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.

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.

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.

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.

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.

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.

Coupling of Cell Surface Biotinylation and SILAC-Based Quantitative Proteomics Identified Myoferlin as a Potential Therapeutic Target for Nasopharyngeal Carcinoma Metastasis.

Distant metastasis is a major cause of treatment failure in nasopharyngeal carcinoma (NPC) patients. Cell surface proteins represent attractive targets for cancer diagnosis or therapy. However, the cell surface proteins associated with NPC metastasis are poorly understood. To identify potential therapeutic targets for NPC metastasis, we isolated cell surface proteins from two isogenic NPC cell lines, 6-10B (low metastatic) and 5-8F (highly metastatic), through cell surface biotinylation. Stable isotope labeling by amino acids in cell culture (SILAC) based proteomics was applied to comprehensively characterize the cell surface proteins related with the metastatic phenotype. We identified 294 differentially expressed cell surface proteins, including the most upregulated protein myoferlin (MYOF), two receptor tyrosine kinases(RTKs) epidermal growth factor receptor (EGFR) and ephrin type-A receptor 2 (EPHA2) and several integrin family molecules. These differentially expressed proteins are enriched in multiple biological pathways such as the FAK-PI3K-mTOR pathway, focal adhesions, and integrin-mediated cell adhesion. The knockdown of MYOF effectively suppresses the proliferation, migration and invasion of NPC cells. Immunohistochemistry analysis also showed that MYOF is associated with NPC metastasis. We experimentally confirmed, for the first time, that MYOF can interact with EGFR and EPHA2. Moreover, MYOF knockdown could influence not only EGFR activity and its downstream epithelial-mesenchymal transition (EMT), but also EPHA2 ligand-independent activity. These findings suggest that MYOF might be an attractive potential therapeutic target that has double effects of simultaneously influencing EGFR and EPHA2 signaling pathway. In conclusion, this is the first study to profile the cell surface proteins associated with NPC metastasis and provide valuable resource for future researches.

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.

IRE1α-XBP1 Affects the Mitochondrial Function of Aß25-35-Treated SH-SY5Y Cells by Regulating Mitochondria-Associated Endoplasmic Reticulum Membranes.

Background: Neurotoxicity induced by the amyloid beta (Aß) peptide is one of the most important pathological mechanisms of Alzheimer's disease (AD). Activation of the adaptive IRE1α-XBP1 pathway contributes to the pathogenesis of AD, making it a potential target for AD therapeutics. However, the mechanism of IRE1α-XBP1 pathway involvement in AD is unclear. We, therefore, investigated the effect of the IRE1α-XBP1 axis in an in vitro AD model and explored its potential mechanism. Methods: The human neuroblastoma cell line, SH-SY5Y, was used. Cells were treated with Aß25-35, with or without 4µ8c, an inhibitor of IRE1α. Cells were collected and analyzed by Western blotting, quantitative real-time PCR, electron microscopy, fluorescence microscopy, calcium imaging, and other biochemical assays. Results: Aß-exposed SH-SY5Y cells showed an increased expression of XBP1s and p-IRE1α. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and calcium imaging analysis showed that the IRE1α inhibitor, 4µ8c, reduced Aß-induced cytotoxicity. Increased levels of ATP, restoration of mitochondrial membrane potential, and decreased production of mitochondrial reactive oxygen species after Aß treatment in the presence of 4µ8c showed that inhibiting the IRE1α-XBP1 axis effectively mitigated Aß-induced mitochondrial dysfunction in SH-SY5Y cells. Furthermore, Aß treatment increased the expression and interaction of IP3R, Grp75, and vdac1 and led to an increased endoplasmic reticulum (ER)-mitochondria association, malfunction of mitochondria-associated ER-membranes (MAMs), and mitochondrial dysfunction. These deficits were rescued by inhibiting the IRE1α-XBP1 axis. Conclusion: These findings demonstrate that Aß peptide induces the activation of the IRE1α-XBP1 axis, which may aggravate cytotoxicity and mitochondrial impairment in SH-SY5Y cells by targeting MAMs. Inhibition of the IRE1α-XBP1 axis provides the protection against Aß-induced injury in SH-SY5Y cells and may, therefore, be a new treatment strategy.

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.

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.

Dissection of Anti-tumor Activity of Histone Deacetylase Inhibitor SAHA in Nasopharyngeal Carcinoma Cells via Quantitative Phosphoproteomics.

Suberoylanilide hydroxamic acid (SAHA), a pan HDAC inhibitor, has been approved by the Food and Drug Administration (FDA) to treat cutaneous T cell lymphoma (CTCL). Nevertheless, the mechanisms underlying the therapeutic effects of SAHA on tumors are yet not fully understood. Protein phosphorylation is one of the most important means to regulate key biological processes (BPs), such as cell division, growth, migration, differentiation, and intercellular communication. Thus, investigation on the impacts of SAHA treatment on global cellular phosphorylation covering major signaling pathways deepens our understanding on its anti-tumor mechanisms. Here we comprehensively identified and quantified protein phosphorylation for the first time in nasopharyngeal carcinoma (NPC) cells upon SAHA treatment by combining tandem mass tags (TMTs)-based quantitative proteomics and titanium dioxide (TiO2)-based phosphopeptide enrichment. In total, 7,430 phosphorylation sites on 2,456 phosphoproteins were identified in the NPC cell line 5-8F, of which 1,176 phosphorylation sites on 528 phosphoproteins were significantly elevated upon SAHA treatment. Gene ontology (GO) analysis showed that SAHA influenced several BPs, including mRNA/DNA processing and cell cycle. Furthermore, signaling pathway analysis and immunoblotting demonstrated that SAHA activated tumor suppressors like p53 and Rb1 via phosphorylation and promoted cell apoptosis in NPC cells but inactivated energetic pathways such as AMPK signaling. Overall, our study indicated that SAHA exerted anti-tumor roles in NPC cells, which may serve as novel therapeutic for NPC patients.

Orexin-A exacerbates Alzheimer's disease by inducing mitochondrial impairment.

Alzheimer's disease (AD) is a progressive neurodegenerative disease which is characterized by the accumulation of amyloid-ß peptide (Aß). Orexin-A is a neuropeptide which has been reported to participate in the pathogenesis of AD. Thus, we aimed to investigate the possible mechanism by which Orexin-A acts in AD. APP/PS1 transgenic mice, an animal model of AD, were intracerebroventricularly injected with Orexin-A. Aß-treated SH-SY5Y cells were used as a cell model of AD and treated with Orexin-A. The Morris water maze test, fluorescence microscopy, enzyme-linked immunosorbent assay (ELISA), electron microscopy, real-time PCR, and other biochemical assays were conducted. The Morris water maze test showed that Orexin-A aggravated cognitive deficit in APP/PS1 mice. Using thioflavine-S staining and ELISA, we found that Orexin-A promoted Aß accumulation in APP/PS1 mice. By evaluating mitochondrial morphology, cytochrome c oxidase activity, ATP level, mitochondrial DNA copy number, and reactive oxygen species, we found that Orexin-A aggravated mitochondrial impairment in APP/PS1 mice and Aß-treated SH-SY5Y cells. Our results indicate that Orexin-A exacerbates AD by inducing mitochondrial impairment. This is a new mechanism that explains how Orexin-A participates in the pathogenesis of AD.

Nanotechnology in cancer diagnosis: progress, challenges and opportunities.

In the fight against cancer, early detection is a key factor for successful treatment. However, the detection of cancer in the early stage has been hindered by the intrinsic limits of conventional cancer diagnostic methods. Nanotechnology provides high sensitivity, specificity, and multiplexed measurement capacity and has therefore been investigated for the detection of extracellular cancer biomarkers and cancer cells, as well as for in vivo imaging. This review summarizes the latest developments in nanotechnology applications for cancer diagnosis. In addition, the challenges in the translation of nanotechnology-based diagnostic methods into clinical applications are discussed.

The Significance of Serum S100A9 and TNC Levels as Biomarkers in Colorectal Cancer.

Purpose: The aim of this study was to evaluate the diagnostic value of S100A9 and tenascin-c (TNC) levels as colorectal cancer (CRC) biomarkers in several ways, including through screening tests, differentiation tests, combination with existing biomarkers (CEA and CA19-9), and serum level measurements before and after surgery. Materials and Methods: In this case-control study, S100A9 and TNC serum levels were measured in 460 participants: 258 CRC patients, 99 patients with benign colonic disease (BCD) and 103 healthy donors (HD). Results: The serum levels of S100A9 were 22.32 (14.88-29.55) ng/ml, 10.02 (5.83-14.15) ng/ml and 10.05 (7.68-15.34) ng/ml in the CRC, BCD and HD groups, respectively. The serum levels of TNC were 4.30 (2.12-6.04) ng/ml, 1.60 (1.06-2.30) ng/ml and 2.00 (1.37-3.00) ng/ml in the CRC, BCD and HD groups, respectively. Significantly higher levels of both biomarkers (S100A9 and TNC) were found in CRC patients (both p<0.001). Both S100A9 and TNC levels were superior to CEA and CA19-9 levels as CRC diagnostic biomarkers; the combination of S100A9, TNC and CEA levels was an excellent biomarker with 79.8% sensitivity and 89.6% specificity. The serum levels of S100A9 and TNC in CRC patients were significantly lower after surgery than before surgery (p<0.01). Conclusion: S100A9 and TNC levels could serve as diagnostic biomarkers of colorectal cancer.

Simultaneous Inhibition of EGFR and HER2 via Afatinib Augments the Radiosensitivity of Nasopharyngeal Carcinoma Cells.

Ionizing radiation (IR) is the central component of the therapeutic scheme for nasopharyngeal carcinoma (NPC) at present. Previous studies show that inhibition of epidermal growth factor receptor (EGFR) enhances the radiosensitivity of NPC; however the effects of EGFR-targeted agents are limited. In this study, we observed that simultaneously inhibition of EGFR and HER2 by afatinib could augment the radiosensitivity of NPC cells; this approach has an advantage over erlotinib-mediated inhibition of EGFR alone. The afatinib-induced augmentation of NPC cell radiosensitivity was associated with increases in apoptosis and accumulation of DNA damage that were induced by radiation. In addition, the crosstalk between radiation-induced activities and EGFR-, and HER2-related downstream pathways may contribute to the enhancement of radiosensitivity. Our findings indicate the potential of repositioning afatinib or other ERBB-family-targeted agents for improving radiation response in NPC cells.

Quantitative proteomic profiling of tumor-associated vascular endothelial cells in colorectal cancer.

To investigate the global proteomic profiles of vascular endothelial cells (VECs) in the tumor microenvironment and antiangiogenic therapy for colorectal cancer (CRC), matched pairs of normal (NVECs) and tumor-associated VECs (TVECs) were purified from CRC tissues by laser capture microdissection and subjected to iTRAQ-based quantitative proteomics analysis. Here, 216 differentially expressed proteins (DEPs) were identified and used for bioinformatics analysis. Interestingly, these proteins were implicated in epithelial mesenchymal transition (EMT), ECM-receptor interaction, focal adhesion, PI3K-Akt signaling pathway, angiogenesis and HIF-1 signaling pathway, which may play important roles in CRC angiogenesis. Among these DEPs we found that Tenascin-C (TNC) was upregulated in TVECs of CRC and correlated with CRC multistage carcinogenesis and metastasis. Furthermore, the reduction of tumor-derived TNC could attenuate human umbilical vein endothelial cell (HUVEC) proliferation, migration and tube formation through ITGB3/FAK/Akt signaling pathway. Based on the present work, we provided a large-scale proteomic profiling of VECs in CRC with quantitative information, a certain number of potential antiangiogenic targets and a novel vision in the angiogenesis bio-mechanism of CRC.

The Radiosensitization of Sodium Glycididazole on Nasopharyngeal Carcinoma Cells via Enhancing DNA Damage and Promoting Apoptosis.

Background: The radioresistance of nasopharyngeal carcinoma (NPC) was the main cause of radiotherapy failure and it was still a challenge in the treatment of advanced NPC patients. Previous clinical studies demonstrated that sodium glycididazole(CMNA) can enhance the radiosensitivity of NPC, but the corresponding cellular mechanisms or processes remains largely unclear. Methods: To clarify the radiosensitizing effects of CMNA on NPC cells and reveal its cellular mechanisms, its effect on cell survival of NPC cells was assessed by MTT and clonogenic assay, with or without radiation. The potential cellular mechanisms such as cell cycle distribution, apoptosis and DNA damage were assessed. A retrospective analysis of the outcome of patients with III-IV stage NPC who undergo same radiochemotherapy with or without concurrent CMNA treatment was performed to elucidate the role of CMNA in the improvement of the curative effects. Results: The treatment with CMNA at the concentration lower or close to the clinical dosage had little effect on cell survival, cell cycle distribution and a weak effect on DNA damage and cell apoptosis of NPC cells. The combination of CMNA and radiation significantly increased the DNA damage and enhanced the apoptosis of NPC cells, but did not significantly alter the cell cycle distribution as compared with the irradiation (IR) alone. A total of 99 patients who underwent radiochemotherapy were categorized into those with (treatment group, n=52) and without (control group, n=47) the treatment with CMNA. The complete response rates of patients in treatment group were significantly higher than in control group. Conclusions: Our results suggested that CMNA enhance the sensitivity of the NPC cells to radiation via enhancing DNA damage and promoting cell apoptosis. It provides clues for further investigation of the molecular mechanism of the radiosensitization of CMNA on NPC cells.

Detection and Analysis of RNAs Expression Profile for Methylated Candidate Tumor Suppressor Genes in Nasopharyngeal Carcinoma.

BACKGROUND: DNA methylation, which acts as an expression regulator for multiple Tumor Suppressor Genes (TSGs), is believed to play an important role in Nasopharyngeal Carcinoma (NPC) development. METHODS: We compared the effects of 5-aza-2-deoxycytidine (decitabine, DAC) on gene expression using RNA sequencing in NPC cells. RESULTS: We analyzed Differentially Expressed Genes (DEGs) in NPC cells using DAC demethylation treatment and found that 2182 genes were significantly upregulated (≥ 2-fold change), suggesting that they may play a key role in cell growth, proliferation, development, and death. For data analysis, we used the Gene Ontology database and pathway enrichment analysis of the DEGs to discover differential patterns of DNA methylation associated with changes in gene expression. Furthermore, we evaluated 74 methylated candidate TSGs from the DEGs in NPC cells and summarized these genes in several important signaling pathways frequently disrupted by promoter methylation in NPC tumorigenesis. CONCLUSION: Our study analyzes the DEGs and identifies a set of genes whose promoter methylation in NPC cells is reversed by DAC. These genes are potential substrates of DNMT inhibitors and may serve as tumor suppressors in NPC cells.

Knockdown of Annexin A1 Enhances Radioresistance and Inhibits Apoptosis in Nasopharyngeal Carcinoma.

Radiotherapy is the primary treatment for nasopharyngeal carcinoma while radioresistance can hinder efficient treatment. To explore the role of annexin A1 and its potential mechanisms in radioresistance of nasopharyngeal carcinoma, human nasopharyngeal carcinoma cell line CNE2-sh annexin A1 (knockdown of annexin A1) and the control cell line CNE2-pLKO.1 were constituted and CNE2-sh annexin A1 xenograft mouse model was generated. The effect of annexin A1 knockdown on the growth of xenograft tumor after irradiation and radiation-induced DNA damage and repair was analyzed. The results of immunohistochemistry assays and Western blotting showed that the level of annexin A1 was significantly downregulated in the radioresistant nasopharyngeal carcinoma tissues or cell line compared to the radiosensitive nasopharyngeal carcinoma tissues or cell line. Knockdown of annexin A1 significantly promoted CNE2-sh annexin A1 xenograft tumor growth compared to the control groups after irradiation. Moreover, the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assays revealed that knockdown of annexin A1 significantly inhibited apoptosis in vivo compared to the control groups. We assessed the intracellular reactive oxygen species levels and the extent of radiation-induced DNA damage and repair using reactive oxygen species assay, comet assays, and immunohistochemistry assay. The results showed that knockdown of annexin A1 remarkedly reduced the intracellular reactive oxygen species levels, level of DNA double-strand breaks, and the phosphorylation level of H2AX and increased the accumulation of DNA-dependent protein kinase in nasopharyngeal carcinoma cells after irradiation. The findings suggest that knockdown of annexin A1 inhibits DNA damage via decreasing the generation of intracellular reactive oxygen species and the formation of γ-H2AX and promotes DNA repair via increasing DNA-dependent protein kinase activity and therefore improves the radioresistance in nasopharyngeal carcinoma cells. Together, our findings suggest that knockdown of annexin A1 promotes radioresistance in nasopharyngeal carcinoma and provides insights into therapeutic targets for nasopharyngeal carcinoma radiotherapy.