[Show abstract][Hide abstract] ABSTRACT: Microarray analysis of promoter hypermethylation provides insight into the role and extent of DNA methylation in the development of colorectal cancer (CRC) and may be co-monitored with the appearance of driver mutations. Colonic biopsy samples were obtained endoscopically from 10 normal, 23 adenoma (17 low-grade (LGD) and 6 high-grade dysplasia (HGD)), and 8 ulcerative colitis (UC) patients (4 active and 4 inactive). CRC samples were obtained from 24 patients (17 primary, 7 metastatic (MCRC)), 7 of them with synchronous LGD. Field effects were analyzed in tissues 1 cm (n = 5) and 10 cm (n = 5) from the margin of CRC. Tissue materials were studied for DNA methylation status using a 96 gene panel and for KRAS and BRAF mutations. Expression levels were assayed using whole genomic mRNA arrays. SFRP1 was further examined by immunohistochemistry. HT29 cells were treated with 5-aza-2' deoxycytidine to analyze the reversal possibility of DNA methylation. More than 85% of tumor samples showed hypermethylation in 10 genes (SFRP1, SST, BNC1, MAL, SLIT2, SFRP2, SLIT3, ALDH1A3, TMEFF2, WIF1), whereas the frequency of examined mutations were below 25%. These genes distinguished precancerous and cancerous lesions from inflamed and healthy tissue. The mRNA alterations that might be caused by systematic methylation could be partly reversed by demethylation treatment. Systematic changes in methylation patterns were observed early in CRC carcinogenesis, occuring in precursor lesions and CRC. Thus we conclude that DNA hypermethylation is an early and systematic event in colorectal carcinogenesis, and it could be potentially reversed by systematic demethylation therapy, but it would need more in vitro and in vivo experiments to support this theory.
PLoS ONE 08/2015; 10(8):e0133836. DOI:10.1371/journal.pone.0133836 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To characterize the regeneration-associated stem cell-related phenotype of hepatocyte-derived growth factor receptor (HGFR)-expressing cells in active ulcerative colitis (UC).
On the whole 38 peripheral blood samples and 38 colonic biopsy samples from 18 patients with histologically proven active UC and 20 healthy control subjects were collected. After preparing tissue microarrays and blood smears HGFR, caudal type homeobox 2 (CDX2), prominin-1 (CD133) and Musashi-1 conventional and double fluorescent immunolabelings were performed. Immunostained samples were digitalized using high-resolution Mirax Desk instrument, and analyzed with the Mirax TMA Module software. For semiquantitative counting of immunopositive lamina propria (LP) cells 5 fields of view were counted at magnification × 200 in each sample core, then mean ± SD were determined. In case of peripheral blood smears, 30 fields of view with 100 μm diameter were evaluated in every sample and the number of immunopositive cells (mean ± SD) was determined. Using 337 nm UVA Laser MicroDissection system at least 5000 subepithelial cells from the lamina propria were collected. Gene expression analysis of HGFR, CDX2, CD133, leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5), Musashi-1 and cytokeratin 20 (CK20) were performed in both laser-microdisscted samples and blood samples by using real time reverse transcription polymerase chain reaction (RT-PCR).
By performing conventional and double fluorescent immunolabelings confirmed by RT-PCR, higher number of HGFR (blood: 6.7 ± 1.22 vs 38.5 ± 3.18; LP: 2.25 ± 0.85 vs 9.22 ± 0.65; P < 0.05), CDX2 (blood: 0 vs 0.94 ± 0.64; LP: 0.75 ± 0.55 vs 2.11 ± 0.75; P < 0.05), CD133 (blood: 1.1 ± 0.72 vs 8.3 ± 1.08; LP: 11.1 ± 0.85 vs 26.28 ± 1.71; P < 0.05) and Musashi-1 (blood and LP: 0 vs scattered) positive cells were detected in blood and lamina propria of UC samples as compared to controls. HGFR/CDX2 (blood: 0 vs 1 ± 0.59; LP: 0.8 ± 0.69 vs 2.06 ± 0.72, P < 0.05) and Musashi-1/CDX2 (blood and LP: 0 vs scattered) co-expressions were found in blood and lamina propria of UC samples. HGFR/CD133 and CD133/CDX2 co-expressions appeared only in UC lamina propria samples. CDX2, Lgr5 and Musashi-1 expressions in UC blood samples were not accompanied by CK20 mRNA expression.
In active UC, a portion of circulating HGFR-expressing cells are committed to the epithelial lineage, and may participate in mucosal regeneration by undergoing mesenchymal-to-epithelial transition.
[Show abstract][Hide abstract] ABSTRACT: Somatostatin (SST) has anti-proliferative and pro-apoptotic effects. Our aims were to analyze and compare the SST expression during normal aging and colorectal carcinogenesis at mRNA and protein levels. Furthermore, we tested the methylation status of SST in biopsy samples, and the cell growth inhibitory effect of the SST analogue octreotide in human colorectal adenocarcinoma cell line.
Colonic samples were collected from healthy children (n1 = 6), healthy adults (n2 = 41) and colorectal cancer patients (CRCs) (n3 = 34) for SST mRNA expression analysis, using HGU133 Plus2.0 microarrays. Results were validated both on original (n1 = 6; n2 = 6; n3 = 6) and independent samples ((n1 = 6; n2 = 6; n3 = 6) by real-time PCR. SST expressing cells were detected by immunohistochemistry on colonic biopsy samples (n1 = 14; n2 = 20; n3 = 23). The effect of octreotide on cell growth was tested on Caco-2 cell line. SST methylation percentage in biopsy samples (n1 = 5; n2 = 5; n3 = 9) was defined using methylation-sensitive restriction enzyme digestion.
In case of normal aging SST mRNA expression did not alter, but decreased in cancer (p<0.05). The ratio of SST immunoreactive cells was significantly higher in children (0.70%±0.79%) compared to CRC (0%±0%) (p<0.05). Octreotide significantly increased the proportion of apoptotic Caco-2 cells. SST showed significantly higher methylation level in tumor samples (30.2%±11.6%) compared to healthy young individuals (3.5%±1.9%) (p<0.05).
In cancerous colonic mucosa the reduced SST production may contribute to the uncontrolled cell proliferation. Our observation that in colon cancer cells octreotide significantly enhanced cell death and attenuated cell proliferation suggests that SST may act as a regulator of epithelial cell kinetics. The inhibition of SST expression in CRC can be epigenetically regulated by promoter hypermethylation.
PLoS ONE 02/2015; 10(2):e0118332. DOI:10.1371/journal.pone.0118332 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Epithelial layer of the intestine relies upon stem cells for maintaining homeostasis and regeneration. Two types of stem cells are currently defined in intestinal crypts: the cycling crypt base columnar cells and quiescent cells. Though several candidate markers and regulators of rapidly cycling and quiescent stem cells have been identified so far, the exact nature of quiescent cells is still questionable since investigations mainly focused on candidate markers rather than the label-retaining population itself. Recent results, however, have strengthened the argument for functional plasticity. Using a lineage tracing strategy label-retaining cells (LRCs) of the intestinal epithelium were marked, then followed by a pulse-chase analysis it was found that during homeostasis, LRCs were Lgr5-positive and were destined to become Paneth and neuroendocrine cells. Nevertheless, it was demonstrated that LRCs are capable of clonogenic growth by recall to the self-renewing pool of stem cells in case of epithelial injury. These new findings highlight on the hierarchical and spatial organization of intestinal epithelial homeostasis and the important plasticity of progenitors during tissue regeneration, moreover, provide a motivation for studying their role in disorders like colorectal cancer.
World Journal of Gastroenterology 02/2015; 21(7):2005-2010. DOI:10.3748/wjg.v21.i7.2005 · 2.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cancer represents a major health problem worldwide, therefore on the basis of current research results constantly more effective therapeutic strategies are expected. Chronic, unchecked inflammation has widely been suggested to trigger carcinogenesis. The innate immune system ensures a first line host defense in which the inflammasome is essential maintaining a delicate balance betweeen pro- and anti-inflammatory signals in order to generate an appropriate immune response without harming the host. Studies have revealed a remarkable, but contradictory link of host inflammatory responses to tumorigenesis. Indeed, activation of the multiprotein complex inflammasome by danger signals seems to play diverse and sometimes conflicting, suppressive or stimulatory role in cancer development and progression with a significant context-dependency. The pleitropic inflammasomes may act at cell-autonomous level to eliminate malignant cells via the programmed cell death type of inflammatory pyroptosis, but on the contrary, may favor the production of gowth and trophic factors for tumor cells and their microenvironment. Further, upon caspase-1 activation the inflammasome can provoke sterile inflammation, and thus facilitate carcinogenesis, though in antigen-presenting cells it can elicit anti-tumor immune responses. Clarifying the exact, context-specific impact of inflammasomes on tumorigenesis represents a new research area with the potential to introduce promising novel targets for cancer therapeutics.
Current Drug Targets 12/2014; 16(3). DOI:10.2174/1389450115666141229154157 · 3.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Determination of methylated Septin 9 (mSEPT9) in plasma has been shown to be a sensitive and specific biomarker for colorectal cancer (CRC). However, the relationship between methylated DNA in plasma and colon tissue of the same subjects has not been reported.
Plasma and matching biopsy samples were collected from 24 patients with no evidence of disease (NED), 26 patients with adenoma and 34 patients with CRC. Following bisulfite conversion of DNA a commercial RT-PCR assay was used to determine the total amount of DNA in each sample and the fraction of mSEPT9 DNA. The Septin-9 protein was assessed using immunohistochemistry.
The percent of methylated reference (PMR) values for SEPT9 above a PMR threshold of 1% were detected in 4.2% (1/24) of NED, 100% (26/26) of adenoma and 97.1% (33/34) of CRC tissues. PMR differences between NED vs. adenoma and NED vs. CRC comparisons were significant (p<0.001). In matching plasma samples using a PMR cut-off level of 0.01%, SEPT9 methylation was 8.3% (2/24) of NED, 30.8% (8/26) of adenoma and 88.2% (30/34) of CRC. Significant PMR differences were observed between NED vs. CRC (p<0.01) and adenoma vs. CRC (p<0.01). Significant differences (p<0.01) were found in the amount of cfDNA (circulating cell-free DNA) between NED and CRC, and a modest correlation was observed between mSEPT9 concentration and cfDNA of cancer (R2 = 0.48). The level of Septin-9 protein in tissues was inversely correlated to mSEPT9 levels with abundant expression in normals, and diminished expression in adenomas and tumors.
Methylated SEPT9 was detected in all tissue samples. In plasma samples, elevated mSEPT9 values were detected in CRC, but not in adenomas. Tissue levels of mSEPT9 alone are not sufficient to predict mSEPT9 levels in plasma. Additional parameters including the amount of cfDNA in plasma appear to also play a role.
PLoS ONE 12/2014; 9(12):e115415. DOI:10.1371/journal.pone.0115415 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Epigenetic changes of stromal-epithelial interactions are of key importance in the regulation of colorectal carcinoma (CRC) cells and morphologically normal, but genetically and epigenetically altered epithelium in normal adjacent tumor (NAT) areas. Here we demonstrated retained protein expression of well-known Wnt inhibitor, secreted frizzled-related protein 1 (SFRP1) in stromal myofibroblasts and decreasing epithelial expression from NAT tissues towards the tumor. SFRP1 was unmethylated in laser microdissected myofibroblasts and partially hypermethylated in epithelial cells in these areas. In contrast, we found epigenetically silenced myofibroblast-derived SFRP1 in CRC stroma. Our results suggest that the myofibroblast-derived SFRP1 protein might be a paracrine inhibitor of epithelial proliferation in NAT areas and loss of this signal may support tumor proliferation in CRC.
PLoS ONE 11/2014; 9(11):e106143. DOI:10.1371/journal.pone.0106143 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the intestine a balance between proinflammatory and repair signals of the immune system is essential for the maintenance of intestinal homeostasis. The innate immunity ensures a primary host response to microbial invasion, which induces an inflammatory process to localize the infection and prevent systemic dissemination of pathogens. The key elements of this process are the germline encoded pattern recognition receptors including Toll-like receptors (TLRs). If pathogens cannot be eliminated, they may elicit chronic inflammation, which may be partly mediated via TLRs. Additionally, chronic inflammation has long been suggested to trigger tissue tumorous transformation. Inflammation, the seventh hallmark of cancer, may affect all phases of tumor development, and evade the immune system. Inflammation acts as a cellular stressor and may trigger DNA damage or genetic instability. Furthermore, chronic inflammation can provoke genetic mutations and epigenetic mechanisms that promote malignant cell transformation. Colorectal cancers in inflammatory bowel disease patients are considered typical examples of inflammation-related cancers. Although data regarding the role of TLRs in the pathomechanism of cancer-associated colitis are rather conflicting, functionally these molecules can be classified as "largely antitumorigenic" and "largely pro-tumorigenic" with the caveat that the underlying signaling pathways are mainly context (i.e., organ-, tissue-, cell-) and ligand-dependent.
World Journal of Gastroenterology 09/2014; 20(36):12713-21. DOI:10.3748/wjg.v20.i36.12713 · 2.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background:
Presence of cell-free-circulating DNA (fcDNA) sequences in sera of patients with inflammatory bowel diseases (IBD) is a well-established phenomenon. Potential roles of fcDNA in diagnosis, prognosis and therapy monitoring of chronic inflammatory colonic disorders have already been examined, albeit its actual biological function still remains unclear.
Aims and methods:
In the present experiment, we studied the immunobiological effects of isolated fcDNA of normal and inflammatory origin administered intravenously to mice prior to induction of dextran sulfate sodium (DSS)-colitis. In addition to evaluate the current disease and histological activity, changes of the gene expression profile in isolated lamina propria cells upon TLR9 ligation were assayed.
A single intravenous dose of fcDNA pretreatment with colitic fcDNA exhibited beneficial response concerning the clinical and histological severity of DSS-colitis as compared to effects of normal fcDNA. Pretreatment with colitic fcDNA substantially altered the expression of several TLR9-related and inflammatory cytokine genes in a clinically favorable manner.
During the process of acute colitis, the subsequent inflammatory environment presumably results in changes of fcDNA with the potential to facilitate the downregulation of inflammation and improvement of regeneration. Thus, preconditioning of mice with colitis-derived fcDNA via TLR9 signaling could exert a tissue-protective effect and influence beneficially the course of DSS-colitis. Elucidating mechanisms of immune response alterations by nucleic acids may provide further insight into the etiology of IBD and develop the basis of novel immunotherapies.
[Show abstract][Hide abstract] ABSTRACT: Carcinoma-associated fibroblast (CAF) as prominent cell type of the tumour microenvironment has complex interaction with both the cancer cells and other non-neoplastic surrounding cells. The CAF-derived regulators and extracellular matrix proteins can support cancer progression by providing a protective microenvironment for the cancer cells via reduction of chemotherapy sensitivity. On the other hand, these proteins may act as powerful prognostic markers as well as potential targets of anticancer therapy. In this review, we summarise the clinical importance of the major CAF-derived signals influencing tumour behaviour and determining the outcome of chemotherapy.
Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.
[Show abstract][Hide abstract] ABSTRACT: Early molecular detection of the colorectal dysplasia-carcinoma transition may augment the accuracy of diagnosis in case of biopsy orientation errors. The combination of high-throughput microarray-based biomarker screening with tissue microarray-based prospective protein biomarker expression analysis could represent an additional test in routine automated diagnostic procedures. Our aim was to test and select protein markers to identify protein expression profile alterations, focusing on the dysplasia-carcinoma transition in sporadic colorectal tumors. Dysplasia-carcinoma transition-specific transcript sets were previously identified using HGU133plus2 microarrays and Taqman RT-PCR cards. Here, 26 potential dysplasia-carcinoma transition-specific markers were tested by immunohistochemistry at the protein level using tissue microarrays in a total of 168 independent colonic biopsy samples. A set of 26 transcripts [including matrix metalloproteinase-3 (MMP3) and chemokine (C-X-C motif) ligand 1 (CXCL1)] has been determined recently, indicating a linear expression correlation with the adenoma-dysplasia-carcinoma sequence, thereby having the potential to discriminate between dysplasia and early malignancy. Currently, we find that high-grade dysplastic sessile adenomatous-stage and early-stage colorectal cancer conditions can be differentiated correctly by the stromal expression of MMP3 and CXCL1, respectively, on tissue microarray-based analysis. Furthermore, in cases of sporadic colorectal tumors, MMP3 protein expression in the lamina propria itself seems to be highly specific for the detection of tumorous transition. Our current and recent results indicate that appropriate antibody marker combinations are highly suitable for tissue microarray-based and digital microscopy-based, automated, high-capacity diagnostic application in tumorous colonic diseases.
European journal of cancer prevention: the official journal of the European Cancer Prevention Organisation (ECP) 07/2014; 23(5). DOI:10.1097/CEJ.0000000000000058 · 3.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Semaphorins and plexins represent a highly conserved group of proteins that have recently been found to exert widespread regulatory functions outside the nervous system, including angiogenesis and immune regulation. Furthermore, these molecules are definitely implicated in the etiology of carcinogenesis and immune disorders. Their expression patterns and levels are deregulated in cancer cells and in cells of the tumor milieu. During the multistep development of cancer, its characteristic features include sustained tumor cell proliferation, resistance to cell death, limitless replicative capacity, activation of angiogenesis along with invasion and metastatic spread, cancer-related smoldering inflammation, and evasion of antitumor immune responses. The diversity of the semaphorin/plexin complexes and, thus, the multiple stimulated molecular interactions allow varied and diverse cell signaling events. The elicited transduction pathways might be involved in modifying the intricate mechanisms of tumorigenesis. Indeed, these pleiotropic signals may influence not only the intrinsic properties of cancer cells but they could also represent a possible link in mediating the cross-talk between tumor cells and the surrounding multiple stromal cells. In tumorigenesis, however, a dual role of different semaphorins is proposed, as some of them may elicit tumor regression, whereas others definitely promote cancer cell survival and progression. The current antitumoral or prosurvival responsiveness to semaphorins is mainly cell context dependent; nevertheless, their precise relation to cancer networks has not yet been fully elucidated. Here, we survey the many faces of a subset of the large semaphorin family, termed immune semaphorins, in carcinogenesis.
European journal of cancer prevention: the official journal of the European Cancer Prevention Organisation (ECP) 06/2014; 23(5). DOI:10.1097/CEJ.0000000000000059 · 3.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Accumulating evidence indicates that the aberrantly altered process of autophagy is definitely involved in carcinogenesis. Nonetheless, Toll-like receptors (TLRs) sensing cell-derived pattern/danger-associated molecules also have the capacity to promote tumor development and immune escape. TLRs are usually expressed in immunocompetent cells, though several types of cancer cells have also been reported to display these innate immune receptors. On the other hand, however, both TLR- and autophagy-related signals may exert tumor suppressor mechanisms mainly in a cell-specific and context-dependent manner. The role of autophagy has been radically expanded, and now this machinery is considered as a fundamental eukaryotic cellular homeostatic process and integral component of the immune system influencing infection, inflammation and immunity. Recent studies have documented that TLRs and autophagy are interrelated in response to danger signals, furthermore there is a controling cross-talk among them to avoid deficient or excessive immunological effects. Although the potential interaction of autophagy and TLRs in cancer cells has not yet been clarified, it seems to be a critical aspect of cancer development and progression. Upon translation of basic knowledge into practice it is reasonable to speculate that modulation of the TLR-autophagy regulatory loop might be relevant for cancer treatment by providing further possible therapeutic targets.
Current Drug Targets 05/2014; 15(8). DOI:10.2174/1389450115666140522120427 · 3.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In inflammatory bowel diseases the presence of free-circulating DNA (fcDNA) sequences in the sera is an established phenomenon, albeit its real biological function still remains unclear. In our study the immunobiologic effects of a single-dose, intravenously administered fcDNA of normal and colitic origin were assayed in DSS-colitic and control mice. In parallel with disease and histological activity evaluations changes of the TLR9 and inflammatory cytokine signaling gene expression profiles were assayed in isolated cells of the lamina propria. Intravenously administered colitis-derived fcDNA displayed a more prominent beneficial action regarding the clinical and histological severity of DSS-colitis than that of fcDNA of normal origin. Systemic administration of colitis-derived fcDNA significantly altered the expression of certain TLR9-related and proinflammatory cytokine genes in a clinically favorable manner. Presumably due to induction of severe colitis, the subsequent marked inflammatory environment may result changes in fcDNA with a potential to promote the downregulation of inflammation and improvement of tissue regeneration. Elucidating mechanisms of innate immune alterations by nucleic acids may provide further insight into the etiology of inflammatory bowel diseases, and develop the basis of novel nucleic acid-based immunotherapies.
[Show abstract][Hide abstract] ABSTRACT: Intratumoral heterogeneity including genetic and nongenetic mechanisms refers to biological differences amongst malignant cells originated within the same tumor. Both, cell differentiation hierarchy and stochasticity in gene expression and signaling pathways may result in phenotypic differences of cancer cells. Since a tumor consists of cancer cell clones that display distinct behaviours, changes in clonal proliferative behavior may also contribute to the phenotypic variability of tumor cells. There is a need to reveal molecular actions driving chemotherapeutic resistance in colon cancer cells. In general, it is widely hypothesized that therapeutic resistance in colorectal cancer is a consequence of the preferential survival of cancer stem cells. However, recent data regarding colorectal cancer suggest that resistance to anticancer therapy and post-therapeutic tumor reappearence could be related to variations of clonal dynamics. Understanding the interaction of genetic and nongenetic determinants influencing the functional diversity and therapy response of tumors should be a future direction for cancer research.
World Journal of Gastroenterology 03/2014; 20(10):2429-2432. DOI:10.3748/wjg.v20.i10.2429 · 2.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To understand the biologic role of self-DNA bound to Toll-like Receptor 9 (TLR9), we assayed its effect on gene and methyltransferase expressions and cell differentiation in HT29 cells. HT29 cells were incubated separately with type-1 (normally methylated/nonfragmented), type-2 (normally methylated/fragmented), type-3 (hypermethylated/nonfragmented), or type-4 (hypermethylated/fragmented) self-DNAs. Expression levels of TLR9-signaling and proinflammatory cytokine-related genes were assayed by qRT-PCR. Methyltransferase activity and cell differentiation were examined by using DNA methyltransferase (DNMT1, -3A, -3B) and cytokeratin (CK) antibodies. Treatment with type-1 DNA resulted in significant increase in TLR9 expression. Type-2 treatment resulted in the overexpression of TLR9-related signaling molecules (MYD88A, TRAF6) and the IL8 gene. In the case of type-3 treatment, significant overexpression of NFkB, IRAK2, and IL8 as well as downregulation of TRAF6 was detected. Using type-4 DNA, TRAF6 and MYD88A gene expression was upregulated, while MYD88B, IRAK2, IL8, and TNFSF10 were all underexpressed. CK expression was significantly higher only after type-1 DNA treatment. DNMT3A expression could also be induced by type-1 DNA treatment. DNA structure may play a significant role in activation of the TLR9-dependent and even independent proinflammatory pathways. There may be a molecular link between TLR9 signaling and DNMT3A. The mode of self-DNA treatment may influence HT29 cell differentiation.
The Scientific World Journal 12/2013; 2013:293296. DOI:10.1155/2013/293296 · 1.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Sporadic colorectal cancer (CRC) development is a sequential process showing age-dependency, uncontrolled epithelial proliferation and decreased apoptosis. During juvenile growth cellular proliferation and apoptosis are well balanced, which may be perturbed upon aging. Our aim was to correlate proliferative and apoptotic activities in aging human colonic epithelium and colorectal cancer. We also tested the underlying molecular biology concerning the proliferation- and apoptosis-regulating gene expression alterations.
Colorectal biopsies from healthy children (n1 = 14), healthy adults (n2 = 10), adult adenomas (n3 = 10) and CRCs (n4 = 10) in adults were tested for Ki-67 immunohistochemistry and TUNEL apoptosis assay. Mitosis- and apoptosis-related gene expression was also studied in healthy children (n1 = 6), adult (n2 = 41) samples and in CRC (n3 = 34) in HGU133plus2.0 microarray platform. Measured alterations were confirmed with RT-PCR both on dependent and independent sample sets (n1 = 6, n2 = 6, n3 = 6).
Mitotic index (MI) was significantly higher (p<0.05) in intact juvenile (MI = 0.33±0.06) and CRC samples (MI = 0.42±0.10) compared to healthy adult samples (MI = 0.15±0.06). In contrast, apoptotic index (AI) was decreased in children (0.13±0.06) and significantly lower in cancer (0.06±0.03) compared to healthy adult samples (0.17±0.05). Eight proliferation- (e.g. MKI67, CCNE1) and 11 apoptosis-associated genes (e.g. TNFSF10, IFI6) had altered mRNA expression both in the course of normal aging and carcinogenesis, mainly inducing proliferation and reducing apoptosis compared to healthy adults. Eight proliferation-associated genes including CCND1, CDK1, CDK6 and 26 apoptosis-regulating genes (e.g. SOCS3) were differently expressed between juvenile and cancer groups mostly supporting the pronounced cell growth in CRC.
Colorectal samples from children and CRC patients can be characterized by similarly increased proliferative and decreased apoptotic activities compared to healthy colonic samples from adults. Therefore, cell kinetic alterations during colorectal cancer development show uncontrolled rejuvenescence as opposed to the controlled cell growth in juvenile colonic epithelium.
PLoS ONE 10/2013; 8(10):e74140. DOI:10.1371/journal.pone.0074140 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: DNA methylation analysis methods have undergone an impressive revolution over the past 15 years. Regarding colorectal cancer (CRC), the localization and distribution of several differently methylated genes have been determined by genome-wide DNA methylation assays. These genes do not just influence the pathogenesis of CRC, but can be used further as diagnostic or prognostic markers. Moreover, the identified four DNA methylation-based subgroups of CRC have important clinical and therapeutic merit. Since genome-wide DNA methylation analyzes result in a large amount of data, there is a need for complex bioinformatic and pathway analysis. Future challenges in epigenetic alterations of CRC include the demand for comprehensive identification and experimental validation of gene abnormalities. By introduction of genome-wide DNA methylation profiling into clinical practice not only the patients' risk stratification but development of targeted therapies will also be possible.