Oridonin-induced Cell Research Articles

Caspase Inhibition Augmented Oridonin-Induced Cell Death in Murine Fibrosarcoma L 929 by Enhancing Reactive Oxygen Species Generation

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 caspase 3 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.

Oridonin induces apoptosis in uveal melanoma cells by upregulation of Bim and downregulation of Fatty Acid Synthase

Oridonin is an orally available drug isolated from Traditional Chinese Medicine. Previous studies with oridonin have demonstrated broad-spectrum anticancer activity in a variety of cancer types. However, the effect of oridonin in uveal melanoma has not been addressed. In this study, we aimed to investigate whether oridonin elicited anticancer activity and its underlying mechanism in human uveal melanoma cells. We demonstrated that oridonin potently reduced cell viability, induced apoptosis and inhibited clonogenic survival and growth with single digit micromolar concentrations in uveal melanoma OCM-1 and MUM2B cell lines. We found that oridonin markedly increased the expression of proapoptotic Bcl-2 family protein Bim in uveal melanoma cells, and knockdown Bim by small interfering RNA significantly attenuated oridonin-induced cell death, indicating an essential role of Bim in oridonin-mediated anticancer activity. Additionally, we observed that oridonin suppressed Fatty Acid Synthase (FAS) expression in uveal melanoma cells, and enforced FAS expression by insulin partially rescued the cells from oridonin-induced apoptosis, showing that inhibition of FAS also contributed to oridonin-mediated apoptosis. Taken together, we reported that oridonin displays potent anticancer effect against uveal melanoma cells through upregulation of Bim and inhibition of FAS. Since oridonin is a popular anticancer agent, our study therefore may have translational implication on the management of patients with uveal melanoma.

Downregulation of Cdk1 and cyclinB1 expression contributes to oridonin-induced cell cycle arrest at G2/M phase and growth inhibition in SGC-7901 gastric cancer cells.

Oridonin isolated from Rabdosia rubescens, a plant used to treat cancer in Chinese folk medicine, is one of the most important antitumor active ingredients. Previous studies have shown that oridonin has anti- tumor activities in vivo and in vitro, but little is known about cell cycle effects of oridonin in gastric cancer. MTT assay was adopted to detect the proliferation inhibition of SGC-7901 cells, the cell cycle was assessed by flow cytometry and protein expression by Western blotting. Oridonin could inhibit SGC-7901 cell proliferation, the IC50 being 15.6 μM, and blocked SGC-7901 cell cycling in the G2/M phase. The agent also decreased the protein expression of cyclinB1 and CDK1. Oridonin may inhibit SGC-7901 growth and block the cells in the G2/M phase by decreasing Cdk1 and cyclinB1 proteins.

Intracellular Glutathione Depletion by Oridonin Leads to Apoptosis in Hepatic Stellate Cells

Proliferation of hepatic stellate cells (HSCs) plays a key role in the pathogenesis of liver fibrosis. Induction of HSC apoptosis by natural products is considered an effective strategy for treating liver fibrosis. Herein, the apoptotic effects of 7,20-epoxy-ent-kaurane (oridonin), a diterpenoid isolated from Rabdosia rubescens, and its underlying mechanisms were investigated in rat HSC cell line, HSC-T6. We found that oridonin inhibited cell viability of HSC-T6 in a concentration-dependent manner. Oridonin induced a reduction in mitochondrial membrane potential and increases in caspase 3 activation, subG1 phase, and DNA fragmentation. These apoptotic effects of oridonin were completely reversed by thiol antioxidants, N-acetylcysteine (NAC) and glutathione monoethyl ester. Moreover, oridonin increased production of reactive oxygen species (ROS), which was also inhibited by NAC. Significantly, oridonin reduced intracellular glutathione (GSH) level in a concentration- and time-dependent fashion. Additionally, oridonin induced phosphorylations of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK). NAC prevented the activation of MAPKs in oridonin-induced cells. However, selective inhibitors of MAPKs failed to alter oridonin-induced cell death. In summary, these results demonstrate that induction of apoptosis in HSC-T6 by oridonin is associated with a decrease in cellular GSH level and increase in ROS production.

Oridonin: targeting programmed cell death pathways as an anti‐tumour agent

Oridonin, an active diterpenoid isolated from traditional Chinese herbal medicine, has drawn rising attention for its remarkable apoptosis- and autophagy-inducing activity and relevant molecular mechanisms in cancer therapy. Apoptosis is a well known type of cell death, whereas autophagy can play either pro-survival or pro-death roles in cancer cells. Accumulating evidence has recently revealed relationships between apoptosis and autophagy induced by oridonin; however, molecular mechanisms behind them remain to be discovered. In this review, we focus on highlighting updated research on oridonin-induced cell death signalling pathways implicated in apoptosis and autophagy, in many types of cancer. In addition, we further discuss cross-talk between apoptosis and autophagy induced by oridonin, in cancer. Taken together, these findings open new perspectives for further exploring oridonin as a potential anti-tumour agent targeting apoptosis and autophagy, in future anti-cancer therapeutics.

The tyrphostin AG1478 augments oridonin-induced A431 cell apoptosis by blockage of JNK MAPK and enhancement of oxidative stress

Oridonin, a diterpenoid compound, extracted and purified from Rabdosia rubescen has been reported to have cytotoxic effect on tumour cells through apoptosis, and tyrosine kinase pathways are involved in these processes. A specific epidermal growth factor receptor (EGFR) inhibitor AG1478 was used to examine the relationship between EGFR signal pathways and oridonin-induced apoptosis and autophagy in EGFR abundant human epidermoid carcinoma A431 cells. Inhibition of EGFRaugmented oridonin-induced A431 cell apoptosis, while the changes of expression of downstream proteins, Bcl-2, Bcl-xL, Bax, cytochrome c, pro-caspase-3, Fas, FADD and pro-caspase-8 suggested that both the intrinsic and extrinsic apoptotic pathways are involved in these processes. Pretreatment with AG1478 aggravated oridonin-induced loss of mitochondrial membrane potential (MMP) and increased ROS generation in A431 cells, while a ROS scavenger, N-acetylcysteine (NAC) completely reversed oridonin- and AG1478-induced ROS generation and apoptosis. Therefore, AG1478 augmented oridonin-induced apoptosis by enhancing oxidative stress. Pretreatment with AG1478 decreased the expression of downstream MAPK proteins ERK, JNK and P38 and their phosphorylated forms to varying degrees compared with oridonin alone treatment. Then after administration of ERK, JNK and P38 inhibitors, only JNK inhibitor SP600125 effectively augmented oridonin-induced apoptosis and ROS generation. Therefore, in EGFR downstream pathways, JNK played a major role in preventing oridonin-induced apoptosis. Autophagy antagonised apoptosis and exerted a protective effect in A431 cells, and both AG1478 and SP600125 decreased oridonin-induced autophagy. Inhibition of EGFR augmented oridonin-induced apoptosis and this was caused by enhanced oxidative stress, and JNK played a major protective role by increasing autophagy, leading to antagonising apoptosis and ROS generation.

Oridonin induces apoptosis and senescence by increasing hydrogen peroxide and glutathione depletion in colorectal cancer cells

We recently demonstrated that oridonin could induce apoptosis and senescence of colon cancer cells in vitro and in vivo. However, the underlying mechanism remains unknown. In this study, the involvement of reactive oxygen species in oridonin-induced cell death and senescence was investigated in colon adenocarcinoma-derived SW1116 cells. Oridonin increased intracellular hydrogen peroxide levels and reduced the glutathione content in a dose-dependent manner. N-acetylcysteine, a reactive oxygen species scavenger, not only blocked the oridonin-induced increase in hydrogen peroxide and glutathione depletion, but also blocked apoptosis and senescence induced by oridonin, as evidenced by the decrease in Annexin V and senescence-associated β-galactosidase- positive cells and the inhibition of oridonin-induced upregulation of p53 and p16 and downregulation of c-Myc. Moreover, exogenous catalase could inhibit the increase in hydrogen peroxide and apoptosis induced by oridonin, but not the glutathione depletion and senescence. Furthermore, thioredoxin reductase (TrxR) activity was reduced by oridonin in vitro and in cells, which may cause the increase in hydrogen peroxide. In conclusion, the increase in hydrogen peroxide and glutathione depletion account for oridonin-induced apoptosis and senescence in colorectal cancer cells, and TrxR inhibition is involved in this process. Given the importance of TrxR as a novel cancer target in colon cancer, oridonin would be a promising clinical candidate. The mechanism of oridonin-induced inhibition of TrxR warrants further investigation.

Interruption of Hepatocyte Growth Factor Signaling Augmented Oridonin-Induced Death in Human Non-small Cell Lung Cancer A549 Cells <i>via</i> c-Met-Nuclear Factor-κB-Cyclooxygenase-2 and c-Met-Bcl-2-Caspase-3 Pathways

The aim of this study was to elucidate the molecular mechanisms mediating hepatocyte growth factor (HGF)-induced protection against oridonin-induced apoptosis in A549 cells. Oridonin induced decrease in Bcl-2/Bax ratio and activation of caspase-3, while these processes were reversed by HGF, suggesting that HGF played an anti-apoptotic role in oridonin-induced A549 cell death. HGF-induced protective effect was partially attributed to the activation of nuclear factor (NF)-κB and cyclooxygenase 2 (COX-2), since the protective effect was abolished by inhibition of NF-κB or interruption of COX-2. Then the activated COX-2 could prevent cells from initiating the apoptotic response by promoting prostaglandin E₂ (PGE₂) release. Activation of NF-κB-COX-2 by HGF-treatment triggered the increase in Bcl-2/Bax ratio, inhibition of procaspase-3 cleavage, promotion of Ca²⁺-independent intracellular phospholipase A2 (iPLA2) expression and augmentation of PGE₂ release, leading to antagonizing oridonin-induced cell death in A549 cells. HGF-induced cell survival in response to oridonin administration was associated with the activation of c-Met-NF-κB-COX-2 and c-Met-Bcl-2-caspase-3 signaling pathways. iPLA2, downstream effector of caspase-3, also participated in these processes.

Hydroxyl Radical (·OH) Played a Pivotal Role in Oridonin-Induced Apoptosis and Autophagy in Human Epidermoid Carcinoma A431 Cells

Oridonin, a diterpenoid compound extracted and purified from Rabdosia rubescen, has been reported to induce tumor cell apoptosis through tyrosine kinase pathway. To further examine the mechanism of oridonin, we selected human epidermoid carcinoma A431 cell as a test object. Besides apoptosis, oridonin also induced A431 cell autophagy, and this autophagy antagonized apoptosis and played a protective role for A431 cells. Reactive oxygen species (ROS) played a pivotal role in induction of cytotoxicity. Therefore, a ROS scavenger, N-acetylcysteine (NAC) combined with oridonin was appiled. Results of morphologic observation, flow cytometric analysis and Western blot analysis showed that NAC could significantly reverse both ROS generation and down-regulation of mitochondrial membrane potential in oridonin treated cells. NAC inhibited oridonin induced apoptosis through both the intrinsic and extrinsic apoptotic pathways. NAC effectively inhibited both oridonin-induced apoptosis and autophagy by reducing intracellular oxidative stress. To further examine the mechanism of ROS, exogenous enzyme antioxidants (superoxide dismutase (SOD), catalase (CAT)) and non-enzyme antioxidants (glutathione (GSH)) were applied to detect the effect of oridonin on ROS generation. Only GSH exerted a similar role with NAC, suggesting that hydroxyl radical (·OH) played the major role in oridonin-induced cell death. Oridonin could decrease the GSH level in A431 cells in a dose-dependent manner. In addition, after treatment with ·OH donor, Fenton reagent, the changes in A431cells were similar to the results of oridonin treatment. All the results proved that ·OH played the pivotal role in oridonin induced apoptosis and autophagy in A431 cells.

Inhibition of EGFR signaling augments oridonin-induced apoptosis in human laryngeal cancer cells via enhancing oxidative stress coincident with activation of both the intrinsic and extrinsic apoptotic pathways

Oridonin, a bioactive diterpenoid isolated from Rabdosia rubescens, has been reported to have anti-tumor effects, while the epidermal growth factor receptor (EGFR) signal pathway has been reported to play a vital role in the biological progression of several tumors and to be a target for therapeutic intervention. In this work, we show that inhibition of EGFR with tyrphostin AG1478 enhances oridonin-induced cell death in human laryngeal cancer cells HEp-2, a cell line characterized by EGFR gene amplification. The enhanced apoptotic effect correlates with high expression and activation of Bax, FADD, caspase-8 as well as caspase-3 and decreased protein levels of Bcl 2 and SIRT1, suggesting that both the extrinsic and intrinsic apoptosis pathways are involved in the apoptotic processes. However, treatment with oridonin and AG1478 greatly enhances nuclear translocation of apoptosis inducing factor (AIF) without caspase-9 activation, indicating that the apoptosis occurs via a caspase-9-independent mitochondrial pathway. Here, it is the active form of caspase-8 but not caspase-9 that activates downstream effector caspase-3, resulting in the cleavage of critical cellular proteins and apoptosis. Furthermore, the combined use of AG1478 and oridonin augments the production of reactive oxygen species (ROS). Incubation of cells with N-Acetylcysteine (NAC) attenuates the apoptosis and the mitochondrial membrane potential ( Δψm) disruption induced by the combination of oridonin and AG1478, which indicates that ROS plays a pivotal role in cell death. In conclusion, targeting EGFR combined with other conventional pro-apoptotic drugs should be a potentially very effective anti-neoplastic therapy for laryngeal cancer.

Autophagy inhibits reactive oxygen species‐mediated apoptosis via activating p38‐nuclear factor‐kappa B survival pathways in oridonin‐treated murine fibrosarcoma L929 cells

Autophagy and apoptosis have been known to be interconnected positively or negatively; however, the molecular mechanisms mediating these two cellular processes are not fully understood. In the present study, we demonstrated that the exposure of L929 cells to oridonin led to intracellular reactive oxygen species generation, followed by lipid peroxidation, as well as decreases in superoxide dismutase and glutathione activities. The reactive oxygen species scavenger N-acetyl-cysteine resulted in the complete inhibition of oridonin-induced apoptosis and mitochondrial membrane potential collapse. We showed that reactive oxygen species triggered apoptosis by Bax translocation, cytochrome c release and extracellular signal-regulated kinase activation. Further data confirmed that oridonin also induced L929 cell autophagy, as demonstrated by extensive autophagic vacuolization and the punctuate distribution of monodansylcadaverine staining and GFP-LC3, as well as the LC3-II/LC3-I proportion and Beclin 1 activation. Subsequently, we found that inhibition of autophagy by 3-methyladenine or small interfering RNA against LC3 and Beclin 1 promoted oridonin-induced cell apoptosis. The effects of p38 and nuclear factor-kappa B in oridonin-induced apoptosis and autophagy were further examined. Interruption of p38 and nuclear factor-kappa B activation by specific inhibitors or small interfering RNAs promoted apoptosis and reactive oxygen species generation, but decreased autophagy. Moreover, we showed that inhibition of autophagy reduced oridonin-induced activation of p38. Additionally, nuclear factor-kappa B activation was inhibited by blocking the p38 pathway. Consequently, these findings indicate that oridonin-induced L929 cell apoptosis is regulated by reactive oxygen species-mediated signaling pathways, and that oridonin-induced autophagy may block apoptosis by up-regulating p38 and nuclear factor-kappa B activation.

Oridonin induces human epidermoid carcinoma A431 cell apoptosis through tyrosine kinase and mitochondrial pathway

Oridonin, a diterpenoid isolated from the plant Rabdosia rubescens, induces human epidermoid carcinoma A431 cell death through apoptosis and tyrosine kinase pathway. To examine the pathway of oridonin-induced A431 cell death, morphologic observation, lactate dehydrogenase activity-based assay, DNA agarose gel electrophoresis and Western blot analysis were carried out. When A431 cells, which overexpress epidermal growth factor receptor (EGFR), were treated with oridonin, caspase-3 was activated followed by the degradation of caspase-3 substrates, inhibitor of caspase-activated DNase (ICAD) and poly(ADP-ribose) polymerase (PARP) in a time-dependent manner. Oridonin promoted the release of cytochrome c and the down-regulation of mitochondrial transmembrane potential (ΔΨm). Oridonin up-regulated the expression ratio of mitochondrial proteins, Bax/Bcl-2. In addition, the total tyrosine kinase activity of A431 cellular proteins and the expression of EGFR were markedly reduced after oridonin treatment. Taken together, oridonin induced apoptosis in A431 cells via mitochondrial pathway, activation of caspase-3 and inhibition of tyrosine kinase activities.

Caspase Inhibition Augmented Oridonin-Induced Cell Death in Murine Fibrosarcoma L929 by Enhancing Reactive Oxygen Species Generation

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.

Oridonin induces apoptosis via PI3K/Akt pathway in cervical carcinoma HeLa cell line

To investigate the apoptosis-inducing effect of oridonin, a diterpenoid isolated from Rabdosia rubescens, in the human cervical carcinoma HeLa cell line. A morphological analysis, nuclear condensation, and fragmentation of chromatin were monitored using Hoechst 33342 staining. Cell viability was assessed using the 3-(4, 5-dimethylthiazol-(2)-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Cell apoptosis and the apoptosis-related activation in the HeLa cell line were evaluated by flow cytometry and Western blotting. Oridonin suppressed the proliferation of the HeLa cell line in a dose- and time-dependent fashion. Oridonin treatment downregulated the activation of protein kinase B (Akt), the expression of forkhead box class O (FOXO) transcription factor, and glycogen synthase kinase 3 (GSK3). Oridonin also induced the release of cytochrome c accompanied by the activation of caspase-3 and poly-adenosine diphosphate- ribose polymerase cleavage. In addition, Z-D(OMe)-E(OMe)-V-D(OMe)- FMK (z-DEVD-fmk), an inhibitor of caspases, prevented caspase-3 activation and abrogated oridonin-induced cell death. Finally, oridonin treatment of the HeLa cell line downregulated the expression of the inhibitor of the apoptosis protein. Our results showed that oridonin-induced apoptosis involved several molecular pathways. Oridonin may suppress constitutively activated targets of phosphatidylinositol 3-kinase (Akt, FOXO, and GSK3) in the HeLa cell line, inhibiting the proliferation and induction of caspase-dependent apoptosis.

Oridonin Inhibited the Tyrosine Kinase Activity and Induced Apoptosis in Human Epidermoid Carcinoma A431 Cells

Oridonin, an active component isolated from the plant Rabdosia rubescens, has been reported to exhibit antitumor effects, but little is known about its molecular mechanism of action. In this study, we first investigated the mechanism involved in oridonin-induced cell death in human epidermoid carcinoma A431 cells, which overexpress epidermal growth factor receptor (EGFR). After treatment with various doses of oridonin for 24 h, the majority of A431 cells underwent apoptosis in a time- and dose-dependent manner as measured by an LDH activity-based assay. Treatment with oridonin at various concentrations for 24 h caused significant inhibition on the total tyrosine kinase activities and downregulation of EGFR expression or EGFR phosphorylation. Oridonin significantly affected the localization of EGFR and phosphorylated EGFR on the cell membrane. However, genistein (a well-known tyrosine kinase inhibitor) did not induce apoptotic A431 cell death. Importantly, oridonin exhibited much stronger inhibitory effect on the total tyrosine kinase activities or EGFR tyrosine phosphorylation as well as much stronger suppression on EGFR and phosphorylated EGFR localization than genistein in A431 cells. Taken together, oridonin exerted a potential inhibitory effect on the tyrosine kinase activity of A431 cells. The decrease in the tyrosine kinase activity and the blockage of EGFR tyrosine phosphorylation might be one of the causes of oridonin-induced A431 cell death.

Fas/FasL Signaling Allows Extracelluar-Signal Regulated Kinase to Regulate Cytochrome c Release in Oridonin-Induced Apoptotic U937 Cells

Previously, we found that human histocytic lymphoma U937 cells possessed high susceptibility to oridonin-induced cell death, but the molecular mechanisms in response to oridonin remain unclear. In this study, U937 cells showed susceptible to apoptosis induced by 27 microM oridonin and an agonistic anti-Fas IgM mAb (CH-11) (500 ng/ml) as a Fas-sensitized positive control. Caspase 8 inhibitor z-IETD, but neither caspase 1 inhibitor Ac-YVAD nor caspase 10 inhibitor z-AEVD, effectively blocked oridonin-induced cell death as well as DNA fragmentation. Western blot analysis showed the up-regulated expression of Fas, FasL, and FADD, and down-regulated expression of procaspase 8, suggesting that Fas/FasL pathway was activated in oridonin-induced cell apoptosis. Further, stimulation of U937 cells with oridonin and CH11 resulted in significant ERK MAPK activation. However, inhibition of ERK by PD98059 reversed oridonin-induced cell death as well as the activation of caspase 8, indicating that ERK-mediated control occured upstream of caspase 8. Simultaneously, ERK activation accounted for the release of cytochrome c, but failed to influence decreased Bcl-2 expression induced by oridonin. Taken together, these results suggest that Fas/FasL signaling pathway-mediated ERK activation sensitized U937 cells to mitochondrial pathway-mediated apoptosis induced by oridonin.

A comparison of the signal pathways between the TNF alpha- and oridonin-induced murine L929 fibrosarcoma cell death.

Oridonin, an active component isolated from Rabdosia rubescences, has been reported to have antitumor effects. In this study, we compared the signal transduction pathways between TNFalpha-and oridonin-induced L929 cell death. Oridonin and TNFalpha initiated apoptotic morphologic changes, but DNA fragmentation was found in TNFalpha-treated L929 cells but not in oridonin-treated ones. The pan-caspase inhibitor (z-VAD-fmk), caspase-8 inhibitor (z-IETD-fmk) and caspase-3 inhibitor (z-DEVD-fmk) augmented oridonin-and TNFalpha-induced cell death. However, the caspase-9 inhibitor (z-LEHD-fmk) only increased oridonin-induced L929 cell death. Moreover, poly (ADPribose) polymerase (PARP) was cleaved in oridonin-treated L929 cells but not in the TNFalpha-treated groups, and the caspase-3 inhibitor (z-DEVD-fmk) failed to inhibit PARP cleavage. These results showed that only oridonin-induced L929 cell death required PARP degradation in a caspase-3 independent manner. In addition, oridonin increased the ratio of Bax/Bcl-2 protein expression, but TNFalpha did not. TNFalpha induced p38 and ERK activation, whereas oridonin triggered only ERK activation. We also investigated the effect of oridonin on intracellular TNFalpha expression, and found that oridonin augmented endogenous pro-TNFalpha expression and its upstream protein IkB phosphorylation. These results indicated that although oridonin promoted endogenous pro-TNFalpha expression, a great difference existed between the signal pathways through which TNFalpha-and oridonin-induced cell death.

Oridonin induces a caspase-independent but mitochondria- and MAPK-dependent cell death in the murine fibrosarcoma cell line L929.

Oridonin, an active component isolated from Rabdosia rubescences, has been reported to exhibit antitumor effects, but little is known about its molecular mechanisms of action. In this study, the growth-inhibitory activity of oridonin for L929 cells is in time- and dose-dependent manner. After treatment with various concentrations of oridonin for 12 h, the majority of L929 cells underwent apoptosis as measured by an LDH activity-based assay. Although apoptotic bodies were observed in oridonin-treated L929 cells, DNA fragmentation as a hallmark of apoptosis was not found. The pan-caspase inhibitor, z-VAD, and caspase-3 inhibitor, z-DEVD, sensitized L929 cells to oridonin, however, a PARP inhibitor (DPQ) effectively blocked oridonin-induced cell death. After 12 h treatment, PARP proenzyme was significantly cleaved. This result indicated that oridonin-induced L929 cell death required PARP degradation in a caspase-independent manner. In addition, an MEK/ERK inhibitor (PD98059) markedly blocked oridonin-induced cell death, whereas a p38 inhibitor (SB203580) and JNK inhibitor (SP600125) weakly protected the cells against death. Treatment with 41.2 microM oridonin for 12 h induced significant and persistent ERK activation and p38 inactivation in L929 cells without evident changes in the protein levels. The responsiveness of ERK and p38 to oridonin suggests the involvement of these kinases in this apoptotic process. Moreover, oridonin increased the ratio of Bax/Bcl-2 protein expression, whereas it had no effect on the expression of Bcl-xL. These results indicate that regulation of the Bcl-2 and MAPK families maybe the effector mechanisms of oridonin-induced L929 cell death, independent of the caspase pathway.