chrysin and Liver-Neoplasms

The study aims to investigate the combined effects of chrysin and cisplatin on hepatoma(HepG2) cell lines in vivo and in vitro.. Studies have suggested that chrysin can enhance the sensitivity of tumor cells to apoptosis. Drug resistance in tumor cells reduced the effectiveness of chemotherapy drugs such as cisplatin. We investigated whether the combination of chrysin and cisplatin can induce more apoptosis than chrysin alone and cisplatin alone.. HepG2 cells were pretreated with chrysin for 2 h, followed by the addition of cisplatin for another 24 h. The morphologic changes were observed under inverted microscope and the cell viability was measured using the MTT test. The protein and cleavage of caspase-3,8,9, PARP, and cFLIP were determined by Western blotting.. The cell viability of the HepG2 cell can be reduced by the combination of chrysin pretreatment for 2 h and cisplatin addition for 24 h; Caspase-3,8,9 and PARP were cleaved after 12 h treatment with chrysin and cisplatin; Pancaspase inhibitor, Z-VAD-fmk, could reverse the apoptosis induced by chrysin and cisplatin in HepG2 cells; cFLIP was down-regulated by the combination of chrysin and cisplatin, and could be reversed by Z-VAD-fmk; the xenografted HepG2 cells formed a tumor in one week; At the end of the experiment, there were significant differences in relative tumor volume (RTV) and relative tumor proliferation rate between the combined group and the control group, the chrysin group and the cisplatin group; Western blotting showed that the levels of PARP, cFLIP, and caspase-3 proteins in isolated tumor tissues also decreased under the combined action of chrysin and cisplatin.. The combination of chrysin and cisplatin induces apoptosis of hepatic tumor in vivo and in vitro. It downregulates cFLIP and then activates caspase-8, which triggers caspase-mediated apoptosis of HepG2 cell.

Topics: Apoptosis; Caspase 3; Caspases; Cell Line, Tumor; Cisplatin; Down-Regulation; Humans; Liver Neoplasms; Poly(ADP-ribose) Polymerase Inhibitors

The aberrant PD-L1 expression on cancer cells was confirmed to participate in immune evasion of hepatocellular carcinoma (HCC). Previous studies had documented that there were anti-tumorigenic effects of chrysin on HCC. However, whether chrysin can act on the over-expressed PD-L1 on HCC cells to exert the therapeutic effectiveness and the involved mechanisms has not yet been deciphered.. Herein, we aimed to explore the regulatory effects of chrysin on the PD-1/PD-L1 immune checkpoint and investigate its possible mechanisms in vivo and in vitro.. H22 xenograft mouse model was used to investigate the effects of chrysin on tumor growth and PD-L1 expression in tumors. In interferon-gamma (IFN-γ)-induced HepG2 cells, the cytotoxicity of chrysin was detected by MTT assay. Flow cytometry, ELISA and RT-PCR were carried out to evaluate the expression of PD-L1, and the expression of proteins in STAT3 and NF-κB pathways was also determined by Western blot. In HepG2 cells and Jurkat T cell co-culture system, ELISA kit was used to detect the level of IL-2, and T cell proliferation was further evaluated by CCK-8 method.. Our data suggested that chrysin could effectively inhibit the progression of tumor, and promote the anti-tumor immunity of mice concomitant with enhanced CD4/CD8-positive T cell proportion in tumor tissues of H22 xenograft mouse model. Additionally, chrysin significantly down-regulated the expression of PD-L1 in vivo and in vitro, which was closely associated with the blockage of STAT3 and NF-κB pathways. Moreover, in the co-culture system, chrysin could increase the proliferation of T cells and the concentration of IL-2.. These results indicate that chrysin may have the potential to be an immune checkpoint inhibitor for preventive or as an adjunctive curative agent for HCC.

Topics: Animals; B7-H1 Antigen; Carcinoma, Hepatocellular; Cell Line, Tumor; Flavonoids; Hep G2 Cells; Humans; Liver Neoplasms; Mice

The natural flavonoid chrysin possesses antiproliferative activity against various types of cancers, including hepatocellular carcinoma (HCC), which is a common malignancy. However, the exact mechanism of chrysin antiproliferative activity remains unclear. This research was executed to explore the impact of chrysin on glypican-3 (GPC3)/sulfatase-2 (SULF2) axis and lncRNA-AF085935 expression in HCC using HepG2 cells. Cisplatin (20, 50, 100 μg/mL), chrysin (15, 30, and 60 μg/mL) and the combination of 50 μg/mL cisplatin with different concentrations of chrysin were applied for 24/48 h. Cell viability was determined by MTT assay. Protein levels of GPC3 and SULF2 were measured by ELISA at 24/48 h. GPC3 immunoreactivity was detected by immunocytochemistry. Moreover, GPC3 and SULF2 mRNA expressions in addition to lncRNA-AF085935 expression were assessed by qPCR at 48 h. The GPC3 protein, immunostaining and mRNA levels, SULF2 protein and mRNA levels, as well as lncRNA-AF085935 expression, were decreased significantly with cisplatin and chrysin alone when compared with the control untreated HepG2 cells. However, the combination treatment exhibited a better chemopreventive effect in a dose- and time-dependent manner. This study demonstrated, for the first time, the antiproliferative activity of chrysin against HCC through the suppression of the GPC3/SULF2 axis along with the downregulation of lncRNA-AF085935 expression. Synergistic effect of chrysin with cisplatin could potentiate their antiproliferative action in a dose- and time-dependent manner.

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Proliferation; Flavonoids; Glypicans; Hep G2 Cells; Humans; Liver Neoplasms; RNA, Long Noncoding; Sulfatases

Flavonoids are natural compounds that show various biological effects, such as the anti-cancer effect. Chrysin is a flavonoid compound found in honey and propolis. Studies have shown that chrysin has anti-cancer activity due to induction of apoptosis signaling. In the present study, we examined the cytotoxic effect of chrysin against liver mitochondria obtained from the hepatocellular carcinoma (HCC) rat model. Diethylnitrosamine (DEN) and 2-acetylaminofluorene (2-AAF) was used for induction of HCC. Mitochondria were isolated from liver hepatocytes using differential centrifugation. Then, hepatocytes and mitochondria markers related to apoptosis signaling were investigated. Our finding indicated an increase in mitochondrial reactive oxygen species (ROS) generation, collapse in the mitochondrial membrane potential (MMP), swelling in mitochondria, and cytochrome c release (about 1.6 fold) after exposure of mitochondria obtained from the HCC rats group with chrysin (10, 20, and 40 µM) compared to the normal rats group. Furthermore, Chrysin was able to increase caspase-3 activity in the HCC rats group (about 2.4 fold) compared to the normal rats group. According to the results, we proposed that chrysin could be considered as a promising complementary therapeutic candidate for the treatment of HCC, but it requires a further in vivo and clinical studies.

Topics: Animals; Carcinoma, Hepatocellular; Cytochromes c; Dose-Response Relationship, Drug; Flavonoids; Gene Expression Regulation; Liver Neoplasms; Male; Mitochondria, Liver; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Succinate Dehydrogenase

Screening active components from Chinese traditional medicine is an effective approach to discover new drugs or active structures. Cell membrane chromatography (CMC), developed rapidly because of its high sensitivity and effectiveness, has achieved a wide application in screening active components on pathological cells or tissues. However, it is hard to clarify the selectivity between pathological and normal tissues through simply using pathological cells. In this study, a novel comparative two-dimensional (2D) cell membrane chromatography system was established. Briefly, hepatic carcinoma HepG2 CMC columns and normal hepatic L02 CMC columns were simultaneously loaded to screen potential selective antitumor components from Scutellariae Radix by comparing the retention behaviors on two kinds of cells. Totally 13 components in Scutellariae Radix retained on both HepG2/ CMC and L02/ CMC columns. Among them, three components, oroxylin A, wogonin and chrysin, were screened out to perform stronger affinity on HepG2 columns, and in further cell proliferation assay, IC

Topics: Antineoplastic Agents, Phytogenic; Carcinoma, Hepatocellular; Cell Membrane; Cell Proliferation; Chromatography, High Pressure Liquid; Drug Screening Assays, Antitumor; Drugs, Chinese Herbal; Flavanones; Flavonoids; Hep G2 Cells; Humans; Inhibitory Concentration 50; Liver Neoplasms; Mass Spectrometry; Scutellaria baicalensis

Sorafenib is now standard treatment for advanced hepatocellular carcinoma (HCC). However, therapeutic efficacy is not as good as was predicted. Many efforts are being made to improve HCC sensitivity to sorafenib. Our previous study demonstrated that co-treatment with chrysin enhanced sorafenib sensitivity through inhibition of ATP-binding cassette super-family G member 2 (ABCG2). Whether there is another mechanism other than inhibition of ABCG2 underlying chrysin-mediated synergistic effect is still not completely elucidated.. Phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) was examined by western blot. Cell viability was examined by crystal violet staining. The importance of ERK1/2 phosphorylation was assessed by overexpression and blockage of mitogen-activated protein kinase kinase 1 (MEK1).. Chrysin induced sustained ERK1/2 phosphorylation of HCC cells in both time- and dose-dependent manners. Overexpression of MEK1 enhanced, whereas blockage of MEK1 led to loss of chrysin-synergized sorafenib effect, through modulating ERK1/2 phosphorylation level.. These results identify another novel mechanism underlying chrysin-mediated synergistic effect on sorafenib activity in HCC cells.

Topics: Adenosine Triphosphate; Antineoplastic Combined Chemotherapy Protocols; ATP Binding Cassette Transporter, Subfamily G, Member 2; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Drug Synergism; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Hep G2 Cells; Humans; Liver Neoplasms; Neoplasm Proteins; Phosphorylation; Sorafenib

Topics: Andrographis; Antineoplastic Agents, Phytogenic; Apigenin; Apocynaceae; Asia, Southeastern; Biflavonoids; Bignoniaceae; Catechin; Diterpenes; Ethnopharmacology; Flavanones; Flavonoids; Hep G2 Cells; Humans; Lamiaceae; Liver Neoplasms; Metabolomics; Plants, Medicinal; Proanthocyanidins

To study the effect of chrysin in inducing apoptosis of human hepatic carcinoma cells and explore the possible mechanism.. Human hepatic carcinoma SMMC-7721 cells treated with DMSO or chrysin at different concentrations (5-200 μg/mL) were examined for changes in the cell proliferation using CCK-8 assay. The morphological changes of SMMC-7721 cells were observed in response to treatment with 5, 10, or 20 μg/mL chrysin, and the changes in the cell nuclei were observed using DAPI nuclear staining. Annexin Ⅴ-FITC/PI flow cytometry was used to determine the cell apoptosis rate. The changes in the apoptosis-related proteins (PARP and caspase-3) and MAPKs signal pathway were detected with Western blotting.. Chrysin treatment obviously suppressed the proliferation of SMMC-7721 cells in a dose-dependent manner below the concentration of 60 μg/mL. Chrysin (20 μg/mL) also caused significantly increased cell apoptosis and significant cleavage of PARP and caspase-3. Chrysin significantly activated MAPKs signaling pathway in a time-and dose-dependent manner, with the peak activation level occurring at 15 min. Pretreatment of the cells with specific inhibitors of the MAPKs pathway obviously inhibited the effect of chrysin in inducing cell apoptosis.. Chrysin inhibits the proliferation and promotes apoptosis of SMMC-7721 cells by regulating the activation of MAPKs signaling.

Topics: Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Dimethyl Sulfoxide; Dose-Response Relationship, Drug; Flavonoids; Humans; Liver Neoplasms; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Signal Transduction

Hexokinase-2(HK-2) plays dual roles in glucose metabolism and mediation of cell apoptosis, making it an attractive target for cancer therapy. Chrysin is a natural flavone found in plant extracts which are widely used as herb medicine in China. In the present study, we investigated the antitumor activity of chrysin against hepatocellular carcinoma (HCC) and the role of HK-2 played for chrysin to exert its function.. The expression of HK-2 in HCC cell line and tumor tissue was examined by western blotting and immunohistochemistry staining. The activities of chrysin against HCC cell proliferation and tumor glycolysis were investigated. Chrysin-induced apoptosis was analyzed by flow cytometry. The effect of chrysin on HK-2 expression and the underlying mechanisms by which induced HCC cell apoptosis were studied. In HK-2 exogenous overexpression cell, the changes of chrysin-induced cell apoptosis and glycolysis suppression were investigated. HCC cell xenograft model was used to confirm the antitumor activity of chrysin in vivo and the effect on HK-2 was tested in chrysin-treated tumor tissue.. In contrast with normal cell lines and tissue, HK-2 expression was substantially elevated in the majority of tested HCC cell lines and tumor tissue. Owing to the decrease of HK-2 expression, glucose uptake and lactate production in HCC cells were substantially inhibited after exposure to chrysin. After chrysin treatment, HK-2 which combined with VDAC-1 on mitochondria was significantly declined, resulting in the transfer of Bax from cytoplasm to mitochondria and induction of cell apoptosis. Chrysin-mediated cell apoptosis and glycolysis suppression were dramatically impaired in HK-2 exogenous overexpression cells. Tumor growth in HCC xenograft models was significantly restrained after chrysin treatment and significant decrease of HK-2 expression was observed in chrysin-treated tumor tissue.. Through suppressing glycolysis and inducing apoptosis in HCC, chrysin, or its derivative has a promising potential to be a novel therapeutic for HCC management, especially for those patients with high HK-2 expression.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Survival; Flavonoids; Gene Expression Regulation, Neoplastic; Glycolysis; Hep G2 Cells; Hexokinase; Humans; Liver Neoplasms; Mice; Xenograft Model Antitumor Assays

A series of flavone-7-phosphoramidate derivatives were synthesized and tested for their antiproliferative activity in vitro against human hepatoma cell line HepG2 and human normal hepatic cell line L-O2. Compound 8d, 16d and 17d, incorporating the amino acid alanine, exhibited high inhibitory activity on HepG2 cell line with IC50 values of 9.0 μmol/L, 5.5 μmol/L and 6.6 μmol/L. The introduction of acyl groups played a pivotal role in the selective inhibition toward human hepatoma HepG2 cells, except for compound 8a, 9a and 16b. Compound 8d, 16d and 17d could significantly induce G2/M arrest in HepG2 cells. Specially, Compound 16d could lead early apoptosis in HepG2 cells.

Topics: Amides; Antineoplastic Agents; Apoptosis; Cell Cycle Checkpoints; Cell Line; Cell Proliferation; Flavones; Hep G2 Cells; Humans; Liver; Liver Neoplasms; Phosphoric Acids

The composition of Morinda citrifolia (M. citrifolia) was determined using high-performance liquid chromatography (HPLC), and the anticancer effects of M. citrifolia extract evaluated in HepG2, Huh7, and MDA-MB-231 cancer cells. M. citrifolia fruit extracts were obtained by using five different organic solvents, including hexane (Hex), methanol (MeOH), ethyl acetate (EtOAc), chloroform (CHCl3), and ethanol (EtOH). The water-EtOAc extracts from M. citrifolia fruits was found to have the highest anticancer activity. HPLC data revealed the predominance of chrysin in water-EtOAc extracts of M. citrifolia fruit. Furthermore, the combined effects of cotreatment with apigenin and chrysin on liver and breast cancer were investigated. Treatment with apigenin plus chrysin for 72-96 h reduced HepG2 and MDA-MB-231 cell viability and induced apoptosis through down-regulation of S-phase kinase-associated protein-2 (Skp2) and low-density lipoprotein receptor-related protein 6 (LRP6) expression. However, the combination treatment for 36 h synergistically decreased MDA-MB-231 cell motility but not cell viability through down-regulation of MMP2, MMP9, fibronectin, and snail in MDA-MB-231 cells. Additionally, chrysin combined with apigenin also suppressed tumor growth in human MDA-MB-231 breast cancer cells xenograft through down-regulation of ki-67 and Skp2 protein. The experimental results showed that chrysin combined with apigenin can reduce HepG2 and MDA-MB-231 proliferation and cell motility and induce apoptosis. It also offers opportunities for exploring new drug targets, and further investigations are underway in this regard.

Topics: Animals; Apigenin; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Drug Synergism; Female; Flavonoids; Fruit; Humans; Liver Neoplasms; Low Density Lipoprotein Receptor-Related Protein-6; Matrix Metalloproteinase 2; Mice; Mice, Inbred BALB C; Mice, Nude; Morinda; Plant Extracts; S-Phase Kinase-Associated Proteins

Chrysin is a natural and biologically active flavonoid with anticancer effects. However, little is known about the adaptive response of cancer cells to chrysin. Chrysin reportedly has proteasome inhibitor activity. Previous studies demonstrated that proteasome inhibitors might induce endoplasmic reticulum (ER) stress response. In this study, we aimed to determine the effects of chrysin on hepatoma cells and roles of the ER-resident protein GRP78 (glucose-regulated protein 78) in its action. Also, we investigated the effects of green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG), a natural GRP78 inhibitor, on the sensitivity of hepatoma cells to chrysin. Here, we report that chrysin inhibits hepatoma cells growth and induces apoptosis in a dose-dependent manner. Chrysin induces GRP78 overexpression, X-box binding protein-1 splicing and eukaryotic initiation factor 2α phosphorylation, hallmarks of the unfolded protein response. GRP78 knockdown potentiates chrysin-induced caspase-7 cleavage in hepatoma cells and enhances chrysin-induced apoptosis. EGCG overcomes chrysin-induced GRP78 expression. Combination of EGCG potentiates chrysin-induced caspase-7 and poly (ADP-ribose) polymerase (PARP) cleavage. Finally, EGCG sensitizes hepatoma cells to chrysin through caspase-mediated apoptosis. These data suggest that chrysin triggers the unfolded protein response. Abrogation of GRP78 induction may improve the anticancer effects of chrysin. Combination of EGCG and chrysin represents a new regimen for cancer chemoprevention and therapeutics.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Caspase 7; Catechin; Cell Line, Tumor; Cell Proliferation; DNA-Binding Proteins; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Eukaryotic Initiation Factor-2; Flavonoids; Heat-Shock Proteins; Hep G2 Cells; Humans; Liver Neoplasms; Phosphorylation; Poly(ADP-ribose) Polymerases; Proteasome Inhibitors; Regulatory Factor X Transcription Factors; RNA Interference; Transcription Factors; Unfolded Protein Response

A series of long-chain derivatives of chrysin (compounds 3-22) were synthesized to evaluate for their antiproliferative activities against the human liver cancer cell line HT-29 and EGFR inhibitory activity. Among the compounds tested, compounds hexadecyl 2-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yloxy)acetate (10) and N-hexadecyl 2-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yloxy)acetamide (20) displayed potent EGFR inhibitory activity with IC(50) values of 0.048 microM and 0.035 microM), comparable to the positive control erlotinib. Docking simulation of compounds 10 and 20 was carried out to illustrate the binding mode of the molecular into the EGFR active site, and the result suggested that compound 10 and 20 can bind the EGFR kinase well. Thus, compounds 10 and 20 with potent EGFR inhibitory activity would be potential anticancer agents.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Drug Screening Assays, Antitumor; ErbB Receptors; Flavonoids; Humans; Liver Neoplasms; Models, Molecular; Protein Binding

The constitutive androstane receptor (CAR) is known as a xeno-sensor that regulates genes involved in xenobiotic excretion and energy metabolism. This study tested a variety of polyphenols for their ability to modulate CAR activity. HepG2 cells were transfected with a CAR expression plasmid and a reporter plasmid containing the human CYP2B6 regulatory region and then treated with flavonoids, catechins, and other bioactive polyphenols. Luciferase assays revealed that baicalein (5,6,7-OH flavone) was a potent activator of both human and mouse CAR. Catechin gallates also activated human and mouse CAR. Wild-type and CAR knockout mice were treated with baicalein and chrysin (5,7-OH flavone), and their liver mRNA was analyzed by real-time polymerase chain reaction (PCR). A significant increase in cyp2b10 mRNA content was observed only in wild-type mice fed chrysin. These results suggest that dietary flavonoids regulate CAR activity and thereby accelerate both detoxification and energy metabolism.

Topics: Androstanes; Animals; Aryl Hydrocarbon Hydroxylases; Carcinoma, Hepatocellular; Catechin; Cell Line, Tumor; Constitutive Androstane Receptor; Cytochrome P450 Family 2; Diet; Flavonoids; Genes, Reporter; Humans; Liver; Liver Neoplasms; Luciferases; Mice; Mice, Knockout; Polymerase Chain Reaction; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Steroid Hydroxylases

Chrysin (5,7-dihydroxyflavone) is a natural flavonoid commonly found in many plants. The anti-cancer property of chrysin has been demonstrated although the molecular mechanisms remain to be further elucidated. In the present study, we found that, pretreatment with chrysin greatly sensitized various human cancer cells to tumor necrosis factor-alpha (TNFalpha)-induced apoptosis. In the search of the molecular mechanisms responsible for the sensitization effect of chrysin, we discovered that such sensitization is closely associated with the inhibitory effect of chrysin on TNFalpha-mediated nuclear transcription factor-kappaB (NF-kappaB) activation. Pretreatment with chrysin inhibited TNFalpha-induced degradation of Inhibitor of kappaB (IkappaB) protein and subsequent nuclear translocation of p65. As a result, chrysin suppressed the expression of NF-kappaB-targeted anti-apoptotic gene, c-FLIP-L. The role of c-FLIP-L was further confirmed by its ectopic expression, which significantly protected cell death induced by combined treatment with chrysin and TNFalpha. Data from this study thus reveal a novel function of chrysin and enhance the value of chrysin as an anti-cancer agent.

Topics: Apoptosis; CASP8 and FADD-Like Apoptosis Regulating Protein; Colorectal Neoplasms; Down-Regulation; Drug Synergism; Flavonoids; HCT116 Cells; Hep G2 Cells; Humans; Immunoblotting; Liver Neoplasms; Nasopharyngeal Neoplasms; Neoplasms; NF-kappa B; Transfection; Tumor Necrosis Factor-alpha

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).. 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.. 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.. BrMC induces apoptosis of HCC cells by ROS generation and sustained JNK activation.

Topics: Acetylcysteine; Anilides; Antineoplastic Agents; Antioxidants; Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Enzyme Activation; Flavonoids; Hep G2 Cells; Humans; JNK Mitogen-Activated Protein Kinases; Liver Neoplasms; Reactive Oxygen Species

The flavonoid chrysin is an important dietary substance and induces UGT1A1 protein expression in cell culture. As a representative of the class of dietary flavonoids, clinical investigations have been considered as a means of inducing hepatic UGT1A1 expression. We demonstrate the necessity of a xenobiotic response element (XRE) in support of chrysin induction of UGT1A1 in the human hepatoma cell line HepG2. Receptor binding assays confirm that chrysin is a ligand for the Ah receptor by competition with [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). However, key differences in Ah receptor recognition and activation of UGT1A1 by chrysin exist when compared with classical mechanisms of UGT1A1 induction by TCDD. Ah receptor degradation, an indicator of Ah receptor activation, does not occur after chrysin treatment, and chrysin cannot transactivate the Ah receptor in a TCDD-dependent fashion. Knock-down of the Ah receptor by siRNA indicates that chrysin uses the Ah receptor in conjunction with other factors through MAP kinase signaling pathways to maximally induce UGT1A1. Most importantly, oral treatment of chrysin to transgenic mice that express the human UGT1 locus is unable to induce UGT1A1 expression in either the small intestine or liver.. Although the implications for chrysin as an atypical agonist of the Ah receptor are intriguing at the molecular level, the relevance of chrysin-induced transcription for the purpose of clinical therapies or to regulate phase 2-dependent glucuronidation is questionable given the lack of in vivo regulation of human UGT1A1 by chrysin in a transgenic animal model.

Topics: Administration, Oral; Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Enzyme Induction; Flavonoids; Gene Expression Regulation; Glucuronosyltransferase; Humans; Ligands; Liver Neoplasms; MAP Kinase Kinase 1; Mice; Mice, Transgenic; Mitogen-Activated Protein Kinase Kinases; Polychlorinated Dibenzodioxins; Promoter Regions, Genetic; Receptors, Aryl Hydrocarbon; Response Elements; RNA, Small Interfering; Signal Transduction; Xenobiotics

Chrysin, a dietary flavonoid, has been shown to markedly induce UGT1A1 expression and activity in HepG2 and Caco-2 cell lines; thus, it has been suggested to have clinical utility in the treatment of UGT1A1-mediated deficiencies, such as unconjugated hyperbilirubinemia or the prevention of 7-ethyl-10-hydroxycamptothecin (SN-38) toxicity. However, little is known about its induction potential in a more physiologically relevant model system, such as primary hepatocyte culture. In this study, induction of UGT1A1 expression (mRNA, protein, and activity) was investigated in primary human hepatocyte cultures after treatment with chrysin and other prototypical inducers. Endogenous nuclear receptor-mediated UGT1A1 induction was studied using transient transfection reporter assays in primary human hepatocytes and HepG2 cells. Results indicated that induction of UGT1A1 expression was minimal in human hepatocytes treated with chrysin compared with that in HepG2 cells (1.2-versus 11-fold, respectively). Subsequent experiments to determine whether the differential response was due to its metabolic stability revealed strikingly different elimination rate constants between the two cell systems (half-life of 13 min in human hepatocytes versus 122 min in HepG2 cell suspensions). Further study demonstrated that UGT1A1 mRNA expression could be induced in human hepatocyte cultures by either increasing the chrysin dosing frequency or by modulating chrysin metabolism, suggesting that the differential induction observed in hepatocytes and HepG2 cells was due to differences in the metabolic clearance of chrysin. In conclusion, this study suggests that the metabolic stability of chrysin likely would limit its ability to induce UGT1A1 in vivo.

Topics: Adolescent; Adult; Aged; Black People; Carcinogens; Carcinoma, Hepatocellular; Cell Line, Tumor; Cells, Cultured; Child, Preschool; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Induction; Female; Flavonoids; Genes, Reporter; Glucuronosyltransferase; Hepatocytes; Humans; Liver Neoplasms; Luciferases; Male; Methylcholanthrene; Middle Aged; RNA, Messenger; White People

1. Chrysin is one of many bioflavonoids with chemopreventive properties in cardiovascular disease and cancer. In an effort better to understand factors that may affect the oral bioavailability of the bioflavonoids from dietary sources, the metabolism of chrysin by cultured intestinal Caco-2 cells and hepatic Hep G2 cells was studied, together modelling human presystemic metabolism. 2. At concentrations that may be achieved in the diet, chrysin was extensively metabolized to two conjugated metabolites, M1 and M2, with no CYP-mediated oxidation. M1 was identified as a glucuronide, and M2 as a sulphate conjugate by LC/MS and other spectroscopic and biochemical techniques. Sulphate conjugation occurred at a rate twice that of glucuronic acid conjugation in both cell types. 3. M1 was catalyzed by UGT1A6 with a Km = 12 microM. M2 was catalyzed both by M- and P-form phenolsulphotransferases (SULT 1A3 and SULT 1A1) with very low Km of 3.1 and 0.05 microM respectively. 4. Pretreatment with 3-methylcholanthrene, interestingly, did not result in oxidation of chrysin but rather in increased glucuronidation. 5. Also, M1 and M2 were the only metabolites formed from chrysin in fresh rat hepatocytes. The metabolism of another flavonoid, apigenin, was very similar to that of chrysin. 6. These observations suggest that both sulphation and glucuronidation are critical determinants of the oral bioavailability of bioflavonoids in humans, although a contribution from CYP-mediated oxidation can not be excluded.

Topics: Animals; Apigenin; Arylsulfotransferase; Caco-2 Cells; Carcinoma, Hepatocellular; Cell Line; Chromatography, High Pressure Liquid; Cytochrome P-450 CYP1A1; Flavonoids; Glucuronidase; Humans; Inactivation, Metabolic; Intestinal Mucosa; Intestines; Isoenzymes; Liver; Liver Neoplasms; Male; Methylcholanthrene; Rats; Rats, Long-Evans; Rats, Sprague-Dawley; Spectrophotometry, Ultraviolet; Sulfotransferases

The common dietary constituent quercetin was a potent inhibitor of sulfoconjugation of acetaminophen and minoxidil by human liver cytosol, partially purified P-form phenolsulfotransferase (PST), and recombinant P-form PST, with IC50 values of 0.025-0.095 microM. Quercetin inhibition of acetaminophen was noncompetitive with respect to acceptor substrate, with a Ki value of 0.067 microM. A number of other flavonoids, such as fisetin, galangin, myricetin, kaempferol, chrysin, and apigenin, were also potent inhibitors of P-form PST-mediated sulfation, with IC50 values < 1 microM. Studies of structural analogs indicated the flavonoid 7-hydroxyl group as particularly important for potent inhibition. Potential human metabolites of quercetin were poor inhibitors. Curcumin, genistein, and ellagic acid (other polyphenolic natural products) were also inhibitors of P-form PST, with IC50 values of 0.38-34.8 microM. Quercetin was also shown to inhibit sulfoconjugation by the human hepatoma cell line Hep G2. Although less potent in this intact cell system (IC50 2-5 microM), quercetin was still more potent than 2,6-dichloro-4-nitrophenol, the classical P-form PST inhibitor that has been shown to be an inhibitor also in vivo. These observations suggest the potential for clinically important drug interactions, as well as a possible role for flavonoids as chemopreventive agents in sulfation-induced carcinogenesis.

Topics: Arylsulfotransferase; Biological Availability; Carcinoma, Hepatocellular; Enzyme Inhibitors; Flavonoids; Humans; Isoenzymes; Liver Neoplasms; Molecular Structure; Quercetin; Tumor Cells, Cultured