Ursodeoxycholic acid induces apoptosis of hepatocellular carcinoma cells in vitro.

OBJECTIVE: Ursodeoxycholic acid (UDCA) is widely used to treat chronic liver diseases, and its cytoprotective effect on normal hepatocytes has been shown. This study aimed to investigate the apoptotic effects of UDCA on hepatocellular carcinoma (HCC) cells and the underlying molecular events in vitro. METHODS: HCC cells were treated by UDCA at different doses and periods of time to assess cell morphology, viability, apoptosis and gene expression using methyl thiazolyl tetrazolium (MTT), Annexin V/propidium iodide (PI) stain, transferase dUTP nick end labeling (TUNEL), enzyme-linked immunosorbent assay (ELISA), immunocytochemistry and quantitative reverse transcription polymerase chain reaction, respectively. RESULTS: UDCA treatment reduced cell viability but induced HCC cell apoptosis in dose-dependent and time-dependent manners. UDCA arrested HepG2 cells at phase S of the cell cycle. At the gene levels, UDCA downregulated Bcl-2 and second mitochondria-derived activator of caspase (Smac) protein expressions, but upregulated Bax and Livin proteins in HCC cells. At the highest concentration, UDCA inhibited Livin mRNA expression but increased Smac and caspase-3 mRNA expressions as well as the activity of caspase-3 in HCC cells. CONCLUSIONS: The induction of HCC cell apoptosis by UDCA was dose-dependent and time-dependent and was mediated by the regulation of Bax to Bcl-2 ratio, the expressions of Smac and Livin, and caspase-3 expression and activity.

[Advanced oxidation protein products promote expression of stromal-cell derived factor-1alpha of ECV304 cells through ERK signal pathway].

OBJECTIVE: To explore the effects of advanced oxidation protein products (AOPP) on expressions of stromal cell-derived factor-1alpha (SDF-1alpha) in ECV304 cells and the signal pathway that mediated the effects. METHODS: AOPP-BSA was made from bovine serum albumin (BSA) and sodium hypochlorite. After treated with AOPP-BSA of different concentrations (50, 100, 200 micromol/L), the expressions of SDF-1alpha mRNA in ECV304 cells were measured by reverse transcription-polymerase chain reaction (RT-PCR) and the expressions of SDF-1alpha protein and the levels of phosphorylated extracellular signal-regulated kinase (ERK) in ECV304 cells were analyzed by Western blot. In inhibition test, U0126, the special inhibitor of ERK of different concentrations (0.1, 1, 10 rmol/L) were added into ECV304 cells culture media for 1 hour, then the cells were treated with AOPP-BSA for 24 hours, at last the protein levels in supernatant were detected by enzyme-linked immunosorbent assay (ELISA). RESULTS: AOPP-BSA obviously promoted the expressions of SDF-1alpha mRNA and increased the levels of SDF-1beta protein of ECV304 cells in dose-dependent manner (all P < 0.01), after 15 minutes treated with 200 micromol/L AOPP-BSA, the levels of phosphorylated ERK of ECV304 cells increased significantly (P < 0.01). When the ERK pathway was blocked by U0126, the promoting effects of AOPP-BSA on expressions of SDF-la protein in ECV304 cells were significantly inhibited in dose-dependent manner (P < 0.05). CONCLUSION: AOPP induced the expression of SDF-la of ECV304 cells, ERK signal pathway is an important pathway that mediated the effects.

[Analysis of immunophenotypes of acute lymphoblastic leukemia by three color flow cytometry].

This study was purposed to investigate the immunophenotype characteristics and their significance in subtypes of acute lymphoblastic leukemia (ALL). The immunophenotypes in 40 cases of ALL were analyzed with three color flow cytometry using CD45/SSC two-parametric gating. The results showed that the three color flow cytometry assay using CD45/SSC two-parametric gating could accurately distinguish each other from lymphocytes, monocytes, granulocytes, erythroblasts and primitive cells in bone marrow and/or peripheral blood. Among 40 cases of ALL, B-ALL was 26 cases, T-ALL was 11 cases, HAL was 3 cases. All of the 26 cases of B-ALL expressed CD19 with positive rate of 100%, meanwhile 11 cases of T-ALL most highly expressed CD17 with positive rate of 100%. 12 cases of ALL with myeloid antigen expression (My-ALL) were involved in ALL, the incidence of these cases was almost 30% (12/40). The CD13 was expressed most highly in myeloid antigens. All 3 cases of HAL coexpressed myeloid and B-lineage antigens, among them CD34 was expressed in 2 cases with positive rate of 66.67%. It is concluded that three color flow cytometry assay using CD45/SSC two-parametric gating can exclude the interference of normal cells, thereby the results are more reliable and more accurate.

Dendroaspis natriuretic peptide relaxes gastric antral circular smooth muscle of guinea-pig through the cGMP/cGMP-dependent protein kinase pathway.

AIM: To systematically investigate if cGMP/cGMP-dependent protein kinase G (PKG) signaling pathway may participate in dendroaspis natriuretic peptide (DNP)-induced relaxation of gastric circular smooth muscle. METHODS: The content of cGMP in guinea pig gastric antral smooth muscle tissue and perfusion solution were measured using radioimmunoassay; spontaneous contraction of gastric antral circular muscles recorded using a 4-channel physiograph; and Ca(2+)-activated K(+) currents (I(K(Ca))) and spontaneous transient outward currents (STOCs) in isolated gastric antral myocytes were recorded using the whole-cell patch clamp technique. RESULTS: DNP markedly enhanced cGMP levels in gastric antral smooth muscle tissue and in the perfusion medium. DNP induced relaxation in gastric antral circular smooth muscle, which was inhibited by KT5823, a cGMP-dependent PKG inhibitor. DNP increased I(K(Ca)). This effect was almost completely blocked by KT5823, and partially blocked by LY83583, an inhibitor of guanylate cyclase to change the production of cGMP. DNP also increased STOCs. The effect of DNP on STOCs was abolished in the presence of KT5823, but not affected by KT-5720, a PKA-specific inhibitor. CONCLUSION: DNP activates I(K(Ca)) and relaxes guinea-pig gastric antral circular smooth muscle via the cGMP/PKG-dependent singling axis instead of cAMP/PKA pathway.

[Inhibition effect of antisense Bmi-1 on Jurkat cells].

OBJECTIVES: To investigate whether antisense Bmi-1 plasmid could inhibit the proliferation of Jurkat cells. METHODS: The antisense plasmid was constructed by PCR amplification of a 171 bp segment spanning Bmi-1 start codon and zinc finger structure and the PCR product was subsequently inserted reversely to plasmid pLNCX2. The final construct was confirmed through restriction enzyme digestion. G418 was added into the medium after the plasmid was successfully introduced into Jurkat cells by using lipofectin-mediated DNA transfection. The proliferation of Jurkat cells were determined by MTT and colony formation assays. Cell cycle was determined by flow cytometry. The p16 expression of Jurkat cells was studied by immunofluorescent histochemistry. RESULTS: The growth rate of antisense Bmi-1 transfected Jurkat cells was significantly lower than that of the controls, and the colony forming capacity of the transfected cells decreased significantly (P < 0.01), the colony numbers being (90.7 +/- 9.07)/10(3) cells, (83.3 +/- 6.11)/10(3) cells and (56.0 +/- 5.56)/10(3) cells for control cells, empty plasmid transfected Jurkat cells and antisense Bmi-1 transfected Jurkat cells, respectively. The percentage of G, phase cells was increased and the p16 expression of antisense Bmi-1 transfected cells was significantly upregulated than that of control cells. CONCLUSION: Antisense Bmi-1 can inhibit the growth and upregulate the expression of p16 of Jurkat cells in vitro.