纳米氧化铈负载槲皮素引起人肝癌细胞自噬阻断

稀土氧化物纳米材料的生物安全性越来越受到关注,这类纳米材料引起的自噬反应对于癌细胞杀伤也具有重要意义。自噬在细胞存活和死亡中发挥双重作用,槲皮素可以促进自噬,稀土氧化物已被证明可引起不同类型的自噬。制备了葡聚糖包被的氧化铈纳米颗粒负载的槲皮素复合材料DCQ,并对其自身性质进行表征,从细胞活力及氧化损伤以及自噬、凋亡机制这几个方面研究了其对人肝癌细胞HepG2的作用。结果表明,此复合材料对HepG2细胞具有更强的毒性(P<0.05),并且对正常细胞人脐静脉血管内皮细胞HUVEC无明显毒害作用,复合材料能够诱发人肝癌细胞产生大量活性氧,引起自噬阻断和诱导癌细胞凋亡。上述结果说明,这种纳米复合材料能有效杀伤人肝癌细胞,为肝癌治疗提供了新思路。     

Nano neodymium oxide induces massive vacuolization and autophagic cell death in non-small cell lung cancer NCI-H460 cells

Neodymium, a rare earth element, was known to exhibit cytotoxic effects and induce apoptosis in certain cancer cells. Here we show that nano-sized neodymium oxide (Nano Nd2O3) induced massive vacuolization and cell death in non-small cell lung cancer NCI-H460 cells at micromolar equivalent concentration range. Cell death elicited by Nano Nd2O3 was not due to apoptosis and caspases were not involved. Electron microscopy and acridine orange staining revealed extensive autophagy in the cytoplasm of the cells treated by Nano Nd2O3. Autophagy induced by Nano Nd2O3 was accompanied by S-phase cell cycle arrest, mild disruption of mitochondrial membrane potential, and inhibition of proteasome activity. Bafilomycin A1, but not 3-MA, induced apoptosis while inhibiting autophagy. Our results revealed a novel biological function for Nano Nd2O3 and may have implications for the therapy of non-small cell lung cancer.     

Autophagy and tumorigenesis

Autophagy, or cellular self-digestion, is activated in cancer cells in response to multiple stresses and has been demonstrated to promote tumor cell survival and drug resistance. Nonetheless, genetic evidence supports that autophagy functions as a tumor suppressor mechanism. Hence, the precise role of autophagy during cancer progression and treatment is both tissue and context dependent. Here, we discuss our current understanding of the biological functions of autophagy during cancer development, overview how autophagy is regulated by cancer-associated signaling pathways, and review how autophagy inhibition is being exploited to improve clinical outcomes.     

Beclin 1 cleavage by caspase-3 inactivates autophagy and promotes apoptosis

Autophagy and apoptosis are both highly regulated biological processes that play essential roles in tissue homeostasis, development and diseases. Autophagy is also described as a mechanism of death pathways, however, the precise mechanism of how autophagy links to cell death remains to be fully understood. Beclin 1 is a dual regulator for both autophagy and apoptosis. In this study we found that Beclin 1 was a substrate of caspase-3 with two cleavage sites at positions 124 and 149, respectively. Furthermore, the autophagosome formation occurred, followed by the appearance of morphological hallmarks of apoptosis after staurosporine treatment. The cleavage products of Beclin 1 reduced autophagy and promoted apoptosis in HeLa cells and the cells in which Beclin 1 was stably knocked down by specific shRNA. In addition, the cleavage of Beclin 1 resulted in abrogating the interaction between Bcl-2 with Beclin 1, which could be blocked by z-VAD-fmk. Thus, our results suggest that the cleavage of Beclin 1 by caspase-3 may contribute to inactivate autophagy leading towards augmented apoptosis.     

Roles of autophagy in cetuximab-mediated cancer therapy against EGFR

Cetuximab is an epidermal growth factor receptor (EGFR)-blocking antibody that is approved to treat several types of solid cancers in patients. We recently showed that cetuximab can induce autophagy in cancer cells by both inhibiting the class I phosphatidylinositol 3-kinase (PtdIns3K)/Akt/mammalian target of rapamycin (mTOR) pathway and activating the class III PtdIns3K (hVps34)/beclin 1 pathway. In the current study, we investigated the relationship between cetuximab-induced autophagy and apoptosis and the biological roles of autophagy in cetuximab-mediated cancer therapy. We found that cetuximab induced autophagy in cancer cells that show strong or weak induction of apoptosis after cetuximab treatment but not in those that show only cytostatic growth inhibition. Inhibition of cetuximab-induced apoptosis by a caspase inhibitor prevented the induction of autophagy. Conversely, inhibition of cetuximab-induced autophagy by silencing the expression of autophagy-related genes (Atg) or treating the cancer cells with lysosomal inhibitors enhanced the cetuximab-induced apoptosis, suggesting that autophagy was a protective cellular response to cetuximab treatment. On the other hand, cotreatment of cancer cells with cetuximab and the mTOR inhibitor rapamycin resulted in an Atg-dependent and lysosomal inhibition-sensitive death of cancer cells that show only growth inhibition or weak apoptosis after cetuximab treatment, indicating that cell death may be achieved by activating the autophagy pathway in these cells. Together, our findings may guide the development of novel clinical strategies for sensitizing cancer cells to EGFR-targeted therapy.Key words: EGFR, cetuximab, autophagy, apoptosis, cancer therapy     

Roles of autophagy in cetuximab-mediated cancer therapy against EGFR

Cetuximab is an epidermal growth factor receptor (EGFR)-blocking antibody that is approved to treat several types of solid cancers in patients. We recently showed that cetuximab can induce autophagy in cancer cells by both inhibiting the class I phosphatidylinositol 3-kinase (PtdIns3K)/Akt/mammalian target of rapamycin (mTOR) pathway and activating the class III PtdIns3K (hVps34)/beclin 1 pathway. In the current study, we investigated the relationship between cetuximab-induced autophagy and apoptosis and the biological roles of autophagy in cetuximab-mediated cancer therapy. We found that cetuximab induced autophagy in cancer cells that show strong or weak induction of apoptosis after cetuximab treatment but not in those that show only cytostatic growth inhibition. Inhibition of cetuximab-induced apoptosis by a caspase inhibitor prevented the induction of autophagy. Conversely, inhibition of cetuximab-induced autophagy by silencing the expression of autophagy-related genes (Atg) or treating the cancer cells with lysosomal inhibitors enhanced the cetuximab-induced apoptosis, suggesting that autophagy was a protective cellular response to cetuximab treatment. On the other hand, cotreatment of cancer cells with cetuximab and the mTOR inhibitor rapamycin resulted in an Atg-dependent and lysosomal inhibition-sensitive death of cancer cells that show only growth inhibition or weak apoptosis after cetuximab treatment, indicating that cell death may be achieved by activating the autophagy pathway in these cells. Together, our findings may guide the development of novel clinical strategies for sensitizing cancer cells to EGFR-targeted therapy.     

Autophagy-dependent EIF2AK3 activation compromises ursolic acid-induced apoptosis through upregulation of MCL1 in MCF-7 human breast cancer cells

Ursolic acid (UA) is a pentacyclic triterpenoid with promising cancer chemopreventive properties. A better understanding of the mechanisms underlying anticancer activity of UA is needed for further development as a clinically useful chemopreventive agent. Here, we found that both endoplasmic reticulum (ER) stress and autophagy were induced by UA in MCF-7 human breast cancer cells. Surprisingly, ER stress was identified as an effect rather than a cause of UA-induced autophagy. Autophagy-dependent ER stress protected the cells from UA-induced apoptosis through EIF2AK3-mediated upregulation of MCL1. Activation of MAPK1/3 but not inhibition of MTOR pathway contributed to UA-induced cytoprotective autophagy in MCF-7 cells. Our findings uncovered a novel cellular mechanism involved in the anticancer activity of UA, and also provided a useful model to study biological significance and mechanisms of autophagy-mediated ER stress.     

自噬与肿瘤

自噬（autophagy）作为细胞一种基本生物学特征,具有独特的形态学改变和特有的调控通路。近年来,自噬在关于对肿瘤的作用的研究已成为热点,在不同的种类肿瘤中,自噬扮演着不同的角色,分为促进和抑制肿瘤两种作用。自噬异常与人类恶性肿瘤的发生、发展联系紧密,其启动及调节与细胞能量代谢、微环境变化、抑癌基因及癌基因家族及复杂的信号调节等有关。清楚了解自噬的特点可为肿瘤治疗提供新的方向。     

Autophagy participants in the dedifferentiation of mouse 3T3-L1 adipocytes triggered by hypofunction of insulin signaling

Our previous data indicate that both insulin and IGF-1 signallings dysfunction promotes the dedifferentiation of primary human and mouse white adipocytes. Based on the fact that insulin activates mTOR and inhibits autophagy, and autophagy deficiency can inhibit the differentiation of white adipocytes, we speculate that autophagy may be related to the dedifferentiation of white adipocytes. We investigated the underlying mechanism of autophagy during dedifferentiation of mouse 3T3-L1 adipocytes. After incomplete inhibition of insulin and IGF-1 signallings, 3T3-L1 adipocytes manifest dedifferentiation accompanied with an increase of autophagy level. If induction only of autophagy in the adipocytes, then the cells also occur somewhat dedifferentiation, and with a slight decrease of insulin signal, while its degree was weaker than insulin signal inhibited cells. Notably, after inhibition of the insulin and IGF-1 signallings and simultaneously inducing autophagy, the dedifferentiation of 3T3-L1 adipocytes was the most obvious compared with other groups, and the insulin and IGF-1 signallings decreases was greater than the cells with inhibition only of insulin signalling. If inhibition of both insulin signal and autophagy simultaneously, the dedifferentiation of the adipocytes reveals similar tendencies to the cells that insulin signal was inhibited. No significant dedifferentiation occurs of 3T3-L1 cells if only inhibition of autophagy. Taken all together, in this study, we proved that autophagy is positively related to the dedifferentiation of 3T3-L1 adipocytes and is regulated through the insulin-PI3K-AKT-mTOCR1-autophagy pathway. Autophagy may also has a certain degree of negative feedback affect on the insulin signalling of 3T3-L1 cells. Our work may help to better understand the biological properties of mature adipocytes and may help formulate anti-obesity strategies by regulating insulin and insulin signaling level.     

Role for nanomaterial-autophagy interaction in neurodegenerative disease

Nanotechnology is the control and manipulation of materials in the size range of 1-100 nm. Due to increasing research into the potential beneficial applications of nanotechnology, there is an urgent need for the study of possible health risks. Several researchers, including those in our laboratory, have demonstrated elevated levels of autophagic vacuoles upon exposure of cells to certain nanomaterials, including carbon- and metal-based nanoparticles. While this apparent increase in autophagic activity may be an appropriate cellular response toward nanomaterial clearance, often the interaction between nanomaterials and the autophagy pathway is disruptive, resulting in severe morphological changes and coincident cell death. Interestingly, epidemiological studies have identified an association between exposure to combustion-derived ambient particles (which are predominantly nanoscale) and neurological conditions with Alzheimer's and Parkinson's disease-like pathologies. Becuse impaired autophagy may play an important role in the pathogenesis of these and other diseases, it is intriguing to speculate about the plausible involvement of nanoscale particulates in this process. The interaction of nanomaterials with the autophagy pathway, and the potential negative consequences of resulting autophagy dysfunction, should be explored further.     

力学刺激诱导细胞自噬的研究进展

自噬是细胞重要的自我保护机制,多种伤害性刺激激活的自噬具有维持细胞稳态和正常功能的作用.此外,自噬还参与调控恶性肿瘤、动脉粥样硬化等多种疾病的发生发展过程.体内细胞处于复杂的力学微环境中,力学刺激参与调控细胞自噬,如压力可诱导心肌细胞的自噬、牵张力调控运动系统多种细胞的自噬、流体剪切力可激活血管内皮细胞和肿瘤细胞的自噬.力学刺激诱导的细胞自噬依赖众多信号通路.细胞骨架作为重要的调节因子,不仅参与细胞力学信号转导,同时可参与调控细胞自噬.因此,细胞骨架与力学刺激诱导的细胞自噬密切相关.本文结合最新的研究成果,综述力学刺激对细胞自噬的影响及其分子机制,以期为研究力学刺激对细胞生物学行为的影响提供新的视角,进而为相关疾病的治疗提供新思路和分子靶点.     

Role for nanomaterial-autophagy interaction in neurodegenerative disease

Nanotechnology is the control and manipulation of materials in the size range of 1-100 nm. Coupled with increasing research into potential beneficial applications of nanotechnology, there is an urgent need for the study of possible health risks. Several researchers, including those in our laboratory, have demonstrated elevated levels of autophagic vacuoles upon exposure of cells to certain nanomaterials, including carbon- and metal-based nanoparticles. While this apparent increase in autophagic activity may be an appropriate cellular response toward nanomaterial clearance, often the interaction between nanomaterials and the autophagy pathway is disruptive, resulting in severe morphological changes and coincident cell death. Interestingly, epidemiological studies have identified an association between exposure to combustion-derived ambient particles (which are predominantly nanoscale) and neurological conditions with Alzheimer�s and Parkinson�s disease-like pathologies. As impaired autophagy may play an important role in the pathogenesis of these and other diseases, it is intriguing to speculate about the plausible involvement of nanoscale particulates in this process. The interaction of nanomaterials with the autophagy pathway, and the potential negative consequences of resulting autophagy dysfunction, should be explored further.

Addendum to: Stern ST, Zolnik BS, McLeland CB, Clogston J, Zheng J, McNeil SE. Induction of autophagy in porcine kidney cells by quantum dots: A common cellular response to nanomaterials? Toxicol Sci 2008; In press; DOI: 10.1093/toxsci/kfn137.     

Autophagy in Yeast: Mechanistic Insights and Physiological Function

Unicellular eukaryotic organisms must be capable of rapid adaptation to changing environments. While such changes do not normally occur in the tissues of multicellular organisms, developmental and pathological changes in the environment of cells often require adaptation mechanisms not dissimilar from those found in simpler cells. Autophagy is a catabolic membrane-trafficking phenomenon that occurs in response to dramatic changes in the nutrients available to yeast cells, for example during starvation or after challenge with rapamycin, a macrolide antibiotic whose effects mimic starvation. Autophagy also occurs in animal cells that are serum starved or challenged with specific hormonal stimuli. In macroautophagy, the form of autophagy commonly observed, cytoplasmic material is sequestered in double-membrane vesicles called autophagosomes and is then delivered to a lytic compartment such as the yeast vacuole or mammalian lysosome. In this fashion, autophagy allows the degradation and recycling of a wide spectrum of biological macromolecules. While autophagy is induced only under specific conditions, salient mechanistic aspects of autophagy are functional in a constitutive fashion. In Saccharomyces cerevisiae, induction of autophagy subverts a constitutive membrane-trafficking mechanism called the cytoplasm-to-vacuole targeting pathway from a specific mode, in which it carries the resident vacuolar hydrolase, aminopeptidase I, to a nonspecific bulk mode in which significant amounts of cytoplasmic material are also sequestered and recycled in the vacuole. The general aim of this review is to focus on insights gained into the mechanism of autophagy in yeast and also to review our understanding of the physiological significance of autophagy in both yeast and higher organisms.     

AMP-activated protein kinase and autophagy

Autophagy is inhibited by TOR-dependent signaling. Interruption of signalling by rapamycin is known to stimulate autophagy, both in mammalian cells and in yeast. However, inactivation of TOR by AMPK has yielded controversial results in the literature with regard to its effect on autophagy: activation of autophagy in yeast but inhibition in hepatocytes. In a recent study, carried out with hepatocytes, HT-29 cells, and HeLa cells, the possible role of AMPK in the control of mammalian autophagy was reexamined. The data suggest that in mammalian cells, as in yeast, AMPK is required for autophagy.     

Autophagy in yeast: mechanistic insights and physiological function.

Unicellular eukaryotic organisms must be capable of rapid adaptation to changing environments. While such changes do not normally occur in the tissues of multicellular organisms, developmental and pathological changes in the environment of cells often require adaptation mechanisms not dissimilar from those found in simpler cells. Autophagy is a catabolic membrane-trafficking phenomenon that occurs in response to dramatic changes in the nutrients available to yeast cells, for example during starvation or after challenge with rapamycin, a macrolide antibiotic whose effects mimic starvation. Autophagy also occurs in animal cells that are serum starved or challenged with specific hormonal stimuli. In macroautophagy, the form of autophagy commonly observed, cytoplasmic material is sequestered in double-membrane vesicles called autophagosomes and is then delivered to a lytic compartment such as the yeast vacuole or mammalian lysosome. In this fashion, autophagy allows the degradation and recycling of a wide spectrum of biological macromolecules. While autophagy is induced only under specific conditions, salient mechanistic aspects of autophagy are functional in a constitutive fashion. In Saccharomyces cerevisiae, induction of autophagy subverts a constitutive membrane-trafficking mechanism called the cytoplasm-to-vacuole targeting pathway from a specific mode, in which it carries the resident vacuolar hydrolase, aminopeptidase I, to a nonspecific bulk mode in which significant amounts of cytoplasmic material are also sequestered and recycled in the vacuole. The general aim of this review is to focus on insights gained into the mechanism of autophagy in yeast and also to review our understanding of the physiological significance of autophagy in both yeast and higher organisms.     

The pancreatitis-induced vacuole membrane protein 1 triggers autophagy in mammalian cells

Autophagy is a degradation process of cytoplasmic cellular constituents, which serves as a survival mechanism in starving cells, and it is characterized by sequestration of bulk cytoplasm and organelles in double-membrane vesicles called autophagosomes. Autophagy has been linked to a variety of pathological processes such as neurodegenerative diseases and tumorigenesis, which highlights its biological and medical importance. We have previously characterized the vacuole membrane protein 1 (VMP1) gene, which is highly activated in acute pancreatitis, a disease associated with morphological changes resembling autophagy. Here we show that VMP1 expression triggers autophagy in mammalian cells. VMP1 expression induces the formation of ultrastructural features of autophagy and recruitment of the microtubule-associated protein 1 light-chain 3 (LC3), which is inhibited after treatment with the autophagy inhibitor 3-methiladenine. VMP1 is induced by starvation and rapamycin treatments. Its expression is necessary for autophagy, because VMP1 small interfering RNA inhibits autophagosome formation under both autophagic stimuli. VMP1 is a transmembrane protein that co-localizes with LC3, a marker of the autophagosomes. It interacts with Beclin 1, a mammalian autophagy initiator, through the VMP1-Atg domain, which is essential for autophagosome formation. VMP1 endogenous expression co-localizes with LC3 in pancreas tissue undergoing pancreatitis-induced autophagy. Finally, VMP1 stable expression targeted to pancreas acinar cell in transgenic mice induces autophagosome formation. Our results identify VMP1 as a novel autophagy-related membrane protein involved in the initial steps of the mammalian cell autophagic process.     

AMP-activated protein kinase and the regulation of autophagic proteolysis

Interruption of mTOR-dependent signaling by rapamycin is known to stimulate autophagy, both in mammalian cells and in yeast. Because activation of AMPK also inhibits mTOR-dependent signaling one would expect stimulation of autophagy by AMPK activation. According to the literature, this is true for yeast but, unexpectedly, not for mammalian cells on the basis of the use of AICAR, a pharmacological activator of AMPK. In the present study, carried out with hepatocytes, HT-29 cells, and HeLa cells, we have reexamined the possible role of AMPK in the control of mammalian autophagy. Inhibition of AMPK activity by compound C or by transfection with a dominant negative form of AMPK almost completely inhibited autophagy. These results suggest that the inhibition of autophagy by AICAR is not related to its ability to activate AMPK. We conclude that in mammalian cells, as in yeast, AMPK is required for autophagy.     

Macroautophagy-aided elimination of chromatin

How tumor cells process damaged or unwanted DNA is a matter of much interest. Recently, Rello-Varona et al. (Cell Cycle 2012; 11:170–76) reported the involvement of macroautophagy (hereon autophagy) in the elimination of micronuclei (MN) from osteosarcoma cells. Prior to that, diminution of whole nuclei from multinucleated TP53-mutant tumor cells was described. Here, we discuss these two kinds of chromatin autophagy evoked after genotoxic stress in the context of the various biological processes involved: (1) endopolyploidy and the ploidy cycle; (2) the timing of DNA synthesis; (3) DNA repair; (4) chromatin:nuclear envelope interactions; and (5) cytoplasmic autophagy. We suggest that whereas some MN can be reunited with the main nucleus (through interactions with envelope-limited chromatin sheets) and participate in DNA repair, failure of repair serves as a signal for the chromatin autophagy of MN. In turn, autophagy of whole sub-nuclei in multi-nucleated cells appears to favor de-polyploidization, mitigation of aneuploidy with its adverse effects, thereby promoting the survival fitness of descendents and treatment resistance. Thus, both kinds of chromatin autophagy provide tumor cells with the opportunity to repair DNA, sort and resort chromatin, reduce DNA content, and enhance survival.     

Autophagy-Related Proteins Are Required for Degradation of Peroxisomes in Arabidopsis Hypocotyls during Seedling Growth

Plant peroxisomes play a pivotal role during postgerminative growth by breaking down fatty acids to provide fixed carbons for seedlings before the onset of photosynthesis. The enzyme composition of peroxisomes changes during the transition of the seedling from a heterotrophic to an autotrophic state; however, the mechanisms for the degradation of obsolete peroxisomal proteins remain elusive. One candidate mechanism is autophagy, a bulk degradation pathway targeting cytoplasmic constituents to the lytic vacuole. We present evidence supporting the autophagy of peroxisomes in Arabidopsis thaliana hypocotyls during seedling growth. Mutants defective in autophagy appeared to accumulate excess peroxisomes in hypocotyl cells. When degradation in the vacuole was pharmacologically compromised, both autophagic bodies and peroxisomal markers were detected in the wild-type vacuole but not in that of the autophagy-incompetent mutants. On the basis of the genetic and cell biological data we obtained, we propose that autophagy is important for the maintenance of peroxisome number and cell remodeling in Arabidopsis hypocotyls.     

Antimony trichloride induces a loss of cell viability via reactive oxygen species-dependent autophagy in A549 cells

Antimony (Sb) is one of the most prevalent heavy metals and frequently leads to biological toxicity. Although autophagy is believed to be involved in metal-associated cytotoxicity, there is no evidence of its involvement following exposure. Moreover, the underlying mechanism of autophagy remains unclear. In this study, treatment with antimony trichloride caused autophagy in a dose- and time-dependent manner in A549 cells but did not affect the level of Atg5 or Atg7 mRNA expression. Furthermore, Sb enhanced autophagic flux while upregulating p62 gene and protein levels. The classic mechanistic target of rapamycin (mTOR) pathway is not involved in Sb-induced autophagy. However, Sb-induced autophagy and the upregulation of p62 were inhibited by treatment with the antioxidant N-acetylcysteine (NAC). Subsequent analyses demonstrated that the inhibition of autophagy protected A549 cells from a loss of cell viability, while the activation of autophagy by rapamycin had the opposite effect. These data suggest that reactive oxygen species-dependent autophagy mediates Sb-stimulated cell viability loss in A549 cells.