cytochrome-c-t and Hepatoblastoma

Isoorientin (ISO) (CAS RN: 4261-42-1) is a flavonoid compound that can be extracted from several plant species, such as Phyllostachys pubescens, Patrinia, and Drosophyllum lusitanicum. ISO is able to induce apoptosis through mitochondrial dysfunction and inhibition of PI3K/Akt signaling pathway in HepG2 cells, however, the effects of ISO on MAPK signaling pathways remain unknown. The present study investigated the effects of ISO on this pathway, and the roles of MAPK kinases on mitochondrial-mediated apoptosis in HepG2 cells. The results showed that ISO induced cell death in a dose- and time-dependent manner, and induction apoptosis is main cause for ISO-induced cytotoxicity in HepG2 cells. ISO significantly inhibited the levels of ERK1/2 kinase and increased the expression of JNK and p38 kinases. Furthermore, U0126 (an ERK1/2 inhibitor) significantly enhanced the ISO-induced the Bax/Bcl-2 ratio, the release of cytochrome c to the cytosol fraction, and the levels of cleaved caspase-3. While SP600125 (a JNK inhibitor) and SB203580 (a p38 inhibitor) markedly prevented the expression of these proteins induced by ISO. Furthermore, the ROS inhibitor (NAC) notably promoted the inhibited effect of ISO on the ERK1/2 kinase. NAC also suppressed the p-JNK and p-p38, but failed to reverse the effects of ISO. These results demonstrated for the first time that ISO induces apoptosis in HepG2 cells through inactivating ERK1/2 kinase and activating JNK and p38 kinases, and ROS stimulated by ISO is able to activate the MAPK singaling pathway as the upstream signaling molecules. Initiating event of the mitochondrial-mediated apoptosis induced by ISO is MAPK signals.

Topics: Anthracenes; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Caspase 3; Cell Survival; Cytochromes c; Electrophoresis, Polyacrylamide Gel; Hep G2 Cells; Hepatoblastoma; Humans; Luteolin; MAP Kinase Signaling System; Mitochondria; p38 Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Phosphorylation; Plant Extracts; Reactive Oxygen Species

Mitochondrial calcium (mCa + 2) overload occurs during cold preservation and is an integral part of mitochondrial-dependent apoptotic pathways. We investigated the role of mCa + 2 overload in cell death following hypothermic storage using HepG2 cells stored in normoxic-hypothermic (4 degrees C) or hypoxic (< 0.1% O2)-hypothermic Belzer storage solution. Cells were stored for 6 h, with or without 10 microM ruthenium red (mCa + 2 uniporter inhibitor) followed by rewarming in oxygenated media at 37 degrees C. Cytoplasmic cytochrome c levels were studied by Western analysis and by fluorescent microscopy after transfection of cytochrome c-GFP expression plasmid. Immunofluorescence determined the intracellular, spatio-temporal distribution of Bax, and TUNEL staining was used to evaluate cell death after 180 min of rewarming. Caspase activation was evaluated using Western analysis and a caspase 3 activity assay. Bax translocation, cytochrome c release, and early rewarming cell death occurred following hypothermic storage and were exacerbated by hypoxia. Caspase 3 activation did not occur following hypothermic storage. Blockade of mCa + 2 uptake prevented Bax translocation, cytochrome c release, and early rewarming cell death. These studies demonstrate that mCa + 2 uptake during hypothermic storage, both hypoxic and normoxic, contributes to early rewarming apoptosis by triggering Bax translocation to mitochondria and cytochrome c release.

Topics: Apoptosis; bcl-2-Associated X Protein; Calcium; Caspase 3; Caspases; Cryopreservation; Cytochromes c; Hepatoblastoma; Humans; Indicators and Reagents; Mitochondria; Protein Transport; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Reperfusion Injury; Ruthenium Red; Tumor Cells, Cultured

Taxol treatment froze the cell cycle in the G(2)/M phase, induced morphological changes characteristic of apoptotic/necrotic cell death and increased CYP3A4 enzymatic activity, CYP3A4 mRNA and protein levels in HepG2 cells overexpressing CYP3A4. Apoptosis was associated with cytochrome c release to the cytosol; however, at higher Taxol levels, cells became relatively resistant to the drug-induced freezing of the cell cycle and saturation thresholds for both antiproliferative and proapoptotic activity of Taxol were observed. P-Glycoprotein expression was only slightly increased by Taxol, however, P-glycoprotein-mediated pumping efficiency was significantly increased. Preincubation of cells with an anti-MDR1 monoclonal antibody prior to the drug treatment, coincubation of cells with a potent CYP3A4 inhibitor--ketoconazole--or with both compounds increased Taxol toxicity and proapoptotic activity, indicating that the P-glycoprotein system has a major role in Taxol disposition in hepatoblastoma cells.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; Carrier Proteins; Cell Line, Tumor; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Cytochromes c; Dose-Response Relationship, Drug; Hepatoblastoma; Humans; Ketoconazole; Necrosis; Paclitaxel; RNA, Messenger