Murine Hepatoma Hepa 1c1c7 Cells Research Articles

Mercury and methylmercury differentially modulate hepatic cytochrome P450 1A1 and 1A2 in vivo and in vitro.

The cytochrome P450 1 A (CYP1A) subfamily enzymes are involved in the metabolic activation of several xenobiotics to toxic metabolites and reactive intermediates, resulting ultimately in carcinogenesis. Mercury and halogenated aromatic hydrocarbons (HAHs), typified by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), are persistent environmental pollutants involved in the modulation of aryl hydrocarbon receptor (AHR) gene battery, including cytochrome P450 (CYP) genes. We previously investigated the effect of coexposure to either inorganic or organic mercury (Hg+2 and MeHg) with TCDD on CYP1A1 in vitro. Thus, we examined the impact of coexposure to Hg+2 or MeHg and TCDD on AHR-regulated genes (Cyp1a1/1a2) in vivo and in vitro. Therefore, male C57BL/6 mice were injected intraperitoneally with MeHg or Hg+2 (2.5 mg/kg) in the absence and presence of TCDD (15 μg/kg) for 6 or 24 h. The concentration-dependent effect of MeHg was examined in murine hepatoma Hepa1c1c7 cells. In vivo, both MeHg and Hg2+ inhibited the TCDD-mediated induction of Cyp1a1/1a2 mRNA levels. However, Only Hg2+ was able to inhibit the TCDD-mediated induction at posttranscriptional levels of CYP1A1/1A2 protein and catalytic activity, suggesting differential modulation effects by Hg+2 and MeHg. In addition, the inhibitory role of HO-1 (Heme oxygenase-1) on CYP1A activity induced by TCDD was investigated using a HO-1 competitive inhibitor, tin-mesoporphyrin, that partially restored the MeHg-mediated decrease in CYP1A1 activity. This study demonstrates that MeHg, alongside Hg2+ , can differentially modulate the TCDD-induced AHR-regulated genes (Cyp1a1/1a2) at different expression levels in C57BL/6 mice liver and Hepa1c1c7 cells.

Anti-Inflammatory Effects of Auranamide and Patriscabratine-Mechanisms and In Silico Studies.

Auranamide and patriscabratine are amides from Melastoma malabathricum (L.) Smith. Their anti-inflammatory activity and nuclear factor erythroid 2-related factor 2 (NRF2) activation ability were evaluated using Escherichia coli lipopolysaccharide (LPSEc)-stimulated murine macrophages (RAW264.7) and murine hepatoma (Hepa-1c1c7) cells, respectively. The cytotoxicity of the compounds was assessed using a 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. The anti-inflammatory activity was determined by measuring the nitric oxide (NO) production and pro-inflammatory cytokines (Interleukin (IL)-1β, Interferon (IFN)-γ, tumour necrosis factor (TNF)-α, and IL-6) and mediators (NF-κB and COX-2). NRF2 activation was determined by measuring the nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) quinone oxidoreductase 1 (NQO1), nuclear NRF2 and hemeoxygenase (HO)-1. In vitro metabolic stability was assessed using the mouse, rat, and human liver microsomes. The compounds were non-toxic to the cells at 10 μM. Both compounds showed dose-dependent effects in downregulating NO production and pro-inflammatory cytokines and mediators. The compounds also showed upregulation of NQO1 activity and nuclear NRF2 and HO-1 levels. The compounds were metabolically stable in mouse, rat and human liver microsomes. The possible molecular targets of NRF2 activation by these two compounds were predicted using molecular docking studies and it was found that the compounds might inhibit the Kelch domain of KEAP1 and GSK-3β activity. The physicochemical and drug-like properties of the test compounds were predicted using Schrodinger small molecule drug discovery suite (v.2022-2).

Three new compounds from the twigs and leaves of Nageia fleuryi Hickel

Two new diterpenoids, 12,15-di-O-acetylhypargenin B (1) and taiwanin F-12-O-β-D-glucopyranoside (2), one new monoterpenoid, (S)-7-methyl-3-methyleneoct-6-ene-1,2-diyl diacetate (3), together with eight known compounds (4−11), were obtained from the twigs and leaves of Nageia fleuryi Hickel. The structures of the new compounds were elucidated by extensive spectroscopic techniques including HR-ESI-MS and 1 D and 2 D NMR experiments. Spectroscopic data of the known compound 4 were provided for the first time. Compounds 1 and 11 exhibited strong inhibitory activity on LPS-stimulated production of NO in RAW 264.7 murine macrophages, while compounds 1, 3, and 5 showed significant quinone reductase inducing activity in Hepa 1c1c7 murine hepatoma cells. Moreover, compounds 7 and 8 showed inhibitory activity against the proliferation of the human prostate carcinoma DU145 cells.

Svalbamides A and B, Pyrrolidinone-Bearing Lipodipeptides from Arctic Paenibacillus sp.

Two new secondary metabolites, svalbamides A (1) and B (2), were isolated from a culture extract of Paenibacillus sp. SVB7 that was isolated from surface sediment from a core (HH17-1085) taken in the Svalbard archipelago in the Arctic Ocean. The combinational analysis of HR-MS and NMR spectroscopic data revealed the structures of 1 and 2 as being lipopeptides bearing 3-amino-2-pyrrolidinone, d-valine, and 3-hydroxy-8-methyldecanoic acid. The absolute configurations of the amino acid residues in svalbamides A and B were determined using the advanced Marfey’s method, in which the hydrolysates of 1 and 2 were derivatized with l- and d- forms of 1-fluoro-2,4-dinitrophenyl-5-alanine amide (FDAA). The absolute configurations of 1 and 2 were completely assigned by deducing the stereochemistry of 3-hydroxy-8-methyldecanoic acid based on DP4 calculations. Svalbamides A and B induced quinone reductase activity in Hepa1c1c7 murine hepatoma cells, indicating that they represent chemotypes with a potential for functioning as chemopreventive agents.

Novel iodinated quinazolinones bearing sulfonamide as new scaffold targeting radiation induced oxidative stress

Reactive oxygen species (ROS) play an integral role in the pathogenesis of most diseases. This work presents the design and synthesis of fourteen new diiodoquinazolinone derivatives bearing benzenesulfonamide moiety with variable acetamide tail and evaluation of their ability to activate nuclear factor erythroid 2-related factor 2 (Nrf2) using its classical target NAD(P)H: quinone oxidoreductase 1 (NQO1) in Hepa1c1c7 murine hepatoma cells. The N-(2-chloropyridin-3-yl)-2-((6,8-diiodo-4-oxo-3-(4-sulfamoylphenyl)-3,4-dihydroquinazolin-2-yl)thio) acetamide 17 was the most potent NQO1 inducer (CD = 25 µM) with free radical scavenging activity (IC50 = 28 µM) and in vivo median lethal dose (LD50) of 500 mg/Kg. The possible radioprotective activity of compound 17 was evaluated in (7 Gy) irradiated mice. Compound 17 showed a reduction in radiation induced oxidative stress as evidenced by the lower levels of ROS, malondialdehyde (MDA) and NQO1 in liver tissues. Moreover, compound 17 showed improvement in the complete blood count (CBC) of irradiated mice and decreased mortality over 30 days following irradiation. Additionally, docking studies inside the Nrf2-binding site of Kelch-like ECH associated protein 1 (Keap1), the main negative regulator of Nrf2, confirmed that 17 revealed the same interactions with the key amino acids as those of the co-crystallized ligand. This study identifies 17 as a novel antioxidant that protects against the harmful effect of radiation.

The synergistic effect of cell wall extracted from probiotic biomass containing Lactobacillus acidophilus CL1285, L. casei LBC80R, and L. rhamnosus CLR2 on the anticancer activity of cranberry juice-HPLC fractions.

Anticancer effects were evaluated on three HPLC fractions obtained from a concentrated cranberry juice and cell wall constituents extracted from a probiotic biomass containing Lactobacillus acidophilus CL1285, Lactobacillus casei LBC80R, and Lactobacillus rhamnosus CLR2. The samples were tested at increasing concentrations for the antiproliferative assay using HT-29 cells' line and for the quinone reductase (QR) assay using Hepa-1c1c7 murine hepatoma cells. Fraction 1 (F1) which is highly concentrated with phenolic acids inhibited the growth of the HT-29 cells' line with IC50 values of 14.80µg Gallic acid equivalent (GAE)/ml. The fraction 3 (F3) which is highly concentrated in flavonols had potency as QR inducer. Furthermore, the results showed that all cranberry fractions combined with cell wall constituents extracted from the probiotic biomass were more effective in inhibiting the growth of HT-29 as compared to the cranberry fractions tested alone, indicating a possible synergy effect between these bio-functional compounds. PRACTICAL APPLICATIONS: This study strongly evidenced that cranberry juice fractions combined with cell wall constituents extracted from the probiotic biomass can be used as a potent preventive functional compound against colorectal cancer. Therefore, this research proposes a natural dietary compound to prevent mutagenesis and carcinogenesis of colorectal cancer. Furthermore, the industry can formulae products containing probiotic and phenolic compounds as colon cancer cell growth preventive and anticancer products.

Beta-ionone-inhibited proliferation of breast cancer cells by inhibited COX-2 activity.

As one of the isoprenoids and widely derived from many fruits and vegetables, β-ionone (BI) has a potent inhibitory proliferation of cancer cells in vitro and in vivo. However, its exact mechanism is still uncompleted understood and needs to be further verified. Cyclooxygenase-2 (COX-2), as a potential target of cancer chemoprevention, has been played pivotal roles in proliferation of tumor cells and carcinogenesis. Thus, the objective of present study was to determine that BI inhibited the activity of COX-2 in breast cancer and related to cancer cell models. Cell proliferation, DNA synthesis, the distribution of cell cycle, apoptosis induction and the expression of P38-MAPK protein were determined in MCF-7 cells by methylene blue, 3H-thymidine (TdR) incorporation, flow cytometry, TUNEL and Western blotting assays. Quinone reductase (QR) activity was determined in murine hepatoma Hepa1c1c7 cells by enzyme-linked immunosorbent assay (ELISA). The expression of COX-2 in a phorbol-12-myristate-13-acetate (PMA)-induced cell model and mammary tumor tissues was examined by Western blotting and immunohistochemistry. The results showed that BI significantly inhibited cell proliferation and DNA synthesis, arrested the distribution of cell cycle at the S phase or decreased proteins related to cell cycle such as cyclin D1 and CDK4, induced apoptosis and increased the expression of p-P38 in MCF-7 cells. BI at low doses (< 50μmol/L) significantly increased QR activity, decreased the expression of COX-2 protein and prostaglandin E2 (PEG2) release in cell models. In addition, BI also significantly decreased the expression of COX-2 protein in rat mammary tumor tissues. Therefore, our findings indicate that BI possesses inhibitory proliferation of breast cancer cells through down-regulation of COX-2 activity.

A CRISPR/Cas9 Whole-Genome Screen Identifies Genes Required for Aryl Hydrocarbon Receptor-Dependent Induction of Functional CYP1A1.

Environmental pollutants including halogenated aromatic hydrocarbons and polycyclic aromatic hydrocarbons, including benzo[a]pyrene, exert their deleterious effects through the activation of the aryl hydrocarbon receptor (AHR) and by the resulting transcription of genes not yet fully identified. Ligand-bound AHR translocates from cytoplasm to nucleus, where it dimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT) protein. The AHR/ARNT dimer binds to enhancer regions of responsive genes to activate transcription. AHR also mediates carcinogenesis caused by PAHs, likely via CYP1A1, CYP1A2, and CYP1B1, which are massively induced by activated AHR in many tissues and generate carcinogenic electrophilic derivatives of PAHs. In the current study, we have used the mouse GeCKOv2 genome-wide CRISPR/Cas9 library to identify novel genes in the AHR pathway by taking advantage of a B[a]P selection assay that we previously used to identify core AHR pathway genes in Hepa-1c1c7 murine hepatoma cells. Besides Ahr, Arnt, and Cyp1a1, we report the identification of multiple additional putative AHR pathway genes including several that we validated. These include cytochrome P450 reductase (Por), which mediates redox regeneration of cytochromes P450, and 5 genes of the heme biosynthesis pathway: delta-aminolevulinate synthase 1 (Alas1), porphobilinogen deaminase (Hmbs), uroporphyrinogen decarboxylase (Urod), coproporphyrinogen oxidase (Cpox), and ferrochelatase (Fech): heme being an essential prosthetic group of cytochrome P450 proteins. Notably, several of these genes were identified by GeCKO screening, despite not being identifiable by reverse genetics approaches. This indicates the power of high-sensitivity genome-wide genetic screening for identifying genes in the AHR pathway.

SEGMENTAL ARTERIAL MEDIOLYSIS: A COMMON MISDIAGNOSIS AND UNDER-RECOGNIZED PHENOMENON

Indole-3-carbinol (I3C) is a secondary plant metabolite produced in vegetables of the Brassica genus, including cabbage, cauliflower, and brussels sprouts. I3C is both an anti-initiator and a promoter of carcinogenesis. Consumption of I3C by humans and rodents can lead to marked increases in activities of cytochrome P-450-dependent monooxygenases and in a variety of phase II drug-metabolizing enzymes. We have reported previously that the enzyme-inducing activity of I3C is mediated through a mechanism requiring exposure of the compound to the low-pH environment of the stomach. We report here the aromatic hydrocarbon responsiveness-receptor Kd values (22 nM-90 nM), determined with C57BL/6J mouse liver cytosol and the in vitro- and in vivo-molar yields (0.1-6%) of the major acid condensation products of I3C. We also show that indolo[3,2-b]carbazole (ICZ) is produced from I3C in yields on the order of 0.01% in vitro and, after oral intubation, in vivo. ICZ has a Kd of 190 pM for aromatic hydrocarbon responsiveness-receptor binding and an EC50 of 269 nM for induction of cytochrome P4501A1, as measured by ethoxyresorufin O-deethylase activity in murine hepatoma Hepa 1c1c7 cells. The binding affinity of ICZ is only a factor of 3.7 x 10(-2) lower than that of the highly toxic environmental contaminant and cancer promoter 2,3,7,8-tetrachlorodibenzo-p-dioxin. ICZ and related condensation products appear responsible for the enzyme-inducing effects of dietary I3C.

Antioxidant and Quinone Reductase Activity of Soyasaponins in Hepa1c1c7 Mouse Hepatocarcinoma Cells.

Saponins have been reported to possess several health beneficial activities including hypocholesterolemic, immune-stimulatory, and anticarcinogenic. The objectives of this study were to determine if soysaponins are radical scavengers and inducers of quinone reductase (QR) activity in Hepa1c1c7 murine hepatoma cell line. The antioxidant capacity of soyasaponin was evaluated using the 1,1′-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging methods. Soyasaponin showed 75.7% radical scavenging activity in the DPPH assay and 81.4% in the ABTS method at 100 μg/mL concentration. Cellular proliferation was determined using the methylthiazolyldiphenyl-tetrazolium bromide colorimetric assay. Soyasaponin inhibited cell growth in a dose-dependent (0.1~100 μg/mL) manner, and growth inhibition was 30% and 39% at 100 μg/mL of saponin after 24 h and 48 h incubation, respectively. Soyasaponin showed QR induction in a dose-dependent manner. Ten, 50, and 100 μg/mL of soyasaponin resulted in a 1.6-, 2.2-, and 2.9-fold induction of QR, respectively. These results provide a basis for the potential of soysaponin as a chemopreventive agent.

3,4-Dihydroxyphenylacetic acid is a potential aldehyde dehydrogenase inducer in murine hepatoma Hepa1c1c7 cells.

3,4-Dihydroxyphenylacetic acid (DOPAC) is one of the major colonic microflora-produced catabolites of quercetin glycosides, such as quercetin 4'-glucoside derived from onion. Here, we investigated whether DOPAC modulates the aldehyde dehydrogenase (ALDH) activity and protects the cells from the acetaldehyde-induced cytotoxicity in vitro. DOPAC was shown to enhance not only the total ALDH activity, but also the gene expression of ALDH1A1, ALDH2 and ALDH3A1 in a concentration-dependent manner. DOPAC simultaneously stimulated the nuclear translocation of NFE2-related factor 2 and aryl hydrocarbon receptor. The pretreatment of DOPAC completely protected the cells from the acetaldehyde-induced cytotoxicity. The present study suggested that DOPAC acts as a potential ALDH inducer to prevent the alcohol-induced abnormal reaction.

Benzyl isothiocyanate ameliorates acetaldehyde-induced cytotoxicity by enhancing aldehyde dehydrogenase activity in murine hepatoma Hepa1c1c7 cells

In the present study, we assessed benzyl isothiocyanate (BITC), an organosulfur compound from cruciferous vegetables, as a potential inducer of aldehyde dehydrogenase (ALDH) activity using murine hepatoma Hepa1c1c7 cells. BITC was shown to enhance not only the total ALDH activity, but also the ALDH activity of the cytosolic/microsomal and mitochondrial fraction. BITC also significantly increased the gene and protein expression of ALDH1A1, ALDH2 and ALDH3A1 in a concentration-dependent manner. Simultaneously, the gene expression of phase 2 drug-metabolizing enzymes, such as NAD(P)H: quinone oxidoreductase 1 and heme oxygenase-1, was increased by the BITC treatment. Western blot experiments revealed that BITC not only up-regulated the Nrf2 protein expression, but also stimulated the nuclear translocation of Nrf2. Furthermore, silencing Nrf2 reduced the basal and BITC-enhanced levels of the total activity and gene expression of ALDHs. The pretreatment of BITC completely mitigated the acetaldehyde-induced cytotoxicity, which was impaired by silencing Nrf2. The present study demonstrated that BITC has been identified as a potential inducer of the total ALDH activity to prevent the acetaldehyde-induced cytotoxicity.

Flavonolignan 2,3-dehydrosilydianin activates Nrf2 and upregulates NAD(P)H:quinone oxidoreductase 1 in Hepa1c1c7 cells

Silybum marianum (milk thistle) is a medicinal plant used for the treatment of various liver disorders. This study examined whether the main flavonolignans from S. marianum (i.e. silybin, silychristin, silydianin) and their 2,3-dehydro derivatives (i.e. 2,3-dehydrosilybin, 2,3-dehydrosilychristin, 2,3-dehydrosilydianin) activate the Nrf2 pathway, which regulates the expression of genes encoding many cytoprotective enzymes, including NAD(P)H:quinone oxidoreductase 1 (NQO1). After 48h of exposure, 2,3-dehydrosilydianin at concentrations of 25μM and higher significantly elevated the activity of NQO1 in murine hepatoma Hepa1c1c7 cells. In contrast, other tested compounds at non-cytotoxic concentrations had a mild or negligible effect on the NQO1 activity. Using a luciferase reporter assay, 2,3-dehydrosilydianin was found to significantly activate transcription via the antioxidant response element in stably transfected human AREc32 reporter cells. Moreover, 2,3-dehydrosilydianin caused the accumulation of Nrf2 and significantly induced the expression of the Nqo1 gene at both the mRNA and protein levels in Hepa1c1c7 cells. We found that 2,3-dehydrosilydianin also increased to some extent the expression of other Nrf2 target genes, namely of the heme oxygenase-1 gene (Hmox1) and the glutamate-cysteine ligase modifier subunit gene (Gclm). We conclude that 2,3-dehydrosilydianin activates Nrf2 and induces Nrf2-mediated gene expression in Hepa1c1c7 cells.

Semisynthetic flavonoid 7-O-galloylquercetin activates Nrf2 and induces Nrf2-dependent gene expression in RAW264.7 and Hepa1c1c7 cells

The natural flavonoid quercetin is known to activate the transcription factor Nrf2, which regulates the expression of cytoprotective enzymes such as heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1). In this study, a novel semisynthetic flavonoid 7-O-galloylquercetin (or quercetin-7-gallate, 3) was prepared by direct galloylation of quercetin, and its effect on the Nrf2 pathway was examined. A luciferase reporter assay showed that 7-O-galloylquercetin, like quercetin, significantly activated transcription via the antioxidant response element in a stably transfected human AREc32 reporter cell line. In addition, 7-O-galloylquercetin caused the accumulation of Nrf2 and induced the expression of HO-1 at both the mRNA and protein levels in murine macrophage RAW264.7 cells. The induction of HO-1 by 7-O-galloylquercetin was significantly suppressed by N-acetyl-l-cysteine and SB203580, indicating the involvement of reactive oxygen species and p38 mitogen-activated protein kinase activity, respectively. HPLC/MS analyses also showed that 7-O-galloylquercetin was not degalloylated to quercetin, but it was conjugated with glucuronic acid and/or methylated in RAW264.7 cells. Furthermore, 7-O-galloylquercetin was found to increase the protein levels of Nrf2 and HO-1, and also the activity of NQO1 in murine hepatoma Hepa1c1c7 cells. Taken together, we conclude that 7-O-galloylquercetin increases Nrf2 activity and induces Nrf2-dependent gene expression in RAW264.7 and Hepa1c1c7 cells.

Screening of traditional Chinese medicines with therapeutic potential on chronic obstructive pulmonary disease through inhibiting oxidative stress and inflammatory response.

BackgroundChronic obstructive pulmonary disease (COPD) is a major public health problem and gives arise to severe chronic morbidity and mortality in the world. Inflammatory response and oxidative stress play dominant roles in the pathological mechanism of COPD, and have been regarded to be two important targets for the COPD therapy. Traditional Chinese medicines (TCMs) possess satisfying curative effects on COPD under guidance of the TCM theory in China, and merit in-depth investigations as a resource of lead compounds.MethodsOne hundred ninety-six of TCMs were collected, and extracted to establish a TCM extract library, and then further evaluated for their potency on inhibitions of oxidative stress and inflammatory response using NADP(H):quinone oxidoreductase (QR) assay and nitric oxide (NO) production assay, respectively.ResultsOur investigation observed that 38 of the tested TCM extracts induced QR activity in hepa 1c1c7 murine hepatoma cells, and 55 of them inhibited NO production in RAW 264.7 murine macrophages at the tested concentrations. Noteworthily, 20 of TCM extracts simultaneously inhibited oxidative stress and inflammatory responses.ConclusionThe observed bioactive TCMs, particularly these 20 TCMs with dual inhibitory effects, might be useful for the treatment of COPD. More importantly, the results of the present research afford us an opportunity to discover new lead molecules as COPD therapeutic agents from these active TCMs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12906-016-1347-y) contains supplementary material, which is available to authorized users.

Electron affinity of tricyclic, bicyclic, and monocyclic compounds containing cyanoenones correlates with their potency as inducers of a cytoprotective enzyme

Tricyclic, bicyclic, and monocyclic compounds containing cyanoenones induce various anti-inflammatory and cytoprotective enzymes through activation of the Keap1/Nrf2/ARE (antioxidant response element) pathway. The potency of these compounds as Nrf2 activators was determined using a prototypic cytoprotective enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) in Hepa1c1c7 murine hepatoma cells. The electron affinity (EA) of the compounds, expressed as the energy of their lowest unoccupied molecular orbital [E (LUMO)], was evaluated using two types of quantum mechanical calculations: the semiempirical (AM1) and the density functional theory (DFT) methods. We observed striking linear correlations [r=0.897 (AM1) and 0.936 (DFT)] between NQO1 inducer potency of these compounds and their E (LUMO) regardless of the molecule size. Importantly and interestingly, this finding demonstrates that the EA is the essentially important factor that determines the reactivity of the cyanoenones with Keap1.

Induction of NAD(P)H:Quinone Oxidoreductase 1 (NQO1) by Glycyrrhiza Species Used for Women's Health: Differential Effects of the Michael Acceptors Isoliquiritigenin and Licochalcone A.

quinone oxidoreductase 1 (NQO1), in vitro and in vivo. Licorice species could also induce NQO1, as they contain the Michael acceptors isoliquiritigenin (LigC) found in Glycyrrhiza glabra (GG), G. uralensis (GU), G. inflata (GI), and licochalcone A (LicA) which is only found in GI. These licorice species and hops induced NQO1 activity in murine hepatoma (Hepa1c1c7) cells; hops ≫ GI > GG ≅ GU. Similar to the known chemopreventive compounds curcumin (turmeric), sulforaphane (broccoli), and XH, LigC and LicA were active dose-dependently; sulforaphane ≫ XH > LigC > LicA ≅ curcumin ≫ liquiritigenin (LigF). Induction of the antioxidant response element luciferase in human hepatoma (HepG2-ARE-C8) cells suggested involvement of the Keap1-Nrf2 pathway. GG, GU, and LigC also induced NQO1 in nontumorigenic breast epithelial MCF-10A cells. In female Sprague-Dawley rats treated with GG and GU, LigC and LigF were detected in the liver and mammary gland. GG weakly enhanced NQO1 activity in the mammary tissue but not in the liver. Treatment with LigC alone did not induce NQO1 in vivo most likely due to its conversion to LigF, extensive metabolism, and its low bioavailability in vivo. These data show the chemopreventive potential of licorice species in vitro could be due to LigC and LicA and emphasize the importance of chemical and biological standardization of botanicals used as dietary supplements. Although the in vivo effects in the rat model after four-day treatment are minimal, it must be emphasized that menopausal women take these supplements for extended periods of time and long-term beneficial effects are quite possible.

Effects of Benzyl Isothiocyanate and Its N-Acetylcysteine Conjugate on Induction of Detoxification Enzymes in Hepa1c1c7 Mouse Hepatoma Cells

The induction of detoxification enzymes by benzyl isothiocyanate (BITC) and its synthetic N-acetyl-L-cysteine (NAC) conjugate (NAC-BITC) was examined in Hepa1c1c7 murine hepatoma cells. BITC and NAC-BITC inhibited Hepa1c1c7 cell growth in a dose-dependent manner. Cell growth was 4.5~57.2% lower in Hepa1c1c7 cells treated with 0.1~10 μM BITC than in control-treated Hepa1c1c7 cells. The NAC-BITC treatment had a similar inhibitory pattern on Hepa1c1c7 cell growth; 0.5 μM and 10 μM NAC-BITC decreased cell growth by 13.6% and 47.4%, respectively. Treatment of Hepa1c1c7 cells with 0.1~2.0 μM BITC also elicited a dose-response effect on the induction of quinone reductase quinone reductase (QR) activity and QR mRNA expression. Treatment with 1 μM and 2 μM BITC caused 1.8- and 2.8-fold inductions of QR mRNA, respectively. By comparison, treatment with 1 μM and 2 μM NAC-BITC caused 1.6- and 1.9-fold inductions of QR mRNA, respectively. Cytochrome P450 (CYP) 1A1 and CYP2E1 induction were lower in 0.1~2 μM BITC-treated cells than in control-treated cells. CYP2E1 activity was 1.2-fold greater in 0.1 μM NAC-BITC-treated cells than in control-treated cells. However, the CYP2E1 activity of cells treated with higher concentrations (i.e., 1~2 μM) of NAC-BITC was similar to the activity of control-treated cells. Considering the potential of isothiocyanatesto prevent cancer, these results provide support for the use of BITC and NAC-BITC conjugates as chemopreventive agents.

Synthesis and biological evaluation of biotin conjugates of (±)-(4bS,8aR,10aS)-10a-ethynyl-4b,8,8-trimethyl-3,7-dioxo-3,4b,7,8,8a,9,10,10a-octahydro-phenanthrene-2,6-dicarbonitrile, an activator of the Keap1/Nrf2/ARE pathway, for the isolation of its protein targets

The tricycle 1 ((±)-(4bS,8aR,10aS))-10a-ethynyl-4b,8,8-trimethyl-3,7-dioxo-3,4b,7,8,8a,9,10,10a-octahydrophenanthrene-2,6-dicarbonitrile), a potent activator of the Keap1/Nrf2/ARE pathway, has the potential to be a first in class drug for the treatment of diabetic nephropathy. To identify the protein targets for the development of 1, the (1:1)-diasteromeric mixture of biotinylated tricycles 3a and 3b were designed and synthesized. For the synthesis of 3a and 3b, a new important precursor, hydroxylated tricycle (±)-16 was synthesized from 4 by a C1 α-methyl group oxidation protocol, which involves cyclopalladation of the C1 α-methyl group from a C2-oxime. For the induction of the phase 2 cytoprotective enzyme NQO1 in Hepa1c1c7 murine hepatoma cells, the diasteromeric mixture 3a and 3b shows high potency (CD, 75nM) although this potency is lower than that of 1 and 16. Thus, biotinylated tricycles 3a and 3b may be promising tools for the isolation of the protein targets of 1.

Bioactive constituents of Indigofera spicata.

Four new flavanones, designated as (+)-5″-deacetylpurpurin (1), (+)-5-methoxypurpurin (2), (2S)-2,3-dihydrotephroglabrin (3), and (2S)-2,3-dihydrotephroapollin C (4), together with two known flavanones (5 and 6), three known rotenoids (7-9), and one known chalcone (10) were isolated from a chloroform-soluble partition of a methanol extract from the combined flowers, fruits, leaves, and twigs of Indigofera spicata, collected in Vietnam. The compounds were obtained by bioactivity-guided isolation using the HT-29 human colon cancer, 697 human acute lymphoblastic leukemia, and Raji human Burkitt's lymphoma cell lines. The structures of 1-4 were established by extensive 1D- and 2D-NMR experiments, and the absolute configurations were determined by the measurement of specific rotations and CD spectra. The cytotoxic activities of the isolated compounds were tested against the HT-29, 697, Raji, and CCD-112CoN human normal colon cells. Also, the quinone reductase induction activities of the isolates were determined using the Hepa 1c1c7 murine hepatoma cell line. In addition, cis-(6aβ,12aβ)-hydroxyrotenone (7) was evaluated in an in vivo hollow fiber bioassay using HT-29, MCF-7 human breast cancer, and MDA-MB-435 human melanoma cells.