plitidepsin and Breast-Neoplasms

Aplidin is an antitumor agent in phase II clinical trials that induces apoptosis through the sustained activation of Jun N-terminal kinase (JNK). We report that Aplidin alters glutathione homeostasis increasing the ratio of oxidized to reduced forms (GSSG/GSH). Aplidin generates reactive oxygen species and disrupts the mitochondrial membrane potential. Exogenous GSH inhibits these effects and also JNK activation and cell death. We found two mechanisms by which Aplidin activates JNK: rapid activation of Rac1 small GTPase and downregulation of MKP-1 phosphatase. Rac1 activation was diminished by GSH and enhanced by L-buthionine (SR)-sulfoximine, which inhibits GSH synthesis. Downregulation of Rac1 by transfection of small interfering RNA (siRNA) duplexes or the use of a specific Rac1 inhibitor decreased Aplidin-induced JNK activation and cytotoxicity. Our results show that Aplidin induces apoptosis by increasing the GSSG/GSH ratio, a necessary step for induction of oxidative stress and sustained JNK activation through Rac1 activation and MKP-1 downregulation.

Topics: Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Calcium; Cell Cycle Proteins; Copper; Depsipeptides; Down-Regulation; Dual Specificity Phosphatase 1; Enzyme Activation; Glutathione Disulfide; Glutathione Peroxidase; Glutathione Reductase; HeLa Cells; Homeostasis; Humans; Immediate-Early Proteins; JNK Mitogen-Activated Protein Kinases; Membrane Potentials; Mice; Mitochondrial Membranes; Oxidative Stress; Peptides, Cyclic; Phosphoprotein Phosphatases; Protein Phosphatase 1; Protein Tyrosine Phosphatases; rac1 GTP-Binding Protein; Reactive Oxygen Species

Aplidin is an antitumor drug that induces apoptosis and activates EGFR, Src, JNK and p38MAPK. Here, we show that Aplidin induces c-JUN, JUN B, JUN D, c-FOS, FRA-1 and FOS B genes of the activator-protein (AP)-1 family, and also p65/RELA, a major component of nuclear factor-kappa B (NF-kappaB). Concordantly, Aplidin increases AP-1 and NF-kappaB activity. c-FOS induction depends on EGFR, Src and JNK/p38MAPK. In contrast, induction of c-JUN does not require EGFR activity and p65/RELA induction is only partially dependent on these kinases. We used several genetically deficient cells to identify the critical target of Aplidin. Mouse embryo fibroblasts (MEFs) deficient for src, yes and fyn, and those lacking all p38MAPK isoforms displayed normal Aplidin sensitivity (IC50=12 nM). In contrast, MEFs lacking jnk1 and jnk2, which do not express any JNK isoform, were much less sensitive (IC50>500 nM). Furthermore, cells lacking c-jun or expressing a c-Jun protein in which JNK targets Ser(63/73) were mutated (c-JunAA) showed intermediate sensitivity (IC50=60 nM). Additionally, Aplidin has higher cytotoxic activity against proliferating than quiescent cells, which is reflected in higher JNK activation. We conclude that phosphorylation by JNK of c-Jun and additional substrate(s) is crucial for Aplidin activity.

Topics: Antibodies, Monoclonal; Antineoplastic Agents; Apoptosis; Blotting, Western; Breast Neoplasms; Cell Division; Cell Line, Tumor; Cell Survival; Depsipeptides; Enzyme Activation; Female; Fibroblasts; Humans; Mitogen-Activated Protein Kinases; NF-kappa B; Peptides, Cyclic; Phosphorylation; Precipitin Tests; Proto-Oncogene Proteins c-jun; Transcription Factor AP-1

We report that Aplidin, a novel antitumor agent of marine origin presently undergoing Phase II clinical trials, induced growth arrest and apoptosis in human MDA-MB-231 breast cancer cells at nanomolar concentrations. Aplidin induced a specific cellular stress response program, including sustained activation of the epidermal growth factor receptor (EGFR), the non-receptor protein-tyrosine kinase Src, and the serine/threonine kinases JNK and p38 MAPK. Aplidin-induced apoptosis was only partially blocked by the general caspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone and was also sensitive to AG1478 (an EGFR inhibitor), PP2 (an Src inhibitor), and SB203580 (an inhibitor of JNK and p38 MAPK) in MDA-MB-231 cells. Supporting a role for EGFR in Aplidin action, EGFR-deficient mouse embryo fibroblasts underwent apoptosis upon treatment more slowly than wild-type EGFR fibroblasts and also showed delayed JNK and reduced p38 MAPK activation. N-Acetylcysteine and ebselen (but not other antioxidants such as diphenyleneiodonium, Tiron, catalase, ascorbic acid, and vitamin E) reduced EGFR activation by Aplidin. N-Acetylcysteine and PP2 also partially inhibited JNK and p38 MAPK activation. The intracellular level of GSH affected Aplidin action; pretreatment of cells with GSH or N-acetylcysteine inhibited, whereas GSH depletion caused, hyperinduction of EGFR, Src, JNK, and p38 MAPK. Remarkably, Aplidin also induced apoptosis and activated EGFR, JNK, and p38 MAPK in two cell lines (A-498 and ACHN) derived from human renal cancer, a neoplasia that is highly refractory to chemotherapy. These data provide a molecular basis for the anticancer activity of Aplidin.

Topics: Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Division; Cell Survival; Cells, Cultured; Depsipeptides; Enzyme Activation; Enzyme Inhibitors; ErbB Receptors; Female; Fibroblasts; Flow Cytometry; Glutathione; Humans; JNK Mitogen-Activated Protein Kinases; Kidney Neoplasms; Mice; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Peptides, Cyclic; Phosphorylation; Proto-Oncogene Proteins pp60(c-src); Receptors, Platelet-Derived Growth Factor; Tumor Cells, Cultured

The cytotoxic agents chlorpheniramine and dehydrodidemnin B decreased the cell growth of estrogen receptor-negative human breast cancer cells MDA-MB231 and estrogen receptor-positive MCF-7, after 48 h treatment. Both agents reduced ornithine decarboxylase activity, but polyamine levels were increased in MDA-MB231 cells treated with dehydrodidemnin B. MCF-7 cells when treated with dehydrodidemnin B showed significant increases in spermidine and spermine contents. The results suggest that besides other effects, the cytotoxicity of DDB could be explained in part by the over-accumulation of spermidine and spermine.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Division; Chlorpheniramine; Depsipeptides; Female; Humans; Ornithine Decarboxylase; Peptides, Cyclic; Spermidine; Spermine; Tumor Cells, Cultured