Byung-Hoon Lee

Antitumor activity of a novel ginseng saponin metabolite in human pulmonary adenocarcinoma cells resistant to cisplatin.

The in vitro antitumor activity of a novel ginseng saponin metabolite, 20-O-beta-D-glucopyranosyl-20(S)-protopanaxadiol (IH-901), was examined against four human cancer cell lines and one subline resistant to cisplatin (CDDP). The growth inhibitory activity of the compound was estimated by MTT tetrazolium assay. The mean concentrations of IH-901 needed to inhibit the proliferation of the cells by 50% (IC50) were 24.3, 25.9, 56.6 and 24.9 microM against human myeloid leukemia (HL-60), pulmonary adenocarcinoma (PC-14), gastric adenocarcinoma (MKN-45) and hepatoma (HepG2) cell lines, respectively. These values are higher than that of CDDP. In the CDDP-resistant PC/DDP cell line, the IC50 values of IH-901 and CDDP were 20.3 and 60.8 microM, respectively. These results suggest that IH-901 is not cross-resistant to CDDP in this cell line and could be a candidate for the treatment of CDDP resistant pulmonary cancer.

Induction of apoptosis by a novel intestinal metabolite of ginseng saponin via cytochrome c-mediated activation of caspase-3 protease.

Ginseng saponins exert various important pharmacological effects with regard to the control of many diseases including cancer. The novel intestinal bacterial metabolites of ginseng protopanaxadiol saponins have recently been found and isolated after the oral administration of ginseng extract in human and rats. 20-O-(beta-D-Glucopyranosyl)-20(S)-protopanaxadiol (IH-901) formed from ginsenosides Rb1, Rb2, and Rc is of particular interest in cancer chemoprevention and treatment. We investigated the effects of IH-901 on the human myeloid leukemia cell line HL-60 in terms of inhibition of proliferation and induction of apoptosis. IH-901 showed a significant cytotoxic activity in HL-60 cells (IC(50) = 24. 3 microM) following a 96-hr incubation. Treatment of HL-60 cells with IH-901 resulted in the formation of internucleosomal DNA fragments. The dose- and time-dependent induction of apoptosis by IH-901 was demonstrated in sandwich enzyme immunoassay and the results were confirmed by flow cytometric analysis. Morphological examination of IH-901-treated samples showed cells with chromatin condensation, cell shrinkage, and nuclear fragmentation, all typical characteristics of apoptotic cells. The treatment of HL-60 cells with IH-901 caused activation of caspase-3 protease and subsequent proteolytic cleavage of poly(ADP-ribose) polymerase. IH-901 did not affect the expression of antiapoptotic protein Bcl-2 but did cause a release of mitochondrial cytochrome c into cytosol. In conclusion, our results demonstrate that IH-901 dramatically suppresses HL-60 cell growth by inducing programed cell death through activation of caspase-3 protease, which occurs via mitochondrial cytochrome c release independently of Bcl-2 modulation. These results may provide a pivotal mechanism for the use of IH-901 in the prevention and treatment of leukemia.

Induction of apoptosis in human hepatoblastoma cells by tetrandrine via caspase-dependent Bid cleavage and cytochrome c release

Tetrandrine, a bis-benzylisoquinoline alkaloid from the root of Stephania tetrandra, induces apoptosis in human T-cell lines, lung carcinoma and hepatoblastoma cells. However, the mechanisms by which tetrandrine inhibits tumor cell growth are poorly understood. The purpose of the present study was to investigate the intracellular signaling mechanism of tetrandrine-induced apoptosis in HepG2 cells. The induction of apoptosis was determined by morphological analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. Treatment of cells with tetrandrine caused the upregulation of p53, downregulation of Bcl-X(L), cleavage of Bid and Bax, and release of cytochrome c, which were accompanied by activation of caspases 9, 3 and 8. The activation of caspases 9 and 3 preceded that of caspase 8. A broad-spectrum caspase inhibitor and a caspase 8-specific inhibitor completely blocked tetrandrine-induced Bid processing, cytochrome c release, activation of caspase 3, and cell death. These findings and data showing the early release of cytochrome c, cleavage of Bid and downregulation of Bcl-X(L) suggest that the mitochondrial pathway is primarily involved in tetrandrine-induced apoptosis. The activation of caspase 8 after early caspases 9 and 3 activation might act as an amplification loop for activation of upstream signals such as Bid cleavage or cytochrome c release. These data suggest that tetrandrine may constitute a plausible therapeutic for hepatocellular carcinoma.

Inhibition of proliferation and induction of apoptosis by tetrandrine in HepG2 cells.

Tetrandrine is a bisbenzylisoquinoline alkaloid derived from the root of Stephania tetrandra S. Moore, which has been reported to elicit in vitro cytotoxic effects on HeLa cells, and in vivo suppressive effects on mouse ascites tumors. In the present study, we examined the antiproliferative and apoptosis-inducing activity of tetrandrine in HepG2 cells, a human hepatoma cell line. Tetrandrine showed potent cytotoxic activity in HepG2 cells (IC(50)=9.0+/-1.0 micro M) following incubation for 48 h. Dose-dependent induction of apoptosis was observed by agarose gel electrophoresis and flow cytometric analysis. Treatment of HepG2 cells with tetrandrine resulted in the activation of caspase-3 protease, and subsequent proteolytic cleavage of poly(ADP-ribose) polymerase. These results suggest that tetrandrine is potentially useful as a chemotherapeutic/chemopreventive agent in hepatocellular carcinoma.

5-Arylamino-2-methyl-4,7-dioxobenzothiazoles as inhibitors of cyclin-dependent kinase 4 and cytotoxic agents

5-Arylamino-2-methyl-4,7-dioxobenzothiazoles were synthesized as inhibitors of cyclin-dependent kinase 4 (CDK4) and cytotoxic agents. Most of the 4,7-dioxobenzothiazoles exhibited selective inhibitory activities for the CDK4 and cytotoxic potential against human cancer cell lines.

Synthesis and antifungal activities of 5/6-arylamino-4,7-dioxobenzothiazoles.

5/6-Arylamino-4,7-dioxobenzothiazoles were synthesized and tested for in vitro antifungal activities against pathogenic fungi. Most of the tested 4,7-dioxobenzothiazoles exhibited potent antifungal activities against Candida species and Aspergillus niger.

Role of the Fas/Fas ligand death receptor pathway in ginseng saponin metabolite-induced apoptosis in HepG2 cells.

This research team found in previous studies, that the ginseng saponin metabolite IH901 induces apoptosis in HepG2 cellsvia a mitochondrial-mediated pathway, which resulted in the activation of caspase-9 and subsequently of caspase-3 and -8. Based on these results, the involvement of the Fas/Fas ligand (FasL) death-receptor pathway, in IH901 -induced apoptosis in HepG2 cells, was investigated. Levels of Fas and the Fas ligand (FasL) mRNA or protein were not increased by IH901, rather they were decreased significantly at 18 h post treatment. Soluble FasL (sFasL) was detectable by immunoprecipitation analysis in the medium of HepG2 cells treated with IH901. Increased levels of sFasL were inversely correlated with the levels of FasL. Preincubation of HepG2 cells with antagonistic anti-Fas antibody showed little protective effect, if any, on IH901-induced cell death. At a 30 u.M (24 and 48 h) and 40 μM (24 h) concentration of IH901, the cytotoxic effect of IH901 was less then 50%, anti-Fas antibody prevented IH901-induced cell death. However, at a 60 μM (24 and 48 h) and 40 uM (48 h) concentration of IH901, cell death rates were about 80% or more and most of the chemopreventive and chemotherapeutic effects of IH901 were manifested. Blocking the Fas receptor did not influence IH901-induced cell death. These results indicate that the Fas/FasL system is engaged, but not required for IH901-induced cell death, at pharmacologically significant concentrations.

N-nitrosocarbofuran, but not carbofuran, induces apoptosis and cell cycle arrest in CHL cells.

Carbofuran (CF) is one of the most widely used carbamate pesticides in the world applied for insect and nematode control. Due to its widespread use in agriculture and households, contamination of food, water, and air has become serious, and consequently adverse health effects are inevitable in humans, animals, wildlife and fish. It has been reported that CF alone or in combination with other carbamate insecticides influences the level of reproductive and metabolic hormones such as thyroxine and corticosterone, and results in impairment of endocrine, immune and behavioral functions. In this study, we evaluated the effects of CF and its metabolite, the N-nitroso derivative N-nitrosocarbofuran (NOCF), on genotoxicity, cell growth, cell cycle and apoptosis of Chinese hamster lung fibroblast (CHL) cells. NOCF, but not CF, induced genotoxicity determined by Ames test. NOCF inhibited the growth of Chinese hamster lung fibroblast (CHL) cells with an IC(50) of 12.8 microM. NOCF induced apoptosis of CHL cells, which was demonstrated by morphological changes, DNA fragmentation and flow cytometric analysis. Treatment of CHL cells with NOCF induced significant G(2)/M cell cycle arrest. Caspase-3, an executioner of apoptosis was also activated by the treatment of CHL cells with NOCF. These results suggest that NOCF, that is an important metabolite of CF, leads to the induction of cell cycle arrest and apoptosis in CHL cells.

Changes in expression and immunolocalization of protein associated with toxic bile salts-induced apoptosis in rat hepatocytes.

Abstract. Cholestatic liver injury results from the accumulation of toxic bile salts within the liver. The aim of the present study was to examine the temporal changes in expression and immunolocalization of protein associated with apoptosis in cholestatic rat liver. Rats were anesthetized and cholestasis was induced by double ligation of the common bile duct and sectioning between the ligatures. The animals were euthanized at dayxa03 and at weeksxa01, 2, 4, and 6 after bile duct ligation (BDL). Apoptotic cell death was increased fivefold after 3xa0days of BDL, decreased over 2xa0weeks, and remained constant thereafter as has been demonstrated by TUNEL staining. Western blot analysis for Bax, Bcl-2, cytochromexa0c, and p53 were performed. Results show that total cellular Bax protein was increased 3xa0days after BDL and decreased over time thereafter. We observed the translocation of Bax to mitochondria and subsequent release of cytochromexa0c. According to our immunohistochemical data, nuclear p53 increased 3xa0days after BDL, but cytoplasmic sequestration of p53 was observed after 1xa0week. The expression of c-Myc was inhibited by 3xa0days, but increased at later stages following BDL. Bcl-2 was increased over time in BDL rats. Our data suggest toxic bile salts-induced hepatocellular apoptosis is related to differential expression of Bcl-2 family member protein and release of cytochromexa0c. Cellular localization of p53 plays an important role in apoptotic death of hepatocytes in BDL rats.

Apoptosis inducing effects of 6-methoxydihydrosanguinarine in HT29 colon carcinoma cells

Abstract6-Methoxydihydrosanguinarine (6ME), a benzophenanthridine alkaloid derived from the methanol extracts ofHylomecon hylomeconoides, showed a dose-dependent effect at 1–10 μM on causing apoptotic cell death in HT29 colon carcinoma cells (IC50 = 5.0 ± 0.2 μM). Treatment of HT-29 cells with 6ME resulted in the formation of internucleosomal DNA fragmentation. Treatment of the cells with 6ME caused activation of caspase-3, -8 and 9 protease and subsequent proteolytic cleavage of poly(ADP-ribose)polymerase. 6ME increased the expression of p53 and Bax and decreased the expression of Bid. These results indicate that p53 and proapoptotic Bcl-2 family proteins might participate in the antiproliferative activity of 6ME in HT29 cells.