[Show abstract][Hide abstract] ABSTRACT: Cerebral ischemia is a leading cause of mortality and morbidity worldwide, which results in cognitive and motor dysfunction, neurodegenerative diseases, and death. Evodiamine (Evo) is extracted from Evodia rutaecarpa Bentham, a plant widely used in Chinese herbal medicine, which possesses variable biological abilities, such as anticancer, anti-inflammation, antiobesity, anti-Alzheimer's disease, antimetastatic, antianoxic, and antinociceptive functions. But the effect of Evo on ischemic stroke is unclear. Increasing data suggest that activation of autophagy, an adaptive response to environmental stresses, could protect neurons from ischemia-induced cell death. In this study, we found that Evo induced autophagy in U87-MG astrocytes. A scavenger of extracellular calcium and an antagonist of transient receptor potential vanilloid-1 (TRPV-1) decreased the percentage of autophagy accompanied by an increase in apoptosis, suggesting that Evo may induce calcium-mediated protective autophagy resulting from an influx of extracellular calcium. The same phenomena were also confirmed by a small interfering RNA technique to knock down the expression of TRPV1. Finally, Evo-induced c-Jun N-terminal kinases (JNK) activation was reduced by a TRPV1 antagonist, indicating that Evo-induced autophagy may occur through a calcium/c-Jun N-terminal kinase (JNK) pathway. Collectively, Evo induced an influx of extracellular calcium, which led to JNK-mediated protective autophagy, and this provides a new option for ischemic stroke treatment.
Preview · Article · Dec 2013 · Evidence-based Complementary and Alternative Medicine
[Show abstract][Hide abstract] ABSTRACT: Glioblastomas, the most common primary gliomas, are characterized by increased invasion and difficult therapy. Major clinical medicines for treating gliomas merely extend the survival time for a number of months. Therefore, development of new agents against gliomas is important. Autophagy, a process for degrading damaged organelles and proteins, is an adaptive response to environmental stress. However, the role of autophagy in glioblastoma development still needs to be further investigated. Evodiamine, a major alkaloid isolated from Evodia rutaecarpa Bentham, has various pharmacological activities, such as inhibiting tumor growth and metastatic properties. However, the effects of evodiamine on glioblastomas and their detailed molecular mechanisms and autophagy formation are not well understood. In this study, we observed that evodiamine induced dose- and time-dependent apoptosis in glioma cells. Blockade of calcium channels in endoplasmic reticulum (ER) significantly reduced evodiamine-induced cytosolic calcium elevation, apoptosis, and mitochondrial depolarization, which suggests that evodiamine induces a calcium-mediated intrinsic apoptosis pathway. Interestingly, autophagy was also enhanced by evodiamine, and had reached a plateau by 24 h. Pharmacological inhibition of autophagy resulted in increased apoptosis and reduced cell viability. Inhibition of ER calcium channel activation also significantly reduced evodiamine-induced autophagy. Inactivation of c-Jun N-terminal kinases (JNK) suppressed evodiamine-mediated autophagy accompanied by increased apoptosis. Furthermore, evodiamine-mediated JNK activation was abolished by BAPTA-AM, an intracellular calcium scavenger, suggesting that evodiamine mediates autophagy via a calcium-JNK signaling pathway. Collectively, these results suggest that evodiamine induces intracellular calcium/JNK signaling-mediated autophagy and calcium/mitochondria-mediated apoptosis in glioma cells.
No preview · Article · Jun 2013 · Chemico-biological interactions
[Show abstract][Hide abstract] ABSTRACT: Autophagy is a crucial process for cells to maintain homeostasis and survival through degradation of cellular proteins and organelles, including mitochondria and endoplasmic reticula (ER). We previously demonstrated that temozolomide (TMZ), an alkylating agent for brain tumor chemotherapy, induced reactive oxygen species (ROS)/extracellular signal-regulated kinase (ERK)-mediated autophagy to protect glioma cells from apoptosis. In this study, we investigated the role of mitochondrial damage and ER stress in TMZ-induced cytotoxicity. Mitochondrial depolarization and mitochondrial permeability transition pore (MPTP) opening were observed as a prelude to TMZ-induced autophagy, and these were followed by the loss of mitochondrial mass. Electron transport chain (ETC) inhibitors, such as rotenone (a complex I inhibitor), sodium azide (a complex IV inhibitor), and oligomycin (a complex V inhibitor), or the MPTP inhibitor, cyclosporine A, decreased mitochondrial damage-mediated autophagy, and therefore increased TMZ-induced apoptosis. TMZ treatment triggered ER stress with increased expression of GADD153 and GRP78 proteins, and deceased pro-caspase 12 protein. ER stress consequently induced autophagy through c-Jun N-terminal kinases (JNK) and Ca(2+) signaling pathways. Combination of TMZ with 4-phenylbutyrate (4-PBA), an ER stress inhibitor, augmented TMZ-induced cytotoxicity by inhibiting autophagy. Taken together, our data indicate that TMZ induced autophagy through mitochondrial damage- and ER stress-dependent mechanisms to protect glioma cells. This study provides evidence that agents targeting mitochondria or ER may be potential anticancer strategies.
[Show abstract][Hide abstract] ABSTRACT: The alkylating agent temozolomide (TMZ) is the major chemotherapeutic drug used clinically in the treatment of malignant gliomas. This study investigated the mechanism behind TMZ-induced cell death and the possibility that resveratrol might increase TMZ efficacy. TMZ induced both apoptotic cell death and cytoprotective autophagy through a reactive oxygen species (ROS) burst and extracellular signal-regulated kinase (ERK) activation, which was suppressed by resveratrol, resulting in a decrease in autophagy and an increase in apoptosis, suggesting that the ROS/ERK pathway plays a crucial role in the fate of cells after TMZ treatment. Isobolographic analysis indicated that the combination of TMZ and resveratrol has a synergistic effect. Moreover, an in vivo mouse xenograft study also showed that coadministration of resveratrol and TMZ reduced tumor volumes by suppressing ROS/ERK-mediated autophagy and subsequently inducing apoptosis. Taken together, our data indicate that TMZ-induced ROS/ERK-mediated autophagy protected glioma cells from apoptosis, and the combination of resveratrol with TMZ could improve the efficacy of chemotherapy for brain tumors.
No preview · Article · Nov 2011 · Free Radical Biology and Medicine
[Show abstract][Hide abstract] ABSTRACT: Cd is an industrial and environmental pollutant that affects many organs in humans and other mammals. However, the molecular mechanisms of Cd-induced nephrotoxicity are unclear. In this study, we show that endoplasmic reticula (ER) played a pivotal role in Cd-induced apoptosis in mesangial cells. Using Fluo-3 AM, the intracellular concentration of calcium ([Ca(2+)](i)) was detected as being elevated as time elapsed after Cd treatment. Co-treatment with BAPTA-AM, a calcium chelator, was able to significantly suppress Cd-induced apoptosis. Calcineurin is a cytosolic phosphatase, which was able to dephosphorylate the inositol-1,4,5-triphosphate receptor (IP(3)R) calcium channel to prevent the release of calcium from ER. Cyclosporine A, a calcineurin inhibitor, increased both [Ca(2+)](i) and the percentage of Cd-induced apoptosis. However, EGTA and the IP(3)R inhibitor, 2-APB, were able to partially modulate Cd cytotoxicity. These results led us to suggest that the extracellular and ER-released calcium plays a crucial role in Cd-induced apoptosis in mesangial cells. Following this line, we further detected the ER stress after Cd treatment since ER is one of the major calcium storage organelles. After Cd exposure, GADD153, a hallmark of ER stress, was upregulated (at 4h of exposure), followed by activation of ER-specific caspase-12 and its downstream molecule caspase-3 (at 16h of exposure). The pan caspase inhibitor, Z-VAD, and BAPTA-AM were able to reverse the Cd-induced cell death and ER stress, respectively. Furthermore, the mitochondrial membrane potential (DeltaPsi(m)) was depolarized significantly and cytochrome c was released after 24h of exposure to Cd and followed by mild activation of caspase-9 at the 36-h time point, indicating that mitochondria stress is a late event. Therefore, we concluded that ER is the major killer organelle in Cd-induced mesangial cell apoptosis and that calcium oscillation plays a pivotal role.
Full-text · Article · Jun 2009 · Chemico-biological interactions
[Show abstract][Hide abstract] ABSTRACT: This study summarizes our most recent findings on the mechanisms underlying the cadmium-induced death of mesangial cells, which leads to nephrotoxicity. Multiple pathways participate in cadmium-induced nephrotoxicity. In the ROS-GSK-3beta autophagy pathway, cadmium induces ROS most likely from the mitochondria, and the ROS consequently activate GSK-3beta leading to autophagic cell death. In the calcium-ERK autophagy and apoptosis pathway, cadmium stimulates calcium release from the endoplasmic reticulum, which activates ERK leading to predominantly autophagic cell death and a minor level of apoptotic cell death. In the calcium-mitochondria-caspase apoptosis pathway, cadmium-induced elevation of calcium depolarizes the mitochondrial membrane potential and then activates caspase signaling leading to apoptosis. A proposed model for cadmium-induced autophagy and apoptosis leading to nephrotoxicity is summarized in Figure 1.
[Show abstract][Hide abstract] ABSTRACT: We previously demonstrated that cadmium (Cd) is able to induce autophagic cell death through a calcium-extracellular signal-regulated kinase pathway. Here, the object of this study is to investigate the role of glycogen synthase kinase-3beta (GSK-3beta) in the induction of autophagy. After treatment with Cd, MES-13 mesangial cells were determined to have undergone autophagy based on the formation of acidic vesicular organelles and autophagosomes as well as on the processing of microtubule-associated protein 1 light chain 3, using flow cytometry with acridine orange staining, electron microscopy, and immunoblot, respectively. Use of the GSK-3beta inhibitor SB 216763 or the small interfering RNA technique to knockdown the expression of GSK-3beta resulted in a decrease of Cd-induced autophagy. In contrast, overexpression of GSK-3beta by transient transfection potentiated Cd toxicity toward the mesangial cells, suggesting that GSK-3beta plays a crucial role in regulating Cd-induced autophagy. Moreover, a decrease of the phosphorylated level at Ser9 of GSK-3beta was observed by immunoblot after treatment with Cd, indicating GSK-3beta was activated by Cd. This phenomenon was reversed by the reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC), demonstrated that ROS might activate GSK-3beta. In fact, intracellular hydrogen peroxide (H(2)O(2)) was 2.6-fold elevated after 3 h of exposure to Cd. Both Cd-induced ROS bursts and autophagy were reduced by NAC and vitamin E. In summary, this study demonstrated that, in MES-13 mesangial cells, Cd-induced autophagy was mediated through the ROS-GSK-3beta signaling pathway.
No preview · Article · Feb 2009 · Toxicological Sciences
[Show abstract][Hide abstract] ABSTRACT: In the presence of neostigmine (0.1 microM), S-isopetasin competitively antagonized cumulative acetylcholine-induced contractions in guinea pig trachealis, because the slope [1.18+/-0.15 (n=6)] of Schild's plot did not significantly differ from unity. The pA2 value of S-isopetasin was calculated to be 4.62+/-0.05 (n=18). The receptor binding assay for muscarinic receptors of cultured human tracheal smooth muscle cells (HTSMCs) was performed using [3H]-N-methylscopolamine ([3H]-NMS). Saturation binding assays were carried out with [3H]-NMS in the presence (non-specific binding) and absence (total binding) of atropine (1 microM). Analysis of the Scatchard plot (y=0.247-1.306x, r2=0.95) revealed that the muscarinic receptor binding sites in cultured HTSMCs constituted a single population (n(H)=1.00). The equilibrium dissociation constant (Kd) and the maximal receptor density (B(max)) for [3H]-NMS binding were 766 pM and 0.189 pmol/mg of protein, respectively. The -logIC50 values of S-isopetasin, methoctramine, and 1,1-Dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP) for displacing 0.4 nM [3H]-NMS-specific binding were 5.05, 6.25, and 8.56, respectively, which suggests that [3H]-NMS binding is predominantly on muscarinic M3 receptors of cultured HTSMCs. The inhibitory effects of S-isopetasin on enhanced pause (P(enh)) value were similar to that of ipratropium bromide, a reference drug. The duration of action of S-isopetasin (20 microM), also similar to that of ipratropium bromide (20 microM), was 3 h. In contrast to ipratropium bromide, which non-selectively acts on muscarinic receptors, S-isopetasin preferentially acts on muscarinic M3 receptors. In conclusion, S-isopetasin may be beneficial as a bronchodilator in the treatment of chronic obstructive pulmonary disease and asthma exacerbations.
Full-text · Article · May 2008 · European Journal of Pharmacology
[Show abstract][Hide abstract] ABSTRACT: Rhamnus nakaharai Hayata (Rhamnaceae) is used as a folk medicine in Taiwan for treating constipation, inflammation, tumors, and asthma. 3-O-Methylquercetin (3-MQ), a main constituent of the plant, has been reported to have potential for use in the treatment of asthma. The mechanisms of anti-inflammation of 3-MQ are still unclear. Nitric oxide (NO) production induced by lipopolysaccharide (LPS) through iNOS expression in RAW 264.7 cells, a mouse macrophage cell line, may reflect the degree of inflammation and may provide a measure for assessing the effect of drugs on the inflammatory process. Therefore, we were interested in investigating the mechanisms of suppression of NO production by 3-MQ in RAW 264.7 cells. 3-MQ (1-10 microM) concentration-dependently inhibited LPS (100 ng/mL)-induced NO production in RAW 264.7 cells. The IC(50) value was calculated to be 4.23 microM. 3-MQ (1-10 microM) significantly and concentration-dependently inhibited LPS (100 ng/mL)-induced iNOS protein and mRNA expressions in cells. The IC(50) values were calculated to be 4.36 and 6.53 microM, respectively. There was no significant difference among these three IC(50) values of 3-MQ. In conclusion, 3-MQ may exert its anti-inflammatory effect through the inhibition of iNOS DNA transcription.
Full-text · Article · Feb 2006 · Journal of Ethnopharmacology
[Show abstract][Hide abstract] ABSTRACT: Mitochondria are believed to be integrators and coordinators of programmed cell death in addition to their respiratory function. Using mitochondrial DNA (mtDNA)-depleted osteosarcoma cells (rho0 cells) as a cell model, we investigated the apoptogenic signaling pathway of cadmium (Cd) under a condition of mitochondrial dysfunction. The apoptotic percentage was determined to be around 58.0% after a 24-h exposure to 25 microM Cd using flow cytometry staining with propidium iodine (PI). Pretreatment with Z-VAD-fmk, a broad-spectrum caspase inhibitor, failed to prevent apoptosis following Cd exposure. Moreover, Cd was unable to activate caspase 3 using DEVD-AFC as a substrate, indicating that Cd induced a caspase-independent apoptotic pathway in rho0 cells. JC-1 staining demonstrated that mitochondrial membrane depolarization was a prelude to apoptosis. On the other hand, the intracellular calcium concentration increased 12.5-fold after a 2-h exposure to Cd. More importantly, the apoptogenic activity of Cd was almost abolished by ruthenium red, a mitochondrial calcium uniporter blocker. This led us to conclude that mtDNA-depleted cells provide an alternative pathway for Cd to conduct caspase-independent apoptosis through a mitochondria-calcium mechanism.
Full-text · Article · Jun 2005 · Annals of the New York Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Rhamnus nakaharai Hayata (Rhamnaceae), has been used as a folk medicine in Taiwan for treating constipation, inflammation, tumors and asthma. 3-O-methylquercetin (3-MQ), a main constituent of the plant, has been reported to inhibit total cAMP- and cGMP-phosphodiesterase (PDE) of guinea pig trachealis. Therefore we were interested in investigating the inhibitory effect of 3-MQ on various PDE isozymes from guinea pig lungs and hearts. Isolated guinea pig lungs and hearts were homogenized and centrifuged. The supernatant was chromatographed over a column of Q-sepharose, and eluted with various concentrations of NaCl. In the following order, PDE subtypes 1, 5, 2, 4 from lungs, and 3 from hearts were separated. The IC 50 values of 3-MQ on these isozymes were 31.9, 86.9, 18.6, 28.5 and 1.6 microM, respectively. 3-MQ (10-100 microM) non-competitively inhibited PDE2, but competitively inhibited PDE4. 3-MQ (1-10 microM) also competitively inhibited PDE3. However, 3-MQ (10-100 microM) did not competitively inhibit PDE1 and 5, although it had a tendency to competitively inhibit PDE1 at concentrations of 10 - 30 microM. The present results showed that K i value of 3-MQ was similar to that of milrinone in PDE3, and was not significantly different from that of Ro 20 - 1724 in PDE4, respectively. In conclusion, 3-MQ was revealed to be a selective and competitive PDE3/PDE4 inhibitor, although its inhibitory effect on PDE4 was not potent. Therefore, 3-MQ may have a potential in the treatment of asthma beside its antiviral activity.
[Show abstract][Hide abstract] ABSTRACT: We investigated the antimuscarinic effect of S-isopetasin in isolated guinea pig atria to clarify whether it preferentially acts on muscarinic M 2 or M 3 receptors. The tension changes of isolated atria were isometrically recorded on a polygraph. S-Isopetasin at 50 and 100 microM significantly inhibited baselines of contractile tension and heart rate, but atropine at 1 microM enhanced both. S-Isopetasin (10 - 100 microM) did not significantly alter the concentration-negative inotropic response curves of carbachol (CCh) in left atria. S-Isopetasin (10 - 100 microM) allosterically antagonized negative inotropic and chronotropic responses induced by CCh in spontaneously beating right atria, based on the slopes of Schild plots significantly differing from unity. On the contrary, atropine (0.01 - 1 microM) competitively antagonized all the above responses to CCh. The pA 2 values of S-isopetasin were significantly less than that of S-isopetasin in guinea pig trachealis, suggesting that S-isopetasin may preferentially act on tracheal muscarinic M 3, but not cardiac muscarinic M 2 receptors. However, atropine preferentially acts neither. This finding reveals that S-isopetasin may have benefit in the treatment of asthma.