This study is to investigate the effects of ceramide on GSTA1 expression in Caco-2 cells. After being exposed to ceramide for a fixed time, GSTA1 protein expression was detected by Western blotting analysis; GSTA1 mRNA expression was detected by real time PCR; dual luciferase assay was used to analyze GSTA1 transcriptional activity and GSTA1 activity was determined toward androstanedione (AD) as substrate. The data showed that ceramide can significantly induce the expression of protein and GSTA1 mRNA, and increase transcriptional activity and enzyme activity of GSTA1. The results demonstrated that ceramide may increase resistance to chemotherapeutics in Caco-2 cells by up-regulating the expression of GSTA1.
Caco-2 Cells ; Enzyme Activation ; drug effects ; Glutathione Transferase ; genetics ; metabolism ; Humans ; RNA, Messenger ; metabolism ; Sphingosine ; analogs & derivatives ; pharmacology ; Transcriptional Activation ; drug effects ; Up-Regulation

OBJECTIVETo study the inhibition effect of HCV NS5A on p53 protein transactivity and its possible mechanism.

METHODSLuciferase reporter gene system was used for the study of p53 transactivity on p21 promoter and electrophorectic mobility-shift assay (EMSA) was applied to observe whether HCV NS5A could suppress the binding ability of p53 protein to its specific DNA sequence.

RESULTSEndogenous p53 protein could stimulate p21 promoter activity, and the relative luciferase activity increased significantly (3.49 x 10(5) vs 0.60 x 10(5), t = 5.92, P<0.01). Exogenous p53 protein also up-regulated p21 promoter driving luciferase expression, comparing to the control group (0.47 x 10(5)), the relative luciferase activity increased (5.63 x 10(5)) obviously (t = 10.12, P<0.01). HCV NS5A protein inhibited both endogenous and exogenous p53 transactivity on p21 promoter in a dose-dependent manner (F > or = 20.71, P<0.01). In the experiment of EMSA, p53 could bind to its specific DNA sequence, but when co-transfected with HCV NS5A expressing vector, the p53 binding affinity to its DNA decreased.

CONCLUSIONHCV NS5A can inhibit p53 protein transactivity on p21 promoter through its inhibiting of p53 binding ability to the specific DNA sequence.

Hepacivirus ; genetics ; Humans ; Promoter Regions, Genetic ; Transcriptional Activation ; drug effects ; Tumor Suppressor Protein p53 ; drug effects ; genetics ; metabolism ; physiology ; Viral Core Proteins ; genetics ; Viral Nonstructural Proteins ; genetics ; pharmacology

The mechanisms by which emodin induces apoptosis and inhibits proliferation of cancer cells remain unclear. In this study, we investigated whether the proapoptotic effect of emodin on human NIH-H460 lung cancer cells and SMMC-7721 liver cancer cells was related to regulating RXR expression and function. MTT assay and DAPI staining were used to detect the anti-proliferative and apoptotic effects of emodin with or without 9-cis-retinoid acid on H460 and SMMC-7721. The reporter assay was used to detect the effect of emodin on RXR homo- and hetero-dimer transactivation. Competitive ligand binding assay was carried out to detect whether emodin could directly bind to RXR. The result showed that emodin could strongly inhibit the proliferation and induce apoptosis of both cancer cell lines, which could be antagonized by 9-cis-RA. The reporter assay showed that emodin could inhibit the transcriptional effect of the homo- and hetero-dimer transactivation of RXRalpha dose-dependently. However, in vitro binding assay did not show that emodin bind to RXRalpha-LBD directly. The findings suggest that exhibition of emodin its anti-cancer activity may be associated with involvement of RXRalpha signal transduction pathways.
Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Emodin ; metabolism ; pharmacology ; Humans ; Retinoid X Receptor alpha ; metabolism ; Signal Transduction ; drug effects ; Transcriptional Activation ; drug effects ; Tretinoin ; antagonists & inhibitors

This study was aimed to investigate the reversing effect of arsenic trioxide (As2O3) on methylation status and the regulatory effect on transcription of malignant lymphoma cell line CA46 p16 gene as well as their possibe mechanisms. The hypermethylated malignant lymphoma cell line CA46 was used as a subject of experiment for studying relation of gene methylation with expression. The effect of As2O3 on the proliferation and viability of CA46 was detected by SRB method, the change of p16 methylation status after exposure to As2O3 was determined by nMSP, the expressions of p16, DNMT1, DNMT3A, DNMT3B mRNA were assayed by RT-PCR, the influence of As2O3 on CA46 cell cycle was analyzed by flow cytometry using analytical method for DNA ploidy. The results showed that the methylation level of p16 gene was obviously reduced after treatment with As2O3 for 72 hours and the hypermethylation of p16 gene was successfully reversed; the expression of p16 gene in untreated (control) group was low while it was enhanced in treated groups; the gray scale ratios of p16 gene to beta-actin in groups treated with As2O3 of concentration 0.5, 1.0 and 2.0 micromol/L were 0.33+/-0.10, 0.57+/-0.11 and 0.67+/-0.09 respectively, exhibiting a significant difference in comparison with 0.73+/-0.13 of positive control (p<0.01); as compared with the untreated group, the expression of DNMT3A and DNMT3B in treated groups was obviously down-regulated in a concentration-dependent manner, while expression of DNMT1 was nearly unchanged; as compared with control, all the 3 different concentrations of As2O3 could inhibit the proliferation of CA46 cells and increase the cell number in G0/G1 phase. It is concluded that the As2O3 may up-regulate the expression of p16 gene, recover the activity of p16 gene, thereby promote the regulatory function on cell cycle resul-ting in arrest of cells in G0/G1 phase and inhibit growth of tumor cells through depressing the expression of DNMT3A and DNMT3B and/or directly reversing the methylation status of p16 gene.
Arsenicals ; pharmacology ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; metabolism ; DNA Methylation ; drug effects ; Genes, p16 ; Humans ; Lymphoma ; genetics ; Oxides ; pharmacology ; Transcriptional Activation

Animals ; Drugs, Chinese Herbal ; pharmacology ; Enzyme Activation ; Humans ; Medicine, Chinese Traditional ; NF-kappa B ; antagonists & inhibitors ; genetics ; metabolism ; Promoter Regions, Genetic ; Signal Transduction ; drug effects ; Transcription, Genetic ; genetics ; Transcriptional Activation ; genetics

Fenofibrate, an agonist for peroxisome proliferator-activated receptor alpha (PPAR-α), lowers blood pressure, but whether this action is mediated via baroreflex afferents has not been elucidated. In this study, the distribution of PPAR-α and PPAR-γ was assessed in the nodose ganglion (NG) and the nucleus of the solitary tract (NTS). Hypertension induced by drinking high fructose (HFD) was reduced, along with complete restoration of impaired baroreceptor sensitivity, by chronic treatment with fenofibrate. The molecular data also showed that both PPAR-α and PPAR-γ were dramatically up-regulated in the NG and NTS of the HFD group. Expression of the downstream signaling molecule of PPAR-α, the mitochondrial uncoupling protein 2 (UCP2), was up-regulated in the baroreflex afferent pathway under similar experimental conditions, along with amelioration of reduced superoxide dismutase activity and increased superoxide in HFD rats. These results suggest that chronic treatment with fenofibrate plays a crucial role in the neural control of blood pressure by improving baroreflex afferent function due at least partially to PPAR-mediated up-regulation of UCP2 expression and reduction of oxidative stress.
Afferent Pathways ; drug effects ; Animals ; Antihypertensive Agents ; pharmacology ; Baroreflex ; drug effects ; Blood Pressure ; drug effects ; Fenofibrate ; pharmacology ; Male ; Oxidative Stress ; drug effects ; PPAR gamma ; drug effects ; metabolism ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects ; Transcriptional Activation ; drug effects ; Uncoupling Protein 2 ; drug effects ; metabolism ; Up-Regulation

OBJECTIVETo study the expression and mechanism of WNK4 gene regulated by aldosterone.

METHODSWistar rats were treated with aldosterone and potassium water. Serum aldosterone and ion as well as urine ion were measured. The expression of WNK4 gene in kidney tissues was detected by real-time PCR. Kidney-derived HEK293 cells were cultured, transfected with pGL3-WNK4, and then stimulated by aldosterone. After 24 h of transfection, luciferase activities of the plasmid were detected.

RESULTSCompared with those of the controls, serum aldosterone and urine K(+) of experimental rats were significantly elevated, whilst urine Na(+) was significantly decreased. And urine Cl(-) was significantly increased only in the group of high K(+). Serum K(+), Na(+) and Cl(-) showed no significant difference. Expression of WNK4 gene in kidney tissues was significantly decreased. The luciferase activity of pGL3-WNK4-484 plasmid has decreased after stimulated with aldosterone, while the activity of pGL3-WNK4-275 showed no change.

CONCLUSIONAldosterone can down-regulate the expression of WNK4 through binding with regulatory element in the upstream of the gene.

Aldosterone ; blood ; pharmacology ; Animals ; Cell Line ; Chlorides ; blood ; Gene Expression Regulation ; drug effects ; Humans ; Kidney ; metabolism ; Male ; Potassium ; blood ; Promoter Regions, Genetic ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; Rats ; Sodium ; blood ; Transcriptional Activation ; drug effects

OBJECTIVETo investigate the effect of ginsenosides of ginseng stem and leaf (GSL) on the transcription activity of glucocorticoid receptor (GR).

METHODSHL7702 cells were transiently cotransfected by GR luciferase reporter plasmid pGRE-tk-Luc and inner referential gene pRL-SV40 plasmid, and the effects of dexamethasone (Dex) and GSL on the reporter gene expression were detected by dual-luciferase reporter assay.

RESULTSThe reporter gene expression Dex-induced in HL7702 was dose- and time-dependent, it could be multiplied to 93 times in maximum, and could be blocked by the GR antagonist RU486. GSL could not induce the expression of reporter gene but could elevate the induction of Dex for 61.4%.

CONCLUSIONGSL could enhance the inducing effect of Dex on transcription activity of GR in vitro.

Cell Line ; Dexamethasone ; pharmacology ; Gene Expression Regulation ; Genes, Reporter ; Ginsenosides ; isolation & purification ; pharmacology ; Humans ; Liver ; cytology ; Mifepristone ; pharmacology ; Panax ; chemistry ; Plant Leaves ; chemistry ; Plant Stems ; chemistry ; Receptors, Glucocorticoid ; drug effects ; physiology ; Transcriptional Activation ; drug effects ; Transfection

In order to determine the involvement of CALM-AF10 fusion transcripted in primary leukaemias with t(10;11) and its chemotherapy sensitivity in vitro, the AF10-CALM fusion transcripts were detected by reverse transcription-polymerase chain reaction (RT-PCR), and the chemotherapy sensitivity testing in vitro was undergone by MTT assay in five t(10;11) leukemia samples from patients with ALL, AML and lymphoblastic lymphoma. The results showed that five different-sized AF10-CALM product and four different-sized CALM-AF10 products were detected. The chemotherapy sensitivity of leukemic cells with t(10;11) in vitro to drugs is lower than that of leukemic cells without t(10;11). 3 out of 5 cases of t(10;11) leukemia were sensitive to chemotherapeutic drugs, while 31 out of 36 cases of leukemia without t(10;11) were sensitive at same condition. There were significant differences (P < 0.01), consistent with clinical features of patients. Apoptosis rate of leukemic cells with t(10;11) induced by chemotherapeutic drugs was lower than that of leukemic cells without t(10;11), (16.37 +/- 2.56)%, and (33.75 +/- 5.59)%, respectively (P < 0.01). It is concluded that the CALM-AF10 fusion transcripts are a common features and are involved in the pathogenesis of haematological malignancies with t(10;11), and are associated with a poor prognosis.
Antineoplastic Agents ; pharmacology ; Cell Survival ; drug effects ; Chromosomes, Human, Pair 10 ; genetics ; Chromosomes, Human, Pair 11 ; genetics ; Humans ; Leukemia ; genetics ; pathology ; Oncogene Proteins, Fusion ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transcriptional Activation ; drug effects ; Translocation, Genetic ; Tumor Cells, Cultured

Retinoid X receptor α (RXRα) and its N-terminally truncated version tRXRα play important roles in tumorigenesis, while some RXRα ligands possess potent anti-cancer activities by targeting and modulating the tumorigenic effects of RXRα and tRXRα. Here we describe NSC-640358 (N-6), a thiazolyl-pyrazole derived compound, acts as a selective RXRα ligand to promote TNFα-mediated apoptosis of cancer cell. N-6 binds to RXRα and inhibits the transactivation of RXRα homodimer and RXRα/TR3 heterodimer. Using mutational analysis and computational study, we determine that Arg316 in RXRα, essential for 9-cis-retinoic acid binding and activating RXRα transactivation, is not required for antagonist effects of N-6, whereas Trp305 and Phe313 are crucial for N-6 binding to RXRα by forming extra π-π stacking interactions with N-6, indicating a distinct RXRα binding mode of N-6. N-6 inhibits TR3-stimulated transactivation of Gal4-DBD-RXRα-LBD by binding to the ligand binding pocket of RXRα-LBD, suggesting a strategy to regulate TR3 activity indirectly by using small molecules to target its interacting partner RXRα. For its physiological activities, we show that N-6 strongly inhibits tumor necrosis factor α (TNFα)-induced AKT activation and stimulates TNFα-mediated apoptosis in cancer cells in an RXRα/tRXRα dependent manner. The inhibition of TNFα-induced tRXRα/p85α complex formation by N-6 implies that N-6 targets tRXRα to inhibit TNFα-induced AKT activation and to induce cancer cell apoptosis. Together, our data illustrate a new RXRα ligand with a unique RXRα binding mode and the abilities to regulate TR3 activity indirectly and to induce TNFα-mediated cancer cell apoptosis by targeting RXRα/tRXRα.
Apoptosis ; drug effects ; Cell Line, Tumor ; Enzyme Activation ; drug effects ; Humans ; Ligands ; Molecular Docking Simulation ; Nuclear Receptor Subfamily 4, Group A, Member 1 ; genetics ; metabolism ; Oximes ; metabolism ; pharmacology ; Protein Conformation ; Proto-Oncogene Proteins c-akt ; metabolism ; Pyrazoles ; metabolism ; pharmacology ; Retinoid X Receptor alpha ; chemistry ; genetics ; metabolism ; Thiazoles ; metabolism ; pharmacology ; Transcription, Genetic ; drug effects ; Transcriptional Activation ; drug effects ; Tumor Necrosis Factor-alpha ; metabolism