cytochrome-c-t and esculetin

Aesculetin (1) is an important coumarin found in various plant materials. It has been shown to have antiproliferative effects on several types of human cancer cells, but its effect on cervical cancer cells in vitro is unknown. In this study, we investigated that the cytotoxic effect of 1 on a non-cancer cell line (293) was smaller than on a tumor cell line (HeLa). This is the first report showing the possible mechanism of antiproliferative effect of 1 for the prevention of cervical cancer in cell culture models. It was found that 1 inhibited cell viability by inducing apoptosis, as evidenced by the formation of apoptotic bodies, generation of reactive oxygen species (ROS), and the accumulation of cells in the sub-G1 phase. Treatment with compound 1 decreased the cell growth in a dose-dependent manner with an IC(50) value of 37.8 microM. Aesculetin-induced apoptosis was correlated with mitochondrial dysfunction (DeltaPsi(m)), leading to the release of cytochrome c from the mitochondria to the cytosol, as well as the proteolytic activation of caspases in HeLa cells. These results indicate that 1 induces apoptosis in HeLa cells through a ROS-mediated mitochondrial dysfunction pathway.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Bisbenzimidazole; Caspases; Cytochromes c; Drug Screening Assays, Antitumor; Female; HeLa Cells; Humans; Mitochondria; Models, Biological; Molecular Structure; Plants, Medicinal; Tumor Cells, Cultured; Umbelliferones; Uterine Cervical Neoplasms

Esculetin is a phenolic compound that is found in various natural plant products and induces apoptosis in several types of human cancer cells. However, the underlying mechanisms of its action are not completely understood. In the present study, we used human leukemia cells to gain further insight into the mechanism of esculetin-induced anti-proliferative action and apoptosis. It was found that esculetin inhibits cell viability by inducing apoptosis, as evidenced by the formation of apoptotic bodies, DNA fragmentation, and the accumulation of cells in the sub-G1 phase. Esculetin-induced apoptosis was correlated with mitochondrial dysfunction, leading to the release of cytochrome c from the mitochondria to the cytosol, as well as the proteolytic activation of caspases. The z-DEVD-fmk caspase-3 inhibitor and the ectopic expression of anti-apoptotic Bcl-2 significantly inhibited esculetin-induced apoptosis, demonstrating the important role of caspase-3 and mitochondrial proteins in the observed cytotoxic effect. Furthermore, esculetin selectively increased the phosphorylation of extracellular-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), but not that of other kinases such as Akt and p38 activation. In addition, an ERK-specific inhibitor, PD98059, and a JNK-specific inhibitor, SP600125, showed inhibited sub-G1 phase DNA content, DNA fragmentation, caspase activation, and mitochondrial dysfunction induced by esculetin treatment. These results indicated that the JNK and ERK pathways were key regulators of apoptosis in response to esculetin in human leukemia U937 cells.

Topics: Antioxidants; Apoptosis; Blotting, Western; Caspase Inhibitors; Cell Cycle; Cell Nucleus; Cell Proliferation; Cell Survival; Cytochromes c; DNA Fragmentation; Extracellular Signal-Regulated MAP Kinases; Genes, bcl-2; Humans; Indicators and Reagents; JNK Mitogen-Activated Protein Kinases; Matrix Metalloproteinases; Oligopeptides; Phospholipase C gamma; U937 Cells; Umbelliferones

The potential use of low dose chemotherapy has been appealing since lower dosages are more attainable during cancer therapy and cause less toxicity in patients. Combination therapy of Taxol, a promising frontline chemotherapy agent, with natural anti-tumor agents that are considerably less toxic with a capability of activating additional apoptotic signals or inhibiting survival signals may provide a rational molecular basis for novel chemotherapeutic strategies. Esculetin, a well-known lipoxygenase inhibitor, showed an inhibitory effect on the cell cycle progression of HL-60 cells in our previous study. In this report, the effects of a concomitant administration of esculetin and Taxol were investigated in human hepatoma HepG2 cells. Firstly, esculetin alone could exert an antiproliferation effect together with an inhibitory effect on the activation of ERKs and p38 MAPK. As compared to the treatment with Taxol only, a co-administration with esculetin and Taxol could result in a further enhancement of apoptosis as revealed by DNA fragmentation assay and Annexin-V-based assay. Meanwhile, immunoblotting analysis also showed that the co-administration of esculetin and Taxol could increase the expression of Bax and the cytosolic release of cytochrome C and enhance the expression of Fas and Fas ligand while the activation of caspase-8 and caspase-3 was also increased. Finally, the ERK cascade was proven to be involved in the enhancement of esculetin on the Taxol-induced apoptosis.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Caspases; Cell Line, Tumor; Cytochromes c; Drug Synergism; Electrophoresis, Polyacrylamide Gel; Fas Ligand Protein; Humans; Immunoblotting; Membrane Glycoproteins; Microscopy, Fluorescence; Mitogen-Activated Protein Kinases; Paclitaxel; Tumor Necrosis Factors; Umbelliferones

Adipose tissue mass is determined by the volume and the number of adipocytes and is subjected to homeostatic regulation involving cell death mechanisms. We investigated the effects of esculetin, a coumarin compound, on apoptotic signaling in 3T3-L1 adipocytes. Esculetin treatment induced an increase in expression of Bax with a concomitant decrease of Bcl-2 in a time-dependent manner. Esculetin treatment also resulted in translocation of cytochrome c from mitochondria to cytosol and cleavage of 116 kDa poly(ADP-ribose) polymerase (PARP)-1, resulting in the accumulation of an 85 kDa cleavage product in a caspase-dependent manner. Furthermore, esculetin selectively altered the phosphorylation state of members of the MAPK superfamily, causing dephosphorylation of extracellular signal-regulating kinase 1/2 (ERK1/2) and hyperphosphorylation of c-Jun-N-terminal kinase (JNK). In addition, an inhibitor of the JNK MAP kinase pathway, SP600125, reduced esculetin-induced cytochrome c release. These results indicate that esculetin mediated adipocyte apoptosis involves the mitochondrial pathway. Esculetin thus decreases adipocyte number by initiating this apoptotic process in 3T3-L1 adipocytes.

Topics: 3T3-L1 Cells; Animals; Anthracenes; Apoptosis; Caspase 3; Cell Survival; Cytochromes c; JNK Mitogen-Activated Protein Kinases; Mice; Mitogen-Activated Protein Kinases; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Umbelliferones