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.     

oridonin部分通过TNFα信号转导途径诱导小鼠成纤维L929细胞凋亡

目的比较冬凌草甲素(oridonin)和TNFα诱导小鼠成纤维L929细胞凋亡的分子机制。方法MTT法、Hoechst33258荧光染色、DNA片断化分析和Western blot分析法。结果Oridonin和TNFα均能诱导L929细胞发生凋亡,其半数有效抑制浓度IC50分别为(24·8±1·2)μmol·L-1和(20·9±1·9)μg·L-1。Oridonin和TNFα均能引起L929细胞凋亡形态学变化,TNFα处理过的细胞在琼脂糖凝胶电泳上可见典型的凋亡DNA梯状条带,但是oridonin处理的细胞却不能,称之为非典型凋亡;caspase-3,-8抑制剂和caspase家族抑制剂能明显得促进25μmol·L-1oridonin和20μg·L-1TNFα诱导的L929细胞凋亡,caspase-9抑制剂只能促进oridonin诱导的L929细胞凋亡;ERK的抑制剂PD98059能明显的抑制oridonin和TNFα诱导的L929细胞凋亡,但是p38的抑制剂SB203580只能抑制TNFα诱导的L929细胞凋亡;在L929细胞中oridonin能促进内源性pro-TNFα的表达和其上游蛋白IκB的磷酸化。结论Oridonin通过激活转录因子NF-κB而促进内源性pro-TNFα的表达,并且部分通过细胞因子TNFα信号转到途径诱导L929细胞凋亡。     

Cytochrome c release from oridonin-treated apoptotic A375-S2 cells is dependent on p53 and extracellular signal-regulated kinase activation

We have reported that oridonin isolated from Rabdosia rubescens induces apoptosis of human melanoma A375-S2 cells within 12 h. In this study, TUNEL assay and flow cytometric analysis also indicate that one of the causes of A375-S2 cell death induced by oridonin was apoptosis. The cell death was preceded by the release of cytochrome c from the mitochondria. Twelve hours after treatment with oridonin, the ratio of Bax/Bcl-xL protein expression was increased and release of cytochrome c was decreased by an extracellular signal-regulated kinase (ERK) MAPK inhibitor (PD98059) and a phosphoinositide 3-kinases (PI3-K) inhibitor (wortmannin). A mitochondrial permeability transition (MPT) inhibitor, decylubiquinone, suppressed the release of cytochrome c without affecting Bax expression. The activation of p53 by oridonin was also blocked by wortmannin. In addtion, PD98059 and wortmannin significantly decreased oridonin-induced DNA fragmentation, but the p38 MAPK inhibitor (SB203580) did not after DNA fragmentation. Oridonin induced A375-S2 cell apoptosis by activating parallel p53 and ERK pathways, increasing the ratio of Bax/Bcl-xL protein expression, and promoting the release of cytochrome c into the cytosol, resulting in apoptotic cell death.     

Roles of Ras and extracellular signal-regulated kinase-dependent IkappaBalpha degradation in oridonin-enhanced phagocytosis of apoptotic cells by human macrophage-like U937 cells

Rapid recognition and ingestion of apoptotic cells by phagocytes are important for the prevention of toxic intracellular contents release, thereby attenuate inflammation and autoimmune diseases such as systemic lupus erythematosus (SLE). We have reported that oridonin isolated from Rabdosia rubescens enhanced phagocytosis of apoptotic U937 cells by macrophage-like U937 cells through TNFalpha and IL-1beta release. In this study, the molecular mechanisms involved in this phagocytic process are investigated. Inhibitors of Ras and Raf1 kinase significantly reduced oridonin-induced phagocytic stimulation as well as extracellular signal-regulated kinase (ERK) phosphorylation. Simultaneously, oridonin-enhanced engulfment was partially blocked by a nuclear factor (NF)-kappaB inhibitor PDTC or proteasome inhibitor MG132. Further studies revealed that oridonin induced IkappaBalpha degradation, which was prevented by Ras inhibitor manumycin A, ERK inhibitor PD98059, but not prevented by c-Jun N-terminal kinase (JNK) MAPK inhibitor SP600125, and up-regulated expression of IL-1beta precursor. These results demonstrate that Ras/Raf1/ERK signaling pathway-dependent IkappaBalpha degradation, resulting in NF-kappaB activation, participates in regulation of oridonin-enhanced phagocytosis, and one of its effector functions is to induce synthesis of IL-1beta, which partially contribute to phagocytic activity of oridonin.     

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.     

Inactivation of ras and changes of mitochondrial membrane potential contribute to oridonin-induced autophagy in a431 cells

We have previously shown that oridonin isolated from Rabdosia rubescens augmented apoptosis while inhibiting autophagy within 24 h in HeLa cells. However, the mechanisms between apoptosis and autophagy induced by oridonin in A431 cells are largely unknown. Here, it was found that autophagic level is significantly upregulated when A431 cells are pretreated with manumycin A (Ras specific inhibitor) compared with oridonin alone treatment, whereas cells precultured with GW5074 (Raf inhibitor) or PD98059 (ERK inhibitor) did not exhibit such an effect. Ras, but not Raf or ERK, was engaged in the control of oridonin-induced autophagy. At the same time, manumycin A contributes to oridonin-induced downregulation of Ras protein expression. Treatment with the combination of oridonin and manumycin A downregulated phosphorylation of Akt, downstream of phosphatidylinositol 3-OH kinase (PI3-K). Preincubation with the PI3-K inhibitor wortmannin and Akt inhibitor KP372-1 enhanced oridonin-induced apoptosis, whereas it inhibited oridonin-induced autophagy. However, under oridonin treatment, the expression of Beclin-1, which has autophagy-inducing activity, was reduced, suggesting that Beclin-1 did not participate in the oridonin-induced autophagy. Morphologic observations, DNA fragmentation analysis, and LDH activity-based assay showed that 3-methyladenine (3-MA), an inhibitor of autophagy, increased the apoptotic sensitivity of A431 cells to oridonin. In addition, manumycin A contributed to oridonin-induced decrease of mitochondrial membrane potential (Deltapsim), consistent with the upregulation of Bax/Bcl-2 ratio. In conclusion, Ras negatively regulated autophagy in oridonin-treated A431 cells, which might be associated with activation of class I PI3-K. Downregulation of Deltapsim and increasing of the ratio of Bax/Bcl-2 might also be partially responsible for the initiation of the autophagic process.     

Activation of phosphoinositide 3-kinase, protein kinase C, and extracellular signal-regulated kinase is required for oridonin-enhanced phagocytosis of apoptotic bodies in human macrophage-like U937 cells

Our previous study showed that oridonin isolated from Rabdosia rubescens enhanced phagocytosis of apoptotic cells by macrophage-like U937 cells through tumor necrosis factor (TNF) alpha and interleukin (IL)-1beta release. In this study, we further investigated signaling events involved in oridonin-augmented phagocytosis. Phagocytic stimulation was significantly suppressed by inhibitors, including a phosphoinositide 3-kinases (PI3K) inhibitor (wortmannin), a protein kinase C (PKC) inhibitor (stauroporine), and a phospholipase C (PLC) inhibitor (U73122). Exposure of U937 cells to oridonin caused an increase in PKC activity time- dependently, which was prevented by pretreatment with inhibitors of PI3K and PLC. Simultaneously, the activation of protein kinase B (PKB/Akt) and the increased expression of PLCgamma2 were also blocked by wortmannin. In addition, an extracellular signal-regulated kinase (ERK) MAPK inhibitor, PD98059, suppressed oridonin-augmented phagocytosis, whereas the p38 MAPK inhibitor (SB203580) and c-Jun N-terminal kinase (JNK) MAPK inhibitor (SP98059) had no inhibitory effect. Furthermore, pretreatment of U937 cells with anti-TNFalpha and anti-IL-1beta antibodies blocked oridonin-induced phagocytic stimulation as well as phosphorylation of ERK, but did not block the activation of PKC, indicating that these signaling events are triggered by oridonin, whereas secreted TNFalpha or IL-1beta only activate the ERK-dependent pathway. Taken together, oridonin is suggested to enhance phagocytosis of apoptotic bodies by activating PI3K, PKC, and ERK-dependent pathways.     

Recombinant TFPI-2 enhances macrophage apoptosis through upregulation of Fas/FasL

Tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type serine proteinase inhibitor with inhibitory activity toward activated factor XI, plasma kallikrein, plasmin, certain matrix metalloproteinases, and the tissue factor-activated factor VII complex. In addition, TFPI-2 has other functions such as promoting cell migration and inducing apoptosis. In the present study, we investigated if TFPI-2 induced apoptosis in cultured U937-derived macrophages and the possible signal pathways that involved in the apoptotic process. Apoptotic DNA fragment detection and caspase-3,9 activity measurements indicated that rTFPI-2 promoted U937-derived macrophage apoptosis. Hoechst 33342 assay and flow cytometry further showed that rTFPI-2 induced apoptosis in cultured macrophages in a dose-dependent manner. Because death receptors of the TNF family such as Fas are the best-understood death pathways that recruit Fas-associated death domain (FADD) and procaspase-8 to the receptor in macrophages, we investigated the expression of Fas and its ligand (FasL) and downstream signal caspase-8 by Western blot analysis. The results indicated that the process of apoptosis triggered by rTFPI-2 was, at least in part, actively conducted by U937-derived macrophages possibly through Fas/FasL signal pathway. In brief, rTFPI-2 may have the potential usefulness in inducing macrophages apoptosis, which suggest TFPI-2 might have antiatherogenic effects.     

Molecular mechanisms of ZD1839 （Iressa）-induced apoptosis in human leukemic U937 cells

Aim： To investigate the molecular mechanisms of ZD 1839-induced apoptosis in human leukemic U937 cells. Methods： The inhibition of human leukemic U937 cell growth was assessed by 3-（4,5-dimethyl-2-thiazolyl）-2,5-diphnyl-2H-tetrazolim bromide （MTT） assays, lactate dehydrogenase （LDH） release, and cell cycle distribution. The expression of anti- and pro-apoptotic proteins was detected by Western blot analysis. Results： This study demonstrated that ZD1839 induced apoptosis in leukemic U937 cells by the downregulation of Bcl-2, caspase activation and subsequent apoptotic features. Cotreatment with ZD 1839 and the caspase- 3 inhibitor z-DEVD-fmk blocked apoptosis, indicating that caspase-3 activation is at least partially responsible for ZD 1839-induced apoptosis. The ectopic expression of Bcl-2 attenuated caspase-3 activation, PARP cleavage, and subsequent indicators of apoptosis, including sub-G1 DNA content and LDH release. These results indicate that the downregulation of Bcl-2 plays a major role in the initiation of ZD1839-induced apoptosis, and that the activation of a caspase cascade is involved in the execution of apoptosis. Furthermore, ZD1839 treatment triggered the activation of p38 mitogen-activated protein kinase （MAPK） and the downregulation of c-Jun-N-terminal kinase （JNK）, extracellular signal-regulated kinase （ERK） and phosphatidyl inositol 3-kinase （PI3K）/Akt. The inhibition of the ERK and PI3K/Akt pathways also significantly increased cellular death. Conclusion： ZD 1839 activated caspase-3 and the inhibited Bcl-2 in human leukemic U937 cells through the downregulation of the ERK and PI3K/Akt pathways.     

Autophagy preceded apoptosis in oridonin-treated human breast cancer MCF-7 cells

Recent studies have shown that MCF-7 cells undergo autophagy under some conditions, such as tamoxifen treatment and starvation. In this study, we investigated autophagy in MCF-7 cells under oridonin treatment and further examined the relationship between autophagy and apoptosis. After 3-MA (the specific inhibitor of autophagy) pre-culture, MCF-7 cells were exposed to oridonin, and the growth inhibitory ratio, morphologic changes, DNA fragmentation, proteins expression, autophagic ratio and apoptotic ratio were evaluated. Oridonin inhibited the proliferation of MCF-7 cells and induced autophagy in vitro. MDC (a specific dye for autophagosome) recruitment and typical apoptotic features, including apoptotic bodies, membrane blebbing as well as nuclear condensation, were induced by oridonin. Oridonin downregulated the phosphorylation of ERK, whereas those of JNK and P38 kinase were upregulated. In the condition of oridonin treatment, 3-MA significantly reduced the autophagic level, and the apoptotic cell ratio was also declined. Furthermore, combined treatment with oridonin and 3-MA upregulated ERK phosphorylation and downregulated JNK and P38 kinases phosphorylation compared with oridonin alone treatment groups, indicating that autophagy facilitated apoptosis in oridonin-induced MCF-7 cells. In addition, 3-MA application downregulated DNA ladder and Bax expression but upregulated Bcl-2 expression, compared with oridonin alone treatment. Taken together, oridonin simultaneously induced MCF-7 cells both apoptosis and autophagy, and in this settings, inhibition of autophagy induced lowered apoptotic level, therefore, autophagy participated in upregulation of apoptosis.     

Involvement of Fas signalling in 7beta-hydroxycholesterol-and cholesterol-5beta,6beta-epoxide-induced apoptosis

The induction of apoptosis in cells of the arterial wall is a critical event in the development of atheroma. 7beta-Hydroxycholesterol (7beta-OH) and cholesterol-5beta,6beta-epoxide (beta-epoxide) are components of oxLDL and have previously been shown to be potent inducers of apoptosis. However, the exact mechanisms through which these oxysterols induce apoptosis remains to be fully elucidated. The specific interaction of the Fas death receptor with Fas ligand (FasL) initiates a caspase cascade culminating in apoptosis. The purpose of the present study was to determine the involvement of Fas signalling in 7beta-OH-and beta-epoxide-induced apoptosis. To this end we employed the Fas/FasL antagonist, Kp7-6, and examined the effect of Fas inhibition on oxysterol-induced cell death in U937 cells. Fas levels were increased following 24 h exposure to 30 micro M 7beta-OH while treatment with 30 micro M beta-epoxide had no effect. Kp7-6 reduced the Fas content of 7beta-OH-treated cells to control levels and partially protected against 7beta-OH-induced apoptosis. This coincided with a decrease in cytochrome c release along with a reduction in caspase-3 and caspase-8 activity. Our data implicate Fas signalling in the apoptotic pathway induced by 7beta-OH and also highlight differences between apoptosis induced by 7beta-OH and beta-epoxide.     

Nanoparticle realgar powders induce apoptosis in u937 cells through caspase mapk and mitochondrial pathways

Nanoparticle realgar powders (NRP) inhibited U937 cell growth in a time and dose-dependent manner. U937 cells treated with NRP showed typical characteristics of apoptosis including the morphological changes and DNA fragmentation. Caspase family inhibitor (z-VAD-fmk), caspase-8, -9 inhibitor (z-IETD-fmk, Ac-LEHD-CHO, respectively) and caspase-3 inhibitor (z-DEVD-fmk) partially prevented NRP -induced apoptosis. Moreover, the classical substrates of caspase-3, poly-ADP ribose polymerase (PARP) was degraded after U937 cells treatment with NRP. In addition, NRP increased the ratio of Bax/Bcl-2 protein expression. Although p38 inhibitor (SB203580) and ERK inhibitor (PD98059) failed to block cell death, JNK inhibitor (SP600125) had marked inhibitory effects on NRP -induced apoptosis. Furthermore, the phosphorylation of JNK was up-regulated, suggesting that JNK was responsible for NRP -induced apoptosis in U937 cells. These results suggested that the caspase, mitochondria and MAPK signal pathways were involved in NRP-induced U937 apoptosis.     

Oridonin-induced A431 cell apoptosis partially through blockage of the Ras/Raf/ERK signal pathway

We have reported that oridonin, a diterpenoid isolated from the plant Rabdosia rubescens, had apoptosis-inducing activities in many cell lines (e.g., human melanoma A375-S2, human cervical cancer HeLa, human breast adenocarcinoma MCF-7, and murine fibrosarcoma L929). In this study, we further investigated signaling events involved in oridonin-induced apoptosis in human epidermoid carcinoma A431 cells. It was found that the total tyrosine kinase activity was inhibited and the protein expressions of epidermal growth factor receptor (EGFR) and phosphorylated EGFR were decreased in oridonin-induced A431 cell apoptosis. Expression of EGFR downstream effector proteins, Grb2, Ras, Raf-1, and extracellular signal-regulated kinase (ERK), was also downregulated by oridonin. Moreover, the oridonin-induced apoptosis was augmented by the Ras inhibitor manumycin A, Raf-1 inhibitor GW5074, or ERK inhibitor PD98059, suggesting that inactivation of Ras, Raf, or ERK participates in oridonin-induced apoptosis. Taken together, oridonin-induced apoptosis in A431 cells might be through blocking EGFR and its downstream Ras/Raf/ERK signal pathway.     

Tetrandrine-induced apoptosis is mediated by activation of caspases and PKC-delta in U937 cells

Tetrandrine, which is isolated from Chinese herb Stephania tetrandrae, possesses anti-inflammatory, immunosuppressive, and cytoprotective properties. Though it was previously shown that tetrandrine causes a G1 blockade and apoptosis in various cell types, however, the mechanism by which tetrandrine initiates apoptosis remains poorly understood. In present study, we investigated the mechanisms of apoptosis induced by tetrandrine in U937 leukemia cells. Tetrandrine inhibited U937 cell growth by inducing apoptosis. After treatment of U937 cells with tetrandrine (10microM) for 24h, alteration of cell morphology, chromatin fragmentation, cytochrome c release, and caspase activation were observed. Tetrandrine also induced early oxidative stress, which resulted in activation of JNK, but not ERK and p38 MAPK. A broad-spectrum caspase inhibitor and antioxidants significantly blocked tetrandrine-induced caspase-3 activation. However, inhibition of the JNK activity with SP600125 did not block tetrandrine-induced apoptosis. Tetrandrine-induced apoptosis of U937 cells also required activity of PKC-delta, because pretreatment with a specific PKC-delta inhibitor greatly blocked tetrandrine-induced caspase-3 activation. In addition, the apoptotic response to tetrandrine was significantly attenuated in dominant-negative PKC-delta transfected MCF-7 cells, suggesting that PKC-delta plays an important role in tetrandrine-induced apoptosis and can induce caspase activation. These results suggest that tetrandrine induces oxidative stress, JNK activation, and caspase activation. However, JNK activation by ROS is not involved in the tetrandrine-induced apoptosis. In addition, tetrandrine induces caspase-dependent generation of a catalytically active fragment of PKC-delta, and this fragment also appears to play a role in the activation of caspases.     

Oridonin induces apoptosis of HeLa cells via altering expression of Bcl-2 ／ Bax and activating caspase-3 ／ ICAD pathway

INTRODUCTIONHerbal medicine, Donglingcao (rabdosiarubescens), has been traditionally used in China for thetreatment of various diseases such as leukemia.Oridonin (Fig 1) is a diterpenoid compound isolated fromRabdosia rubescens (hemsl). It has various pharmaco-logical and physiological effects such as anti-inflammation, anti-bacteria, anti-tumor[1-3] and has beenused for the treatment of human cancers, especiallyFig 1. Chemical structure of oridonin.esophageal carcinoma[4]. This comp…     

冬凌草甲素增强U937细胞吞噬凋亡小体过程中ERK途径的调节作用

目的研究冬凌草甲素促进U937人淋巴瘤细胞分化的巨噬细胞吞噬凋亡小体的机制。方法光学显微镜下计数检测吞噬率,PKC活力检测盒测定PKC活力,吖啶橙染色,Hoechst 33258染色及W estern b lot法。结果酪氨酸蛋白激酶(PTK)抑制剂gen iste in和蛋白激酶C(PKC)广泛的抑制剂stauroporine均不同程度地抑制了冬凌草甲素诱导U937分化的巨噬细胞对凋亡小体的吞噬增强效果。2.7μmol.L-1的冬凌草甲素处理U937细胞后,时间依赖性地增加了PKC活力。ERK磷酸化抑制剂PD98059阻断了冬凌草甲素的吞噬增强作用。免疫印迹结果显示冬凌草甲素作用U937细胞后,ERK磷酸化程度增加,而PD98059逆转了ERK磷酸化。结论冬凌草甲素增强U937细胞对凋亡小体的吞噬作用,其吞噬机制是通过激活PTK和PKC激酶,导致下游ERK途径活化,从而增强吞噬过程。     

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.     

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 (DeltaPsim). 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.     

Fibroblast growth factor-2 suppresses oridonin-induced L929 apoptosis through extracellular signal-regulated kinase-dependent and phosphatidylinositol 3-kinase-independent pathway

Oridonin, isolated from Rabdosia rubescences, has been reported to exert cytotoxic effects on L929 cells. In this study, we investigated the mechanisms of FGF-2 protection of L929 cells from oridonin-induced apoptosis. Phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB) signal did not mediate this effect because the PI3K inhibitor wortmannin failed to reverse this protection and PKB activation was not observed in this process. In contrast, the extracellular signal-regulated kinase (ERK) was responsible for this rescue because its inhibition abolished the protective effect of fibroblast growth factor (FGF)-2. ERK had dual regulatory functions: mediating cell apoptosis or preventing cells from initiating the apoptotic response by phosphorylation or promoting expression of Bcl-2 in dependence of different stimuli. In L929 cells treated with oridonin alone, the activated ERK decreased the ratio of Bcl-2/Bax by mediating the phosphorylation of Bcl-2, resulting in apoptosis; the Ras inhibitor manumycin A and Raf inhibitor GW5074 failed to inhibit this apoptosis, indicating that there is a signal other than Ras/Raf pathway activated ERK. However, in the presence of FGF-2, Bcl-2 phosphorylation was blocked, and the Ras/Raf/ERK signal pathway was activated and protected against the oridonin-induced apoptosis by the alternative function of promoting of Bcl-2 expression.