denbinobin and Adenocarcinoma

In the present study, we explore the role of apoptosis signal-regulating kinase 1 (ASK1) in denbinobin-induced apoptosis in human lung adenocarcinoma (A549) cells. Denbinobin-induced cell apoptosis was attenuated by an ASK1 dominant-negative mutant (ASK1DN), two antioxidants (N-acetyl-L-cysteine (NAC) and glutathione (GSH)), a c-Jun N-terminal kinase (JNK) inhibitor (SP600125), and an activator protein-1 (AP-1) inhibitor (curcumin). Treatment of A549 cells with denbinobin caused increases in ASK1 activity and reactive oxygen species (ROS) production, and these effects were inhibited by NAC and GSH. Stimulation of A549 cells with denbinobin caused JNK activation; this effect was markedly inhibited by NAC, GSH, and ASK1DN. Denbinobin induced c-Jun phosphorylation, the formation of an AP-1-specific DNA-protein complex, and Bim expression. Bim knockdown using a bim short interfering RNA strategy also reduced denbinobin-induced A549 cell apoptosis. The denbinobin-mediated increases in c-Jun phosphorylation and Bim expression were inhibited by NAC, GSH, SP600125, ASK1DN, JNK1DN, and JNK2DN. These results suggest that denbinobin might activate ASK1 through ROS production to cause JNK/AP-1 activation, which in turn induces Bim expression, and ultimately results in A549 cell apoptosis.

Topics: Adenocarcinoma; Anthraquinones; Antineoplastic Agents; Apoptosis; Cell Death; Cell Line, Tumor; Humans; Lung Neoplasms; MAP Kinase Kinase Kinase 5; Phenanthrenes; Reactive Oxygen Species

Human pancreatic carcinoma is a highly malignant cancer. Previous studies have shown that the decoy receptor 3 (DcR3) for Fas ligand (FasL) plays significant roles in tumour progression and immune suppression. In the present study, we evaluated the anti-cancer activity of a natural compound, denbinobin (5-hydroxy-3,7-dimethoxy-1,4-phenanthraquinone), through decreasing DcR3 levels in human pancreatic adenocarcinoma cell lines.. We used immunoprecipitation and ELISA assays to examine DcR3 levels, and used FACS to determine the percentage of cells with a sub-G1 DNA content.. AsPC-1 and BxPC-3 human pancreatic cancer cells express high levels of DcR3. Denbinobin concentration-dependently decreased DcR3 levels in BxPC-3 cells. MTT and flow cytometry assays indicated that BxPC-3 was FasL-resistant because high concentrations (100 ng.mL(-1)) of soluble FasL did not inhibit cell growth. However, combinations of denbinobin (3 micromol.L(-1)) with lower concentrations of soluble FasL (10, 30 and 50 ng.mL(-1)) or membrane-bound FasL, were synergistic on cell growth inhibition and apoptosis. Exogenous excess DcR3 reversed this synergistic effect. We observed no significant increase in the levels of surface Fas, cleaved forms of caspase-8, -3, -9, Bax, Bid, Bcl-xL, cytochrome c or mitochondrial membrane potentials following denbinobin treatment. However, denbinobin treatment increased the levels of apoptosis-inducing factor.. Denbinobin and FasL trigger a synergistic cytotoxic effect in human pancreatic adenocarcinoma cells. Denbinobin mediated a decrease in levels of DcR3, which played a major role in this synergistic effect, and also increased caspase-independent apoptosis, via apoptosis-inducing factor.

Topics: Adenocarcinoma; Anthraquinones; Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Inducing Factor; Cell Line, Tumor; Cell Survival; Drug Synergism; Fas Ligand Protein; Humans; Pancreatic Neoplasms; Phenanthrenes; Receptors, Tumor Necrosis Factor, Member 6b

The bane of anti-cancer therapy is usually the development of resistance to apoptosis in malignant cells. Identification of strategies to re-sensitize cancer cells to apoptosis has now become a top priority in anti-cancer research. Denbinobin is a novel, naturally occurring phenathroquinone isolated from orchids of the genus Dendrobium that has remarkable anti-cancer activities demonstrated both in vitro and in vivo. Recently denbinobin has been shown to diminish the levels of expression of the decoy receptor-3 and also to act synergistically with Fas ligand to induce apoptosis in a pancreatic adenocarcinoma cell line. There is hope that denbinobin could be developed as an adjuvant in combination therapies aimed at killing cancers that rely on decoy receptors to evade the host's immune surveillance.

Topics: Adenocarcinoma; Anthraquinones; Antineoplastic Agents, Phytogenic; Apoptosis; Cell Line, Tumor; Drug Synergism; Fas Ligand Protein; Humans; Pancreatic Neoplasms; Phenanthrenes; Receptors, Tumor Necrosis Factor, Member 6b; Tumor Escape

Increasing evidence demonstrated that denbinobin, isolated from Ephemerantha lonchophylla, exert cytotoxic effects in cancer cells. The purpose of this study was to investigate whether denbinobin induces apoptosis and the apoptotic mechanism of denbinobin in human lung adenocarcinoma cells (A549). Denbinobin (1-20microM) caused cell death in a concentration-dependent manner. Flow cytometric analysis and annexin V labeling demonstrated that denbinobin increased the percentage of apoptotic cells. A549 cells treated with denbinobin showed typical characteristics of apoptosis including morphological changes and DNA fragmentation. Denbinobin induced caspase 3 activation, and N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), a broad-spectrum caspase inhibitor, prevented denbinobin-induced cell death. Denbinobin induced the loss of the mitochondrial membrane potential and the release of mitochondrial apoptotic proteins including cytochrome c, second mitochondria derived activator of caspase (Smac), and apoptosis-inducing factor (AIF). In addition, denbinobin-induced Bad activation was accompanied by the dissociation of Bad with 14-3-3 and the association of Bad with Bcl-xL. Furthermore, denbinobin induced Akt inactivation in a time-dependent manner. Transfection of A549 cells with both wild-type and constitutively active Akt significantly suppressed denbinobin-induced Bad activation and cell apoptosis. These results suggest that Akt inactivation, followed by Bad activation, mitochondrial dysfunction, caspase 3 activation, and AIF release, contributes to denbinobin-induced cell apoptosis.

Topics: Adenocarcinoma; Amino Acid Chloromethyl Ketones; Anthraquinones; Antineoplastic Agents, Phytogenic; Apoptosis; Apoptosis Inducing Factor; Apoptosis Regulatory Proteins; bcl-Associated Death Protein; Caspase 3; Caspase Inhibitors; Cell Count; Cell Line, Tumor; Cell Survival; Cysteine Proteinase Inhibitors; DNA Fragmentation; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Humans; Intracellular Signaling Peptides and Proteins; Lung Neoplasms; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Membranes; Mitochondrial Proteins; Oncogene Protein v-akt; Phenanthrenes