A pairwise radiomics algorithm-lesion pair relation estimation model for distinguishing multiple primary lung cancer from intrapulmonary metastasis.

Background: Distinguishing multiple primary lung cancer (MPLC) from intrapulmonary metastasis (IPM) is critical for their disparate treatment strategy and prognosis. This study aimed to establish a non-invasive model to make the differentiation pre-operatively. Methods: We retrospectively studied 168 patients with multiple lung cancers (307 pairs of lesions) including 118 cases for modeling and internal validation, and 50 cases for independent external validation. Radiomic features on computed tomography (CT) were extracted to calculate the absolute deviation of paired lesions. Features were then selected by correlation coefficients and random forest classifier 5-fold cross-validation, based on which the lesion pair relation estimation (PRE) model was developed. A major voting strategy was used to decide diagnosis for cases with multiple pairs of lesions. Cases from another institute were included as the external validation set for the PRE model to compete with two experienced clinicians. Results: Seven radiomic features were selected for the PRE model construction. With major voting strategy, the mean area under receiver operating characteristic curve (AUC), accuracy, sensitivity, and specificity of the training versus internal validation versus external validation cohort to distinguish MPLC were 0.983 versus 0.844 versus 0.793, 0.942 versus 0.846 versus 0.760, 0.905 versus 0.728 versus 0.727, and 0.962 versus 0.910 versus 0.769, respectively. AUCs of the two clinicians were 0.619 and 0.580. Conclusions: The CT radiomic feature-based lesion PRE model is potentially an accurate diagnostic tool for the differentiation of MPLC and IPM, which could help with clinical decision making.

Gut microbiota interactions with antitumor immunity in colorectal cancer: From understanding to application.

Colorectal cancer (CRC) is one of highly prevalent cancer. Immunotherapy with immune checkpoint inhibitors (ICIs) has dramatically changed the landscape of treatment for many advanced cancers, but CRC still exhibits suboptimal response to immunotherapy. The gut microbiota can affect both anti-tumor and pro-tumor immune responses, and further modulate the efficacy of cancer immunotherapy, particularly in the context of therapy with ICIs. Therefore, a deeper understanding of how the gut microbiota modulates immune responses is crucial to improve the outcomes of CRC patients receiving immunotherapy and to overcome resistance in nonresponders. The present review aims to describe the relationship between the gut microbiota, CRC, and antitumor immune responses, with a particular focus on key studies and recent findings on the effect of the gut microbiota on the antitumor immune activity. We also discuss the potential mechanisms by which the gut microbiota influences host antitumor immune responses as well as the prospective role of intestinal flora in CRC treatment. Furthermore, the therapeutic potential and limitations of different modulation strategies for the gut microbiota are also discussed. These insights may facilitate to better comprehend the interplay between the gut microbiota and the antitumor immune responses of CRC patients and provide new research pathways to enhance immunotherapy efficacy and expand the patient population that could be benefited by immunotherapy.

ERK/MAPK Signalling Pathway Regulates MMP2 Through ETS1 in Renal Clear Cell Carcinoma.

BACKGROUND: The c-ETS-1 (ETS1) expression is high in clear cell renal cell carcinoma (ccRCC) tissues; however, how it impacts ccRCC is currently unknown. METHOD: The online STRING web source was used to construct a protein network interacting with ETS1. The Cell Counting Kit-8 was used to detect the cell viability. A clonogenic assay, a wound-healing assay, and a Transwell assay were used to detect cell proliferation, invasion and migration abilities. Western blot was used to detect the expression of proteins. RESULT: The data showed the expression of ETS1 in ccRCC tissues to be significantly increased compared to adjacent tissues (p<0.05). The positive expression of ETS1 in ccRCC patients aged 20-100 was statistically significant compared to adjacent normal tissues (p<0.05). The grade of ETS1 positive expression (1-4) and lymph node metastasis (N1) in ccRCC were significantly higher than those in adjacent normal tissues (p<0.05). The tumour stage (stages 1-4) in ccRCC patients with positive ETS1 expression was significantly higher than that in adjacent normal tissues (p<0.05). Knockdown of ETS1 and PERK inhibitors significantly inhibited the proliferation, migration and invasion of ccRCC cells. Knockdown of ETS1 inhibited MMP-2 expression, and an extracellular signal-related kinase (ERK) inhibitor inhibited both ETS1 and MMP-2 expression. CONCLUSION: A high expression of ETS1 is associated with the progression of ccRCC. This study suggests that ETS1 promotes proliferation by increasing MMP2 expression in ccRCC, and combined knockdown of ETS1 and inhibition of ERK can significantly inhibit the proliferation, migration and invasion of ccRCC. ETS1 may be a therapeutic and prognostic target for renal cell carcinoma.

A bibliometric and visual analysis of cancer-associated fibroblasts.

Background: Cancer-associated fibroblasts (CAFs) represent the predominant stromal component within the tumour microenvironment (TME), exhibiting considerable heterogeneity and plasticity that significantly impact immune response and metabolic reprogramming within the TME, thereby influencing tumour progression. Consequently, investigating CAFs is of utmost importance. The objective of this study is to employ bibliometric analysis in order to evaluate the current state of research on CAFs and predict future areas of research and emerging trends. Methods: Conduct a comprehensive search for scholarly publications within the Web of Science Core Collection database, encompassing the time period from January 1, 2001, to December 31, 2022. Apply VOSviewer, CiteSpace, R software and Microsoft Excel for bibliometric analysis and visualisation. Results: This study involved a comprehensive analysis of 5,925 publications authored by 33,628 individuals affiliated with 4,978 institutions across 79 countries/regions. These publications were published in 908 journals, covering 14,495 keywords and 203,947 references. Notably, there was a significant increase in articles published between 2019 and 2022. China had the highest count of articles, while the United States emerged as the most frequently cited country. The primary research institutions in this field were Shanghai Jiao Tong University, Harvard University, and the University of Texas MD Anderson Cancer Center. Sotgia, Federica and Lisanti, Michael P from the University of Manchester, and Martinet, Wim from the University of Antwerp were the most prolific and highly cited authors. The journal Cancers had the highest number of publications, while Cancer Research was the most frequently cited journal. Molecular, biology, immunology, medicine and genetics were the main research disciplines in the field of CAFs. Key directions in CAFs research encompassed the study of transforming growth factor-ß, Fibroblast Activation Protein, breast cancer, as well as growth and metastasis. The findings from the analysis of keyword co-occurrence and literature co-citation have revealed several emerging hotspots and trends within the field of CAFs. These include STAT3, multidrug resistance, pancreatic ductal adenocarcinoma, pan-cancer analysis, preclinical evaluation, ionizing radiation, and gold nanoparticles. Conclusion: Targeting CAFs is anticipated to be a novel and effective strategy for cancer treatment. This study provides a comprehensive overview of the existing research on CAFs from 2001 to 2022, utilizing bibliometric analysis. The study identified the prominent areas of investigation and anticipated future research directions, with the aim of providing valuable insights and recommendations for future studies in the field of CAFs.

NOTCH1 mutation associates with impaired immune response and decreased relapse-free survival in patients with resected T1-2N0 laryngeal cancer.

Background: Patients with early-stage laryngeal cancer, even stage T1-2N0, are at considerable risk of recurrence and death. The genetic and immunologic characteristics of recurrent laryngeal cancer remain unclear. Methods: A total of 52 T1-2N0 laryngeal cancer patients were enrolled. Of these, 42 tissue samples were performed by targeted DNA sequencing, and 21 cases were performed by NanoString immuno-oncology targeted RNA sequencing to identify the distinct molecular bases and immunologic features associated with relapse in patients with early laryngeal cancer, respectively. Results: To the best to our knowledge, we present for the first time an overview of the genomic mutation spectrum of early-stage laryngeal cancers. A total of 469 genomic alterations were detected in 211 distinct cancer-relevant genes, and the genes found to be mutated in more than five patients (>10%) included tumor protein p53 (TP53, 78.5%), FAT atypical cadherin 1 (FAT1, 26%), LDL receptor related protein 1B (LRP1B, 19%), cyclin dependent kinase inhibitor 2A (CDKN2A, 17%), tet methylcytosine dioxygenase 2 (TET2, 17%), notch receptor 1 (NOTCH1, 12%) and neuregulin 1 (NRG1, 12%). Recurrent laryngeal cancer demonstrated a higher tumor mutation burden (TMB), as well as higher LRP1B mutation and NOTCH1 mutation rates. Univariate and multivariate analyses revealed that high TMB (TMB-H) and NOTCH1 mutation are independent genetic factors that are significantly associated with shorter relapse-free survival (RFS). Simultaneously, the results of the transcriptome analysis presented recurrent tumors with NOTCH1 mutation displayed upregulation of the cell cycle pathway, along with decreased B cells score, T cells score, immune signature score and tumor-infiltrating lymphocytes (TILs) score. The Cancer Genome Atlas (TCGA)-laryngeal cancer dataset also revealed weakened immune response and impaired adhesion functions in NOTCH1-mutant patients. Conclusions: Genomic instability and impaired immune response are key features of the immunosurveillance escape and recurrence of early laryngeal cancer after surgery. These findings revealed immunophenotypic attenuation in recurrent tumors and provided valuable information for improving the management of these high-risk patients. Due to the small number of patients in this study, these differences need to be further validated in a larger cohort.

Novel Plasma Proteomic Biomarkers for Early Identification of Induction Chemotherapy Beneficiaries in Locoregionally Advanced Nasopharyngeal Carcinoma.

Background: Induction chemotherapy (IC) can alleviate locoregionally advanced nasopharyngeal carcinoma (LA-NPC), but effectiveness differs between patients, toxicity is problematic, and effective blood-based IC efficacy predictors are lacking. Here, we aimed to identify biomarkers for early identification of IC beneficiaries. Methods: Sixty-four pairs of matched plasma samples collected before and after IC from LA-NPC patients including 34 responders and 30 non-responders, as well as 50 plasma samples of healthy individuals, were tested using data-independent acquisition mass spectrometry. The proteins associated with clinical traits or IC benefits were investigated by weighted gene co-expression network analysis (WGCNA) and soft cluster analysis. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes functional annotations were performed to determine the potential function of the identified proteins. The area under the receiver operating characteristic curve (AUC) was used to evaluate the performance of candidate biomarkers in predicting IC beneficiaries. Results: Compared with healthy individuals, 1027 differentially expressed proteins (DEPs) were found in the plasma of LA-NPC patients. Based on feedback from IC outcomes, 463 DEPs were identified in the pre-IC plasma between responders and non-responders. A total of 1212 DEPs represented the proteomic changes before and after IC in responders, while 276 DEPs were identified in post-IC plasma between responders and non-responders. WGCNA identified nine protein co-expression modules correlated with clinical traits. Soft cluster analysis identified four IC benefits-related protein clusters. Functional enrichment analysis showed that these proteins may play a role in IC via immunity, complement, coagulation, glycosaminoglycan and serine. Four proteins differentially expressed in all group comparisons, paraoxonase/arylesterase 1 (PON1), insulin-like growth factor-binding protein 3 (IGFBP-3), rheumatoid factor D5 light chain (v-kappa-3) and RNA helicase (DDX55), were associated with clinical traits or IC benefits. A four-protein model accurately identified potential IC beneficiaries (AUC=0.95) while diagnosing LA-NPC (AUC=0.92), and the prediction performance was verified using the models to confirm the effective IC (AUC=0.97) and evaluate IC outcome (AUC=0.94). Conclusion: The plasma protein profiles among IC responders and non-responders were different. PON1, IGFBP3, v-kappa-3 and DDX55 could serve as potential biomarkers for early identification of IC beneficiaries for individualised treatment of LA-NPC.

A novel three-microRNA signature for predicting survival in patients with nasopharyngeal carcinoma.

BACKGROUND/PURPOSE: Nasopharyngeal carcinoma (NPC) is a malignant neoplasm of the head and neck. This study aims to use integrated bioinformatics technologies to develop a predictive miRNA-signature correlated with the prognosis of NPC. MATERIALS AND METHODS: Initially, the differentially expressed miRNAs (DEMs) in NPC were identified, and then DEMs related to the prognosis of NPC were further screened. Subsequently, the relatively important DEMs identified by random forest algorithm were used to construct a predictive signature by multivariate COX regression analysis. Moreover, PCA, Kaplan-Meier analysis, time-dependent ROC analysis, and univariate and multivariate COX regression analysis were performed to evaluate the ability of the signature in risk identification and prognosis prediction in NPC. RESULTS: Hsa-miR-29c, hsa-miR-30e and hsa-miR-93 were selected from DEMs to construct a signature, and their abnormal expression was significantly associated with poor prognosis of NPC. The average AUC values of 1- to 5-year OS, DFS and DMFS predicted by the signature were all above 0.7, and showed better clinical independence than other indexes. In addition, 295 differentially expressed mRNAs could be used as potential target genes of the 3 DEMs. Among them, 56 differentially expressed mRNAs were related to PFS. GO and KEGG enrichment analysis indicated that the poor prognosis of NPC was related to the abnormality of chromosomes, cytokines, and chemokines. CONCLUSION: We constructed a three-miRNA signature with good independent performance in predicting the prognosis for NPC. This study may lay the foundation for exploring new therapeutic targets and improving survival outcomes in NPC patients.

Research status and prospects of acupuncture for prevention and treatment of chemo- and radiotherapy-induced salivary gland dysfunction in head and neck cancer.

Salivary gland dysfunction (SGD) induced by chemo- and radiotherapy for head and neck cancer (HNC) has always been a difficult problem in modern medicine. The quality of life of a large number of HNC patients is severely impaired by SGD such as xerostomia and dysphagia. In recent years, several studies have found that acupuncture can improve patients' salivary secretion, but it has not yet been approved as an alternative therapy for SGD. For this reason, we collected the clinical study reports on acupuncture in the treatment of SGD induced by chemo- and radiotherapy in HNC patients in the past 20 years, and analyzed and discussed the advantages and disadvantages of these studies with respect to tumor types, group setting, intervention modality, acupoints selection, outcome evaluation, and safety. We believed that acupuncture is beneficial for SGD, but the existing objective evidence is insufficient to support its effectiveness. Therefore, improving the Standards for Reporting Interventions in Clinical Trials of Acupuncture, selecting the optimal combination of acupoints through scientific and rigorous study design, and exploring the potential mechanism of acupuncture in the treatment of diseases combined with the meridian theory may be effective ways to promote the acceptance of acupuncture as an alternative therapy for SGD in future. The significance of this review is to provide a reference for researchers to carry out high-quality clinical trials of acupuncture in the treatment of SGD in future from the perspective of the combination of modern medicine and traditional Chinese medicine.

Celastrol induces caspase-dependent apoptosis of hepatocellular carcinoma cells by suppression of mammalian target of rapamycin.

OBJECTIVE: To investigate the efficacy of celastrol treatment of hepatocellular carcinoma (HCC) cells in vitro and in vivo and to propose a mechanism of action. METHODS: A human HepG2 liver cancer cell line and a xenograft tumor model were used to investigate the effects of celastrol on HCC in vitro and in vivo. A CCK-8 kit was used to detect cell viability. Flow cytometry and terminal-deoxynucleoitidyl transferase mediated nick end labeling staining were used to detect apoptosis. Western blotting and immunohistochemistry were used to detect the expression of cleaved-caspase-3, cleaved-caspase-8, cleaved-caspase-9, cleaved-PARP, mammalian target of rapamycin (mTOR), and p-mTOR. Hematoxylin-eosin staining was used to observe the tissue morphology. RESULTS: Celastrol decreased the viability of HepG2 cells and induced apoptosis. Western blot assays indicated that celastrol up-regulated cleaved-caspase-3, cleaved-caspase-8, cleaved-caspase-9, and cleaved-PARP by inhibiting the phosphorylation of mTOR in HepG2 cells. Moreover, celastrol inhibited the tumor growth in a xenograft model. Celastrol also induced caspase-dependent apoptosis (up-regulation of cleaved-caspase- 3, -8, -9, and cleaved-PARP) and inhibited the activation of mTOR in vivo. CONCLUSION: Celastrol induces caspase-dependent apoptosis in HCC cells by inhibiting the activation of mTOR.

Research status and prospects of biomarkers for nasopharyngeal carcinoma in the era of high­throughput omics (Review).

As a malignant tumor type, nasopharyngeal carcinoma (NPC) is characterized by distinct geographical, ethnic and genetic differences; presenting a major threat to human health in many countries, especially in Southern China. At present, no accurate and effective methods are available for the early diagnosis, efficacious evaluation or prognosis prediction for NPC. As such, a large number of patients have locoregionally advanced NPC at the time of initial diagnosis. Many patients show toxic reactions to overtreatment and have risks of cancer recurrence and distant metastasis owing to insufficient treatment. To solve these clinical problems, high­throughput '­omics' technologies are being used to screen and identify specific molecular biomarkers for NPC. Because of the lack of comprehensive descriptions regarding NPC biomarkers, the present study summarized the research progress that has been made in recent years to discover NPC biomarkers, highlighting the existing problems that require exploration. In view of the lack of authoritative reports at present, study design factors that affect the screening of biomarkers are also discussed here and prospects for future research are proposed to provide references for follow­up studies of NPC biomarkers.

Natural selection and genetic diversity of domain I of Plasmodium falciparum apical membrane antigen-1 on Bioko Island.

BACKGROUND: Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) is a promising candidate antigen for a blood-stage malaria vaccine. However, antigenic variation and diversity of PfAMA-1 are still major problems to design a universal malaria vaccine based on this antigen, especially against domain I (DI). Detail understanding of the PfAMA-1 gene polymorphism can provide useful information on this potential vaccine component. Here, general characteristics of genetic structure and the effect of natural selection of DIs among Bioko P. falciparum isolates were analysed. METHODS: 214 blood samples were collected from Bioko Island patients with P. falciparum malaria between 2011 and 2017. A fragment spanning DI of PfAMA-1 was amplified by nested polymerase chain reaction and sequenced. Polymorphic characteristics and the effect of natural selection were analysed using MEGA 5.0, DnaSP 6.0 and Popart programs. Genetic diversity in 576 global PfAMA-1 DIs were also analysed. Protein function prediction of new amino acid mutation sites was performed using PolyPhen-2 program. RESULTS: 131 different haplotypes of PfAMA-1 were identified in 214 Bioko Island P. falciparum isolates. Most amino acid changes identified on Bioko Island were found in C1L. 32 amino acid changes identified in PfAMA-1 sequences from Bioko Island were found in predicted RBC-binding sites, B cell epitopes or IUR regions. Overall patterns of amino acid changes of Bioko PfAMA-1 DIs were similar to those in global PfAMA-1 isolates. Differential amino acid substitution frequencies were observed for samples from different geographical regions. Eight new amino acid changes of Bioko island isolates were also identified and their three-dimensional protein structural consequences were predicted. Evidence for natural selection and recombination event were observed in global isolates. CONCLUSIONS: Patterns of nucleotide diversity and amino acid polymorphisms of Bioko Island isolates were similar to those of global PfAMA-1 DIs. Balancing natural selection across DIs might play a major role in generating genetic diversity in global isolates. Most amino acid changes in DIs occurred in predicted B-cell epitopes. Novel sites mapped on a three dimensional structure of PfAMA-1 showed that these regions were located at the corner. These results may provide significant value in the design of a malaria vaccine based on this antigen.

[Rectal toxicity prediction based on accurate rectal surface dose summation for cervical cancer radiotherapy].

OBJECTIVE: To propose arectal toxicity prediction method based on deformable surface dose accumulation. METHODS: The clinical data were collected retrospectively from 42patients receiving radiotherapy for cervical cancer. With the first fraction as the reference, the other fractions of rectum surface were registered to the reference fraction to obtain the deformation vector fields (DVFs), which were used to deform and sum the fractional rectal doses to yield the cumulative rectal dose. The cumulative rectal dose was flattened via 3D-2D mapping to generate a 2D rectum surface dose map. Two dosimetric features, namely DVPs and DGPs were extracted. Logistic regression embedded with sequential forward feature selection was used as the prediction model. The predictive performance was evaluated in terms of the accuracy, sensitivity, specificity, and the area under the receiver operating characteristic (ROC) curve (AUC). RESULTS: Significant improvements for rectum surface DIR were achieved. The best predictive results were achieved by using both DVPs and DGPs as the features with a sensitivity of 79.5%, a specificity of 81.3% and an AUC of 0.88. CONCLUSION: The proposed method is feasible for predicting clinical rectal toxicity in patients undergoing radiotherapy for cervical cancer.

[Effect of Xiaoai Jiedu Recipe on mIRNA Expression Profiles in H22 Tumor-bearing Mice].

Objective To observe the mechanism of Xiaoai Jiedu Recipe (XJR) for fighting a- gainst hepatoma by detecting tumor miRNAs expression profiles in H22tumor-bearing mice. Methods To- tally 50 H22tumor-bearing mice were randomly divided into the model group, the low dose XJR group, the medium dose XJR group, the high dose XJR group, the Cisplatin group, 10 in each group. Different expressions of tumor tissues in H22 tumor-bearing mice under light microscope were detected using histopathological technique. Differentially expressed miRNAs of tumor tissue in H22tumor-bearing mice were detected by using miRNA chip technique. Differentially expressed miRNAs associated with antitumor mechanism of XJR were found out by statistical analysis. Results Histopathological results showed that reduced pathologic mitosis, smaller cancer cells, and obviously enhanced anti-cancer effect along with increased XJP dose. Results of miRNA chip analyses indicated XJP could significantly up-regulate the ex- pressions of miRNAs, such as miR-1298-5p, miR-874-3p, miR-721, miR-298-5p, miR-551b-5p, miR-346- 5p, miR-105, and so on. It could also down-regulate the expressions of miR-24-3p, miR-3963, miR-127- 3p, miR-434-5p, miR-1187, miR-468-3p, miR-221-5p, and miR-6695-5p. Conclusion XJP could fight a- gainst tumor possibly by regulating expressions of multiple miRNAs.

Meta-Analysis of Randomized Controlled Trials Comparing Risk of Major Adverse Cardiac Events and Bleeding in Patients With Prasugrel Versus Clopidogrel.

The use of prasugrel in patients with coronary artery disease (CAD) has been associated with decreased major adverse cardiac events (MACEs) compared with clopidogrel but with an increased risk of bleeding. However, it remains unclear if the risks of bleeding outweigh those of MACEs in patients on prasugrel treatment. We systematically reviewed randomized controlled trials comparing prasugrel with clopidogrel in patients with CAD. We performed a literature search of PubMed, EMBASE, and Cochrane Central Register of Controlled Trial databases from inception to November 25, 2014, and reviewed the reference lists of retrieved articles. A comparative estimate was made for the combined rates of MACEs and bleeding from the same trials in the framework of this meta-analysis and expressed as odds ratios (ORs) and 95% confidence intervals (CIs) in both random- and fixed-effects models. Nine studies involving 25,214 patients were included in our meta-analysis. In both the random- and fixed-effects models, the risks of MACEs outweighed those of major bleeding (OR 7.48, 95% CI 3.75 to 14.94, p <0.0001, random effects) and of minor bleeding (OR 3.77, 95% CI 1.73 to 8.22, p = 0.009, random effects). Results were corroborated in a standard-dose clopidogrel subgroup analysis (OR 7.46, 95% CI 3.54 to 15.68, p <0.0001, and OR 6.44, 95% CI 2.80 to 14.80, p <0.0001, random effects, respectively). In conclusion, despite the increased risk of bleeding associated with prasugrel treatment compared with clopidogrel, the risk of MACEs far outweighed the risk of bleeding.

Microarray analysis on gene regulation by estrogen, progesterone and tamoxifen in human endometrial stromal cells.

Epithelial stromal cells represent a major cellular component of human uterine endometrium that is subject to tight hormonal regulation. Through cell-cell contacts and/or paracrine mechanisms, stromal cells play a significant role in the malignant transformation of epithelial cells. We isolated stromal cells from normal human endometrium and investigated the morphological and transcriptional changes induced by estrogen, progesterone and tamoxifen. We demonstrated that stromal cells express appreciable levels of estrogen and progesterone receptors and undergo different morphological changes upon hormonal stimulation. Microarray analysis indicated that both estrogen and progesterone induced dramatic alterations in a variety of genes associated with cell structure, transcription, cell cycle, and signaling. However, divergent patterns of changes, and in some genes opposite effects, were observed for the two hormones. A large number of genes are identified as novel targets for hormonal regulation. These hormone-responsive genes may be involved in normal uterine function and the development of endometrial malignancies.

Development of an intracellular, DNA methyltransferase-specific, and gene-specific assay for studying dynamic DNA methylation.

A growing body of evidence supports that DNA methylation-mediated silencing of tumor suppressor genes plays a significant role in cancer development. DNA methylatransferase (DNMT) is the enzyme catalyzing the methylation modification of cytosines in a CpG dinucleotide context. In humans, this reaction is highly selective for certain gene promoters and/or genomic DNA domains. Elucidation of the intracellular targeting mechanism by DNMT has become the key task for understanding epigenetic regulation in cancers. Unfortunately, no suitable method is available to explore this important cell function. This study focuses on the development of an efficient technique for measuring the intracellular, DNMT isoform-specific, and methylated gene-specific, DNA methylation alterations. The technique, designated IMA for Intracellular DNA Methylation Assay, takes advantage of covalent arresting of active DNMT molecules by aza-deoxycytidine (ADC), a modified cytosine homologue readily incorporated into genomic DNA at the cytosine position. The DNMT-DNA complex was isolated by a modified ETOH precipitation procedure to remove cellular proteins including free DNMT. Chromatin immunoprecipitation using DNMT isoform-specific antibody was subsequently performed to collect DNMT-bound DNA fragments. PCR amplification was used to detect and quantify the isolated gene fragment. Validation of the IMA was performed by manipulating the DNMT activity, by treating with antisense oligonucleotides against DNMT1 and DNMT3B, and repeating the IMA experiment. One of the main discoveries with this technique was the observation of DNMT3B maintenance methylation activity. This new technique can be applied to examine the dynamic DNMT-specific action on diversified methylation-silenced genes, in a variety of cell culture conditions.

Hypomethylation and activation of syncytin-1 gene in endometriotic tissue.

Syncytin-1 plays a critical role in the maintenance of normal pregnancy by mediating the formation of syncytiotrophoblasts through a fosugenic action. Encoded by the human endogenous retrovirus envelope gene HERV-W, syncytin-1 trophoblast-specific expression is controlled by epigenetic mechanisms. In non-placental tissues, the syncytin-1 gene is suppressed by hypermethylation in the LTR promoter region. Hypomethylated and activated syncytin-1 gene is found in placental trophoblast lineages and malignant cells. We here demonstrate that while syncytin-1 gene remains silenced in the eutopic endometrium from endometriotic patients, syncytin-1 mRNA and protein are detected in ectopic, endometriotic lesions; particularly the endometrioid glandular endothelial cells. LINE-1 COBRA assay and immunohistochemistry using the 5-MC-specific antibody did not detect any changes in global DNA methylation in the endometriotic tissues. However, results from COBRA and bisulfite sequencing indicated that the LTR region of the syncytin-1 promoter is hypomethylated in endometriotic tissues, highlighting the significance of DNA demethylation in syncytin-1 gene activation. Analysis of DNA methyltransferase 3B (DNMT3B) mRNA levels revealed that DNMT3B3, an isoform carrying methyltransferase activity, is downregulated; whereas DNMT3B7, the isoform without enzymatic activity, is upregulated in the endometriotic tissues, pointing to positive and negative regulatory functions, respectively, of these isoforms on syncytin-1 methylation. These results have provided the first evidence supporting the involvement of epigenetic mechanisms for syncytin-1 upregulation in endometriotic tissues. Considering recent findings on the nonfusogenic activity of syncytin-1, its expression in endometriotic tissues suggests that this multifunctional protein may be implicated in the pathogenesis and/or progression of endometriosis.

Decreased expression and altered methylation of syncytin-1 gene in human placentas associated with preeclampsia.

Syncytin-1 is a protein coded by a human endogenous retrovirus (HERV) gene of the HERV-W family (HERVWE1). Syncytin- 1 mediates formation of syncytiotrophoblasts through fusion of cytotrophoblasts, a hallmark of terminal differentiation of placental trophoblast linage. Syncytin-1 also possesses nonfusogenic functions and regulates cell cycle progression. While decreased syncytin-1 expression and syncytium deficiency are considered important pathological changes in preeclampsia, the molecular mechanism(s) underlying syncytin-1 downregulation remains unclear. In this study, we confirmed that expression levels of syncytin-1 mRNA and protein were significantly lower in preeclamptic placentas compared to normal controls. Human chorionic somatomammotropin expression, a marker for syncytium function, was also decreased in preeclamptic placentas. The mRNA levels of ASCT2, the syncytin-1 receptor involved in cell fusion process, and GCMa, a transcriptional factor known to regulate syncytin-1 expression, were not significantly altered. Methylation in the 5'LTR of syncytin-1 promoter was quantified by COBRA, methylation-specific PCR, and DNA sequencing. Results from all three assays indicated significantly hypermethylated syncytin-1 promoter in preeclamptic placentas compared to normal controls. Methylation levels were inversely correlated with syncytin-1 mRNA levels, suggesting that hypermethylation may lead to syncytin-1 downregulation. Further experiments indicated that DNMT1 and DNMT3B3 mRNA and protein levels were increased in preeclamptic placentas, suggesting that higher DNA methyltransferase activity may contribute to the hypermethylation of syncytin-1 in preeclamptic placentas. These results indicated that aberrant hypermethylation is involved in downregulation of syncytin-1, and epigenetic alterations may play a significant role in the development of preeclampsia.

Cytostatic and apoptotic effects of DNMT and HDAC inhibitors in endometrial cancer cells.

DNA methyltransferase (DNMT) and histone deacetylase are key enzymes mediating the epigenetic regulation of gene expression. DNA hypermethylation and/or histone deacetylation in promoter regions is often associated with downregulation or silencing of transcription. Epigenetic silencing of tumor suppressor genes plays an important role in malignant transformation. DNMT and HDAC inhibitors induce DNA demethylation and histone acetylation, respectively, leading to reactivation of silenced genes, and dramatic morphological and functional changes in cancer cells. In this study, we have conducted a series of experiments to characterize the effects of epigenetic inhibitors in endometrial cancer cells. Using cell lines representing different stages of endometrioid cancers, we examined the impact of DNMT inhibitor, ADC, and HDAC inhibitor, TSA, on cell cycle and apoptosis. We found that while both reagents were capable of inhibiting cell proliferation and inducing cell apoptosis, TSA appeared to be a more potent apoptosis inducer, but with a smaller effect on cell cycle. On the other hand, ADC exhibited strong effects on cell cycle regulation, but had smaller impact on cell apoptosis. We subsequently confirmed the presence of a strong synergism between DNMT and HDAC inhibitors. Thus, ADC and TSA exhibited strong cytostatic and apoptotic effects in endometrial cancer cell lines and the combined application may deliver the highest response in the clinical setting.

Neutrophil gelatinase-associated lipocalin in gastric carcinoma cells and its induction by TPA are controlled by C/EBPß.

Neutrophil gelatinase-associated lipocalin (NGAL) expression has been found to be upregulated in a variety of tumors, but the mechanism of NGAL elevation in gastric carcinoma remains unknown. Here, immunohistochemistry was applied to analyze NGAL expression in gastric carcinoma patients. Reverse transcription PCR, Western blot, and enzyme-linked immunosorbent assay (ELISA) were performed to evaluate NGAL mRNA and protein levels before and after 12-O-tetradecanoylphorbol-13-acetate (TPA) induction. Luciferase reporter assay was carried out to identify the core cis element in NGAL promoter. The binding ability and specificity of transcription factors were analyzed by electrophoretic mobility-shift assay (EMSA) and chromatin immunoprecipitation (ChIP), respectively. Results showed that NGAL was overexpressed in gastric tumor tissues. Gastric cancer cells treated with TPA resulted in the transactivation of NGAL promoter and the upregulation of its mRNA and protein levels. We identified the -110 to -79 sequence segment upstream from the transcription initiation site of NGAL as a TPA responsive element (TRE) and confirmed that C/EBPß was able to bind to the -87 to -79 segment. Forced expression of C/EBPß significantly increased the promoter activity of NGAL as well as its mRNA level. These results suggest that NGAL is overexpressed in gastric cancer, the binding of C/EBPß to the TRE of its gene promoter mediates its TPA-induced overexpression in gastric carcinoma cells.