Inhibition of anlotinib-induced autophagy attenuates invasion and migration by regulating epithelial-mesenchymal transition and cytoskeletal rearrangement through ATG5 in human osteosarcoma cells

Abstract The cure rates for osteosarcoma have remained unchanged in the past three decades, especially for patients with pulmonary metastasis. Thus, a new and effective treatment for metastatic osteosarcoma is urgently needed. Anlotinib has been reported to have antitumor effects on advanced osteosarcoma. However, both the effect of anlotinib on autophagy in osteosarcoma and the mechanism of anlotinib-mediated autophagy in pulmonary metastasis are unclear. The effect of anlotinib treatment on the metastasis of osteosarcoma was investigated by transwell assays, wound healing assays, and animal experiments. Related proteins were detected by western blotting after anlotinib treatment, ATG5 silencing, or ATG5 overexpression. Immunofluorescence staining and transmission electron microscopy were used to detect alterations in autophagy and the cytoskeleton. Anlotinib inhibited the migration and invasion of osteosarcoma cells but promoted autophagy and increased ATG5 expression. Furthermore, the decreases in invasion and migration induced by anlotinib treatment were enhanced by ATG5 silencing. In addition, Y-27632 inhibited cytoskeletal rearrangement, which was rescued by ATG5 overexpression. ATG5 overexpression enhanced epithelial-mesenchymal transition (EMT). Mechanistically, anlotinib-induced autophagy promoted migration and invasion by activating EMT and cytoskeletal rearrangement through ATG5 both in vitro and in vivo. Our results demonstrated that anlotinib can induce protective autophagy in osteosarcoma cells and that inhibition of anlotinib-induced autophagy enhanced the inhibitory effects of anlotinib on osteosarcoma metastasis. Thus, the therapeutic effect of anlotinib treatment can be improved by combination treatment with autophagy inhibitors, which provides a new direction for the treatment of metastatic osteosarcoma.

Autophagy enhanced by curcumin ameliorates inflammation in atherogenesis via the TFEB-P300-BRD4 axis

Disturbance of macrophage-associated lipid metabolism plays a key role in atherosclerosis. Crosstalk between autophagy deficiency and inflammation response in foam cells (FCs) through epigenetic regulation is still poorly understood. Here, we demonstrate that in macrophages, oxidized low-density lipoprotein (ox-LDL) leads to abnormal crosstalk between autophagy and inflammation, thereby causing aberrant lipid metabolism mediated through a dysfunctional transcription factor EB (TFEB)-P300-bromodomain-containing protein 4 (BRD4) axis. ox-LDL led to macrophage autophagy deficiency along with TFEB cytoplasmic accumulation and increased reactive oxygen species generation. This activated P300 promoted BRD4 binding on the promoter regions of inflammatory genes, consequently contributing to inflammation with atherogenesis. Particularly, ox-LDL activated BRD4-dependent super-enhancer associated with liquid-liquid phase separation (LLPS) on the regulatory regions of inflammatory genes. Curcumin (Cur) prominently restored FCs autophagy by promoting TFEB nuclear translocation, optimizing lipid catabolism, and reducing inflammation. The consequences of P300 and BRD4 on super-enhancer formation and inflammatory response in FCs could be prevented by Cur. Furthermore, the anti-atherogenesis effect of Cur was inhibited by macrophage-specific Brd4 overexpression or Tfeb knock-out in Apoe knock-out mice via bone marrow transplantation. The findings identify a novel TFEB-P300-BRD4 axis and establish a new epigenetic paradigm by which Cur regulates autophagy, inhibits inflammation, and decreases lipid content.

Lonicerin targets EZH2 to alleviate ulcerative colitis by autophagy-mediated NLRP3 inflammasome inactivation

Aberrant activation of NLRP3 inflammasome in colonic macrophages strongly associates with the occurrence and progression of ulcerative colitis. Although targeting NLRP3 inflammasome has been considered to be a potential therapy, the underlying mechanism through which pathway the intestinal inflammation is modulated remains controversial. By focusing on the flavonoid lonicerin, one of the most abundant constituents existed in a long historical anti-inflammatory and anti-infectious herb

Chikusetsu saponin Ⅳa ameliorates myocardial hypertrophy of rats through regulating expression of miR199a-5p/Atg5 / 中国中药杂志

The present study investigated the effects of chikusetsu saponin Ⅳa(CHS Ⅳa) on isoproterenol(ISO)-induced myocardial hypertrophy in rats and explored the underlying molecular mechanism. ISO was applied to establish a rat model of myocardial hypertrophy, and CHS Ⅳa(5 and 15 mg·kg~(-1)·d~(-1)) was used for intervention. The tail artery blood pressure was measured. Cardiac ultrasound examination was performed. The ratio of heart weight to body weight(HW/BW) was calculated. Morphological changes in the myocardial tissue were observed by HE staining. Collagen deposition in the myocardial tissue was observed by Masson staining. The mRNA expression of myocardial hypertrophy indicators(ANP and BNP), autophagy-related genes(Atg5, P62 and beclin1), and miR199 a-5 p was detected by qRT-PCR. Atg5 protein expression was detected by Western blot. The results showed that the model group exhibited increased tail artery blood pressure and HW/BW ratio, thickened left ventricular myocardium, enlarged myocardial cells, disordered myocardial fibers with widened interstitium, and a large amount of collagen aggregating around the extracellular matrix and blood vessels. ANP and BNP were largely expressed. Moreover, P62 expression was up-regulated, while beclin1 expression was down-regulated. After intervention by CHS Ⅳa at different doses, myocardial hypertrophy was ameliorated and autophagy activity in the myocardial tissue was enhanced. Meanwhile, miR199 a-5 p expression declined and Atg5 expression increased. As predicted by bioinformatics, Atg5 was a target gene of miR199 a-5 p. CHS Ⅳa was capable of preventing myocardial hypertrophy by regulating autophagy of myocardial cells through the miR-199 a-5 p/Atg5 signaling pathway.

Effect of total saponins from Panax japonicus on non-alcoholic steatohepatitis by regulating autophagy / 中国中药杂志

Non-alcoholic steatohepatitis(NASH) was induced by high-sugar and high-fat diet in mice to investigate the intervention effect of total saponins from Panax japonicus(TSPJ) and explore its possible mechanism. Mice were fed with high-sugar and high-fat diet to establish NASH model, and intervened with different doses of TSPJ(15, 45 mg·kg~(-1)). The animals were fed for 26 weeks. The histomorphology and pathological changes of liver tissues were observed by HE staining. The transcriptional expression levels of miR-199 a-5 p, autophagy related gene 5(ATG5) and inflammatory cytokines interleukin-6(IL-6), interleukin-1β(IL-1β) and tumor necrosis factor α(TNF-α) in mouse liver were measured by quantitative Real-time polymerase chain reaction(qRT-PCR). Western blot was used to detect the expression of autophagy-related proteins ATG5, P62/SQSTM1(P62), and microtubule-associated protein light chain 3(LC3)-I/Ⅱ proteins in mouse liver. The expression of P62 protein was detected by immunofluorescence staining. In order to verify the targeting regulation relationship between miR-199 a-5 p and ATG5, miR mimic/inhibitor NC and miR-199 a-5 p mimic/inhibitor were transfected into Hepa 1-6 cells, and the expression of ATG5 mRNA and protein was detected. pMIR-reportor ATG5-3'UTR luciferase reporter gene plasmid was constructed and co-transfected with miR mimic/inhibitor NC and miR-199 a-5 p mimic/inhibitor into Hepa 1-6 cells to detect luciferase activity. In vivo, HE staining in the model group showed typical fatty degeneration and inflammatory infiltration, with increased expression of miR-199 a-5 p and decreased expression of ATG5 mRNA and protein. The expression of autophagy-associated protein P62 increased significantly, the ratio of LC3Ⅱ/Ⅰ decreased, and the transcriptional expression of inflammatory factors increased significantly. After the intervention by TSPJ, the pathological performance of liver tissue was significantly improved, the expression of miR-199 a-5 p decreased and the expression of ATG5 mRNA and protein increased, the expression of autophagy-associated protein P62 decreased significantly, the ratio of LC3Ⅱ/Ⅰ increased, and the transcriptional expression of inflammatory cytokines IL-6, IL-1β and TNF-α decreased significantly. In vitro, it was found that the expression of ATG5 mRNA and protein and luciferase activity decreased significantly in miR-199 a-5 p overexpression cells, while after inhibition of miR-199 a-5 p expression, the expression level of ATG5 mRNA and protein and luciferase activity increased. The results showed that TSPJ can improve NASH in mice fed with high-sugar and high-fat diet, and its mechanism may be related to the regulation of miR-199 a-5 p/ATG5 signal pathway, the regulation of autophagy activity and the improvement of inflammatory response of NASH.

Effect of Qiyu Sanlong Decoction in Inhibiting Growth of Lung Cancer by Regulating Expressions of Beclin1, Atg5 and LC3B / 中国实验方剂学杂志

Objective::To study the effect of Qiyu Sanlong decoction on the growth of subcutaneous tumor in lung cancer mice and the expressions of key autophagy molecule, yeast Atg6 homologous (Beclin1), autophagy related genes5 (Atg5), and microtubule-associated protein1 light chain3 (LC3B). Method::Lewis lung carcinoma cells (LLC) were used to reproduce the lung cancer mice transplanted model. After the modeling, the mice were randomly divided into model group, Qiyu Sanlong decoction group, chemotherapy group and combination group, with 18 transplanted mice in each group. In model group, mice were fed with 0.9% saline 20 mL·kg-1 daily. In Qiyu Sanlong decoction group, mice were fed with Qiyu Sanlong decoction 80.48 g·kg-1 daily. The chemotherapy group was intraperitoneally injected with 0.4 mL cisplatin solution (DDP) at the 1st, 3rd and 5th day. The combination group was orally given the drugs at the concentration of 80.48 g·kg-1, and 0.4 mL DDP solution was intraperitoneally injected at the 1st, 3rd and 5th day. After 21 days of continuous treatment, tumor tissue was exfoliated and weighed, and the tumor inhibition rate was calculated. Hematoxylin-eosin (HE) staining was used to observe the histological changes of tumor. The expressions and localizations of Beclin1 and LC3B in tumor tissues were detected by immunohistochemical staining. Protein expressions of Beclin1, Atg5, LC3B-Ⅰand LC3B-Ⅱ were determined by Western blot, and the ratio of LC3B-Ⅱ/LC3B-Ⅰ was calculated. The transcription levels of Beclin1, Atg5 mRNA in tumor tissues were detected by Real-time PCR. Result::Qiyu Sanlong decoction had a mild inhibitory effect on transplanted tumor, with an inhibitory rate of 31.2%. Under microscope, patchy necrotic tumor cells were observed in the tumor tissues of Qiyu Sanlong decoction group. Immunohistochemical staining and Western blot analysis showed that Qiyu Sanlong decoction could up-regulate the expressions of Beclin1, Atg5 and LC3B protein (P<0.01), and promote the conversion from LC3B-Ⅰ into LC3-Ⅱ compared with the model group. Real-time PCR results showed that Qiyu Sanlong decoction could promote the transcription of Beclin1 mRNA and Atg5 mRNA compared with the model group (P<0.01). Conclusion::Qiyu Sanlong decoction has a mild inhibitory effect on lung tumors, and its mechanism may be related to up-regulating the expressions of autophagy key proteins Beclin1, Atg5 and LC3B, and promoting the conversion from LC3B-Ⅰ to LC3B-Ⅱ.

miR-183-5p enhances the radioresistance of colorectal cancer by directly targeting ATG5

Intracellular trafficking is a field that has been intensively studied for years and yet there remains much to be learned. Part ofthe reason that there is so much obscurity remaining in this field is due to all the pathways and the stages that define cellulartrafficking. One of the major steps in cellular trafficking is fusion. Fusion is defined as the terminal step that occurs when acargo-laden vesicle arrives at the proper destination. There are two types of fusion within a cell: homotypic and heterotypicfusion. Homotypic fusion occurs when the two membranes merging together are of the same type such as vacuole tovacuole fusion. Heterotypic fusion occurs when the two membranes at play are of different types such as when anendosomal membrane fuses with a Golgi membrane. In this review, we will focus on all the protein components – Rabs,Golgins, Multisubunit tethers, GTPases, protein phosphatases and SNAREs – that have been known to function in both ofthese types of fusion. We hope to develop a model of how all of these constituents function together to achieve membranefusion. Membrane fusion is a biological process absolutely necessary for proper intracellular trafficking. Due to the degreeof importance multiple proteins are required for it to be properly carried through. Whether we are talking about heterotypicor homotypic fusion, any defects in the fusion machinery can result in disease states such as Parkinson’s and Alzheimer’sdisease. Although much research has significantly expanded our knowledge of fusion, there is still much more to belearned.

The autophagy Protein Atg5 Plays a Crucial Role in the Maintenance and Reconstitution Ability of Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) in bone marrow are pluripotent cells that can constitute the hematopoiesis system through self-renewal and differentiation into immune cells and red blood cells. To ensure a competent hematopoietic system for life, the maintenance of HSCs is tightly regulated. Although autophagy, a self-degradation pathway for cell homeostasis, is essential for hematopoiesis, the role of autophagy key protein Atg5 in HSCs has not been thoroughly investigated. In this study, we found that Atg5 deficiency in hematopoietic cells causes survival defects, resulting in severe lymphopenia and anemia in mice. In addition, the absolute numbers of HSCs and multiple-lineage progenitor cells were significantly decreased, and abnormal erythroid development resulted in reduced erythrocytes in blood of Vav_Atg5(−/−) mice. The proliferation of Lin⁻Sca-1⁺c-Kit⁺ HSCs was aberrant in bone marrow of Vav_Atg5(−/−) mice, and mature progenitors and terminally differentiated cells were also significantly altered. Furthermore, the reconstitution ability of HSCs in bone marrow chimeric mice was significantly decreased in the presence of Atg5 deficiency in HSCs. Mechanistically, impairment of autophagy-mediated clearance of damaged mitochondria was the underlying cause of the HSC functional defects. Taken together, these results define the crucial role of Atg5 in the maintenance and the reconstitution ability of HSCs.

Regulatory roles of rno-miR-30b-5p in the expression of Atg5, Atg12 and Becn1 autophagy genes in rats with experimental autoimmune uveitis / 眼科新进展

Objective To investigate the regulatory roles of rno-miR-30b-5p in the expression of Atg5,Atg12 and Becn1 autophagy genes and their expressions in rats with experimental autoimmune uveitis (EAU).Methods Application of dual luciferase report system was conducted to detect the regulatory roles of rno-miR-30b-5p in Atg5,Atg12 and Becn1 gene expression.Lewis rats were randomly divided into control group and EAU group,with 6 rats in each group.Next,rats in EAU group were immunized to establish EAU model.After treatments,Genesis-D fundus camera was used to observe the fundus inflammation every day.After immunization for 12 days,the pathological features of rat ciliary body and retina were detected,and meanwhile,the spleen and lymph nodes in both groups were isolated to detect the expressions of rno-miR-30b-5p,Atg5,Atg12 and Becn1 genes by quantitative PCR (Q-PCR);the levels of autophagy related proteins were determined by ELISA.Results Dual luciferase report gene expression assay confirmed that Atg5,Atg12 and Becn1 were target genes of rno-miR-30b-5p.Twelve days after immunization,compared with the control group,rats in the EAU group had severe iris adhesions and severe blood vessel swelling,and pathological examination revealed massive infiltration of inflammatory cells in the ciliary body and retina.Furthermore,rno-miR-30b-5p mRNA was 0.46 ±0.01,0.29 ±0.17in the spleen and lymph nodes in EAU group,respectively,which was down-regulated when compared with the control group (P ＜0.01);whereas the expressions of Atg5,Atg12 and Becn1 were significantly upregulated,and the differences were statistically significant (all P ＜ 0.05).ELISA results showed that Atg5,Atg 12 and Becn 1 protein expression levels in the spleen and lymph nodes in EAU rats were significantly higher than those in the control group (all P ＜ 0.05).Conclusion rno-miR-30b-5p can regulate the expressions of Atg5,Atg12 and Becn1 autophagy-related genes.The down-regulation of rno-miR-30b-5p expression in the spleen and lymph nodes in EAU rats can significantly up-regulate the expressions of Atg5,Atg12 and Becn1 genes,thereby regulating the pathogenesis of uveitis.

Andrographolide protects mouse astrocytes against hypoxia injury by promoting autophagy and S100B expression

Andrographolide (ANDRO) has been studied for its immunomodulation, anti-inflammatory, and neuroprotection effects. Because brain hypoxia is the most common factor of secondary brain injury after traumatic brain injury, we studied the role and possible mechanism of ANDRO in this process using hypoxia-injured astrocytes. Mouse cortical astrocytes C8-D1A (astrocyte type I clone from C57/BL6 strains) were subjected to 3 and 21% of O2 for various times (0-12 h) to establish an astrocyte hypoxia injury model in vitro. After hypoxia and ANDRO administration, the changes in cell viability and apoptosis were assessed using CCK-8 and flow cytometry. Expression changes in apoptosis-related proteins, autophagy-related proteins, main factors of JNK pathway, ATG5, and S100B were determined by western blot. Hypoxia remarkably damaged C8-D1A cells evidenced by reduction of cell viability and induction of apoptosis. Hypoxia also induced autophagy and overproduction of S100B. ANDRO reduced cell apoptosis and promoted cell autophagy and S100B expression. After ANDRO administration, autophagy-related proteins, S-100B, JNK pathway proteins, and ATG5 were all upregulated, while autophagy-related proteins and s100b were downregulated when the jnk pathway was inhibited or ATG5 was knocked down. ANDRO conferred a survival advantage to hypoxia-injured astrocytes by reducing cell apoptosis and promoting autophagy and s100b expression. Furthermore, the promotion of autophagy and s100b expression by ANDRO was via activation of jnk pathway and regulation of ATG5.

Knock-down ATG5 gene inhibits autophagyand enhances celastrol-induced apoptosis in human lung cancer cell H1299 / 基础医学与临床

Objective To establish the lung cancer cell strain with low ATG5 expression and to detect the effect of celastrol on lung cancer cell apoptosis after downregulation of autophagy.Methods H1299 was infected by lentivirus-mediated ATG5 shRNA.RT-qPCR and Western blot assays were applied to confirm the effect of ATG5 knock down.Autophagy was measured by Western blot and RFP-LC3 transfection.Cell apoptosis of ATG5 normal expression group and of ATG5 low expression group of H1299 cells was detected by FACS.Finally, Western blot was used to detect the expression of apoptosis-related proteins Bcl-2, Bax and cleaved caspase-3.Results The expression of ATG mRNA and protein significantly decreased after ATG5 knockdown in H1299 cells (P<0.05).The autophagy marker of LC3-Ⅱ level was downregulated and P62 expression was upregulated after inhibition of ATG5, and the RFP-LC3 puncta reduced significantly after ATG5 knockdown (P<0.05).Compared with control group,the apoptosis rate in ATG5 downregulation group increased significantly after celastrol treatment (P<0.01).Pro-apoptotic proteins of Bax and cleaved caspase-3 levels were upregulated and anti-apoptotic protein of Bcl-2 level decreased after ATG5 inhibition (P<0.05).ConclusionsThe effect of celastrol-induced apoptosis of lung cancer cells was enhanced after downregulation of autophagy, demonstrating inhibition autophay may be a new target of lung cancer treatment.

Expression of Beclin1, p62 and Atg5 in tissue of serous ovarian cancer and its significance / 中华内分泌外科杂志

Objective To investigate the expression of Beclin1,p62 and Atg5 in serous ovarian cancer and its clinical significance.Methods The expression of Beclin1,p62 and Atg5 was detected in 50 serous ovarian cancer tissues by immunohistochemical method,and the relationship between their expression levels and the clinical parameters of serous ovarian cancer patients were analyzed.Results The expression of Beclin1 and Atg5 in serous ovarian cancer tissue was significantly lower than that in normal ovarian tissue,but p62 was significantly higher than that in normal ovarian tissue,and the difference had statistically significance (P＜0.05).The expression of Beclin1 and Atg5 were correlated with degree of cell differentiation and clinical stage (P＜0.05),while the expression of p62 was associated with clinical stages and lymph node metastasis (P＜0.05).Conclusions The abnormal expression of Beclin1,p62,Atg5 in serous ovarian cancer,which can lead to reduced autophagy body formation and autophagy activity,may be involved in the occurrence and development of serous ovarian cancer.

Suppression of autophagy activity enhances the radiosensitivity of human nasopharyngeal carcinoma CNE-2 cells / 中华放射医学与防护杂志

Objective To investigate the effect of autophagy in radiosensitivity of nasopharyngeal carcinoma CNE-2 cells.Methods The expression of ATG5 in CNE-2 cells was inhibited by lentiviral mediated RNA interference.The cells were divided into three groups:control group,NC group and ATG5 group.Cell proliferation,apoptosis and radiosensitivity were detected by CCK-8 method,flow cytometry and colony-forming assay,respectively.Results Compared with the NC group and control group,the survival of ATG5-interfected cells was reduced (F =3.755,46.086,8.609,44.160,P ＜ 0.05).After 6 Gy X-ray irradiation,the apoptosis index of the ATG5 group significantly higher than that of NC group and control group (F =394.876,P ＜ 0.05).In addition,the radiosensitivity of ATG5 group was also higher than that of control cells.Conclusions Suppression of autophagy activity enhances the radiosensitivity of human nasopharyngeal carcinoma cells.

Expression and clinical significance of Atg3 and Atg5 genes in bone marrow mononuclear cells isolated from children with acute leukemia / 中华实用儿科临床杂志

Objective To detect the expression of autophagy-related gene Atg3 and Atg5 in bone marrow mononuclear cells (BMMNCs) from children with acute leukemia(AL),so as to explore the relationship between autophagy and the pathogenesis of AL in children.Methods Seventy-four bone marrow specimens were obtained from children with AL in the First Affiliated Hospital of Zhengzhou University Pediatrics Hematology Ward,including 37 cases of initially diagnosed AL without any treatment,28 cases of AL in complete remission,9 cases of refractory or relapse AL and 28 bone marrow specimens from children without tumor were also collected as the control group.BMMNCs were separated by Lydroxypropylmethyl Cellulose.After BMMNCs were stained by Monodansylcadaverine,the autophagy phenomenon was observed by using fluorescence microscope,and the ratio of autophagy was detected by using flow cytometry.Reverse transcription polymerase chain reaction(RT-PCR) was used to detect the expression of Atg3 mRNA and Atg5 mRNA in each group.Results It was found that autophagy phenomenon was more common in the initially diagnosed group and the refractory/relapse group,and the autophagy ratio in both groups was respectively (17.07 ±2.31) ％,(15.37 ± 1.59) ％,respectively,which were obviously higher than that of the control group (2.71 ± 1.57) ％ and that of the complete remission group.The differences were statistically significant (t =28.29,20.96,all P ＜ 0.01).The autophagy ratio in complete remission group was (3.48 ± 1.94) ％,and compared with the control group,the difference was of no statistical significance(t =1.634,P ＞ 0.05).The autophagy ratio in the refractory/relapse group higher than that in the complete remission group (t =16.61,P ＜ 0.05).The expressions of Atg3 mRNA and Atg5 mRNA in initially diagnosed group and refractory/relapse group were higher than those of the complete remission group and control group,and the differences were statistically significant (F =67.592,106.160,all P ＜ 0.008) ; the difference between complete remission group and control group was of no statistical significance (P ＞ 0.008).Conclusions The autophagy ratio and the expressions of Atg3 mRNA and Atg5 mRNA in initially diagnosed group and the refractory/ relapse group were both obviously higher.It was revealed that higher autophagy activity,which was caused by upregulated expressions of Atg3 mRNA and Atg5 mRNA,had a closely connection with the mechanism of occurrence,development and resistance of AL in children.

Molecules and their functions in autophagy

Autophagy is a self-degradation system of cellular components through an autophagosomal-lysosomal pathway. Over the last 15 yr, yeast genetic screens led to the identification of a number of genes involved in the autophagic pathway. Most of these autophagy genes are present in higher eukaryotes and regulate autophagy process for cell survival and homeostasis. Significant progress has recently been made to better understand the molecular mechanisms of the autophagy machinery. Especially, autophagy process, including the regulation of autophagy induction through mTOR and the nucleation and elongation in autophagosome formation through class III phosphatidylinositol 3-kinase complex and ubiquitin-like conjugation systems, became evident. While many unanswered questions remain to be answered, here, we summarize the recent process of autophagy with emphasis on molecules and their protein complexes along with advanced molecular mechanisms that regulate the autophagy machinery.

Mutational and Expressional Analysis of ATG5 Gene in Non-Small Cell Lung Cancers

PURPOSE: Several lines of evidence have indicated that perturbations of autophagy are involved in the development of many human diseases, including cancer. The autophagy-related genes (ATG) encode proteins that play important roles in autophagic processes. The aim of this study was to see whether alterations of the ATG5 protein expression and somatic mutations of the ATG5 gene are present in human non-small cell lung cancers (NSCLCs). MATERIALS AND METHODS: We analyzed the ATG5 somatic mutations in 45 NSCLCs by performing single-strand conformation polymorphism (SSCP). We examined the ATG5 protein expression in 45 NSCLCs by performing immunohistochemistry. RESULTS: The SSCP analysis revealed no evidence of somatic mutation in the DNA sequences encoding the ATG5 gene in the 45 NSCLCs. On the immunohistochemistry, ATG5 protein was expressed in the normal bronchial epithelial cells, while it was lost in 9 (20%) of the NSCLCs. CONCLUSION: Our data indicates that ATG5 is altered in NSCLC at the expressional level, but not at the mutational level. The data also suggests that the loss of expression of ATG5 might play a role in the pathogenesis of NSCLC by altering autophagic and apoptotic cell death.

Calpain-mediated Cleavage of Atg5 Determine Autophagy or Apoptosis of PMN, and C5a's Role / 生物化学与生物物理进展

Apoptosis of neutrophils controls the duration and the intensity of an inflammatory response and therefore the extent of neutrophil- mediated tissue damage, disturbance of neutrophil apoptosis has been associated with many diseases, underlying mechanism is not elucidated. C5a is a complement fragment that has multifunctional properties, which induces neutrophil chemoattraction, an oxidative burst, enhancement of phagocytosis, release of granule enzymes, and suppress neutrophil apoptosis. Several studies have reported calpain is involved in both neutrophil functions and apoptosis and it might play a more specific role in the regulation of neutrophil apoptosis. Diffenrent isoform of calpains is activted by diffenrent stimuli through different transduction pathway. It was reported previously that calpain is required for neutrophil migration and chemotaxis induced by C5a. In addition, autophagy is a ubiquitous physiological process that occurs in all eukaryotic cells and is considered to be a survival mechanism. Atg5 promotes autophagy and is indispensable to autophagosome formation. Upon calpain activation, Atg5 is cleaved and the resulting 24 ku Atg5 mediates apoptosis while losting the property of autophagy. Therefore, Atg5 represents a molecular switch between autophagy and apoptosis. The interaction among the C5a, calpain and Atg5 was introduced and new direction for further research was provided.