Autophagic Levels Research Articles

Prognostic Role of Circulating Exosomal miR-425-3p for the Response of NSCLC to Platinum-Based Chemotherapy.

Platinum-based doublets with a third-generation agent are the recommended option for many patients with non-small cell lung cancer (NSCLC) with no contraindications to platinum compounds. Unfortunately, the clinical effectiveness of such chemotherapy is limited by intrinsic or acquired resistance. Circulating exosomal miRNAs were isolated and used to perform HiSeq deep-sequencing analyses on serum pool samples from platinum-resistant or platinum-sensitive patients, and six exosomal miRNAs were further validated for their predictive utility by qRT-PCR in 170 serum samples of patients with advanced NSCLC. Gain- and loss-of-function experiments clarified the responsiveness regulating role of the clinically relevant miRNA. IHC analyses were performed to evaluate the association between basal autophagy in lung cancer tissues and responsiveness in 203 patients with NSCLC receiving platinum-based chemotherapy. Six circulating exosomal miRNAs (miR-425-3p, miR-1273h, miR-4755-5p, miR-9-5p, miR-146a-5p, and miR-215-5p) were found to be differentially expressed with the largest fold change in platinum-resistant patients compared with platinum-sensitive patients. High miR-425-3p proved to be a potent predictive biomarker for low responsiveness and poor progression-free survival (PFS). Mechanistically, miR-425-3p upregulated the autophagic levels via targeting AKT1, leading to the decrease in therapeutic response. Concordantly, high levels of basal autophagy in lung cancer tissues correlate with low responsiveness in patients with NSCLC within the early and advanced disease stages. Our study highlights circulating exosomal miR-425-3p as a potential biomarker for improved predictions of the clinical response to platinum-based chemotherapy in patients with NSCLC. This study provides the first evidence that miR-425-3p in NSCLC patient-derived exosomes can be a marker for predicating the clinical response to platinum-based chemotherapy.

Sestrin-like protein from Dictyostelium discoideum is involved in autophagy under starvation stress

Sestrins are highly conserved; stress inducible proteins that help maintain metabolic homeostasis and protect cells under stress conditions. They are up-regulated during stress and influence AMPK and mTOR pathways. Our objective was to find the role of Sestrin protein from Dictyostelium discoideum (Dd), a lower eukaryote where starvation stress initiates multicellular development. The single DdSesn-like gene was expressed and its endogenous functions were characterized. Both, the knockout and constitutively expressing strains were made and their involvement in starvation-induced autophagy was analyzed. Autophagic fluxes and ROS levels were also monitored. Additionally, overexpression of DdSesn decreased cell growth and showed a longer lag phase. Upon starvation both DdSesn and ROS levels increased. SesnOE showed reduced ROS levels while sesn− showed increased ROS levels when compared to the wild type. Therefore, we suggest that increased sesn expression may be beneficial in reducing ROS levels during starvation. Deletion of sesn showed reduced autophagic flux and increased p4EBP1 levels. We show that DdSesn promotes autophagy in D. discoideum upon starvation.

Basal autophagy is required for promoting dendritic terminal branching in Drosophila sensory neurons.

Dendrites function as the primary sites for synaptic input and integration with impairments in dendritic arborization being associated with dysfunctional neuronal circuitry. Post-mitotic neurons require high levels of basal autophagy to clear cytotoxic materials and autophagic dysfunction under native or cellular stress conditions has been linked to neuronal cell death as well as axo-dendritic degeneration. However, relatively little is known regarding the developmental role of basal autophagy in directing aspects of dendritic arborization or the mechanisms by which the autophagic machinery may be transcriptionally regulated to promote dendritic diversification. We demonstrate that autophagy-related (Atg) genes are positively regulated by the homeodomain transcription factor Cut, and that basal autophagy functions as a downstream effector pathway for Cut-mediated dendritic terminal branching in Drosophila multidendritic (md) sensory neurons. Further, loss of function analyses implicate Atg genes in promoting cell type-specific dendritic arborization and terminal branching, while gain of function studies suggest that excessive autophagy leads to dramatic reductions in dendritic complexity. We demonstrate that the Atg1 initiator kinase interacts with the dual leucine zipper kinase (DLK) pathway by negatively regulating the E3 ubiquitin ligase Highwire and positively regulating the MAPKKK Wallenda. Finally, autophagic induction partially rescues dendritic atrophy defects observed in a model of polyglutamine toxicity. Collectively, these studies implicate transcriptional control of basal autophagy in directing dendritic terminal branching and demonstrate the importance of homeostatic control of autophagic levels for dendritic arbor complexity under native or cellular stress conditions.

Carvedilol attenuates liver fibrosis by suppressing autophagy and promoting apoptosis in hepatic stellate cells.

Carvedilol has been identified as a promising agent for the treatment of liver fibrosis. Meanwhile, autophagy and apoptosis have been reported to play key roles in the activation of hepatic stellate cells (HSCs), which can contribute to the progression of liver fibrosis. However, the effects of carvedilol on autophagy and apoptosis in HSCs remain unclear. Our study aimed to detect these effects and identify the underlying mechanisms by which carvedilol mediates HSC autophagy and apoptosis. For this purpose, the LX-2 cell line was used in this study, and the cells were exposed to various concentrations of carvedilol for specific times. First, we found that carvedilol increased autophagic marker levels, the number of GFP-LC3-containing puncta and LC3B-II levels in LX-2 cells. Interestingly, the addition of chloroquine (CQ) failed to enhance the effects on GFP-LC3 puncta and LC3B-II levels, and carvedilol treatment resulted in a significant increase in p62 protein levels. Moreover, carvedilol treatment led to the accumulation of yellow dots only in GFP-RFP-LC3-LX-2 cells, similar to the results following CQ treatment, indicating that carvedilol inhibited autophagic flux. Next, we found evidence that carvedilol inhibited autophagic flux by increasing lysosomal pH and not by impairing the fusion of autophagosomes with lysosomes. Moreover, carvedilol substantially reduced the viability of LX-2 cells and noticeably induced cell apoptosis, as observed by flow cytometry. In addition, increased levels of cleaved caspase-3, cleaved caspase-8 and cleaved PARP, increased Bax activity and decreased Bcl-2 expression were detected in LX-2 cells. Finally, the carvedilol treatment inhibited autophagy and subsequently induced apoptosis in vitro. In conclusion, carvedilol suppresses autophagy and promotes apoptosis in HSCs and the late-stage inhibition of autophagy preceded the induction of apoptosis.

Andrographolide simultaneously augments Nrf2 antioxidant defense and facilitates autophagic flux blockade in cigarette smoke-exposed human bronchial epithelial cells

Cigarette smoking is the leading cause of chronic obstructive pulmonary disease (COPD). Cigarette smoke heightens oxidative stress and impairs autophagy, advancing COPD progression. Andrographolide is a bioactive diterpenoid lactone isolated from the plant Andrographis paniculata which has been a traditional medicinal herb for respiratory diseases. As airway epithelial cells form the first interface to be exposed to cigarette smoke, this study aimed to explore the modulatory effects of andrographolide on oxidative stress and autophagy in human bronchial epithelial BEAS-2B cells exposed to cigarette smoke extract (CSE). CSE (2%) exposure increased autophagic markers p62 and LC3B-II levels in BEAS-2B cells. Andrographolide alone increased p62 and p-p62 (S349) but not LC3B-II in BEAS-2B cells. However, in the presence of CSE, andrographolide was able to simultaneously increase LC3B-II level and enhance antioxidant defense by decreasing oxidative stress and increasing total antioxidant capacity, through upregulation of nuclear Nrf2 via the p62-Nrf2 positive feedback loop. Using RFP-GFP-LC3B transfected BEAS-2B cells exposed to CSE, andrographolide was found to impair autophagosome fusion with lysosome, which may account for the moderate increase in activated caspase 3/7 and annexin V levels. Our findings revealed for the first time that andrographolide simultaneously upregulated antioxidant defense through the p62-Nrf2 loop and moderately induced apoptosis through impairment of autophagic flux in CSE-exposed bronchial epithelium. Andrographolide facilitated cigarette smoke-induced apoptosis may be a potential toxicological outcome or may protect against chronic inflammation and aberrant DNA repair. Validation of these in-vitro findings in an experimental COPD model by andrographolide is warranted.

RB1CC1-enhanced autophagy facilitates PSCs activation and pancreatic fibrogenesis in chronic pancreatitis

Chronic pancreatitis (CP) is described as a progressive fibro-inflammatory disorder of the exocrine disease, which eventually leads to damage of the gland. Excessive activation of pancreatic stellate cells (PSCs) is a critical participant in the initiation of CP. Autophagy is involved in multiple degeneration and inflammation in acute pancreatitis and CP. In our study, we report that retinoblastoma coiled coil protein 1 (RB1CC1) expression and the autophagic level are elevated in activated PSCs. RB1CC1 is positively correlated with pancreatic fibrogenesis in tissues and plasma of CP patients. Knockdown of RB1CC1 restrains alpha smooth muscle actin (α-SMA) and collagen expressions, and autophagy in activated PSCs in vitro. Furthermore, we show that RB1CC1 induces PSC activation via binding to ULK1 promoter and the direct interaction with ULK1 protein. These suppress ULK1 expression and its kinase activity. In mice, knockdown of RB1CC1 blocks autophagy and then inhibits the pancreatic duct ligation-induced pancreatic fibrosis. Consequently, our study highlights that RB1CC1-mediated autophagy is a key event for the activation of PSCs. Inhibition of RB1CC1 alleviates autophagy, which plays a critical role in anti-fibrotic activation in PSCs and CP progression. RB1CC1 could be a novel strategy for the treatment of pancreatic fibrosis.

IP3 receptor blockade restores autophagy and mitochondrial function in skeletal muscle fibers of dystrophic mice

Duchenne muscular dystrophy (DMD) is characterized by a severe and progressive destruction of muscle fibers associated with altered Ca2+ homeostasis. We have previously shown that the IP3 receptor (IP3R) plays a role in elevating basal cytoplasmic Ca2+ and that pharmacological blockade of IP3R restores muscle function. Moreover, we have shown that the IP3R pathway negatively regulates autophagy by controlling mitochondrial Ca2+ levels. Nevertheless, it remains unclear whether IP3R is involved in abnormal mitochondrial Ca2+ levels, mitochondrial dynamics, or autophagy and mitophagy observed in adult DMD skeletal muscle. Here, we show that the elevated basal autophagy and autophagic flux levels were normalized when IP3R was downregulated in mdx fibers. Pharmacological blockade of IP3R in mdx fibers restored both increased mitochondrial Ca2+ levels and mitochondrial membrane potential under resting conditions. Interestingly, mdx mitochondria changed from a fission to an elongated state after IP3R knockdown, and the elevated mitophagy levels in mdx fibers were normalized. To our knowledge, this is the first study associating IP3R1 activity with changes in autophagy, mitochondrial Ca2+ levels, mitochondrial membrane potential, mitochondrial dynamics, and mitophagy in adult mouse skeletal muscle. Moreover, these results suggest that increased IP3R activity in mdx fibers plays an important role in the pathophysiology of DMD. Overall, these results lead us to propose the use of specific IP3R blockers as a new pharmacological treatment for DMD, given their ability to restore both autophagy/mitophagy and mitochondrial function.

Intermittent high glucose-induced oxidative stress modulates retinal pigmented epithelial cell autophagy and promotes cell survival via increased HMGB1

BackgroundIn this study, we evaluated the effects of intermittent high glucose on oxidative stress production in retinal pigmented epithelial (RPE) cells and explored whether the mechanisms of autophagy and apoptosis in oxidative stress are associated with high-mobility group box 1 (HMGB1) protein.MethodsCultured human RPE cell line ARPE-19 cells were exposed to intermittent high glucose-induced oxidative stress. Reactive oxygen species (ROS) was determined by 2′, 7′-dichlorofluorescin diacetate (DCFH-DA); and malonyldialdehyde (MDA), superoxide dismutase (SOD) by commercial kits. Transmission electron microscopy was used to observe the generation of autophagosome. And MTT assay was used to examine the effect of autophagy on cell viability. For the inhibition experiments, cells were pre-incubated with lysosomal inhibitors NH4Cl or N-acetyl cysteine (NAC).Western blot was used to measure the expression patterns of autophagic markers, including LC3 and p62. The expression of HMGB1 was detected by immunohistochemistry.Cells were pre-incubated with HMGB1 inhibitor ethyl pyruvate (EP) ,then detected the expression pattern of autophagic markers and level of cellular ROS.ResultsWe found that intermittent high glucose significantly increased oxidative stress levels (as indicated by ROS, MDA, SOD), increased in the generation of autophagosome, decreased the level of p62, induced conversion of LC3 I to LC3 II. We further demonstrated that the NH4Cl/NAC inhibited intermittent high glucose-induced autophage by altered level of LC3 and p62. Intermittent high glucose-induced autophagy is independent of HMGB1 signaling, inhibition of HMGB1 release by EP decreased expression pattern of autophagic markers and level of cellular viability.ConclusionsUnder intermittent high glucose condition, autophagy may be required for preventing oxidative stress-induced injury in RPE. HMGB1 plays important roles in signaling for both autophagy and oxidative stress.

Emodin Attenuates Autophagy Response to Protect the Pancreas From Acute Pancreatitis Failure.

The aim of this study was to investigate the effects of emodin on attenuating autophagy response in acute pancreatitis (AP) models. Acute pancreatitis was induced in Wistar rats by injecting 3% sodium taurocholate into the biliopancreatic duct. Emodin (40 mg/kg per day) was then given intragastrically, administrated 2 hours after AP induction. Rats were killed 24 hours after AP induction. The pancreatic injury was assessed using biochemical and histological approaches. Autophagosomes in pancreatic acinar cells were observed by electron microscopy. The expression levels of microtubule-associated protein 1 light chain 3 (LC3) B/A, beclin-1, and p62/SQSTM1 (p62) were detected by Western blotting, quantitative real-time polymerase chain reaction, and immunohistochemistry in pancreatic tissues. Compared with non-emodin-treated rats, the pathological injuries of the pancreas of emodin-treated rats were significantly alleviated, and autophagy vacuole formation was reduced within pancreatic acinar cells. Administration of emodin led to a reduction in the autophagy-associated protein level of LC3 (B/A) and p62 but not beclin-1. The transcript levels of LC3B, beclin-1, and p62 were decreased in the emodin-treated rats compared with non-emodin-treated rats. Our data demonstrate that emodin plays a critical role in ameliorating AP, possibly by down-regulating autophagic protein levels.

Inhibiting autophagy promotes collagen degradation by regulating matrix metalloproteinases in pancreatic stellate cells

AimsAutophagy is an intracellular metabolic process that degrades and recycles own constituents to maintain homeostasis and supply substrates. Disruption of collagen degradation is one of the pathogenesis of pancreatic fibrosis. In this study, we investigated the effects of inhibiting autophagy on the collagen degradation of PSCs. Main methodsRats were injected dibutyltin dichloride (DBTC) to induce chronic pancreatitis (CP) model. The expression of LC3B was measured by western blotting. Rat PSCs were isolated from pancreas tissues, and the experiments used the primary PSCs. Autophagosome was confirmed by transmission electron microscope. Immunofluorescence for LC3B and α-SMA were applied to assess autophagy and activated PSCs. The effects of autophagy inhibition of 3-MA on the expressions of LC3B, Atg5, and Beclin-1 were investigated by real-time PCR and Western blotting, as well as the α-SMA, TGF-β1, ColI, Col III, FN, MMP-2, MMP-13, TIMP-1 and TIMP-2. Meanwhile, the secretion of ColI, Col III and FN were investigated by ELISA. Key findingsThe LC3-II/I ratio was increased in rat CP model. Autophagosomes and an increased autophagic level were observed during PSCs activation. Inhibiting autophagy could down-regulate the expressions of α-SMA, TGF-β1, FN, ColI, Col III, TIMP-1 and TIMP-2, while the expressions of MMP-2 and MMP-13 were increased. SignificanceThis study confirmed that autophagic level is increased during PSCs activation in vivo and in vitro. Inhibiting autophagy prevents the activation of PSCs, and suppresses fibrosis through promoting extracellular matrix (ECM) degradation by decreasing the expression of TGF-β1 and increasing MMPs/TIMPs ratio.

PARP-1 inhibition attenuates cardiac fibrosis induced by myocardial infarction through regulating autophagy

Post-MI heart failure is characterized by structural remodeling, in which intramyocardial fibrosis takes a important part. Poly(ADP-ribose) polymerase 1 (PARP-1) is a extensive nuclear enzyme and plays a critical role in various diseases. It was shown that PARP-1 inhibition could alleviate heart failure and dowregulate autophagy, but whether PARP-1 regulates autophagy and thus impacts the activities of CFs remain unknown. We transfected cultured cardiac fibroblasts (CFs) with small interfere RNA-PARP-1 (siPARP-1) to downregulate PARP-1 and analyzed the ability of proliferation, migration, differentiation, and autophagy levels of CFs under different treatments using CCK8 assays, transwell migration assays, immunofluorescence assays detecting expression of α-SMA, western blot assays detecting autophagy-related proteins respectively. Furthermore, rat models of myocardial infarction (MI) were induced by ligation of left anterior descending coronary artery and PARP-1 inhibitor, 4-aminobenzamide (4-AB), was injected intraperitoneally after MI, followed by echocardiography detection, masson assays, immunohistochemistry assays detecting expression of α-SMA and western blot assays detecting autophagy-related proteins to investigate whether PARP-1 inhibition could regulate autophagy, alleviate cardiac fibrosis and improve cardiac function in vivo. In cultured CFs, siPARP-1 repressed TGF-β1-induced proliferation, migration, and differentiation through regulating autophagic levels. The in vitro results was verified by the in vivo study, indicating that PARP-1 inhibition partially decreased autophagy, abrogated cardiac fibrosis and significantly improved cardiac function post-MI. In conclusion, this work demonstrated the vital connection of PARP-1 and autophagy in the activation of CFs, and provided solid evidence supporting PARP-1 inhibition as a feasible strategy for the treatment of post-MI heart failure.

Expression of urotensin II is\xa0associated with placental autophagy in patients with severe preeclampsia

The aims of this study are to explore the correlation between the expressions of urotensin II (UII) and autophagic markers (LC3 and P62) in patients with severe preeclampsia (SPE). A total of 64 pregnant subjects were recruited, including 29 healthy pregnancies and 35 preeclamptic patients (7 mild preeclamptic (MPE) patients and 28 SPE patients). UII and autophagic markers expression in placenta specimens was investigated by immunohistochemistry (IHC), RT-qPCR, and western blot. IHC analysis manifested that the expressions of UII and autophagic markers were mainly located in the placental cytotrophoblast and syncytiotrophoblast. Western blot and IHC analysis both indicated that the expression of UII was significantly correlated with autophagic marker LC3II (by western blot) or LC3 (by IHC) (r = 0.495, P = 0.010; r = 0.816, P = 0.007). Moreover, SPE group had higher expression of UII and LC3II, lower expression of P62 than that of normal controls. The expression of LC3II was positively related with systolic blood pressure (SBP) and urinary protein level (SBP (r = 0.501, P = 0.003) and urine protein quantitation (r = 0.509, P = 0.022)), whereas P62 had negative correlation with SBP. We first verify that UII has positive correlation with autophagic marker LC3 in placentas of preeclampsia patients; besides, autophagic levels are positively correlated with SBP and urine protein in patients with SPE.

Increased Macroautophagy in Interferon-Gamma-Producing T Cells from Patients with Newly Diagnosed Systemic Lupus Erythematosus.

Background:Imbalance of interferon-gamma (IFN-γ), interleukin (IL)-4, and IL-17 producing by T cells is confirmed to contribute to the pathogenesis of systemic lupus erythematosus (SLE). Autophagy is now emerging as a core player in the development and the function of the immune system. Therefore, we investigated the autophagic behavior in IFN-γ-, IL-4-, and IL-17-producing T cells from patients with SLE.Methods:Thirty patients with SLE and 25 healthy controls matched for gender and age were recruited between September 2016 and May 2017. The autophagic levels in IFN-γ+ T cells, IL-4+ T cells, and IL-17+ T cells from patients with newly diagnosed SLE and healthy controls were measured using flow cytometry. The plasma levels of IFN-γ were determined by enzyme-linked immunosorbent assay in SLE patients and healthy controls. Unpaired t-tests and the nonparametric Mann-Whitney U-test were used to compare data from patients with SLE and controls. Spearman's rank correlation coefficient was applied for calculation of the correlation between parallel variables in single samples.Results:Our results showed increased percentage of autophagy in IFN-γ+ T cells from patients with SLE and healthy controls ([8.07 ± 2.72]% vs. [3.76 ± 1.67]%, t = 5.184, P < 0.001), but not in IL-4+ T cells or IL-17+ T cells (P > 0.05) as compared to healthy donors. Moreover, the plasma levels of IFN-γ in SLE patients were significantly higher than those in healthy controls ([68.9 ± 29.1] pg/ml vs. [24.7 ± 17.6] pg/ml, t = 5.430, P < 0.001). Moreover, in SLE patients, the percentage of autophagy in IFN-γ+ T cells was positively correlated with the plasma levels of IFN-γ (r = 0.344, P = 0.046), as well as the disease activity of patients with SLE (r = 0.379, P = 0.039).Conclusion:The results indicate that autophagy in IFN-γ+ T cells from SLE patients is activated, which might contribute to the persistence of T cells producing IFN-γ, such as Th1 cells, and consequently result in the high plasma levels of IFN-γ, and then enhance the disease activity of SLE.

Glutamine catabolism promotes autophagy and radioresistance in nasopharyngeal carcinoma

Objective To study the relationship between glutamine metabolism and radiotherapy resistance in nasopharyngeal carcinoma (NPC). Methods The glutaminase 1 (GLS1) knockout CNE1, 6-10B cell lines and the control cell lines were established by lentivirus transfection technology. The CNE2 cell lines with GLS1 overexpression and control cell lines were established by liposome transfection technology. BPTES, one of Glutamine enzyme inhibitors, was used to inhibit the activity of GLS1. Glutamine/Glutamate(Gln/Glu) and Glu kits were used to detect glutamine metabolism in NPC cells; in vitro radiation clone formation experiment and flow cytometre mediated apoptosis assay were adopted to detect the effects of glutamine metabolism modulation on NPC radiosensitivity. Western blot and autophagic puncta monitor assay were carried out to detect the effects of glutamine metabolism modulation on autophagy status in NPC cells. Results Inhibiting the glutamine metabolism led to decreased clone formation, increased apoptotic rates, and decreased autophagic level in NPC cells; while activating the glutamine metabolism led to increased clone formation, decreased apoptotic rates and increased autophagic level in NPC cells. Conclusions The reduction of glutamine metabolism inhibits autophagy and increases radio sensitivity of NPC. Key words: Nasopharyngeal neoplasms; Glutamine; Catabolite repression; Autophagy; Radiotherapy

The effect of depression on fracture healing and osteoblast differentiation in rats.

BackgroundDepressive disorder has been proven to be associated with disturbed bone metabolism. However, the effect of depression on fracture healing still lacks evidence.Materials and methodsA rat depressive model was first established by exposing the animals to chronic unpredictable stress, which was assessed using the sucrose preference test, forced swimming test, and open field test. Subsequently, the bone repairing ability was detected by micro-computed tomography and histological analysis of the femoral condyle defect rats with or without depression. To further investigate the potential mechanisms of depression on fracture healing, the osteogenic differentiation and autophagic level were compared between the bone marrow mesenchymal stem cells (BMSCs) derived from depressive and normal rats.ResultsOur results showed that rats with depressive disorder significantly slowed the healing process at 4 and 8 weeks postinjury. Furthermore, the osteogenic potential and autophagy remarkably decreased in BMSCs from the depressive rats, suggesting an inherent relationship between autophagy and osteogenic differentiation. Finally, rapamycin, an autophagic agonist, significantly improved osteogenic differentiation of depressive BMSCs through autophagy activation.ConclusionThe present study indicated that depression had a negative effect on fracture healing with low osteoblast differentiation of BMSCs. Also, autophagy activation in BMSCs offers a novel therapeutic target for depressive patients with poor fracture healing.

Characterization of imatinib-resistant K562 cell line displaying resistance mechanisms

Chronic myeloid leukemia (CML) is a hematopoietic malignancy characterized by the t(9; 22) and the related oncogene, BCR-ABL. Tyrosine kinase activity of fusion protein BCR-ABL is the main cause of CML. Even if imatinib is used as a tyrosine kinase inhibitor (TKI) for CML therapy, drug resistance may occur in patients and the clinical failure of imatinib treatment in resistant patients had resulted with the use of another alternative TKIs. BCR-ABL dependent and independent molecular mechanisms have crucial roles in drug resistance. To reveal the underlying molecular mechanisms which play significant roles in imatinib resistance in CML, we established K562 imatinib-resistant cell line (K562r5) which was continuously exposed to (5µM) imatinib to investigate molecular mechanisms which play significant roles in drug resistance. First of all, we analyzed T315I, M351T, F315L and F359C/L/V mutations with DNA sequencing as a BCR-ABL dependent mechanism in our cell lines. Moreover, we investigated BCR-ABL independent mechanisms such as apoptosis, autophagy, drug transport and DNA repair which affect drug resistance in these cell lines. In vitro cell viability was determined by MTT assay. DNA sequencing analysis was performed to detect BCR-ABL mutations. The apoptotic effect of imatinib on CML cell lines was tested by flow cytometric Annexin V-PE staining and caspase activation assays. Apoptotic, autophagic, drug transporter and DNA repair genes expression levels were determined by RT-PCR. The conventional cytogenetic analysis was performed on K562s and K562r cells. Our results indicate that inhibition of apoptosis, induction of autophagy, overexpression of efflux gene MDR1 and down-regulation of influx gene OCT1 play crucial roles in the progression of imatinib resistance.

LPS at low concentration promotes the fracture healing through regulating the autophagy of osteoblasts via NF-κB signal pathway.

To investigate the effect of low-concentration lipopolysaccharide (LPS) on proliferation and apoptosis of osteoblasts and to discover the mechanism of low-concentration LPS in facilitating the proliferation of osteoblasts. MC3T3-E1 osteoblasts were treated with LPS, 3-methyladenine (3-MA, autophagy inhibitor), and BAY11-7082 (inhibitor of nuclear factor-kappa b, NF-κB), respectively. The cell cycles were detected using a flow cytometer. Cell proliferation and activity of MC3T3-E1 osteoblasts were explored by cell counting kit-8. Western blotting and immunofluorescence assay were performed to detect the protein level. RNA expression was measured through polymerase chain reaction (PCR) and immunofluorescence assay. At the third day after cell culture, cell infusion reached 80%, and cells were taken as the subjects. At 6 h after treatment with low-concentration LPS, the proliferation and activity of cells were higher than those at 1 h and 12 h after treatment, and the apoptotic level was significantly lower than that in cells at 12 h after treatment. The proliferation and activity of cells in the low-concentration LPS group were significantly higher than those in the control group, 3-MA group and BAY11-7082 group, and the apoptotic level was lower than those in these groups. Compared with those of cells in control group and BAY11-7082 group, the messenger RNA (mRNA) and protein expressions and nuclear transfer of cells in low-concentration LPS group were significantly elevated, but there were no statistically significant differences in comparisons with the 3-MA group. In the experiment of cell autophagy, the autophagic level in cells in low-concentration LPS group was higher than those in the control group, 3-MA group and BAY11-7082 group. Through the NF-κB signaling pathway in osteoblasts, low-concentration LPS can activate the autophagy and promote cell proliferation, thereby inhibiting cell apoptosis and accelerating the fracture healing.

The novel involvement of podocyte autophagic activity in the pathogenesis of lupus nephritis.

Lupus nephritis (LN) is one of the most common and severe complications in Systemic lupus erythematosus patients, and the mechanism underlining the pathogenesis of LN is still unknown. Autophagy plays vital roles in maintaining cell homeostasis and is involved in the pathogenesis of many diseases. In this study, we investigated the role of autophagy in the progression of LN. Autophagic activities in podocytes of both LN patients (Class IV and V) and mice were evaluated. Podocytes were observed by electron microscopy, and autophagic activity was evaluated by immunofluorescence staining and western blot analysis. Apoptotic activity was evaluated by immunohistochemistry, TUNEL assays and flow cytometric analysis. Significantly greater podocyte injury and discrepant autophagic levels were observed in LN patients. Differentiated mouse podocytes in the LN group showed reduced nephrin expression and increased apoptosis, as well as significantly higher levels of apoptosis-related proteins (cleaved caspase-3 and Bax). In the mice LN group, the increased number of autophagosomes was accompanied by increased LC3-II/LC3-I ratios and decreased p62, suggesting increased autophagic and apoptotic activity in podocytes. Blockade of autophagic activity by 3-MA or siRNA-mediated silencing of Atg5 resulted in decreases in LC3-II/LC3-I ratios, podocyte apoptosis and damage in the mice LN group. Futhermore, Rapamycin treatment increased LC3-II/LC3-I ratios, and enhanced LN-induced apoptosis in podocyte from modal animal. This study demonstrates that autophagic activity of podocytes is a crucial factor in renal injury by directly affecting the function of podocyte; thus, inhibiting this activity during the early stages of LN is implicated as a potential therapeutic strategy for delaying the progression of LN. Also, clinical application in LN needs to consider patients' pathological type and drugs' comprehensive effectiveness.

MicroRNA-let-7a regulates cell autophagy by targeting Rictor in gastric cancer cell lines MGC-803 and SGC-7901.

miR-let-7a is the most widely studied miRNA, whose functions have been well-established by scientists in both carcinogenesis and progression of human cancer, including gastric cancer (GC). However, to date there is a lack of information concerning the relationship between miR-let-7a and cellular autophagy. Using western blotting and immunofluorescence, we determined that upregulation of miR-let-7a led to increased cellular autophagic level, whereas miR-let-7a suppression decreased autophagy activity in GC cells. To further elucidate the mechanisms underlying this, we screened potential targets of miR-let-7a using bioinformatics analyses, validated by a series of assays. Our results indicated that Rptor independent companion of mTOR complex2 (Rictor) was a direct target of miR-let-7a. In addition, rescue experiments invitro showed that miR-let-7a promoted cellular autophagic level by inhibiting Rictor expression in GC cells. Furthermore, as an upstream executor of Akt-mTOR signaling pathway, we found that Rictor elaborated its effect on autophagy by phosphorylating Akt and mTOR, and this regulatory process could also be mediated by miR-let-7a. Taken together, our results present a novel role for miR-let-7a in GC which modulates autophagy by targeting Rictor, following the regulation of Akt-mTOR signal pathway.

The Anti-atherosclerotic Effect of Paeonol against Vascular Smooth Muscle Cell Proliferation by Up-regulation of Autophagy via the AMPK/mTOR Signaling Pathway.

Introduction: Paeonol (2′-hydroxy-4′-methoxyacetophenone), isolated from moutan cortex, is an active component and has been shown to have anti-atherosclerotic and anti-proliferation effects on vascular smooth muscle cells (VSMCs). However, the possible role of Paeonol in protecting against VSMC proliferation as related to autophagy has yet to be elucidated.Materials and Methods: The athero-protective effects of Paeonol were evaluated in apoE-/- mice. The effects of Paeonol on VSMC proliferation and autophagy were examined by staining α-SMA and LC3II spots in the media layer of apoE-/- mice, respectively. CCK8 and BrdU assays were used to investigate the effects of Paeonol on cell proliferation in vitro. The autophagic levels in VSMCs were evaluated by detecting LC3II accumulation and p62 degradation by immunoblot analysis. To investigate if Paeonol could prevent VSMCs proliferation through autophagy induction, we tested the change in autophagy and cell proliferation by inhibition of autophagy. The levels of the AMPK/mTOR pathway in autophagy regulation were detected by immunoblot analysis. An AMPK inhibitor and si-AMPK transfection in VSMCs was used to confirm whether AMPK activity plays a key role in autophagy regulation of Paeonol.Results: In vivo experiments confirmed that Paeonol restricted atherosclerosis development and decreased the amount of VSMCs in the media layer of apoE-/- mice. Paeonol increased protein levels of LC3II and the presence of autophagosomes in the media layer of arteries, which implies that Paeonol may induce VSMCs autophagy in vivo. Paeonol showed potential in inhibiting ox-LDL-induced proliferation in vitro experiments. Paeonol dose-dependently enhanced the formation of acidic vesicular organelles and autophagosmomes, up-regulated the expression of LC3II and increased p62 degradation. The autophagy inhibitor CQ obviously attenuated Paeonol-induced autophagy and the anti-proliferation effect in VSMCs. In addition, Paeonol induced phosphorylation of AMPK and reduced phosphorylation of mTOR. An AMPK inhibitor reversed the Paeonol-induced p-mTOR/mTOR decrease. Paeonol induced LC3II conversion, increased p62 degradation and inhibited cell proliferation in VSMCs, the effects of which were abolished by si-AMPK.Conclusion: These results imply that Paeonol inhibits proliferation of VSMCs by up-regulating autophagy, and activating the AMPK/mTOR signaling pathway, providing new insights into the anti-atherosclerosis activity of Paeonol.