benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and ajoene

Ajoene, a garlic-derived organosulfur compound, exerts anti-tumorigenic effect against various cancers. However, little is known about the biological effect of ajoene on lung adenocarcinoma, an aggressive malignancy with dismal prognosis. We investigated the biological effect of ajoene on lung adenocarcinoma and the underlying pathway. Lung adenocarcinoma cells A549, NCI-H1373, and NCI-H1395, along with the noncancerous lung bronchus cells BEAS-2B, were used. MTT test showed that ajoene (25 μM) reduces viability of lung adenocarcinoma cells but not the noncancerous BEAS-2B cells. Bromodeoxyuridine incorporation assay revealed that ajoene inhibits proliferation of lung adenocarcinoma cells. Treatment of lung adenocarcinoma cells with ajoene enhances apoptosis and ROS generation in a time- and dose-dependent fashion. Abrogation of caspase activation by zVAD-fmk completely prevents the ajoene-induced apoptosis; whereas block of ROS generation by N-acetylcysteine partly abolishes the ajoene-induced apoptosis. ROS-mediated induction of apoptosis contributes partially to the anti-tumorigenic property of ajoene observed, a phenomenon also confirmed by xenograft tumor study. Mitogen activated protein kinases (MAPKs), pivots of ROS-mediated signaling pathway, are activated upon ajoene treatment; Jun-N-terminal kinase (JNK)/p38 activations are required for signaling pathway underlying the ajoene-induced apoptosis. Our results suggest that ROS-mediated activation of JNK/p38 contributes partially to the pro-apoptotic action of ajoene on cells of lung adenocarcinoma. Ajoene may be a promising chemotherapeutic agent for lung adenocarcinoma.

Topics: A549 Cells; Adenocarcinoma; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase Inhibitors; Caspases; Cell Line; Cell Line, Tumor; Cell Survival; Disulfides; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Humans; Immunoblotting; JNK Mitogen-Activated Protein Kinases; Lung Neoplasms; Male; Mice, Nude; p38 Mitogen-Activated Protein Kinases; Reactive Oxygen Species; Signal Transduction; Sulfoxides; Time Factors; Xenograft Model Antitumor Assays

The organosulfur compound ajoene, a constitutent of garlic, has been shown to induce apoptosis in a leukemic cell line as well as in blood cells of a leukemic patient. The mechanisms of action of ajoene, however, are unknown. The present study aims to characterize the molecular events leading to ajoene-triggered apoptosis. We show here that ajoene (20 microM) leads to a time-dependent activation of caspase-3-like activity as well as to the proteolytic processing of procaspase-3 and -8. Activation of caspases was necessary for ajoene-induced apoptosis since the broad-range caspase inhibitor zVAD-fmk completely abrogated ajoene-mediated DNA fragmentation. Although the initiator caspase-8 was activated, the CD95 death receptor was not involved in death signaling since the HL-60 clone used was shown to express a functionally inactive CD95 receptor. Furthermore, ajoene induced the release of cytochrome c, which was not inhibited by zVAD-fmk indicating that cytochrome c release precedes caspase activation. Ajoene also led to a dissipation of the mitochondrial transmembrane potential. Overexpression of Bcl-x(L) clearly diminished ajoene-induced caspase activation as well as apoptosis. These results indicate that apoptosis in leukemia cells triggered by ajoene is based on the activation of a mitochondria-dependent caspase cascade which includes also the activation of the initiator caspase-8.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-X Protein; Caspase 3; Caspase 8; Caspase 9; Caspases; Cysteine Proteinase Inhibitors; Cytochrome c Group; Disulfides; DNA Fragmentation; DNA, Neoplasm; Enzyme Activation; Enzyme Precursors; Fas Ligand Protein; fas Receptor; Gene Expression Regulation, Leukemic; HL-60 Cells; Humans; Intracellular Membranes; Jurkat Cells; Membrane Glycoproteins; Membrane Potentials; Mitochondria; Neoplasm Proteins; NF-kappa B; Oxidative Stress; Permeability; Plant Extracts; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Sulfoxides