piperidines and benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone

We previously reported that flavopiridol-induced apoptosis of B cell chronic lymphocytic leukemia (CLL) patients' cells ex vivo is associated with downregulation of both the inducible nitric oxide (NO) synthase (iNOS) that produces the antiapoptotic molecule NO, and the CDK inhibitor p27kip1 that is thought to block the cell cycle of CLL cells. Here, we show that iNOS downregulation is caspase-dependent and thus can be considered as one of the effector mechanisms of apoptosis, but not a primary triggering event induced by flavopiridol. Furthermore, we also find that this flavone favors the entry into the S and G2 phases of the cell cycle of a subpopulation of the leukemic cells, confirming that flavopiridol might be useful for improving the efficacy of cell cycle-dependent cytostatic agents in the therapy of CLL.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Caspases; Cell Cycle; Cells, Cultured; Down-Regulation; Flavonoids; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Nitric Oxide Synthase Type II; Piperidines

Selective modulation of cell death is important for rational chemotherapy. By depleting Hsp90-client oncoproteins, geldanamycin (GA) and 17-allylamino-17-demethoxy-GA (17-AAG) (heat-shock protein-90-active drugs) render certain oncoprotein-addictive cancer cells sensitive to chemotherapy. Here we investigated effects of GA and 17-AAG in apoptosis-prone cells such as HL60 and U937. In these cells, doxorubicin (DOX) caused rapid apoptosis, whereas GA-induced heat-shock protein-70 (Hsp70) (a potent inhibitor of apoptosis) and G1 arrest without significant apoptosis. GA blocked caspase activation and apoptosis and delayed cell death caused by DOX. Inhibitors of translation and transcription and siRNA Hsp70 abrogated cytoprotective effects of GA. Also GA failed to protect HL60 cells from cytotoxicity of actinomycin D and flavopiridol (FL), inhibitors of transcription. We next compared cytoprotection by GA-induced Hsp70, caspase inhibitors (Z-VAD-fmk) and cell-cycle arrest. Whereas cell-cycle arrest protected HL60 cells from paclitaxel (PTX) but not from FL and DOX, Z-VAD-fmk prevented FL-induced apoptosis but was less effective against DOX and PTX. Thus, by inducing Hsp70, GA protected apoptosis-prone cells in unique and cell-type selective manner. Since GA does not protect apoptosis-reluctant cancer cells, we envision a therapeutic strategy to decrease side effects of chemotherapy without affecting its therapeutic efficacy.

Topics: Amino Acid Chloromethyl Ketones; Antibiotics, Antineoplastic; Apoptosis; Benzoquinones; Caspase 9; Caspase Inhibitors; Cell Cycle; Cell Line, Tumor; Cytoprotection; Dactinomycin; Doxorubicin; Enzyme Activation; Flavonoids; HSP70 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Paclitaxel; Piperidines; Protein Biosynthesis; RNA, Small Interfering; Transcriptional Activation

Flavopiridol was developed as a drug for cancer therapy due to its ability to inhibit cell cycle progression by targeting cyclin-dependent kinases (CDKs). In this study, we show that flavopiridol may also have a neuroprotective action. We show that at therapeutic dosage (or at micromolar range), flavopiridol almost completely prevents colchicine-induced apoptosis in cerebellar granule neurones. In agreement with this, flavopiridol inhibits both the release of cyt c and the activation of caspase-3 induced in response to colchicine treatment. We demonstrate that in this cellular model for neurotoxicity, neither re-entry in the cell cycle nor activation of stress-activated protein kinases, such as c-Jun N-terminal kinase (JNK) or p38 MAP kinase, is involved. In contrast, we show that colchicine-induced apoptosis correlates with a substantial increase in the expression of cdk5 and Par-4, which is efficiently prevented by flavopiridol. Accordingly, a cdk5 inhibitor such as roscovitine, but not a cdk4 inhibitor such as 3-ATA, was also able to protect neurons from apoptosis as well as prevent accumulation of cdk5 and Par-4 in response to colchicine. Our data suggest a potential therapeutic use of flavopiridol in disorders of the central nervous system in which cytoskeleton alteration mediated by cdk5 activation and Par-4 expression has been demonstrated, such as Alzheimer's disease.

Topics: Amino Acid Chloromethyl Ketones; Animals; Animals, Newborn; Anthracenes; Anti-Bacterial Agents; Apoptosis; Apoptosis Regulatory Proteins; Blotting, Western; Bromodeoxyuridine; Carrier Proteins; Caspase 3; Caspases; CDC2-CDC28 Kinases; Cell Count; Cell Survival; Cells, Cultured; Cerebellum; Chromatin; Colchicine; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 5; Cyclin-Dependent Kinases; Cytochromes c; Dose-Response Relationship, Drug; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Flavonoids; Flow Cytometry; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; JNK Mitogen-Activated Protein Kinases; Kainic Acid; MAP Kinase Kinase 4; Microtubules; Minocycline; Mitogen-Activated Protein Kinase Kinases; Neurons; Neuroprotective Agents; Piperidines; Purines; Rats; Rats, Sprague-Dawley; Roscovitine; Time Factors; Tubulin

Many chemotherapeutic agents induce apoptosis in tumor cells, but killing of normal cells remains a major obstacle. Development of multidrug resistance further limits chemotherapy in cancer. Here, I show that multidrug resistance can be exploited for selective killing of multidrug-resistant cells by a combination of an apoptosis-inducing agent that is not a substrate of either Pgp or MRP (e.g. flavopiridol) with a caspase inhibitor that is a substrate (e.g. Z-DEVD-fmk). In normal cells, treatment with caspase inhibitors prevented PARP cleavage, nuclear fragmentation, and cell death caused by flavopiridol or epothilone B. In contrast, Pgp- and MRP-expressing cells were not rescued by caspase inhibitors. Furthermore, reversal of drug resistance renders Pgp cells sensitive to caspase inhibitors abolishing therapeutic advantage. Thus, caspase inhibitors, that are inactive in multidrug-resistant cells, protect normal but not multidrug-resistant cells against chemotherapy, permitting selective eradication of multidrug-resistant cells. Clinical application of this approach may diminish the toxic side-effects of chemotherapy in patients with multidrug-resistant tumors.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP-Binding Cassette Transporters; Boronic Acids; Bortezomib; Cell Cycle; Cell Survival; Cyclosporins; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; DNA Fragmentation; Doxorubicin; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Epothilones; Epoxy Compounds; Flavonoids; Hematopoietic Stem Cells; HL-60 Cells; Humans; Jurkat Cells; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Oligopeptides; Paclitaxel; Piperidines; Poly(ADP-ribose) Polymerases; Pyrazines; Substrate Specificity; Thiazoles