cytochrome-c-t and sanguinarine

Sanguinarine is a benzophenanthridine alkaloid that is known to have antimicrobial, anti-inflammatory, antioxidant and anticancer properties. In this study, we examined the effects of this compound on reactive oxygen species (ROS) production and the association of these effects with apoptotic tumor cell death using a human breast carcinoma MDA-MB-231 cell line.. Cytotoxicity was evaluated by trypan blue exclusion methods. Apoptosis was detected using DAPI staining, agarose gel electrophoresis and flow cytometer. The expression levels of proteins were determined by Western blot analyses and caspase activities were measured using colorimetric assays. The ROS production and mitochondrial membrane potential (MMP, Delta Psi(m)) changes were measured fluorimetrically.. The results of this study demonstrate that sanguinarine mediates ROS production, and that this mediation is followed by a decrease in MMP, the release of cytochrome c, activation of caspase-9 and caspase-3, and downregulation of antiapoptosis factor XIAP and cIAP-1. Sanguinarine also promoted the activation of caspase-8 and truncation of Bid (tBid). Moreover, the quenching of ROS generation by N-acetyl-L-cysteine administration, a scavenger of ROS, reversed the sanguinarine-induced apoptosis effects via inhibition of ROS production, MMP collapse, tBid expression and the subsequent activation of caspases. These observations clearly indicate that ROS are involved in the early molecular events in the sanguinarine-induced apoptotic pathway.. Our data imply that sanguinarine-induced ROS are key mediators of MMP collapse, which leads to the release of cytochrome c followed by caspase activation, culminating in apoptosis.

Topics: Apoptosis; Benzophenanthridines; BH3 Interacting Domain Death Agonist Protein; Breast Neoplasms; Caspases; Cell Line, Tumor; Cytochromes c; Enzyme Activation; Humans; Isoquinolines; Membrane Potential, Mitochondrial; Mitochondria; Molecular Structure; Reactive Oxygen Species

Apoptogenic and DNA damaging effects of chelidonine (CHE) and sanguinarine (SAN), two structurally related benzophenanthridine alkaloids isolated from Chelidonium majus L. (Papaveraceae), were compared. Both alkaloids induced apoptosis in human acute T-lymphoblastic leukaemia MT-4 cells. Apoptosis induction by CHE and SAN in these cells was accompanied by caspase-9 and -3 activation and an increase in the pro-apoptotic Bax protein. An elevation in the percentage of MT-4 cells possessing caspase-3 in active form after their treatment with CHE or SAN was in parallel to a corresponding increase in the fraction of apoptotic cells. The involvement of mitochondria in apoptosis induction by both alkaloids was supported by cytochrome C elevation in cytosol, with an accompanying decrease in cytochrome C content in the mitochondrial fraction. At the same time, two alkaloids under study differed drastically in their cell cycle phase-specific effects, since only CHE arrested MT-4 cells in G(2)/M phase. It was shown earlier, that CHE, in contrast to SAN, does not interact directly with DNA. This fact is in line with DNA damaging effects of the alkaloids detected in the COMET assay. Nevertheless, apoptosis-inducing activity of CHE even slightly exceeded that of SAN.

Topics: Alkaloids; Apoptosis; bcl-2-Associated X Protein; Benzophenanthridines; Blotting, Western; Caspase 9; Cell Line, Tumor; Cell Nucleus; Chelidonium; Comet Assay; Cytochromes c; DNA Damage; DNA, Neoplasm; Flow Cytometry; Humans; Intercalating Agents; Isoquinolines; Leukemia-Lymphoma, Adult T-Cell

Primary effusion lymphoma (PEL) is an incurable, aggressive B-cell malignancy that develops rapid resistance to conventional chemotherapy. In efforts to identify novel approaches to block proliferation of PEL cells, we found that sanguinarine, a natural compound isolated from the root plant Sanguinaria canadendid, inhibits cell proliferation and induces apoptosis in a dose-dependent manner in several PEL cell lines. Our data show that sanguinarine treatment of PEL cells results in up-regulation of death receptor 5 (DR5) expression via generation of reactive oxygen species (ROS) and causes activation of caspase-8 and truncation of Bid (tBid). Subsequently, tBid translocates to the mitochondria causing conformational changes in Bax, leading to loss of mitochondrial membrane potential and release of cytochrome c to the cytosol. Sanguinarine-induced release of cytochrome c results in activation of caspase-9 and caspase-3 and poly(ADP-ribose) polymerase (PARP) cleavage, leading to induction of caspase-dependent apoptosis. In addition, we show that pretreatment of PEL cells with carbobenzoxy-Val-Ala-Asp-fluoromethylketone, a universal inhibitor of caspases, abrogates caspase and PARP activation and prevents cell death induced by sanguinarine. Moreover, treatment of PEL cells with sanguinarine down-regulates expression of inhibitor of apoptosis proteins (IAP). Finally, N-acetylcysteine, an inhibitor of ROS, inhibits sanguinarine-induced generation of ROS, up-regulation of DR5, Bax conformational changes, activation of caspase-3, and down-regulation of IAPs. Taken together, our findings suggest that sanguinarine is a potent inducer of apoptosis of PEL cells via up-regulation of DR5 and raise the possibility that this agent may be of value in the development of novel therapeutic approaches for the treatment of PEL.

Topics: Alkaloids; Apoptosis; bcl-2-Associated X Protein; Benzophenanthridines; BH3 Interacting Domain Death Agonist Protein; Caspases; Cell Growth Processes; Cell Line, Tumor; Collagen Type XI; Cytochromes c; Dose-Response Relationship, Drug; Enzyme Activation; Exudates and Transudates; Humans; Isoenzymes; Isoquinolines; Lymphoma, B-Cell; Membrane Potential, Mitochondrial; Mitochondria; Molecular Conformation; Reactive Oxygen Species; Receptors, TNF-Related Apoptosis-Inducing Ligand; Signal Transduction; Up-Regulation

Sanguinarine, derived from the root of Sanguinaria canadensis and other poppy fumaria species, possesses strong antimicrobial, anti-inflammatory, and antioxidant properties. We earlier showed that sanguinarine kills human epidermoid carcinoma A431 cells via an induction of apoptosis [N. Ahmad et al., Clin. Cancer Res., 6: 1524-1528, 2000]. In this study, using immortalized human keratinocytes (HaCaT cells), we provide information about mechanism of the antiproliferative effect of sanguinarine. Sanguinarine [0.1 (M-2 (M)] treatment to HaCaT cells was found to inhibit in a dose-dependent manner the cell proliferation and induce apoptosis, as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and ELISA, respectively. Sanguinarine treatment also resulted in a significant cleavage of poly(ADP-ribose) polymerase in HaCaT cells. Because mitochondrial pathway is critical for the regulation of apoptosis, we studied the involvement and regulation of mitochondrial events in sanguinarine-mediated apoptosis of HaCaT cells. As shown by the immunoblot analysis, our data clearly demonstrated that sanguinarine treatment to HaCaT cells resulted in a dose-dependent (a) increase in the level of Bax with a concomitant decrease in Bcl-2 levels and (b) increase in Bax/Bcl-2 ratio. Sanguinarine also resulted in significant increases in the proapoptotic members of Bcl-2 family proteins, i.e., Bak and Bid. This was accompanied by increase in (a) protein expression of cytochrome c and apoptotic protease-activating factor-1 and (b) activity and protein expression of caspase-3, caspase-7, caspase-8, and caspase-9. Taken together, our data showed the involvement of mitochondrial pathway and Bcl-2 family proteins during sanguinarine-mediated apoptosis of immortalized keratinocytes. We suggest that sanguinarine could be developed as a drug for the management of hyperproliferative skin disorders, including skin cancer.

Topics: Alkaloids; Antioxidants; Apoptosis; bcl-2-Associated X Protein; Benzophenanthridines; Caspase 3; Caspase 7; Caspase 8; Caspase 9; Caspases; Cell Division; Cell Line, Tumor; Cell Survival; Cytochromes c; Cytosol; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Humans; Immunoblotting; Isoquinolines; Keratinocytes; Mitochondria; Models, Biological; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2