroartemisinin (DHA) possessed antitumor activity in many human tumor cells through the induction of apoptosis. The aim of this study was to investigate the effects of DHA on apoptosis in the human hepatocellular carcinoma cell line HepG2 and the possible molecular mechanisms involved., Methods: The inhibitory effect of DHA on HepG2 cells was measured by MTT assay. The percentage of apoptotic cells was detected by flow cytometry with double staining of fluorescein isothiocyanate-annexin V/propidium iodide. The intracellular production of reactive oxygen species (ROS) and intracellular Ca2+ concentration ([Ca2+]i) were detected by fluorescence spectrophotometry. Protein expression of GADD153, Bcl-2 and Bax in HepG2 cells was examined by Western blot and immunocytochemistry., Results: DHA significantly inhibited proliferation of HepG2 cells in a dose- and time-dependent manner. The apoptosis rates in HepG2 cells treated with 0, 50, 100 and 200 μmol/L DHA for 24 hours were 2.53 ± 0.88%, 24.85 ± 3.63%, 35.27 ± 5.92% and 48.53 ± 7.76%, respectively. Compared with the control group, DHA significantly increased ROS generation and [Ca2+]i level (P <0.05), with the generation of ROS preceding the increase in [Ca2+]i. An increase in GADD153 and Bax expression and a decrease in Bcl-2 were observed in DHA-treated cells. Pretreatment with the antioxidant N-acetylcysteine could attenuate the effects of DHA in the experiments., Conclusion: DHA could inhibit proliferation and induce apoptosis in HepG2 cell lines through increasing the intracellular production of ROS and [Ca2+]i. Endoplasmic reticulum stress-induced apoptosis may contribute to this effect by regulating the expression of GADD153, proapoptotic Bax, and antiapoptotic Bcl-2.

Objective: To observe the effect of SSd on reversing the malignant phenotype of HepG2 cells and to investigate its mechanism in order to prove that SSd is a new choice to prevent and treat HCC., Methods: HepG2 cells were cultured and treated by different concentration (0 mg/L, 2.5 mg/L, 5.0 mg/L, 10.0 mg/L and 20.0 mg/L) of SSd for 24 h, and treated by 10 mg/L of SSd for 0 h, 6 h, 12 h, 24 h, 48 h and 72h respectively. The cell inhibition rates were measured by MTT assay. Then cells were treated by 10 mg/L SSd for 48 hr in experimental group and treated by no SSd as a control, their morphological changes were observed by contrast phase microscope. The concentrations of ALB and AFP in clear supernatant liquid of cells were detected by radio-immunity and chemiluminescence. The cell migration rates were observed by transwell method, the relative expression levels of p27 mRNA were measured by RT-PCR., Results: The inhibitive effect of 10 mg/L SSd was the most significant among different concentrations ( F = 265.06, P less than 0.01). The shape of HepG2 from experimental group turned into small and round, and their volume ratios of nucleus to plasma decreased. ALB in supernatant liquid of HepG2 was higher ( t = 7.83, P less than 0.05, and its AFP was lower ( t = -10.72, P less than 0.01) as compared to control group. Cells migrated were fewer and p27 mRNA expression of HepG2 was higher in experimental group than that in control group (t = 22.00, P less than 0.05)., Conclusion: SSd could reverse the malignant phenotype of HepG2 cells. It was suggested that the up-regulation of p27 mRNA expression play an important role in the differentiation of HepG2 cells treated by SSd.

Aim: To investigate whether the apoptotic activities of 8-bromo-7-methoxychrysin (BrMC) involve reactive oxygen species (ROS) generation and c-Jun N-terminal kinase (JNK) activation in human hepatocellular carcinoma cells (HCC)., Methods: HepG2, Bel-7402 and L-02 cell lines were cultured in vitro and the apoptotic effects of BrMC were evaluated by flow cytometry (FCM) after propidium iodide (PI) staining, caspase-3 activity using enzyme-linked immunosorbent assay (ELISA), and DNA agarose gel electrophoresis. ROS production was evaluated by FCM after dichlorodihydrofluorescein diacetate (DCHF-DA) probe labeling. The phosphorylation level of JNK and c-Jun protein was analyzed by Western blotting., Results: FCM after PI staining showed a dose-dependent increase in the percentage of the sub-G1 cell population (P < 0.05), reaching 39.0% +/- 2.8% of HepG2 cells after 48 h of treatment with BrMC at 10 micromol/L. The potency of BrMC to HepG2 and Bel-7402 (32.1% +/- 2.6%) cells was found to be more effective than the lead compound, chrysin (16.2% +/- 1.6% for HepG2 cells and 11.0% +/- 1.3% for Bel-7402 cell) at 40 micromol/L and similar to 5-fluorouracil (33.0% +/- 2.1% for HepG2 cells and 29.3% +/- 2.3% for Bel-7402 cells) at 10 micromol/L. BrMC had little effect on human embryo liver L-02 cells, with the percentage of sub-G1 cell population 5.4% +/- 1.8%. Treatment of HepG2 cells with BrMC for 48 h also increased the levels of active caspase-3, in a concentration-dependent manner. z-DEVD-fmk, a caspase-3-specific inhibitor, prevented the activation of caspase-3. Treatment with BrMC at 10 micromol/L for 48 h resulted in the formation of a DNA ladder. Treatment of cells with BrMC (10 micromol/L) increased mean fluorescence intensity of DCHF-DA in HepG2 cells from 7.2 +/- 1.12 at 0 h to 79.8 +/- 3.9 at 3 h and 89.7 +/- 4.7 at 6 h. BrMC did not affect ROS generation in L-02 cells. BrMC treatment failed to induce cell death and caspase-3 activation in HepG2 cells pretreated with N-acetylcysteine (10 mmol/L). In addition, in HepG2 cells treated with BrMC (2.5, 5.0, 10.0 micromol/L) for 12 h, JNK activation was observed. Peak JNK activation occurred at 12 h post-treatment and this activation persisted for up to 24 h. The expression of phosphorylated JNK and c-Jun protein after 12 h with BrMC-treated cells was inhibited by N-acetylcysteine and SP600125 pre-treatment, but GW9662 had no effect. SP600125 substantially reduced BrMC-induced cell death and caspase-3 activation of HepG2 cells. N-acetylcysteine and GW9662 also attenuated induction of cell death and caspase-3 activation in HepG2 cells treated with BrMC., Conclusion: BrMC induces apoptosis of HCC cells by ROS generation and sustained JNK activation.

Hepatocellular carcinoma (HCC) is resistant to chemotherapy. Recently, however, several oxaliplatin-based combinatorial treatments have shown a promising anti-tumor activity in patients with HCC. Presently, we demonstrate that oxaliplatin triggers necrosis more than apoptosis in HepG2, SK-Hep1, SNU-423 and Hep3B HCC cells, while mainly inducing apoptosis in HCT116 and HT29 colon cancer cells. Interestingly, ursodeoxycholic acid (UDCA), a less hydrophobic bile acid that can suppress carcinogenesis, shifted oxaliplatin-induced necrosis to apoptosis in HepG2 cells. The same effect was produced by hydrophilic bile acids (tauroursodeoxycholic acid and taurohyodeoxycholic acid), but not by highly hydrophobic bile acids (deoxycholic acid and chenodeoxycholic acid). UDCA also triggered the necrosis-to-apoptosis switch when cotreated with other platinum-based chemotherapeutic drugs including cisplatin and carboplatin, suggesting that the cell death mode switching effect of UDCA is a general phenomenon when combined with platinum drugs. Oxaliplatin produced high level of reactive oxygen species (ROS) in HepG2 cells and UDCA significantly reduced oxaliplatin-induced ROS generation. In addition, N-acetyl-L-cysteine and the superoxide scavengers butylated hydroxyanisole and dihydroxybenzene-3,5-disulfonic acid attenuated necrosis, indicating a critical role(s) of ROS in occurrence of necrotic death. Apoptosis induced by combined treatment appeared to be mediated by p53-caspase 8-caspase 3 pathway. In conclusion, UDCA switches oxaliplatin-induced necrosis to apoptosis via inhibition of ROS production and activation of the p53-caspase 8 pathway in HepG2 cells. As necrosis and subsequent inflammation are implicated in tumor progression and malignancy, our results imply a potential improved efficacy of UDCA-combined chemotherapy in HCC by reducing inflammatory responses that may be triggered by oxaliplatin.

Background/aims: The use of chemotherapy in hepatocellular carcinoma is still controversial. The aim of this study was to investigate whether the combined use of epirubicin and progesterone has a synergistic effect on cell proliferation and apoptosis in HepG2 cells., Methodology: Different concentrations of epirubicin (0.1 microg/ml, 0.25 microg/ml and 0.5microg/ml) or progesterone (12.5 microM, 25 microM and 50 microM) were added to HepG2 cells either alone or in combinations consisting of different concentrations of the two. Their effects on HepG2 cells were studied by (1) XTT assay for analysis of cell proliferation, (2) 3H-Thymidine incorporation for DNA synthesis, (3) annexin V-FITC/ propidium iodide (PI) flowcytometery for cell apoptosis, (4) flowcytometry for cell cycle distributions, and (5) reverse transcription-polymerase chain reaction for expression of cell cycle modulator, cyclin D1., Results: 50 microM progesterone increased both the cytotoxic and apoptotic effects of 0.1 microg/ml epirubicin on HepG2 cells at 48 hr culture due to 50 microM progesterone accumulated cells in S phase of the cell cycle and subsequently reduced cyclin D1 expression. These effects on HepG2 cells induced by this combination were comparable to those induced by 0.5 microg/ml epirubicin alone., Conclusions: In vitro, progesterone can increase the cytotoxicity and apoptosis induced by epirubicin on HepG2 cells.