OBJECTIVE: To investigate the effects of melatonin on the growth and metastasis of MDA-MB-231 breast cancer cells and explore the mechanism. METHODS: MDA-MB-231 cells were treated with 1, 3 or 5 mmol/L melatonin, and the changes in cell proliferation were examined using CCK-8 assay. Colony-forming assay and wound healing assay were used to assess the effects of melatonin treatmnent on colony-forming ability and migration of the cells. Flow cytometry and immunofluoresnce assay were employed to examine apoptosis and positive staining for autophagy-related proteins in the cells treated with 3 mmol/L melatonin. The effects of melatonin treatment alone or in combination with 3-methyladenine (3-MA) on the expressions of the proteins associated with autophagy (LC3, P62 and Beclin1), apoptosis (Bcl2 and Bax) and epithelial-mesenchymal transition (E-cadherin and Snail) were examined with Western blotting. RESULTS: Melatonin treatment significantly inhibited the proliferation of breast cancer cells in a concentration- and time-dependent manner (P < 0.05), suppressed colony-forming ability and migration (P < 0.01), and promoted apoptosis of the cells (P < 0.01). Melatonin treatment alone significantly increased the expressions of Bax (P < 0.05), E-cadherin, LC3-II/LC3-I, and Beclin1 and lowered the expressions of Bcl2 (P < 0.05), Snail, P62 (P < 0.05), and Bcl2/Bax ratio (P < 0.01) in the cells, and caused enhanced positive staining of Beclin1 protein and attenuated staining of P62 protein. Compared with melatonin treatment alone, melatonin treatment combined with 3-MA significantly decreased the expressions of Beclin1 (P < 0.001), LC3-II/LC3-I (P < 0.05), Bax (P < 0.01), and E-cadherin (P < 0.001) and increased the expressions of Bcl2 (P < 0.05), Snail, and Bcl2/Bax ratio (P < 0.01). CONCLUSION Melatonin can induce autophagy of MDA-MB-231 breast cancer cells to inhibit cell proliferation and metastasis and promote cell apoptosis, and suppressing autophagy can weaken the inhibitory effect of melatonin on the growth and metastasis of breast cancer cells.
Autophagy ; Autophagy-Related Proteins/metabolism* ; Breast Neoplasms ; Cell Line, Tumor ; Female ; Humans ; Melatonin/pharmacology*

OBJECTIVETo study the effect of calcitonin gene-related peptide (CGRP) on apoptosis and autophagy of mouse MC3T3-E1 osteoblast and their interaction and to further clarify protective mechanism of CGRP on osteoblasts.

METHODSMC3T3-E1 osteoblasts of mouse were cultured in vitro. Western blot and flow cytometry were used to detect expressions of microtubule-associated protein 1 light chain 3 (LC3) and P62 protein of MC3T3-E1 osteoblasts cultured with serum culture and serum-free (serum starvation) culture. Western blot was also used to detect expressions of LC3 and P62 protein of MC3T3-E1 osteoblast cultured at different concentrations (10⁻¹⁰, 10⁻⁹, 10⁻⁸, and 10⁻⁷ mol·L⁻¹) or without added CGRP. MC3T3-E1 osteoblasts were treated with 10⁻⁸ mol·L⁻¹ CGRP at different times (2, 6, 12, 24, 48, and 72 h), protein expression levels of LC3 were assessed by Western blot and flow cytometry, and changes in autophagosome in cells were detected by monodansylcadaverin staining. Autophagy inhibitor 3-methyladenine (3-MA) was used to pretreat MC3T3-E1 osteoblasts. Cells were then treated with or without CGRP for 24 h. Flow cytometry was used to detect apoptosis level.

RESULTSUnder serum starvation conditions, LC3Ⅱ expression and apoptosis of osteoblasts increased compared with that of serum culture. Under 3-MA pretreatment and serum starvation conditions, LC3Ⅱ expression of osteoblasts increased compared with that of serum culture (P<0.01). Compared with serum culture, serum starvation culture with or without CGRP significantly increased expression level of LC3 and reduced expression level of P62. LC3Ⅱ/Ⅰ of osteoblasts was the highest under serum starvation and 10⁻⁸ mol·L⁻¹ CGRP conditions. Serum starvation and 10⁻⁸ mol·L⁻¹ CGRP culture inhibited apoptosis of osteoblasts and promoted synthesis of autophagosome. Apoptosis of osteoblasts increased after 3-MA pretreatment, and CGRP reversed inhibitory effects of 3-MA CGRP on apoptosis.

CONCLUSIONSCGRP can increase autophagy of MC3T3-E1 osteoblasts under serum starvation conditions. CGRP may also inhibit apoptosis of MC3T3-E1 osteoblasts by promoting autophagy.
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Animals ; Apoptosis ; Autophagy ; Calcitonin ; Calcitonin Gene-Related Peptide ; Mice ; Microtubule-Associated Proteins ; Osteoblasts

OBJECTIVE: To explore the genetic etiology of Vici syndrome in a Chinese family. METHODS: Whole exome sequencing (WES) technology was used to detect gene variants in a fetus of abnormal ultrasonic structure without abnormalities in routine chromosome karyotype analysis and SNP-array. Sanger sequencing and bioinformatics prediction were performed for the suspected variants of the fetus and parents. RESULTS: The fetus and the elder sister have carried c. 2427delC (p.T809fs) and c.1886A>T (p.E629V) compound heterozygous variants of the EPG5 gene, which were respectively inherited from their mother and father. Neither variant was reported previously. According to ACMG guidelines, the c.2427delC variant was predicted as pathogenic, while the c.1886A>T variant was of uncertain significance. PolyPhen-2 and PROVEAN software indicated that c.1886A>T variant was probably damaging. CONCLUSION The c.2427delC and c.1886A>T variants of the EPG5 gene probably underlie the pathogenesis of the Vici syndrome in this family. Above finding has enriched the variational spectrum of EPG5 gene and provided a basis for genetic counseling and prenatal diagnosis for the family.
Aged ; Agenesis of Corpus Callosum ; Autophagy-Related Proteins ; Cataract ; Female ; Humans ; Mutation ; Pregnancy ; Vesicular Transport Proteins/genetics* ; Whole Exome Sequencing

OBJECTIVETo understand the relationship between the susceptibility to inflammatory bowel disease (IBD) and ATG16L1 gene single nucleotide polymorphism (SNP) site, rs2241880.

METHODSPeripheral blood samples were collected from 80 IBD patients (including 40 with Crohn's disease and 40 with ulcerative colitis) and 50 healthy controls, and the genomic DNA was extracted from the white blood cells. Specific primers were designed according to the target gene sequence for PCR amplification of the target gene fragment, and the PCR products were purified followed by sequence analysis of the target region of ATG16L1 gene. The results of the sequence analysis were compared with the BenBank data to analyze the relationship between the allele gene polymorphisms and the susceptibility to Crohn's disease.

RESULTSNo significant differences were noted in the ATG16L1 gene SNP site rs2241880 polymorphisms among the patients with Crohn's disease, ulcerative colitis and the control subjects (Chi(2)=4.94, P=0.293).

CONCLUSIONATG16L1 gene polymorphisms in the SNP site rs2241880 are not found to correlate to the susceptibility to Crohn's disease as reported in literature. The SNP site associated with Crohn's disease susceptibility identified in foreign populations does not seem to be identical with that in Chinese population.

Adult ; Autophagy ; genetics ; Autophagy-Related Proteins ; Base Sequence ; Carrier Proteins ; genetics ; Colitis, Ulcerative ; genetics ; Crohn Disease ; genetics ; Female ; Genetic Predisposition to Disease ; genetics ; Humans ; Inflammatory Bowel Diseases ; genetics ; Male ; Middle Aged ; Phagosomes ; genetics ; Polymorphism, Single Nucleotide ; Sequence Analysis, DNA

Objective To identify the relationship between nephritis activity, autophagy and inflammation in patients with SLE. Methods Western blot analysis was used to detect the expression of microtubule-associated protein 1 light chain 3 (LC3) and P62 in peripheral blood mononuclear cells (PBMCs) of SLE patients with lupus nephritis and non-lupus nephritis patients. Tumor necrosis factor α (TNF-α) and interferon γ (IFN-γ) in the serum of SLE patients were determined by ELISA. The correlation between LC3II/LC3I ratio and SLE disease activity score (SLEDAI), urinary protein, TNF-α and IFN-γ levels was analyzed by Pearson method. Results The expression of LC3 was increased and P62 was decreased in SLE patients. TNF-α and IFN-γ were increased in the serum of SLE patients. LC3II/LC3I ratio was positively correlated with SLEDAI (r=0.4560), 24 hour urine protein (r=0.3753), IFN-γ (r=0.5685), but had no correlation with TNF-α (r=0.04 683). Conclusion Autophagy is found in PBMCs of SLE, and the autophagy is correlated with renal damage and inflammation in patients with lupus nephritis.
Humans ; Tumor Necrosis Factor-alpha/metabolism* ; Leukocytes, Mononuclear/metabolism* ; Autophagy-Related Proteins/metabolism* ; Lupus Nephritis/urine* ; Kidney ; Interferon-gamma/metabolism* ; Inflammation/metabolism* ; Lupus Erythematosus, Systemic/metabolism*

OBJECTIVETo investigate the correlation of the autophagy-associated gene Atg5 with the pathogenesis of prostate cancer.

METHODSUsing real-time fluorescent quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and immunohistochemistry, we detected the expression of Atg5 in 50 cases of prostate intraepithelial neoplasm (PIN), 69 cases of prostate cancer (PCa), and 30 cases of benign prostatic hyperplasia (BPH).

RESULTSThe expression level of Atg5 mRNA was significantly higher in PIN (5.270 ± 0.230) and PCa (5.131 ± 0.252) than in the BPH tissue (1.723 ± 0.017) (P <0.01), and so was the positive rate of the Atg5 expression in the patients of the PIN group (94%) and PCa group (88.4%) than in those of the BPH group (6.7%) (P<0.01), but with no statistically significant differences between the PIN and PCa groups (P >0.05). No significant correlation was observed between the expression of Atg5 and the Gleason score of PCa (P >0.05).

CONCLUSIONThe upregulated expression of Atg5 might play a role in the tumorigenesis of prostate cancer.

Aged ; Autophagy ; Autophagy-Related Protein 5 ; Humans ; Immunohistochemistry ; Male ; Microtubule-Associated Proteins ; genetics ; Middle Aged ; Prostatic Hyperplasia ; genetics ; Prostatic Neoplasms ; genetics ; RNA, Messenger ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Up-Regulation

Anoikis ; Autophagy ; Autophagy-Related Protein 5 ; genetics ; metabolism ; Blotting, Western ; Cell Line, Tumor ; Humans ; MicroRNAs ; genetics ; metabolism ; Microscopy, Fluorescence ; Microtubule-Associated Proteins ; genetics ; metabolism ; Poly (ADP-Ribose) Polymerase-1 ; metabolism ; Transfection

OBJECTIVE: To investigate the effect of estradiol (E2)/estrogen receptor 1 (ESR1) on the proliferation of human chondrocytes and explore the molecular mechanism. METHODS: The Ad-Easy adenovirus packaging system was used to construct and package the ESR1-overexpressing adenovirus Ad-ESR1. Western blotting and qPCR were used to detect the expression of ESR1 protein and mRNA in human chondrocyte C28I2 cells. In the cells treated with different adenoviruses, the effects of E2 were tested on the expressions of proteins related with cell autophagy and apoptosis and the phosphorylation of ERK signaling pathway using Western blotting. Immunofluorescence assay was used to observe the intracellular autophagic flow, flow cytometry was performed to analyze the cell apoptosis rate and the cell cycle changes, and qPCR was used to detect the expressions of PCNA, cyclin B1 and cyclin D1 mRNAs. The inhibitory effect of the specific inhibitor of ERK on the expressions of autophagy- and apoptosis-related genes at both the protein and mRNA levels were detected using Western blotting and qPCR. RESULTS: Transfection with the recombinant adenovirus overexpressing ESR1 and E2 treatment of C28I2 cells significantly enhanced the expressions of autophagy-related proteins LC3, ATG7, promoted the colocalization of LC3 and LAMP1 in the cytoplasm, increased the expressions of the proliferation-related marker genes PCNA, cyclin B1 and cyclin D1, and supressed the expressions of cleaved caspase-3, caspase-12 and pERK. RNA interference of ESR1 obviously lowered the expression levels of autophagy-related proteins in C28I2 cells, causing also suppression of the autophagic flow, increments of the expressions of apoptosis-related proteins and pERK, and down-regulated the expressions of the proliferation marker genes. Blocking ERK activation with the ERK inhibitor obviously inhibited the effects of E2/ESR1 on autophagy, proliferationrelated gene expressions and cell apoptosis. CONCLUSIONS The targeted binding of E2 with ESR1 promotes the proliferation of human chondrocytes possibly by inhibiting the activation of ERK signaling pathway to promote cell autophagy and induce cell apoptosis.
Adenoviridae ; metabolism ; Apoptosis ; Autophagy ; Autophagy-Related Protein 7 ; metabolism ; Cell Line ; Cell Proliferation ; Chondrocytes ; cytology ; metabolism ; Estradiol ; metabolism ; Estrogen Receptor alpha ; metabolism ; Humans ; Lysosome-Associated Membrane Glycoproteins ; metabolism ; MAP Kinase Signaling System ; Microtubule-Associated Proteins ; metabolism ; Transfection

Autophagy is fundamental to maintain cellular homeostasis. As one kind of the most well-studied selective autophagy, autophagy of mitochondria (mitophagy)is crucial for the clearance of damaged mitochondria. Mitophagy dysfunction has been proved to be closely associated with many human diseases. Nix is a key protein for mitophagy during the maturation of reticulocytes. However, the detailed molecular mechanisms underlying Nix-mediated mitophagy are not fully understood. This article summarizes three possible working models of Nix in mitophagy induction. Firstly, Nix can interplay with Parkin, another important protein for mitophagy, to initiate mitophagy. Secondly, Nix can serve as a receptor for autophagy machinery by interacting with Atg8 family through its LIR motif. Finally, as a BH3-only protein, Nix can compete with Beclin-1 to bind other members of Bcl-2 family resulting in increased free Beclin-1 in cytosol, which further promotes autophagy flux.
Autophagy ; genetics ; physiology ; Autophagy-Related Protein 8 Family ; physiology ; Beclin-1 ; physiology ; Membrane Proteins ; physiology ; Mitochondria ; genetics ; physiology ; Mitochondrial Degradation ; genetics ; physiology ; Protein Interaction Domains and Motifs ; Proto-Oncogene Proteins ; physiology ; Proto-Oncogene Proteins c-bcl-2 ; antagonists & inhibitors ; Tumor Suppressor Proteins ; physiology ; Ubiquitin-Protein Ligases ; physiology

Autophagy is an evolutionarily conserved cellular process which degrades intracellular contents. The Atg17-Atg31-Atg29 complex plays a key role in autophagy induction by various stimuli. In yeast, autophagy occurs with autophagosome formation at a special site near the vacuole named the pre-autophagosomal structure (PAS). The Atg17-Atg31-Atg29 complex forms a scaffold for PAS organization, and recruits other autophagy-related (Atg) proteins to the PAS. Here, we show that Atg31 is a phosphorylated protein. The phosphorylation sites on Atg31 were identified by mass spectrometry. Analysis of mutants in which the phosphorylated amino acids were replaced by alanine, either individually or in various combinations, identified S174 as the functional phosphorylation site. An S174A mutant showed a similar degree of autophagy impairment as an Atg31 deletion mutant. S174 phosphorylation is required for autophagy induced by various autophagy stimuli such as nitrogen starvation and rapamycin treatment. Mass spectrometry analysis showed that S174 is phosphorylated constitutively, and expression of a phosphorylation-mimic mutant (S174D) in the Atg31 deletion strain restores autophagy. In the S174A mutant, Atg9-positive vesicles accumulate at the PAS. Thus, S174 phosphorylation is required for formation of autophagosomes, possibly by facilitating the recycling of Atg9 from the PAS. Our data demonstrate the role of phosphorylation of Atg31 in autophagy.
Alanine ; chemistry ; metabolism ; Amino Acid Motifs ; Aspartic Acid ; chemistry ; metabolism ; Autophagy ; genetics ; Autophagy-Related Proteins ; Carrier Proteins ; chemistry ; metabolism ; Gene Expression Regulation, Fungal ; Membrane Proteins ; chemistry ; metabolism ; Models, Molecular ; Molecular Sequence Data ; Nitrogen ; deficiency ; Phagosomes ; chemistry ; drug effects ; metabolism ; Phosphorylation ; Protein Transport ; Saccharomyces cerevisiae ; drug effects ; genetics ; metabolism ; Saccharomyces cerevisiae Proteins ; chemistry ; genetics ; metabolism ; Serine ; chemistry ; metabolism ; Signal Transduction ; Sirolimus ; pharmacology