leupeptins and caffeic-acid-phenethyl-ester

The ability to modulate balance between cell survival and death is recognized for its great therapeutic potential. Therefore, research continues to focus on elucidation of cell machinery and signaling pathways that control cell proliferation and apoptosis. Conventional chemotherapeutic agents often have a cytostatic effect over tumor cells. New natural or synthetic chemotherapeutic agents have a wider spectrum of interesting antitumor activities that merit in-depth studies. In the present work, we aimed at characterizing the molecular mechanism leading to induction of cell death upon treatment of the lymphoblastoid cell line PL104 with caffeic acid phenylethyl ester (CAPE), MG132 and two conventional chemotherapeutic agents, doxorubicine (DOX) and vincristine (VCR). Our results showed several apoptotic hallmarks such as phosphatidylserine (PS) exposure on the outer leaflet of the cell membrane, nuclear fragmentation, and increase sub-G1 DNA content after all treatments. In addition, all four drugs downregulated survivin expression. CAPE and both chemotherapeutic agents reduced Bcl-2, while only CAPE and MG132 significantly increased Bax level. CAPE and VCR treatment induced the collapse of mitochondrial membrane potential (∆ψm). All compounds induced cytochrome c release from mitochondrial compartment to cytosol. However, only MG132 caused the translocation of Smac/DIABLO. Except for VCR treatment, all other drugs increased reactive oxygen species (ROS) production level. All treatments induced activation of caspases 3/7, but only CAPE and MG132 led to the activation of caspase 9. In conclusion, our results indicate that CAPE and MG132 treatment of PL104 cells induced apoptosis through the mitochondrial intrinsic pathway, whereas the apoptotic mechanism induced by DOX and VCR may proceed through the extrinsic pathway.

Topics: Adolescent; Adult; Antineoplastic Agents; Apoptosis; Caffeic Acids; Child; Child, Preschool; Drugs, Investigational; Female; Humans; Leukemia; Leupeptins; Male; Middle Aged; Mitochondria; Phenylethyl Alcohol; Tumor Cells, Cultured; Young Adult

The production of reactive nitrogen, nitric oxide (NO), has previously been demonstrated to be a major mechanism by which the innate immune system defends against microbial invasion. The induction of many antimicrobial mechanisms is regulated by numerous components during the transduction of the signal from the cell surface to the cell nucleus where response genes are upregulated. Toll-like cell surface receptor activation often leads to sequential modulation of protein tyrosine kinases (PTK), mitogen activated protein kinases (MAPK), degradation of I kappa B (IkappaB) regulatory molecules which, in turn, release the nuclear factor-kappa B (NF-kappaB) family proteins for translocation into the nucleus and subsequent gene transcription. The purpose of this study was to investigate components of the upstream signal transduction pathway induced by bacterial and viral-like stimulation of NO for antimicrobial defense by the transformed chicken macrophage cell line, HD11. We quantified the production of nitrite by chicken macrophages after exposure to selective pharmacological inhibitors of specific signal transduction components prior to stimulation by polyinosinic polycytidylic acid (poly I:C), formalin-fixed Enterococcus gallinarum (EG) or formalin-fixed Klebsiella pneumoniae (KP). We found that NO production induced by dsRNA or bacteria was reduced in a dose dependent manner by specific inhibitors of PTK, p38 MAPK, IkappaB, and NF-kappaB. Inhibition efficacy varied dependent on stimulation by bacterial or viral-like ligands. In general, NO production induced by bacterial stimulation was most effectively reduced by inhibition of p38 MAPK and least effectively reduced by inhibition of IkappaB. NF-kappaB and IkappaB inhibition affected NO production induced by dsRNA more than that induced by bacterial stimulation.

Topics: Animals; Caffeic Acids; Cell Line; Chickens; Enterococcus; Enzyme Inhibitors; Genistein; I-kappa B Proteins; Klebsiella pneumoniae; Leupeptins; Macrophage Activation; Macrophages; NF-kappa B; Nitric Oxide; p38 Mitogen-Activated Protein Kinases; Phenylethyl Alcohol; Poly I-C; Poultry Diseases; Protein-Tyrosine Kinases; RNA, Double-Stranded; Signal Transduction

The molecular mechanisms that regulate the expression of genes involved in parturition are poorly understood. The mRNA expression of the prostaglandin F(2alpha) receptor (PTGFR), a uterine activating gene, is increased at labor and is required for uterine contractile activity in numerous animal models, although the signaling pathways responsible for this increased expression have not been identified. Proinflammatory cytokines have been proposed to regulate the expression of the uterine activating genes via activation of the nuclear transcription factor, NFkappaB, and initiate labor. However, it is uncertain whether uterine PTGFR is regulated this way. In this report, we demonstrate for the first time that treatment of immortalized human myometrial-derived ULTR cells with the proinflammatory cytokine IL1beta causes an increase in PTGFR mRNA levels. Furthermore, IL1beta treatment increased the nuclear levels of the RELA subunit of NFkappaB and increased binding of RELA to the NFkappaB DNA-binding site. Inhibition of NFkappaB activation with either the proteasome inhibitor MG132 or phenethyl caffeiate reduced PTGFR mRNA levels, which indicates that this transcription factor is important for basal transcription. Furthermore, this inhibition prevented IL1beta induction ofPTGFRmRNA, which confirms that NFkappaB is required for the IL1beta-induced increase inPTGFR. These results are consistent with the proposal that proinflammatory cytokines directly regulate uterine activation genes and that the transcription factor NFkappaB is involved in both basal and IL1beta-stimulated transcription of the PTGFR gene.

Topics: Caffeic Acids; Cell Line; Cell Nucleus; Female; Humans; Interleukin-1beta; Leupeptins; Myometrium; NF-kappa B; Phenylethyl Alcohol; Proteasome Inhibitors; Receptors, Prostaglandin; Response Elements; RNA, Messenger; Transcription Factor RelA

The possible involvement of nuclear factor (NF)-kappa B in mediating the regulation of interleukin (IL)-6 biosynthesis in response to E. coli-derived lipopolysaccharide-endotoxin (LPS) was investigated in vitro. In alveolar epithelial cells, irreversible inhibition of the proteasome complex by carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG-132; 1-50 muM) did not affect LPS-mediated IL-6 secretion. Whereas the selective inhibition of the NF-kappa B pathway by the action of caffeic acid phenyl ethyl ester (CAPE; 1-100 microM) attenuated LPS-dependent IL-6 production at 100 muM, sulfasalazine (SSA; 0.1--10 mM), a potent and irreversible inhibitor of NF-kappa B, did not inhibit LPS-dependent IL-6 secretion. Incorporation of a selectively permeant inhibitor of NF-kappa B, SN-50 (1-20 microM), a peptide which contains the nuclear localization sequence (NLS) for the p50 NF-kappa B subunit and the amino-terminal sequence of Kaposi fibroblast growth factor to promote cell permeability, did not reduce LPS-mediated release of IL-6. These data indicate a NF-kappa B-independent pathway mediating LPS-dependent regulation of IL-6 biosynthesis in the airway epithelium.

Topics: Animals; Caffeic Acids; Cells, Cultured; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Interleukin-6; Leupeptins; Lipopolysaccharides; Multienzyme Complexes; NF-kappa B; Peptides; Phenylethyl Alcohol; Proteasome Endopeptidase Complex; Pulmonary Alveoli; Rats; Rats, Sprague-Dawley; Respiratory Mucosa; Sulfasalazine