Small extracellular vesicle DNA-mediated horizontal gene transfer as a driving force for tumor evolution: Facts and riddles.

The basis of the conventional gene-centric view on tumor evolution is that vertically inherited mutations largely define the properties of tumor cells. In recent years, however, accumulating evidence shows that both the tumor cells and their microenvironment may acquire external, non-vertically inherited genetic properties via horizontal gene transfer (HGT), particularly through small extracellular vesicles (sEVs). Many phases of sEV-mediated HGT have been described, such as DNA packaging into small vesicles, their release, uptake by recipient cells, and incorporation of sEV-DNA into the recipient genome to modify the phenotype and properties of cells. Recent techniques in sEV separation, genome sequencing and editing, as well as the identification of new secretion mechanisms, shed light on a number of additional details of this phenomenon. Here, we discuss the key features of this form of gene transfer and make an attempt to draw relevant conclusions on the contribution of HGT to tumor evolution.

Global DNA hypomethylation of colorectal tumours detected in tissue and liquid biopsies may be related to decreased methyl-donor content.

BACKGROUND: Hypomethylation of long interspersed nuclear element 1 (LINE-1) is characteristic of various cancer types, including colorectal cancer (CRC). Malfunction of several factors or alteration of methyl-donor molecules' (folic acid and S-adenosylmethionine) availability can contribute to DNA methylation changes. Detection of epigenetic alterations in liquid biopsies can assist in the early recognition of CRC. Following the investigations of a Hungarian colon tissue sample set, our goal was to examine the LINE-1 methylation of blood samples along the colorectal adenoma-carcinoma sequence and in inflammatory bowel disease. Moreover, we aimed to explore the possible underlying mechanisms of global DNA hypomethylation formation on a multi-level aspect. METHODS: LINE-1 methylation of colon tissue (n = 183) and plasma (n = 48) samples of healthy controls and patients with colorectal tumours were examined with bisulfite pyrosequencing. To investigate mRNA expression, microarray analysis results were reanalysed in silico (n = 60). Immunohistochemistry staining was used to validate DNA methyltransferases (DNMTs) and folate receptor beta (FOLR2) expression along with the determination of methyl-donor molecules' in situ level (n = 40). RESULTS: Significantly decreased LINE-1 methylation level was observed in line with cancer progression both in tissue (adenoma: 72.7 ± 4.8%, and CRC: 69.7 ± 7.6% vs. normal: 77.5 ± 1.7%, p ≤ 0.01) and liquid biopsies (adenoma: 80.0 ± 1.7%, and CRC: 79.8 ± 1.3% vs. normal: 82.0 ± 2.0%, p ≤ 0.01). However, no significant changes were recognized in inflammatory bowel disease cases. According to in silico analysis of microarray data, altered mRNA levels of several DNA methylation-related enzymes were detected in tumours vs. healthy biopsies, namely one-carbon metabolism-related genes-which met our analysing criteria-showed upregulation, while FOLR2 was downregulated. Using immunohistochemistry, DNMTs, and FOLR2 expression were confirmed. Moreover, significantly diminished folic acid and S-adenosylmethionine levels were observed in parallel with decreasing 5-methylcytosine staining in tumours compared to normal adjacent to tumour tissues (p ≤ 0.05). CONCLUSION: Our results suggest that LINE-1 hypomethylation may have a distinguishing value in precancerous stages compared to healthy samples in liquid biopsies. Furthermore, the reduction of global DNA methylation level could be linked to reduced methyl-donor availability with the contribution of decreased FOLR2 expression.

Folic Acid Treatment Directly Influences the Genetic and Epigenetic Regulation along with the Associated Cellular Maintenance Processes of HT-29 and SW480 Colorectal Cancer Cell Lines.

Folic acid (FA) is a synthetic form of vitamin B9, generally used as a nutritional supplement and an adjunctive medication in cancer therapy. FA is involved in genetic and epigenetic regulation; therefore, it has a dual modulatory role in established neoplasms. We aimed to investigate the effect of short-term (72 h) FA supplementation on colorectal cancer; hence, HT-29 and SW480 cells were exposed to different FA concentrations (0, 100, 10,000 ng/mL). HT-29 cell proliferation and viability levels elevated after 100 ng/mL but decreased for 10,000 ng/mL FA. Additionally, a significant (p ≤ 0.05) improvement of genomic stability was detected in HT-29 cells with micronucleus scoring and comet assay. Conversely, the FA treatment did not alter these parameters in SW480 samples. RRBS results highlighted that DNA methylation changes were bidirectional in both cells, mainly affecting carcinogenesis-related pathways. Based on the microarray analysis, promoter methylation status was in accordance with FA-induced expression alterations of 27 genes. Our study demonstrates that the FA effect was highly dependent on the cell type, which can be attributed to the distinct molecular background and the different expression of proliferation- and DNA-repair-associated genes (YWHAZ, HES1, STAT3, CCL2). Moreover, new aspects of FA-regulated DNA methylation and consecutive gene expression were revealed.

The Behavior of MMP-2, MMP-7, MMP-9, and Their Inhibitors TIMP-1 and TIMP-2 in Adenoma-Colorectal Cancer Sequence.

BACKGROUND: We and others have previously shown that matrix metalloproteinases (MMPs) play a role in colorectal cancer (CRC) invasion and metastasis. However, the serum changes of various MMPs and their inhibitors (TIMPs) have scarcely been concomitantly investigated in identical blood samples in the normal colon-adenoma-CRC sequence. METHODS: The MMP-2, MMP-7, MMP-9, TIMP-1, and TIMP-2 serum antigen concentrations were determined concomitantly in 19 tumor-free control patients, 19 patients with high-risk colorectal adenoma, and 47 patients with CRC by ELISA technique. The analyzed parameters were also investigated in correlation with CRC stages. Statistical analysis with one-way ANOVA and Student's t test was performed. p values <0.05 were considered significant. RESULTS: Serum antigen levels of MMPs and TIMPs were significantly increased in patients with CRC and adenomas compared to controls (mean values, ng/mL) (MMP-7: 5.88, 4.44, and 2.89, respectively, p = 0.001; MMP-9: 1,075.81, 999.22, and 845.97, respectively, p = 0.01; TIMP-1: 241.80, 205.98, and 166.53, respectively, p = 0.001; TIMP-2: 83.40, 80.30, and 69.62, respectively, p = 0.01). The elevated serum MMP-7, MMP-9, TIMP-1, and TIMP-2 levels significantly correlated with advanced tumor stages (p < 0.05). No statistically significant differences were observed in MMP-2 levels. CONCLUSIONS: We demonstrate that serum antigen concentrations of MMP-7, MMP-9, TIMP-1, and TIMP-2 were significantly increased in patients with CRC and adenomas compared to controls. These results suggest that MMPs and their inhibitors TIMP-1 and TIMP-2 play an important role in CRC invasion; however, they are also activated in premalignant adenomas. Furthermore, MMP-7, MMP-9, TIMP-1, and TIMP-2 may have a potential prognostic impact in CRC.

S-Adenosylmethionine Treatment of Colorectal Cancer Cell Lines Alters DNA Methylation, DNA Repair and Tumor Progression-Related Gene Expression.

Global DNA hypomethylation is a characteristic feature of colorectal carcinoma (CRC). The tumor inhibitory effect of S-adenosylmethionine (SAM) methyl donor has been described in certain cancers including CRC. However, the molecular impact of SAM treatment on CRC cell lines with distinct genetic features has not been evaluated comprehensively. HT-29 and SW480 cells were treated with 0.5 and 1 mmol/L SAM for 48 h followed by cell proliferation measurements, whole-genome transcriptome and methylome analyses, DNA stability assessments and exome sequencing. SAM reduced cell number and increased senescence by causing S phase arrest, besides, multiple EMT-related genes (e.g., TGFB1) were downregulated in both cell lines. Alteration in the global DNA methylation level was not observed, but certain methylation changes in gene promoters were detected. SAM-induced γ-H2AX elevation could be associated with activated DNA repair pathway showing upregulated gene expression (e.g., HUS1). Remarkable genomic stability elevation, namely, decreased micronucleus number and comet tail length was observed only in SW480 after treatment. SAM has the potential to induce senescence, DNA repair, genome stability and to reduce CRC progression. However, the different therapeutic responses of HT-29 and SW480 to SAM emphasize the importance of the molecular characterization of CRC cases prior to methyl donor supplementation.

Promoter Hypomethylation and Increased Expression of the Long Non-coding RNA LINC00152 Support Colorectal Carcinogenesis.

Up-regulation of the long non-coding RNA LINC00152 can contribute to cancer development, proliferation and invasion, including colorectal cancer, however, its mechanism of action in colorectal carcinogenesis and progression is only insufficiently understood. In this work we correlated LINC00152 expression with promoter DNA methylation changes in colorectal tissues along the normal-adenoma-carcinoma sequence and studied the effects of LINC00152 silencing on the cell cycle regulation and on the whole transcriptome in colon carcinoma cells using cell and molecular biology techniques. LINC00152 was significantly up-regulated in adenoma and colorectal cancer (p < 0.001) compared to normal samples, which was confirmed by real-time PCR and in situ hybridization. LINC00152 promoter hypomethylation detected in colorectal cancer (p < 0.01) was strongly correlated with increased LINC00152 expression (r=-0.90). Silencing of LINC00152 significantly suppressed cell growth, induced apoptosis and decreased cyclin D1 expression (p < 0.05). Whole transcriptome analysis of LINC00152-silenced cells revealed significant down-regulation of oncogenic and metastasis promoting genes (e.g. YES proto-oncogene 1, PORCN porcupine O-acyltransferase), and up-regulation of tumour suppressor genes (e.g. DKK1 dickkopf WNT signalling pathway inhibitor 1, PERP p53 apoptosis effector) (adjusted p < 0.05). Pathway analysis confirmed the LINC00152-related activation of oncogenic molecular pathways including those driven by PI3K/Akt, Ras, WNT, TP53, Notch and ErbB. Our results suggest that promoter hypomethylation related overexpression of LINC00152 can contribute to the pathogenesis of colorectal cancer by facilitating cell progression through the up-regulation of several oncogenic and metastasis promoting pathway elements.

[Potential role of estrogens in colorectal tumour development]. / Az ösztrogének lehetséges szerepe a vastagbéldaganatok kialakulásában.

Colorectal cancer (CRC) is one of the most common types of cancers worldwide. The incidence of sporadic CRC is lower in individuals below 50 years and increases with age, furthermore, it shows typical clinical, macroscopic and molecular differences between females and males. According to the results of epidemiological and molecular biology studies, the estradiol-regulating signaling pathway plays an important role in the development and prognosis of CRC, predominantly through estrogen receptor beta (ERß), which is dominant in the colonic epithelium. Estradiol has multiple gastrointestinal effects, which were confirmed by in vitro and in vivo studies on histologically intact and cancerous cells as well. In contrast to estrogen receptor alpha (ERα), the activation of ERß inhibits cell proliferation and enhances apoptosis, nevertheless, the expression of estrogen receptor beta can change both during physiological ageing and in colorectal disorders. The ERß-mediated antitumour effects of estradiol may be exerted through inhibition of cell proliferation, stimulation of apoptosis, inhibition of metastasis formation and its anti-inflammatory activity. Based on the results of cell culture and animal studies, selective modulators of estrogen receptor beta (selective estrogen receptor modulator [SERM]) and phytoestrogens can be new, additional therapeutic options in the treatment of colorectal diseases characterized by chronic inflammation and uncontrolled cell proliferation. Orv Hetil. 2020; 161(14): 532-543.

Microscopic Colitis: A Challenging Disorder.

BACKGROUND: The clinical importance of microscopic colitis (MC) is increasing. This is explained by both the increasing incidence and the challenges posed by the disease. However, recent MC data also reveal a number of doubts and uncertainties. SUMMARY: This review focuses on current knowledge of MC and highlights the various controversies and criticisms regarding the clinical data about definitions, subtypes, pathogenesis, diagnosis, and treatment of this condition. Key Messages: The diagnosis of MC is based on histology, which distinguishes 2 subtypes. However, transitional forms often cause misclassification, which calls into question the reality (specificity, meaning) of the distinction between the 2 forms. The location of the colon biopsy is not defined by international consensus. There is no credible, clear explanation for the incidence increase. The pathogenesis is unknown, probably multifactorial, but the importance of the immunological background is increasing. The natural history of the disease and the underlying cause of relapses are unclear. It is suggested that MC would be the prelude of IBD. Further data collection is needed to clarify these issues.

Genome-wide expression profiling in colorectal cancer focusing on lncRNAs in the adenoma-carcinoma transition.

BACKGROUND: Long non-coding RNAs (lncRNAs) play a fundamental role in colorectal cancer (CRC) development, however, lncRNA expression profiles in CRC and its precancerous stages remain to be explored. We aimed to study whole genomic lncRNA expression patterns in colorectal adenoma-carcinoma transition and to analyze the underlying functional interactions of aberrantly expressed lncRNAs. METHODS: LncRNA expression levels of colonic biopsy samples (20 CRCs, 20 adenomas (Ad), 20 healthy controls (N)) were analyzed with Human Transcriptome Array (HTA) 2.0. Expression of a subset of candidates was verified by qRT-PCR and in situ hybridization (ISH) analyses. Furthermore, in silico validation was performed on an independent HTA 2.0, on HGU133Plus 2.0 array data and on the TCGA COAD dataset. MiRNA targets of lncRNAs were predicted with miRCODE and lncBase v2 algorithms and miRNA expression was analyzed on miRNA3.0 Array data. MiRNA-mRNA target prediction was performed using miRWALK and c-Met protein levels were analyzed by immunohistochemistry. Comprehensive lncRNA-mRNA-miRNA co-expression pattern analysis was also performed. RESULTS: Based on our HTA results, a subset of literature-based CRC-associated lncRNAs showed remarkable expression changes already in precancerous colonic lesions. In both Ad vs. normal and CRC vs. normal comparisons 16 lncRNAs, including downregulated LINC02023, MEG8, AC092834.1, and upregulated CCAT1, CASC19 were identified showing differential expression during early carcinogenesis that persisted until CRC formation (FDR-adjusted p < 0.05). The intersection of CRC vs. N and CRC vs. Ad comparisons defines lncRNAs characteristic of malignancy in colonic tumors, where significant downregulation of LINC01752 and overexpression of UCA1 and PCAT1 were found. Two candidates with the greatest increase in expression in the adenoma-carcinoma transition were further confirmed by qRT-PCR (UCA1, CCAT1) and by ISH (UCA1). In line with aberrant expression of certain lncRNAs in tumors, the expression of miRNA and mRNA targets showed systematic alterations. For example, UCA1 upregulation in CRC samples occurred in parallel with hsa-miR-1 downregulation, accompanied by c-Met target mRNA overexpression (p < 0.05). CONCLUSION: The defined lncRNA sets may have a regulatory role in the colorectal adenoma-carcinoma transition. A subset of CRC-associated lncRNAs showed significantly differential expression in precancerous samples, raising the possibility of developing adenoma-specific markers for early detection of colonic lesions.

Diagnostic and prognostic potential of tissue and circulating long non-coding RNAs in colorectal tumors.

Long non-coding RNAs (lncRNAs) are members of the non-protein coding RNA family longer than 200 nucleotides. They participate in the regulation of gene and protein expression influencing apoptosis, cell proliferation and immune responses, thereby playing a critical role in the development and progression of various cancers, including colorectal cancer (CRC). As CRC is one of the most frequently diagnosed malignancies worldwide with high mortality, its screening and early detection are crucial, so the identification of disease-specific biomarkers is necessary. LncRNAs are promising candidates as they are involved in carcinogenesis, and certain lncRNAs (e.g., CCAT1, CRNDE, CRCAL1-4) show altered expression in adenomas, making them potential early diagnostic markers. In addition to being useful as tissue-specific markers, analysis of circulating lncRNAs (e.g., CCAT1, CCAT2, BLACAT1, CRNDE, NEAT1, UCA1) in peripheral blood offers the possibility to establish minimally invasive, liquid biopsy-based diagnostic tests. This review article aims to describe the origin, structure, and functions of lncRNAs and to discuss their contribution to CRC development. Moreover, our purpose is to summarise lncRNAs showing altered expression levels during tumor formation in both colon tissue and plasma/serum samples and to demonstrate their clinical implications as diagnostic or prognostic biomarkers for CRC.

[Physiological and pathophysiological significance of vitamin B9. Summary on the occasion of the 30-year introduction of folic acid as a dietary supplement]. / A B9-vitamin élettani és kórélettani jelentosége. Összegzés a folsav táplálékkiegészítoként történo alkalmazásának 30. évfordulójára.

Vitamin B9, also known as folate, can be found in natural and synthetic forms, mostly in vegetables or folic acid containing food supplements. By participating in the proper cell development and division, its presence is indispensable for certain basic metabolic processes. The decreased folate level of the body, mainly caused by environmental and hereditary factors as well as aging, can lead to genetic, epigenetic and metabolic changes. It can be related to the development of megaloblastic anemia, various cardiovascular diseases (such as atherosclerosis, stroke) obstetrical complications (such as abruption of the placentae, spontaneous abortion, preterm delivery, neural tube defect), neuropsychiatric diseases (such as Alzheimer's disease, Parkinson's disease, depression) and tumors. The vitamin has a preventive effect in all the above-mentioned diseases, however, in the case of tumor existence, its therapeutic use requires great care, as it may promote the progression of certain precancerous lesions. Food fortification with folic acid is currently being carried out in more than 60 countries in order to ensure a minimum vitamin B9 requirement for the population and therefore to prevent the development of the diseases that are connected to folic acid deficiency. Due to its assumable role in carcinogenesis, an initial concern had taken place when fortification was implemented (1998), however, the present statistical data do not confirm such adverse health effects. On the other hand, several beneficial properties can be connected to the vitamin, that can be the reason why more and more countries are considering to join this program. Besides the fact that folic acid is a widely used food supplement, it is also applied in oncological medicine (leucovorin) to increase the effectiveness of certain chemotherapeutical drugs (e.g. methotrexate, 5-fluorouracil). Orv Hetil. 2019; 160(28): 1087-1096.

[Characteristics and diagnostic applications of circulating cell-free DNA in colorectal cancer]. / A plazmában keringo szabad DNS jellemzoi és diagnosztikai alkalmazási lehetoségei vastagbélrák esetén.

The incidence and mortality of colorectal cancer (CRC) are considerably high in Central European countries, it is the second most common cancer in both men and women in Hungary with 10,000 newly registered patients per year. These data indicate the necessity of new screening methods that are more comfortable for patients, hence the compliance can be increased. Cell-free DNA (cfDNA) level in blood is elevated in certain physiological conditions, such as pregnancy or high physical activity. Furthermore, cfDNA concentration alterations can also be detected in some pathological processes; increased cfDNA amount was observed in autoimmune and inflammatory diseases, as well as in various cancers including CRC. Numerous studies about origin, function, and mechanism of cfDNA can be found in the scientific literature. In this review, we aimed to describe the quantitative and qualitative changes of cfDNA, to present its functions, and to provide an overview of the available diagnostic applications for CRC. CfDNA can be released to the circulatory system via apoptosis, necrosis or by direct secretions by living cells. In cancer patients, cfDNA can originate from healthy and cancer cells, hence genetic (e.g. mutations in APC, KRAS, BRAF) and epigenetic (e.g. methylation in SEPT9, SFRP1) alterations of tumor cells can be examined in cfDNA fraction. Several high-throughput, sensitive and even automated methods are available providing opportunity to perform standardized sample preparation and to analyse biomarker candidates quantitatively. These enhancements can help to develop alternative screening methods that can be easily integrated into the clinical practice and can contribute to early cancer detection. Orv Hetil. 2019; 160(30): 1167-1177.

Circulating cell-free nucleic acids as biomarkers in colorectal cancer screening and diagnosis - an update.

Introduction: Screening methods for one of the most frequently diagnosed malignancy, colorectal cancer (CRC), have limitations. Circulating cell-free nucleic acids (cfNA) hold clinical relevance as screening, prognostic and therapy monitoring markers. Area covered: In this review, we summarize potential CRC-specific cfNA biomarkers, the recently developed sample preparation techniques, their applications, and pitfalls. Expert opinion: Automated extraction of cfDNA is highly reproducible, however, cfDNA yield is less compared to manual isolation. Quantitative and highly sensitive detection techniques (e.g. digital PCR, NGS) can be applied to analyze genetic and epigenetic changes. Detection of DNA mutations or methylation in cfDNA and related altered levels of mRNA, miRNA, and lncRNA may improve early cancer recognition, based on specific, CRC-related patterns. Detection of cfDNA mutations (e.g. TP53, KRAS, APC) has limited diagnostic sensitivity (40-60%), however, methylated DNA including SEPT9, SFRP1, SDC2 can be applied with higher sensitivity (up to 90%) for CRC. Circulating miRNAs (e.g. miR-21, miR-92, miR-141) provide comparably high sensitivity for CRC as the circulating tumor cell mRNA markers (e.g. EGFR, CK19, CK20, CEA). Automation of cfNA isolation coupled with quantitative analysis of CRC-related, highly sensitive biomarkers may enhance CRC screening and early detection in the future.

The Diagnosis and Therapy of Helicobacter pylori Infection in Hungary: Comparison of Strategies Applied by Family Physicians and Internists.

BACKGROUND AIMS: Most patients with Helicobacter pylori infection are consulted for the first time by family physicians. We aimed to survey the adherence to the newest guidelines of the management of H. pylori infection in the primary and secondary care settings in Hungary. METHODS: From a total of 793 physicians, 94 trainees in family medicine, 334 family physicians without and 195 with board certification in internal medicine, 87 internists, 78 family paediatricians were invited to take part in the study. Diagnostic and therapeutic attitudes towards H. pylori infection were compared by a voluntary and anonymous questionnaire. RESULTS: Participants test for H. pylori infection in 92.8% of cases with a family history of peptic ulcer or 76.9% of gastric cancer, 68.9% of dyspepsia and 49.9% of non-specific abdominal complaints, before initiation of non-steroidal anti-inflammatory drug (NSAID; 17.3%) and antiplatelet treatment (14.5%), respectively. They confirm the success of eradication therapy in 88.1% mainly by urea breath test. Most of them initiate eradication therapy by themselves and only 22.4% refer their patients to a gastroenterologist. Clarithromycin-based standard triple therapy is the most preferred (62.1%) and only 3.7% choose quadruple combination with bismuth as first-line and 48.1% as second-line therapy. We found significant differences between groups with respect to the physicians' own infection, localization of practice, and sources of information on H. pylori infection. Internists are more likely to clarify H. pylori status before the initiation of NSAID and antiplatelet therapies, initiate second-line therapies and use bismuth compared to the other groups. Family physicians with board certification in internal medicine are also prone to start eradication therapy and less prone to refer patients to a gastroenterologist. Family paediatricians prefer stool antigen determination, screen family members and prefer gastroenterologist consultation more often, and use bismuth less frequently than the other groups. Family physicians with previous infection check for H. pyloriinfection more frequently before the initiation of NSAID treatment and are more likely to use histology to detect H. pylori. Postgraduate trainings were the most popular source of information. CONCLUSION: The adherence to the recent recommendations of current guidelines is moderate. There is a need to increase adherence to current recommendations by family physicians and internists.

En bloc release of MVB-like small extracellular vesicle clusters by colorectal carcinoma cells.

Small extracellular vesicles (EVs) are membrane enclosed structures that are usually released from cells upon exocytosis of multivesicular bodies (MVBs) as a collection of separate, free EVs. In this study, we analysed paraffin embedded sections of archived human colorectal cancer samples. We studied 3D reconstructions of confocal microscopic images complemented by HyVolution and STED imaging. Unexpectedly, we found evidence that large, MVB-like aggregates of ALIX/CD63 positive EV clusters were released en bloc by migrating tumour cells. These structures were often captured with partial or complete extra-cytoplasmic localization at the interface of the plasma membrane of the tumour cell and the stroma. Their diameter ranged between 0.62 and 1.94 µm (mean±S.D.: 1.17 ± 0.34 µm). High-resolution 3D reconstruction showed that these extracellular MVB-like EV clusters were composed of distinguishable internal particles of small EV size (mean±S.D.: 128.96 ± 16.73 nm). In vitro, HT29 colorectal cancer cells also showed the release of similar structures as confirmed by immunohistochemistry and immune electron microscopy. Our results provide evidence for an en bloc transmission of MVB-like EV clusters through the plasma membrane. Immunofluorescent-based detection of the MVB like small EV clusters in archived pathological samples may represent a novel and unique opportunity which enables analysis of EV release in situ in human tissues.

Modified Genomic Self-DNA Influences In Vitro Survival of HT29 Tumor Cells via TLR9- and Autophagy Signaling.

In relation of immunobiology, the consequence of the crosstalk between TLR9-signaling and autophagy is poorly documented in HT29 cancer cells. To assess the TLR9-mediated biologic effects of modified self-DNA sequences on cell kinetics and autophagy response HT29 cells were incubated separately with intact genomic (g), hypermethylated (m), fragmented (f), and hypermethylated/fragmented (m/f) self-DNAs. Cell viability, apoptosis, cell proliferation, colonosphere-formation were determined. Moreover, the relation of TLR9-signaling to autophagy response was assayed by real-time RT-PCR, immunocytochemistry and transmission electron microscopy (TEM). After incubation with g-, m-, and m/f-DNAs cell viability and proliferation decreased, while apoptosis increased. F-DNA treatment resulted in an increase of cell survival. Methylation of self-DNA resulted in decrease of TLR9 expression, while it did not influence the positive effect of DNA fragmentation on MyD88 and TRAF6 overexpression, and TNFα downregulation. Fragmentation of DNA abrogated the positive effect of methylation on IRAK2, NFκB and IL-8 mRNA upregulations. In case of the autophagy genes and proteins, g- and f-DNAs caused significant upregulation of Beclin1, Atg16L1, and LC3B. According to TEM analyses, autophagy was present in each group of tumor cells, but to a varying degree. Incubation with m-DNA suppressed tumor cell survival by inducing features of apoptotic cell death, and activated mitophagy. F-DNA treatment enhanced cell survival, and activated macroautophagy and lipophagy. Colonospheres were only present after m-DNA incubation. Our data provided evidence for a close existing interplay between TLR9-signaling and the autophagy response with remarkable influences on cell survival in HT29 cells subjected to modified self-DNA treatments.

Blood Collection and Cell-Free DNA Isolation Methods Influence the Sensitivity of Liquid Biopsy Analysis for Colorectal Cancer Detection.

During colorectal cancer (CRC) development tumor-derived cell-free DNA (cfDNA) can be released into the bloodstream. Many different cfDNA isolation methods and specific blood collection tubes preventing the release of genomic DNA and stabilizing cfDNA with preservative reagents became available. These factors may affect greatly on the further liquid biopsy analyses. Our aim was to test different blood collection tubes and cfDNA isolation methods to determine whether these factors influence the cfDNA amount and the promoter methylation of four previously described hypermethylated biomarkers. Three manual isolation methods (High Pure Viral Nucleic Acid Large Volume Kit; Epi proColon 2.0 Kit; Quick-cfDNA™ Serum & Plasma Kit) and automated sample preparation systems (InviGenius and InviGenius PLUS) were examined. Furthermore, K3EDTA Vacuette tubes and Streck Cell-Free DNA BCT® tubes were compared. After cfDNA isolation and bisulfite conversion of samples, the methylation level of SFRP1, SFRP2, SDC2, and PRIMA1 were defined with MethyLight assays. We have ascertained that there are differences between the cfDNA amounts depending on the isolation methods. Higher cfDNA yield was observed using InviGenius system than column-based manual isolation method; however, InviGenius PLUS has produced lower cfDNA amounts. No remarkable variance could be found between K3EDTA and Streck tubes; slightly higher cfDNA quantity was detected in 60% of plasma samples using Streck tubes. In point of methylation level and frequency, manual column-based isolation produced more consistent results. Automated cfDNA extraction systems are easy-to-use and high-throughput; however, further improvements in the isolation protocols might lead to the increase of the sensitivity of further methylation analysis.

Comparison of Circulating miRNAs Expression Alterations in Matched Tissue and Plasma Samples During Colorectal Cancer Progression.

MicroRNAs (miRNAs) have been found to play a critical role in colorectal adenoma-carcinoma sequence. MiRNA-specific high-throughput arrays became available to detect promising miRNA expression alterations even in biological fluids, such as plasma samples, where miRNAs are stable. The purpose of this study was to identify circulating miRNAs showing altered expression between normal colonic (N), tubular adenoma (ADT), tubulovillous adenoma (ADTV) and colorectal cancer (CRC) matched plasma and tissue samples. Sixteen peripheral plasma and matched tissue biopsy samples (N n = 4; ADT n = 4; ADTV n = 4; CRC n = 4) were selected, and total RNA including miRNA fraction was isolated. MiRNAs from plasma samples were extracted using QIAamp Circulating Nucleic Acid Kit (Qiagen). Matched tissue-plasma miRNA microarray experiments were conducted by GeneChip® miRNA 3.0 Array (Affymetrix). RT-qPCR (microRNA Ready-to-use PCR Human Panel I + II; Exiqon) was used for validation. Characteristic miRNA expression alterations were observed in comparison of AD and CRC groups (miR-149*, miR-3196, miR-4687) in plasma samples. In the N vs. CRC comparison, significant overexpression of miR-612, miR-1296, miR-933, miR-937 and miR-1207 was detected by RT-PCR (p < 0.05). Similar expression pattern of these miRNAs were observed using microarray in tissue pairs, as well. Although miRNAs were also found in circulatory system in a lower concentration compared to tissues, expression patterns slightly overlapped between tissue and plasma samples. Detected circulating miRNA alterations may originate not only from the primer tumor but from other cell types including immune cells.

[The role of gut microbiota in chronic liver diseases, and treatment possibilities]. / A bélflóra patofiziológai jelentosége és szerepe mint terápiás célpont májbetegségekben.

The community of microorganisms in the intestine, namely gut microbiome lives in symbiosis with the host, contributing to its homeostasis and influencing it simultaneously. It can be suspected that gut microbiome plays a central role in the pathophysiology of intestinal and extraintestinal diseases: determining their development, progress and complications. Recently, intestinal microbiome has become a highlighted field of interest and important topic in research, especially in hepatology. It is in the focus of relevant research as the liver is the organ which meets nutrients, bacterial components, toxins and metabolites at first, as a filter. The evolvement of different liver diseases - just like alcoholic and non-alcoholic fatty liver disease, steatohepatitis, cirrhosis or hepatocellular carcinoma - correlates with the changed composition and activity of gut microbiome. Thus, it can be hypothesized that pre-, pro- and antibiotics could have an impact on the treatment of these diseases. In our review article, the relationship between intestinal flora and liver diseases with different etiologies as well as therapeutic possibilities are discussed. Orv Hetil. 2018; 159(36): 1465-1474.

Perspective: bidirectional exosomal transport between cancer stem cells and their fibroblast-rich microenvironment during metastasis formation.

Carcinomas are complex structures composed of hierarchically organized distinct cell populations such as cancer stem cells and non-stem (bulk) cancer cells. Their genetic/epigenetic makeup and the dynamic interplay between the malignant cell populations and their stromal fibroblasts are important determinants of metastatic tumor invasion. Important mediators of these interactions are the small, membrane-enclosed extracellular vesicles, in particular exosomes. Both cancer cell and fibroblast-derived exosomes carry a set of regulatory molecules, including proteins and different species of RNA, which cooperatively support metastatic tumor spread. Here, we briefly overview potential links between cancer stem cells and the exosome-mediated fibroblast-enriched metastatic niche formation to discuss their role in the promotion of tumor growth and metastatic expansion in breast carcinoma models.