manumycin and Lymphoma

Numerous compounds for treating human disease have been discovered in nature. Manumycin-A (Man-A) is a natural, well-tolerated microbial metabolite and a potent experimental tumoricide. We recently showed that Man-A stimulated reactive oxygen species (ROS) which were upstream of serine/threonine (Ser/Thr) dephosphorylation and caspase-dependent cleavage of MEK and Akt in lymphoma apoptosis. Conversely, activation-specific, Ser/Thr phosphorylation of MEK and Akt proteins was stable in Man-A-resistant tumors suggesting that stimulation of Ser/Thr PPase activity might be required for Man-A tumoricidal activity. Pre-treatment with Calyculin-A, an equipotent inhibitor of PP1 and PP2A, blocked all downstream effects of Man-A whereas, the PP2A-selective inhibitor, Okadaic acid did not, suggesting that PP1 and not PP2A played a role in Man-A action. Phosphorylation of PP1α on Thr320 inhibits its activity. Hence, we posited that if PP1α was important for Man-A action, then Man-A treatment should promote dephosphorylation of PP1α on Thr320. Indeed, T320 was only dephosphorylated in the tumors that underwent apoptosis. Lastly, stable over-expression of a constitutively active PP1α mimetic (PP1αT320A mutant), elevated basal ROS levels and enhanced Man-A-stimulated apoptosis. Taken together, we conclude that PP1α is an important proximal effector of Man-A mediated lymphoma apoptosis and that the mechanisms of Man-A action warrant further investigation.

Topics: Animals; Antineoplastic Agents; Apoptosis; Humans; Hydrogen Peroxide; Jurkat Cells; K562 Cells; Lymphoma; MAP Kinase Kinase Kinases; Marine Toxins; Membrane Potential, Mitochondrial; Mice; Oxazoles; Phosphorylation; Polyenes; Polyunsaturated Alkamides; Protein Phosphatase 1; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt; Vanadates

Manumycin-A (Man-A) is a farnesyltransferase inhibitor (FTI), which was originally identified as an effective tumoricide against several cancers, especially ones harboring constitutively active Ras. However, it is becoming apparent that Man-A can stimulate tumor death independently of FTases. Antioxidant treatment blocked Man-A-stimulated DNA damage and reversed Man-A-inhibited tumor growth. However, the precise molecular details of how these reactive oxygen species (ROS) influence cell signaling modules are poorly understood. We examined how ROS may modulate death and survival pathways in a panel of tumor cells. Man-A treatment resulted in a massive induction of superoxide anion (.O(2) (-)) only in Man-A-sensitive tumors. Within 1 hr, Man-A caused the ROS-dependent activation of caspases 9 and 3. In this time-frame, the Ras-Raf target, MEK, and the survival protein Akt were dephosphorylated in ROS-dependent fashions and then cleaved in ROS and caspase-dependent manners. Pretreatment with ROS scavengers blocked the adverse effects of Man-A, including the processing of caspases and the cleavage of MEK and Akt. These events were noted before any losses in Ras activity or changes in its maturation could be detected. Finally, transfection with cDNAs encoding the antioxidant enzymes catalase, superoxide dismutase and thioredoxin reductase inhibited superoxide induction and apoptosis. Together, our data suggest that the elimination of tumors by Man-A can be independent of the inhibiting of Ras. However, one universal feature observed is the generation of death-triggering intracellular oxidants that appear to directly participate in the select targeting of growth and survival proteins that then either augment or ensure tumor cell death.

Topics: Apoptosis; Blotting, Western; Caspases; Cell Line, Tumor; Cell Survival; Enzyme Activation; Enzyme Inhibitors; Humans; Lymphoma; Mitogen-Activated Protein Kinase Kinases; Multiple Myeloma; Phosphorylation; Polyenes; Polyunsaturated Alkamides; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Signal Transduction; Superoxides