cytochrome-c-t and Leukemia-P388

The work presented here was initiated to explore the mechanisms underlying vinorelbine resistance in two previously established murine leukemia P388 cell lines (N.63 and N2.5). IC(50) measurements demonstrated that the vinorelbine-resistant cell line N.63 was sensitive to both vinblastine and vinflunine. In addition, vinorelbine-resistant cell line N2.5 retained sensitivity to vinflunine. We used flow cytometry with propidium iodide to measure G2/M arrest in response to drug treatment. Annexin V labeling was used as a marker of apoptosis and JC-1 dye labeling as a marker of mitochondrial membrane depolarization to explore differential responses that might help explain the absence of cross resistance to vinflunine. At equipotent (10X IC(50)) doses, after 8 h of drug treatment, vinflunine induced G2/M arrest in a significantly larger fraction of vinorelbine- resistant cells compared to vinorelbine. At the same drug doses, at 16 h after initiation of drug treatment, vinflunine induced a statistically significant greater apoptotic response and mitochondrial depolarization. The mitochondrial depolarization at 16 h was confirmed by Western blotting that showed release of cytochrome c. Comparison of apoptotic and mitochondrial depolarization responses in vinorelbine-resistant cells upon exposure to vinorelbine, vinblastine and vinflunine demonstrated the following pattern of drug activity: vinflunine > vinblastine > vinorelbine, confirming the importance of a antimitotic-induced mitochondria-mediated pathways in these P388 cell lines. We conclude that vinflunine may be preferred for treatment of specific cancers compared to other vinca alkaloids due to its enhanced effects on apoptotic pathways that follow G2/M arrest.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Cell Division; Cell Line, Tumor; Cytochromes c; Drug Resistance, Neoplasm; Flow Cytometry; G2 Phase; Inhibitory Concentration 50; Leukemia P388; Mice; Mitochondrial Membranes; Vinblastine; Vinorelbine

Lamellarin D is a marine alkaloid with a pronounced cytotoxicity against a large panel of cancer cell lines and is a potent inhibitor of topoisomerase I. However, lamellarin D maintains a marked cytotoxicity toward cell lines resistant to the reference topoisomerase I poison camptothecin. We therefore hypothesized that topoisomerase I is not the only cellular target for the drug. Using complementary cell-based assays, we provide evidence that lamellarin D acts on cancer cell mitochondria to induce apoptosis. Lamellarin D, unlike camptothecin, induces early disruption of the inner mitochondrial transmembrane potential (Deltapsi(m)) in the P388 leukemia cell line. The functional alterations are largely prevented by cyclosporin A, an inhibitor of the mitochondrial permeability transition (MPT), but not by the inhibitor of caspases, benzyloxycarbonyl-Val-Ala-Asp(Ome)-fluoromethylketone. Deltapsi(m) disruption is associated with mitochondrial swelling and cytochrome c leakage. Using a reliable real-time flow cytometric monitoring of Deltapsi(m) and swelling of mitochondria isolated from leukemia cells, we show that lamellarin D has a direct MPT-inducing effect. Furthermore, mitochondria are required in a cell-free system to mediate lamellarin D-induced nuclear apoptosis. The direct mitochondrial effect of lamellarin D accounts for the sensitivity of topoisomerase I-mutated P388CPT5 cells resistant to camptothecin. Interestingly, a tumor-active analogue of lamellarin D, designated PM031379, also exerts a direct proapoptotic action on mitochondria, with a more pronounced activity toward mitochondria of tumor cell lines compared with nontumor cell lines. Altogether, this work reinforces the pharmacologic interest of the lamellarins and defines lamellarin D as a lead in the search for treatments against chemoresistant cancer cells.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Camptothecin; Cell Line, Tumor; Cell Membrane Permeability; Cell-Free System; Coumarins; Cytochromes c; Heterocyclic Compounds, 4 or More Rings; Humans; Isoquinolines; Leukemia P388; Lung Neoplasms; Membrane Potentials; Mice; Mitochondria; Mitochondrial Membranes; NIH 3T3 Cells; Rats

alpha-Hederin, a pentacyclic triterpene saponin isolated from the seeds of Nigella sativa, was recently reported to have potent in vivo antitumor activity against LL/2 (Lewis Lung carcinoma) in BDF1 mice. In this study we observed that alpha-hederin caused a dose- and time-dependent increase in apoptosis of murine leukemia P388 cells. In order to evaluate the possible mechanisms for apoptosis, the effects of alpha-hederin on intracellular thiol concentration, including reduced glutathione (GSH), and protein thiols, and the effects of pretreatment with N-acetlycysteine (NAC), a precursor of intracellular GSH synthesis, or buthionine sulfoxime (BSO), a specific inhibitor of intracellular GSH synthesis, on alpha-hederin-induced apoptosis were investigated. It was found that alpha-hederin rapidly depleted intracellular GSH and protein thiols prior to the occurrence of apoptosis. NAC significantly alleviated alpha-hederin-induced apoptosis, while BSO augmented alpha-hederin-induced apoptosis significantly. The depletion of cellular thiols observed after alpha-hederin treatment caused disruption of mitochondrial membrane potential (deltapsi(m)) and subsequently increased the production of reactive oxygen species (ROS) in P388 cells at an early time point. Bongkrekic acid (BA), a ligand of the mitochondrial adenine nucleotide translocator, and cyclosporin (CsA) attenuated the alpha-hederin-induced loss of deltapsi(m), and ROS production. Thus, oxidative stress after alpha-hederin treatment is an important event in alpha-hederin-induced apoptosis. As observed in this study, permeability transition of mitochondrial membrane occurs after depletion of GSH and precedes a state of reactive oxygen species (ROS) generation. Further, we observed that alpha-hederin caused the release of cytochrome c from the mitochondria to cytosol, leading to caspase-3 activation. Our findings thus demonstrate that changes in intracellular thiols and redox status leading to perturbance of mitochondrial functions are important components in the mechanism of alpha-hederin-induced cell death.

Topics: Animals; Apoptosis; Cytochromes c; Dose-Response Relationship, Drug; Enzyme Inhibitors; Free Radical Scavengers; Glutathione; Humans; Intracellular Membranes; Leukemia P388; Mice; Mitochondria; Nigella sativa; Oleanolic Acid; Oxidative Stress; Plant Extracts; Reactive Oxygen Species; Saponins; Tumor Cells, Cultured