Ji-Chang Yoo

Antiproliferative effects of alkaloids from Sedum sarmentosum on murine and human hepatoma cell lines.

The whole plant of Sedum sarmentosum (SS) has been traditionally used for the treatment of chronic viral hepatitis in China and South Korea. Certain hepatitis virus causes acute and chronic hepatitis and induces hepatocellular carcinoma (HC). In the present study, we examined whether the crude alkaloid fraction (CAF) of SS had any anticancer effects on hepatoma cell lines. Murine hepatoma (BNL CL. 2) and human hepatoma (HepG2) cell lines were cultured in the presence of CAF of SS at various doses (50-150 microg/ml) for 24 or 48 h. CAF caused a dose-dependent inhibition of cell proliferation without necrosis or apoptosis. Antiproliferative effects of CAF of SS were associated with an increase in the number of cells in the G1 phase of cell cycle. This study suggests that SS may improve survival of hepatoma patients via the inhibition of excessive growth of tumor cells.

Inhibition of nitric oxide synthesis by butanol fraction of the methanol extract of Ulmus davidiana in murine macrophages

Since there is increasing evidence that nitric oxide (NO) plays a crucial role in the pathogenesis of inflammatory diseases, this study was undertaken to address whether the methanol (MeOH) extract and its fractions of the bark of Ulmus davidiana Planch (Ulmaceae) could modulate the expression of inducible NO synthase (iNOS) in thioglycollate-elicited murine peritoneal macrophages and murine macrophage cell line, RAW264.7 cells. Stimulation of the peritoneal macrophages and RAW264.7 cells with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) resulted in increased production of NO in the medium. However, the butanol (BuOH) fraction of the MeOH extract of U. davidiana barks showed marked inhibition of NO synthesis in a dose-dependent manner. The inhibition of NO synthesis was reflected in the decreased amount of iNOS protein, as determined by Western blotting. The BuOH fraction did not affect the viability of RAW264.7 cells, as assessed by methylthiazol-2-yl-2, 5-diphenyl tetrazolium bromide (MTT) assay; rather, it reduced endogenous NO-induced apoptotic cell death via inhibition of NO synthesis in RAW264.7 cells. On the other hand, the BuOH fraction showed no inhibitory effect on the synthesis of NO by RAW264.7 cells, when iNOS was already expressed by the stimulation with IFN-gamma and LPS. Collectively, these results demonstrate that the BuOH fraction inhibits NO synthesis by inhibition of the induction of iNOS in murine macrophages.

Cyclic Adenosine Monophosphate Inhibits Ursolic Acid-Induced Apoptosis via Activation of Protein Kinase A in Human Leukaemic HL-60 Cells

This study was designed to investigate the effect of cAMP on ursolic acid-induced apoptosis of HL-60 cells. Ursolic acid decreased the viability of the cells in a dose-dependent manner, which was revealed as an apototic process characterized by ladder-pattern DNA fragmentation in agarose gel electrophoresis and segmented nuclei in DAPI-sulpharhodamin 101 staining. Ursolic acid-induced apoptosis of the cells was markedly inhibited by the addition of cAMP-elevating agents including DB-cAMP, CPT-cAMP, 8-Br-cAMP and forskolin. These results were further evidenced by the fact that inhibitors of cAMP-dependent protein kinase including H89 and KT5720 completely inhibited the cAMP-mediated rescue of HL-60 cells from ursolic acid-induced apoptosis. In addition, differentiating agents of the cells such as dimethyl sulfoxide and retinoic acid did not affect the ursolic acid-induced apoptosis of HL-60 cells. These results suggest that signaling pathway of cAMP-dependent activation of protein kinase A may affect the responsiveness of tumor cells upon ursolic acid.

Ionizing radiation potentiates the induction of nitric oxide synthase by interferon-γ (Ifn-γ) or Ifn-γ and lipopolysaccharide in Bnl Cl.2 murine embryonic liver cells: role of hydrogen peroxide

The effects of ionizing irradiation on the nitric oxide (NO) production in murine embryonic liver cell line, BNL CL.2 cells, were investigated. Various doses (5-40 Gy) of radiation made BNL CL.2 cells responsive to interferon-gamma alone for the production of NO in a dose-dependent manner. Small amounts of lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-alpha) synergized with IFN-gamma in the production of NO from irradiated BNL CL.2 cells, even though LPS or TNF-alpha alone did not induce NO production from the same cells. Immunoblots showed parallel induction of inducible nitric oxide synthase (iNOS). NO production in irradiated BNL CL.2 cells by IFN-gamma or IFN-gamma plus LPS was decreased by the addition of catalase, suggesting that H(2)O(2) produced by ionizing irradiation primed the cells to trigger NO production in response to IFN-gamma or IFN-gamma plus LPS. Furthermore, the treatment of nongamma-irradiated BNL CL.2 cells with H(2)O(2) made the cells responsive to IFN-gamma or IFN-gamma plus LPS for the production of NO. This study shows that ionizing irradiation has the ability to induce iNOS gene expression in responsive to IFN-gamma via the formation of H(2)O(2) in BNL CL.2 murine embryonic liver cells.

12-O-tetradecanoyl phorbol 13-acetate, protein kinase C (PKC) activator, protects human leukemia HL-60 cells from taxol-induced apoptosis: possible role for extracellular signal-regulated kinase.

Abstract The purpose of this study was to evaluate whether the mitogen-activated protein kinase / extracellular signal-regulated kinase (MEK) signaling pathway contributes to 12-O-tertadecanoyl phorbol 13-acetate (TPA)mediated protection from taxol-induced apoptosis of human leukemia HL-60 cells. Treatment of cells with taxol for 12 h resulted in apoptosis of HL-60 cells. TPA was protective against taxol-induced apoptosis and this anti-apoptotic effect was reversible when TPA was used in conjunction with staurosporine and H-7, PKC inhibitors, suggesting that TPA may protect HL-60 cells against taxol-induced apoptosis via the PKC-dependent pathway. Since TPA stimulates MEK signal transduction pathway in HL-60 cells, we postulated that MEK pathway may be playing a role in the ability of TPA to inhibit taxol-induced apoptosis. PD098059, a specific MEK kinase inhibitor, abolished the ability of TPA to inhibit taxol-induced apoptosis. These results suggest that activation of PKC in HL-60 cells confers protection against taxol-induced apoptosis and that MEK mediates anti-apoptotic signaling of PKC.

Apoptotic cell death induced by taxol is inhibited by nitric oxide in human leukemia HL-60 cells.

Taxol, an antineoplastic drug, increases the fraction of cells in G2/M phases of cell cycle, induces apoptosis of leukemic cells, and activates macrophages to produce nitric oxide (NO) in response to interferon-gamma. NO has been found to play roles as pro-apoptotic or anti-apoptotic effector molecules. In this study, we investigate effects of NO on taxol-induced apoptosis in human myeloid leukemia cell, HL-60. Incubation of the cells with taxol for 24 hr induced marked DNA fragmentation of HL-60 cells. Treatment of the cells with S-nitrosogluthathione (GSNO), a NO-generating agent, protected the cells against taxol-induced apoptosis. Cell cycle analysis showed that treatment of the cells with 100 nM taxol for 12 hr rendered the cells to be accumulated in G2/M phase, but the cotreatment of the cells with taxol and 0.1 mM GSNO decreased the accumulation of the cell in G2/M phases, suggesting that NO might interfere entering of taxol-treated cells into G2/M phases. Deferoxamine or mimosine, which can arrest cells mainly at G1/S phases, also decreased taxol-induced apoptosis and reduced the number of the taxol-treated cells arresting in G2/M phases. Thus, we conclude that a protective effect of NO on taxol-treated cells from apoptosis may be partially caused by interfering entering of the taxol-treated cells into G2/M phases.

Increased Intracellular cAMP Renders HL-60 Cells Resistant to Cytotoxicity of Taxol

The treatment of advanced cancers with paclitaxel (taxol) is hindered by the development of drug resistance. Resistance to taxol is known to be associated with multidrug resistance (MDR) and a mutation affecting either the alpha- or beta-subunit of tubulin. In this study, we demonstrated that an intracellular cAMP level may also play an important role in resistance to taxol in HL-60, acute promyelocytic leukemia cells. Exposure of HL-60 cells to various doses of taxol for 18 hr resulted in cell death. However, pretreatment of the cells with cAMP analogs such as N6:O2-dibutyl cAMP (Db-cAMP), 8-(4-chlorophenylthio) cAMP (CPT-cAMP) and 8-bromo-cAMP (8-Br-cAMP) or an intracellular cAMP elevating agent such as forskolin apparently rendered HL-60 cells more resistant to taxol, but not with dimethyl sulfoxide (DMSO) or retinoic acid (RA), well known differentiating agents. To investigate whether protein kinase A (PKA) activated by an increase in intracellular cAMP level could be involved in increased taxol resistance of the cells, we examined the effects of PKA inhibitors, including H-89 and KT5720, on taxol resistance induced by Db-cAMP. The PKA inhibitors significantly abolished Db-cAMP-induced taxol resistance. These results suggest that cAMP analogs may render tumor cells more resistant to taxol via PKA activation.

Interferon-Gamma Alone Triggers the Production of Nitric Oxide from Serum-Starved BNL CL.2, Murine Embryonic Liver Cells

A previous study has demonstrated that both interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) were needed to induce the production of nitric oxide (NO) in BNL CL.2 cells, murine embryonic liver cells. We here demonstrate that when BNL CL.2 cells were cultured with serum-free medium, they were induced to produce NO by the stimulation of IFN-gamma alone. BNL CL.2 cells were cultured with serum-free or serum-containing medium for 1-3 days and then stimulated to synthesize NO by IFN-gamma. Surprisingly, only serum-starved cells showed significant amount of nitrite accumulation and iNOS protein expression in response to IFN-gamma in dose- and time-dependent manners, but serum-supplied cells did not. When the cells were stimulated with IFN-gamma, tumor necrosis factor-alpha (TNF-alpha), or LPS in combinations, only the combination of IFN-gamma and LPS produced more NO than that produced by IFN-gamma alone. The production of NO by the cells stimulated with IFN-gamma or IFN-gamma plus LPS was blocked by the addition of N(G)-monomethyl-L-arginine (N(G)MMA), a NO synthesis inhibitor. To address the intracellular signal pathway responsible for the production of NO by the cells stimulated with IFN-gamma aloneor IFN-gamma plus LPS, we examined the effects of several protein kinase inhibitors on the production of NO from the cells. The production of NO was significantly inhibited by protein tyrosine kinase (PTK) inhibitors, genistein and herbimycin A, but not by protein kinase A or C inhibitors. These results suggest that the deprivation of serum from BNL CL.2 cell culture medium might prime the cells to induce NO synthesis when the cells are triggered by IFN-gamma and the involvement of PTK signal transduction pathway in the expression of inducible NO synthase gene in murine hepatoma cells.

Protective Effects of Glucocorticoids on Taxol-Induced Cytotoxicity in Human Leukemia Hl-60 Cells

Although the therapeutic actions of glucocorticoids are largely attributed to anti-inflammatory and immunosuppressive effects, they have been implicated in enhancing tissue and cellular protections. In this study, we examined whether glucocorticoids including dexamethasone (Dex) and hydroxycortisone could diminish the cytotoxic effects of anti-microtubule agents including taxol (paclitaxel), microtubule stabilizing agent, and colchicine, microtubule disrupting agent, on human leukemia HL-60 cells. Taxol or colchicine decreased the viability of HL-60 cells in a dose-dependent manner. However, micromolar concentrations of glucocorticoids rendered HL-60 cells resistant against the cytotoxic activity of anti-microtubule agents. Pretreatments of the glucocorticoids were more effective than simultaneous treatments with antimicrotuble agents. The fact that actinomycin D or cycloheximide reversed the cytoprotective effects of glucocorticoids on cytotoxicities in HL-60 cells induced by antimicrotuble agents suggests glucocorticoids cytoprotection might be mediated via newly synthesized protein. Collectively, these data showed that micromolar concentrations of dexamethasone or hydrocortisone could attenuate the cytotoxic effects of taxol or colchicine on human leukemia HL-60 cells via protein synthesis.

In vitro and in vivo modulation of transforming growth factor beta 1 gene expression by antisense oligomer.

Publisher Summary Antisense technology is emerging as an effective means of lowering the levels of specific gene products. It is based on the findings that these antisense sequences hybridize to specific ribonucleic acid (RNA) transcripts, thereby disrupting normal RNA processing, stability, and translation and preventing the expression of a targeted gene. Administration of antisense oligonucleotides, or transfer of expression constructs capable of producing intracellular antisense sequences complementary to the messenger RNA (mRNA) of interest, blocks the translation of specific genes in vitro and in vivo. Because antisense oligomer inhibits expression of specific genes, this approach provides the opportunity to examine the role of the expression of transforming growth factor beta (TGF- β 1 ) during macrophage activation or wound healing processes. Thus, the approach introduces in vitro and in vivo modulation of TGF- β 1 gene expression by antisense oligomer complementary to TGF- β 1 mRNA. The materials with which the antisense approach can be applied are classified as antisense oligodeoxynucleotides (ODNs), antisense RNAs, and catalytic RNAs or ribozymes. Antisense sequences can be engineered either chemically (antisense ODNs) or genetically (antisense RNAs). This chapter discusses antisense ODNs, which offer important stability and synthetic advantages over the use of antisense RNAs, and is intended as an introduction to practical approaches in the use of antisense ODNs. Evidence of the cellular uptake of antisense TGF- β 1 and its effect on TGF- β 1 expression in cultured macrophages is presented and the skin absorption of antisense and scar-removing effect by the inhibition of TGF- β 1 expression on the skin are described in the chapter.