benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and oridonin

Oridonin, an active diterpenoid compound from Rabdosia rubescens, has anti-tumor effects. Rheumatoid arthritis fibroblast-like synoviocytes (RAFLS), a pathological hallmark of RA, exhibits "tumor-like" phenotype. Here, we investigated the effects of oridonin on the proliferation and apoptosis of RAFLS. Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Apoptosis and mitochondrial membrane potential were detected by flow cytometry. Western blot analysis was performed to examine the phosphorylation of extra-cellular regulated kinases (ERK1/2), C-Jun N-Terminal Kinase (JNK), and p38 mitogen-activated protein kinases and the expression of apoptosis-related proteins. Oridonin inhibited cell proliferation and induced cell apoptosis in interleukin-1β (IL-1β)-treated FLS. z-VAD-fmk, a pan-caspase inhibitor, significantly (P < 0.05) attenuated oridonin-induced apoptosis of FLS. Oridonin suppressed IL-1β-mediated phosphorylation of ERK1/2 and JNK in a dose-dependent manner. Meanwhile, oridonin alone dose-dependently suppressed FLS proliferation, triggered cell apoptosis, and reduced mitochondrial membrane potential (ΔΨm) through activating caspase-3, caspase-9, and PARP, leading to translocation of cytochrome c into cytoplasm. z-VAD-fmk significantly (P < 0.05) inhibited oridonin-induced apoptosis. The accumulation of cellular reactive oxygen species (ROS) was about sevenfold increase in oridonin-treated cells. Pretreatment of N-acetylcysteine (NAC), an inhibitor of ROS, significantly attenuated oridonin-triggered apoptosis, indicating the involvement of ROS production in oridonin-induced mitochondrial apoptosis. Oridonin inhibits cell proliferation, induces cell apoptosis, and decreases the phosphorylation of ERK1/2 and JNK in IL-1β-exposed RAFLS. Oridonin induces mitochondrial apoptosis by enhancing the production of ROS in FLS.

Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Apoptosis; Arthritis, Rheumatoid; Caspase 3; Caspase 9; Cell Line; Cell Proliferation; Cell Survival; Cytochromes c; Diterpenes, Kaurane; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Humans; Interleukin-1beta; JNK Mitogen-Activated Protein Kinases; Membrane Potential, Mitochondrial; Mitochondria; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Poly(ADP-ribose) Polymerases; Reactive Oxygen Species; Synoviocytes

Herein, inhibition of hepatocyte growth factor receptor, c-Met, significantly increased cytochrome c release and Bax/Bcl-2 ratio, indicating that c-Met played an anti-apoptotic role. The following experiments are to elucidate this anti-apoptotic mechanism, then the effect of c-Met on autophagy has also been discussed.. Investigated was the influence of c-Met on apoptosis, autophagy and loss of mitochondrial transmembrane potential (Δψm), and the relevant proteins were examined.. First, we found that activation of extracellular signal-regulated kinase (ERK), p53 was promoted by c-Met interference. Subsequent studies indicated that ERK was the upstream effector of p53, and this ERK-p53 pathway mediated release of cytochrome c and up-regulation of Bax/Bcl-2 ratio. Secondly, the inhibition of c-Met augmented oridonin-induced loss of mitochondrial transmembrane potential (Δψm), resulting apoptosis. Finally, the inhibition of c-Met increased oridonin-induced A549 cell autophagy accompanied by Beclin-1 activation and conversion from microtubule-associated protein light chain 3 (LC3)-I to LC3-II. Activation of ERK-p53 was also detected in autophagy process and could be augmented by inhibition of c-Met. Moreover, suppression of autophagy by 3-methyladenine (3-MA) or small interfering RNA against Beclin-1 or Atg5 decreased oridonin-induced apoptosis. Inhibition of apoptosis by pan-caspase inhibitor (z-VAD-fmk) decreased oridonin-induced autophagy as well and Loss of Δψm also occurred during autophagic process.. Thus, inhibiting c-Met enhanced oridonin-induced apoptosis, autophagy and loss of Δψm in A549 cells.

Topics: Adenine; Amino Acid Chloromethyl Ketones; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Autophagy-Related Protein 5; bcl-2-Associated X Protein; Beclin-1; Cytochromes c; Diterpenes, Kaurane; Extracellular Signal-Regulated MAP Kinases; Humans; Isodon; Lung Neoplasms; Membrane Potential, Mitochondrial; Membrane Proteins; Microtubule-Associated Proteins; Mitochondria; Phytotherapy; Plant Extracts; Proto-Oncogene Proteins c-met; RNA, Small Interfering; Tumor Suppressor Protein p53

Oridonin, a diterpenoid isolated from Rabdosia rubescences, has been reported to have antitumor effects. In this study, the growth-inhibitory activity of oridonin for L929 cells was exerted in a time-and dose-dependent manner. After treatment with oridonin for 24 h, L929 cells underwent both apoptosis and necrosis as measured by an lactate dehydrogenase (LDH) activity-based assay. A rapid generation of reactive oxygen species (ROS) was triggered by oridonin, and subsequently up-regulation of phospho-p53 (ser 15) expression and an increased expression ratio of Bax/Bcl-2 was observed. Furthermore, there was a significant fall in mitochondrial membrane potential (MMP) and increase in caspase-3 activity after exposure to oridonin for 24 h. Surprisingly, the pan-caspase inhibitor z-VAD-fmk and caspase3 inhibitor z-DEVD-fmk rendered L929 cells more sensitive to oridonin, rather than preventing oridonin-induced cell death. Oridonin and z-VAD-fmk co-treatment not only resulted in an even higher ROS production, but also made a more significant reduction in the MMP. Pretreatment of ROS scavenger N-acetylcysteine (NAC) led to a complete inhibition of oridonin-induced cell death, intracellular ROS generation, and MMP collapse. NAC treatment also reversed the potentiation of cell death by the pan-caspase inhibitor z-VAD-fmk. Taken together, these observations showed that oridonin-induced cell death in L929 cells involved intracellular ROS generation, activation of phospho-p53 (ser 15), and up-regulation of the Bax/Bcl-2 ratio; and the augmented cell death by z-VAD-fmk was dependent on an increased ROS production.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Caspase Inhibitors; Cell Death; Cell Line; Diterpenes, Kaurane; Enzyme Inhibitors; Fibroblasts; Genes, bcl-2; Indicators and Reagents; L-Lactate Dehydrogenase; Membrane Potentials; Mice; Mitochondrial Membranes; Reactive Oxygen Species; Tumor Suppressor Protein p53

To study the caspase-3-independent mechanisms in oridonin-induced MCF-7 human breast cancer cell apoptosis in vitro.. The viability of oridonin-treated MCF-7 cells was measured by MTT (thiazole blue) assay. Apoptotic cells with condensed nuclei were visualized by phase contrast microscopy. Nucleosomal DNA fragmentation was assayed by agarose gel electrophoresis. The apoptotic ratio was determined by lactate dehydrogenase assay. Cell cycle alternation and mitochondrial membrane potential were measured by flow cytometric analysis. Bax, Bcl-2, caspase-3, caspase-9, heat shock protein (Hsp)90, p53, p-p53, p21, Poly (ADP-ribose) polymerase (PARP), and the inhibitor of caspase-activated DNase (ICAD) protein expressions were detected by Western blot analysis.. Oridonin inhibited cell growth in a time- and dose-dependent manner. Cell cycle was altered through the upregulation of p53 and p21 protein expressions. Pancaspase inhibitor Z-VAD-fmk and calpain inhibitor II both decreased cell death ratio. Nucleosomal DNA fragmentation and the downregulation of DeltaPhimit were detected in oridonin-induced MCF-7 cell apoptosis, which was involved in a postmitochondrial caspase-9-dependent pathway. Decreased Bcl-2 and Hsp90 expression levels and increased Bax and p21 expression levels were positively correlated with elevated levels of phosphorylated p53 phosphorylation. Moreover, PARP was partially cleaved by calpain rather than by caspase-3.. DNA damage provoked alternations in the mitochondrial and caspase-9 pathways as well as p53-mediated cell cycle arrest, but was not related to caspase-3 activity in oridonin-induced MCF-7 cells.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Breast Neoplasms; Caspase 3; Caspase 9; Cell Cycle; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; Cysteine Proteinase Inhibitors; Diterpenes, Kaurane; Drugs, Chinese Herbal; Female; HSP90 Heat-Shock Proteins; Humans; Medicine, Chinese Traditional; Mitochondria; Poly(ADP-ribose) Polymerases; Signal Transduction; Tumor Suppressor Protein p53