Light Chain 3 Levels Research Articles

Effect of calcitonin gene-related peptide on MC3T3-E1 osteoblast apoptosis and autophagy induced by serum starvation

To 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. MC3T3-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. Under 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. CGRP can increase autophagy of MC3T3-E1 osteoblasts under serum starvation conditions. CGRP may also inhibit apoptosis of MC3T3-E1 osteoblasts by promoting autophagy. .

Near-infrared photothermal therapy using EGFR-targeted gold nanoparticles increases autophagic cell death in breast cancer

Although triple negative breast cancer (TNBC) is a small percentage of all breast cancers, to date, TNBC is one of the most challenging types of breast cancer for basic and clinic research because TNBC patients display a high risk of relapse, shorter overall survival and limited therapeutic options after completion of conventional chemotherapy compared with patients with other breast cancer subtypes. The epidermal growth factor receptor (EGFR) is a promising target for TNBC treatment. Although near infrared-photothermal therapy (NIR-PTT) using anti-EGFR antibody-conjugated gold nanorods (anti-EGFR-GNs), has attracted considerable interest for non-invasive and targeted TNBC treatment through an activation of apoptotic pathway, it is unclear whether anti-EGFR-GNs-combined NIR-PTT modulates the induction of autophagy contributing to cell death. Therefore, we investigated the autophagic cell death in cultured TNBC cells and mouse xenograft tumors during anti-EGFR-GNs-combined NIR-PTT. We here found that the cytotoxicity induced by anti-EGFR-GNs-combined NIR-PTT was rescued by treatment with autophagy inhibitor, 3-methyladenine (3-MA). Anti-EGFR-GNs-combined NIR-PTT induced remarkable levels of autophagy activity as evidenced by a large number of autophagic vesicles and a significant increase in autophagy-specific proteins; microtubule-associated protein light chain 3 (LC3), p62, beclin-1, and autophagy-related gene5 (Atg5), accompanying the inhibition of AKT-mTOR signaling pathway responsible for inducing autophagy. Moreover, in mouse xenograft tumors, anti-EGFR-GNs-combined NIR-PTT also increased LC3 and beclin-1 levels. Our findings, for the first time, demonstrate that anti-EGFR-GNs-combined NIR-PTT remarkably induces autophagy leading to EGFR-targeted cancer cell death.

BAMBI overexpression together with β-sitosterol ameliorates NSCLC via inhibiting autophagy and inactivating TGF-β/Smad2/3 pathway.

Non-small cell lung cancer(NSCLC) has the highest mortality rate among all solid tumors with a poor prognosis. The BMP and activin receptor membrane bound inhibitor(BAMBI) has been identified as a hallmark of NSCLC and β-sitosterol possesses antitumor potentiality. This study explores the effect of BAMBI overexpression and β-sitosterol in the context of NSCLC. The results revealed that the transfection of pcDNA‑BAMBI and β-sitosterol treatment significantly reduced the levels of autophagy markers light chain3(LC3)II and Beclin1, whereas the levels of LC3I and p62 were promoted. The reduced punctate accumulations of GFP-LC3 were detected in pcDNA-BAMBI and β-sitosterol groups, especially in pcDNA-BAMBI+β-sitosterol group. BAMBI overexpression and β-sitosterol induced G0/G1 cell cycle arrest and inhibted cell proliferation in A549 cells. In addition, the levels of transforming growth factor-β(TGF‑β)/p-Smad2/3/c-Myc pathway proteins were decreased. The TGF-β overexpression further confirmed that BAMBI overexpression and β-sitosterol treatment suppre-ssed autohagy and viability of A549cells was through TGF-β/Smad2/3/c-Myc pathway. Finally, the tumor growth was suppressed in NSCLC xenografts, and the inhibitory effect was stronger under treatment of pcDNA-BAMBI toge-ther with β-sitosterol. These results indicate that BAMBI overexpression and β-sitosterol may serve as novel targets for the treatment of NSCLC.

Vitamin D induces autophagy of pancreatic β-cells and enhances insulin secretion.

Epidemiological evidence indicates that vitamin D is involved in defense against diabetes; however, the precise underlying mechanism remains to be elucidated. In the present study, the effect of vitamin D on the pathogenesis of diabetes was investigated, with an emphasis on its direct effect on pancreatic β‑cells. A streptozotocin (STZ)‑induced type 1 diabetes mellitus (T1DM) mouse model and MIN6 mouse insulinoma β‑cells were subjected to vitamin D treatment. Histopathological analysis of pancreatic islets was performed to investigate insulitis, and reverse transcription-quantitative polymerase chain reaction and western blotting were used to determine the mRNA and protein expression levels of markers of autophagy [microtubule-associated protein 1A/1B‑light chain 3 (LC3) and Beclin 1] and regulation of apoptosis [B-cell lymphoma 2 (Bcl-2)]. Apoptosis of MIN6 cells was examined by flow cytometry following annexin V/propidium iodide labeling. The secretion of insulin was measured by ELISA. The results revealed that vitamin D reduced the incidence of T1DM, enhanced insulin secretion and relieved pancreatic inflammation in STZ‑treated mice. Furthermore, vitamin D increased the mRNA expression levels of LC3 and Beclin 1, and increased Bcl‑2 protein expression levels in STZ‑treated MIN6 cells, while decreasing the apoptosis rate. The results of the present study demonstrated, for the first time to the best of our knowledge, that vitamin D induces autophagy and suppresses apoptosis of pancreatic β‑cells, as well as preventing insulitis. These findings regarding vitamin D provide insights into its involvement in diabetes, and suggest a potential novel strategy for the treatment of diabetes via agents enhancing autophagy in pancreatic β-cells.

Prognostic value of the autophagy markers LC3 and p62/SQSTM1 in early-stage non-small cell lung cancer.

Autophagy is a cellular degrading process that promotes tumor cell survival or cell death in cancer, depending on the progress of oncogenesis. Protein light chain 3 (LC3) and p62/SQSTM1 (p62) are associated with autophagosomal membranes that engulf cytoplasmic content for subsequent degradation. We studied LC3 and p62 expression using immunohistochemistry in a large cohort of 466 stage I/II non-small cell lung cancer (NSCLC) using a tissue microarray. We evaluated dot-like cytoplasmic expression of LC3 and dot-like, cytoplasmic and nuclear staining for p62 in relation to clinico-pathological parameters.LC3 expression correlated with all p62 patterns, as those correlated among each other (p < 0.001 each). There was no correlation with stage, age or gender. A combination of high LC3/high p62 dot-like staining (suggesting impaired autophagy) showed a trend for better outcome (p = 0.11). Interestingly, a combined low cytoplasmic/low nuclear p62 expression regardless of dot-like staining was an independent prognostic factor for longer survival (p = 0.006; HR=1.96), in addition to tumor stage (p = 0.004; HR=1.4).The autophagy markers LC3 and p62 are differentially expressed in NSCLC, pointing towards a biologically significant role. High LC3 levels seem to be linked to lower tumor aggressiveness, while high general p62 expression was significantly associated with aggressive tumor behavior.

Effects of bone marrow-derived mesenchymal stem cells on the autophagic activity of alveolar macrophages in a rat model of silicosis.

The aim of the present study was to evaluate the effects of bone marrow-derived mesenchymal stem cells (BMSCs) on the expression of the autophagy-associated proteins, microtubule-associated protein light chain 3 (LC-3) and autophagy-related gene Beclin-1 (Beclin-1), in alveolar macrophages (AMs) in a rat model of silicosis. Furthermore, the study investigated the molecular mechanisms underlying the effects of BMSC treatment. A population of 60 adult female Sprague-Dawley (SD) rats were allocated at random into three groups, namely the control, model and BMSC treatment groups (n=20 per group). BMSCs were isolated from five male SD rats (age, 6-8 weeks) and cultured in vitro. The silicosis model was established using a single 1.0-ml infusion of silicon dioxide suspension administered via non-exposed tracheal intubation. Rats in the BMSC treatment group received a 1.0-ml transplantation of BMSCs (1×106/ml). The rats were sacrificed on days 1, 7, 14 and 28 after modeling, and AMs were extracted from the rats using bronchoalveolar lavage. Third-generation BMSCs were identified using flow cytometry with fluorescein isothiocyanate staining, and the morphological characteristics of the AMs were observed using hematoxylin and eosin staining. The expression levels of LC-3 and Beclin-1 were determined using immunocytochemistry sand western blot analysis. The expression levels of LC-3 and Beclin-1 were found to be increased at all the time points in the model group. LC-3 and Beclin-1 levels began to increase at day 1, peaked at day 14 and decreased after day 28; however, the levels remained elevated compared with the basal expression levels. The AMs of the BMSC treatment group exhibited significantly alleviated pathological symptoms compared with the model group AMs, as indicated by significantly decreased expression levels of LC-3 and Beclin-1 at each time point. Therefore, the results indicated that autophagy was promoted in the AMs of the silicosis model rats. Furthermore, treatment with BMSCs was demonstrated to reduce the expression levels of LC-3 and Beclin-1, subsequently inhibiting autophagic activity and mitigating the damage associated with silicosis.

Downregulation of Choline Kinase-Alpha Enhances Autophagy in Tamoxifen-Resistant Breast Cancer Cells.

Choline kinase-α (Chk-α) and autophagy have gained much attention, as they relate to the drug-resistance of breast cancer. Here, we explored the potential connection between Chk-α and autophagy in the mechanisms driving to tamoxifen (TAM) resistance, in estrogen receptor positive (ER+) breast cancer cells (BCCs). Human BCC lines (MCF-7 and TAM-resistant MCF-7 (MCF-7/TAM) cells) were used. Chk-α expression and activity was suppressed by the transduction of shRNA (shChk-α) with lentivirus and treatment with CK37, a Chk-α inhibitor. MCF-7/TAM cells had higher Chk-α expression and phosphocholine levels than MCF-7 cells. A specific downregulation of Chk-α by the transduction of shChk-α exhibited a significant decrease in phosphocholine levels in MCF-7 and MCF-7/TAM cells. The autophagy-related protein, cleaved microtubule-associated protein light chain 3 (LC3) and autophagosome-like structures were significantly increased in shChk-α-transduced or CK37-treated MCF-7 and MCF-7/TAM cells. The downregulation of Chk-α attenuated the phosphorylation of AKT, ERK1/2, and mTOR in both MCF-7 and MCF-7/TAM cells. In MCF-7 cells, the downregulation of Chk-α resulted in an induction of autophagy, a decreased proliferation ability and an activation of caspase-3. In MCF-7/TAM cells, despite a significant decrease in proliferation ability and an increase in the percentage of cells in the G0/G1 phase of the cell cycle, the downregulation of Chk-α did not induced caspase-dependent cell death and further enhanced autophagy and G0/G1 phase arrest. An autophagy inhibitor, methyladenine (3-MA) induced death and attenuated the level of elevated LC3 in MCF-7/TAM cells. Elucidating the interplay between choline metabolism and autophagy will provide unique opportunities to identify new therapeutic targets and develop novel treatment strategies that preferentially target TAM-resistance.

Defective autophagy is a key feature of cerebral cavernous malformations.

Cerebral cavernous malformation (CCM) is a major cerebrovascular disease affecting approximately 0.3–0.5% of the population and is characterized by enlarged and leaky capillaries that predispose to seizures, focal neurological deficits, and fatal intracerebral hemorrhages. Cerebral cavernous malformation is a genetic disease that may arise sporadically or be inherited as an autosomal dominant condition with incomplete penetrance and variable expressivity. Causative loss-of-function mutations have been identified in three genes, KRIT1 (CCM1), CCM2 (MGC4607), and PDCD10 (CCM3), which occur in both sporadic and familial forms. Autophagy is a bulk degradation process that maintains intracellular homeostasis and that plays essential quality control functions within the cell. Indeed, several studies have identified the association between dysregulated autophagy and different human diseases. Here, we show that the ablation of the KRIT1 gene strongly suppresses autophagy, leading to the aberrant accumulation of the autophagy adaptor p62/SQSTM1, defective quality control systems, and increased intracellular stress. KRIT1 loss-of-function activates the mTOR-ULK1 pathway, which is a master regulator of autophagy, and treatment with mTOR inhibitors rescues some of the mole-cular and cellular phenotypes associated with CCM. Insufficient autophagy is also evident in CCM2-silenced human endothelial cells and in both cells and tissues from an endothelial-specific CCM3-knockout mouse model, as well as in human CCM lesions. Furthermore, defective autophagy is highly correlated to endothelial-to-mesenchymal transition, a crucial event that contributes to CCM progression. Taken together, our data point to a key role for defective autophagy in CCM disease pathogenesis, thus providing a novel framework for the development of new pharmacological strategies to prevent or reverse adverse clinical outcomes of CCM lesions.

Metformin synergistically sensitizes FLT3-ITD-positive acute myeloid leukemia to sorafenib by promoting mTOR-mediated apoptosis and autophagy

Mutations of Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD), accounting for approximately 30% of patients with acute myeloid leukemia (AML), results in poor therapeutic efficacy and short survival. Sorafenib, an oral multikinase inhibitor, can inhibit FLT3 and improve clinical outcome of FLT3 mutated leukemia. Our current studies have shown that, the antidiabetic drug metformin also exerts anti-leukemic effect by activating p-AMPK and synergistically sensitizes FLT3 mutated AML to sorafenib. Both agents suppress cell proliferation in a dose-dependent manner and induce apoptosis via cell cycle arrest, but does not obviously modulate autophagy marker, light chain 3 (LC3). Mechanistically, in the presence of metformin, the anticancer potential of sorafenib, accompanying with increased LC3 levels, is found to be synergistically enhanced with the remarkably reduced protein expression of the mTOR/p70S6K/4EBP1 pathway, while not appreciably altering cell cycle. Overall, these results show metformin in aid of sorafenib may represent a promising and attractive strategy for the treatment of FLT3-ITD mutated AML.

Ghrelin Attenuates Liver Fibrosis through Regulation of TGF-β1 Expression and Autophagy.

Ghrelin is a stomach-derived growth hormone secretagogue that promotes various physiological effects, including energy metabolism and amelioration of inflammation. The purpose of this study was to investigate the protective mechanism of ghrelin against liver fibrosis. Liver fibrosis was induced in C57BL/6 mice by intraperitoneal injection of CCl4 (2.0 mL/kg of 10% CCl4 v/v solution in peanut oil) two times per week for eight weeks. Ghrelin (10 μg/kg) was intraperitoneally injected two times per week for eight weeks. A second murine liver fibrosis model was induced by bile duct ligation (BDL) and concurrent ghrelin administration for four weeks. Hematoxylin eosin (H&E), and Masson’s trichrome were used to detect pathological changes to liver tissue. Western blotting was used to detect protein levels of transforming growth factor (TGF)-β1, phosphorylated Smad3 (p-Smad3), I-collage, α-smooth muscle actin (α-SMA), matrix metalloproteinases (MMPs) 2, tissue inhibitor of matrix metalloproteinases (TIMPs) 1, phosphorylated NF-κB (p-NF-κB), and microtubule-associated protein light chain 3 (LC3). In addition, qRT-PCR was used to detect mRNA levels of TGF-β1, I-collage, α-SMA, MMP2, TIMP1 and LC3, while levels of TGF-β1, p-Smad3, I-collage, α-SMA, and LC3 were detected immunohistochemically. Levels of aspartate aminotransferase and alanine aminotransferase were significantly decreased by ghrelin treatment. Ghrelin administration also significantly reduced the extent of pathological changes in both murine liver fibrosis models. Expression levels of I-collage and α-SMA in both models were clearly reduced by ghrelin administration. Furthermore, ghrelin treatment decreased protein expression of TGF-β1 and p-Smad3. The protein levels of NF-κB and LC3 were increased in the CCl4- and BDL-treatment groups but were significantly reduced following ghrelin treatment. In addition, ghrelin inhibited extracellular matrix formation by decreasing NF-κB expression and maintaining the balance between MMP2 and TIMP1. Our results demonstrated that ghrelin attenuates liver fibrosis via inhibition of the TGF-β1/Smad3 and NF-κB signaling pathways, as well as autophagy suppression.

HMGB1 overexpression correlates with poor prognosis in early-stage squamous cervical cancer.

High mobility group box 1 (HMGB1) is associated with tumor progression and a poor prognosis; microtubule-associated protein 1 light chain 3 (LC3) plays a critical role in autophagy. However, the roles of HMGB1 and LC3 in squamous cervical cancer (SCC) remain unclear. An array of 166 early-stage SCC, 62 cervical intraepithelial neoplasia (CIN), and 50 normal cervical tissue samples was assessed. HMGB1 and LC3 protein levels were examined by immunohistochemistry, and the associations of HMGB1 and LC3 levels with clinicopathological characteristics evaluated, to assess their prognosis significance. High nuclear HMGB1 levels were detected in 72.9% SCC cases; 16% cases showed cytoplasmic expression of HMGB1 in cancer cells with low nuclear expression. Interestingly, HMGB1 levels in SCC samples were significantly higher than CIN and control specimens, while lower LC3 expression was found in SCC samples (P < 0.001). Nuclear HMGB1 expression was weakly negatively correlated to LC3 amounts (r = -0.254, P = 0.001). High nuclear HMGB1 levels were associated with vascular metastasis (P < 0.05). In addition, cytoplasmic HMGB1 expression was associated with lymph node metastasis (P < 0.05). Furthermore, high nuclear HMGB1 levels and cytoplasmic HMGB1 expression predicted poor overall survival (OS) and disease-free survival (DFS). Meanwhile, high LC3 expression was associated with favorable prognosis. Multivariate analysis showed that both nuclear and cytoplasmic HMGB1 expressions were independent prognostic factors for overall- and disease-free survival, along with nodule metastasis. HMGB1 overexpression plays a significant role in SCC progression. Both nuclear and cytoplasmic HMGB1 are independent factors for poor prognosis in early-stage SCC.

Basal level of autophagy is increased in aging human skin fibroblasts in vitro, but not in old skin.

Intracellular autophagy (AP) is a stress response that is enhanced under conditions of limitation of amino acids, growth factors and other nutrients, and also when macromolecules become damaged, aggregated and fibrillated. Aging is generally accompanied by an increase in intracellular stress due to all the above factors. Therefore, we have compared the basal levels of AP in serially passaged human facial skin fibroblasts undergoing aging and replicative senescence in vitro, and ex vivo in the skin biopsies from the photo-protected and photo-exposed area of the arms of 20 healthy persons of young and old ages. Immunofluorescence microscopy, employing antibodies against a specific intracellular microtubule-associated protein-1 light chain-3 (LC3) as a well established marker of AP, showed a 5-fold increase in the basal level of LC3 in near senescent human skin fibroblasts. However, no such age-related increase in LC3 fluorescence and AP could be detected in full thickness skin sections from the biopsies obtained from 10 healthy young (age 25 to 30 yr) and 10 old (age 60 to 65 yr) donors. Furthermore, there was no difference in the basal level of LC3 in the skin sections from photo-protected and photo-exposed areas of the arm. Thus, in normal conditions, the aging phenotype of the skin cells in culture and in the body appears to be different in the case of AP.

Moderate Hypothermia Significantly Decreases Hippocampal Cell Death Involving Autophagy Pathway after Moderate Traumatic Brain Injury.

Here, we evaluated changes in autophagy after post-traumatic brain injury (TBI) followed by moderate hypothermia in rats. Adult male Sprague-Dawley rats were randomly divided into four groups: sham injury with normothermia group (37 °C); sham injury with hypothermia group (32 °C); TBI with normothermia group (TNG; 37 °C); and TBI with hypothermia group (THG; 32 °C). Injury was induced by a fluid percussion TBI device. Moderate hypothermia (32 °C) was achieved by partial immersion in a water bath (0 °C) under general anesthesia for 4 h. All rats were killed at 24 h after fluid percussion TBI. The ipsilateral hippocampus in all rats was analyzed with hematoxylin and eosin staining; terminal deoxynucleoitidyl transferase-mediated nick end labeling staining was used to determine cell death in ipsilateral hippocampus. Immunohistochemistry and western blotting of microtubule-associated protein light chain 3 (LC3), Beclin-1, as well as transmission electron microscopy performed to assess changes in autophagy. At 24 h after TBI, the cell death index was 27.90 ± 2.36% in TNG and 14.90 ± 1.52% in THG. Expression level of LC3 and Beclin-1 were significantly increased after TBI and were further up-regulated after post-TBI hypothermia. Further, ultrastructural observations showed that there was a marked increase of autophagosomes and autolysosomes in ipsilateral hippocampus after post-TBI hypothermia. Our data demonstrated that moderate hypothermia significantly attenuated cell death and increased autophagy in ipsilateral hippocampus after fluid percussion TBI. In conclusion, autophagy pathway may participate in the neuroprotective effect of post-TBI hypothermia.

Routine Western blot to check autophagic flux: Cautions and recommendations

At present, the analysis of autophagic flux by Western blotting (WB), which measures two of the most important markers of autophagy, i.e., microtubule-associated protein 1 light chain 3 (LC3) and p62, is widely accepted in the scientific community. In this study, we addressed the possible disadvantages and limitations that this method presents for a correct interpretation of the results according to the lysis buffer used for extracting proteins. Here, we tested the LC3 and p62 protein levels by WB in four cell models (mouse embryonic and human fibroblasts (MEFs and HFs, respectively), N27 rat mesencephalic dopaminergic neurons and SH-SY5Y human neuroblastoma cells). The cells were exposed to the autophagy inhibitor bafilomycin A1 (Baf. A1) in combination (or not) with nutrient deprivation to induce autophagy, and they were lysed by using four different buffers (nonyl phenoxypolyethoxylethanol (NP-40), radioimmunoprecipitation assay (RIPA), Triton X-100, and sample buffer (SB) 1×). Based on our observations, we want to highlight that this technique is not always appropriate for analyzing and monitoring autophagy. In this report, we show conflicting data that hinder the correct interpretation of the results, especially in relation to p62 protein levels, at least in the models studied in this work.

Down-regulation of connexin43 and connexin32 in keratocystic odontogenic tumours: potential association with clinical features.

The objective of this study was to explore the potential involvement of connexin43 (Cx43) and connexin32 (Cx32), two vital members of the connexin families, in the pathogenesis of keratocystic odontogenic tumours (KCOT). The expression levels of Cx43 and Cx32 in human KCOT and normal oral mucosa (OM) tissues were measured using immunohistochemistry and real-time quantitative polymerase chain reaction (qPCR). The relationship between Cx43 and Cx32 expression and markers of proliferation [proliferating cell nuclear antigen (PCNA), cyclin D1], anti-apoptosis [B cell lymphoma 2 (Bcl-2)] and autophagy [light chain 3 (LC3), Sequestosome 1 p62 (p62)] was then investigated in the KCOT samples. The results showed that Cx43 and Cx32 expression was down-regulated significantly in KCOT samples relative to OM samples. Meanwhile, the expression levels of Cx43 and Cx32 were correlated negatively with the expression levels of PCNA, cyclin D1, Bcl-2, LC3 and p62, as confirmed further by double-labelling immunofluorescence analyses. This study reveals for the first time that Cx43 and Cx32 are down-regulated in KCOT and suggests an association with growth regulation, anti-apoptosis and autophagy in KCOT.

Involvement of autophagy inhibition in Brucea javanica oil emulsion-induced colon cancer cell death

Brucea javanica oil emulsion (BJOE), the petroleum ether extract of B. javanica emulsified by phospholipid, is widely used in China as an anticancer agent. The extracts from B. javanica induce cancer cell death by various mechanisms; however, it is not known whether these mechanisms involve autophagy, which is an important process in cancer development and treatment. Thus, the current study aimed to investigate whether BJOE modulates autophagy in HCT116 human colon cancer cells and whether modulation of autophagy is an anticancer mechanism of BJOE. Immunoblotting was employed to analyze the protein expression levels of microtubule-associated protein light-chain 3 (LC3), a specific protein marker of autophagy, in HCT116 cancer cells following exposure to BJOE. The apoptosis rate of the HCT116 cancer cells was detected by performing an Annexin V-fluorescein isothiocyanate/propidium iodide assay. According to the effect of BJOE administration on autophagy in the HCT116 cancer cells (induction or suppression), a functionally opposite agent (autophagy suppressor or inducer) was applied to counteract this effect, and the apoptosis rate of the cancer cells was detected again. The role of autophagy (pro-survival or pro-death) was demonstrated by comparing the rates of apoptotic cancer cells prior to and following the counteraction. The results revealed that BJOE suppressed the protein expression levels of LC3, including the LC3-I and LC3-II forms, and induced apoptosis in the HCT116 cancer cells with a high level of basal LC3. The apoptosis-inducing activity of BJOE was significantly attenuated when autophagy was induced by the administration of trehalose, an autophagy inducer. The data indicates that autophagy inhibition is involved in BJOE-induced cancer cell death, and that this inhibition may be a potential anticancer mechanism of BJOE.

A quick signal of starvation induced autophagy: Transcription versus post-translational modification of LC3

Autophagy is the major intracellular lysosomal bulk degradation pathway induced by nutrient starvation and contributes to the elimination of damaged organelles and protein aggregates to recycle building block and is essential for cell survival. Microtubule-associated protein 1 light chain 3 (LC3) plays an indispensable role in macroautophagy formation and is a molecular marker for the process. Here, we show that autophagy increased through quick robust signaling from starvation by enhanced levels of LC3, LC3–EGFP (enhanced green fluorescent protein) punctate, and bulk proteolysis in rat hepatoma H4-II-E cells and fresh rat hepatocytes. After the addition of amino acids to the starvation condition, a similar quick signal appeared by significant reduction of the LC3 ratio and bulk proteolysis. Interestingly, we observed that post-translational modification of LC3 conversion occurred even long before the changes happened in the level of LC3–mRNA (messenger RNA) expression. A similar coordinated but diverse effect on LC3 was confirmed by using autophagy and lysosomal inhibitors. These results indicated that during starvation the initial robust signal to the cytoplasm can induce autophagy activity through modification at the protein level, whereas after depleting readily available autophagy proteins the signal goes to the DNA transcription level to maintain the autophagy capacity of cells.

Transforming growth factor‑β1 reduces apoptosis via autophagy activation in hepatic stellate cells.

Autophagy is a metabolic process that is important in fibrogenesis, in which cellular components are degraded by lysosomal machinery. Transforming growth factor β1 (TGF-β1) is a potent fibrogenic cytokine involved in liver fibrosis; however, it remains elusive whether autophagy is regulated by TGF-β1 in this process. In the present study, the function of TGF-β1-mediated autophagy in the proliferation and apoptosis of hepatic stellate cells (HSCs) was investigated. A rat HSC cell line (HSC-T6) was incubated with or without TGF-β1 followed by bafilomycin A1, and microtubule-associated proteins 1A/1B light chain 3 (LC3) small interfering (si)RNA was used to inhibit autophagy in order to assess the association between TGF-β1 and autophagy. HSC-T6 cell transient transfection was accomplished with a pLVX-AcGFP-N1-rLC3B-encoding plasmid. An MTS assay and flow cytometry were utilized to detect proliferation and apoptosis of HSC-T6 cells. Quantitative polymerase chain reaction, immunofluorescence and western blot analysis were used to detect the presence of activation markers. Proliferation was increased and apoptosis was reduced in HSC-T6 cells treated with TGF-β1 compared with cells subjected to serum deprivation. However, when HSC-T6 cells were treated with bafilomycin A1 and LC3 siRNA, increased apoptosis and reduced proliferation were observed. In addition, protein and mRNA expression levels of the autophagy marker LC3 were significantly increased. GFP-LC3 punctate markings were more prolific following TGF-β1 treatment of HSC-T6 cells, indicating that TGF-β1 may rescue HSC-T6 cells from serum deprivation and reduce apoptosis via autophagy induction. The present study elucidated the possible functions of TGF-β1-mediated autophagy in the pathological process of liver fibrosis.

Clinical significance of autophagic protein LC3 levels and its correlation with XIAP expression in hepatocellular carcinoma.

Autophagy is a cellular recycling process to enable cell survival in less favorable conditions through degradation of their unnecessary cellular components and utilization of the breakdown components. Autophagy also plays an important role in tumor pathology. In this study, we detected autophagy protein light chain 3 (LC3) and X-linked inhibitor of apoptosis protein (XIAP) in hepatocellular carcinoma (HCC) tissue specimens to assess their role in HCC tumorigenesis and progression. We analyzed expression of LC3, XIAP, and Ki-67 proteins immunohistochemically in surgical specimen of 150 HCC and 136 nontumor hepatic tissues. The levels of LC3 and XIAP proteins were compared between tumor and nontumoral parenchyme. The data showed that LC3 expression was increased in HCC compared with nontumoral parenchyma. LC3 expression was significantly associated with male gender, large tumor size, advanced tumor stages, and worse relapse-free and overall survival (OS). In contrast, XIAP expression was associated with small tumor size, early tumor stage, and better relapse-free and OS. In contrast, XIAP expression was associated with small tumor size, early tumor stage, and better relapse-free and OS. Furthermore, expression of LC3 and XIAP was inversely associated in HCC tissue specimens. In conclusion, increase in autophagic LC3 activity and low XIAP expression could be useful to predict the worse HCC prognosis.

Effects of suppressed autophagy on mitochondrial dynamics and cell cycle of N2a cells

Autophagy dysregulation, mitochondrial dynamic abnormality and cell cycle re-entry are implicated in the vulnerable neurons of patients with Alzheimer's disease. This study was designed to testify the association among autophagy, mitochondrial dynamics and cell cycle in dividing neuroblastoma (N2a) cells. The N2a cells were cultured in vitro and treated with different concentrations of 3-methyladenine (3-MA). The cell viability was detected by methyl thiazolyl tetrazolium (MTT) assay. They were randomly divided into control group (cells cultured in normal culture medium) and 3-MA group (cells treated with 10 mmol/L 3-MA). The cell cycle was analyzed in the two groups 3, 6, 12, and 24 h after treatment by flow cytometry. Western blotting was used to evaluate the expression levels of mitofission 1 (Fis1), mitofusin 2 (Mfn2), microtubule-associated protein 1 light chain 3 (LC3), cell cycle-dependent kinase 4 (CDK4) and cdc2. The flow cytometry revealed that the proportion of cells in G(2)/M was significantly increased, and that in G0/G1 was significantly reduced in the 3-MA group as compared with the control group. Western blotting showed that the expression levels of Fis1, LC3, and CDK4 were significantly up-regulated in the 3-MA group at the four indicated time points as compared with the control group. Mfn2 was initially decreased in the 3-MA group, and then significantly increased at 6 h or 12 h. Cdc2 was significantly increased in the 3-MA group at 3 h and 6 h, and then dropped significantly at 12 h and 24 h. Our data indicated that 3-MA-induced suppressed autophagy may interfere with the cell cycle progression and mitochondrial dynamics, and cause cell death. There are interactions among cell cycle, mitochondrial dynamics and autophagy in neurons.