Kinesin family member 23 exerts a protumor function in breast cancer via stimulation of the Wnt/ß-catenin pathway.

Kinesin family member 23 (KIF23) has been described as one of the main genes that are associated with malignant transformation in numerous cancers. However, the exact significance of KIF23 in breast cancer has not been well-addressed. The present study was dedicated to the comprehensive investigation of KIF23 in breast cancer. Initial expression analysis through The Cancer Genome Atlas (TCGA) demonstrated high KIF23 levels in breast cancer compared with normal controls. These in silico data showing high levels of KIF23 in breast cancer were verified by assessing clinical specimens using real-time quantitative PCR and immunoblot assays. Moreover, a high KIF23 level was correlated with adverse clinical outcomes in breast cancer patients. Cellular functional experiments showed that the down-regulation of KIF23 affected the malignant behaviors of breast cancer cells in vitro, whereas the forced expression of KIF23 stimulated them. Mechanistic studies revealed that KIF23 restraint down-regulated the levels of phosphorylated glycogen synthetase kinase-3ß (GSK-3ß), ß-catenin, cyclin D1 and c-myc in breast cancer cells, showing an inhibitory effect on the Wnt/ß-catenin pathway. The suppression of GSK-3ß was able to reverse KIF23-silencing-induced inactivation of the Wnt/ß-catenin pathway. Inhibition of the Wnt/ß-catenin pathway abolished KIF23 overexpression-mediated protumor effects in breast cancer. A xenograft assay confirmed the in vivo antitumor function of KIF23 inhibition. In conclusion, these findings suggest that KIF23 may exert a protumor function in breast cancer by stimulating the Wnt/ß-catenin pathway. This work suggests that KIF23 has potential values for targeted therapy and prognosis in breast cancer.

Cinnamaldehyde induces autophagy-mediated cell death through ER stress and epigenetic modification in gastric cancer cells.

Previous reports suggested that cinnamaldehyde (CA), the bioactive ingredient in Cinnamomum cassia, can suppress tumor growth, migratory, and invasive abilities. However, the role and molecular mechanisms of CA in GC are not completely understood. In the present study, we found that CA-induced ER stress and cell death via the PERK-CHOP axis and Ca2+ release in GC cells. Inhibition of ER stress using specific-siRNA blocked CA-induced cell death. Interestingly, CA treatment resulted in autophagic cell death by inducing Beclin-1, ATG5, and LC3B expression and by inhibiting p62 expression whereas autophagy inhibition suppressed CA-induced cell death. We showed that CA induces the inhibition of G9a and the activation of LC3B. Moreover, CA inhibited G9a binding on Beclin-1 and LC3B promoter. Overall, these results suggested that CA regulates the PERK-CHOP signaling, and G9a inhibition activates autophagic cell death via ER stress in GC cells.

in Vitro Effect of Methamphetamine on Proliferation, Differentiation and Apoptosis of Adipose Tissue Stem Cells.

PURPOSE: Among abused substances, methamphetamine is a psychostimulant drug widely used recreationally with public health importance. This study investigated the effect of methamphetamine on proliferation, differentiation, and apoptosis of human adipose tissue stem cells (AdSCs). METHODS: AdSCs were isolated from human abdominal adipose tissue and were characterized for mesenchymal properties and growth kinetics. MTT assay was undertaken to assess methamphetamine toxicity on proliferation and differentiation properties and apoptosis of hAdSCs. RESULTS: Isolated cells were shown to have mesenchymal properties and a population doubling time (PDT) of 40.1 h. Following methamphetamine treatment, expressions of KI-67 and TPX2 as proliferation genes and Col1A1 and PPARg as differentiation genes decreased. Methamphetamine administration increased the expression of Bax and decreased Bcl-2 genes responsible for apoptosis. CONCLUSIONS: Our data suggested when AdSCs were exposed to methamphetamine, it decreased proliferation and differentiation properties of stem cells together with an increase in apoptosis. These findings can be added to the literature, especially when methamphetamine is used recreationally for weight loss purposes.

Berberine Inhibits MDA-MB-231 Cells as an Agonist of G Protein-Coupled Estrogen Receptor 1.

G protein-coupled estrogen receptor 1 (GPER1) is a potential therapeutic target for treating triple-negative breast cancers (TNBC). However, modulators for GPER1 that can be used to treat TNBC have not appeared. Berberine (BBR) is a bioactive isoquinoline alkaloid with high oral safety. In recent years, BBR has shown an inhibitory effect on TNBC tumors such as MDA-MB-231, but the molecular target remains unclear, which hinders related clinical research. Our work proved that BBR is a modulator of GPER1 that can inhibit cell viability, migration, and autophagy of MDA-MB-231 cells. The inhibitory effect of BBR on MDA-MB-231 cells has a dependence on estrogen levels. Although BBR promoted the proteasome, which is a major factor in the degradation of GPER1, it could still induce the protein level of GPER1. Correspondingly, the transcription of cellular communication network factor 2 (CCN2) was promoted. BBR could bind to GPER1 directly and change the secondary structure of GPER1, as in the case of 17ß-estradiol (E2). In addition, BBR induced not only a high degree of co-localization of GPER1 and microtubule-associated protein 1 light chain 3 (MAP1LC3), but also the accumulation of sequestosome 1 (SQSTM1/p62) by the inhibition of the nuclear translocation of the nuclear factor-kappa B (NF-κB) subunit (RELA/p65), which indicates NF-κB inhibition and anti-cancer effects. This result proved that the promotional effect of BBR on the GPER1/NF-κB pathway was closely related to its inhibitory effect on autophagy, which may serve as a new mechanism by which to explain the inhibitory effect of BBR on MDA-MB-231 cells and expand our understanding of the function of both BBR and GPER1.

Circ-SKA3 Enhances Doxorubicin Toxicity in AC16 Cells Through miR-1303/TLR4 Axis.

Doxorubicin (DOX) is a widely used anticancer drug, but its cardiotoxicity largely limits its clinical utilization. Circular RNA spindle and kinetochore-associated protein 3 (circ-SKA3) were found to be differentially expressed in heart failure patients. In this study, we investigated the role and mechanism of circ-SKA3 in DOX-induced cardiotoxicity.The quantitative real-time polymerase chain reaction and western blot assays were applied to measure the expression of circ-SKA3, microRNA (miR) -1303, and toll-like receptor 4 (TLR4). The viability and apoptosis of AC16 cells were analyzed using cell counting kit-8, flow cytometry, and western blot assays. The interaction between miR-1303 and circ-SKA3 or TLR4 was verified using dual-luciferase reporter and RNA immunoprecipitation assays. Exosomes were collected from culture media by the use of commercial kits and then qualified by transmission electron microscopy.The expression of circ-SKA3 and TLR4 was increased, whereas miR-1303 expression was decreased in DOX-treated AC16 cells. DOX treatment promoted cell apoptosis and inhibited cell viability in AC16 cells in vitro, which was partially reversed by circ-SKA3 knockdown, TLR4 silencing, or miR-1303 overexpression. Mechanistically, circ-SKA3 served as a sponge for miR-1303 to upregulate TLR4, which was confirmed to be a target of miR-1303. Additionally, circ-SKA3 contributed to DOX-induced cardiotoxicity through the miR-1303/TLR4 axis. Further studies suggested that circ-SKA3 was overexpressed in exosomes extracted from DOX-mediated AC16 cells, which could be internalized by surrounding untreated AC16 cells.Circ-SKA3 enhanced DOX-induced toxicity in AC16 cells through the miR-1303/TLR4 axis. Extracellular circ-SKA3 was packaged into exosomes, and exosomal circ-SKA3 could function as a mediator in intercellular communication between AC16 cells.

Melatonin ameliorates excessive PINK1/Parkin-mediated mitophagy by enhancing SIRT1 expression in granulosa cells of PCOS.

Mitochondrial injury in granulosa cells is associated with the pathogenesis of polycystic ovary syndrome (PCOS). However, the protective effects of melatonin against mitochondrial injury in the granulosa cells of PCOS remain unclear. In this study, decreased mitochondrial membrane potential and mtDNA content, increased number of autophagosomes were found in the granulosa cells of PCOS patients and the dihydrotestosterone (DHT)-treated KGN cells, with decreased protein level of the autophagy substrate p62 and increased levels of the cellular autophagy markers Beclin 1 and LC3B-II, while the protein levels of PTEN-induced kinase-1 (PINK1) and Parkin were increased and the level of sirtuin 1 (SIRT1) was decreased. DHT-induced PCOS-like mice also showed enhanced mitophagy and decreased SIRT1 mRNA expression. Melatonin treatment significantly increased the protein level of SIRT1 and decreased the levels of PINK1/Parkin, whereas it ameliorated the mitochondrial dysfunction and PCOS phenotype in vitro and in vivo. However, when the KGN cells were treated with SIRT1 siRNA to knock down SIRT1 expression, melatonin treatment failed to repress the excessive mitophagy. In conclusion, melatonin protects against mitochondrial injury in granulosa cells of PCOS by enhancing SIRT1 expression to inhibit excessive PINK1/Parkin-mediated mitophagy.

The preventive effect of liraglutide on the lipotoxic liver injury via increasing autophagy.

INTRODUCTION AND OBJECTIVES: The incidence of non-alcoholic fatty liver disease (NAFLD) is increasing. Previous studies indicated that Liraglutide, glucagon-like peptide-1 analogue, could regulate glucose homeostasis as a valuable treatment for Type 2 Diabetes. However, the precise effect of Liraglutide on NAFLD model in rats and the mechanism remains unknown. In this study, we investigated the molecular mechanism by which Liraglutide ameliorates hepatic steatosis in a high-fat diet (HFD)-induced rat model of NAFLD in vivo and in vitro. MATERIALS AND METHODS: NALFD rat models and hepatocyte steatosis in HepG2 cells were induced by HFD and palmitate fatty acid treatment, respectively. AMPK inhibitor, Compound C was added in HepG2 cells. Autophagy-related proteins LC3, Beclin1 and Atg7, and AMPK pathway-associated proteins were evaluated by Western blot and RT-PCR. RESULTS: Liraglutide enhanced autophagy as showed by the increased expression of the autophagy markers LC3, Beclin1 and Atg7 in HFD rats and HepG2 cells treated with palmitate fatty acid. In vitro, The AMPK inhibitor exhibited an inhibitory effect on Liraglutide-induced autophagy enhancement with the deceased expression of LC3, Beclin1 and Atg7. Additionally, Liraglutide treatment elevated AMPK levels and TSC1, decreased p-mTOR expression. CONCLUSIONS: Liraglutide could upregulate autophagy to decrease lipid over-accumulation via the AMPK/mTOR pathway.

Aqua-soluble DDQ reduces the levels of Drp1 and Aß and inhibits abnormal interactions between Aß and Drp1 and protects Alzheimer's disease neurons from Aß- and Drp1-induced mitochondrial and synaptic toxicities.

The purpose of our study was to develop a therapeutic target that can reduce Aß and Drp1 levels, and also can inhibit abnormal interactions between Aß and Drp1 in AD neurons. To achieve this objective, we designed various compounds and their 3-dimensional molecular structures were introduced into Aß and Drp1 complex and identified their inhibitory properties against Aß-Drp1 interaction. Among all, DDQ was selected for further investigation because of 1) its best docking score and 2) its binding capability at interacting sites of Drp1 and Aß complex. We synthesized DDQ using retro-synthesis and analyzed its structure spectrally. Using biochemical, molecular biology, immunostaining and transmission electron microscopy (TEM) methods, we studied DDQ's beneficial effects in AD neurons. We measured the levels of Aß and Drp1, Aß and Drp1 interaction, mRNA and protein levels of mitochondrial dynamics, biogenesis and synaptic genes, mitochondrial function and cell viability and mitochondrial number in DDQ-treated and untreated AD neurons. Our qRT-PCR and immunoblotting analysis revealed that reduced levels of mitochondrial fission and increased fusion, biogenesis and synaptic genes in DDQ-treated AD neurons. Our immunoblotting and immunostaining analyses revealed that Aß and Drp1 levels were reduced in DDQ-treated AD neurons. Interaction between Aß and Drp1 is reduced in DDQ-treated AD neurons. Aß42 levels were significantly reduced in DDQ-treated mutant APPSwe/Ind cells. Mitochondrial number is significantly reduced and mitochondrial length is significantly increased. Mitochondrial function and cell viability were maintained in AD neurons treated with DDQ. These observations indicate that DDQ reduces excessive mitochondrial fragmentation, enhances fusion, biogenesis and synaptic activity and reduces Aß42 levels and protects AD neurons against Aß-induced mitochondrial and synaptic toxicities.

Pharmacological inhibition of mTORC1 but not mTORC2 protects against human disc cellular apoptosis, senescence, and extracellular matrix catabolism through Akt and autophagy induction.

OBJECTIVE: The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that integrates nutrients to execute cell growth. We hypothesized that mTOR is influential in the intervertebral disc-largest avascular, low-nutrient organ. Our objective was to identify the optimal mTOR inhibitor for treating human degenerative disc disease. DESIGN: mTOR complex 1 (mTORC1) regulates p70/ribosomal S6 kinase (p70/S6K), negatively regulates autophagy, and is controlled by Akt. Akt is controlled by phosphatidylinositol 3-kinase (PI3K) and mTOR complex 2 (mTORC2). mTORC1 inhibitors-rapamycin, temsirolimus, everolimus, and curcumin, mTORC1&mTORC2 inhibitor-INK-128, PI3K&mTOR inhibitor-NVP-BEZ235, and Akt inhibitor-MK-2206-were applied to human disc nucleus pulposus (NP) cells. mTOR signaling, autophagy, apoptosis, senescence, and matrix metabolism were evaluated. RESULTS: mTORC1 inhibitors decreased p70/S6K but increased Akt phosphorylation, promoted autophagy with light chain 3 (LC3)-II increases and p62/sequestosome 1 (p62/SQSTM1) decreases, and suppressed pro-inflammatory interleukin-1 beta (IL-1ß)-induced apoptotic terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positivity (versus rapamycin, 95% confidence interval (CI) -0.431 to -0.194; temsirolimus, 95% CI -0.529 to -0.292; everolimus, 95% CI -0.477 to -0.241; curcumin, 95% CI -0.248 to -0.011) and poly (ADP-ribose) polymerase (PARP) and caspase-9 cleavage, senescent senescence-associated beta-galactosidase (SA-ß-gal) positivity (versus rapamycin, 95% CI -0.437 to -0.230; temsirolimus, 95% CI -0.534 to -0.327; everolimus, 95% CI -0.485 to -0.278; curcumin, 95% CI -0.210 to -0.003) and p16/INK4A expression, and catabolic matrix metalloproteinase (MMP) release and activation. Meanwhile, dual mTOR inhibitors decreased p70/S6K and Akt phosphorylation without enhanced autophagy and suppressed apoptosis, senescence, and matrix catabolism. MK-2206 counteracted protective effects of temsirolimus. Additional disc-tissue analysis found relevance of mTOR signaling to degeneration grades. CONCLUSION: mTORC1 inhibitors-notably temsirolimus with an improved water solubility-but not dual mTOR inhibitors protect against inflammation-induced apoptosis, senescence, and matrix catabolism in human disc cells, which depends on Akt and autophagy induction.

Recombinant Buckwheat Trypsin Inhibitor Improves the Protein and Mitochondria Homeostasis in Caenorhabditis elegans Model of Aging and Age-Related Disease.

BACKGROUND: With the acceleration of aging process in human society, improvements of the physical functionality and life quality in the elderly population are more meaningful than pure longevity. Buckwheat trypsin inhibitor is a low molecular weight polypeptide extracted from buckwheat, which is a beneficial food for improving the health in the elderly. OBJECTIVES: The aim of the current study was to evaluate the potential beneficial effects of recombinant buckwheat trypsin inhibitor (rBTI) on age-dependent function decline and the primary mechanism. METHOD: Day 10 N2 Caenorhabditis elegans and day 6 AM140 C. elegans cultured at 25°C were used as models of aging and age-related disease, respectively. Motor function was as an indicator of age-dependent function. ATP content and damage mitochondrial DNA mass were detected to assess mitochondrial damage and function by ATP Assay Kit and agarose gel electrophoresis, respectively. Soluble protein content was quantified by SDS polyacrylamide gel electrophoresis. Autophagy-related genes transcription levels, autophagy marker proteins lgg-1, and lysosomal content were analyzed to quantify autophagy levels by qRT-PCR, transgenic C. elegans, and lysosomal staining. Autophagy inhibitor chloroquine, daf-16 mutant, and RNA Interference were used to determine the roles of autophagy and DAF-16 in rBTI-mediated effects. RESULTS: In this study, we found that rBTI could decrease the proportions of insoluble protein and impaired mitochondria, finally reduce motility deficits in both models. Further study indicated that rBTI activated the autophagy, and the inhibition of autophagy reduced rBTI-mediated beneficial effects. Genetic analyses showed the transcriptional activity of DAF-16 was increased by rBTI and was required for rBTI-mediated beneficial effects. CONCLUSIONS: These data indicated that rBTI might promote the autophagy to alleviate the age-related functional decline via DAF-16 in C. elegans and suggested a potential role of rBTI as a nutraceutical for the improvement of age-related complications.

Selective interference of mTORC1/RAPTOR protects against human disc cellular apoptosis, senescence, and extracellular matrix catabolism with Akt and autophagy induction.

OBJECTIVE: The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that integrates nutrients to execute cell growth and protein synthesis. We hypothesized that mTOR is essential for the intervertebral disc, the largest avascular, low-nutrient organ. Our objective was to elucidate roles of mTOR signaling in human disc cells. DESIGN: The mTOR exists in two complexes: mTORC1 containing the regulatory-associated protein of mTOR (RAPTOR) and mTORC2 containing the rapamycin-insensitive companion of mTOR (RICTOR). To analyze their functions in human disc nucleus pulposus cells, RNA interference (RNAi) of mTOR targeting mTORC1 and mTORC2, RAPTOR targeting mTORC1, or RICTOR targeting mTORC2 or rapamycin, a pharmacological mTORC1 inhibitor, was applied. First, mTOR signaling including Akt, p70/ribosomal S6 kinase (p70/S6K), and autophagy were assessed. Then, apoptosis, senescence, and matrix metabolism were evaluated under pro-inflammatory interleukin-1 beta (IL-1ß) stimulation. RESULTS: Western blotting showed significant decreases in specific proteins by each RNAi (all P < 0.0001). In mTOR signaling, RNAi of mTOR and RICTOR decreased p70/S6K and Akt phosphorylation, whereas RAPTOR RNAi decreased p70/S6K but increased Akt phosphorylation. All RNAi treatments increased light chain 3 (LC3)-II and decreased p62/sequestosome 1 (p62/SQSTM1), indicating enhanced autophagy. In apoptosis, IL-1ß-induced terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells and poly (ADP-ribose) polymerase (PARP) and caspase-9 cleavage decreased by RAPTOR RNAi. In senescence, IL-1ß-induced senescence-associated beta-galactosidase (SA-ß-gal)-positive cells and p16/INK4A expression also decreased by RAPTOR RNAi. In matrix metabolism, RAPTOR RNAi reduced IL-1ß-induced catabolic matrix metalloproteinase (MMP) release and activation and up-regulated anabolic gene expression. These findings were all consistent with rapamycin administration. Additional disc-tissue analysis detected expression and phosphorylation of mTOR-signaling molecules in varying ages. CONCLUSION: Selective interference of mTORC1/RAPTOR protects against inflammation-induced apoptosis, senescence, and matrix catabolism possibly through Akt and autophagy induction in human disc cells.

Autophagy plays beneficial effect on diabetic encephalopathy in type 2 diabetes: studies in vivo and in vitro.

OBJECTIVES: The hypothalamus regulates metabolism and feeding behavior by perceiving the levels of peripheral insulin. However, little is known about the hypothalamic changes after aberrant metabolism. In this study, we investigated the changes of insulin and autophagy relevant signals of hypothalamus under diabetes mellitus. METHODS: C57B/L mice were injected with low-dose streptozotocin (STZ) and fed with high-fat diet to induce type 2 diabetes mellitus. In vitro, PC12 cells were treated with oleic acid to mimic lipotoxicity. RESULTS: Results showed that the cholesterol level in the hypothalamus of the diabetic mice was higher than that of the normal mice. The expression of insulin receptors and insulin receptor substrate-1 were downregulated and the number of Fluoro-Jade C positive cells significantly increased in the hypothalamic arcuate nucleus of the diabetic mice. Furthermore, Upregulation of mammalian target of rapamycin (mTOR) and downregulation of LC 3II were obvious in the hypothalamus of the diabetic mice. In vitro, results showed that high-lipid caused PC12 cell damage and upregulated LC3 II expression. Pretreatment of cells with 3-methyladenine evidently downregulated LC3 II expression and aggravated PC12 cell death under high lipid conditions. By contrast, pretreatment of cells with rapamycin upregulated LC3 II expression and ameliorated PC12 cell death caused by lipotoxicity. CONCLUSION: These results demonstrate that autophagy activation confers protection to neurons under aberrant metabolism and that autophagy dysfunction in the hypothalamus occurs in the chronic metabolic disorder such as T2DM.

Centrosomal Protein 70 Is a Mediator of Paclitaxel Sensitivity.

Centrosome aberrations have been implicated in the development and progression of breast cancer. Our previous worked show that centrosomal protein 70 (Cep70) regulates breast cancer growth and metastasis. However, it remains elusive whether Cep70 is implicated in the sensitivity of the anti-microtubule drug paclitaxel in breast cancer. Here we provide evidence that Cep70 is a mediator of paclitaxel sensitivity in breast cancer. Cell proliferation assays show that Cep70 expression correlates with paclitaxel sensitivity in breast cancer cell lines. In addition, paclitaxel sensitivity varies when altering Cep70 expression level. Mechanistic studies reveal that Cep70 interacts with tubulin, and promotes the ability of paclitaxel to stimulate microtubule assembly. These data demonstrate that Cep70 mediates paclitaxel sensitivity in breast cancer.

14-Deoxy-11,12-didehydroandrographolide induces DDIT3-dependent endoplasmic reticulum stress-mediated autophagy in T-47D breast carcinoma cells.

14-Deoxy-11,12-didehydroandrographolide (14-DDA), a major diterpenoid isolated from Andrographis paniculata (Burm.f.) Nees, is known to be cytotoxic and elicits a non-apoptotic cell death in T-47D breast carcinoma cells. In this study, the mechanistic toxicology properties of 14-DDA in T-47D cells were further investigated. 14-DDA is found to induce the formation of endoplasmic reticulum (ER) vacuoles and autophagosomes, with concurrent upregulation of LC3-II in the breast carcinoma cells. It stimulated an increase in cytosolic calcium concentration and caused a collapse in mitochondrial membrane potential in these cells. In addition, both DDIT3 and GADD45A, molecules implicated in ER stress pathway, were significantly upregulated. DDIT3 knockdown suppressed the formation of both ER vacuoles and autophagosomes, indicating that 14-DDA-induced ER stress and autophagy is dependent on this transcription factor. Collectively, it is possible that GADD45A/p38 MAPK/DDIT3 pathway is involved in the 14-DDA-induced ER-stress-mediated autophagy in T-47D cells.

Honokiol induces autophagic cell death in malignant glioma through reactive oxygen species-mediated regulation of the p53/PI3K/Akt/mTOR signaling pathway.

Honokiol, an active constituent extracted from the bark of Magnolia officinalis, possesses anticancer effects. Apoptosis is classified as type I programmed cell death, while autophagy is type II programmed cell death. We previously proved that honokiol induces cell cycle arrest and apoptosis of U87 MG glioma cells. Subsequently in this study, we evaluated the effect of honokiol on autophagy of glioma cells and examined the molecular mechanisms. Administration of honokiol to mice with an intracranial glioma increased expressions of cleaved caspase 3 and light chain 3 (LC3)-II. Exposure of U87 MG cells to honokiol also induced autophagy in concentration- and time-dependent manners. Results from the addition of 3-methyladenine, an autophagy inhibitor, and rapamycin, an autophagy inducer confirmed that honokiol-induced autophagy contributed to cell death. Honokiol decreased protein levels of PI3K, phosphorylated (p)-Akt, and p-mammalian target of rapamycin (mTOR) in vitro and in vivo. Pretreatment with a p53 inhibitor or transfection with p53 small interfering (si)RNA suppressed honokiol-induced autophagy by reversing downregulation of p-Akt and p-mTOR expressions. In addition, honokiol caused generation of reactive oxygen species (ROS), which was suppressed by the antioxidant, vitamin C. Vitamin C also inhibited honokiol-induced autophagic and apoptotic cell death. Concurrently, honokiol-induced alterations in levels of p-p53, p53, p-Akt, and p-mTOR were attenuated following vitamin C administration. Taken together, our data indicated that honokiol induced ROS-mediated autophagic cell death through regulating the p53/PI3K/Akt/mTOR signaling pathway.

Hydroxycamptothecin shows antitumor efficacy on HeLa cells via autophagy activation mediated apoptosis in cervical cancer.

OBJECTIVES: To investigate the effect and the mechanism of anti-tumor agent hydroxycamptothecin (HCPT) on HeLa cells in cervical cancer. MATERIALS AND METHODS: Autophagy and apoptosis were detected by western blotting and the transfection of GFP-LC3 shRNA as well as Hoechst staining. RESULTS: The authors found that the expression of the regulators of Beclin-1, p62, and microtubule-associated protein 1 light chain 3 (LC3) upregulated and then triggered the occurrence of cell autophagy. On the other hand, HCPT could induce to the formation of autophagy and resulted in cell apoptosis after autophagy. CONCLUSION: HCPT can alter cell autophagy and then trigger cell apoptosis to achieve antitumor effects.

Phellinus linteus extract induces autophagy and synergizes with 5-fluorouracil to inhibit breast cancer cell growth.

Phellinus linteus (PL) is a medicinal mushroom due to its several biological properties, including anticancer activity. However, the mechanisms of its anticancer effect remain to be elucidated. We evaluated the inhibitory effects of the ethanolic extract from the PL combined with 5-FU on MDA-MB-231 breast cancer cell line and to determine the mechanism of cell death. Individually, PL extract and 5-FU significantly inhibited the proliferation of MDA-MB-231 cells in a dose-dependent manner. PL extract (30 mg/mL) in combination with 5-FU (10 µg/mL) synergistically inhibited MDA-MB-231 cells by 1.8-fold. PL did not induce apoptosis, as demonstrated by the DNA fragmentation assay, the sub-G1 population, and staining with annexin V-FITC and propidium iodide. The exposure of MDA-MB-231 cells to PL extracts resulted in several confirmed characteristics of autophagy, including the appearance of autophagic vacuoles revealed by monodansylcadaverine staining, the formation of acidic vesicular organelles, autophagosome membrane association of microtubule-associated protein light chain 3 (LC3) characterized by cleavage of LC3 and its punctuate redistribution, and ultrastructural observation of autophagic vacuoles by transmission electron microscopy. We concluded that PL extracts synergized with low doses of 5-FU to inhibit triple-negative breast cancer cell growth and demonstrated that PL extract can induce autophagy-related cell death.

Corticosteroids block autophagy protein recruitment in Aspergillus fumigatus phagosomes via targeting dectin-1/Syk kinase signaling.

Aspergillus fumigatus is the predominant airborne fungal pathogen in immunocompromised patients. Genetic defects in NADPH oxidase (chronic granulomatous disease [CGD]) and corticosteroid-induced immunosupression lead to impaired killing of A. fumigatus and unique susceptibility to invasive aspergillosis via incompletely characterized mechanisms. Recent studies link TLR activation with phagosome maturation via the engagement of autophagy proteins. In this study, we found that infection of human monocytes with A. fumigatus spores triggered selective recruitment of the autophagy protein LC3 II in phagosomes upon fungal cell wall swelling. This response was induced by surface exposure of immunostimulatory ß-glucans and was mediated by activation of the Dectin-1 receptor. LC3 II recruitment in A. fumigatus phagosomes required spleen tyrosine kinase (Syk) kinase-dependent production of reactive oxygen species and was nearly absent in monocytes of patients with CGD. This pathway was important for control of intracellular fungal growth, as silencing of Atg5 resulted in impaired phagosome maturation and killing of A. fumigatus. In vivo and ex vivo administration of corticosteroids blocked LC3 II recruitment in A. fumigatus phagosomes via rapid inhibition of phosphorylation of Src and Syk kinases and downstream production of reactive oxygen species. Our studies link Dectin-1/Syk kinase signaling with autophagy-dependent maturation of A. fumigatus phagosomes and uncover a potential mechanism for development of invasive aspergillosis in the setting of CGD and corticosteroid-induced immunosupression.

[Effects of polygala on the neurogenesis of manganese poisoned mice].

OBJECTIVE: To investigate the effects of polygala on leaning and memory and the expression of Microtubule associated protein on manganese poisoned mice. METHODS: 60 female Kunming mice were randomly and equally divided into 5 group. They are normal control group (CG), manganese poisoned group (MG), manganese poisoned with polygala high dose group (MHG), manganese poisoned with polygala middle dose group (MMG), manganese poisoned with polygala low dose group (MLG). The model of manganese poisoned mice was prepared of the way of intraperitoneal injection of manganese chloride (MnCl2 15 mg/kg), the spatial learning and memory ability was tested by Morris water maze, the Doublecortin (DCX) was tested by the way of immunofluorescent staining in the SVZ and SGZ. RESULT: In the navigation test, compared with MG, the escape latency of MHG, MMG and MLG were significantly decreased (P < 0.05), in space exploration experiments, MHG, MMG, MLG compared with MG, the number increased significantly across platforms (P < 0.05). compared with MG, the DCX expression of MHG, MMG and MLG were significantly increased (P < 0.05). CONCLUTION: The leaning and memory ability of manganese poisoned mice can be improved by the polygala, and the mechanism may be related to promote the expression of DCX and neurogenesis in the brain.

Glucosamine activates autophagy in vitro and in vivo.

OBJECTIVE: Aging-associated changes in articular cartilage represent a main risk factor for osteoarthritis (OA). Autophagy is an essential cellular homeostasis mechanism. Aging-associated or experimentally induced defects in autophagy contribute to organismal- and tissue-specific aging, while enhancement of autophagy may protect against certain aging-related pathologies such as OA. The objective of this study was to determine whether glucosamine can activate autophagy. METHODS: Chondrocytes from normal human articular cartilage were treated with glucosamine (0.1- 10 mM). Autophagy activation and phosphorylation levels of Akt, FoxO3, and ribosomal protein S6 were determined by Western blotting. Autophagosome formation was analyzed by confocal microscopy. Reporter mice systemically expressing green fluorescent protein (GFP) fused to light chain 3 (LC3) (GFP-LC3-transgenic mice) were used to assess changes in autophagy in response to starvation and glucosamine treatment. RESULTS: Glucosamine treatment of chondrocytes activated autophagy, as indicated by increased LC3-II levels, formation of LC3 puncta, and increased LC3 turnover. This was associated with glucosamine-mediated inhibition of the Akt/FoxO3/mammalian target of rapamycin pathway. Administration of glucosamine to GFP-LC3-transgenic mice markedly activated autophagy in articular cartilage. CONCLUSION: Glucosamine modulates molecular targets of the autophagy pathway in vitro and in vivo, and the enhancement of autophagy is mainly dependent on the Akt/FoxO/mTOR pathway. These findings suggest that glucosamine is an effective autophagy activator and should motivate future studies on the efficacy of glucosamine in modifying aging-related cellular changes and supporting joint health.