| Summary: | 博士 === 國立臺灣大學 === 生化學研究所 === 87 === Prolinedithiocarbamate (PDTC) and diethyldithiocarbamate (DDTC) are cancer chemopreventive agents and can be biotransformed to prolinethiuramdisulfide (PTDS) and tetraethylthiuramdisulfide (disulfiram: DTDS). We found that the reactive metabolites, PTDS and DTDS, induced apoptosis following G1/S arrest. Phosphorylation of cyclin E, inhibition of CDK2 kinase activity and degradation of cyclin E were found in human hepatoma Hep G2 cells during apoptotic process. Moreover, PTDS and DTDS decreased the level of Bcl-2 but increased p53. In contrast, PDTC, DDTC and ammonium dithiocarbamate (ADTC) did not induce apoptosis but led to the induction of p53 and p21 followed by G1/S arrest. They arrested cells in G1 phase. We then examined the effects of PTDS and DTDS on signal transduction mechanisms leading to apoptosis. While transcription factors NFkB, and AP-1 cooperatively decreased their DNA binding activities to the kB, TRE, respectively and p53 increased DNA binding activity in early stage but decreased in latter stage after treatment with PTDS, the human Hep G2 cells were undergoing apoptosis. In summary, our results indicated that 1). PTDS and DTDS induced apoptosis as well as G1/S arrest mediated by p53 whereas PDTC, DDTC and ADTC induced p53 dependent p21 expression leading to G1/S arrest; 2). PDTC, DDTC and ADTC induced p21/KIP1/CIP1 expression in a p53 dependent pathway leading to G1/S arrest; 3). NFkB, AP-1 and Bcl-2 were downregulated during PTDS and DTDS induced apoptosis. These results suggested that PTDS and DTDS induced p53-dependent apoptosis whereas PDTC, DDTC, and ADTC induced G1/S arrest. Apoptosis is regulated by the modulation of intracellular effectors of apoptosis such as NFkB, AP-1, Bcl-2 and activation of p53 in early stage.
1,2-Dithiol-3-thiones, constituents of cruciferous vegetables and cabbage, are five cyclic sulfur compounds with chemopreventive and chemotherapeutic activities. Oltipraz, [4-methyl-5-(2-pyrazinyl)-1,2-dithiole-3-thione] a synthetic 1,2-dithiol-3-thione chemopreventive agent, was able to induce apoptosis in human promyelocytic leukemia HL 60 cells. Characteristic morphology of apoptosis could be observed after 6 h when HL 60 cells were treated with high dose, 100 and 200 mM oltipraz, but not with 1,3-dithiol-2-thione. HL 60 cells exposed to increase concentrations of oltipraz from 10 mM to 200 mM confirmed the concentration dependence of apoptosis as evidenced by: 1). the DNA fragmentation assay of HL 60 cells; 2). the chromatin condensation appearance by acridine orange staining; 3). the evidence of apoptotic peak by flow cytometry. In addition, oltipraz induced NFkB and AP-1 binding to kB and 12-O-tetradecanoylphorbol-13-acetate-responsive elements (TRE) when treated with 10 mM, for 1, 3, and 12 h, respectively but not influence in xenobiotic responsive element (XRE) and antioxidant responsive element (ARE) in the same condition. We predicted that oltipraz induced apoptosis was mediated by NFkB and AP-1 pathway. On the other hand, NFkB family c-Rel (p75), Rel A (p65), p50 and MAPKs (mitogen-activated protein kinases) family, JNK1 antisense oligonucleotides significantly increased the cell apoptosis, but not sense oligonucleotides. Moreover, HL 60 cells were inhibited the endogenous c-Rel (p75), Rel A (p65), p50 and JNK1 proteins and the NFkB and AP-1 DNA binding activities by antisense oligonucleotides which were confirmed our hypothesis "Oltipraz inducing apoptosis mediated by NFkB and JNK1". Our results indicated that NFkB and AP-1 prevented the progress of cell death, but when these two transcription factors were inhibited by antisense oligonucleotides that would trigger cell apoptosis.
Peroxyacetyl nitrate (PAN), an ubiquitous air pollutant, was able to induce apoptosis in human leukemia HL 60, human chronic myelogenous leukemia K 562 and mouse monocyte-macrophage RAW 264.7 cell lines. Characteristic morphology of apoptosis could be observed 4 h after HL 60 cells were treated with 50 mM PAN. HL 60 cells exposed to increase concentrations of PAN from 1 mM to 100 mM confirmed the concentration dependence of apoptosis as evidenced by: a). the DNA fragmentation assay of HL 60 cells; b). the chromatin condensation appearance by acridine orange staining; c). the evidence of apoptotic peak by flow cytometry. In addition, PAN suppressed the tyrosine kinase activity through peroxynitrite-mediated nitration of tyrosine residues and we detected 3-nitrotyrosine and 3,5-dinitrotyrosine during apoptotic process of HL 60 cell by HPLC and LC-MS-MS. We predicted that PAN through peroxynitrite which induced human leukemia cell death. Moreover, exogenous superoxide dismutase (SOD) promoted PAN-induced apoptosis. In contrast, both SOD/catalase (CAT) repressed that PAN-induced apoptosis. The generation reactive oxygen species (ROS) of during PAN-induced apoptosis of HL 60 cells could activate SAPK/JNK kinase activity. The formation of H2O2 produced from the dismutation of PAN-elicited O2.- contributed to apoptotic mechanism in HL 60 cells through ROS pathways. These direct evidences suggested that air pollutant PAN-induced apoptosis were through the releases of both reactive nitrogen species (RNS) and ROS.
Peroxyacetyl nitrate (PAN), an ubiquitous air pollutant, was able to induce apoptosis in human leukemia HL 60, human chronic myelogenous leukemia K 562 and mouse monocyte-macrophage RAW 264.7 cell lines. Characteristic morphology of apoptosis could be observed 4 h after HL 60 cells were treated with 50 mM PAN. HL 60 cells exposed to increase concentrations of PAN from 1 mM to 100 mM confirmed the concentration dependence of apoptosis as evidenced by: a). the DNA fragmentation assay of HL 60 cells; b). the chromatin condensation appearance by acridine orange staining; c). the evidence of apoptotic peak by flow cytometry. In addition, PAN suppressed the tyrosine kinase activity through peroxynitrite-mediated nitration of tyrosine residues and we detected 3-nitrotyrosine and 3,5-dinitrotyrosine during apoptotic process of HL 60 cell by HPLC and LC-MS-MS. We predicted that PAN through peroxynitrite which induced human leukemia cell death. Moreover, exogenous superoxide dismutase (SOD) promoted PAN-induced apoptosis. In contrast, both SOD/catalase (CAT) repressed that PAN-induced apoptosis. The generation reactive oxygen species (ROS) of during PAN-induced apoptosis of HL 60 cells could activate SAPK/JNK kinase activity. The formation of H2O2 produced from the dismutation of PAN-elicited O2.- contributed to apoptotic mechanism in HL 60 cells through ROS pathways. These direct evidences suggested that air pollutant PAN-induced apoptosis were through the releases of both reactive nitrogen species (RNS) and ROS.
1). Naphthoquinone derivatives, the group natural products isolated from Diospyros; 2). Distyrylketones and Curcuminods, the group natural products isolated from the yellow pigment in curry; 3). Phlorophenone derivatives, the group natural products isolated from Dryopteris; 4). antiinflammatory drugs; 5). Prof. Th. Eicher and 6). C. Herhaus synthesized compounds were shown previously to mediate strong inhibition of 12-0- tetradecanoylphorbol-13-acetate (TPA)-induced ornithine decarboxylase (ODC) activity in cell culture. As reported currently, Naphthoquinone derivatives and phlorophenone derivatives evaluated for chemopreventive activity in the reducing the tumor marker: Epstein-Barr early antigen activation and we discovered those compounds inhibited the other tumor marker: ornithine decarboxylase. Curcumin derivative, including six methyl ether groups, was more effective than curcumin to inhibit the ornithine decarboxylase. The IC50 was lower than 10 mM. The IC50 of antiinflammatory drugs all were higher than 10 mM. At the same time, we have discovered the synthesized compounds from Prof. Th. Eicher (7/45) and C. Herhaus (11/31) which inhibited the ODC and IC50 was lower than 10 mM. These data indicate that those natural and snythetic compounds exhibits cancer chemopreventive effects in tumorigenesis marker: ODC and we need additional studies to characterize the cancer chemopreventive or chemotherapeutic potential of this substances more fully.
|
|---|
