Tephrosin-induced autophagic cell death in A549 non-small cell lung cancer cells

Anticancer effect of tephrosin (1) has been documented; however, the molecular mechanisms underlying the cytotoxicity of tephrosin in cancer cells remain unclear. In the present paper, the proliferation inhibition rate of several cancer cells was tested using the MTT assay; cell cycle, reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) were determined by flow cytometry; poly(ADP-ribose) polymerase (PARP) cleavage and heat shock protein 90 (Hsp90) expression were evaluated by Western blotting; autophagy was examined by confocal microscopy and light chain 3 (LC3) conversion assay. The results showed that exposure of the cells to tephrosin induced significant proliferation inhibition in a dose-dependent manner, especially on A549 with G(2)/M being arrested. Tephrosin was not found to induce cell apoptosis as PARP cleavage was not detected after 24?h treatment, but the formation of acidic vesicular organelle of autophagy character was found, and autophagy was further confirmed by the increase in the ratio of LC3-II to LC3-I. It was observed that tephrosin induced ROS generation and Hsp90 expression inhibition. These results indicate that tephrosin induces A549 cancer cell death via the autophagy pathway, and the roles of ROS generation and Hsp90 expression inhibition in this process need further study in the future.     

Activation of ERK-p53 and ERK-mediated phosphorylation of Bcl-2 are involved in autophagic cell death induced by the c-Met inhibitor SU11274 in human lung cancer A549 cells

SU11274, a small molecule inhibitor of c-Met, was reported to induce apoptosis in human non-small-cell lung cancer (NSCLC) cells. However, SU11274-mediated autophagy in NSCLC cells has rarely been reported. The aim of this study was to elucidate the molecular mechanisms mediating SU11274-induced autophagy in NSCLC A549 cells. Here we reported that SU11274-induced autophagy was accompanied with an increase in the conversion of LC3-I to LC3-II and up-regulation of Beclin-1 expression. Subsequently, we also found that small interfering RNA against c-Met induced A549 cell autophagy while promotion of c-Met by hepatocyte growth factor (HGF) suppressed A549 cell autophagy. Inhibition of autophagy by 3-methyladenine (3-MA) suppressed SU11274-induced cell death, suggesting that SU11274-induced autophagy caused cell death. Further study showed that ERK and p53 were activated after SU11274 treatment. Interruption of ERK and p53 activities decreased SU11274-induced autophagy, and blocking of ERK by the specific inhibitor PD98059 suppressed SU11274-induced p53 activation. Moreover, ERK activation upregulated Beclin-1 expression through induction of Bcl-2 phosphorylation, but p53 did not induce Bcl-2 phosphorylation. In conclusion, inhibition of c-Met induced autophagic cell death, which was associated with ERK-p53 activation and ERK-mediated Bcl-2 phosphorylation in A549 cells.     

植物化学物诱导肿瘤细胞自噬性死亡及对核受体调节作用研究进展

自噬性细胞死亡是一种非依赖性细胞程序性死亡。核受体可以调控下游基因的转录和翻译,影响自噬相关基因的表达,调节肿瘤细胞的自噬。目前研究发现,一些植物化学物,如异硫氰酸盐、黄酮类化合物、皂苷、植物雌激素、蒽醌类和生物碱等,可诱导特定的肿瘤细胞自噬性死亡。异硫氰酸盐、黄酮类化合物和植物雌激素等植物化学物与核受体结合可使核受体活化。除此之外,一些通过核受体发挥抗肿瘤作用的药物如他莫昔芬、15-脱氧Δ12,14-前列腺素J2、维生素D类似物EB1089、倍半萜烯紫杉醇类似物AGS 115和EFDAC也可以诱导肿瘤细胞发生自噬性死亡。上述研究为了解植物化学物、核受体和自噬三者之间的联系提供了线索。     

Gangliosides induce autophagic cell death in astrocytes

Background and purpose:

Gangliosides, sialic acid-containing glycosphingolipids, abundant in brain, are involved in neuronal function and disease, but the precise molecular mechanisms underlying their physiological or pathological activities are poorly understood. In this study, the pathological role of gangliosides in the extracellular milieu with respect to glial cell death and lipid raft/membrane disruption was investigated.

Experimental approach:

We determined the effect of gangliosides on astrocyte death or survival using primary astrocyte cultures and astrocytoma/glioma cell lines as a model. Signalling pathways of ganglioside-induced autophagic cell death of astrocytes were examined using pharmacological inhibitors and biochemical and genetic assays.

Key results:

Gangliosides induced autophagic cell death in based on the following observations. Incubation of the cells with a mixture of gangliosides increased a punctate distribution of fluorescently labelled microtubule-associated protein 1 light chain 3 (GFP-LC3), the ratio of LC3-II/LC3-I and LC3 flux. Gangliosides also increased the formation of autophagic vacuoles as revealed by monodansylcadaverine staining. Ganglioside-induced cell death was inhibited by either a knockdown of beclin-1/Atg-6 or Atg-7 gene expression or by 3-methyladenine, an inhibitor of autophagy. Reactive oxygen species (ROS) were involved in ganglioside-induced autophagic cell death of astrocytes, because gangliosides induced ROS production and ROS scavengers decreased autophagic cell death. In addition, lipid rafts played an important role in ganglioside-induced astrocyte death.

Conclusions and implications:

Gangliosides released under pathological conditions may induce autophagic cell death of astrocytes, identifying a neuropathological role for gangliosides.     

Investigation of anticancer mechanism of thiadiazole-based compound in human non-small cell lung cancer A549 cells

In this study, we have synthesized several compounds and examined their cytotoxic effects on human non-small cell lung cancer A549 cells. We found that GO-13 ((E,E)-2,5-bis[4-(3-dimethyl-aminopropoxy)styryl]-1,3,4-thiadiazole) is the most effective one by the MTT assay. Furthermore, the GO-13-induced apoptotic reaction was identified based on several criteria, such as negative release reaction of lactate dehydrogenase and positive labeling of annexin V and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) techniques. GO-13 induced the apoptosis in A549 cells in a concentration- and time-dependent manner. The data demonstrate that the regulations of p38 mitogen-activated protein kinase and protein kinase C was not involved in the GO-13-mediated mechanism. However, GO-13 significantly induced a down-regulation of Bcl-X(L) expression in a short-term treatment (less than 3hr), whereas stimulated up-regulation of Bax expression in a long-term treatment (24hr) indicating their involvement in GO-13 action. GO-13-mediated apoptosis is also positively correlated with the increase in caspase-3 activity. Worth noting is the fact that GO-13 did not modify the phosphorylation level of Akt/protein kinase B (PKB) until a 24-hr exposure was carried out indicating that the inhibition of Akt/PKB activation was involved in the late-phase apoptosis. Besides the anticancer activity, GO-13 also showed equivalent anti-angiogenic activity in the nude mice angiogenesis model.In summary, we conclude that GO-13 is the most effective anticancer compound in our screening tests. It induced the early-phase apoptosis in A549 cells via the Bcl-X(L) down-regulation, and that of the late-phase through up-regulation of Bax expression as well as inhibition of Akt/PKB activation.     

Chloroquine rescues A549 cells from paraquat-induced death

Paraquat (PQ) is a widely used herbicide associated with a high mortality rate, yet, there are no effective treatments for PQ poisoning. PQ may damage alveolar type II cells leading to moderate to severe acute respiratory distress syndrome (ARDS). The present study was undertaken to show that PQ causes alveolar type II (A549) cell death and to evaluate whether chloroquine (CQ) can protect A549 cells against PQ-induced cell death. The results showed that high concentrations of PQ resulted in toxicity, as indicated by a decrease in cell viability. More importantly, for the first time, CQ was found to improve cell viability of PQ treated A549 cells. Moreover, our data demonstrated that CQ increased lysosome-associated membrane protein-1, lysosome-associated membrane protein-2 and light chain-3 expressions, suggesting that the mechanism by which CQ rescues PQ-induced cytotoxicity may be through protection of the lysosomal membrane or up-regulation of autophagy. In conclusion, our study indicates that CQ may be used as a potential drug to rescue PQ-induced ARDS.     

Ganetespib for small cell lung cancer

Introduction: Heat shock proteins (Hsps) are part of a complex network of chaperone proteins that are critically involved in the conformational maturation of intracellular proteins and regulate their degradation via the proteasome system Hsps (especially Hsp70 and Hsp90) are upregulated in many cancers and are potentially attractive therapeutic targets. Ganetespib is a potent non-geldanamycin analogue, and avoids the toxicities associated with older analogues due to its small molecular weight, lipophilicity and the absence of the benzoquinone moiety; strong pre-clinical data support its evaluation in lung cancer, especially small cell lung cancer (SCLC).

Areas covered: The chemical structure of ganetespib, the biology of Hsp90 in cancer and the pharmacokinetic and pharmacodynamic data related to ganetespib are summarized; data from preclinical studies and multiple Phase I-III clinical trials, with a focus on its evaluation in SCLC are reviewed.

Expert opinion: Recent progress made in the treatment of refractory SCLC with immune checkpoint inhibitors and DLL3-directed antibody-drug conjugate have made the development of ganetespib particularly challenging in SCLC. Hsp90 remains a critical therapeutic target. Hsp90 inhibitors with a wider therapeutic index and combinations with drugs targeting iHsp90 co-chaperones such as Cdc37 or Protein Kinase 2 may need to be explored in the future.     

Effects of fullerene C60 nanoparticles on A549 cells

Fullerene C₆₀ nanoparticles (C₆₀ NPs) have been widely applied in many fields due to their excellent physical and chemical properties. As production and applications of C₆₀ NPs expand, public concern about the potential risk to human health has also risen. The toxicity of C₆₀ NPs was evaluated by the CCK-8 assay using the cultured human epithelial cell line A549. Cellular uptake of the C₆₀ NPs was observed by TEM imaging. In our findings, C₆₀ NPs could readily enter A549 cells and showed no significant toxicity. Exposure of cultured A549 cells to C₆₀ NPs led to an increase of intracellular reactive oxygen species (ROS) while glutathione reductase activity was probably activated to generate more GSH to maintain a cellular oxidation–reduction equilibrium. The A549 cells responded to the ROS increases through the inauguration of autophagic responses, aimed at restoring cellular health and equilibrium.     

XIAP inhibitor embelin induces autophagic and apoptotic cell death in human oral squamous cell carcinoma cells

Embelin is an active ingredient of traditional herbal remedies for cancer and other diseases. Recently, it has been suggested that autophagy may play an important role in cancer therapy. However, little data are available regarding the role of autophagy in oral cancers. Therefore, we conducted this study to examine whether Embelin modulates autophagy in Ca9‐22. Our results showed that Embelin had anticancer activity against the Ca9‐22 human tongue squamous cell, and we observed that autophagic vacuoles were formed by MDC and AO. We also analyzed Embelin‐treated Ca9‐22 cells for the presence of biochemical markers and found that it directly affected the conversion of LC3‐II, the degradation of p62/SQSTM1, full‐length cleavage formation of ATG5‐ATG12 complex and Beline‐1, and caspase activation. Rescue experiments using an autophagy inhibitor showed Embelin‐induced cell death in Ca9‐22, confirming that autophagy acts as a pro‐death signal. Furthermore, Embelin exhibited anticancer activity against Ca9‐22 via both autophagy and apoptosis. These findings suggest that Embelin may potentially contribute to oral cancer treatment and provide useful information for the development of a new therapeutic agent.     

Antiproliferative activity of aucubin is through cell cycle arrest and apoptosis in human non-small cell lung cancer A549 cells

Aucubin, an iridoid glycoside isolated from the leaves of Aucuba japonica, inhibits human non-small cell lung cancer A549 cells by blocking cell cycle progression in the G(0)/G(1) phase and inducing apoptosis. An ELISA showed that the G(0)/G(1) phase arrest is due to p53-mediated induction of p21. Enhancement of Fas and its two ligands, membrane-bound and soluble Fas ligand, may be responsible for the apoptotic effect induced by aucubin. The present study shows, for the first time, that the induction of p53 and activity of the Fas/Fas ligand apoptotic system may participate in the antiproliferative activity of aucubin in A549 cells.     

Ethanol extract of Dunaliella salina induces cell cycle arrest and apoptosis in A549 human non-small cell lung cancer cells

The ethanol extract of Dunaliella salina (EDS) on proliferation and apoptosis in the A549 human lung cancer cell line and their associated protein expressions were investigated. After 24 and 48 h treatment, MTT assay showed that 25 microg/ml of EDS significantly reduced A549 cell proliferation by 25.2% (p<0.05) and 48.3% (p<0.01), respectively. To explore its molecular mechanisms in regulating cell proliferation, we first showed that EDS markedly reduced A549 proliferation via inhibition of BrdU incorporation at 25 microg/ml by 65.8% (p<0.001). By cytometric analysis, EDS was found to induce apoptosis and cell cycle arrest in the G0/G1 phase. In the DNA gel electrophoresis assay, EDS (25, 50 and 100 microg/ml) induced significant apoptosis at 48 h. Annexin V/Propodium iodide double staining demonstrated that administration of EDS (25 microg/ml) in 12, 24 and 48 h induces apoptosis of 27.7%, 30.7%, and 38.7%. Western blotting assay demonstrated that EDS significantly increased the expression of cyclin-dependent kinase (CDK) inhibitors p53 and p21 and death-receptor proteins Fas and FasL. Bax expression was also elevated by treatment with EDS. Our data suggested that EDS could influence the antiproliferative effects and induce cell cycle G0/G1 arrest and apoptosis of A549 lung cancer cells.     

麦冬皂苷B促进细胞铁死亡抑制非小细胞肺癌A549细胞增殖的机制

目的：研究麦冬皂苷B对A549细胞增殖的影响及其作用机制。方法：细胞计数试剂盒（CCK-8）检测不同浓度麦冬皂苷B处理后A549细胞的存活率;将A549细胞分为对照组和麦冬皂苷B低、中、高（12,24,48 μmol·L^-1）剂量组干预24 h,分析细胞克隆形成情况;DCFH-DA、C11-BODIPY、FeRhonox-1荧光探针分别检测细胞内活性氧、脂质过氧化水平、Fe²⁺浓度的荧光强度变化;GSH和MDA试剂盒分别检测细胞内GSH和MDA水平;Real-time PCR法和Western blot法分别检测SLC7A11、GPX4、SLC40A1、Transferrin、Nrf2、HO-1的mRNA水平和蛋白表达情况。结果：与对照组比较,A549细胞存活率随麦冬皂苷B药物浓度增大逐渐降低,其IC₅₀为 23.7 μmol·L^-1。麦冬皂苷B低、中、高（12,24,48 μmol·L^-1）剂量组显著细胞克隆形成（P<0.05）。与对照组比较,OP B低、中、高（12,24,48 μmol·L^-1）剂量组显著增强细胞内脂质过氧化水平的荧光强度和脂质过氧化产物MDA 积累（P<0.05）,且呈剂量依赖性,OP B中、高（24,48 μmol·L^-1）剂量组显著增强细胞内ROS和亚铁离子的荧光强度（P<0.05）,明显降低细胞内GSH含量（P<0.05）。RT-PCR结果显示,与对照组比较,OP B中、高（24,48 μmol·L^-1）剂量组显著降低GPX4、SLC7A11 mRNA 水平（P<0.05）,只有OP B高（48 μmol·L^-1）剂量组显著降低SLC40A1 的mRNA水平;Western blot结果显示,与对照组比较,OP B低、中、高（12,24,48 μmol·L^-1）剂量组显著降低SLC7A11的蛋白水平（P<0.05）,OP B中、高（24,48 μmol·L^-1）剂量组显著降低GPX4、SLC7A11的蛋白水平（P<0.05）。与对照组比较,OP B中、高（24,48 μmol·L^-1）剂量组显著降低Nrf2的mRNA水平和Nrf2和HO-1的蛋白水平（P<0.05）,而OP B高（48 μmol·L^-1）剂量组显著降低HO-1的mRNA水平（P<0.05）。结论：麦冬皂苷B能够抑制非小细胞肺癌A549细胞增殖,其机制可能与抑制Nrf2/HO-1抗氧化通路促进细胞铁死亡有关。     

Sesquiterpene lactones from Ixeris sonchifolia Hance and their cytotoxicities on A549 human non-small cell lung cancer cells

A new sesquiterpene lactone glucoside, 11,13-dihydroixerinoside (1), together with the five known sesquiterpene lactones, ixerinoside (2), ixerin Z (3), 11,13α-dihydroixerin Z (4), ixerin Z1 (5), and 3-hydroxydehydroleucodin (6), respectively, were isolated from the whole plants of Ixeris sonchifolia Hance. The compounds were identified by spectral analysis and comparison with spectroscopic data reported in the literatures. When the in vitro cytotoxic activities of compounds 1–6 were evaluated against A549 human non-small cell lung cancer cells, all six compounds exhibited cytotoxic activity against A549 cells, with compounds 2, 3, and 6 showing good activities (inhibitory concentration (IC₅₀ values < 30 μg/ml) that are comparable with well-established chemotherapeutic drug, 5-fluorouracil.     

麦角甾醇与吉非替尼联合用药协同抑制非小细胞肺癌A549和PC-9细胞增殖

目的研究麦角甾醇(ERG)与吉非替尼(GEF)联合用药协同对非小细胞肺癌A549(GEF耐药细胞)和PC-9(GEF敏感细胞)细胞的抑制作用。方法将人肺癌细胞A549和PC-9细胞分别分为对照组、ERG 5～160μmol·L^-1单药组、GEF 5～160μmol·L^-1(A549)或0.625～20μmol·L^-1(PC-9)单药组及不同浓度ERG+GEF联合用药组,MTT法测定细胞增殖;Hoechst 33258观察细胞核形态变化;流式细胞术测定细胞凋亡率及细胞周期;Western印迹法检测蛋白质丝氨酸苏氨酸激酶(Akt),磷酸化AKT(p-Akt),表皮生长因子受体(EGFR),磷酸化EGFR(p-EGFR)蛋白的表达。结果在不同作用时间点,与ERG和GEF单药组相比,相应浓度两药联用组对A549和PC-9细胞的增殖抑制率提高(P<0.01),表现出明显的协同效应(q>1.15);Hoechst 33258凋亡染色结果显示,与单药组相比,联用组A549细胞和PC-9细胞各给药组细胞出现明显核分叶、碎裂、...     

Chloroquine potentiates the anti-cancer effect of lidamycin on non-small cell lung cancer cells in vitro

Aim： To assess the synergistic actions of lidamycin （LDM） and chloroquine （CQ）, a lysosomal enzyme inhibitor, in human non-small cell lung cancer （NSCLC） cells, and to elucidate the potential mechanisms. Methods： Human NSCLC cell lines A549 and H460 were treated with CQ and/or LDM. Cell proliferation was analyzed using MTI- assay and apoptosis was quantified using flow cytometry. Western blotting was used to detect the protein levels of caspase 3, PARP, Bcl-2, Bax, p53, LC3-1 and LC3-11. A H460 cell xenograft model in BALB/c nude mice was used to evaluate the anticancer efficacy of CQ and LDM in vivo. Results： Both LDM and CQ concentration-dependently suppressed the proliferation of A549 and H460 ceils in vitro （the ICso values of LDM were 1.70±0.75 and 0.043±0.026 nmol/L, respectively, while the IC50 values of CQ were 71.3±6.1 and 55.6±12.5 pmol/L, respectively）. CQ sensitized both NSCLC cell lines to LDM, and the majority of the coefficients of drug interaction （CDIs） for combination-doses were less than 1. The ratio of apoptosis of H460 cells induced by a combined treatment of CQ and LDM （77.0%±5.2%） was significantly higher than those caused by CQ （23.1%±4.2%） or by LDM （65.1%±4.1%） alone. Furthermore, the combined treatment markedly increased the cleaved PARP and cleaved caspase 3 in H460 cells, which were partly reversed by pretreatment with the caspase inhibitor zVAD.fmk, zVAD.fmk also partially reversed the inhibitory effect of the combination treatment on the proliferation of H460 cells. The combination therapy group had a notable increase in expression of Bax and a very slight decrease in expression of Bcl-2 and p53 protein. LDM alone scarcely affected the level of LC3-11 in H460 cells, but slightly reduced CQ-induced LC3-11 expression. 3-MA, an autophagy inhibitor also sensitized H460 cells to LDM. In nude mice bearing H460 cell xenograft, administration of LDM （25 pg/kg, iv） and CQ （60 mg/kg, ip） suppressed tumor growth by 57.14% and 73.02%, respectively. Conclusion： The synergistic anticancer effect of LDM and CQ in vitro results from activation of a caspase-dependent and p53- independent apoptosis pathway as well as inhibition of cytoprotective autophagy.     

Acute exposure to ZnO nanoparticles induces autophagic immune cell death

Abstract

The increasing risk of incidental exposure to nanomaterials has led to mounting concerns regarding nanotoxicity. Zinc oxide nanoparticles (ZnO NPs) are produced in large quantities and have come under scrutiny due to their capacity to cause cytotoxicity in vitro and potential to cause harm in vivo. Recent evidence has indicated that ZnO NPs promote autophagy in cells; however, the signaling pathways and the role of ion release inducing toxicity remain unclear. In this study, we report that ZnO NPs are immunotoxic to primary and immortalized immune cells. Importantly, such immunotoxicity is observed in mice in vivo, since death of splenocytes is seen after intranasal exposure to ZnO NPs. We determined that ZnO NPs release free Zn²⁺ that can be taken up by immune cells, resulting in cell death. Inhibiting free Zn²⁺ ions in solution with EDTA or their uptake with CaCl₂ abrogates ZnO NP-induced cell death. ZnO NP-mediated immune cell death was associated with increased levels of intracellular reactive oxygen species (ROS). ZnO NP death was not due to apoptosis, necroptosis or pyroptosis. Exposure of immune cells to ZnO NPs resulted in autophagic death and increased levels of LC3A, an essential component of autophagic vacuoles. Accordingly, ZnO NP-mediated upregulation of LC3A and induction of immune cell death were inhibited by blocking autophagy and ROS production. We conclude that release of Zn²⁺ from ZnO NPs triggers the production of excessive intracellular ROS, resulting in autophagic death of immune cells. Our findings suggest that exposure to ZnO NPs has the potential to impact host immunity.     

双氢青蒿素对非小细胞肺癌A549细胞增殖及放疗增敏的影响

目的观察双氢青蒿素(DHA)对非小细胞肺癌A549细胞的影响。方法通过CCK-8法测定双氢青蒿素的IC10,选择低毒剂量IC10作实验浓度,CCK-8法测定DHA对A549细胞增殖的影响,流式细胞术检测DHA对细胞周期及凋亡的影响,平板克隆形成实验检测DHA对放疗敏感性的影响。结果双氢青蒿素的IC10为23.47μmol·L-1,双氢青蒿素能抑制A549细胞增殖,阻滞细胞周期,将细胞周期控制在S期,增加放疗敏感性。结论青蒿素能明显抑制细胞增殖,增加放疗敏感性。     

人程序化死亡因子5（PDCD5）在非小细胞肺癌细胞中的表达及相关性研究

目的：研究PDCD5在非小细胞肺癌中的表达及其相关性．探讨其在非小细胞肺癌发生、发展中的作用。方法：采用免疫组织化学方法,研究非小细胞肺癌组织中PDCD5的表达。结果：PDCD5阳性表达率随非小细胞肺癌组织学分级的降低和临床分期的上升而下降．染色强度也减弱。结论：PDCD5为非小细胞肺癌的负性调节因子,对抑制非小细胞肺癌的恶性转化和进展可能有重要的意义。     

5-氮杂脱氧胞苷对非小细胞肺癌细胞A549中抑癌基因FHIT表达的影响

DNA甲基化是表观遗传修饰的主要方式之一,启动子CpG的高甲基化是除突变和缺失外肿瘤中抑癌基因失活的重要机制[1].脆性组氨酸三联体(fragile histidine triad,FHIT)是第一个将脆性位点和肿瘤联系起来的抑癌基因[2],并且FHIT基因甲基化异常是肿瘤发生和发展过程中的早期事件.去甲基化治疗已成为一种新的抑制细胞增殖和诱导细胞分化的肿瘤治疗方法.5-氮杂脱氧胞苷(5-Aza-dC)是一种脱甲基化试剂,实验采用5-Aza-dC处理非小细胞肺癌细胞株A549细胞,观察其FHIT基因甲基化改变及对A549生长的影响.