Machine learning based on metabolomics unveils neutrophil extracellular trap-related metabolic signatures in non-small cell lung cancer patients undergoing chemoimmunotherapy.

BACKGROUND: Non-small cell lung cancer (NSCLC) is the primary form of lung cancer, and the combination of chemotherapy with immunotherapy offers promising treatment options for patients suffering from this disease. However, the emergence of drug resistance significantly limits the effectiveness of these therapeutic strategies. Consequently, it is imperative to devise methods for accurately detecting and evaluating the efficacy of these treatments. AIM: To identify the metabolic signatures associated with neutrophil extracellular traps (NETs) and chemoimmunotherapy efficacy in NSCLC patients. METHODS: In total, 159 NSCLC patients undergoing first-line chemoimmunotherapy were enrolled. We first investigated the characteristics influencing clinical efficacy. Circulating levels of NETs and cytokines were measured by commercial kits. Liquid chromatography tandem mass spectrometry quantified plasma metabolites, and differential metabolites were identified. Least absolute shrinkage and selection operator, support vector machine-recursive feature elimination, and random forest algorithms were employed. By using plasma metabolic profiles and machine learning algorithms, predictive metabolic signatures were established. RESULTS: First, the levels of circulating interleukin-8, neutrophil-to-lymphocyte ratio, and NETs were closely related to poor efficacy of first-line chemoimmunotherapy. Patients were classed into a low NET group or a high NET group. A total of 54 differential plasma metabolites were identified. These metabolites were primarily involved in arachidonic acid and purine metabolism. Three key metabolites were identified as crucial variables, including 8,9-epoxyeicosatrienoic acid, L-malate, and bis(monoacylglycerol)phosphate (18:1/16:0). Using metabolomic sequencing data and machine learning methods, key metabolic signatures were screened to predict NET level as well as chemoimmunotherapy efficacy. CONCLUSION: The identified metabolic signatures may effectively distinguish NET levels and predict clinical benefit from chemoimmunotherapy in NSCLC patients.

Clinical significance and biological function of interferon regulatory factor 1 in non-small cell lung cancer.

The clinical application and biological function of interferon regulatory factor 1 (IRF1) in non-small cell lung cancer (NSCLC) patients undergoing chemoimmunotherapy remain elusive. The aim of this study was to investigate the predictive and prognostic significance of IRF1 in NSCLC patients. We employed the cBioPortal database to predict frequency changes in IRF1 and explore its target genes. Bioinformatic methods were utilized to analyze the relationship between IRF1 and immune regulatory factors. Retrospective analysis of clinical samples was conducted to assess the predictive and prognostic value of IRF1 in chemoimmunotherapy. Additionally, A549 cells with varying IRF1 expression levels were constructed to investigate its effects on NSCLC cells, while animal experiments were performed to study the role of IRF1 in vivo. Our findings revealed that the primary mutation of IRF1 is deep deletion and it exhibits a close association with immune regulatory factors. KRAS and TP53 are among the target genes of IRF1, with interferon and IL-2 being the predominantly affected pathways. Clinically, IRF1 levels significantly correlate with the efficacy of chemoimmunotherapy. Patients with high IRF1 levels exhibited a median progression-free survival (mPFS) of 9.5 months, whereas those with low IRF1 levels had a shorter mPFS of 5.8 months. IRF1 levels positively correlate with PD-L1 distribution and circulating IL-2 levels. IL-2 enhances the biological function of IRF1 and recapitulates its role in vivo in the knockdown group. Therefore, IRF1 may possess predictive and prognostic value for chemoimmunotherapy in NSCLC patients through the regulation of the IL-2 inflammatory pathway.

Research progress on novel antibody drug conjugates in cancer therapy. / ç™Œç—‡æ²»ç–—ä¸­æ–°åž‹æŠ—ä½“å¶è”è¯ç‰©çš„ç ”ç©¶è¿›å±•.

Traditional antibody drug conjugates (ADC) combine monoclonal antibodies with cytotoxic drugs to accurately strike cancer cells, but there are still many shortcomings in stability, targeting, efficacy, and safety. Novel ADC, such as bi-specific, site-specific, dual-payload, and pro-drug type ADC, can be optimized by simultaneously binding 2 different antigens or epitopes, selecting more stable linkers, coupling with specific amino acid sites of antibodies, carrying different drug payloads, and adopting prodrug strategies, while retaining the characteristics of traditional ADC. Significantly improving the stability, targeting, efficacy and safety of drugs can better meet the needs of clinical treatment. Novel ADC will play a more important role in cancer treatment in the future. Discussing the progress of novel ADC in cancer treatment and analyzing their advantages and challenges can provide theoretical support for the development of anti-cancer strategies and provide directions for drug research and development.

A deep learning-based automatic image quality assessment method for respiratory phase on computed tomography chest images.

Background: Computed tomography (CT) chest scans have become commonly used in clinical diagnosis. Image quality assessment (IQA) for CT images plays an important role in CT examination. It is worth noting that IQA is still a manual and subjective process, and even experienced radiologists make mistakes due to human limitations (fatigue, perceptual biases, and cognitive biases). There are also kinds of biases because of poor consensus among radiologists. Excellent IQA methods can reliably give an objective evaluation result and also reduce the workload of radiologists. This study proposes a deep learning (DL)-based automatic IQA method, to assess whether the image quality of respiratory phase on CT chest images are optimal or not, so that the CT chest images can be used in the patient's physical condition assessment. Methods: This retrospective study analysed 212 patients' chest CT images, with 188 patients allocated to a training set (150 patients), validation set (18 patients), and a test set (20 patients). The remaining 24 patients were used for the observer study. Data augmentation methods were applied to address the problem of insufficient data. The DL-based IQA method combines image selection, tracheal carina segmentation, and bronchial beam detection. To automatically select the CT image containing the tracheal carina, an image selection model was employed. Afterward, the area-based approach and score-based approach were proposed and used to further optimize the tracheal carina segmentation and bronchial beam detection results, respectively. Finally, the score about the image quality of the patient's respiratory phase images given by the DL-based automatic IQA method was compared with the mean opinion score (MOS) given in the observer study, in which four blinded experienced radiologists took part. Results: The DL-based automatic IQA method achieved good performance in assessing the image quality of the respiratory phase images. For the CT sequence of the same patient, the DL-based IQA method had an accuracy of 92% in the assessment score, while the radiologists had an accuracy of 88%. The Kappa value of the assessment score between the DL-based IQA method and radiologists was 0.75, with a sensitivity of 85%, specificity of 91%, positive predictive value (PPV) of 92%, negative predictive value (NPV) of 93%, and accuracy of 88%. Conclusions: This study develops and validates a DL-based automatic IQA method for the respiratory phase on CT chest images. The performance of this method surpassed that of the experienced radiologists on the independent test set used in this study. In clinical practice, it is possible to reduce the workload of radiologists and minimize errors caused by human limitations.

Surgical margins and prognosis of borderline and malignant phyllodes tumors.

PURPOSE: To investigate the optimal surgical margin and prognostic risk factors for borderline and malignant phyllodes tumors (PTs). METHODS: A retrospective analysis was conducted on patients with borderline and malignant PTs at our hospital from 2011 to 2022. Univariate and multivariate Cox proportional hazard models were employed to analyze the effects of various variables on local recurrence-free survival (LRFS) and disease-free survival (DFS). RESULTS: This study comprised 150 patients, 85 classified as borderline and 65 as malignant. During a median follow-up of 66 months (range: 3-146 months), 34 cases (22.7%) experienced local recurrence, 9 cases (6.0%) exhibited distant metastasis, and 7 cases (4.7%) resulted in death. Irrespective of the histological subtypes, patients with surgical margins ≥ 1 cm exhibit significantly higher 5-year LRFS and 5-year DFS rates compared to those with margins < 1 cm. Among patients with initial margins < 1 cm, LRFS (P = 0.004) and DFS (P = 0.003) were improved in patients reoperated to achieve margins ≥ 1 cm. Surgical margin < 1 cm (HR = 2.567, 95%CI 1.137-5.793, P = 0.023) and age < 45 years (HR = 2.079, 95%CI 1.033-4.184, P = 0.040) were identified as independent risk factors for LRFS. Additionally, surgical margin < 1 cm (HR = 3.074, 95%CI 1.622-5.826, P = 0.001) and tumor size > 5 cm (HR = 2.719, 95%CI 1.307-5.656, P = 0.007) were determined to be independent risk factors for DFS. CONCLUSIONS: A negative surgical margin of at least 1 cm (with secondary resection if necessary) should be achieved for borderline and malignant PTs. Tumor size > 5 cm and age < 45 years were predictive of recurrence, suggesting multiple therapy modalities may be considered for these high-risk patients.

The Evaluation of Prognostic Value and Immune Characteristics of Ferroptosis-Related Genes in Lung Squamous Cell Carcinoma.

Background The purpose of our study was to construct a prognostic model based on ferroptosis-related gene signature to improve the prognosis prediction of lung squamous carcinoma (LUSC). Methods The mRNA expression profiles and clinical data of LUSC patients were downloaded. LUSC-related essential differentially expressed genes were integrated for further analysis. Prognostic gene signatures were identified through random forest regression and univariate Cox regression analyses for constructing a prognostic model. Finally, in a preliminary experiment, we used the reverse transcription-quantitative polymerase chain reaction assay to verify the relationship between the expression of three prognostic gene features and ferroptosis. Results Fifty-six ferroptosis-related essential genes were identified by using integrated analysis. Among these, three prognostic gene signatures (HELLS, POLR2H, and POLE2) were identified, which were positively affected by LUSC prognosis but negatively affected by immune cell infiltration. Significant overexpression of immune checkpoint genes occurred in the high-risk group. In preliminary experiments, we confirmed that the occurrence of ferroptosis can reduce three prognostic gene signature expression. Conclusions The three ferroptosis-related genes could predict the LUSC prognostic risk of antitumor immunity.

A real-world study and network pharmacology analysis of EGFR-TKIs combined with ZLJT to delay drug resistance in advanced lung adenocarcinoma.

OBJECTIVE: This study aimed to explore the efficacy and safety of combining epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) with ZiLongJin Tablet (ZLJT) in delaying acquired resistance in advanced EGFR-mutant lung adenocarcinoma (LUAD) patients. Furthermore, we employed network pharmacology and molecular docking techniques to investigate the underlying mechanisms. METHODS: A retrospective comparative study was conducted on stage IIIc/IV LUAD patients treated with EGFR-TKIs alone or in combination with ZLJT at the Second Affiliated Hospital of the Air Force Medical University between January 1, 2017, and May 1, 2023. The study evaluated the onset of TKI resistance, adverse reaction rates, safety indicators (such as aspartate aminotransferase, alanine aminotransferase, and creatinine), and inflammatory markers (neutrophil-to-lymphocyte ratio and platelet-to-lymphocyte ratio) to investigate the impact of EGFR-TKI combined with ZLJT on acquired resistance and prognostic indicators. Additionally, we utilized the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, the Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine, PubChem, UniProt, and Swiss Target Prediction databases to identify the active ingredients and targets of ZLJT. We obtained differentially expressed genes related to EGFR-TKI sensitivity and resistance from the Gene Expression Omnibus database using the GSE34228 dataset, which included sensitive (n = 26) and resistant (n = 26) PC9 cell lines. The "limma" package in R software was employed to detect DEGs. Based on this, we constructed a protein‒protein interaction network, performed gene ontology and KEGG enrichment analyses, and conducted pathway network analysis to elucidate the correlation between the active ingredients in ZLJT and signaling pathways. Finally, molecular docking was performed using AutoDockVina, PYMOL 2.2.0, and Discovery Studio Client v19.1.0 software to simulate spatial and energy matching during the recognition process between predicted targets and their corresponding compounds. RESULTS: (1) A total of 89 patients were included, with 40 patients in the EGFR-TKI combined with ZLJT group (combination group) and 49 patients in the EGFR-TKI alone group (monotherapy group). The baseline characteristics of the two groups were comparable. There was a significant difference in the onset of resistance between the combination group and the monotherapy group (P < 0.01). Compared to the monotherapy group, the combination group showed a prolongation of 3.27 months in delayed acquired resistance. There was also a statistically significant difference in the onset of resistance to first-generation TKIs between the two groups (P < 0.05). (2) In terms of safety analysis, the incidence of adverse reactions related to EGFR-TKIs was 12.5% in the combination group and 14.3% in the monotherapy group, but this difference was not statistically significant (P > 0.05). There were no statistically significant differences in serum AST, ALT, CREA, TBIL, ALB and BUN levels between the two groups after medication (P > 0.05). (3) Regarding inflammatory markers, there were no statistically significant differences in the changes in neutrophil-to-lymphocyte Ratio(NLR) and Platelet-to-lymphocyte Ratio(PLR) values before and after treatment between the two groups (P > 0.05). (4) Network pharmacology analysis identified 112 active ingredients and 290 target genes for ZLJT. From the GEO database, 2035 differentially expressed genes related to resistant LUAD were selected, and 39 target genes were obtained by taking the intersection. A "ZLJT-compound-target-disease" network was successfully constructed using Cytoscape 3.7.0. GO enrichment analysis revealed that ZLJT mainly affected biological processes such as adenylate cyclase-modulating G protein-coupled receptor. In terms of cellular components, ZLJT was associated with the cell projection membrane. The molecular function primarily focused on protein heterodimerization activity. KEGG enrichment analysis indicated that ZLJT exerted its antitumor and anti-drug resistance effects through pathways such as the PI3K-Akt pathway. Molecular docking showed that luteolin had good binding activity with FOS (-9.8 kJ/mol), as did tanshinone IIA with FOS (-9.8 kJ/mol) and quercetin with FOS (-8.7 kJ/mol). CONCLUSION: ZLJT has potential antitumor progression effects. For patients with EGFR gene-mutated non-small cell LUAD, combining ZLJT with EGFR-TKI treatment can delay the occurrence of acquired resistance. The underlying mechanisms may involve altering signal transduction pathways, blocking the tumor cell cycle, inhibiting tumor activity, enhancing cellular vitality, and improving the bioavailability of combination therapy. The combination of EGFR-TKI and ZLJT represents an effective approach for the treatment of tumors using both Chinese and Western medicine.

Change of Peri-mitral Atrial Flutter into Single-loop Biatrial Flutter During Ablation.

Anterior line ablation for peri-mitral atrial flutter (AFL) is associated with biatrial flutter due to disruption of the electrical conduction in the left atrial septum. An AFL case with valvular disease, cardiac surgery, and prior ablation was confirmed to be counterclockwise peri-mitral flutter with isthmus on the left atrial septum. Ablation on the septum of the left atrium (LA) targeting the isthmus prolonged the tachycardia cycle length (TCL) from 266 to 286 ms. Left atrial mapping during AFL with a TCL of 286 ms showed that the activation remained peri-mitral counterclockwise, but there was interruption of the local activation time (LAT) sequence. Combined mapping of the LA and the right atrium (RA) showed a counterclockwise single-loop biatrial flutter, involving the whole LA and the RA septum, with Bachmann's bundle and the posteroinferior septum being the interatrial connections. The AFL was terminated by ablation at the right superior cavoatrial junction. RA mapping should be considered if there is prolongation of TCL but without termination of the peri-mitral AFL, and if there is interruption of the continuity of the LAT sequence during AFL with a longer TCL. The biatrial flutter can be terminated by ablation targeting the interatrial connections.

Low-frequency acoustic radiation from a flanged circular pipe at an inclined angle.

The generic problem of low-frequency acoustic radiation through quiescent air from a circular pipe that is inclined with respect to its exit flange is studied in this work. The exit flange is taken to extend as an infinite plane away from the pipe opening. The analysis implements a hybrid method that combines modal expansions with the boundary element method. The reflection coefficient and pipe end correction for Helmholtz numbers (based on the pipe radius) less than 2.5 are calculated for various inclination angles up to 75°. Calculations are validated using simulations from the finite-element solver of the commercial software package COMSOL. The reflection coefficient and end correction predictions agree closely with the validation simulations yet differ notably from the results available in the literature. The solution obtained from the hybrid method is subsequently used to analyse the acoustic field at the pipe exit and in the downstream space. The key aspects of the governing physics pertaining to practical engineering applications at low frequencies are captured in a low-order approximation, which significantly reduces the degrees of freedom of the problem and provides generally good estimates of the reflection coefficient and end correction, as well as the downstream acoustic field.

GOLPH3 Promotes Angiogenesis of Lung Adenocarcinoma by Regulating the Wnt/ß-Catenin Signaling Pathway.

PURPOSE: We aim to investigate the role of Golgi phosphoprotein 3 (GOLPH3) and the possible regulation mechanism underlying lung adenocarcinoma (LADC). METHODS: The level of GOLPH3 was performed by quantitative real time (qRT)-PCR, Western blot and immunohistochemistry. Patient survival rate was analyzed by Kaplan-Meier method. MTT was used to detect cell viability. The levels of p-serine/threonine-protein kinase (Akt), Akt, p-p65, p65 and ß-catenin were determined by Western blot. Cell apoptosis was tested using flow cytometry. Angiogenesis was determined by in vitro angiogenesis assay. qPCR and Western blot were performed to identify apoptotic protein B-cell lymphoma 2 (Bcl-2)/Bcl-2-associated X protein (Bax) and vascular endothelial growth factor (VEGF). RESULTS: GOLPH3 was highly expressed in LADC cell lines and tissues and was significantly correlated with poor overall survival among patients with LADC. Furthermore, GOLPH3 expression was reduced in A549 and H23 cells in a cisplatin-dependent manner. Silencing of GOLPH3 enhanced inhibition of A549 and H23 cells by cisplatin and suppressed the protein expression of p-Akt, while p-p65 expression remained stable. However, overexpression of GOLPH3 weakened the inhibition of A549 and H23 cells by cisplatin and improved the protein expression of p-Akt, while p-p65 expression remained stable. XAV939, an inhibitor of Wnt/ß-catenin signaling pathway, decreased GOLPH3 overexpression-induced proliferation and enhanced cisplatin-induced angiogenesis inhibition and apoptosis, which was supported by the changes of VEGF, Bax and Bcl-2. CONCLUSION: GOLPH3 promotes proliferation capacity in LADC through activating the Wnt/ß-catenin signaling pathway.

Dynamic m6A mRNA methylation reveals the role of METTL3-m6A-CDCP1 signaling axis in chemical carcinogenesis.

N6-methyladenosine (m6A) is the most abundant internal modification in mammalian mRNAs. Despite its functional importance in various physiological events, the role of m6A in chemical carcinogenesis remains largely unknown. Here we profiled the dynamic m6A mRNA modification during cellular transformation induced by chemical carcinogens and identified a subset of cell transformation-related, concordantly modulated m6A sites. Notably, the increased m6A in 3'-UTR mRNA of oncogene CDCP1 was found in malignant transformed cells. Mechanistically, the m6A methyltransferase METTL3 and demethylases ALKBH5 mediate the m6A modification in 3'-UTR of CDCP1 mRNA. METTL3 and m6A reader YTHDF1 preferentially recognize m6A residues on CPCP1 3'-UTR and promote CDCP1 translation. We further showed that METTL3 and CDCP1 are upregulated in the bladder cancer patient samples and the expression of METTL3 and CDCP1 is correlated with the progression status of the bladder cancers. Inhibition of the METTL3-m6A-CDCP1 axis resulted in decreased growth and progression of chemical-transformed cells and bladder cancer cells. Most importantly, METTL3-m6A-CDCP1 axis has synergistic effect with chemical carcinogens in promoting malignant transformation of uroepithelial cells and bladder cancer tumorigenesis in vitro and in vivo. Taken together, our results identify dynamic m6A modification in chemical-induced malignant transformation and provide insight into critical roles of the METTL3-m6A-CDCP1 axis in chemical carcinogenesis.

Disabled homolog 2 interactive protein functions as a tumor suppressor in osteosarcoma cells.

The disabled homolog 2 interactive protein (DAB2IP) gene is a member of the family of Ras GTPases and functions as a tumor suppressor in many types of carcinoma; however, its function in osteosarcoma remains unclear. The aim of the present study was to determine the function of DAB2IP in osteosarcoma and normal bone cells in vitro. The expression of DAB2IP protein was assessed in osteoblast and osteosarcoma cell lines by western blot analysis. The effects of DAB2IP expression on cell proliferation, colony formation, apoptosis, cell cycle, and cell migration and invasion were evaluated by in vitro studies. DAB2IP expression was lower in osteosarcoma cell lines than in normal osteoblast cell lines. DAB2IP expression affected cell proliferation, apoptosis and cell cycle distribution. In addition, DAB2IP inhibited the migration and invasion of osteosarcoma and normal osteoblast cells. Therefore, DAB2IP may function as a tumor suppressor in osteosarcoma cell lines by inhibiting cell proliferation and invasion.

A novel regulatory circuit of miR­152 and DNMT1 in human bladder cancer.

Downregulation of microRNA­152 (miR­152) has been observed in various types of human malignancies, including Bladder cancer (BC). However, the role of miR­152 in the development and progression of BC is still unclear. In our previous study, we identified a functional crosstalk between miR­152 and DNA methyltransferase 1 (DNMT1) involved in Nis­induced malignant transformation. In the present study, we found that the expression of miR­152 was specifically downregulated in BC cells and tissues via the DNA hypermethylation of the miR­152 promoter. The overexpression of miR­152 in BC cells resulted in a reduction of DNMT1, whereas the inhibition of the expression of miR­152 induced an elevated level of DNMT1. Further studies revealed that miR­152 directly downregulated the expression of DNMT1 by targeting the 3'­UTR of its transcript in BC cells. In addition, ectopic expression of miR­152 in BC cells significantly inhibited cell proliferation, whereas the inhibition of miR­152 expression led to increased cell proliferation. These findings indicated a novel regulatory circuit of miR­152/DNMT1 in BC, and more importantly, the combination of miR­152 and DNMT1 may function as promising therapeutic modalities and early biomarkers for BC.

Long non-coding RNA DBCCR1-003 regulate the expression of DBCCR1 via DNMT1 in bladder cancer.

BACKGROUND: Many long non coding RNAs have been identified as key modulators in cancer development. A lncRNA, DBCCR1-003, derived from the locus of tumor suppressor gene DBCCR1 (deleted in bladder cancer chromosome region 1), has unknown function. In the present study, we explored function and molecular mechanism of DBCCR1-003 in bladder cancer (BC) development. METHODS: We evaluated the expression levels of DBCCR1-003 in tissues and cells with western blot and quantitative real-time polymerase chain reaction. Multiple approaches including chromatin immunoprecipitation assay and RNA immunoprecipitation were used to confirm the direct binding of DBCCR1-003 to DNMT1. The recombinant vector overexpressing DBCCR1-003 was constructed. Cell proliferation assay, colony formation assay and flow cytometric analysis were employed to measure the role of DBCCR1-003 in regulation of cell proliferation, cycle and apoptosis. RESULTS: Firstly we detected the expression of DBCCR1-003, DBCCR1, DNMT1 (DNA methyltransferase 1) and DNA methylation in the promoter of DBCCR1. We found low expression of DBCCR1-003, same as DBCCR1, while high expression of DNMT1 and hypermethylation of DBCCR1 gene promoter in BC tissues and T24 cells line. Further studies revealed that treatment of DNMT inhibitor, 5-aza-2-deoxycytidine(DAC), or overexpression of DBCCR1-003 led to increased DBCCR1 expression by reversion of promoter hypermethylation and DNMT1 binding to DBCCR1 promoter in T24 cells. Importantly, RNA immunoprecipitation (RIP) showed that DBCCR1-003 physically associates with DNMT1. The binding of them was increased with the inhibition of DBCCR1 promoter methylation, indicating that DBCCR1-003 may bind to DNMT1 and prevent DNMT1-mediated the methylation of DBCCR1. Furthermore, overexpression of DBCCR1-003 resulted in significant inhibition of T24 cells growth through the inducing G0/G1 arrest and apoptosis. CONCLUSIONS: Taken together, these findings demonstrated that a novel tumor suppressor DBCCR1-003 regulates the expression of DBCCR1 via binding to DNMT1 and preventing DNMT1-mediated the methylation of DBCCR1 in BC. LncRNA DBCCR1-003 may serve as a novel biomarker and therapeutic target for BC in future cancer clinic.

Hyperthermia Induces Apoptosis of 786-O Cells through Suppressing Ku80 Expression.

Hyperthermia as an anticancer method has been paid increasing attention in recent years. Several studies have shown that hyperthermia can kill tumor cells by inducing apoptosis. However, the underlying molecular mechanisms of hyperthermia-induced apoptosis are largely unknown. To investigate the effects and molecular mechanism of hyperthermia on the apoptosis in renal carcinoma 786-O cells, we firstly examined apoptosis and Ku expression in 786-O cell line treated with heat exposure (42°C for 0-4 h). The results showed that hyperthermia induced apoptosis of 786-O cells, and suppressed significantly Ku80 expression, but not Ku70 expression. Next, we knock-down Ku80 in 786-O cells, generating stable cell line 786-O-shKu80, and detected apoptosis, cell survival and cell cycle distribution. Our data showed higher apoptotic rate and lower surviving fraction in the stable cell line 786-O-shKu80 compared with those in control cells, exposed to the same heat stress (42°C for 0-4 h). Moreover, the results also showed suppression of Ku80 led to G2/M phase arrest in the stable cell line 786-O-shKu80 following heat treatment. Together, these findings indicate that Ku80 may play an important role in hyperthermia-induced apoptosis and heat-sensitivity of renal carcinoma cells through influencing the cell cycle distribution.

The relationship between promoter methylation of p16 gene and bladder cancer risk: a meta-analysis.

PURPOSE: Many scientific evidences suggested that the methylation of p16INK4a (p16) was associated with bladder cancer, but some existing studies have yielded inconclusive results about the relationship between p16 promoter methylation and pathological features or the tumor grade of bladder cancer. This meta-analysis of studies aims to evaluate the clinical and prognostic significance of p16 methylation in bladder carcinogenesis. METHODS: Studies were systemically searched via PubMed and Google Scholar in English up to Sept 2015 and a total of ten appropriate studies (693 cases and 290 controls) with an average NOS score of 6.8 were included. The quality of the appropriate studies was measured by the Newcastle-Ottawa Scale (NOS) assessment. RESULTS: The meta-analysis results revealed that the methylation state of p16 was statistically significantly associated with an increased risk of bladder cancer (OR=6.71, 95% CI=3.79-11.87) compared to control, and there is no statistically significantly association between the p16 methylation and the tumor pTNM staging (OR=0.59, 95% CI=0.22-1.60) or the tumor grade (OR=1.01, 95% CI=0.52-1.94) in p16 methylated patients compared to unmethylated patients. CONCLUSIONS: our meta-analysis indicates that p16 promoter methylation may be a promising biomarker for the diagnosis of bladder cancer and the inactivation of p16 may be an early event in bladder carcinogenesis. More studies with larger numbers of participants worldwide are needed to further identify the obvious association above.

Impaired regulator of G protein signaling 2 transcription facilitates vascular remodeling in injured rat aorta.

BACKGROUND: The activation of Gq-protein-coupled receptors induces proliferation of vascular smooth muscle cells (VSMCs) proliferation and is involved in vascular remodeling. The regulator of G protein signaling 2 (RGS2), which accelerates the termination of Gq protein signaling, may play a role in vascular remodeling. However, this role remains unclear. METHODS: Aortic balloon injury or sham operation was produced in male Wistar rats. Histological examination and gene expression analysis were performed after surgery. In cultured VSMCs after modulation of RGS2 expression, cell proliferation was also evaluated. RESULTS: At day 3 after injury, RGS2 transcription was reduced by 52.8% (P <0.05 vs. sham group) with vascular remodeling. In cultured VSMCs stimulated by endothelin-1, phenylephrine or angiotensin II, the proliferation of RGS2 overexpressed cells was significantly inhibited; the proliferation of RGS2 downregulated cells was significantly promoted, compared with that of RGS2 normal cells. Moreover, after incubation with angiotensin II of high concentration (>10 µmol/l) or long term (>8 h), the RGS2 expression was clearly downregulated in cultured VSMCs. Administration of an angiotensin receptor blocker, valsartan (20 mg/kg per day) starting from 1 week preballoon injury to 3 days after injury, restored aortic RGS2 transcription and improved vascular remodeling. CONCLUSION: These results suggested that the inhibiting effect of RGS2 on VSMC proliferation is downregulated in vascular remodeling of injured rat aorta, and this effect is likely to be mediated by angiotensin II signaling.

Adrenomedullin and adrenotensin increase the transcription of regulator of G­protein signaling 2 in vascular smooth muscle cells via the cAMP­dependent and PKC pathways.

The regulator of G­protein signaling 2 (RGS2) has been shown to be crucial in the regulation of vascular tone and blood pressure. The vascular activities of adrenomedullin (ADM) and adrenotension (ADT), two natural peptides, are dependent upon the modulation of RGS2 expression. However, the effects and pathways involved in their modulation remain unknown. This study aimed to observe the changes of RGS2 expression in response to ADM and ADT in cultured vascular smooth muscle cells and to clarify the potential signaling pathways in vitro. In the present study, vascular smooth muscle cells (VSMCs) were cultured with ADM and ADT of various concentrations for different time periods, and the gene expression of RGS2 was analyzed by PCR. ADM significantly increased the gene expression at 0.5 h to ~35­fold of that at baseline, whereas ADT marginally increased the expression after 1­2 h. SQ22,536 and chelerythrine were used to block the protein kinase A (PKA) and PKC pathways activated by incubation with ADM. The gene expression of RGS2 was reduced by SQ22,536 only. Furthermore, when SQ22,536 and chelerythrine were added to the cells incubated with ADT, the gene expression was markedly reduced by both SQ22,536 and chelerythrine. In conclusion, ADM immediately showed a marked increase in the gene expression of RGS2 in cultured VSMCs via a cAMP­dependent pathway and ADT gradually showed a marginal increase in the gene expression via a cAMP­dependent pathway and a PKC pathway.

Regulator of G protein signaling 2 is a functionally important negative regulator of angiotensin II-induced cardiac fibroblast responses.

Cardiac fibroblasts play a key role in fibrosis development in response to stress and injury. Angiotensin II (ANG II) is a major profibrotic activator whose downstream effects (such as phospholipase Cß activation, cell proliferation, and extracellular matrix secretion) are mainly mediated via G(q)-coupled AT(1) receptors. Regulators of G protein signaling (RGS), which accelerate termination of G protein signaling, are expressed in the myocardium. Among them, RGS2 has emerged as an important player in modulating G(q)-mediated hypertrophic remodeling in cardiac myocytes. To date, no information is available on RGS in cardiac fibroblasts. We tested the hypothesis that RGS2 is an important regulator of ANG II-induced signaling and function in ventricular fibroblasts. Using an in vitro model of fibroblast activation, we have demonstrated expression of several RGS isoforms, among which only RGS2 was transiently upregulated after short-term ANG II stimulation. Similar results were obtained in fibroblasts isolated from rat hearts after in vivo ANG II infusion via minipumps for 1 day. In contrast, prolonged ANG II stimulation (3-14 days) markedly downregulated RGS2 in vivo. To delineate the functional effects of RGS expression changes, we used gain- and loss-of-function approaches. Adenovirally infected RGS2 had a negative regulatory effect on ANG II-induced phospholipase Cß activity, cell proliferation, and total collagen production, whereas RNA interference of endogenous RGS2 had opposite effects, despite the presence of several other RGS. Together, these data suggest that RGS2 is a functionally important negative regulator of ANG II-induced cardiac fibroblast responses that may play a role in ANG II-induced fibrosis development.