benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and cepharanthine

We previously showed that cepharanthine (CEP), a biscoclaurine alkaloid, induces caspase-dependent and Fas-independent apoptosis in Jurkat and K562 human leukemia cells. In the present study, we investigated the effect of CEP on three groups of human mitogen-activated protein kinases (MAPKs) in relation to CEP-induced apoptosis. CEP, at the concentration required for and at the time of induction of apoptosis, activated MAPKs p38 in both Jurkat and K562 cells and activated extracellular signal-regulated kinases (ERKs) only in K562 cells. However, CEP treatment did not trigger c-Jun NH(2)-terminal kinases (JNKs) activation. CEP increased the expression and phosphorylation levels of c-Jun and ATF-2 transcription factors. zVAD-fmk, a general caspase inhibitor, did not inhibit CEP-triggered p38 activation in Jurkat and K562 cells or ERK activation in K562 cells. Unexpectedly, pretreatment with a specific p38 inhibitor, SB203580, promoted CEP-induced apoptosis and caspase activation in Jurkat and K562 cells, whereas pretreatment with an MEK-1 inhibitor PD98059 inhibited CEP-induced apoptosis and caspase activation in K562 cells. A selective tyrosine kinase inhibitor, herbimycin A, which completely inhibited CEP-triggered ERKs activation, clearly promoted CEP-induced c-Jun expression and phosphorylation. Our results suggest that each of the three groups of MAP family members is uniquely involved in the CEP-mediated signal cascades in two different leukemia cell lines for inducing/regulating caspase activation and DNA fragmentation.

Topics: Alkaloids; Amino Acid Chloromethyl Ketones; Antineoplastic Agents, Phytogenic; Apoptosis; Benzylisoquinolines; Caspase Inhibitors; Cysteine Proteinase Inhibitors; Enzyme Activation; Enzyme Inhibitors; Humans; Jurkat Cells; K562 Cells; Kinetics; Leukemia; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein-Tyrosine Kinases; Transcription Factors

Cepharanthine (CEP) is a known membrane stabilizer that has been widely used in Japan for the treatment of several disorders such as anticancer therapy-provoked leukopenia. We here report that apoptosis was induced by low concentrations (1-5 microM) of CEP in a human leukemia T cell line, Jurkat, and by slightly higher concentrations (5-10 microM) in a human chronic myelogenous leukemia (CML) cell line K562, which expresses a p210 antiapoptotic Bcr-Abl fusion protein. Induction of apoptosis was confirmed in both Jurkat and K562 cells by DNA fragmentation and typical apoptotic nuclear change, which were preceded by disruption of mitochondrial membrane potential and were induced through a Fas-independent pathway. CEP treatment induced activation of caspase-9 and -3 accompanied by cleavage of PARP, Bid, lamin B1, and DFF45/ICAD in both Jurkat and K562 cells, whereas caspase-8 activation and Akt cleavage were observed only in Jurkat cells. The CEP-induced apoptosis was completely blocked by zVAD-fmk, a broad caspase inhibitor. Interestingly, CEP treatment induced remarkable degradation of the Bcr-Abl protein in K562 cells, and this degradation was prevented partially by zVAD-fmk. When used in combination with a nontoxic concentration of herbimycin A, lower concentrations (2-5 microM) of CEP induced obvious apoptosis in K562 cells with rapid degradation or decrease in the amount of Bcr-Abl and Akt proteins. Our results suggest that CEP, which does not have bone marrow toxicity, may possess therapeutic potential against human leukemias, including CML, which is resistant to anticancer drugs and radiotherapy.

Topics: Alkaloids; Amino Acid Chloromethyl Ketones; Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Regulatory Proteins; Benzoquinones; Benzylisoquinolines; BH3 Interacting Domain Death Agonist Protein; Carrier Proteins; Caspases; Cysteine Proteinase Inhibitors; DNA Fragmentation; Drug Screening Assays, Antitumor; Drug Synergism; Enzyme Activation; Enzyme Inhibitors; Fas Ligand Protein; fas Receptor; Fusion Proteins, bcr-abl; Humans; Intracellular Membranes; Jurkat Cells; K562 Cells; Lactams, Macrocyclic; Lamin Type B; Lamins; Membrane Glycoproteins; Mitochondria; Neoplasm Proteins; Nuclear Proteins; Poly(ADP-ribose) Polymerases; Protein Serine-Threonine Kinases; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Quinones; Rifabutin