lamellarin-d and Lung-Neoplasms

Non-small cell lung carcinomas (NSCLCs) are typically resistant against apoptosis induced by standard chemotherapy. We evaluated the effects of the two potential antitumor agents of the lamellarin class on a highly apoptosis-resistant NSCLC cell line. Both the marine alkaloid lamellarin-D and its synthetic amino derivative PM031379 induced the activation of Bax, the mitochondrial release of cytochrome c and apoptosis-inducing factor (AIF), as well as the activation of caspase-3. However, only PM031379 triggered cell death and sign of nuclear apoptosis coupled to the nuclear translocation of AIF. Depletion of AIF with small interfering RNA or microinjection of a neutralizing anti-AIF antibody largely prevented PM031379-induced cytotoxicity, underscoring the essential contribution of AIF to NSCLC killing. Using NSCLC cells lacking mitochondrial DNA, we showed that the generation of mitochondrial reactive oxygen species (ROS) was crucial for the PM031379-induced translocation of AIF to the nucleus and subsequently cell death. Pretreatment of NSCLC cells with menadione, a mitochondrial ROS generator, was able to restore the deficient chemotherapy-induced apoptosis of NSCLC cells. Altogether, these data suggest that mitochondrial ROS generation is crucial for overriding the chemoresistance of NSCLC cells. Moreover, this study delineates the unique mechanism of action of lamellarins as potential anticancer agents.

Topics: Active Transport, Cell Nucleus; Apoptosis; Apoptosis Inducing Factor; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Coumarins; DNA, Mitochondrial; Drug Resistance, Neoplasm; Heterocyclic Compounds, 4 or More Rings; Humans; Isoquinolines; Lung Neoplasms; Mitochondria; Reactive Oxygen Species; RNA, Small Interfering; Vitamin K 3

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