Decreased expression of autophagy-related proteins in malignant epithelial ovarian cancer

Some studies have shown that the change of autophagic capacity may correlate with malignant transformation. Our study was designed to investigate the expression and significance of two autophagy-related proteins, microtubule-associated protein 1 light chain 3 (LC3) and Beclin 1 in the tumorigenesis and development of epithelial ovarian carcinoma. We observed that the positive expression of LC3 and Beclin 1 was significantly higher in the samples of benign and borderline ovarian tumors than those in malignant epithelial ovarian cancers. The expression of LC3 and Beclin 1 was associated with FIGO stage and histological grade. No significant relationship was observed between age and histological grade. However, we observed that the expression of LC3 was not related to Beclin 1. Therefore, the decrease of autophagic capacity may be related to tumorigenesis and development of epithelial ovarian cancer.

Addendum: Shen Y, Liang LZ, Hong MH, Xiong Y, Wei M, Zhu XF. Expression and clinical significance of microtubule-associated protein 1 light chain 3 (LC3) and Beclin1 in epithelial ovarian cancer. Ai Zheng 2008; 27:595-9.     

TRPM3表达上调通过调控Beclin1的表达促进卵巢癌细胞自噬

目的:探讨瞬时受体电位离子通道3(TRPM3)和Beclin1在卵巢癌中的表达和对卵巢癌细胞自噬的影响。方法:收集6例正常卵巢组织标本和20例卵巢癌组织标本,应用免疫组织化学染色法检测TRPM3和Beclin1在卵巢癌组织中的表达,采用western blotting法检测TRPM3和Beclin1在正常卵巢上皮细胞Moody和卵巢癌细胞Hey、ES-2中的表达差异。用TRPM3-siRNA瞬时转染细胞Hey和ES-2,通过western blotting法检测TRPM3基因沉默情况及Beclin1、p62和LC3的蛋白表达变化。结果:在正常卵巢组织和卵巢癌组织中,TRPM3的阳性表达率分别为33.3%、80.0%(P0.05),而Beclin1的阳性表达率分别为33.3%、65.0%(P0.05)。与正常卵巢上皮细胞Moody相比,卵巢癌细胞Hey和ES-2中TRPM3、Beclin1蛋白表达水平明显较高(P0.05)。沉默TRPM3基因表达的卵巢癌细胞中Beclin1和LC3蛋白表达与对照组相比明显降低,而p62表达升高(P0.05)。结论:卵巢癌组织和细胞中TRPM3蛋白呈高表达,可能通过调控Beclin1促进卵巢癌细胞的自噬。     

Decreased Expression of Beclin 1 Correlates Closely with Bcl-xL Expression and Poor Prognosis of Ovarian Carcinoma

Background

It has been suggested that autophagy-related Beclin 1 plays a critical role in the regulation of tumor development and/or progression, but its prognostic significance and relationship with Bcl-xL expression in ovarian carcinoma are unclear.

Methodology/Principal Findings

In the present study, the methods of Western blotting and immunohistochemistry (IHC) were utilized to investigate the expression status of Beclin 1 and Bcl-xL in fresh ovarian tissues and paraffin-embedded epithelial ovarian tumor tissues. Decreased expression of Beclin 1 was examined by IHC in 8.3% of normal ovaries, in 15.4% of cystadenomas, in 20.0% of borderline tumors, and in 55.6% of ovarian carcinomas, respectively. In ovarian carcinomas, decreased expression of Beclin 1 was correlated closely with ascending histological grade, later pT/pN/pM status and/or advanced clinical stage (P<0.05). In univariate survival analysis, a highly significant association between low-expressed Beclin 1 and shortened patient survival was evaluated in ovarian carcinoma patients (P<0.01), and Beclin 1 expression was an independent prognostic factor as evidenced by multivariate analysis (P = 0.013). In addition, decreased expression of Beclin 1 was inversely correlated with altered expression of Bcl-xL in ovarian carcinoma cohort, and combined analysis further showed that the low Beclin 1/high Bcl-xL group had the lowest survival rate.

Conclusions/Significance

Our findings suggest that Beclin 1 expression, as examined by IHC, could be served as an additional tool in identifying ovarian carcinoma patients at risk of tumor progression, and predicting patient survival in ovarian carcinomas with increased expression of Bcl-xL.     

p27kip1、Cyclin D1在卵巢癌中的表达及临床意义

目的探讨p27kip1、Cyclin D1在卵巢癌发生方面的意义.方法应用免疫组织化学方法及半定量分析方法,检测50例卵巢癌、19例卵巢良性上皮肿瘤、 13例正常卵巢组织中的p27kip1和Cyclin D1表达,并分析它们与良恶性肿瘤、病理学分级、临床分期的相关性. 结果正常卵巢和卵巢良性肿瘤间,p27和Cyclin D1的各自表达无明显差异;p27在正常卵巢组织和卵巢良性上皮肿瘤中高表达,在卵巢癌中表达降低(P<0.05),且随着肿瘤分级、分期增高(恶性程度增高),阳性表达率逐渐下降;而Cyclin D1的表达则相反;两者在肿瘤中的表达呈负相关.结论 p27kip表达下降、Cyclin D1过表达可能在卵巢癌的发生中起重要作用,检测p27kip1、Cyclin D1在卵巢癌中的表达可预测肿瘤生物学行为特征,可以作为判断预后的指标.     

FLIP、FADD在上皮性卵巢癌中表达及其临床意义

目的：研究FLIP、FADD在上皮性卵巢癌组织中的表达情况,探讨二者在上皮性卵巢癌发生发展中的作用及其临床意义。方法：采用免疫组化SP法及RT—PCR法检测44例上皮性卵巢癌及17例正常卵巢组织中FLIP、FADD的表达,并检测上皮性卵巢癌中PCNA的表达,分析FLIP,FADD表达与上皮性卵巢癌临床病理参数及PCNA表达的关系。结果：FLIP、FADD在上皮性卵巢癌组织中的阳性表达率,与正常卵巢组织相比,差异有统计学意义（P〈0．05）。FLIP,FADD表达与上皮性卵巢癌病理分级及临床分期有关,FLIP、FADD在I—II期中的阳性表达率,与III．IV期相比,差异有统计学意义（P〈0．05）。FLIP、FADD在病理组织学-1级中的阳性表达率,与2—3级相比,差异有统计学意义（P〈0．05）。FLIP、FADD的表达与患者年龄、组织学分型及淋巴结转移无关。上皮性卵巢癌组织中FLIP表达与PCNA表达成线性正相关,r分别0．880和0．564;FADD表达与PCNA表达成线性负相关,r分别为-0．591和-0．683。结论：FLIP、FADD与上皮性卵巢癌的发生发展密切相关,可能是治疗上皮性卵巢癌的潜在靶点。     

Autophagic proteins regulate cigarette smoke-induced apoptosis: protective role of heme oxygenase-1

Cigarette smoke-induced cell death contributes to the pathogenesis of chronic obstructive pulmonary disease, though the relative roles of apoptosis and autophagy remain unclear. The inducible stress protein heme oxygenase-1 (HO-1) confers cytoprotection against oxidative stress. We examined the relationships between these processes in human bronchial epithelial cells (Beas-2b) exposed to cigarette smoke extract (CSE). CSE induced morphological and biochemical markers of autophagy in Beas-2b cells and induced autophagosome formation as evidenced by formation of GFP-LC3 puncta and electron microscopic analysis. Furthermore, CSE increased the processing of microtubule-associated protein-1 light chain-3 (LC3B-I) to LC3B-II, within 1 hr of exposure. Increased LC3B-II was associated with increased autophagy, since inhibitors of lysosomal proteases and of autophagosome-lysosome fusion further increased LC3B-II levels during CSE exposure. CSE concurrently induced extrinsic apoptosis in Beas-2b cells involving early activation of death-inducing-signaling-complex (DISC) formation and downstream activation of caspases (-8,-9,-3). The induction of extrinsic apoptosis by CSE was dependent in part on autophagic proteins. Reduction of Beclin 1 levels with beclin 1 siRNA inhibited DISC formation and caspase-3/8 activation in response to CSE. LC3B siRNA also inhibited caspase-3/8 activation. The stress protein HO-1 protected against CSE-induced cell death by concurrently downregulating apoptosis and autophagy-related signaling. Adenoviral mediated expression of HO-1 inhibited DISC formation and caspase-3/9 activation in CSE-treated epithelial cells, diminished the expression of Beclin 1, and partially inhibited the processing of LC3B-I to LC3B-II. Conversely, transfection of Beas-2b with ho-1 siRNA augmented CSE-induced DISC formation and increased intracellular reactive oxygen species formation. HO-1 expression augmented CSE-induced phosphorylation of NFkappaB p65 in Beas-2b cells. Consistently, expression of IkappaB, the inhibitor of NFkappaB, increased CSE-induced DISC formation. LC3B siRNA also enhanced p65 phosphorylation. In fibroblasts from beclin 1 heterozygous knockout mice, p65 phosphorylation was dramatically upregulated, while CSE-induced DISC formation was inhibited, consistent with an anti-apoptotic role for NFkappaB and a pro-apoptotic role for Beclin 1. These studies demonstrated an interdependence of autophagic and apoptogenic signaling in CSE-induced cell death, and their coordinated downregulation by HO-1. An understanding of the regulation of cell death pathways during smoke exposure may provide therapeutic strategies in smoke-related illness.     

Acute Phase Expression Pattern of ZnTs,LC3, and Beclin-1 in Rat Hippocampus and Its Regulation by 3-Methyladenine Following Recurrent Neonatal Seizures

It has been reported that autophagy and zinc transporters (ZnTs) both play the key roles in excitotoxicity, which is associated with cognitive deficits following developmental seizures. However, the influence of autophagy on acute phase ZnTs expression has never been studied. The present study sought to investigate the contribution of an autophagy inhibitor (3-methyladenine, 3-MA) on the regulation of ZnTs, microtubule-associated protein 1A/1B light chain 3 (LC3), and beclin-1 expression in rat hippocampus following recurrent neonatal seizures. We examined the expression of ZnT1∼ZnT3, LC3, and beclin-1 at 1.5, 3, 6, and 24 h after the last seizures using real-time RT-PCR and Western blot methods, respectively. The results showed that there were upregulated expressions of ZnT-1, ZnT-2, LC3, and beclin-1 of RS group. Pretreatment with 3-MA remarkably attenuated seizure-induced ZnT-1, ZnT-2, LC3, and beclin-1 increase. Additionally, linear correlations could be observed between LC3–Beclin1, LC3–ZnT-2, Beclin1–ZnT2, Beclin1–ZnT3, and among ZnT1∼ZnT3 in control group, while the linear correlations could be observed between LC3–Beclin1, Beclin1–ZnT2, and Beclin1–ZnT3 in RS group. These results demonstrate, for the first time, that there exists an interaction of Zn²⁺ with autophagic signals that are immediately activated in hippocampus after recurrent neonatal seizures, which might play a key role in neonatal seizure-induced excitotoxicity.     

Localization of Beclin1 in mouse developing tooth germs: possible implication of the interrelation between autophagy and apoptosis

Our previous study identified the appearance of autophagy in developing tooth germs, and suggested its possible association with apoptosis in odontogenesis. Beclin1 was recently indicated to play a central role in bridging autophagy and apoptosis, and occupied a key position in the process of development. This study hypothesized that Beclin1 may be involved, and act as the molecular basis of the connection between autophagy and apoptosis in odontogenesis. Immunohistochemical analysis showed the spatiotemporal expression pattern of Beclin1 in odontogenesis from embryonic (E) day 13.5 to postnatal (P) day 5.5. At E stages, Beclin1 was mainly immunolocalized in the cytoplasm of the cells in the enamel organ. Meanwhile, the nucleus localization of Beclin1 was detected in part of the stellate reticulum, outer and inner enamel epithelium, especially at E16.5 and E18.5. At P stages, Beclin1 was detected in the cytoplasm of the odontoblasts, besides the dental epithelium cells. Triple immunofluorescence analysis showed the partial colocalization of Beclin1, autophagic marker LC3, or activated caspase-3 in the E14.5 tooth germs, especially the Beclin1⁺LC3⁺Caspase-3⁺ cells in the PEK. Furthermore, western blot analysis revealed that the full-length (60 kDa) and/or cleaved (50, 37, and 35 kDa) Beclin1 in the developing tooth germs. Taken together, our findings indicate that Beclin1 is involved, and might be responsible for the crosstalk between autophagy and apoptosis in mouse odontogenesis.     

Upregulation of Beclin 1 in the ischemic penumbra

Here we discuss the probable role of autophagy in cerebral ischemia based on our own recent data and the literature. We examined the protein level of Beclin 1 (Bcl-2 interacting protein) and microtubule-associated protein 1 light chain 3 (LC3) which were previously found to promote autophagy. We found a dramatic elevation in Beclin 1 levels and LC3 in the penumbra of rats challenged by cerebral ischemia. We found also that a subpopulation of Beclin 1-upregulating cells is also expressing the active form of caspase-3, and that all Beclin 1 upregulating cells display dense staining of LC3. Neuronal cells that overexpress Beclin 1 may exhibit damaged DNA but without changes in nuclear morphology, which indicates that not all the Beclin 1-upregulating cells are predestined to die. We conclude that the cell death in the penumbra bears a resemblance not only to necrosis, apoptosis, or a compromise between the two, but exhibits also biochemical and morphological characteristics of autophagic cell death. The question that constantly arises, however, is whether autophagic activity in damaged cells is the cause of death or is actually an attempt to prevent it as a part of an endogenous neuroprotective response.     

Phenethyl caffeate benzoxanthene lignan is a derivative of caffeic acid phenethyl ester that induces bystander autophagy in WiDr cells

We recently reported that Phenethyl caffeate benzoxanthene lignan (PCBL), a semisynthetic compound derived from Caffeic Acid Phenethyl Ester (CAPE), induces DNA damage and apoptosis in tumor cells. In this study, we further investigated whether PCBL induces autophagy in WiDr cells. We also analyzed the pathways regulating autophagy and the role of autophagy in PCBL-induced cell death. Our acridine orange staining and LC3 II expression results suggest that PCBL induces autophagosomes in WiDr cells. The levels of LC3 II expression we observed after co-treatment of PCBL with bafilomycin A1 and the reductions in p62 expression we observed after PCBL treatment in WiDr cells demonstrate increased autophagic flux, a reliable indicator of autophagic induction. The increased Beclin 1 expression in PCBL-treated cells and the incapacity of PCBL to induce LC3 II in 3-methyladenine (3-MA)-treated cells we observed suggests that PCBL-induced autophagy is class III PI3-kinase dependent. PCBL did not alter phosphorylation of the mTOR substrate p70 S6 kinase, indicating that PCBL-induced autophagy was not mTOR regulated. Two autophagy related proteins, Atg5 and Atg12, also remained uninduced during PCBL treatment. The increased caspase activity and expression levels of LC3 II and p62 we observed in response to PCBL treatment in primary glioma cells demonstrates that PCBL-induced apoptosis and autophagy were not cell line specific. Pharmacological inhibition of autophagy did not alter the antitumor efficacy of PCBL in WiDr cells. This attests to the bystander nature of PCBL-induced autophagy (in terms of cell death). In toto, these data suggest that PCBL induces a class III kinase dependent, but mTOR independent, bystander mode of autophagy in WiDr cells.     

Upregulation of Beclin 1 in the ischemic penumbra

Here we discuss the probable role of autophagy in cerebral ischemia based on our own recent data and the literature. We examined the protein level of Beclin 1 (Bcl-2 interacting protein) and microtubule-associated protein 1 light chain 3 (LC3) which were previously found to promote autophagy. We found a dramatic elevation in Beclin 1 levels and LC3 in the penumbra of rats challenged by cerebral ischemia. We found also that a subpopulation of Beclin 1-upregulating cells is also expressing the active form of caspase-3, and that all Beclin 1 upregulating cells display dense staining of LC3. Neuronal cells that overexpress Beclin 1 may exhibit damaged DNA but without changes in nuclear morphology, which indicates that not all the Beclin 1-upregulating cells are predestined to die. We conclude that the cell death in the penumbra bears a resemblance not only to necrosis, apoptosis, or a compromise between the two, but exhibits also biochemical and morphological characteristics of autophagic cell death. The question that constantly arises, however, is whether autophagic activity in damaged cells is the cause of death or is actually an attempt to prevent it as a part of an endogenous neuroprotective response.

Addendum to: Rami A et al. Focal cerebral ischemia induces upregulation of Beclin 1 and autophagy-like cell death. Neurobiol Dis 2007; In press.     

Beclin 1 knockdown inhibits autophagic activation and prevents the secondary neurodegenerative damage in the ipsilateral thalamus following focal cerebral infarction

Cerebral infarction can cause secondary degeneration of thalamus and delay functional recovery. However, the mechanisms underlying secondary degeneration are unclear. The present study aimed to determine the occurrence and contribution of autophagy to the thalamic degeneration after cerebral infarction. Focal cerebral infarction was induced by distal middle cerebral artery occlusion (MCAO). Autophagic activation, Beclin 1 expression and amyloid β (Aβ) deposits were determined by immunofluorescence, immunoblot and electron microscopy. Secondary damage to thalamus was assessed with Nissl staining and immunofluorescence analysis. Apoptosis was determined using TUNEL staining. The contribution of autophagy to the secondary damage was evaluated by shRNA-mediated downregulation of Beclin 1 and the autophagic inhibitor, 3-methyladenine (3-MA). The potential role of Aβ in autophagic activation was determined with N-[N-(3, 5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). The results showed that the conversion of LC3-II, the formation of autophagosomes, and the levels of activated cathepsin B and Beclin 1 were significantly increased in the ipsilateral thalamus at 7 and 14 days after MCAO (p < 0.05 or 0.01). Both Beclin 1 knockdown and 3-MA treatment significantly reduced LC3-II conversion and autophagosome formation, which were accompanied by obvious decreases in neuronal loss, gliosis and apoptosis in the ipsilateral thalamus (p < 0.05 or 0.01). Additionally, DAPT treatment markedly reduced Aβ deposits, which coincided with decreases in LC3-II conversion and autophagosome formation (p < 0.01). These results suggest that inhibition of autophagy by Beclin 1 knockdown can attenuate the secondary thalamic damage after focal cerebral infarction. Furthermore, Aβ deposits may be involved in the activation of autophagy.     

POSTN promotes diabetic vascular calcification by interfering with autophagic flux

Autophagy is a lysosomal degradative process that is closely related to the pathogenesis of vascular calcification. Recent evidence suggests that periostin (POSTN) is a unique extracellular matrix protein that is associated with diabetic vascular complications. The aim of current study is to investigate the role of POSTN in diabetic vascular calcification and the underlying mechanisms. Results showed that POSTN was highly upregulated in both calcified arteries of diabetic rats and AGEs-BSA mediated vascular smooth muscle cell (VSMC) calcification. POSTN blocked autophagic flux during the diabetic calcification process, as evidenced by increased protein expression of Beclin1, LC3-II, and P62, as well as the co-localization of LC3-II and LAMP1. Inhibition of POSTN alleviated AGEs-BSA-induced autophagic flux blockade, thereby attenuating AGEs-BSA-induced VSMC calcification. Mechanistically, the upregulation of POSTN impaired the fusion of autophagosomes and lysosome and resulted in the autophagic flux blockade in AGEs-BSA-treated VSMC. Furthermore, this autophagic blockade was intracellular ROS-dependent. In summary, this study uncovered a novel mechanism of POSTN in autophagy regulation of diabetic vascular calcification.     

Prognostic significance of Beclin 1-dependent apoptotic activity in hepatocellular carcinoma

Autophagy is believed to be important in tumorigenesis and tumor progression. However, the role of autophagy in hepatocellular carcinoma (HCC), and especially the prognostic value of autophagic proteins, has not been investigated. Our studies described here show decreased basal expression of autophagic genes and their corresponding autophagic activity under conditions of starvation in HCC cell lines, and the autophagy defect correlated well with the highly malignant phenotype of HCC. In addition, in a tissue microarray study of HCC patients who underwent resection, the expression of the autophagy-related protein Beclin 1 was extremely low in tumors, where Beclin 1 could predict the prognosis of HCC patients only in a Bcl-X_L-positive expression background. Based on our results, we propose that autophagy defects that synergize with altered apoptotic activity might facilitate tumor progression and poor prognosis of HCC, due to the fact that autophagy may interact with apoptosis in the regulation of HCC.     

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.     

FTY720 induces necrotic cell death and autophagy in ovarian cancer cells: a protective role of autophagy

FTY720, a sphingosine analog, is a novel immunosuppressant currently undergoing multiple clinical trials for the prevention of organ transplant rejection and treatment of various autoimmune diseases. Recent studies indicate an additional cytotoxic effect of FTY720 and its preclinical efficacy in a variety of cancer models, yet the underlying mechanisms remain unclear. We demonstrate here for the first time that FTY720 exhibits a potent, dose- and time-dependent cytotoxic effect in human ovarian cancer cells, even in the cells that are resistant to cisplatin, a commonly prescribed chemotherapeutic drug for treatment of ovarian cancer. In contrast to the previously reported cytotoxicity of FTY720 in many other cancer cell types, FTY720 kills ovarian cancer cells independent of caspase 3 activity and induces cellular swelling and cytoplasmic vacuolization with evident features of necrotic cell death. Furthermore, the presence of autophagic hallmarks, including an increased number of autophagosomes and the formation and accumulation of LC3-II, are observed in FTY720-treated cells before cell death. FTY720 treatment enhances autophagic flux as reflected in the increased LC3 turnover and p62 degradation. Notably, blockade of autophagy by either specific chemical inhibitors or siRNAs targeting Beclin 1 or LC3 resulted in aggravated necrotic cell death in response to FTY720, suggesting that FTY720-induced autophagy plays a self-protective role against its own cytotoxic effect. Thus, our findings not only demonstrate a new death pathway underlying the cytotoxic effect of FTY720, but also suggest that targeting autophagy could augment the anticancer potency, providing the framework for further development of FTY720 as a new chemotherapeutic agent for ovarian cancer treatment.     

FTY720 induces necrotic cell death and autophagy in ovarian cancer cells: A protective role of autophagy

FTY720, a sphingosine analog, is a novel immunosuppressant currently undergoing multiple clinical trials for the prevention of organ transplant rejection and treatment of various autoimmune diseases. Recent studies indicate an additional cytotoxic effect of FTY720 and its preclinical efficacy in a variety of cancer models, yet the underlying mechanisms remain unclear. We demonstrate here for the first time that FTY720 exhibits a potent, dose- and time-dependent cytotoxic effect in human ovarian cancer cells, even in the cells that are resistant to cisplatin, a commonly prescribed chemotherapeutic drug for treatment of ovarian cancer. In contrast to the previously reported cytotoxicity of FTY720 in many other cancer cell types, FTY720 kills ovarian cancer cells independent of caspase 3 activity and induces cellular swelling and cytoplasmic vacuolization with evident features of necrotic cell death. Furthermore, the presence of autophagic hallmarks, including an increased number of autophagosomes and the formation and accumulation of LC3-II, are observed in FTY720-treated cells before cell death. FTY720 treatment enhances autophagic flux as reflected in the increased LC3 turnover and p62 degradation. Notably, blockade of autophagy by either specific chemical inhibitors or siRNAs targeting Beclin 1 or LC3 resulted in aggravated necrotic cell death in response to FTY720, suggesting that FTY720-induced autophagy plays a self-protective role against its own cytotoxic effect. Thus, our findings not only demonstrate a new death pathway underlying the cytotoxic effect of FTY720, but also suggest that targeting autophagy could augment the anticancer potency, providing the framework for further development of FTY720 as a new chemotherapeutic agent for ovarian cancer treatment.     

Ambra1 modulates starvation-induced autophagy through AMPK signaling pathway in cardiomyocytes

Recent research has revealed a role for Ambra1, an autophagy-related gene-related (ATG) protein, in the autophagic pro-survival response, and Ambra1 has been shown to regulate Beclin1 and Beclin1-dependent autophagy in embryonic stem cells and cancer cells. However, whether Ambra1 plays an important role in the autophagy pathway in cardiomyocytes is unknown. In this study, we hypothesized that Ambra1 is an important regulator of autophagy and apoptosis in cardiomyocytes. To test this hypothesis, we confirmed autophagic activity in serum-starved cardiomyocytes by assessing endogenous microtubule-associated protein 1 light chain 3 (LC3) localization, the presence of autophagosomes and LC3 protein levels. Cell apoptosis and viability were measured by annexin-V and PI staining and MTT assays. We determined that serum deprivation-induced autophagy was associated with Ambra1 upregulation in cardiomyocytes. When Ambra1 expression was reduced by siRNA, the cardiomyocytes were more sensitive to staurosporine-induced apoptosis. In addition, co-immunoprecipitation of Ambra1 and Beclin1 demonstrated that Ambra1 and Beclin1 interact in serum-starved or rapamycin-treated cardiomyocytes, suggesting that Ambra1 regulates autophagy in cardiomyocytes by interacting with Beclin1. Finally, we determined that starvation stress-induced activation of Ambra1 contributes to the attenuation of adaptive AMP-activated protein kinase (AMPK) signaling. In conclusion, Ambra1 is a crucial regulator of autophagy and apoptosis through AMPK signaling pathway in cardiomyocytes that maintains the balance between autophagy and apoptosis.     

大负荷运动诱导大鼠骨骼肌损伤对其自噬超微结构及Beclin1和LC3-II/I的影响*

目的: 观察大负荷离心运动对大鼠骨骼肌自噬超微结构及自噬相关蛋白Beclin1和LC3II/I的影响。方法: 48只SD雄性大鼠适应性训练后随机分成对照组(C,n=8)和大负荷离心运动组(E,n=40)。E组于跑台进行90 min下坡跑,运动后0 h、12 h、24 h、48 h和72 h取比目鱼肌,透射电镜观察其自噬体超微结构变化;Western blot检测Beclin1和LC3II/I蛋白表达;免疫荧光观测LC3的定位及含量变化。结果: E组比目鱼肌自噬体数量在运动后0 h、12 h和24 h均有增加,并伴LC3自噬荧光明显增强(P＜0.01),同时运动后48 h自噬荧光仍有显著性升高(P＜0.05);Beclin1和LC3II/I在大负荷离心干预后表达升高(P＜0.05),运动后12 h～24 h达到峰值(P＜0.01),直至运动后72 h完全恢复。结论: 大负荷离心运动可诱导骨骼肌自噬超微结构变化,自噬蛋白表达增强,以上可能是运动损伤的骨骼肌功能下降的原因之一。     

CCR9和CCL25蛋白在上皮性卵巢癌中表达及临床意义

目的:探讨CCR9和CCL25蛋白在不同卵巢组织中的表达及其与上皮性卵巢癌患者临床病理因素之间的关系。方法:通过组织芯片结合免疫组织化学法检测78例上皮性卵巢癌组织和30例正常卵巢组织中CCR9和CCL25表达水平,结合上皮性卵巢癌病人的临床病理资料,进行统计分析。结果:CCR9和CCL25在上皮性卵巢癌中高表达,在正常卵巢组织中低表达,二者的表达与上皮性卵巢癌的组织类型、患者年龄无显著相关(P0.05),而与淋巴结转移、组织学分级和临床分期有显著相关(P0.05);上皮性卵巢癌组织中CCR9与CCL25表达相关(P0.05)。结论:CCR9和CCL25在上皮性卵巢癌的发生发展中可能起重要作用,二者可能是上皮性卵巢癌治疗的一个潜在的分子靶点。