Effect of orexin-A on mitochondrial biogenesis, mitophagy and structure in HEK293-APPSWE cell model of Alzheimer's disease.

Mitochondrial dysfunction plays a key role in the pathogenesis and progression of Alzheimer's Disease (AD). Our previous studies showed that over expression of AD-associated mutant ß-amyloid precursor protein (APP) led to abnormalities of mitochondrial biogenesis and mitophagy, leading to mitochondrial dysfunction. However, the mechanism remains unclear. In this study, we investigated the effect of orexin-A on mitochondrial biogenesis, mitophagy and mitochondrial structure in overexpression of AD-associated mutant APP cells. We used 20E2 cells as the AD cell model. 20E2 cells were treated with orexin-A (50, 100 nmol/L). The effect of different concentrations of orexin-A on cell activity was detected by MTT. As compared with the non-treated 20E2 cells, orexin-A-treated 20E2 cells showed increased expression of APP, decreased cell viability and decreased adenosine triphosphate (ATP) level, decreased levels of regulatory proteins of mitochondrial biogenesis (peroxisome proliferator-activated receptor gamma coactivator 1-alpha [PGC-1α], nuclear respiratory factor 1/2 [NRF1/2], mitochondrial transcription factor A [TFAM]), increased levels of regulatory proteins of mitophagy (Parkin, PTEN-induced putative kinase 1 [PINK1], microtubule-associated protein light chain 3 II/I [LC3-II/LC3-I]) and decreased p62 level, with damaged mitochondrial structure. Orexin-A may reduce mitochondrial biogenesis, enhance mitophagy and damage mitochondrial structure in AD.

Vascular endothelial growth factor alleviates mitochondrial dysfunction and suppression of mitochondrial biogenesis in models of Alzheimer's disease.

PURPOSE: Mitochondrial dysfunction is a prominent feature of Alzheimer's disease (AD). As vascular endothelial growth factor (VEGF) has been shown to be protective in AD, the aim of this study was to investigate the effects of VEGF on mitochondrial function in models of AD. MATERIALS AND METHODS: Adeno associated virus (AAV)-VEGF was injected into the hippocampus of APP/PS1 mice. Cognitive function was assessed in these mice with use of the Morris water maze (MWM) and ß-amyloid (Aß) levels in the hippocampus were also measured. Cell viability and reactive oxygen species (ROS) levels were determined in the SH-SY5Y cells treated with Aß25-35 which served as a cell model of AD. Transmission electron microscopy (TEM) was used to evaluate structural changes in mitochondria and mitochondrial DNA (mtDNA) copy number and mitochondrial membrane potential (MMP) were also recorded. Finally, we investigated the effects of VEGF upon mitochondrial biogenesis, autophagy and mitochondrial autophagy (mitophagy) as determined both in vivo and in vitro with western blots. RESULTS: VEGF treated mice showed improvements in spatial learning and memory along with reduced Aß levels. VEGF protected SH-SY5Y cells against Aß25-35 induced neurotoxicity as demonstrated by increased cell viability and decreased ROS production. Associated with these effects were improvements in mitochondrial structure and function, and increased numbers of mitochondria resulting from stimulation of mitochondrial biogenesis. CONCLUSIONS: VEGF alleviates Aß related patholoy in models of AD. In part, these beneficial effects of VEGF result from protection of mitochondria and stimulation of mitochondrial biogenesis.

[Establishment and differential protein identification of two-dimensional gel electrophoresis for proteomics in the spinal cord of morphine-tolerant rats].

OBJECTIVE: To establish a two-dimensional gel electrophoresis (2-DE) map for comparative proteomic analysis of rat spinal cord with chronic morphine tolerance, and to detect differentially expression proteins that are associated with chronic morphine tolerance.
 Methods: Sixteen male SD rats received the intrathecal catheterization operation and they were randomly divided into a morphine tolerance group (MT group, n=8) and a saline group (NS group, n=8). The lumbar enlargement segments of the MT group and the NS group spinal cord were harvested and proteins were separated by 2-DE. Differential proteome profiles were established and analyzed by means of immobilized pH gradient-based two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The 2-DE maps were visualized after coomassie blue staining and analyzed using PDQuest analysis software. Identification of differential protein spots was conducted by MALDI-TOF-MS, and the Mascot query software was used to search Swiss-Prot database for bioinformatics analysis. Western blotting was used to verify the expression of some differentially expressed proteins.
 Results: A total of 1 000 spots were identified in 2-DE maps of rat spinal cord tissues from the MT group and the NS group, and 36 proteins were significantly differentially expressed in the MT group compared with the NS group. Identification was conducted by MALDI-TOF-MS and Swiss-Prot database through Mascot query software, and a total of 14 proteins were obtained. Among them, 2 protein spots were down-regulated in the MT group compared with that in the NS group, and 12 protein spots were up-regulated in the MT group compared with that in the NS group. Two kinds of proteins (NUDAA, ENOG) were verified by Western blotting and the results were consistent with proteomics data.
 Conclusion: The optimized 2-DE profiles for the proteome of spinal cord tissue in rats with chronic morphine tolerance is established preliminarily, which showed that morphine tolerance can cause changes in the expression of various proteins in the spinal cord.

How many proteins can be identified in a 2DE gel spot within an analysis of a complex human cancer tissue proteome?

Two-dimensional gel electrophoresis (2DE) in proteomics is traditionally assumed to contain only one or two proteins in each 2DE spot. However, 2DE resolution is being complemented by the rapid development of high sensitivity mass spectrometers. Here we compared MALDI-MS, LC-Q-TOF MS and LC-Orbitrap Velos MS for the identification of proteins within one spot. With LC-Orbitrap Velos MS each Coomassie Blue-stained 2DE spot contained an average of at least 42 and 63 proteins/spot in an analysis of a human glioblastoma proteome and a human pituitary adenoma proteome, respectively, if a single gel spot was analyzed. If a pool of three matched gel spots was analyzed this number further increased up to an average of 230 and 118 proteins/spot for glioblastoma and pituitary adenoma proteome, respectively. Multiple proteins per spot confirm the necessity of isotopic labeling in large-scale quantification of different protein species in a proteome. Furthermore, a protein abundance analysis revealed that most of the identified proteins in each analyzed 2DE spot were low-abundance proteins. Many proteins were present in several of the analyzed spots showing the ability of 2DE-MS to separate at the protein species level. Therefore, 2DE coupled with high-sensitivity LC-MS has a clearly higher sensitivity as expected until now to detect, identify and quantify low abundance proteins in a complex human proteome with an estimated resolution of about 500 000 protein species. This clearly exceeds the resolution power of bottom-up LC-MS investigations.

Identification of the proteomic variations of invasive relative to non-invasive non-functional pituitary adenomas.

The incomplete surgery section of invasive non-functional pituitary adenomas (NFPAs) carries the increased risks of complications and requires adjuvant radiotherapy and medications. It is necessary to clarify the molecular mechanisms and markers of invasiveness to guide the management of NFPA patients. The study aimed to proteomic variations of invasive and non-invasive NFPAs and sought the protein markers for invasive NFPAs. Invasive (n = 4) and non-invasive (n = 4) NFPA tissues were analyzed (n = 3-5/each tissue) with 2DE and PDQuest software. Twenty-four high-resolution 2DE gels were quantitatively compared to determine differentially expressed proteins (DEPs) between invasive and non-invasive NFPAs. Approximately 1200 protein spots were detected in each 2DE map, and 103 differential spots (64 upregulated and 39 downregulated) were identified. Among those 103 differential spots, 57 DEPs (30 upregulated and 27 downregulated) were characterized with peptide mass fingerprint and MS/MS. Gene-ontology (GO) and ingenuity pathway analyses of those DEPs revealed pathway networks including mitochondrial dysfunction, oxidative stress, mitogen-activated protein kinase signaling abnormality, TR/RXR activation, proteolysis abnormality, ketogenesis and ketolysis, cyclin-dependent kinase C signaling abnormality, and amyloid processing that were significantly associated with invasive characteristics of invasive NFPA. Those data demonstrate that proteomic variations exist between invasive and non-invasive NFPAs. 2DE-based comparative proteomics is an effective approach to identify proteomic variations and pathway network variations. Those findings will serve as a basis to understand the molecular mechanisms of invasive NFPAs and to discover protein markers to effectively manage patients with invasive NFPAs.

Identification of candidate biomarkers for early detection of human lung squamous cell cancer by quantitative proteomics.

To discover novel biomarkers for early detection of human lung squamous cell cancer (LSCC) and explore possible mechanisms of LSCC carcinogenesis, iTRAQ-tagging combined with two dimensional liquid chromatography tandem MS analysis was used to identify differentially expressed proteins in human bronchial epithelial carcinogenic process using laser capture microdissection-purified normal bronchial epithelium (NBE), squamous metaplasia (SM), atypical hyperplasia (AH), carcinoma in situ (CIS) and invasive LSCC. As a result, 102 differentially expressed proteins were identified, and three differential proteins (GSTP1, HSPB1 and CKB) showing progressively expressional changes in the carcinogenic process were selectively validated by Western blotting. Immunohistochemistry was performed to detect the expression of the three proteins in an independent set of paraffin-embedded archival specimens including various stage tissues of bronchial epithelial carcinogenesis, and their ability for early detection of LSCC was evaluated by receiver operating characteristic analysis. The results showed that the combination of the three proteins could perfectly discriminate NBE from preneoplastic lesions (SM, AH and CIS) from invasive LSCC, achieving a sensitivity of 96% and a specificity of 92% in discriminating NBE from preneoplatic lesions, a sensitivity of 100% and a specificity of 98% in discriminating NBE from invasive LSCC, and a sensitivity of 92% and a specificity of 91% in discriminating preneoplastic lesions from invasive LSCC, respectively. Furthermore, we knocked down GSTP1 in immortalized human bronchial epithelial cell line 16HBE cells, and then measured their susceptibility to carcinogen benzo(a)pyrene-induced cell transformation. The results showed that GSTP1 knockdown significantly increased the efficiency of benzo(a)pyrene-induced 16HBE cell transformation. The present data first time show that GSTP1, HSPB1 and CKB are novel potential biomarkers for early detection of LSCC, and GSTP1 down-regulation is involved in human bronchial epithelial carcinogenesis.

Proteomic analysis identifies PFKP lactylation in SW480 colon cancer cells.

Aerobic glycolysis is a pivotal hallmark of cancers, including colorectal cancer. Evidence shows glycolytic enzymes are regulated by post-translational modifications (PTMs), thereby affecting the Warburg effect and reprograming cancer metabolism. Lysine lactylation is a PTM reported in 2019 in histones. In this study, we identified protein lactylation in FHC cells and SW480 colon cancer cells through mass spectrometry. Totally, 637 lysine lactylation sites in 444 proteins were identified in FHC and SW480 cells. Lactylated proteins were enriched in the glycolysis pathway, and we identified lactylation sites in phosphofructokinase, platelet (PFKP) lysine 688 and aldolase A (ALDOA) lysine 147. We also showed that PFKP lactylation directly attenuated enzyme activity. Collectively, our study presented a resource to investigate proteome-wide lactylation in SW480 cells and found PFKP lactylation led to activity inhibition, indicating that lactic acid and lactylated PFKP may form a negative feedback pathway in glycolysis and lactic acid production.

Association of the High-Sensitivity C-Reactive Protein-to-Albumin Ratio with Carotid Atherosclerotic Plaque: A Community-Based Cohort Study.

Background: The inflammatory response is a pivotal factor in accelerating the progression of atherosclerosis. The high-sensitivity C-reactive protein-to-albumin ratio (CAR) has emerged as a novel marker of systemic inflammation. However, few studies have shown the CAR to be a promising prognostic marker for carotid atherosclerotic disease. This study aimed to analyse the predictive role of the CAR in carotid atherosclerotic disease. Methods: This community-based cohort study recruited 2003 participants from the Rose asymptomatic IntraCranial Artery Stenosis (RICAS) study who were free of stroke or transient ischemic attack. Carotid atherosclerotic plaques and their stability were identified via carotid ultrasound. Logistic regression models were utilized to investigate the association between CAR and the presence of carotid atherosclerotic plaques. Results: The prevalence of carotid atherosclerotic plaques was 38.79% in this study. After adjusting for clinical risk factors, including sex, age, dyslipidemia, hypertension, diabetes mellitus (DM), and smoking and drinking habits, a high CAR-level was independently associated with carotid plaque (odds ratio [OR] of upper: 1.46, 95% confidence interval [CI]: 1.13-1.90, P = 0.004; P for trend = 0.011). The highest CAR tertile was still significantly associated with carotid plaques among middle-aged (40-64 years) or female participants. Notably, an elevated CAR may be an independent risk factor for vulnerable carotid plaques (OR of upper: 2.06, 95% CI: 1.42-2.98, P < 0.001; P for trend <0.001). Conclusion: A high CAR may be correlated with a high risk of carotid plaques, particularly among mildly aged adults (40-64 years) or females. Importantly, the CAR may be associated with vulnerable carotid plaques, suggesting that the CAR may be a new indicator for stroke prevention.

Identification of stromal differentially expressed proteins in the colon carcinoma by quantitative proteomics.

Tumor microenvironment plays very important roles in the carcinogenesis. A variety of stromal cells in the microenvironment have been modified to support the unique needs of the malignant state. This study was to discover stromal differentially expressed proteins (DEPs) that were involved in colon carcinoma carcinogenesis. Laser capture microdissection (LCM) was captured and isolated the stromal cells from colon adenocarcinoma (CAC) and non-neoplastic colon mucosa (NNCM) tissues, respectively. Seventy DEPs were identified between the pooled LCM-enriched CAC and NNCM stroma samples by iTRAQ-based quantitative proteomics. Gene Ontology (GO) relationship analysis revealed that DEPs were hierarchically grouped into 10 clusters, and were involved in multiple biological functions that were altered during carcinogenesis, including extracellular matrix organization, cytoskeleton, transport, metabolism, inflammatory response, protein polymerization, and cell motility. Pathway network analysis revealed 6 networks and 56 network eligible proteins with Ingenuity pathway analysis. Four significant networks functioned in digestive system development and its function, inflammatory disease, and developmental disorder. Eight DEPs (DCN, FN1, PKM2, HSP90B1, S100A9, MYH9, TUBB, and YWHAZ) were validated by Western blotting, and four DEPs (DCN, FN1, PKM2, and HSP90B1) were validated by immunohistochemical analysis. It is the first report of stromal DEPs between CAC and NNCM tissues. It will be helpful to recognize the roles of stromas in the colon carcinoma microenvironment, and improve the understanding of carcinogenesis in colon carcinoma. The present data suggest that DCN, FN1, PKM2, HSP90B1, S100A9, MYH9, TUBB, and YWHAZ might be the potential targets for colon cancer prevention and therapy.

Mapping the interactome of overexpressed RAF kinase inhibitor protein in a gastric cancer cell line.

BACKGROUND: Gastric cancer (GC) is a threat to human health with increasing incidence and mortality worldwide. Down-regulation or absence of RAF kinase inhibitor protein (RKIP) was associated with the occurrence, differentiation, invasion, and metastasis of GC. This study aims to investigate the molecular mechanisms and biological functions of RKIP in the GC biology. METHODS: The fusion expression plasmid pcDNA3.1-RKIP-3xFLAG was transfected into SGC7901 cells, the RKIP fusion proteins were purified with anti-flag M2 magnetic beads, and the RKIP-interacting proteins were identified with tandem mass spectrometry (MS/MS), and were analyzed with bioinformatics tools. Western blot and co-immunoprecipitation were used to confirm the interaction complex. RESULTS: A total of 72 RKIP-interacting proteins were identified by MS/MS. Those proteins play roles in enzyme metabolism, molecular chaperoning, biological oxidation, cytoskeleton organization, signal transduction, and enzymolysis. Three RKIP-interaction protein network diagrams were constructed with Michigan Molecular Interactions, functional linage network, and Predictome analysis to address the molecular pathways of the functional activity of RKIP. The MS/MS-characterized components of the existing interaction complex (RKIP, HSP90, 14-3-3ε, and keratin 8) were confirmed by Western blot analysis and co-immunoprecipitation. CONCLUSION: This study is the first discovery of the interaction of RKIP with HSP90, 14-3-3, and keratin. The present data would provide insight into the molecular mechanisms of how RKIP inhibits the occurrence and development of GC.

Lactylation-Related Gene Signature Effectively Predicts Prognosis and Treatment Responsiveness in Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a malignant tumor associated with high morbidity and mortality. Therefore, it is of great importance to develop effective prognostic models and guide clinical treatment in HCC. Protein lactylation is found in HCC tumors and is associated with HCC progression. METHODS: The expression levels of lactylation-related genes were identified from the TCGA database. A lactylation-related gene signature was constructed using LASSO regression. The prognostic value of the model was assessed and further validated in the ICGC cohort, with the patients split into two groups based on risk score. Glycolysis and immune pathways, treatment responsiveness, and the mutation of signature genes were analyzed. The correlation between PKM2 expression and the clinical characteristics was investigated. RESULTS: Sixteen prognostic differentially expressed lactylation-related genes were identified. An 8-gene signature was constructed and validated. Patients with higher risk scores had poorer clinical outcomes. The two groups were different in immune cell abundance. The high-risk group patients were more sensitive to most chemical drugs and sorafenib, while the low-risk group patients were more sensitive to some targeted drugs such as lapatinib and FH535. Moreover, the low-risk group had a higher TIDE score and was more sensitive to immunotherapy. PKM2 expression correlated with clinical characteristics and immune cell abundance in the HCC samples. CONCLUSIONS: The lactylation-related model exhibited robust predictive efficiency in HCC. The glycolysis pathway was enriched in the HCC tumor samples. A low-risk score indicated better treatment response to most targeted drugs and immunotherapy. The lactylation-related gene signature could be used as a biomarker for the effective clinical treatment of HCC.

Effects of advance care planning on end-of-life decisions among community-dwelling elderly people and their relatives: a systematic review and meta-analysis.

BACKGROUND: Currently, more and more older people are inevitably facing the final stages of life and their need for end-of-life care is becoming more prominent. It is therefore important to understand in advance what older people expect from their approaching end-of-life care and attention. We conducted a meta-analysis to explore the influence of advance care planning (ACP) on end-of-life decision-making among older adults living in community settings and their family members. METHODS: We searched databases including PubMed, Embase, Cochrane Library, and Web of Science through 10 August 2022, to locate randomized controlled trials (RCTs) that investigated the effects of ACP on the end-of-life decision-making of community-dwelling elderly individuals and their family members. Studies we obtained from the databases were screened based on specific inclusion and exclusion criteria. The software Stata 15.0 was used for combining and analyzing data. RESULTS: A total of 8 RCTs were eligible for meta-analysis. They involved 1,292 community-dwelling elderly people. The meta-analysis results revealed the incidence of the following items among participants after the intervention of the ACP: cardiopulmonary resuscitation (CPR) [rate =26%, 95% confidence interval (CI): 11-41%], life-sustaining treatment (rate =12%, 95% CI: 6-18%), gastric gavage (rate =34%, 95% CI: 18-50%), mechanical ventilation (rate =34%, 95% CI: 14-54%), death at home (rate =7%, 95% CI: 3-12%), and death in hospital (rate =6%, 95% CI: 3-10%). The systematic review protocol was prespecified and registered in the international prospective register of systematic reviews (PROSPERO; CRD42022348900). CONCLUSIONS: According to current research, ACP is a promising treatment that can improve the end of life of elderly people living in the community and their families. However, considering the heterogeneity of the included studies, multi-center RCTs with high quality and larger sample sizes need to be conducted to confirm our conclusions.

Structural insight into the macrocyclic inhibitor TPX-0022 of c-Met and c-Src.

c-Met has been an attractive target of prognostic and therapeutic studies in various cancers. TPX-0022 is a macrocyclic inhibitor of c-Met, c-Src and CSF1R kinases and is currently in phase I/II clinical trials in patients with advanced solid tumors harboring MET gene alterations. In this study, we determined the co-crystal structures of the c-Met/TPX-0022 and c-Src/TPX-0022 complexes to help elucidate the binding mechanism. TPX-0022 binds to the ATP pocket of c-Met and c-Src in a local minimum energy conformation and is stabilized by hydrophobic and hydrogen bond interactions. In addition, TPX-0022 exhibited potent activity against the resistance-relevant c-Met L1195F mutant and moderate activity against the c-Met G1163R, F1200I and Y1230H mutants but weak activity against the c-Met D1228N and Y1230C mutants. Overall, our study reveals the structural mechanism underlying the potency and selectivity of TPX-0022 and the ability to overcome acquire resistance mutations and provides insight into the development of selective c-Met macrocyclic inhibitors.

Suberoylanilide Hydroxamic Acid (SAHA) Treatment Reveals Crosstalk Among Proteome, Phosphoproteome, and Acetylome in Nasopharyngeal Carcinoma Cells.

Suberoylanilide hydroxamic acid (SAHA), a famous histone deacetylase (HDAC) inhibitor, has been utilized in clinical treatment for cutaneous T-cell lymphoma. Previously, the mechanisms underlying SAHA anti-tumor activity mainly focused on acetylome. However, the characteristics of SAHA in terms of other protein posttranslational modifications (PTMs) and the crosstalk between various modifications are poorly understood. Our previous work revealed that SAHA had anti-tumor activity in nasopharyngeal carcinoma (NPC) cells as well. Here, we reported the profiles of global proteome, acetylome, and phosphoproteome of 5-8 F cells upon SAHA induction and the crosstalk between these data sets. Overall, we detected and quantified 6,491 proteins, 2,456 phosphorylated proteins, and 228 acetylated proteins in response to SAHA treatment in 5-8 F cells. In addition, we identified 46 proteins exhibiting both acetylation and phosphorylation, such as WSTF and LMNA. With the aid of intensive bioinformatics analyses, multiple cellular processes and signaling pathways involved in tumorigenesis were clustered, including glycolysis, EGFR signaling, and Myc signaling pathways. Taken together, this study highlighted the interconnectivity of acetylation and phosphorylation signaling networks and suggested that SAHA-mediated HDAC inhibition may alter both acetylation and phosphorylation of viral proteins. Subsequently, cellular signaling pathways were reprogrammed and contributed to anti-tumor effects of SAHA in NPC cells.

pH indicator-based sensor array in combination with hyperspectral imaging for intelligent evaluation of withering degree during processing of black tea.

Withering is one of the most critical steps in the processing of black tea. The degree of withering affects the aroma quality of the finished tea. In this study, we used a pH indicator-based colorimetric sensor array in combination with hyperspectral imaging to intelligently evaluate the withering degree. After analyzing the difference between images taken before and after the reaction of pH indicators with withered leaves, six pH indicators were selected to build a sensor array. Then, the hyperspectral image of each pH indicator was obtained at wavelengths between 400 and 1000 nm. Nonlinear support vector machine (SVM) and least-squares (LS) SVM models were established to determine the degree of withering. Results revealed that the spectral information from single pH indicator failed to accurately evaluate the withering degree. The LS-SVM model achieved satisfactory discriminant results with the low-level data fusion of six pH indicators followed by principal component analysis for dimensionality reduction. The optimal model yielded accuracies of 93.75% and 90.00% for the calibration and prediction sets, respectively. The results indicated that colorimetric sensor array in combination with hyperspectral imaging can effectively determine the withering degree, thus providing a novel method for the intelligent processing of food and tea.

Structure-based design of a dual-warhead covalent inhibitor of FGFR4.

The fibroblast growth factor 19 (FGF19)/fibroblast growth factor receptor 4 (FGFR4) signaling pathways play critical roles in a variety of cancers, such as hepatocellular carcinoma (HCC). FGFR4 is recognized as a promising target to treat HCC. Currently, all FGFR covalent inhibitors target one of the two cysteines (Cys477 and Cys552). Here, we designed and synthesized a dual-warhead covalent FGFR4 inhibitor, CXF-009, targeting Cys477 and Cys552 of FGFR4. We report the cocrystal structure of FGFR4 with CXF-009, which exhibits a dual-warhead covalent binding mode. CXF-009 exhibited stronger selectivity for FGFR4 than FGFR1-3 and other kinases. CXF-009 can also potently inhibit the single cystine mutants, FGFR4(C477A) and FGFR4(C552A), of FGFR4. In summary, our study provides a dual-warhead covalent FGFR4 inhibitor that can covalently target two cysteines of FGFR4. CXF-009, to our knowledge, is the first reported inhibitor that forms dual-warhead covalent bonds with two cysteine residues in FGFR4. CXF-009 also has the potential to overcome drug induced resistant FGFR4 mutations and might serve as a lead compound for future anticancer drug discovery.

Activation of EGFR promotes squamous carcinoma SCC10A cell migration and invasion via inducing EMT-like phenotype change and MMP-9-mediated degradation of E-cadherin.

EGFR is a potent stimulator of invasion and metastasis in head and neck squamous cell carcinomas (HNSCC). However, the mechanism by which EGFR may stimulate tumor cell invasion and metastasis still need to be elucidated. In this study, we showed that activation of EGFR by EGF in HNSCC cell line SCC10A enhanced cell migration and invasion, and induced loss of epitheloid phenotype in parallel with downregulation of E-cadherin and upregulation of N-cadherin and vimentin, indicating that EGFR promoted SCC10A cell migration and invasion possibly by an epithelial to mesenchymal transition (EMT)-like phenotype change. Interestingly, activation of EGFR by EGF induced production of matrix metalloproteinase-9 (MMP-9) and soluble E-cadherin (sE-cad), and knockdown of MMP-9 by siRNA inhibited sE-cad production induced by EGF in SCC10A. Moreover, both MMP-9 knockdown and E-cadherin overexpression inhibited cell migration and invasion induced by EGF in SCC10A. The results indicate that EGFR activation promoted cell migration and invasion through inducing MMP-9-mediated degradation of E-cadherin into sE-cad. Pharmacologic inhibition of EGFR, MEK, and PI3K kinase activity in SCC10A reduced phosphorylated levels of ERK-1/2 and AKT, production of MMP-9 and sE-cad, cell migration and invasion, and expressional changes of EMT markers (E-cadherin and N-cadherin) induced by EGF, indicating that EGFR activation promotes cell migration and invasion via ERK-1/2 and PI3K-regulated MMP-9/E-cadherin signaling pathways. Taken together, the data suggest that EGFR activation promotes HNSCC SCC10A cell migration and invasion by inducing EMT-like phenotype change and MMP-9-mediated degradation of E-cadherin into sE-cad related to activation of ERK-1/2 and PI3K signaling pathways.

Enhancing the stability of ¹8O-labeled peptides through removal of immobilized trypsin by ZipTips.

Trypsin-catalyzed ¹8O labeling is increasingly used in shotgun proteomics for relative peptide/protein quantitation. However, precise quantitative measurements are often complicated by the instability of ¹8O-labeled peptides caused mainly by oxygen back-exchange. Although a number of attempts have been made to reduce or prevent oxygen back-exchange, there is still room for improvement. Here we demonstrate that the removal of immobilized trypsin by filtration using ZipTips can efficiently minimize oxygen back-exchange and enhance the stability of ¹8O-labeled peptides under various pH conditions. The ¹8O-labeled peptides processed by the approach were successfully separated by immobilized pH gradient-isoelectric focusing (IPG-IEF), and no marked decrease in the extent of labeling was observed. The results also demonstrated that there was no correlation between the extent of ¹8O labeling and molecular weight or isoelectric point (pI). The approach presented here is especially applicable to microscale samples. Its ability to generate stably ¹8O-labeled samples without back-exchange should expand the application scope of the ¹8O-labeling technique.

Analysis of EGFR signaling pathway in nasopharyngeal carcinoma cells by quantitative phosphoproteomics.

BACKGROUND: The epidermal growth factor receptor (EGFR) is usually overexpressed in nasopharyngeal carcinoma (NPC) and is associated with pathogenesis of NPC. However, the downstream signaling proteins of EGFR in NPC have not yet been completely understood at the system level. The aim of this study was identify novel downstream proteins of EGFR signaling pathway in NPC cells. RESULTS: We analyzed EGFR-regulated phosphoproteome in NPC CNE2 cells using 2D-DIGE and mass spectrometry analysis after phosphoprotein enrichment. As a result, 33 nonredundant phosphoproteins including five known EGFR-regulated proteins and twenty-eight novel EGFR-regulated proteins in CNE2 were identified, three differential phosphoproteins were selectively validated, and two differential phosphoproteins (GSTP1 and GRB2) were showed interacted with phospho-EGFR. Bioinformatics analysis showed that 32 of 33 identified proteins contain phosphorylation modification sites, and 17 identified proteins are signaling proteins. GSTP1, one of the EGFR-regulated proteins, associated with chemoresistance was analyzed. The results showed that GSTP1 could contribute to paclitaxel resistance in EGF-stimulated CNE2 cells. Furthermore, an EGFR signaling network based on the identified EGFR-regulated phosphoproteins were constructed using Pathway Studio 5.0 software, which includes canonical and novel EGFR-regulated proteins and implicates the possible biological roles for those proteins. CONCLUSION: The data not only can extend our knowledge of canonical EGFR signaling, but also will be useful to understand the molecular mechanisms of EGFR in NPC pathogenesis and search therapeutic targets for NPC.

Nanomaterials for cancer therapy: current progress and perspectives.

Cancer is a disease with complex pathological process. Current chemotherapy faces problems such as lack of specificity, cytotoxicity, induction of multi-drug resistance and stem-like cells growth. Nanomaterials are materials in the nanorange 1-100 nm which possess unique optical, magnetic, and electrical properties. Nanomaterials used in cancer therapy can be classified into several main categories. Targeting cancer cells, tumor microenvironment, and immune system, these nanomaterials have been modified for a wide range of cancer therapies to overcome toxicity and lack of specificity, enhance drug capacity as well as bioavailability. Although the number of studies has been increasing, the number of approved nano-drugs has not increased much over the years. To better improve clinical translation, further research is needed for targeted drug delivery by nano-carriers to reduce toxicity, enhance permeability and retention effects, and minimize the shielding effect of protein corona. This review summarizes novel nanomaterials fabricated in research and clinical use, discusses current limitations and obstacles that hinder the translation from research to clinical use, and provides suggestions for more efficient adoption of nanomaterials in cancer therapy.