Stress-mediated Pathway Research Articles

An Oxygen-Chelate Complex, Palladium Bis-acetylacetonate, Induces Apoptosis in H460 Cells via Endoplasmic Reticulum Stress Pathway Rather than Interacting with DNA

Current precious-metal-containing anticancer agents are mostly chelated with N-containing ligands and function by interacting with DNA. In the present study, Pd(acac)2, a Pd(II) complex containing four O-donor ligands, has been evaluated as an active anticancer agent. Pd(acac)2 showed no interaction with N-ligand-containing DNA and the S-ligand-containing DMSO, probably because of the two six-member chelate rings that limit the release of the central Pd nuclei to bind to other ligands. Importantly, we found that Pd(acac)2 exhibited better growth inhibitory effects than cisplatin in several cancer cells. Treatment with Pd(acac)2 significantly induced apoptosis in H460 cells. Mechanistically, Pd(acac)2 induced the activation of a series of key components in ER stress-mediated apoptotic pathway, followed by caspase cleavage and activation, while cisplatin showed no similar effects. CHOP knockdown by specific siRNA significantly attenuated Pd(acac)2-induced cell apoptosis. Finally, Pd(acac)2 significantly inhibits H460 cell growth in xenograft mouse models. Taken together, these mechanistic insights on Pd(acac)2 provide us with a novel mechanism and strategy for the development of precious-metal-based anticancer drugs.

Downregulation of Hsp70 inhibits apoptosis induced by sialic acid-binding lectin (leczyme)

Heat shock proteins (Hsps) are molecular chaperones that maintain homeostasis of organisms. In regards to the Hsps, many studies have investigated the structure, expression, localization and functions of Hsp70 and Hsc70 including expression in the glycosphingolipid-enriched microdomain (GEM) on the cell surface and involvement in cell death. Sialic acid-binding lectin (SBL) isolated from oocytes of Rana catesbeiana is a multifunctional protein which has lectin activity, ribonuclease activity and antitumor activity. SBL has potential as a new type of anticancer drug, since it causes cancer-selective induction of apoptosis by multiple signaling pathways in which RNA is its target; and the participation of the mitochondrial pathway and the endoplasmic reticulum (ER) stress-mediated pathway has been suggested. It has also been suggested that receptor(s) for SBL (SBLR) may exist in the GEM on the cell surface. In the present study, we studied the possible involvement of Hsp70 and Hsc70 in SBL-induced apoptosis. We showed that Hsp70 and Hsc70 were expressed on the P388 cell surface similar to SBLR, and their distribution in cells dramatically changed immediately prior to the execution of apoptosis following stimulation of SBL. Functional study of Hsp70 revealed that decreased expression of Hsp70 diminished the apoptosis induced by SBL. It is suggested that Hsp70 participates in the antitumor effect of SBL.

Fenretinide Targets Chronic Myeloid Leukemia Stem/Progenitor Cells by Regulation of Redox Signaling

We have recently shown that fenretinide preferentially targets CD34(+) cells of acute myeloid leukemia (AML), and here, we test whether this agent exerts the effect on CD34(+) cells of chronic myeloid leukemia (CML), which are refractory to imatinib. As tested by colony-forming cell assays using clinical specimens, both number and size of total colonies derived from CD34(+) CML cells were significantly reduced by fenretinide, and by combining fenretinide with imatinib. In particular, colonies derived from erythroid progenitors and more primitive pluripotent/multipotent progenitors were highly sensitive to fenretinide/fenretinide plus imatinib. Accordantly, fenretinide appeared to induce apoptosis in CD34(+) CML cells, particularly with regard to the cells in the subpopulation of CD34(+)CD38(-). Through cell quiescent assays, including Ki-67 negativity test, we added evidence that nonproliferative CD34(+) CML cells were largely eliminated by fenretinide. Transcriptome and molecular data further showed that mechanisms underlying the apoptosis in CD34(+) CML cells were highly complex, involving multiple events of oxidative stress responses. As compared with CD34(+) AML cells, the apoptotic effects of fenretinide on CD34(+) CML cells were more prominent whereas less varied among the samples of different patients, and also various stress-responsive events appeared to be more robust in fenretinide-treated CD34(+) CML cells. Thus, the combination of fenretinide with imatinib may represent a more sophisticated strategy for CML treatment, in which imatinib mainly targets leukemic blast cells through the intrinsic pathway of apopotosis, whereas fenretinide primarily targets CML stem/progenitor cells through the oxidative/endoplasmic reticulum stress-mediated pathway.

Involvement of ER stress in apoptosis induced by sialic acid-binding lectin (leczyme) from bullfrog eggs

Sialic-acid binding lectin (SBL) isolated from bullfrog (Rana catesbeiana) oocytes is a multifunctional protein which has lectin activity, ribonuclease activity and cancer-selective antitumor activity. It has been reported that SBL induces apoptosis accompanied by rigid mitochondrial perturbation, which indicates mediation of the intrinsic pathway. However, the mechanism of the antitumor effect of SBL has not been fully elucidated. We report, here, that ER stress is evoked in SBL-treated cells. We show that caspase-4, an initiator caspase of ER stress-mediated apoptosis was activated, and inhibition of caspase-4 resulted in significant attenuation of apoptosis induced by SBL. We analyzed the precise mechanism of activation of the caspase cascade induced by SBL, and found that caspase-9 and -4 are activated upstream of activation of caspase-8. Further study revealed that SBL induces the mitochondrial and ER stress-mediated pathways independently. It is noteworthy that SBL can induce cancer-selective apoptosis by multiple apoptotic signaling pathways, and it can serve as a candidate molecule for anticancer drugs in a novel field.

Genome-wide identification and analysis of Japonica and Indica cultivar-preferred transcripts in rice using 983 Affymetrix array data

BackgroundAccumulation of genome-wide transcriptome data provides new insight on a genomic scale which cannot be gained by analyses of individual data. The majority of rice (O. sativa) species are japonica and indica cultivars. Genome-wide identification of genes differentially expressed between japonica and indica cultivars will be very useful in understanding the domestication and evolution of rice species.ResultsIn this study, we analyzed 983 of the 1866 entries in the Affymetrix array data in the public database: 595 generated from indica and 388 from japonica rice cultivars. To discover differentially expressed genes in each cultivar, we performed significance analysis of microarrays for normalized data, and identified 490 genes preferentially expressed in japonica and 104 genes in indica. Gene Ontology analyses revealed that defense response-related genes are significantly enriched in both cultivars, indicating that japonica and indica might be under strong selection pressure for these traits during domestication. In addition, 36 (34.6%) of 104 genes preferentially expressed in indica and 256 (52.2%) of 490 genes preferentially expressed in japonica were annotated as genes of unknown function. Biotic stress overview in the MapMan toolkit revealed key elements of the signaling pathway for defense response in japonica or indica eQTLs.ConclusionsThe percentage of screened genes preferentially expressed in indica was 4-fold higher (34.6%) and that in japonica was 5-fold (52.2%) higher than expected (11.1%), suggesting that genes of unknown function are responsible for the novel traits that distinguish japonica and indica cultivars. The identification of 10 functionally characterized genes expressed preferentially in either japonica or indica highlights the significance of our candidate genes during the domestication of rice species. Functional analysis of the roles of individual components of stress-mediated signaling pathways will shed light on potential molecular mechanisms to improve disease resistance in rice.Electronic supplementary materialThe online version of this article (doi:10.1186/1939-8433-6-19) contains supplementary material, which is available to authorized users.

Phenethyl isothiocyanate triggers apoptosis in human malignant melanoma A375.S2 cells through reactive oxygen species and the mitochondria-dependent pathways

We have reported previously that phenethyl isothiocyanate (PEITC) induces apoptosis in human osteosarcoma U-2 OS cells. Cytotoxic activity of PEITC towards other cancer cells such as human malignant melanoma and skin cancer cells has not been reported. In this study, the anticancer activity of PEITC towards human malignant melanoma cancer A375.S2 cells was investigated. To determine the mechanisms of PEITC inhibition of cell growth, the following end points were determined in A375.S2 cells: cell morphological changes, cell cycle arrest, DNA damage and fragmentation assays and morphological assessment of nuclear change, reactive oxygen species (ROS) and Ca(2+) generations, mitochondrial membrane potential disruption, and nitric oxide and 10-N-nonyl acridine orange productions, expression and activation of caspase-3 and -9, B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax), Bcl-2, poly (adenosine diphosphate-ribose) polymerase, and cytochrome c release, apoptosis-inducing factor and endonuclease G. PEITC induced morphological changes in time- and dose-dependent manner. PEITC induced G2/M phase arrest and induced apoptosis via endoplasmic reticulum stress-mediated mitochondria-dependent pathway. Western blot analysis showed that PEITC promoted Bax expression and inhibited Bcl-2 expression associated with the disintegration of the outer mitochondrial membrane causing cytochrome c release, and activation of caspase-9 and -3 cascade leading to apoptosis. We conclude that PEITC-triggered apoptotic death in A375.S2 cells occurs through ROS-mediated mitochondria-dependent pathways.

AB0138 Altered er stress-induced autophagic reactions are associated with increased apoptosis of t lymphocytes in systemic lupus erythematosus

BackgroundThe autophagic responses to ER stress are involved in the regulation of the maintenance of lymphocyte homeostasis and has been implicated in the pathogenesis of autoimmunity. However, there were no...

Mitomycin C-induced apoptosis of human fibroblasts via CCAAT/enhancer binding protein-homologous protein pathway

Objective To investigate the role of CCAAT/enhancer binding protein(C/EBP)-homologous protein (CHOP) in mitomycin C (MMC)-induced apoptosis in human fibroblasts.Methods Cells were exposed to various concentrations of MMC (0,0.1,0.2,0.4,0.6,0.8,1.0 g/L) for5 min,then refreshed with new medium and maintained for different periods (0,12,24,48,60 and 72 h).Cell viability was mneasured by usingMTT assay and cell apoptosis was analyzed by using TdT-mediated dUTP nick end labeling (TUNEL) staining.The expression of CHOP,B lymphocytes/leukemia-2 (bcl-2) and bcl-2 associated X protein (bax) was detected by using Western blotting.The CHOP expression was downregulated by using CHOP siRNA,and the effects of MMC on cell viability and apoptosis were observed.Results MMC could induce a loss in the viability of fibroblast cells in time-and dose-dependent pattern.The median effective dose (ED50) was 0.4 g/L.MMC (0.4 g/L) could significantly induce apoptosis of human fibroblasts [(40.0 ± 5.2) ％] as comnpared with the control group [(3.5 ± 1.8) ％].In addition,down-regulation of CHOP expression with CHOP siRNA attenuated MMC-induced loss of cell viability and apoptosis.Conclusion Endoplasmic reticulum stress-mediated CHOP apoptosis pathway is involved in MMCinduced fibyroblast apoptosis,and CHOP plays a provital role in MMC-induced apoptosis. Key words: Fibroblasts; Mitomycin C; Apoptosis; CCAAT/enhancer binding protein-homologous protein

Antiapoptotic effects of cerium oxide and yttrium oxide nanoparticles in isolated rat pancreatic islets

Type I diabetes mellitus is a metabolic disease caused by the impairment of pancreatic β-cells mainly mediated through oxidative stress and related apoptosis. Islets transplantation seems a promising treatment for these patients, but during islets transplant, various types of stresses related to the isolation and transplantation procedure compromise the function and viability of islets. We recently hypothesized that the combination of cerium oxide (CeO2) and yttrium oxide (Y2O3) nanoparticles with a potential free radical scavenger behavior should be useful to make isolated islets survive until transplanted. In the present study, oxidative stress-induced apoptosis in isolated rat pancreatic islets exposed to hydrogen peroxide (H2O2) and the protective effects of CeO2 and Y2O3 nanoparticles were investigated. Exposure of islets to H2O2 (50 µm, 2 h) increased intracellular oxidant formation such as reactive oxygen species and subsequently apoptosis and decreased viability, glucose-induced adenosine triphosphate (ATP) production and glucose-stimulated insulin secretion. Pretreatment with CeO2 and/or Y2O3 nanoparticles reduced the oxidant formation and apoptosis and increased viability, glucose-induced ATP production and glucose-stimulated insulin secretion. These results suggest that this combination may protect β-cell apoptosis by improving the oxidative stress-mediated apoptotic pathway.

Endoplasmic Reticulum Stress Plays a Pivotal Role in Cell Death Mediated by the Pan-Deacetylase Inhibitor Panobinostat in Human Hepatocellular Cancer Cells

Panobinostat, a pan-deacetylase inhibitor, represents a novel therapeutic option for cancer diseases. Besides its ability to block histone deacetylases (HDACs) by promoting histone hyperacetylation, panobinostat interferes with several cell death pathways providing a potential efficacy against tumors. We have previously demonstrated that panobinostat has a potent apoptotic activity in vitro and causes a significant growth delay of hepatocellular carcinoma (HCC) tumor xenografts in nude mice models. Here, we show that treatment with panobinostat is able to induce noncanonical apoptotic cell death in HepG2 and in Hep3B cells, involving the endoplasmic reticulum (ER) stress by up-regulation of the molecular chaperone binding immunoglobulin protein/glucose-regulated protein 78, activation of eukaryotic initiation factor 2α-activating transcription factor 4 (tax-responsive enhancer element B67) and inositol requiring 1α-X-box binding protein 1 factors, strong increase and nuclear translocation of the transcription factor C/EBP homologous protein/growth arrest and DNA damage-inducible gene 153, and involvement of c-Jun N-terminal kinase. These signaling cascades culminate into the activation of the ER-located caspase-4/12 and of executioner caspases, which finally lead to cell demise. Our results clearly show that panobinostat induces an alternative ER stress-mediated cell death pathway in HCC cells, independent of the p53 status.

RNA Interference of GADD153 Protects Photoreceptors from Endoplasmic Reticulum Stress-Mediated Apoptosis after Retinal Detachment

BackgroundApoptosis of photoreceptors plays a critical role in the vision loss caused by retinal detachment (RD). Pharmacologic inhibition of photoreceptor cell death may prevent RD. This study investigated the role of GADD153 that participates in endoplasmic reticulum (ER) stress-mediated apoptosis of photoreceptor cells after RD.MethodsRetinal detachment was created in Wistar rats by subretinal injection of hyaluronic acid. The rats were then randomly divided into four groups: normal control group, RD group, GADD153 RNAi group and vehicle group. RNA interference of GADD153 was performed using short hairpin RNA (shRNA). Expressions of GADD153 mRNA and protein were examined by RT-PCR and Western blotting analysis, respectively. GADD153 protein distribution in the retinal cells was observed using immunofluorescence confocal laser scanning microscopy. Apoptosis of retinal cells was determined by TdT-mediated fluorescein-16-dUTP nick-end labeling (TUNEL) assay.ResultsLentivirus GADD153 shRNA with the most effective silencing effect was chosen for in vivo animal study and was successfully delivered into the retinal tissues. GADD153 mRNA and protein expressions in GADD153 RNAi group were significantly lower than those in the RD group. Silencing of GADD153 by RNAi protected photoreceptors from ER stress-induced apoptosis.ConclusionER stress-mediated pathway is involved in photoreceptor cell apoptosis after RD. GADD153 is a key regulatory molecule regulating ER-stress pathways and plays a crucial role in the apoptosis of photoreceptor cells after RD.

Vitamin D reduces deposition of advanced glycation end-products in the aortic wall and systemic oxidative stress in diabetic rats

AimsVitamin D may have an important role in reducing the risk of cardiovascular disease. Advanced glycation end-products (AGEs) such as Nε-(carboxymethyl)lysine (CML), have been implicated in diabetic vascular complications via oxidative stress-mediated pathways. We investigated the potential protective effect of vitamin D on CML accumulation in the diabetic aortic wall. To test the effects of vitamin D on systemic oxidative stress we also assessed liver oxidative stress index (OSI) and serum total antioxidant capacity (TAC). MethodsMale Wistar rats were assigned to three groups: control, untreated diabetes, and diabetes+cholecalciferol. Diabetes was induced by streptozotocin, followed by oral administration of cholecalciferol (500IU/kg) for 10 weeks in the treatment group. Aortic CML accumulation was determined by ELISA and immunohistochemical assays. OSI was assessed by measuring TAC and the level of total peroxides in the liver and serum using colorimetric assays. ResultsUntreated diabetes was associated with significantly elevated CML levels in the aortic wall (19.5±3.3 vs 10.2±4.7ng/mL), increased liver OSI (6.8±1.9 vs 3.1±0.7), and reduced serum TAC (0.4±0.1 vs 0.8±0.3mmol Trolox/L), in comparison with the control group. Cholecalciferol significantly blocked the accumulation of CML in the aortic wall (10.4±8.4 vs 19.5±3.3ng/mL), decreased liver OSI (4.2±1.4 vs 6.8±1.9), and improved serum TAC (1.0±0.2 vs 0.4±0.1mmol Trolox/L), compared with the untreated diabetic group. ConclusionsStreptozotocin-diabetes resulted in increased deposition of AGEs and increased oxidative stress in the serum and liver. Vitamin D supplementation may provide significant protection against oxidative stress-mediated vascular complications in diabetes.

Astragaloside IV suppresses collagen production of activated hepatic stellate cells via oxidative stress-mediated p38 MAPK pathway

Oxidative stress is involved in hepatic fibrogenesis. Activation of hepatic stellate cells (HSCs), the key effectors in hepatic fibrogenesis, is characterized by overproduction of extracellular matrix. Astragaloside IV, the active component of Radix Astragali, has antioxidant properties and antifibrotic potential in renal fibrosis. Little is known about the role of astragaloside IV in liver and its involvement in hepatic fibrosis. This study aims at evaluating the antifibrotic potential of astragaloside IV and characterizing involved signal transduction pathways in culture-activated HSCs. Our results show that astragaloside IV attenuates oxidative stress in culture-activated HSCs, as demonstrated by scavenging reactive oxygen species and reducing lipid peroxidation, and elevates the level of cellular glutathione by stimulating Nrf2gene expression. Depletion of cellular glutathione by buthionine sulfoximine or abrogation of p38 MAPK by SB-203580 evidently eliminates the inhibitory effects of astragaloside IV on genes relevant to HSC activation. These results demonstrate that astragaloside IV inhibits HSC activation by inhibiting generation of oxidative stress and associated p38 MAPK activation and provide novel insights into the mechanisms of astragaloside IV as an antifibrogenic candidate in the prevention and treatment of liver fibrosis.

N-acetylcysteine protects against cadmium-induced germ cell apoptosis by inhibiting endoplasmic reticulum stress in testes

Cadmium (Cd) is a reproductive toxicant that induces germ cell apoptosis in the testes. Previous studies have demonstrated that endoplasmic reticulum (ER) stress is involved in Cd-induced germ cell apoptosis. The aim of the present study was to investigate the effects of N-acetylcysteine (NAC), an antioxidant, on Cd-induced ER stress and germ cell apoptosis in the testes. Male CD-1 mice were intraperitoneally injected with CdCl2 (2.0mg kg(-1)). As expected, acute Cd exposure induced germ cell apoptosis in the testes, as determined by terminal dUTP nick-end labelling (TUNEL). However, the administration of NAC alleviated Cd-induced germ cell apoptosis in the testes. Further analysis showed that NAC attenuated the Cd-induced upregulation of testicular glucose-regulated protein 78 (GRP78), an important ER molecular chaperone. Moreover, NAC inhibited the Cd-induced phosphorylation of testicular eukaryotic translation initiation factor 2α (eIF2α), a downstream target of the double-stranded RNA-activated kinase-like ER kinase (PERK) pathway. In addition, NAC blocked the Cd-induced activation of testicular X binding protein (XBP)-1, indicating that NAC attenuates the Cd-induced ER stress and the unfolded protein response (UPR). Interestingly, NAC almost completely prevented the Cd-induced elevation of C/EBP homologous protein (CHOP) and phosphorylation of c-Jun N-terminal kinase (JNK), two components of the ER stress-mediated apoptotic pathway. In conclusion, NAC protects against Cd-induced germ cell apoptosis by inhibiting endoplasmic reticulum stress in the testes.

Human Leptin Protein Induces Proliferation of A549 Cells via Inhibition of PKR-Like ER Kinase and Activating Transcription Factor-6 Mediated Apoptosis

PurposeTo investigate the anti-apoptotic mechanism of leptin in non-small cell lung cancer.Materials and MethodsThe influences of leptin on apoptosis were investigated, analyzing the mechanism that triggers growth of A549 cells. The effects of leptin on cell proliferation were examined by XTT analysis. Leptin, C/EBP homologous protein (CHOP), phosphorylated-PKR-like ER kinase (p-Perk), inositol requiring proteins-1, spliced X-box transcription factor-1 (XBP1), cleaved activating transcription factor-6 (ATF6), eukaryotic translation initiation factor-2α, caspase-12 and CHOP protein were detected in four groups by western blot, and endoplasmic reticulum (ER) stress related mRNA were detected by reverse transcription PCR.ResultsThe expression of leptin in A549 and leptin transfected cells inhibited cisplatin activated ER stress-associated mRNA transcription and protein activation. Two ER stress unfolded protein response pathways, PERK and ATF6, were involved, and XBP1 and tumor necrosis factor receptor-associated factor 2 (TRAF2) were increased significantly when treated with cisplatin in A549-siRNA against leptin cells. Furthermore, CHOP expression was inhibited upon leptin expression in A549, LPT-PeP and LPT-EX cells.ConclusionLeptin serves as an important factor that promotes the growth of A549 cells through blocking ER stress-mediated pathways. This blocking is triggered by p-Perk and ATF6 via inhibition of CHOP expression.

Human gastrin-releasing peptide triggers growth of HepG2 cells through blocking endoplasmic reticulum stress-mediated apoptosis

Gastrin-releasing peptide (GRP) is a kind of neural peptide that plays an important role in the growth of various human cancer cells. However, very little is known about the relationship between GRP and apoptosis in human hepatocellular carcinoma cells. This study investigated the influences of GRP on apoptosis, as well as the mechanism that triggers HepG2 growth. The effects of GRP on cell proliferation were examined by analysis of lactate dehydrogenase. The GRP, caspase 12, and CHOP protein were detected in HepG2 and HL-7702 cells by Western blot, and endoplasmic reticulum (ER) stress-related mRNA transcription was detected by reverse transcription polymerase chain reaction. To explore the specific pathway by which GRP induces the cell growth, we investigated the apoptosis-related pathway. The expression of GRP in HL-7702 cells inhibited tunicamycin triggered ER stress-associated XBP1, ATF4, and TRAF2 mRNA transcription. Three main ER stress-unfolded protein response pathway proteins, including spliced XBP1, cleaved ATF6, IRE1-α, PERK, and eIF2-α, were increased significantly. Furthermore, the cleaved caspase 12 activation was blocked and CHOP expression was inhibited when GRP was expressed either in HepG2 or HL-7702 cells. In conclusion, GRP triggers the growth of HepG2 cells through blocking the ER stress-mediated pathway.

Identification of the Black Tea Polyphenol Theaflavin-3, 3'-Digallate From Screen of Natural Product Inducers of Endoplasmic Reticulum Stress

Abstract 5021 Background:A distinguishing characteristic of myeloma plasma cells is the large quantity of paraprotein that they synthesize and secrete, rendering them especially sensitive to the effects of endoplasmic reticulum (ER) stress. Consistent with this notion, the proteasome inhibitor bortezomib disrupts protein equilibrium in the ER by preventing misfolded proteins from being properly degraded. Given the clinically validated importance of targeting ER stress mediated pathways in the treatment of multiple myeloma (MM), we sought to identify natural products that modulate pathways known to be an effective therapeutic target for MM for potential use to inhibit progression of asymptomatic MM to symptomatic MM without the limiting side effects of current targeted therapies. Methods:Using decreased protein processing in the secretory pathway as a measurable hallmark of ER stress, our screen employed the naturally secreted Gaussia luciferase (Gluc) as a reporter that can be easily monitored through extracellular release of luciferase activity in real time. KMS11 and ARP-1 MM cells expressing Gluc were exposed to compounds in our natural products library in order to identify those which potentially induce ER stress as measured by inhibition of Gluc secretion. The growth inhibitory activity of theaflavin-3, 3’–digallate (TF3) was further characterized by MTS assay. Mechanistic studies of ER stress related pathways including the unfolded protein response (UPR) and apoptotic cascades were analyzed by standard Western blotting techniques. Results:Our screen identified the black tea polyphenol TF3 as a significant inhibitor of GLUC secretion in ARP-1 and KMS-11 cells. TF3 at 0. 5 μM inhibits GLUC secretion by 73 and 68% in ARP-1 and KMS-11 cells, respectively. This inhibition observed is on par with that observed for bortezomib and tunacamycin (a well known inducer of ER stress). TF-3 effectively inhibits cellular proliferation and induces apoptosis in a panel of MM cell lines at physiologically achievable concentrations. Apoptotic induction is at least partially mediated by ER stress mediated pathways as upregulation of the protein chaperone HSP90 and phosphorylation of eIF2-a, a key mediator of the UPR pathway, occurs prior to caspase and PARP cleavage. Conclusions:Our results suggest that TF-3 inhibits protein secretion and MM cell growth through promotion of ER stress generating mechanisms. Based upon these promising results, further mechanistic evaluation and characterization of this safe, natural product as a prophylactic agent in the treatment of asymptomatic conditions like monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) is warranted. Disclosures:No relevant conflicts of interest to declare.

Effect of Dicycloplatin, a Novel Platinum Chemotherapeutical Drug, on Inhibiting Cell Growth and Inducing Cell Apoptosis

Dicycloplatin, a new supramolecular platinum-based antitumor drug, has been approved by the State Food and Administration (SFDA) of China. In this study, we investigated the anticancer activity of dicycloplatin in cancer cells and signaling pathways involved in dicycloplatin-induced apoptosis. Dicycloplatin inhibited the proliferation of cancer cells and increased the percentage of apoptosis in a concentration-dependent manner. Besides, some apoptosis related events were observed after treatment with dicycloplatin, including increase of reactive oxygen species (ROS), collapse of mitochondrial membrane potential (Δψm), release of cytochrome c from the mitochondria to the cytosol, upregulation of p53, which were accompanied by activation of caspase-9, caspase-3, caspase-8, and poly (ADP-ribose) polymerase cleavage in a concentration-dependent manner. The role of apoptosis in dicycloplatin-mediated cell death was further confirmed by the concomitant treatment with caspase-8 or caspase-9 inhibitors, which inhibited apoptosis and PARP cleavage. Intracellular glutathione (GSH) was also found to inhibit the cytotoxic effect of dicycloplatin. In conclusion, these findings suggest that dicycloplatin induces apoptosis through ROS stress-mediated death receptor pathway and mitochondrial pathway which is similar to carboplatin.

Chloroacetic acid induced neuronal cells death through oxidative stress-mediated p38-MAPK activation pathway regulated mitochondria-dependent apoptotic signals

Chloroacetic acid (CA), a toxic chlorinated analog of acetic acid, is widely used in chemical industries as an herbicide, detergent, and disinfectant, and chemical intermediates that are formed during the synthesis of various products. In addition, CA has been found as a by-product of chlorination disinfection of drinking water. However, there is little known about neurotoxic injuries of CA on the mammalian, the toxic effects and molecular mechanisms of CA-induced neuronal cell injury are mostly unknown. In this study, we examined the cytotoxicity of CA on cultured Neuro-2a cells and investigated the possible mechanisms of CA-induced neurotoxicity. Treatment of Neuro-2a cells with CA significantly reduced the number of viable cells (in a dose-dependent manner with a range from 0.1 to 3mM), increased the generation of ROS, and reduced the intracellular levels of glutathione depletion. CA also increased the number of sub-G1 hypodiploid cells; increased mitochondrial dysfunction (loss of MMP, cytochrome c release, and accompanied by Bcl-2 and Mcl-1 down-regulation and Bax up-regulation), and activated the caspase cascades activations, which displayed features of mitochondria-dependent apoptosis pathway. These CA-induced apoptosis-related signals were markedly prevented by the antioxidant N-acetylcysteine (NAC). Moreover, CA activated the JNK and p38-MAPK pathways, but did not that ERK1/2 pathway, in treated Neuro-2a cells. Pretreatment with NAC and specific p38-MAPK inhibitor (SB203580), but not JNK inhibitor (SP600125) effectively abrogated the phosphorylation of p38-MAPK and attenuated the apoptotic signals (including: decrease in cytotoxicity, caspase-3/-7 activation, the cytosolic cytochrome c release, and the reversed alteration of Bcl-2 and Bax mRNA) in CA-treated Neuro-2a cells. Taken together, these data suggest that oxidative stress-induced p38-MAPK activated pathway-regulated mitochondria-dependent apoptosis plays an important role in CA-caused neuronal cell death.

Perturbation of sphingolipid metabolism induces endoplasmic reticulum stress‐mediated mitochondrial apoptosis in budding yeast

Sphingolipids are a class of membrane lipids conserved from yeast to mammals which determine whether a cell dies or survives. Perturbations in sphingolipid metabolism cause apoptotic cell death. Recent studies indicate that reduced sphingolipid levels trigger the cell death, but little is known about the mechanisms. In the budding yeast Saccharomyces cerevisiae, we show that reduction in complex sphingolipid levels causes loss of viability, most likely due to the induction of mitochondria-dependent apoptotic cell death pathway, accompanied by changes in mitochondrial and endoplasmic reticulum morphology and endoplasmic reticulum stress. Elevated cytosolic free calcium is required for the loss of viability. These results indicate that complex sphingolipids are essential for maintaining endoplasmic reticulum homeostasis and suggest that perturbation in complex sphingolipid levels activates an endoplasmic reticulum stress-mediated and calcium-dependent pathway to propagate apoptotic signals to the mitochondria.