Expression Of Autophagy-related Proteins Beclin-1 Research Articles

Effect of Periostin Silencing on the Autophagy of Osteoblasts.

The objective of the present work was to investigate the effect of Periostin (POSTN) silencing on autophagy in osteoblasts, and provide an experimental basis for studying the mechanism of dental eruption. The cells were divided into the following four groups according to their viral number: the NC group, pFU-GW-016PSC53349-1; group KD1, LVpFU-GW-016PSC66471-1; group KD2, LVpFU-GW-016PSC66472-1; and group KD3, LVpFU-GW-016PSC66473-1. The lentiviral vector was infected at MOI = 100 in the ENi.S medium containing 5 g/mL Polybrene. The target gene expression was observed by a Celigo® Image Cytometer at 72 hours after infection, and the positive rate of fluorescence was noted. A two-step method of quantitative real-time PCR (qRT-PCR) was used to detect the silencing effect of POSTN. Western blotting was then performed to assess the expression of autophagy-related proteins Beclin-1 and LC3 in the group showing the best gene silencing effects. The experimental results showed that there was strong green fluorescence in group KD3. As confirmed via qRT-PCR analysis, the POSTN silencing efficiency in group KD3 reached 92.1%. The Western blotting revealed that the expression of Beclin-1 protein in group KD3 was significantly higher than that in the NC group. However, the LC3 protein expression was not significantly different from that of the control group. The lentiviral vector targeting POSTN in osteoblasts was constructed successfully. In addition, the expression of autophagy protein in mouse osteoblasts increased after POSTN silencing. This finding may provide new approaches for understanding the molecular signal transduction of POSTN during the tooth eruption process.

Disparate expression of autophagy in corneas of C57BL/6 mice and BALB/c mice after Aspergillus fumigatus infection.

To determine the disparate expression of autophagy in the Aspergillus fumigatus (A. fumigatus) keratitis between susceptible C57BL/6 mice and resistant BALB/c mice. C57BL/6 and BALB/c mice were used to establish fungal keratitis models. Disease severity and inflammatory response were observed by slit lamp microscopy in A. fumigatus-infected corneas of C57BL/6 and BALB/c mice at 1, 3 and 5d. Hematoxylin-eosin (H&E) staining was used to detect pathological changes of corneas. The expression of autophagy-related proteins Beclin-1, LC3, SQSTM1/p62, and LAMP-1 was assessed by Western blot in C57BL/6 and BALB/c mice at 1, 3 and 5d post infection (p.i.). Immunofluorescent staining was used to test the expression of LC3 in corneas after A. fumigatus infection. Keratitis severity was higher in C57BL/6 mice versus BALB/c mice at 1, 3 and 5d p.i. H&E staining showed that the number of inflammatory cells was larger and the severity of ulcer was higher in C57BL/6 mice than in BALB/c mice after stimulation with A. fumigatus. Higher expression of LAMP-1, Beclin-1, and LC3 was shown in C57BL/6 mice corneas than in BALB/c mice corneas at 1, 3 and 5d p.i., while the expression of p62 was lower in C57BL/6 mice. The fluorescence of LC3 was significantly increased in corneas of C57BL/6 mice compared with BALB/c mice after A. fumigatus infection. The expression of autophagy is higher in corneas of C57BL/6 mice than in BALB/c mice after A. fumigatus infection. Autophagy may be positively correlated with keratitis severity and pathological changes.

Expression of autophagy-related protein Beclin-1 and microtubule-associated protein 2 light chain 3 in periodontal ligament cells in orthodontic tooth pressure areas

To investigate the expression of autophagy-related protein Beclin-1 and microtubule-associated protein 2 light chain 3 (LC3Ⅱ) in periodontal ligament cells in orthodontic tooth pressure areas. Sixty male SD rats were randomly divided into a blank control group and nine experimental groups. In the experimental groups, 0.392 N orthodontic force was used to move the first right upper molars for 15 min, 30 min, 1 h, 2 h, 4 h, 12 h, 1 d, 3 d, or 7 d. The blank control group did not receive any treatment. The rats were euthanized. Changes in the morphology of the periodontal membrane in the pressure areas were observed through hematoxylin and eosin (HE) staining. The expression levels of Beclin-1 and LC3Ⅱ were detected by immunohistochemical staining, and tartrate-resistant acid phosphatase (TRAP) staining was performed for the counting of osteoclasts. The HE stains showed that the hyalinization of the periodontal ligament appeared in the pressure areas after 1 day of exertion and was gradually aggravated. The immunohistochemical stains showed that the expression levels of Beclin-1 and LC3Ⅱ in the experimental groups gradually increased, peaked after 1 h, and then gradually decreased. The expression levels peaked again after 1 d, then decreased to baseline levels at 7 d of exertion. Beclin-1 and LC3Ⅱ were expressed in the osteoclasts. The TRAP stains indicated that the number of osteoclasts started to increase after 1 day. Autophagy may participate in the process of periodontal ligament reconstruction in orthodontic tooth pressure areas by mediating the hyalinization of periodontal ligament and affecting the biological effects of osteoclasts.

Pyridoxamine Treatment of HK-2 Human Proximal Tubular Epithelial Cells Reduces Oxidative Stress and the Inhibition of Autophagy Induced by High Glucose Levels.

BackgroundDiabetic nephropathy is a predominant cause of renal failure, which is an important chronic complication of diabetes. Pyridoxamine (PM) has been reported to protect renal tubular epithelial cells against oxidative damage and delay or inhibit the development and generation of glucose-induced renal insufficiency at the early stage of disease. In this study, we attempted to explore the protection mechanism of PM on human proximal tubular epithelial cells (HK-2 cells) induced by high glucose.Material/MethodsHK-2 cells were cultivated by high glucose medium in the absence or presence of PM. Cell Counting Kit-8 was used to investigate the most appropriate drug concentration of PM by detecting the cell viability of HK-2 cells. The expression of autophagy-related protein Beclin-1, LC-3II, and p62 was measured by western blot analysis, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and immunofluorescence. The expression and localization of Beclin-1 and p62 were also detected via immunofluorescence. The intracellular reactive oxygen species generation was detected using the reactive oxygen species assay kit. The effects of PM on antioxidant defenses were evaluated with glutathione peroxidase (GPx), manganese superoxide dismutase (MnSOD) activity, and glutathione/glutathione disulfide (GSH/GSSG) ratio.ResultsHigh glucose levels were able to upregulate the expression of oxidative stress associated protein and inhibit autophagy-associated changes verified by western blotting, RT-qPCR and immunofluorescence. Administration of PM reversed the high glucose-induced low-expressed Beclin-1 and LC-3II, and overexpressed p62 and intracellular reactive oxygen species levels. Furthermore, non-enzymatic antioxidant defenses and enzymatic antioxidant defenses were turned on by the application of PM.ConclusionsTreatment with PM could reverse high glucose-induced inhibition of autophagy and oxidative stress.

Supercritical CO2 fluid extraction of croton crassifolius Geisel root: Chemical composition and anti-proliferative, autophagic, apoptosis-inducing, and related molecular effects on A549 tumour cells

BackgroundThe use of plant essential oils as pharmaceuticals is a fast-growing market especially in China. Throughout the 20th century, a rapid increase took place in the use of many essential oil-derived products in the medicinal industry as nutraceuticals, medicinal supplements, and pharmaceuticals. PurposeThe objective of this study was to explore the chemical composition of Croton crassifolius essential oil as well as its potential anti-tumour properties and related anti-proliferative, autophagic, and apoptosis-inducing effects. MethodsSupercritical CO2 fluid extraction technology was used to extract CCEO and the chemical constituents of the essential oil were identified by comparing the retention indices and mass spectra data taken from the NIST library with those calculated based on the C7-C40 n-alkanes standard. The cytotoxic activity and anti-proliferative effects of CCEO were evaluated against five cancer cell lines and one normal human cell line via CCK-8 assays. In addition, flow cytometry was used to detect cell cycle arrest. The efficacy of CCEO treatments in controlling cancer cell proliferation was assessed by cell cycle analysis, clonal formation assays, RT-qPCR, and western blot analysis. Autophagic and apoptosis-inducing effects of oils and the associated molecular mechanisms were assessed by flow cytometry, cell staining, reactive oxygen species assays, RT-qPCR, and western blot analysis. ConclusionForty compounds representing 92.90% of the total oil were identified in CCEO. The results showed that CCEO exerted a measurable selectivity for cancer cell lines, especially for A549 with the lowest IC50 value of 25.00 ± 1.62 μg/mL. Assessment of the anti-proliferative effects of CCEO on A549 cells showed that the oil inhibited cell proliferation and colony formation in a dose- and time-dependent manner. Investigation of the molecular mechanisms of cell cycle regulation confirmed that the oil arrested A549 cells in G2/M phase by decreasing the expression of cyclin B1-CDK1 and cyclin A-CDK1 and increasing the expression of cyclin-dependent kinase inhibitor (CKI) P21 at both the transcriptional and translational levels. Autophagy staining assays and western blot analysis revealed that CCEO promoted the formation of autophagic vacuoles in A549 cells and increased the expression of autophagy-related proteins beclin-1 and LC3-II in a dose-dependent manner. A series of apoptosis analyses indicated that CCEO induces apoptosis through a mitochondria-mediated intrinsic pathway. This study revealed that CCEO is a promising candidate for development into an anti-tumour drug of the future.

Killing effect of oncolytic adenovirus Ad11 containing human telomerase reverse transcriptase combined with ifosfamide on human osteosarcoma cells and possible mechanism

Objective To study the killing effect of oncolytic adenovirus Ad11 combined with ifosfamide on human osteosarcoma cells and the possible mechansim, and provide cytological basis for oncolytic adenovirus Ad116 in the treatment of osteosarcoma. Methods The human osteosarcoma cell line MG63 was cultured in vitro, and the 50% maximal effect concentration (EC50) of Ad11 and the 25% inhibitory concentration (IC25) and 50% inhibitory concentration (IC50) of adrephosphamide were detected by cytotoxicity assay (MTS method). The MTS method was used to detect the cell viability of the control group, Ad11 group [multiplicity of infection (MOI)=5], ifosfamide group 1 (IC25=15 μmol/ml), ifosfamide group 2 (IC50=25 μmol/ml), combined group 1 (Ad11+ IC25) and combined group 2 (Ad11+ IC50). Western blotting was used to detect the expression of autophagy-related proteins Beclin-1 and LC3A/B in the control and experimental groups. Results The MTS assay showed that Ad11 treatment of osteosarcoma cells had an EC50 value of 5.26 MOI, and combined with ifosfamide had synergistic killing effect on osteosarcoma cell MG63. Western blotting results showed that autophagy induced by ifosfamide was inhibited when Ad11 was treated with ifosfamide. Conclusion Ad11 has a killing effect on osteosarcoma cell line MG63. The synergistic effect is enhanced when Ad11 is combined with ifosfamide. The synergistic effect may be due to the weakened protective autophagy of MG63 cells. Key words: Oncolytic adenovirus; Ad11; Osteosarcoma; Autophagy; Ifosfamide

Secreted Phosphoprotein 1 Promotes the Development of Small Cell Lung Cancer Cells by Inhibiting Autophagy and Apoptosis.

This study aimed to investigate the expression of secreted phosphoprotein 1 (SPP1) on lung cancer cells and explore its underlying mechanism on autophagy and apoptosis which effect the development of lung cancer cells. GSE19804 related to lung cancer cells was screened from Gene Expression Omnibus (GEO) database, and we screened the 47 pairs of differential expressed mRNAs in lung cancer cells and adjacent tissues using microarray analysis. The expression of the core gene SPP1 was detected by qRT-PCR and western-blot. The transfection efficiency of lung cancer cells was detected by qRT-PCR and the expression of transfected group was tested by western-blot. Cell proliferation after transfection was tested by MTT assay and plate cloning experiment. The apoptosis rate of each transfection group was detected by flow cytometry. We use western-blot to test protein expression of autophagy-related proteins Beclin-1, LC3-I, LC3-II and p62 of each transfected group. Through analysis of GSE19804,the heat map showed SPP1 was the highest expressed in tumor tissues. qRT-PCR and western-blot detected SPP1 expression in lung cancer tissues was higher than that in normal adjacent tissues and was significantly increased in lung cancer cell lines. After transfection with pcDNA3.1-SPP1 (p-SPP1 group), siRNA1-SPP1 (siRNA1 group) and siRNA2-SPP1 (siRNA2 group), showed different expression of SPP1. Up-regulation of SPP1 enhanced cell viability and promoted tumor cell proliferation, while knockdown of SPP1 inhibited tumor cell proliferation. From the results of apoptosis rate, SPP1 inhibited the tumor cell apoptosis. However, in normal lung cell, SPP1 had no effect on cell proliferation and apoptosis. And to test autophagy-related proteins, we found that overexpression of SPP1 inhibited autophagy. High expression of SPP1 inhibited autophagy and apoptosis to promote the development of small cell lung cancer cells.

Importance of Autophagy in the Recovery of Muscle Function After Injury in an Ovariectomized Model

The lack of ovarian hormones accentuates the loss of muscle contractility after muscle injury. Mitochondria are required to meet the energetic demands of muscle contractility, but whether mitochondrial function is affected by muscle injury and impacts repair is unclear in the context of ovarian hormone depletion. PURPOSE: To test mitochondrial dysfunction after muscle injury in the context of ovarian hormone depletion and to investigate autophagy, a cellular process for degrading damaged and dysfunctional mitochondria, as a mechanism of mitochondrial remodeling during regeneration. METHODS: We subjected sham surgery wildtype (WT) and ovariectomized (OVX) mice to traumatic muscle injury and assessed the recovery of in vivo muscle strength (i.e. ankle dorsiflexion) and state 3 respiration from permeabilized muscle fibers at 7 and 14 days post-injury. To investigate autophagy, expression of autophagy-related proteins Beclin1 (Atg6) and LC3 were assessed. To determine if an interaction exists between ovarian hormones and autophagy, muscle strength and state 3 respiration were assessed 14 days post-injury following sham or OVX surgeries on Ulk1 deficient mice, a necessary protein for mitochondrial-specific autophagy, and littermate controls. RESULTS: OVX resulted in a 10% reduction in muscle strength (p=0.045) pre-injury compared to sham. This was exacerbated by muscle injury (14 days post-injury: OVX 25% vs. sham 35% of pre-injury, p=0.038). For state 3 respiration, there was a main effect of injury demonstrating a substantial reduction in mitochondrial function at 7 and 14 days post-injury (p<0.0001), independent of ovarian hormones. There was a large induction of autophagy as indicated by greater Beclin1 and LC3 expression at 7 and 14 days post-injury, independent of ovarian hormones (p=0.001, p=0.014 respectfully). Interestingly, OVX-Ulk1-deficient mice demonstrated less recovery of muscle strength at 14 days post-injury compared to OVX-LM (p= p=0.016). CONCLUSIONS: After muscle injury a robust autophagic response is required to recover muscle function in a timely manner and this occurs in the presence and absence of ovarian hormones. However, decreased strength recovery in OVX-Ulk deficient mice suggests an interaction between ovarian hormones and autophagy during muscle regeneration.

Astragalus polysaccharide may increase sensitivity of cervical cancer HeLa cells to cisplatin by regulating cell autophagy

This study aimed to investigate the possible sensitivity of Astragalus polysaccharides, in order to improve the chemosensitivity of cervical cancer HeLa cells to cisplatin by regulating the cell autophagy, and explore its possible mechanism. In this study, HeLa cells were divided into control group, cisplatin group, Astragalus polysaccharide group, and Astragalus polysaccharide combined with cisplatin group. MTT assay was used to detect the proliferation of cervical cancer HeLa cells. Flow cytometry was used to detect the apoptosis and cycle of HeLa cells in each experimental group. RT-PCR was used to detect the mRNA expression of autophagy-related proteins beclin1, LC3Ⅱ and p62. The expression levels of autophagy-related proteins beclin1, LC3Ⅱ, LC3Ⅰ and p62 were detected by WB method. MTT results showed that compared with the control group, the proliferation of HeLa cells was significantly inhibited in each administration group(P<0.05), and the inhibitory effect of the combination group was more significant(P<0.01). The apoptotic rate of HeLa cells was significantly increased(P<0.05), and the apoptotic rate of the combination group was significantly increased(P<0.01) compared with the control group(P<0.05).In conclusion, G₀/G₁ phase showed the most significant differences between the two groups. RT-PCR and WB results showed that the gene and protein expressions of beclin1 and LC3Ⅱ were up-regulated, while the gene and protein expressions of p62 were down-regulated compared with the control group. The above-mentioned changes in the combination group were more significant. Through the analysis of the above experimental results, it is speculated that Astragalus polysaccharides may increase the sensitivity of cervical cancer HeLa cells to cisplatin by regulating the cell autophagy. Its possible mechanism of action is correlated with the up-regulation of autophagy-related proteins beclin1, the promote the conversion from LC3Ⅰ to LC3Ⅱ, the down-regulation of labeled protein p62, and the enhancement of HeLa cell autophagic activity, thereby increasing the sensitivity of HeLa cells to cisplatin chemotherapy.

Receptor for advanced glycation end product blockade enhances the chemotherapeutic effect of cisplatin in tongue squamous cell carcinoma by reducing autophagy and modulating the Wnt pathway.

Tongue squamous cell carcinoma (TSCC) is one of the most severe types of cancer with poor outcomes. Cisplatin is used widely to treat cancer cells, but many patients develop acquired drug resistance. The receptor for advanced glycation end products (RAGE) is expressed widely in TSCC and associated with drug-induced chemotherapy resistance. However, the effect of RAGE and cisplatin on Tca-8113 cells remains unknown. We assayed the combined use of RAGE blockade and cisplatin effect on Tca-8113 cells' viability by MTT and apoptosis rate of Tca-8113 cells on RAGE blockade+cisplatin treatment; cisplatin alone; or RAGE blockade alone by flow cytometry. We observed the expressions of autophagy-related proteins beclin1, LC3II, p62; Wnt signaling-related proteins β-catenin, GSK3β, WNT5A, ROR-2; and apoptosis-related protein cleaved caspase-3, bcl-2-associated X proteins using western blot. We determined WNT5A and beclin1 expression on Tca-8113 cells by immunofluorescence. We further observed autophagy vacuoles by monodansylcadaverine staining. We found that RAGE blockade and cisplatin significantly decreased cell viability and increased the cell apoptosis rate compared with cisplatin alone. Furthermore, RAGE blockade suppressed the canonical Wnt pathway proteins β-catenin and GSK-3β, but upregulated noncanonical WNT5A and receptor ROR-2. We show that RAGE blockade suppressed the levels of autophagy-related protein LC3II/I, beclin1, accelerated degradation of autophagy for the increasing p62 expression, and increased cell apoptosis for the increasing expressions of cleaved caspase-3 and bcl-2-associated X proteins. We observed the location of WNT5A and beclin1 expressions on cells by immunofluorescence and their trends were consistent with western blotting. Taken together, our findings suggested that RAGE blockade+cisplatin improved chemotherapeutic effects by reducing autophagy and regulating Wnt/β-catenin to suppress the progression of TSCC.

Electroacupuncture Ameliorates Cerebral Ischemia-Reperfusion Injury by Regulation of Autophagy and Apoptosis.

Background. The therapeutic mechanisms of cerebral ischemia treatment by acupuncture are yet not well addressed. Objective. We investigated the effects of electroacupuncture (EA) at GV26 observing the expression of autophagy-related proteins Beclin-1 and LC3B and proportion of apoptotic cells and Bcl-2 positive cells in MCAO/R model rats. Methods. Sprague-Dawley (SD) male rats were randomly assigned to 7 groups: model groups (M6h, M24h, and M72h), EA treatment groups (T6h, T24h, and T72h), and sham operation group (S). Neurological deficit and cerebral infarction volume were measured to assess the improvement effect, while the expression of Beclin-1 and LC3B and proportion of Tunel-positive and Bcl-2 positive cells were examined to explore EA effect on autophagy and apoptosis. Results. EA significantly decreased neurological deficit scores and the volume of cerebral infarction. Beclin-1 was significantly decreased in T24h, while LC3B-II/LC3B-I ratio markedly reduced in 6th hour. EA groups markedly reduced the number of Tunel positive cells, especially in T24h. Meanwhile, the number of Bcl-2 positive cells obviously increased after EA treatment, especially in T6h and T24h. Conclusions. The alleviation of inadequate autophagy and apoptosis may be a key mechanism involved in the reflex regulation of EA at GV26 to treat cerebral ischemia.

Clinical significance of expression of autophagy-related proteins Beclin1, LC3 and P62 in advanced pancreatic cancer

Clinical significance of expression of autophagy-related proteins Beclin1, LC3 and P62 in advanced pancreatic cancer

Expression of autophagy-related protein beclin-1 in malignant canine mammary tumors

BackgroundAutophagy is a self-catabolic mechanism that degrades unnecessary cellular components through lysosomal enzymes. Beclin-1, an autophagy-related protein, establishes the first connection between autophagy and tumorigenesis. The purpose of this study is to assess the Beclin-1 expression pattern and to determine its prognostic significance in patients with malignant canine mammary tumor (CMT).ResultsWe examined Beclin-1 expression in 70 cases of malignant CMTs by immunohistochemistry. Cytoplasmic Beclin-1 expression was significantly weaker in cancer cells than in nearby normal mammary glands (p < 0.001). Low cytoplasmic expression (57.14%) was associated with older age, lower degree of tubular formation, increased mitotic activity, higher histologic grade, and extensive necrosis. Low nuclear expression (40%) was connected with older age, lower degree of tubular formation, extensive necrosis, and negative for Her2/neu overexpression. Univariate survival analysis showed that Beclin-1 cytoplasmic expression was a poor prognostic factor for overall survival rate (p < 0.001). Multivariate survival analysis demonstrated that Beclin-1 cytoplasmic expression is an independent prognostic factor (p = 0.016).ConclusionsLoss of Beclin-1 is associated with aggressive clinicopathologic features and poor overall survival. The results suggest that Beclin-1 plays an important role in tumor progression of malignant CMTs.