Becn1通过线粒体凋亡途径参与调控肺癌细胞的凋亡过程

自噬相关基因Becn1在肺癌等多种肿瘤中处于低表达状态,具有抑制肿瘤发生发展的作用。目前,已有研究发现Becn1可以通过自噬途径参与调控肺癌的发生发展过程,且细胞自噬还与凋亡关系密切。但是,Becn1在调控肺癌发生发展过程中涉及的凋亡过程和相关机制尚未完全阐明。本研究选用肺癌细胞系PC9和A549,建立Becn1高表达的肺癌细胞模型,采用蛋白质免疫共沉淀实验和GFP-BECLIN1、DsRed-Mit荧光共定位实验首次证实了Becn1可通过线粒体途径参与调控肺癌细胞的凋亡过程。     

Defects in Mitochondrial Fission Protein Dynamin-Related Protein 1 Are Linked to Apoptotic Resistance and Autophagy in a Lung Cancer Model

Evasion of apoptosis is implicated in almost all aspects of cancer progression, as well as treatment resistance. In this study, resistance to apoptosis was identified in tumorigenic lung epithelial (A549) cells as a consequence of defects in mitochondrial and autophagic function. Mitochondrial function is determined in part by mitochondrial morphology, a process regulated by mitochondrial dynamics whereby the joining of two mitochondria, fusion, inhibits apoptosis while fission, the division of a mitochondrion, initiates apoptosis. Mitochondrial morphology of A549 cells displayed an elongated phenotype–mimicking cells deficient in mitochondrial fission protein, Dynamin-related protein 1 (Drp1). A549 cells had impaired Drp1 mitochondrial recruitment and decreased Drp1-dependent fission. Cytochrome c release and caspase-3 and PARP cleavage were impaired both basally and with apoptotic stimuli in A549 cells. Increased mitochondrial mass was observed in A549 cells, suggesting defects in mitophagy (mitochondrial selective autophagy). A549 cells had decreased LC3-II lipidation and lysosomal inhibition suggesting defects in autophagy occur upstream of lysosomal degradation. Immunostaining indicated mitochondrial localized LC3 punctae in A549 cells increased after mitochondrial uncoupling or with a combination of mitochondrial depolarization and ectopic Drp1 expression. Increased inhibition of apoptosis in A549 cells is correlated with impeded mitochondrial fission and mitophagy. We suggest mitochondrial fission defects contribute to apoptotic resistance in A549 cells.     

Gambogenic Acid Kills Lung Cancer Cells through Aberrant Autophagy

Lung cancer is one of the most common types of cancer and causes 1.38 million deaths annually, as of 2008 worldwide. Identifying natural anti-lung cancer agents has become very important. Gambogenic acid (GNA) is one of the active compounds of Gamboge, a traditional medicine that was used as a drastic purgative, emetic, or vermifuge for treating tapeworm. Recently, increasing evidence has indicated that GNA exerts promising anti-tumor effects; however, the underlying mechanism remains unclear. In the present paper, we found that GNA could induce the formation of vacuoles, which was linked with autophagy in A549 and HeLa cells. Further studies revealed that GNA triggers the initiation of autophagy based on the results of MDC staining, AO staining, accumulation of LC3 II, activation of Beclin 1 and phosphorylation of P70S6K. However, degradation of p62 was disrupted and free GFP could not be released in GNA treated cells, which indicated a block in the autophagy flux. Further studies demonstrated that GNA blocks the fusion between autophagosomes and lysosomes by inhibiting acidification in lysosomes. This dysfunctional autophagy plays a pro-death role in GNA-treated cells by activating p53, Bax and cleaved caspase-3 while decreasing Bcl-2. Beclin 1 knockdown greatly decreased GNA-induced cell death and the effects on p53, Bax, cleaved caspase-3 and Bcl-2. Similar results were obtained using a xenograft model. Our findings show, for the first time, that GNA can cause aberrant autophagy to induce cell death and may suggest the potential application of GNA as a tool or viable drug in anticancer therapies.     

Mitochondrial Uncoupling as a Therapeutic Target Following Neuronal Injury

Mitochondrial dysfunction is a prominent feature of excitotoxic insult and mitochondria are known to play a pivotal role in neuronal cell survival and death following injury. Following neuronal injury there is a well-documented increase in cytosolic Ca(2+), reactive oxygen species (ROS) production and oxidative damage. In vitro studies have demonstrated these events are dependent on mitochondrial Ca(2+) cycling and that a reduction in membrane potential is sufficient to reduce excitotoxic cell death. This concept has gained additional support from experiments demonstrating that the overexpression of endogenous mitochondrial uncoupling proteins (UCP), which decrease the mitochondrial membrane potential, decreases cell death following oxidative stress. Our group has demonstrated that upregulation of UCP activity can reduce excitotoxic-mediated ROS production and cell death whereas a reduction in UCP levels increases susceptibility to neuronal injury. These findings raise the possibility that mitochondrial uncoupling could be a potential novel treatment for acute CNS injuries.     

A Plasma Long Noncoding RNA Signature for Early Detection of Lung Cancer

The early detection of lung cancer is a major clinical challenge. Long noncoding RNAs (lncRNAs) have important functions in tumorigenesis. Plasma lncRNAs directly released from primary tumors or the circulating cancer cells might provide cell-free cancer biomarkers. The objective of this study was to investigate whether the lncRNAs could be used as plasma biomarkers for early-stage lung cancer. By using droplet digital polymerase chain reaction, we determined the diagnostic performance of 26 lung cancer–associated lncRNAs in plasma of a development cohort of 63 lung cancer patients and 33 cancer-free individuals, and a validation cohort of 39 lung cancer patients and 28 controls. In the development cohort, 7 of the 26 lncRNAs were reliably measured in plasma. Two (SNHG1 and RMRP) displayed a considerably high plasma level in lung cancer patients vs. cancer-free controls (all P?<?.001). Combined use of the plasma lncRNAs as a biomarker signature produced 84.13% sensitivity and 87.88% specificity for diagnosis of lung cancer, independent of stage and histological type of lung tumor, and patients' age and sex (all P?>?.05). The diagnostic value of the plasma lncRNA signature for lung cancer early detection was confirmed in the validation cohort. The plasma lncRNA signature may provide a potential blood-based assay for diagnosing lung cancer at the early stage. Nevertheless, a prospective study is warranted to validate its clinical value.     

Rnd3 Regulates Lung Cancer Cell Proliferation through Notch Signaling

Rnd3/RhoE is a small Rho GTPase involved in the regulation of different cell behaviors. Dysregulation of Rnd3 has been linked to tumorigenesis and metastasis. Lung cancers are the leading cause of cancer-related death in the West and around the world. The expression of Rnd3 and its ectopic role in non-small cell lung cancer (NSCLC) remain to be explored. Here, we reported that Rnd3 was down-regulated in three NSCLC cell lines: H358, H520 and A549. The down-regulation of Rnd3 led to hyper-activation of Rho Kinase and Notch signaling. The reintroduction of Rnd3 or selective inhibition of Notch signaling, but not Rho Kinase signaling, blocked the proliferation of H358 and H520 cells. Mechanistically, Notch intracellular domain (NICD) protein abundance in H358 cells was regulated by Rnd3-mediated NICD proteasome degradation. Rnd3 regulated H358 and H520 cell proliferation through a Notch1/NICD/Hes1 signaling axis independent of Rho Kinase.     

布雷菲德菌素A通过线粒体凋亡途径增强顺铂抗肺癌GLC-82细胞的作用

本研究证明了线粒体凋亡途径在布雷菲德菌素A（brefeldin A,BFA）联合顺铂（cis-dichlorodiamine platinum,CDDP）抗非小细胞肺癌（non-small cell lung cancer,NSCLC）中的作用。MTT结果显示,BFA对肺癌GLC-82和NCI-H1299细胞的半数有效抑制浓度（half maximal inhibitory concentration,IC50）分别是100 ng/mL和400 ng/mL,CDDP对GLC-82和NCI-H1299细胞的IC50分别是4 μg/mL和15 μg/mL;而分别采用半量的BFA和CDDP联合处理GLC-82或NCI-H1299细胞后,抑制作用均进一步加强。DAPI染色结果进一步证明了二者的协同作用——与单独用药组相比,细胞核染色质固缩加剧,核裂解碎片增多,乃至形成凋亡小体,表明细胞凋亡的发生。与单药组比较,联合用药导致肺癌GLC-82细胞线粒体膜电位显著下降;q-RT-PCR及Western印迹结果显示,在联合用药早期（24 h）,GLC-82细胞可能通过提高Bcl2表达以促进存活;而在联合用药晚期（48 h）,细胞已发生不可逆转的凋亡,Bcl2表达受抑制,同时二者通过促进Bax表达来诱导细胞色素C释放,使胱天蛋白酶 3发生剪切激活,最终诱导细胞凋亡发生。提示线粒体凋亡途径可能是BFA协同CDDP抗非小细胞肺癌的分子机制之一,为肺癌的临床治疗方案提供了更多的理论依据。     

Anthraquinone G503 Induces Apoptosis in Gastric Cancer Cells through the Mitochondrial Pathway

G503 is an anthraquinone compound isolated from the secondary metabolites of a mangrove endophytic fungus from the South China Sea. The present study elucidates the anti-tumor activity and the underlying mechanism of G503. Cell viability assay performed in nine cancer cell lines and two normal cell lines demonstrated that the gastric cancer cell line SGC7901 is the most G503-sensitive cancer cells. G503 induced SGC7901 cell death via apoptosis. G503 exposure activated caspases-3, -8 and -9. Pretreatment with the pan-caspase inhibitor Z-VAD-FMK and caspase-9 inhibitor Z-LEHD-FMK, but not caspase-8 inbibitor Z-IETD-FMK, attenuated the effect of G503. These results suggested that the intrinsic mitochondrial apoptosis pathway, rather than the extrinsic pathway, was involved in G503-induced apoptosis. Furthermore, G503 increased the ratio of Bax to Bcl-2 in the mitochondria and decreased the ratio in the cytosol. G503 treatment resulted in mitochondrial depolarization, cytochrome c release and the subsequent cleavage of caspase -9 and -3. Moreover, it is reported that the endoplasmic reticulum apoptosis pathway may also be activated by G503 by inducing capase-4 cleavage. In consideration of the lower 50% inhibitory concentration for gastric cancer cells, G503 may serve as a promising candidate for gastric cancer chemotherapy.     

Mitochondrial Fragmentation Due to Inhibition of Fusion Increases Cyclin B through Mitochondrial Superoxide Radicals

During the cell cycle, mitochondria undergo regulated changes in morphology. Two particularly interesting events are first, mitochondrial hyperfusion during the G₁-S transition and second, fragmentation during entry into mitosis. The mitochondria remain fragmented between late G₂- and mitotic exit. This mitotic mitochondrial fragmentation constitutes a checkpoint in some cell types, of which little is known. We bypass the ‘mitotic mitochondrial fragmentation’ checkpoint by inducing fragmented mitochondrial morphology and then measure the effect on cell cycle progression. Using Drosophila larval hemocytes, Drosophila S2R⁺ cell and cells in the pouch region of wing imaginal disc of Drosophila larvae we show that inhibiting mitochondrial fusion, thereby increasing fragmentation, causes cellular hyperproliferation and an increase in mitotic index. However, mitochondrial fragmentation due to over-expression of the mitochondrial fission machinery does not cause these changes. Our experiments suggest that the inhibition of mitochondrial fusion increases superoxide radical content and leads to the upregulation of cyclin B that culminates in the observed changes in the cell cycle. We provide evidence for the importance of mitochondrial superoxide in this process. Our results provide an insight into the need for mitofusin-degradation during mitosis and also help in understanding the mechanism by which mitofusins may function as tumor suppressors.     

Estimating a Path through a Map of Decision Making

Studies of the evolution of collective behavior consider the payoffs of individual versus social learning. We have previously proposed that the relative magnitude of social versus individual learning could be compared against the transparency of payoff, also known as the “transparency” of the decision, through a heuristic, two-dimensional map. Moving from west to east, the estimated strength of social influence increases. As the decision maker proceeds from south to north, transparency of choice increases, and it becomes easier to identify the best choice itself and/or the best social role model from whom to learn (depending on position on east–west axis). Here we show how to parameterize the functions that underlie the map, how to estimate these functions, and thus how to describe estimated paths through the map. We develop estimation methods on artificial data sets and discuss real-world applications such as modeling changes in health decisions.     

A Novel Class of Mitochondria-Targeted Soft Electrophiles Modifies Mitochondrial Proteins and Inhibits Mitochondrial Metabolism in Breast Cancer Cells through Redox Mechanisms

Despite advances in screening and treatment over the past several years, breast cancer remains a leading cause of cancer-related death among women in the United States. A major goal in breast cancer treatment is to develop safe and clinically useful therapeutic agents that will prevent the recurrence of breast cancers after front-line therapeutics have failed. Ideally, these agents would have relatively low toxicity against normal cells, and will specifically inhibit the growth and proliferation of cancer cells. Our group and others have previously demonstrated that breast cancer cells exhibit increased mitochondrial oxygen consumption compared with non-tumorigenic breast epithelial cells. This suggests that it may be possible to deliver redox active compounds to the mitochondria to selectively inhibit cancer cell metabolism. To demonstrate proof-of-principle, a series of mitochondria-targeted soft electrophiles (MTSEs) has been designed which selectively accumulate within the mitochondria of highly energetic breast cancer cells and modify mitochondrial proteins. A prototype MTSE, IBTP, significantly inhibits mitochondrial oxidative phosphorylation, resulting in decreased breast cancer cell proliferation, cell attachment, and migration in vitro. These results suggest MTSEs may represent a novel class of anti-cancer agents that prevent cancer cell growth by modification of specific mitochondrial proteins.     

Gefitinib-mediated Reactive Oxygen Specie (ROS) Instigates Mitochondrial Dysfunction and Drug Resistance in Lung Cancer Cells

Therapeutic benefits offered by tyrosine kinase inhibitors (TKIs), such as gefitinib (Iressa) and erlotinib (Tarceva), are limited due to the development of resistance, which contributes to treatment failure and cancer-related mortality. The aim of this study was to elucidate mechanistic insight into cellular perturbations that accompany acquired gefitinib resistance in lung cancer cells. Several lung adenocarcinoma (LAD) cell lines were screened to characterize epidermal growth factor receptor (EGFR) expression and mutation profile. To circumvent intrinsic variations between cell lines with respect to response to drug treatments, we generated gefitinib-resistant H1650 clone by long-term, chronic culture under gefitinib selection of parental cell line. Isogenic cells were analyzed by microarray, Western blot, flow cytometry, and confocal and transmission electron microscope. We observed that although chronic gefitinib treatment provided effective action against its primary target (aberrant EGFR activity), secondary effects resulted in increased cellular reactive oxygen species (ROS). Gefitinib-mediated ROS correlated with epithelial-mesenchymal transition, as well as striking perturbation of mitochondrial morphology and function. However, gefitinib treatment in the presence of ROS scavenger provided a partial rescue of mitochondrial aberrations. Furthermore, withdrawal of gefitinib from previously resistant clones correlated with normalized expression of epithelial-mesenchymal transition genes. These findings demonstrate that chronic gefitinib treatment promotes ROS and mitochondrial dysfunction in lung cancer cells. Antioxidants may alleviate ROS-mediated resistance.     

Genetic Variants in Inflammation-Related Genes Are Associated with Radiation-Induced Toxicity Following Treatment for Non-Small Cell Lung Cancer

Treatment of non-small cell lung cancer (NSCLC) with radiotherapy or chemoradiotherapy is often accompanied by the development of esophagitis and pneumonitis. Identifying patients who might be at increased risk for normal tissue toxicity would help in determination of the optimal radiation dose to avoid these events. We profiled 59 single nucleotide polymorphisms (SNPs) from 37 inflammation-related genes in 173 NSCLC patients with stage IIIA/IIIB (dry) disease who were treated with definitive radiation or chemoradiation. For esophagitis risk, nine SNPs were associated with a 1.5- to 4-fold increase in risk, including three PTGS2 (COX2) variants: rs20417 (HR:1.93, 95% CI:1.10–3.39), rs5275 (HR:1.58, 95% CI:1.09–2.27), and rs689470 (HR:3.38, 95% CI:1.09–10.49). Significantly increased risk of pneumonitis was observed for patients with genetic variation in the proinflammatory genes IL1A, IL8, TNF, TNFRSF1B, and MIF. In contrast, NOS3:rs1799983 displayed a protective effect with a 45% reduction in pneumonitis risk (HR:0.55, 95% CI:0.31–0.96). Pneumonitis risk was also modulated by polymorphisms in anti-inflammatory genes, including genetic variation in IL13. rs20541 and rs180925 each resulted in increased risk (HR:2.95, 95% CI:1.14–7.63 and HR:3.23, 95% CI:1.03–10.18, respectively). The cumulative effect of these SNPs on risk was dose-dependent, as evidenced by a significantly increased risk of either toxicity with an increasing number of risk genotypes (P<0.001). These results suggest that genetic variations among inflammation pathway genes may modulate the development of radiation-induced toxicity and, ultimately, help in identifying patients who are at an increased likelihood for such events.     

Arctigenin Enhances Chemosensitivity to Cisplatin in Human Nonsmall Lung Cancer H460 Cells through Downregulation of Survivin Expression

Arctigenin, a dibenzylbutyrolactone lignan, enhances cisplatin‐mediated cell apoptosis in cancer cells. Here, we sought to investigate the effects of arctigenin on cisplatin‐treated non‐small‐cell lung cancer (NSCLC) H460 cells. The 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay and annexin‐V/propidium iodide staining were performed to analyze the proliferation and apoptosis of H460 cells. Arctigenin dose‐dependently suppressed cell proliferation and potentiated cell apoptosis, coupled with increased cleavage of caspase‐3 and poly(ADP‐ribose) polymerase. Moreover, arctigenin sensitized H460 cells to cisplatin‐induced proliferation inhibition and apoptosis. Arctigenin alone or in combination with cisplatin had a significantly lower amount of survivin. Ectopic expression of survivin decreased cell apoptosis induced by arctigenin (P < 0.05) or in combination with cisplatin (P < 0.01). Moreover, arctigenin (P < 0.05) or in combination with cisplatin (P < 0.01) induced G1/G0 cell‐cycle arrest. Our data provide evidence that arctigenin has a therapeutic potential in combina‐tion with chemotherapeutic agents for NSLC.     

Mitochondrial precursor proteins are imported through a hydrophilic membrane environment

We have analyzed how translocation intermediates of imported mitochondrial precursor proteins, which span contact sites, interact with the mitochondrial membranes. F1-ATPase subunit beta (F1 beta) was trapped at contact sites by importing it into Neurospora mitochondria in the presence of low levels of nucleoside triphosphates. This F1 beta translocation intermediate could be extracted from the membranes by treatment with protein denaturants such as alkaline pH or urea. By performing import at low temperatures, the ADP/ATP carrier was accumulated in contact sites of Neurospora mitochondria and cytochrome b2 in contact sites of yeast mitochondria. These translocation intermediates were also extractable from the membranes at alkaline pH. Thus, translocation of precursor proteins across mitochondrial membranes seems to occur through an environment which is accessible to aqueous perturbants. We propose that proteinaceous structures are essential components of a translocation apparatus present in contact sites.     

光敏剂HpD和单态氧探针FCLA的跨膜效率及其亚细胞定位研究

化学发光探针分子FCLA是一种海萤荧光素类似物分子,它可以选择性地与1O2及O2.反应产生化学发光,近年来已被成功用于在组织水平上进行光动力学和声动力学的肿瘤诊断中。但是FCLA在生物样品中能否进入细胞以及在细胞内的定位等问题目前尚不清楚。本文中报道利用激光共焦扫描显微镜进行FCLA和HpD的跨膜效率以及细胞内定位的形态学研究初步结果。结果表明,在37℃培养箱中用完全培养液进行培养时发现,HpD和FCLA都可以有效地跨膜,并定位在细胞质中。虽然FCLA与HpD的分子量大小相近,但是其进入肿瘤细胞的效率却并不相同。与HpD相比FCLA更容易进入细胞,对细胞没有明显的毒性。实验中未观测到FCLA和HpD进入细胞核的证据。本研究为利用1O2和O2.探针FCLA动态观测细胞内1O2或O2.的产生和定位建立了实验基础,并将推动在细胞或亚细胞水平上进行光动力学机制以及光敏过程引起细胞凋亡机制的研究。