Enrichment of Phosphopeptides Based on Zirconium Phthalocyanine-Modified Magnetic Nanoparticles.

Highly selective extraction of phosphopeptides is necessary before mass spectrometry (MS) analysis. Herein, zirconium phthalocyanine-modified magnetic nanoparticles were prepared through a simple method. The Fe-O groups on Fe3O4 and the zirconium ions on phthalocyanine had a strong affinity for phosphopeptides based on immobilized metal ion affinity chromatography (IMAC). The enrichment platform exhibited low detection limit (0.01 fmol), high selectivity (α-/ß-casein/bovine serum albumin, 1/1/5000), good reusability (10 circles), and recovery (91.1 ± 1.1%) toward phosphopeptides. Nonfat milk, human serum, saliva, and A549 cell lysate were employed as actual samples to assess the applicability of the enrichment protocol. Metallo-phthalocyanine will be a competitive compound for designing highly efficient adsorbents and offers a new approach to phosphopeptide analysis.

Exosomes derived from ITGB1 modified Telocytes alleviates LPS-induced inflammation and oxidative stress through YAP1/ROS axis.

Aims: Previous studies have demonstrated a significant upregulation of Integrin Beta 1 (ITGB1) in Telocytes. This study aims to explore the roles and underlying mechanisms of ITGB1 in inflammation and oxidative stress following Lipo-polysaccharide (LPS) administration in Telocytes. Methods: We observed an increase in reactive oxygen species (ROS) production, accompanied by a reduction in ITGB1 levels post-LPS treatment. Results: Notably, inhibiting ROS synthesis markedly reduced LPS-induced ITGB1 expression. Additionally, ectopic ITGB1 expression mitigated LPS-induced inflammation and oxidative stress, evident through decreased levels of pro-inflammatory markers such as Tumor Necrosis Factor-alpha (TNF-α), Interleukin (IL)-1ß, IL-6, and Monocyte Chemoattractant Protein (MCP)-1. Depletion of endothelial Yes-Associated Protein 1 (YAP1) notably diminished the levels of inflammatory markers and ROS production. Furthermore, exosomes secreted by ITGB1-modified Telocytes promoted Human Umbilical Vein Endothelial Cells (HUVECs) proliferation and inhibited apoptosis. In vivo experiments revealed that exosomes from ITGB1-modified Telocytes modulated functional and structural changes, as well as inflammatory responses in Acute Lung Injury (ALI). Conclusion: These findings highlight the critical role of the YAP1/ROS axis in LPS-induced Telocyte injuries, underlining the therapeutic potential of targeting ITGB1 for mitigating inflammation and oxidative stress in these cells.

Zirconium-rich magnetic polyoxometalate-based metal-organic framework: Tailored for phosphopeptide analysis from lung cancer A549 cells.

Polyoxometalate-based metal-organic frameworks (POMOFs) have become a promising affinity material for separation and enrichment. The analysis of protein phosphorylation represents a challenge for the development of efficient enrichment materials. Here, a novel zirconium-rich magnetic POMOF was successfully designed and prepared for the enrichment of phosphopeptides. The binding affinity of the nanomaterial partly came from Fe-O clusters in the MOF. The Lewis acid-base interactions between V-O clusters and zirconium ions in V10O28-Zr4+ and phosphate groups in phosphopeptides further strengthened the enrichment ability. The zirconium-rich magnetic POMOF was employed to capture phosphopeptides from non-fat milk, human saliva, and serum. Additionally, 748 unique phosphopeptide peaks were detected from the tryptic digests of lung cancer A549 cell proteins with a high specificity (86.9 %). POMOFs will become an active competitor for the design of protein affinity materials and will provide a new approach for phosphopeptide analysis.

Preparation of high-efficiency titanium ion immobilized magnetic graphite nitride nanocomposite for phosphopeptide enrichment.

BACKGROUND: Protein phosphorylation has been implicated in life processes including molecular interaction, protein structure transformation, and malignant disease. An in-depth study of protein phosphorylation may provide vital information for the discovery of early biomarkers. Mass spectrometry (MS)-based techniques have become an important method for phosphopeptide identification. Nevertheless, direct detection remains challenging because of the low ionization efficiency of phosphopeptides and serious interference from non-phosphopeptides. There is a great need for an efficient enrichment strategy to analyze protein phosphorylation prior to MS analysis. RESULTS: In this study, a novel nanocomposite was prepared by introducing titanium ions into two-dimensional magnetic graphite nitride. The nanocomposite was combined with immobilized metal ion affinity chromatography (IMAC) and anion-exchange chromatography mechanisms for phosphoproteome research. The nanocomposite had the advantages of a large specific surface (412.9 m2 g-1), positive electricity (175.44 mV), and excellent magnetic property (35.7 emu g-1). Moreover, it presented satisfactory selectivity (α-casein:ß-casein:bovine serum albumin = 1:1:5000), a low detection limit (0.02 fmol), great recyclability (10 cycles), and high recovery (92.8%). The nanocomposite demonstrated great practicability for phosphopeptides from non-fat milk, human serum, and saliva. Further, the nanocomposite was applied to enrich phosphopeptides from a more complicated specimen, A549 cell lysate. A total of 890 phosphopeptides mapping to 564 phosphoproteins were successfully detected with nano LC-MS. SIGNIFICANCE: We successfully designed and developed an efficient analysis platform for phosphopeptides, which includes protein digestion, phosphopeptide enrichment, and MS detection. The MS-based enrichment platform was further used to analyze phosphopeptides from complicated bio-samples. This work paves the way for the design and preparation of graphite nitride-based IMAC materials for phosphoproteome analysis.

Integrin beta1 mediates the effect of telocytes on mesenchymal stem cell proliferation and migration in the treatment of acute lung injury.

Co-transplantation of mesenchymal stem cells (MSCs) with telocytes (TCs) was found to have therapeutic effects, although the mechanism of intercellular communication is still unknown. Our current studies aim at exploring the potential molecular mechanisms of TCs interaction and communication with MSCs with a focus on integrin beta1 (ITGB1) in TCs. We found that the co-culture of MSCs with ITGB1-deleted TCs (TCITGB1-ko ) changed the proliferation, differentiation and growth dynamics ability of MSC in responses to LPS or PI3K inhibitor. Changes of MSC proliferation and apoptosis were accompanied with the dysregulation of cytokine mRNA expression in MSCs co-cultured with TCITGB1-ko during the exposure of PI3Kα/δ/ß inhibitor, of which IL-1ß, IL-6 and TNF-α increased, while IFN-γ, IL-4 and IL-10 decreased. The responses of PI3K p85, PI3K p110 and pAKT of MSCs co-cultured with TCITGB1-ko to LPS or PI3K inhibitor were opposite to those with ITGB1-presented TCs. The intraperitoneal injection of TCITGB1-ko , TCvector or MSCs alone, as well as the combination of MSCs with TCITGB1-ko or TCvector exhibited therapeutic effects on LPS-induced acute lung injury. Thus, our data indicate that telocyte ITGB1 contributes to the interaction and intercellular communication between MSCs and TCs, responsible for influencing other cell phenomes and functions.

Effects of perioperative exercise on cardiorespiratory endurance in children with congenital heart disease in plateau areas after surgical repair.

We aimed to explore the effects of perioperative exercise on cardiorespiratory endurance in children with congenital heart disease (CHD) in plateau areas after surgical repair. Fifty children with CHD in the plateau admitted to our hospital were randomly divided into the exercise and control groups. The exercise group received a perioperative exercise intervention beginning within 24 h postoperatively, while the control group received routine nursing and treatment alone. To assess the 6 min walk distance (6MWD) at baseline and at end of intervention, children participated in a 6-min walk test before cardiac repair and at 1 week after general ward transfer. A subset of children in the study underwent the cardiopulmonary exercise test pre-operatively. The 6MWD of children with CHD at baseline was positively correlated with the peak oxygen uptake pre-operatively. No significant difference was reported in the preoperative baseline data of both groups. The 6MWD of the exercise group was significantly higher than that of the control group. Early exercise therapy after cardiac repair could significantly improve the cardiorespiratory endurance and exercise capacity of children with CHD in plateau areas.

Cerium ions immobilized magnetic graphite nitride decorated with L-Alanyl-L-Glutamine as new chelator for enrichment of phosphopeptides.

Cerium ions immobilized magnetic graphite nitride material have been prepared using L-Alanyl-L-Glutamine as the new chelator. The resulting Fe3O4/g-C3N4-L-Ala-L-Gln-Ce4+, as an immobilized metal ion affinity chromatography (IMAC) sorbent, was reusable. This is due to the strong coordination interaction between L-Alanyl-L-Glutamine and cerium ions. After a series of characterizations, the magnetic nanocomposite showed high surface area, good hydrophilicity, positive electricity, and magnetic response. Fe3O4/g-C3N4-L-Ala-L-Gln-Ce4+ had high sensitivity (0.1 fmol), selectivity (α-/ß-casein/bovine serum albumin, 1:1:5000), and good recyclability (10 cycles). A total of 647 unique phosphopeptides mapped to 491 phosphoproteins were identified from A549 cell lysate by nano LC-MS analysis.

Effective Enrichment of Phosphopeptides Using Magnetic Polyoxometalate-Based Metal-Organic Frameworks.

In this study, magnetic polyoxometalate-based metal-organic frameworks (Fe3O4-POMOFs) were designed and applied to the enrichment of phosphopeptides. Thanks to the abundant metal oxide and metal ion sites, the material had a strong affinity for phosphopeptides. Simultaneously, the high amount of amino and guanidyl groups provided hydrophilicity and positive charge for phosphopeptide capture. By coupling with MS detection, the established platform possessed good reusability, high sensitivity (0.01 fmol), and high selectivity (α-casein/ß-casein/bovine serum albumin = 1:1:5000). Furthermore, the method was successfully used for the detection of phosphopeptides in nonfat milk, human serum, saliva, and A549 cell lysate, showing great potential for practical application.

Roles of pulmonary telocytes in airway epithelia to benefit experimental acute lung injury through production of telocyte-driven mediators and exosomes.

BACKGROUND: Telocytes (TCs) are experimentally evidenced as an alternative of cell therapies for organ tissue injury and repair. The aims of the present studies are to explore direct roles of TCs and the roles of TC-derived exosomes in support of experimental acute lung injury (ALI) in vivo or in vitro. MATERIALS AND METHODS: The roles of TCs in experimental ALI were firstly estimated. Phosphoinositide 3-kinase (PI3K) p110δ and α/δ/ß isoform inhibitors were used in study dynamic alterations of bio-behaviors, and in expression of functional factors of TCs per se and TC-co-cultured airway epithelial cells during the activation with lipopolysaccharide (LPS). TC-driven exosomes were furthermore characterized for intercellular communication by which activated or non-activated TCs interacted with epithelia. RESULTS: Our results showed that TCs mainly prevented from lung tissue edema and hemorrhage and decreased the levels of VEGF-A and MMP9 induced by LPS. Treatment with CAL101 (PI3K p110δ inhibitor) and LY294002 (PI3Kα/δ/ß inhibitor) could inhibit TC movement and differentiation and increase the number of dead TCs. The expression of Mtor, Hif1α, Vegf-a, or Mmp9 mRNA increased in TCs challenged with LPS, while Mtor, Hif1α, and Vegf-a even more increased after adding CAL101 or Mtor after adding LY. The rate of epithelial cell proliferation was higher in co-culture of human bronchial epithelial (HBE) and TCs than that in HBE alone under conditions with or without LPS challenge or when cells were treated with LPS and CAL101 or LY294002. The levels of mTOR, HIF1α, or VEGF-A significantly increased in mono-cultured or co-cultured cells, challenged with LPS as compared with those with vehicle. LPS-pretreated TC-derived exosomes upregulated the expression of AKT, p-AKT, HIF1α, and VEGF-A protein of HBE. CONCLUSION: The present study demonstrated that intraperitoneal administration of TCs ameliorated the severity of lung tissue edema accompanied by elevated expression of VEGF-A. TCs could nourish airway epithelial cells through nutrients produced from TCs, increasing epithelial cell proliferation, and differentiation as well as cell sensitivity to LPS challenge and PI3K p110δ and α/δ/ß inhibitors, partially through exosomes released from TCs.

Design of two-dimensional molybdenum trioxide-immobilized magnetic graphite nitride nanocomposites with multiple affinity sites for phosphopeptide enrichment.

Here, two-dimensional magnetic graphite nitride nanosheets were synthesized, over which molybdenum trioxide was distributed evenly. The magnetic nanocomposite served as an enrichment platform for phosphopeptides based on metal oxide affinity chromatography and anion-exchange chromatography. Owing to the abundant affinity sites, positive electricity, and large surface area, the nanocomposite had high selectivity (α-casein/ß-casein/bovine serum albumin, 1:1:5000), sensitivity (0.1 fmol), quantitative recovery (91.3%), and good recyclability (10 cycles). The nanocomposite also showed good enrichment performance for phosphopeptides from non-fat milk, human serum, saliva, and A549 cell lysate. The results demonstrated the value of this material in phosphorylated proteomics.

Layer-by-layer assembly of multilayered double hydroxides/polyoxometalate-coated magnetic nanoparticles for highly efficient phosphopeptide enrichment.

A layer-by-layer (LbL) assembly strategy was developed to prepare multilayered double hydroxide/polyoxometalate shell-coated magnetic nanoparticles. The introduction of functional shells not only offered abundant affinity sites of metal oxide and metal ions but also increased the surface area for the contact with targets. By combining the enrichment strategies of immobilized metal ion affinity chromatography and metal oxide affinity chromatography, the nanomaterial can capture phosphopeptides via a synergistic effect. The method presented a low detection limit of 0.1 fmol in combination with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis. The nanomaterial showed satisfactory selectivity (1:1:5000 M ratio of α-/ß-casein/bovine serum albumin), good recovery (92.07%), high adsorption capacity (117.6 mg g-1), and ten times reusability for capturing phosphopeptides.

Metabolomics profiling in prediction of chemo-immunotherapy efficiency in advanced non-small cell lung cancer.

Background: To explore potential metabolomics biomarker in predicting the efficiency of the chemo-immunotherapy in patients with advanced non-small cell lung cancer (NSCLC). Methods: A total of 83 eligible patients were assigned to receive chemo-immunotherapy. Serum samples were prospectively collected before the treatment to perform metabolomics profiling analyses under the application of gas chromatography mass spectrometry (GC-MS). The key metabolites were identified using projection to latent structures discriminant analysis (PLS-DA). The key metabolites were used for predicting the chemo-immunotherapy efficiency in advanced NSCLC patients. Results: Seven metabolites including pyruvate, threonine, alanine, urea, oxalate, elaidic acid and glutamate were identified as the key metabolites to the chemo-immunotherapy response. The receiver operating characteristic curves (AUC) were 0.79 (95% CI: 0.69-0.90), 0.60 (95% CI: 0.48-0.73), 0.69 (95% CI: 0.57-0.80), 0.63 (95% CI: 0.51-0.75), 0.60 (95% CI: 0.48-0.72), 0.56 (95% CI: 0.43-0.67), and 0.67 (95% CI: 0.55-0.80) for the key metabolites, respectively. A binary logistic regression was used to construct a combined biomarker model to improve the discriminating efficiency. The AUC was 0.86 (95% CI: 0.77-0.94) for the combined biomarker model. Pathway analyses showed that urea cycle, glucose-alanine cycle, glycine and serine metabolism, alanine metabolism, and glutamate metabolism were the key metabolic pathway to the chemo-immunotherapy response in patients with advanced NSCLC. Conclusion: Metabolomics analyses of key metabolites and pathways revealed that GC-MS could be used to predict the efficiency of chemo-immunotherapy. Pyruvate, threonine, alanine, urea, oxalate, elaidic acid and glutamate played a central role in the metabolic of PD patients with advanced NSCLC.

Exosomal lncRNA KLF3-AS1 derived from bone marrow mesenchymal stem cells stimulates angiogenesis to promote diabetic cutaneous wound healing.

AIMS: We focused on BMSC-derived exosomal lncRNA KLF3-AS1 and its significance in diabetic cutaneous wound healing. METHODS: Potential interaction between KLF3-AS1 and miR-383, miR-383 and VEGFA were predicted using bioinformatic analysis and validated by luciferase reporter, RIP, and FISH assays. The proliferation, apoptosis, migration and tube formation of HUVECs were evaluated by CCK-8, flow cytometry, wound healing, and tube formation assays, respectively. A murine diabetic cutaneous wound model was used to investigate therapeutic effects of exosomal KLF3-AS1 in vivo. Histological alterations in skin tissues were examined using HE, Masson staining, and immunostaining of CD31. RESULTS: BMSC-derived exosomal KLF3-AS1 sufficiently promoted proliferation, migration, and tube formation, while inhibited apoptosis of HUVECs challenged by high glucose. The protective effects of exosomal KLF3-AS1 were achieved at least partially by down-regulating miR-383, and boosting the expression of its target, VEGFA. In vivo, exosomes from KLF3-AS1-expressing BMSCs demonstrated the best effects in promoting cutaneous wound healing in diabetic mice, which were associated with minimal weight loss, increased blood vessel formation, reduced inflammation, decreased miR-383 expression, and up-regulated VEGFA. CONCLUSIONS: Exosomal lncRNA KLF3-AS1 derived from BMSCs induces angiogenesis to promote diabetic cutaneous wound healing.

Combining multiple Bacillus spp. with fish protein hydrolysates mitigates root rot (Fusarium solani) and improves cucumber seedlings growth and substrate nutrients.

AIMS: The effect of Bacillus strains combined with fish protein hydrolysates (FPHs) on cucumber root rot disease, seedlings growth and substrate nutrients was investigated. METHODS AND RESULTS: We isolated three strains capable of mitigating cucumber root rot disease, XY-1 and XY-13 strains were identified as B. amyloliquefaciens, and XY-53 strain as B. subtilis. In the absence of bacteria, The 200×dilution (5 ml L-1 ) of FPHs was the optimum concentration for improving cucumber seedlings growth. In vivo antibiosis tests showed that combined bacteria alongside FPHs inhibited the pathogen growth by 85%~90%, higher than individual bacteria. The FPHs combined either with XY-1 and XY-53 strains or with XY-13 and XY-53 strains promoted seedlings growth under infection, whereas FPHs combined with a mixture of XY-1, XY-13 and XY-53 strains showed the highest total phosphorus and organic matter content in substrate. Moreover, FPHs combined with XY-53 strain increased urease activity, while combined either with XY-13 and XY-53 strains or with XY-1, XY-13 and XY-53 strains increased sucrase activity under infection. CONCLUSIONS: FPHs combined with B. amyloliquefaciens and B. subtilis had great potential to suppress growth of root rot and promote cucumber seedlings and increase substrate nutrient content. SIGNIFICANCE AND IMPACT OF THE STUDY: Co-inoculation of B. amyloliquefaciens and B. subtilis with addition of FPHs is a good strategy for maintaining healthy crops.

Potential biomarkers and targets of mitochondrial dynamics.

Mitochondrial dysfunction contributes to the imbalance of cellular homeostasis and the development of diseases, which is regulated by mitochondria-associated factors. The present review aims to explore the process of the mitochondrial quality control system as a new source of the potential diagnostic biomarkers and/or therapeutic targets for diseases, including mitophagy, mitochondrial dynamics, interactions between mitochondria and other organelles (lipid droplets, endoplasmic reticulum, endosomes, and lysosomes), as well as the regulation and posttranscriptional modifications of mitochondrial DNA/RNA (mtDNA/mtRNA). The direct and indirect influencing factors were especially illustrated in understanding the interactions among regulators of mitochondrial dynamics. In addition, mtDNA/mtRNAs and proteomic profiles of mitochondria in various lung diseases were also discussed as an example. Thus, alternations of mitochondria-associated regulators can be a new category of biomarkers and targets for disease diagnosis and therapy.

MiR-302a-5p suppresses cell proliferation and invasion in non-small cell lung carcinoma by targeting ITGA6.

MicroRNA-302a-5p (miR-302a-5p) has been implicated in several cancers; however, its role in human non-small cell lung carcinoma (NSCLC) remains unknown. In this study, we showed that miR-302a-5p is downregulated in NSCLC tissues and cell lines. Cell Counting Kit-8 and 5-ethynyl-2'-deoxyuridine assays showed that overexpression of a miR-302a-5p mimic suppressed NSCLC cell proliferation, which was confirmed by the results of a cell cycle assay. Overexpression of miR-302a-5p also reduced the migration and invasion of NSCLC cells. Additionally, miR-302a-5p overexpression significantly inhibited NSCLC growth and metastasis in a mouse xenograft model. With regard to the underlying mechanism, integrin α6 (ITGA6) mRNA was shown to be a novel target of miR-302a-5p, and overexpression of ITGA6 attenuated the inhibitory effects of miR-302a-5p on the proliferation and migration of NSCLC cells. In clinical NSCLC samples, miR-302a-5p expression was negatively correlated with ITGA6 expression, which was high in the samples. Collectively, these results indicate that miR-302a-5p acts as a tumor suppressor in NSCLC by directly targeting ITGA6 mRNA and may be useful as a theranostic biomarker of NSCLC.

Influence of gene modification in biological behaviors and responses of mouse lung telocytes to inflammation.

BACKGROUND: Telocytes play key roles in maintenance of organ/tissue function and prevention of organ injury. However, there are great challenges to investigate telocytes functions using primary telocytes, due to the difficulties of isolation, identification, and stability. The present study aims at constructing continuous cell strain of mouse lung telocyte cell line with stable characters by gene modification and investigating biological behaviors and responses of gene-modified telocytes to inflammation. METHODS: Mouse primary lung telocytes were isolated and identified using immune-labeling markers and immunoelectron microscopy. Primary telocytes were transformed with Simian vacuolating virus 40 small and large T antigen (SV40). Biological characters, behaviors morphology, and proliferation of those gene-modified telocytes were defined and monitored dynamically for 50 generations, as compared with primary lung telocytes. Cell cycle of mouse primary lung telocytes or gene-modified telocytes was detected by flow cytometry. RESULTS: Gene modified telocytes of generations 5, 10, 30 and 50 were observed with telopodes and also showed CD34 and ckit positive. Multiple cellular morphology were also observed on telocyte cell-line under monitor of celliq and enhanced cell proliferation were showed. SV40 transduction was also reduced apoptosis and increased the ratio of S and G2 phases in telocyte cell-line. CONCLUSION: We successfully constructed mouse lung telocyte cell-line which maintained the biological properties and behaviors as primary telocytes and could responses to inflammation induced by LPS. Thus, gene-modified lung telocytes, Telocyte Line, would provide a cell tool for researchers exploring the roles and applications of telocytes involved in physiological and pathological states in future.

Small interfering RNA targeting S100A4 sensitizes non-small-cell lung cancer cells (A549) to radiation treatment.

OBJECTIVE: This study aimed to investigate the impact of S100A4-small interfering RNA (S100A4-siRNA) on apoptosis and enhanced radiosensitivity in non-small-cell lung cancer (A549) cells. We also explored the mechanisms of radiosensitization and identified a new target to enhance radiosensitivity and gene therapy for non-small-cell lung cancer. METHODS: RNA interference is a powerful tool for gene silencing. In this study, we constructed an effective siRNA to knock down S100A4. A549 cells were randomly divided into three groups: blank, negative control, and S100A4-siRNA. To investigate the effect of S100A4-siRNA, the expression of S100A4, E-cadherin, and p53 proteins and their messenger RNA (mRNA) was detected by Western blot and quantitative real-time polymerase chain reaction. Transwell chambers were used to assess cell invasion. Cell cycle and apoptosis were analyzed by flow cytometry. Radiosensitivity was determined by colony formation ability. RESULTS: Our results demonstrate that S100A4-siRNA effectively silenced the S100A4 gene. When siRNA against S100A4 was used, S100A4 protein expression was downregulated, whereas the expressions of E-cadherin and p53 were upregulated. In addition, a clear reduction in S100A4 mRNA levels was noted compared with the blank and negative control groups, whereas E-cadherin and p53 mRNA levels increased. Transfection with S100A4-siRNA significantly reduced the invasiveness of A549 cells. S100A4 silencing induced immediate G2/M arrest in cell cycle studies and increased apoptosis rates in A549 cells. In clonogenic assays, we used a multitarget, single-hit model to detect radiosensitivity after S100A4 knockdown. All parameters (D0, Dq, α, ß) indicated that the downregulation of S100A4 enhanced radiosensitivity in A549 cells. Furthermore, S100A4-siRNA upregulated p53 expression, suggesting that S100A4 may promote A549 cell proliferation, invasion, and metastasis by regulating the expression of other proteins. Therefore, siRNA-directed S100A4 knockdown may represent a viable clinical therapy for lung cancer. CONCLUSION: S100A4 downregulation potentially enhances the sensitivity of human A549 cells to radiotherapy.

Knocking Down Nucleolin Expression Enhances the Radiosensitivity of Non-Small Cell Lung Cancer by Influencing DNA-PKcs Activity.

Nucleolin (C23) is an important anti-apoptotic protein that is ubiquitously expressed in exponentially growing eukaryotic cells. In order to understand the impact of C23 in radiation therapy, we attempted to investigate the relationship of C23 expression with the radiosensitivity of human non-small cell lung cancer (NSCLC) cells. We investigated the role of C23 in activating the catalytic subunit of DNA-dependent protein kinase (DNA- PKcs), which is a critical protein for DNA double-strand breaks (DSBs) repair. As a result, we found that the expression of C23 was negatively correlated with the radiosensitivity of NSCLC cell lines. In vitro clonogenic survival assays revealed that C23 knockdown increased the radiosensitivity of a human lung adenocarcinoma cell line, potentially through the promotion of radiation-induced apoptosis and adjusting the cell cycle to a more radiosensitive stage. Immunofluorescence data revealed an increasing quantity of γ-H2AX foci and decreasing radiation-induced DNA damage repair following knockdown of C23. To further clarify the mechanism of C23 in DNA DSBs repair, we detected the expression of DNA-PKcs and C23 proteins in NSCLC cell lines. C23 might participate in DNA DSBs repair for the reason that the expression of DNA-PKcs decreased at 30, 60, 120 and 360 minutes after irradiation in C23 knockdown cells. Especially, the activity of DNA-PKcs phosphorylation sites at the S2056 and T2609 was significantly suppressed. Therefore we concluded that C23 knockdown can inhibit DNA-PKcs phosphorylation activity at the S2056 and T2609 sites, thus reducing the radiation damage repair and increasing the radiosensitivity of NSCLC cells. Taken together, the inhibition of C23 expression was shown to increase the radiosensitivity of NSCLC cells, as implied by the relevance to the notably decreased DNA-PKcs phosphorylation activity at the S2056 and T2609 clusters. Further research on targeted C23 treatment may promote effectiveness of radiotherapy and provide new targets for NSCLC patients.

[Inverse correlation of S100A4 and E-cad protein expression and their clinical significance in non-small cell lung cancer].

OBJECTIVE: The purpose of the present study was to explore the relationship between the expression of S100A4 and E-cad protein and some clinicopathological factors such as histological types, TNM stages, lymph node metastasis and prognosis in non-small cell lung cancer (NSCLC), and analyze whether there is a correlation between them. METHODS: The expression of S100A4 protein and E-cad protein was detected with immunohistochemical SP technique in 116 non-small-cell lung cancers. RESULTS: The positive immunohistochemical staining for S100A4 protein was observed in 64 out of the 116 patients, with a positive rate of 55.2%. The expression rate of S100A4 protein was associated with age, tumor size, lymph node metastasis and prognosis of NSCLC. The expression of S100A4 protein was not significantly related with histological types, sex and TNM stages. The positive rate of E-cad protein expression was 65.5%. The expression of E-cad protein was associated with TNM staging, lymph node metastasis and prognosis of NSCLC. The expression of E-cad protein had no significant correlation with histological types, patient age and sex. An inverse correlation between the levels of S100A4 and E-cad protein expression was found (P < 0.005). CONCLUSION: Expression of S100A4 protein and loss of E-cad protein expression are significantly associated with tumor progression, and may become valuable markers in prediction of biological behavior and tendency of metastasis of non-small-cell lung cancers.