To study the mechanisms of total flavonoid from Glycyrrhizae Radix et Rhizoma (TFGR) and its ingredient isoliquiritigenin (ISL) on their regulation of M2 phenotype polarization of macrophages. IL-4 (60 μg x L(-1)) induced RAW264.7 cells for 6 h to establish the M2 macrophage model. TFGR and ISL restrained breast cancer cells migration with the aid of M2 macrophages in vitro. TFGR and ISL inhibited gene and protein expression of Arg-1, up-regulated gene of HO-1 and protein expression of iNOS, enhanced the expression of microRNA 155 and its target gene SHIP1, meanwhile down-regulated.the phosphorylation of STAT3 and STAT6. So TFGR and ISL were the bioactive fraction and ingredient in Glycyrrhizae Radix et Rhizoma to reverse M2 phenotype macrophages polarization. TFGR and ISL inhibited the promotion of M2 macrophages to breast cancer cells migration in vitro, STAT signal pathways and miR155 were partly involved.
Animals ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Polarity ; drug effects ; Chalcones ; pharmacology ; Flavonoids ; pharmacology ; Glycyrrhiza ; chemistry ; Interleukin-4 ; genetics ; metabolism ; Macrophages ; cytology ; drug effects ; metabolism ; Mice ; RAW 264.7 Cells ; Rhizome ; chemistry

AIM: To investigate the active constituents of Lignum Sappan (Caesalpinia sappan L.) on growth-related signaling and cell mitosis. METHOD: The influence of the ethyl acetate (EtOAc) extract of Lignum Sappan and its constituents on growth-related signaling were evaluated by a luciferase assay in cells stably-transfected with NF-κB, STAT1, or STAT3 responsive luciferase reporter plasmid. The inhibitory effect on the cell cycle was determined by flow cytometric analysis. The anti-tumor activities were assessed in vitro and in vivo. RESULTS: The EtOAc extract of Lignum Sappan had inhibitory activities on growth-related signaling and cell mitosis. Three major active compounds were sappanchalcone, brazilin, and butein. Sappanchalcone blocked cell cycle progression in the G2/M phase, brazilin inhibited TNFα/NF-κB signaling, while butein inhibited IL-6/STAT3 signaling, as well as TNFα/NF-κB signaling. The three compounds all demonstrated cytotoxic activities against human tumor cells in vitro. In a S180 tumor cell-bearing mice model, the anti-tumor efficacy of the EtOAc extract was better than the individual compounds acting alone. CONCLUSION These results indicate that Lignum Sappan contains multiple active compounds with different antitumor activities, which act synergistically to enhance their anti-tumor effects. The EtOAc extract of Lignum Sappan may be better than individual active constituent as a novel medicine for the treatment of cancer.
Animals ; Antineoplastic Agents, Phytogenic ; pharmacology ; therapeutic use ; Benzopyrans ; pharmacology ; therapeutic use ; Caesalpinia ; Cell Cycle Checkpoints ; drug effects ; Chalcones ; pharmacology ; therapeutic use ; Hep G2 Cells ; Humans ; Interleukin-6 ; metabolism ; Male ; Mice, Inbred BALB C ; Mitosis ; drug effects ; NF-kappa B ; metabolism ; Phytotherapy ; Plant Extracts ; pharmacology ; therapeutic use ; STAT3 Transcription Factor ; metabolism ; Sarcoma ; drug therapy ; metabolism ; Signal Transduction ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVEIsoliquiritigenin (ISL), a licorice chalconoid, is considered to be a bioactive agent with chemopreventive potential. This study investigates the mechanisms involved in ISL-induced apoptosis in human cervical carcinoma HeLa cells.

METHODSCell viability was evaluated using a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay. Apoptosis was determined by flow cytometry using an Annexin V-FITC Apoptosis Detection Kit. The intracellular ROS levels were assessed using a 2, 7-dichlorofluorescein probe assay. The mitochondrial membrane potential was measured with the dual-emission potential-sensitive probe 5, 5', 6, 6'-tetra-chloro-1, 1', 3, 3'-tetraethyl-imidacarbocyanine iodide (JC-1). The degradation of poly-ADP-ribose polymerase (PARP) protein, the phosphorylation of PKR-like ER kinase (PERK), the phosphorylation of the α-subunit of eukaryotic initiation factor 2 (eIF2α), the expression of the 78 kD glucose-regulated protein (GRP 78), and the activation of caspase-12 were analyzed via western blot analysis.

RESULTSISL significantly inhibited the proliferation, the increase in ROS levels and apoptotic rates of HeLa cells in a concentration-dependent manner. Moreover, ISL induced mitochondrial dysfunction, caspase activation, and PARP cleavage, which displayed features of mitochondria dependent on apoptotic signals. Besides, exposure of HeLa cells to ISL triggered endoplasmic reticulum (ER) stress, as indicated by the increase in p-eIF2α and GRP78 expression, ER stress-dependent apoptosis is caused by the activation of ER-specific caspase-12.

CONCLUSIONThe findings from our study suggest that ISL-induced oxidative stress causes HeLa cell apoptosis via the mitochondrion-dependent and the ER stress-triggered signaling pathways.

Aldehyde Reductase ; antagonists & inhibitors ; Apoptosis ; drug effects ; Cell Survival ; drug effects ; Chalcones ; pharmacology ; therapeutic use ; Chemoprevention ; Drug Screening Assays, Antitumor ; Endoplasmic Reticulum Stress ; drug effects ; HeLa Cells ; Humans ; Mitochondria ; drug effects ; Neoplasms ; prevention & control ; Reactive Oxygen Species ; metabolism

OBJECTIVETo investigate the antiproliferation effect of cardamonin (CAR) and its possible mechanisms on human umbilical artery smooth muscle cells (HUASMCs) cultured in the mimicking insulin resistance (IR) medium.

METHODProliferation of HUASMCs was assayed by MTT method. The mRNA expression of mTOR, Raptor and Rictor was detected by a real-time PCR. The expression content was calculated by Livak method using internal control of beta-actin.

RESULTThe proliferation of HUASMCs cultured in the mimicking IR medium was significantly increased. Both in normal and mimic IR culture medium, cells proliferation was inhibited by CAR (1 x 10(-5), 1 x 10(-4) mol x L(-1)). Pretreated with PD98059 and LY294002, cell proliferation induced by phosphatidic acid (PA) was inhibited, and the mRNA expression of mTOR, Raptor and Rictor was significantly decreased by CAR in the mimic IR medium.

CONCLUSIONIt is implicated that antiproliferation of CAR is involved in mRNA expression decrease of mTOR and its relative protein Raptor and Rictor.

Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Carrier Proteins ; genetics ; metabolism ; Cell Proliferation ; drug effects ; Cells, Cultured ; Chalcones ; pharmacology ; Gene Expression Regulation ; drug effects ; Growth Inhibitors ; pharmacology ; Humans ; Myocytes, Smooth Muscle ; cytology ; drug effects ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rapamycin-Insensitive Companion of mTOR Protein ; Regulatory-Associated Protein of mTOR ; TOR Serine-Threonine Kinases ; genetics ; metabolism

OBJECTIVETo study the in-vitro inducing apoptosis mechanism of human hepatoma SMMC-7721 cells by 2',4'-di- hydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC), a chalcone compound from Cleistocalyx operculatus.

METHODQuantitative DNA fragmentation assay was carried out to detect the effect of DMC of different concentrations on SMMC-7721 cells, according to the method of Sellins and Cohen with some modifications. Telomerase activities of the cells were determined by PCR-ELISA methods. The expression quantity of c-myc and hTERT mRNA were determined by semi-quantitative RT-PCR The effect of DMC on expression levels of cmyc and hTERT protein were measured by western blot.

RESULTThe percentage of DNA fragmentation increased with notable concen- tration dependence, after treatment with DMC for 48 h. Compared with that of control group, the telomerase activity of the cells de- creased by (66.2 ± 2.1)% after 48 h treatment with 20 μmol x L(-1) DMC, the mRNA expression of c-myc and hTERT decreased by (67.3 ± 2.1)% and (64.4 ± 2.3)%, respectively, and the protein expression of c-myc and hTERT decreased by (69.6 ± 1.9)% and (71.3 ± 2.4)%, respectively.

CONCLUSIONDMC can induce SMMC-7721 cell apoptosis and the apoptosis mechanism may be related to the decreased mRNA and protein expression of c-myc and hTERT.

Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; pathology ; Cell Line, Tumor ; Chalcones ; pharmacology ; DNA Fragmentation ; drug effects ; Dose-Response Relationship, Drug ; Humans ; Liver Neoplasms ; pathology ; Proto-Oncogene Proteins c-myc ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Syzygium ; chemistry ; Telomerase ; genetics ; metabolism

Ornithine decarboxylase (ODC) is the rate-limiting enzyme in polyamine biosynthesis and a target for chemoprevention. Hydroxydibenzoylmethane (HDB), a derivative of dibenzoylmethane of licorice, is a promising chemopreventive agent. In this paper, we investigated whether HDB would inhibit the ODC pathway to enhance apoptosis in human promyelocytic leukemia HL-60 cells. We found ODC enzyme activity was reduced during HDB treatment. Overexpression of ODC in HL-60 parental cells could reduce HDB-induced apoptosis, which leads to loss of mitochondrial membrane potential (Deltapsim), through lessening intracellular ROS. Furthermore, ODC overexpression protected cytochrome c release and the activation of caspase-3 following HDB treatment. The results demonstrated HDB-induced apoptosis was through a mechanism of down-regulation of ODC and occurred along a ROS-dependent mitochondria-mediated pathway.
Apoptosis/*drug effects ; Caspase 3/metabolism ; Chalcones/metabolism/*pharmacology ; Chemoprevention ; Cytochromes c/biosynthesis/secretion ; Down-Regulation ; Gene Expression ; HL-60 Cells ; Humans ; Immunoblotting ; Leukemia, Myeloid/*enzymology/pathology ; Membrane Potential, Mitochondrial/drug effects ; Mitochondria/enzymology ; Ornithine Decarboxylase/antagonists & inhibitors/genetics/*metabolism ; Reactive Oxygen Species/analysis/metabolism ; Reverse Transcriptase Polymerase Chain Reaction

Microglial cells are the resident innate immune cells that sense pathogens and tissue injury in the central nervous system (CNS). Microglial activation is critical for neuroinflammatory responses. The synthetic compound 2-hydroxy-3',5,5'-trimethoxychalcone (DK-139) is a novel chalcone-derived compound. In this study, we investigated the effects of DK-139 on Toll-like receptor 4 (TLR4)-mediated inflammatory responses in BV2 microglial cells. DK-139 inhibited lipopolysaccharide (LPS)-induced TLR4 activity, as determined using a cell-based assay. DK-139 blocked LPS-induced phosphorylation of IkappaB and p65/RelA NF-kappaB, resulting in inhibition of the nuclear translocation and trans-acting activity of NF-kappaB in BV2 microglial cells. We also found that DK-139 reduced the expression of NF-kappaB target genes, such as those for COX-2, iNOS, and IL-1beta, in LPS-stimulated BV2 microglial cells. Interestingly, DK-139 blocked LPS-induced Akt phosphorylation. Inhibition of Akt abrogated LPS-induced phosphorylation of p65/RelA, while overexpression of dominant-active p110CAAX enhanced p65/RelA phosphorylation as well as iNOS and COX2 expression. These results suggest that DK-139 exerts an anti-inflammatory effect on microglial cells by inhibiting the Akt/IkappaB kinase (IKK)/NF-kappaB signaling pathway.
Animals ; Binding Sites ; Cell Line ; Chalcones/chemistry/*pharmacology ; Cyclooxygenase 2/metabolism ; I-kappa B Kinase/metabolism ; Inflammation/*drug therapy ; Interleukin-1beta/metabolism ; Lipopolysaccharides/immunology ; Microglia/*drug effects/immunology/metabolism ; Molecular Dynamics Simulation ; NF-kappa B/*antagonists & inhibitors ; Nitric Oxide Synthase Type II/metabolism ; Phosphorylation/drug effects ; Protein Binding ; Proto-Oncogene Proteins c-akt/*antagonists & inhibitors ; Rats ; Signal Transduction ; Toll-Like Receptor 4/*antagonists & inhibitors/metabolism ; Transcription Factor RelA/metabolism