Appearance Of Autophagic Vacuoles Research Articles

Autophagy induced by Vip3Aa has a pro-survival role in Spodoptera frugiperda Sf9 cells

ABSTRACT Vip3Aa is an insecticidal protein that can effectively control certain lepidopteran pests and has been used widely in biological control. However, the mechanism of action of Vip3Aa is unclear. In the present study, we showed that Vip3Aa could cause autophagy in Sf9 cells, which was confirmed by the increased numbers of GFP-Atg8 puncta, the appearance of autophagic vacuoles, and an elevated Atg8-II protein level. Moreover, we found that the AMPK-mTOR-ULK1 pathway is involved in Vip3Aa-induced autophagy, which might be associated with the destruction of ATP homeostasis in Vip3Aa-treated cells. Both the elevated p62 level and the increased numbers of GFP-RFP-Atg8 yellow fluorescent spots demonstrated that autophagy in Sf9 cells was inhibited at 24 h after Vip3Aa treatment. With the prolongation of Vip3Aa treatment time, this inhibition became more serious and led to autophagosome accumulation. Genetic knockdown of ATG5 or the use of the autophagy inhibitor 3-MA further increased the sensitivity of Sf9 cells to Vip3Aa. Overexpression of ATG5 reduced the cell mortality of Vip3Aa-treated cells. In summary, the results revealed that autophagy induced by Vip3Aa has a pro-survival role, which might be related to the development of insect resistance.

Antitumor and apoptotic effects of 5-methoxypsoralen in U87MG human glioma cells and its effect on cell cycle, autophagy and PI3K/Akt signaling pathway.

IntroductionThe main purpose of the current study was to investigate the antitumor effects of 5-methoxypsoralen in U87MG human glioma cells along with studying its effects on cell cycle progression, autophagy and the PI3K/Akt signaling pathway.Material and methodsThe cytotoxic effects of the drug were demonstrated by the MTS cell viability assay while its effects on cellular morphology associated with cell apoptosis were evaluated by phase contrast and fluorescence microscopic techniques. Effects of 5-methoxypsoralen on the cell cycle were studied by flow cytometry while effects on PI3K/Akt proteins were evaluated by western blot assay.ResultsThe results indicate that 5-methoxypsoralen led to time-dependent as well as dose-dependent inhibitory effects on U-87MG human glioma cells. 5-Methoxypsoralen led to a substantial decrease of cell count along with distorted cell morphology. The molecule also led to DNA ladder formation which increased with increasing doses of 5-methoxypsoralen. 5-methoxypsoralen also led to dose-dependent G2/M phase cell cycle arrest. 5-Methoxypsoralen-treated cells also exhibited altered cell ultrastructure with the appearance of autophagic vacuoles and the number of these vacuoles increased with increasing drug dose.ConclusionsIn brief, the results indicate that 5-methoxypsoralen exerted potent anticancer and apoptotic effects in U-87MG human glioma cells along with inducing cell cycle arrest, autophagy and m-TOR/PI3K/Akt signaling pathway inhibition.

P57‐mediated autophagy promotes the efficacy of EGFR inhibitors in hepatocellular carcinoma

Resistance to EGFR-targeted therapy is a major obstacle in hepatocellular carcinoma (HCC) treatment, but its underlying mechanism remains unclear. Autophagy plays a vital role in antitumour treatment. Our previous study suggested that p57 is associated with autophagy and cisplatin resistance. The present study aimed to investigate whether p57 can enhance the sensitivity of HCC cells to Erlotinib (Er)/Cetuximab(C-225) and further explore the potential mechanisms of Er/C-225 resistance. HCC cells were transfected with pIRES2-EGFP-p57 and pIRES2-EGFP-nc, accompanied by Er/C-225 treatment. Cell viability was detected by an Annexin apoptosis kit and MTT assay. Xenograft experiments were performed to study the function of p57 in the treatment of Er/C-225 in vivo. The level of autophagy was determined by analysis of the appearance of autophagic vacuoles. Western blotting was used to investigate the potential pathways involved. Up-regulation of p57 decreased the level of Er/C-225-induced autophagy and enhanced the decrease in Er/C-225-induced cell viability. P57 overexpression combined with CQ treatment further enhanced the therapeutic efficiency of Er/C-225. The xenograft experiment verified that p57 up-regulation sensitizes HCC cells to Er/C-225. Moreover, a mechanistic investigation demonstrated that the up-regulation of p57 resulted in a decrease of LC3B-II and beclin-1, and an increase in p-PI3K, p-AKT and p-mTOR protein expressions. Through activating the PI3K/AKT/mTOR signalling pathway, p57 can reverse Er/C-225-induced autophagy, and thereby increase the therapeutic efficiency of Er/C-225 treatment. Given these results, p57 up-regulation may be applicable as a therapeutic strategy to improve EGFR-targeted therapy in HCC.

Abstract 354: Accelerated Autophagy is Required for Doxorubicin-induced Oxidative Damage and Cardiomyopathy

Doxorubicin (DOX) is a highly effective chemotherapeutic agent used in the treatment of a broad spectrum of malignancies. However, clinical use of DOX is limited by the risk of severe cardiotoxicity. In patients, the acute onset of cardiomyopathy often precludes chemotherapy treatment, resulting in reduced quality of life and increased morbidity. While the mechanisms by which DOX promotes cardiac dysfunction are not well understood, our previous findings show that intracellular metabolism of DOX elicits mitochondrial reactive oxygen species (ROS) generation, and subsequent activation of the autophagy/lysosomal system. Equally, evidence suggests that elimination of vital cellular organelles and proteins within the cell as a result of pathologically elevated autophagy may in turn stimulate an increase in ROS generation. Therefore, we hypothesize that DOX-induced autophagy promotes cardiac dysfunction as a result of an autophagy-induced increase in oxidative stress in the heart. To prevent elevated activity of the autophagy/lysosomal system, we administered a dominant negative mutation of the autophagy protein ATG5 (rAAV-dnATG5) to female Sprague-Dawley rats via tail vein administration four weeks prior to either DOX (20 mg/kg ip) or saline treatment. Two days following treatment, assessment of cardiac function showed significant alterations to the myocardial performance index and fractional shortening percentage in control, DOX treated animals. These changes corresponded with deficits to mitochondrial respiration, increased mitochondrial ROS emission and increased appearance of autophagic vacuoles within the heart. In contrast, rAAV-dnATG5 administration in DOX treated animals prevented the conjugation of ATG12 to ATG5 thereby reducing autophagosome formation and attenuating cardiac dysfunction. Interestingly, protein expression of the antioxidant enzymes catalase and superoxide dismutase 2 (SOD2) was elevated in these animals, which may account for improved mitochondrial respiration and reduced mitochondrial free radical production. These findings indicate that accelerated autophagy promotes DOX-induced cardiac dysfunction and is an underlying cause of chemotherapy-induced oxidative damage to the heart.

Autophagy promotes cancer chemotherapy‐induced oxidative stress and skeletal muscle dysfunction

Chemotherapy‐induced cachexia is a debilitating condition characterized by fatigue and muscle wasting. The onset of muscle weakness in patients often limits treatment, resulting in reduced quality of life and increased morbidity. Our previous findings show that the chemotherapeutic agent doxorubicin (DOX) causes myotoxicity as a result of reactive oxygen species (ROS) generation during intracellular metabolism of DOX by the mitochondria, and subsequent activation of the autophagy/lysosomal system. We hypothesize that DOX‐induced accelerated autophagy results in the elimination of vital cellular organelles and proteins, resulting in functional deficits to the muscle tissue. To prevent elevated activity of the autophagy/lysosomal system, we administered a dominant negative mutation of the autophagy protein ATG5 (rAAV‐dnATG5) via intramuscular injection of the rat soleus muscle four weeks prior to treatment. Animals received either DOX (20 mg/kg ip) or saline. Two days after chemotherapy, soleus muscle specific force production and fiber cross‐sectional area were significantly reduced in control animals. These changes corresponded with deficits to mitochondrial respiration, increased mitochondrial ROS emission and the increased appearance of autophagic vacuoles within the muscle. In contrast, rAAV‐dnATG5 administration in DOX treated animals prevented the conjugation of ATG12 and ATG5 thereby reducing autophagosome formation within the soleus muscle. Interestingly, the protein expression of catalase and superoxide dismutase 2 (SOD2) was elevated in these animals, which may account for improved mitochondrial respiration and reduced mitochondrial free radical production. Finally, DOX‐induced muscle fiber atrophy and contractile dysfunction were prevented in rAAV‐dnATG5 animals. These findings indicate that accelerated autophagy is an underlying cause of chemotherapy‐induced oxidative damage and skeletal muscle weakness.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Anti-osteoclastogenic activity of isoliquiritigenin via inhibition of NF-κB-dependent autophagic pathway

Previous studies, including those from our laboratory, have demonstrated that the natural flavonoid isoliquiritigenin (ISL) is a promising agent for bone destructive diseases. However, the mechanisms underlying its anti-osteoclastogenic effects are still far from clear. Here, we evaluated the potential alterations of autophagy and nuclear factor-κB (NF-κB) during anti-osteoclastogenic effects by ISL in vitro and in vivo. We observed that ISL inhibited the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis and suppressed autophagic microtubule-associated protein light chain 3 (LC3)-II and Beclin 1 accumulation. ISL treatment resulted in the interruption of several specific features for autophagy in osteoclast precursors, including acidic vesicular organelle formation, LC3-II accumulation, and appearance of autophagic vacuoles. The RANKL-stimulated expression levels of autophagy-related genes and proteins also diminished in ISL-treated osteoclast precursors. The reactivation of autophagy by rapamycin almost reversed the ISL-elicited anti-osteoclastogenic effects. Interestingly, ISL inhibited the RANKL-stimulated NF-κB expression and nuclear translocation, whereas the NF-κB inhibitor Bay 11-7082 markedly suppressed the RANKL-induced autophagic activation. Consistent with the in vitro results, the administration of ISL could attenuate osteoclastogenic cathepsin K, autophagic LC3, and NF-κB expression to protect against inflammatory calvarial bone erosion in vivo. Our findings highlight the inhibition of NF-κB-dependent autophagy as an important mechanism of ISL-mediated anti-osteoclastogenic activity.

YL4073 is a potent autophagy-stimulating antitumor agent in an in vivo model of Lewis lung carcinoma.

Cancer cells activate autophagy in response to anticancer therapies. Autophagy induction is a promising therapeutic approach to treat cancer. In a previous study, YL4073 inhibited the growth of liver cancer and induced liver cancer cell apoptosis. Here, we demonstrated the anticancer activity and specific mechanisms of YL4073 in Lewis lung carcinoma LL/2 cells. Our results show that YL4073-induced autophagy was followed by apoptotic cell death. The anticancer and autophagy stimulating efficacy was confirmed by several factors, including the appearance of autophagic vacuoles, formation of acidic vesicular organelles, recruitment of microtubule-associated protein 1 light chain 3 II (LC3-II) to the autophagosomes, conversion and cleavage of LC3-I to LC3-II, upregulation of Beclin 1 expression, and formation of the Atg12-Atg5 conjugate in LL/2 cells after YL4073 treatment for 24 or 48 h. Furthermore, P53 activation and p-histone H3 phosphorylation occurred after cell exposure to YL4073 for 48 h, suggesting that cell apoptosis had occurred. Pharmacological inhibition of autophagy using 3-methyladenine increased cell apoptosis. Molecular level studies revealed that YL4073 inhibited survival signalling by blocking the activation of Akt and mTOR phosphorylation and reduced the expression of p-mTOR downstream targets for phosphorylation, including p70S6K, p-TSC, p-MAPK, and p-AMPK. This suggests that the Akt/mTOR/p70S6K and TSC/MAPK/AMPK pathways are involved in the effects of YL4073 treatment in LL/2 cells. In addition, YL4073 significantly inhibited LL/2 tumor growth and induced apoptosis in vivo. These data suggest that YL4073 has a significant anticancer effect, with a pathway-specific mechanism of autophagy both in vitro and in vivo.

Optimization of adipose tissue-derived mesenchymal stem cells by rapamycin in a murine model of acute graft-versus-host disease.

IntroductionMesenchymal stem cells (MSCs) can protect bone marrow transplantation (BMT) recipients from the lethal acute graft-versus-host disease (aGVHD) development. However, the mechanisms underlying the anti-inflammatory properties of MSCs in aGVHD remain to be elucidated. The immunoregulatory properties of MSCs are mediated by their production of anti-inflammatory molecules, including IL-10 and TGF-β. On the other hand, MSCs can also produce proinflammatory cytokines during their normal growth, such as IL-1β and IL-6. These opposite actions may limit their therapeutic application in aGVHD. Therefore, optimization of the functional properties of MSCs can increase their benefits.MethodsThe expressions of mRNA and protein were analyzed by real-time PCR and western blotting, respectively. Expression of MSC markers was assessed by flow cytometry. An animal model of aGVHD was established by transplanting C57BL/6 donor bone marrow cells and spleen cells into lethally irradiated BALB/c recipient mice. The recipient mice were divided into the control group and the therapy [adipose tissue-derived human MSCs (Ad-hMSCs) or rapamycin-treated Ad-hMSCs] groups. The survival, body weight and clinical score of aGVHD in transplanted mice were monitored.ResultsRapamycin pre-treatment of Ad-hMSCs increased mRNA synthesis of IL-10, indoleamine 2,3-dioxygenase, and TGF-β compared with untreated Ad-hMSCs. Rapamycin-treated Ad-hMSCs suppressed clonal expansion of interleukin-17-producing CD4+ T (Th17) cells more effectively than untreated cells. mRNA expression of autophagy markers such as ATG5, LC3A and LC3B was significantly increased in the rapamycin-treated Ad-hMSCs compared with untreated Ad-hMSCs. Transmission electron microscopy revealed that Ad-hMSCs exposure to rapamycin resulted in the appearance of autophagic vacuoles. Interestingly, in vitro migration efficiency of rapamycin-treated Ad-hMSCs toward the CD4+ T cells was increased significantly compared with the untreated cells. And, these effects were associated with autophagy induction capacity of rapamycin. In vivo, the inhibiting properties of MSCs on the clinical severities of aGVHD were greater in the mice receiving rapamycin-treated Ad-hMSCs compared with untreated Ad-hMSCs. The beneficial effects of rapamycin treatment in Ad-hMSCs shown in vivo were associated with a reduction of Th17 cells and an increase in regulatory T cells.ConclusionsRapamycin can optimize the immunomodulatory potential of Ad-hMSCs, suggesting a promising strategy of MSC use in aGVHD treatment.

Abstract B15: Targeting SQSTM1/p62 induces cargo-loading failure and converts autophagy to apoptosis via NBK/Bik in human multiple myeloma cells.

Abstract Purpose: The purpose of this study was to determine whether transcriptional repression of the adaptor protein SQSTM1/p62, which plays a critical role in recognizing/loading cargo (e.g., malfolded proteins) into autophagosomes for lysosomal degradation, could potentiate multiple myeloma (MM) cell death in response to agents (e.g., BH3-mimetics) that elicit a cytoprotective autophagic response. Methods: To this end, various multiple myeloma cell lines (e.g., U266, RPMI8226, and bortezomib-resistant PS-R U266 cells) were exposed to the pan BH3-mimetic obatoclax (Teva) in the presence or absence of pan-CDK inhibitors e.g., flavopiridol (Telero), dinaciclib (Merck), after which the induction of autophagy (LC3-II expression, GFP-LC3 puncta by confocal microscopy) and apoptosis (Annexin V staining by flow cytometry) were monitored. Autophagic flux was determined using cells transfected with a pBABE mCherry-EGFP-LC3B plasmid, followed by observation of autophagosomes and autolysosomes. Accumulation of ubiquitinated protein aggregates was assessed by filter trap (retardation) assays and immunofluorescent staining using an anti-ubiquitin antibody. The functional significance of perturbations in protein expression (e.g., p62, CDK9, cyclin T, Bik) was determined using cells in which target gene expression was down-regulated by shRNA. Alterations in the appearance of autophagic vacuoles (AV) were observed using electron microscopy. Lysosome biogenesis was analyzed by LysoTracker Probe. Gene expression was determined by quantitative real-time PCR (qPCR), and protein-protein interactions characterized by co-immunoprecipitation. The in vivo effects of combined treatment were assessed in mouse MM flank xenograft models. Results: Here we report that whereas p62 levels fluctuated in a time-dependent manner during autophagy induced by obatoclax, inhibition (e.g., by CDK inhibitors administered at sub-micromolar concentrations) or knockdown of CDK9 or cyclin T1 transcriptionally suppressed p62 expression, but did not affect autophagic flux or modify lysosome biogenesis. These interventions, or shRNA directly targeting SQSTM1/p62, resulted in cargo-loading failure and inefficient autophagy, phenomena recently described in Huntington's disease neurons. Inefficient autophagy was characterized by the formation of “empty” AVs accompanied by the accumulation of ubiquitinated proteins, presumably due to failure of loading into autophagosomes for degradation. These events led to accumulation of the BH3-only protein NBK/Bik on ER, most likely by blocking loading and autophagic degradation of Bik. Whereas Bik up-regulation was further enhanced by disruption of distal autophagic events (e.g., autophagosome maturation) by chloroquine or Lamp2 shRNA, it was substantially diminished by inhibition of autophagy initiation (e.g., by shRNA targeting Ulk1, Beclin-1, or Atg5, or pharmacologically by 3-MA or spautin-1), arguing that Bik accumulation primarily stems from inefficient autophagy. Moreover, NBK/Bik knock-down markedly attenuated apoptosis in vitro and in in vivo. Finally, similar interactions between a CDK inhibitor and obatoclax were observed in vivo in MM mouse models. Together, these findings identify a novel cross-link between autophagy and apoptosis, wherein targeting p62 converts cytoprotective autophagy to an inefficient form due to cargo loading failure, leading to NBK/Bik accumulation which triggers apoptosis. Conclusions: Collectively, these findings highlight a novel approach to target the cargo-loading process during cytoprotective autophagy by the transcriptional down-regulation of the adaptor protein p62, rather than direct inhibition of autophagy at either early or late stages (e.g., by chloroquine). Together, these findings provide new mechanistic insights into and a theoretical foundation for developing a autophagy-targeting therapeutic strategy based on induction of inefficient autophagy in MM. Citation Format: Yun Dai, Shuang Chen, Liang Zhou, Yu Zhang, Yun Leng, Xin-Yan Pei, Hui Lin, Richard Jones, Orlowski Robert, Steven Grant. Targeting SQSTM1/p62 induces cargo-loading failure and converts autophagy to apoptosis via NBK/Bik in human multiple myeloma cells. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B15.

Phellinus Linteus Extract Induces Autophagy and Synergizes With 5-Fluorouracil to Inhibit Breast Cancer Cell Growth

Phellinus linteus (PL) is a medicinal mushroom due to its several biological properties, including anticancer activity. However, the mechanisms of its anticancer effect remain to be elucidated. We evaluated the inhibitory effects of the ethanolic extract from the PL combined with 5-FU on MDA-MB-231 breast cancer cell line and to determine the mechanism of cell death. Individually, PL extract and 5-FU significantly inhibited the proliferation of MDA-MB-231 cells in a dose-dependent manner. PL extract (30 mg/mL) in combination with 5-FU (10 μg/mL) synergistically inhibited MDA-MB-231 cells by 1.8-fold. PL did not induce apoptosis, as demonstrated by the DNA fragmentation assay, the sub-G1 population, and staining with annexin V-FITC and propidium iodide. The exposure of MDA-MB-231 cells to PL extracts resulted in several confirmed characteristics of autophagy, including the appearance of autophagic vacuoles revealed by monodansylcadaverine staining, the formation of acidic vesicular organelles, autophagosome membrane association of microtubule-associated protein light chain 3 (LC3) characterized by cleavage of LC3 and its punctuate redistribution, and ultrastructural observation of autophagic vacuoles by transmission electron microscopy. We concluded that PL extracts synergized with low doses of 5-FU to inhibit triple-negative breast cancer cell growth and demonstrated that PL extract can induce autophagy-related cell death.

Autophagy in resin monomer-initiated toxicity of dental mesenchymal cells: a novel therapeutic target of N-acetyl cysteine.

Dental restorative biomaterials are commonly used to restore the teeth impaired by caries, erosion, or fracture. Resin monomers released from polymerized restorative composite materials could disturb cell viability and cause toxicity of oral eukaryotic cells, which remains a medical challenge. However, the intracellular processes or underlying mechanisms of resin monomer-mediated toxicity and the potential preventive/therapeutic strategy are still far from clear. The present study aimed to determine the role of autophagy in resin monomer triethylene glycol dimethacrylate (TEGDMA)-induced cytotoxicity and explore autophagy as a potential therapeutic target of anti-oxidant N-acetyl cysteine (NAC) in vitro and ex vivo. The results showed that TEGDMA exposure resulted in several specific features of autophagy in human dental mesenchymal cells (DMCs), including the formation of acidic vesicular organelles, appearance of autophagic vacuoles, and LC3-II accumulation. By pharmacological and genetic approaches, the inhibition of autophagy significantly prevented TEGDMA-induced apoptosis in DMCs. Moreover, the autophagy activated by TEGDMA occurred via the AMPK/mTOR pathway, which could be abrogated by NAC pretreatment. More importantly, the tooth slice organ culture model provided further evidence of autophagy involvement in TEGDMA-triggered dental mesenchymal tissue toxicity and as a therapeutic target of NAC ex vivo. Our findings provide novel insights into the mechanisms of resin monomer-mediated toxicity and highlight autophagy as a promising therapeutic target of NAC for improving dental restorative biomaterials that enable dental tissue protection.

Cytotoxic effects of Euterpe oleracea Mart. in malignant cell lines.

BackgroundEuterpe oleracea Mart., a plant from the Amazon region, is commonly known as açaí or juçara; it has high nutritional value and elevated levels of lipids, proteins, and minerals. Açaí is an abundant and much consumed fruit by the Amazon local population, and studies have demonstrated that it is rich in phytochemicals with antioxidant, anti-inflammatory, and anticancer activities. Therefore, the aim of this study was to test this plant for anticancer activity in different human malignant cell lines.MethodsCell lines derived from breast and colorectal adenocarcinomas were treated with 10, 20, and 40 μg/mL of bark, seed, and total açaí fruit hydroalcoholic extracts for 24 and 48 h. After treatment, cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, and cell morphological features were observed by light and transmission electron microscopy. The type of cell death was also evaluated. The data were analyzed statistically by one-way analysis of variance (ANOVA), followed by Dunnett’s or Tukey’s post hoc tests, as appropriate.ResultsWe observed that of all the cell lines tested, MCF-7 was the only line that responded to açaí treatment. The extracts caused significant reduction (p < 0.01) in cell viability and altered cell morphological features by inducing the appearance of autophagic vacuoles, as observed by transmission electron microscopy. Furthermore, increased expression of LC3BII, a protein marker of autophagosome formation, was observed by western blotting. Caspase Glo™ assays and morphologic observations by DAPI nuclear staining and transmission electron microscopy did not indicate any apoptotic events.ConclusionsThe present study demonstrated that açaí possesses antitumorigenic potential in the MCF-7 cell line. Further studies are needed to identify the compound (s) responsible for this cytotoxic activity and the molecular target in the cell. This discovery of the anticancer potential of açaí may help in the development of chemopreventive drugs and may have therapeutic effects in the treatment of breast cancer.

Cadmium‐induced cell death in BY‐2 cell culture starts with vacuolization of cytoplasm and terminates with necrosis

Cadmium is a potent inducer of programmed cell death (PCD) in plants but the morphological changes in cells exposed to cadmium are poorly characterized. Using light and transmission electron microscopy (TEM) we have investigated the changes in ultrastructure of tobacco BY-2 cells treated with 50 µM CdSO4. The cadmium-induced alterations in cell morphology occurred gradually over a period of 3-4 days and the first stages of the response resembled vacuolar type of cell death. The initial formation of numerous small cytoplasmic vacuoles and dilation of endoplasmic reticulum was followed first by fusion of smaller vacuoles with each other and with big vacuoles, and then by the appearance of autophagic vacuoles containing autophagic bodies. The final stages of cell death were accompanied by necrotic features including loss of plasmalemma integrity, shrinkage of the protoplast and unprocessed cellular components. In addition, we observed a gradual degradation of nuclear material. Our results demonstrate that cadmium-induced plant cell death is a slow process featuring elements of vacuolar cell death and terminating with necrosis.

Dendropanoxide Induces Autophagy through ERK1/2 Activation in MG-63 Human Osteosarcoma Cells and Autophagy Inhibition Enhances Dendropanoxide-Induced Apoptosis

Anticancer effects of dendropanoxide (DP) newly isolated from leaves and stem of Dendropanax morbifera Leveille were firstly investigated in this study. DP inhibited cell proliferation and induced apoptosis in dose- and time-dependent manner in MG-63 human osteosarcoma cells, which was dependent on the release of cytochrome c to the cytosol and the activation of caspases. Moreover, the DP-treated cells exhibited autophagy, as characterized by the punctuate patterns of microtubule-associated protein 1 light chain 3 (LC3) by confocal microscopy and the appearance of autophagic vacuoles by MDC staining. The expression levels of ATG7, Beclin-1 and LC3-II were also increased by DP treatment. Inhibition of autophagy by 3-methyladenine (3-MA) and wortmannin (Wort) significantly enhanced DP-induced apoptosis. DP treatment also caused a time-dependent increase in protein levels of extracellular signal-regulated kinase 1 and 2 (ERK1/2), and inhibition of ERK1/2 phosphorylation with U0126 resulted in a decreased DP-induced autophagy that was accompanied by an increased apoptosis and a decreased cell viability. These results indicate a cytoprotective function of autophagy against DP-induced apoptosis and suggest that the combination of DP treatment with autophagy inhibition may be a promising strategy for human osteosarcoma control. Taken together, this study demonstrated for the first time that DP could induce autophagy through ERK1/2 activation in human osteosarcoma cells and autophagy inhibition enhanced DP-induced apoptosis.

NF-κB activation was involved in reactive oxygen species-mediated apoptosis and autophagy in 1-oxoeudesm-11(13)-eno-12,8α-lactone-treated human lung cancer cells

1-oxoeudesm-11(13)-eno-12,8α-lactone (OEL), a novel eudesmane-type sesquiterpene compound, has been shown to inhibit the growth of some cancer cell lines and induce significant apoptosis. Here, we investigated the anti-cancer activities of OEL in human lung cancer cells. Our studies demonstrated that OEL induced both apoptosis and autophagy in A549 and H460 cells. OEL-induced autophagy was assessed by appearance of autophagic vacuoles, formation of acidic vesicular organelles, conversion of LC3-I to LC3-II, recruitment of LC3-II to the autophagosomes, and activation of autophagy genes. Furthermore, administration of autophagic inhibitor 3-methyladenine augments OEL-induced apoptotic cell death. The induction of autophagy and apoptosis by OEL links to NF-κB activation and the generation of reactive oxygen species (ROS). Interruption of NF-κB activation by specific inhibitor promotes apoptosis, but decreases autophagy. ROS antioxidants (N-acetylcysteine) attenuated both OEL-induced autophagy and apoptosis. Further experiments confirmed that OEL-induced activation of ROS was increased by NF-κB inhibitor whereas NF-κB activation was not affected by ROS inhibition. These findings suggest that OEL-elicited autophagic response plays a protective role that impedes cell death, and inhibition of autophagy could be an adjunctive strategy for enhancing the chemotherapeutic effect of OEL as an antitumor agent.

Abstract A17: ME-344, a novel mitochondrial oxygenase inhibitor: Results from a first-in-human Phase I study.

Abstract Background: ME-344 is a second generation isoflavone-derived drug candidate that causes caspase-independent cell death in multiple human tumor cell lines including CD44+/MyD88+ ovarian cancer stem cells by interfering with mitochondrial energy generation. Treatment with ME-344 results in decreased ATP production, an increase in reactive oxygen species and disruption of tumor cell mitochondrial integrity. Decreased ATP also lead to inhibition of mTOR1 and mTOR2-dependent signaling pathways via the activation of AMP kinase and the appearance of autophagic vacuoles. We report clinical and pharmacokinetic (PK) results from the first-in-human phase I study of ME-344. Methods: A 3+3 dose escalation design enrolled patients with refractory solid tumors in 6 cohorts at 1.25, 2.5, 5, 10, 15, and 20mg/kg IV weekly times 3, followed by 1 week of rest, then continuous weekly dosing. Restaging occurred every 2 cycles and PK was assessed at day 1 and 15 of cycle 1. Results: Safety: Thirty patients were enrolled. The MTD was established at 10 mg/kg. DLT's were observed at 15 mg/kg (2) and 20 mg/kg (2), all grade 3 neuropathy, and one patient at 10 mg/kg with cardiac chest pain. Possibly related grade 1/2 AE's occurring in &amp;gt;1 patient included: dizziness (5), fatigue (5), nausea (5), asthenia (3), diarrhea (3), dyspnea (3), vomiting (3), abdominal pain (2), chest pain (2), dysphonia (2), headache (2), and neuropathy (2). Pharmacokinetics: ME[[Unable to Display Character: &amp;#8208;]]344 plasma concentrations declined in a multi[[Unable to Display Character: &amp;#8208;]]exponential fashion, with a mean half-life of 6 hrs. There was a linear relationship between both Cmax and AUC as a function of dose (mg/kg), with no evidence of accumulation between Day 1 and Day 15. At the MTD of 10 mg/kg, Cmax was 25.78μg/mL and AUC∞ was 25.9 hr*μg/mL. Volume of distribution, half-life and clearance parameters appear dose[[Unable to Display Character: &amp;#8208;]]independent. Efficacy: Median time on treatment is 56 days (range 1 to 372+). To date, 1 confirmed PR (SCLC, duration 52+ weeks) and 7 prolonged SD (carcinoid (1), cervical (1), leiomyosarcoma (2), NSCLC (1), urothelial (1), uterine (1), 8 - 40+ weeks) have been observed in 23 patients evaluable for efficacy. Conclusions: ME-344 demonstrated dose-limiting toxicity of neuropathy, a known effect of mitochondrial respiratory chain inhibitors. PK data at the 10 mg/kg MTD dose level suggests a sufficient therapeutic index. Evidence of clinical activity at the MTD was observed and supports continued clinical development. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A17. Citation Format: Johanna C. Bendell, Manish Patel, Jeffrey R. Infante, Carla Kurkjian, Suzanne Jones, Shubham Pant, Howard A. Burris, John D. Hainsworth, Ofir Moreno, Kathleen Moore. ME-344, a novel mitochondrial oxygenase inhibitor: Results from a first-in-human Phase I study. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A17.

Evaluation of Praziquantel effects on Echinostoma paraensei ultrastructure

Echinostomiasis is a food-borne, intestinal, zoonotic, snail-mediated helminthiasis caused by digenean trematodes of the family Echinostomatidae with seven species of the genus Echinostoma infecting humans or domestic and wildlife animals. Echinostoma paraensei is a peristomic 37-collar-spined echinostome belonging to the “revolutum group”.Praziquantel (PZQ) is the drug of choice for treatment and control of human schistosomiasis and food-borne trematodiasis. In the present study we used scanning and transmission electron microscopy to further elucidate the trematocidal effect of PZQ on adult E. paraensei and confirmed that this trematode is a suitable model to study anthelmintic drugs. Hamsters infected with E. paraensei were treated with a single dose of 30mgkg−1 of PZQ. The worms were recovered 15, 30, 90 and 180min after drug administration. There was a significant decrease in worm burden in the small intestine in the hamster-E. paraensei model at the intervals of 30, 90 and 180min after the treatment. The worms displayed damage of the peristomic collar with internalization of the spines and erosion of the tegument of the circumoral head-collar of spines. Ultrastructural analysis demonstrated an intense vacuolization of the tegument, appearance of autophagic vacuoles and swelling of the basal infolds of the tegumental syncytium. There was no change in the morphology of cells from the excretory system of adult E. paraensei, however, there was an apparent decrease of stores of glycogen particles in parenchymal cells in PZQ-treated worms. Our results demonstrated that PZQ promotes surface and ultrastructural damage of the tegument of adult E. paraensei supporting the idea that this trematode may constitute a good model to investigate drug effects mechanisms in vitro and in vivo.

Simultaneous induction of autophagy and toll-like receptor signaling pathways by graphene oxide

Graphene oxide (GO) nanosheets have sparked growing interests in biological and medical applications. This study examined how macrophage, the primary immune cell type engaging microbes, responded to GO treatment. We uncovered that incubation of macrophage cell RAW264.7 with GO elicited autophagy in a concentration-dependent manner, as evidenced by the appearance of autophagic vacuoles and activation of autophagic marker proteins. Such GO-induced autophagy was observed in various cell lines and in macrophage treated with GO of different sizes. Strikingly, GO treatment of macrophage provoked the toll-like receptor (TLR) signaling cascades and triggered ensuing cytokine responses. Molecular analysis identified that TLR4 and TLR9 and their downstream signaling mediators MyD88, TRAF6 and NF-κB played pivotal roles in the GO-induced inflammatory responses. By silencing individual genes in the signaling pathway, we further unveiled that the GO-induced autophagy was modulated by TLR4, TLR9 and was dependent on downstream adaptor proteins MyD88, TRIF and TRAF6. Altogether, we demonstrated that GO treatment of cells simultaneously triggers autophagy and TLR4/TLR9-regulated inflammatory responses, and the autophagy was at least partly regulated by the TLRs pathway. This study thus suggests a mechanism by which cells respond to nanomaterials and underscores the importance of future safety evaluation of nanomaterials.

Effects of (−) mammea A/BB isolated from Calophyllum brasiliense leaves and derivatives on mitochondrial membrane of Leishmania amazonensis

We have previously demonstrated antileishmanial activity on Leishmania amazonensis of the natural (1–2), synthetic (7) and derivatives of coumarin (−) mammea A/BB (3–6) isolated from the dichloromethane extract of Calophyllum brasiliense leaves. The aim of the present study was to evaluate morphological and ultrastructural alterations in Leishmania amazonensis induced by these compounds. In promastigote forms, all seven compounds produced significant morphological and ultrastructural alterations, as revealed by scanning and transmission electron microscopy. The compound 5,7-dihydroxy-8-(2-methylbutanoyl)-6-(3-methylbutyl)-4-phenyl-chroman-2-one (3), the most active antileishmanial with LD50 of 0.9μM), induced cell shrinkage and a rounded appearance of the cells. Parasites incubated in the presence of compound (3) showed ultrastructural changes, such as the appearance of mitochondrial swelling with a reduction in the density of the mitochondrial matrix and the presence of vesicles inside the mitochondrion, indicating damage and significant change in this organelle; abnormal chromatin condensation, alterations in the nuclear envelope, intense atypical cytoplasmic vacuolization, and the appearance of autophagic vacuoles were also observed. In addition, the compound (3) may be acting to depolarize the mitochondrial membrane potential of the cells, leading to death of the parasite.

Resveratrol induces apoptosis via ROS-triggered autophagy in human colon cancer cells

Resveratrol (Res; 3,4′,5-trihydroxy-trans-stilbene), which is a polyphenol found in grapes, can block cell proliferation and induce growth arrest and/or cell death in several types of cancer cells. However, the precise mechanisms by which Res exerts anticancer effects remain poorly understood. Res blocked both anchorage-dependent and -independent growth of HT-29 and COLO 201 human colon cancer cells in a dose- and time-dependent manner. Annexin V staining and Western blot analysis revealed that Res induced apoptosis accompanied by an increase in Caspase-8 and Caspase-3 cleavage. In HT-29 cells, Res caused autophagy as characterized by the appearance of autophagic vacuoles by electron microscopy and elevation of microtubule-associated protein 1 light chain 3 (LC3)-II by immunoblotting, which was associated with the punctuate pattern of LC3 detected by fluorescein microscopy. Inhibition of Res-induced autophagy by the autophagy inhibitor 3-methyladenine caused a significant decrease in apoptosis accompanied by decreased cleavage of Casapse-8 and Caspase-3, indicating that Res-induced autophagy was cytotoxic. However, inhibition of Res-induced apoptosis by the pan-caspase inhibitor Z-VAD(OMe)-FMK did not decrease autophagy but elevated LC3-II levels. Interestingly, Res increased the intracellular reactive oxygen species (ROS) level, which correlated to the induction of Casapse-8 and Caspase-3 cleavage and the elevation of LC3-II; treatment with ROS scavenger N-acetyl cysteine diminished this effect. Therefore, the effect of Res on the induction of apoptosis via autophagy is mediated through ROS in human colon cancer cells.