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.

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.

Az öregedés emésztőrendszeri hatásainak vizsgálata, molekuláris hátterének megismerése és bizonyos betegségek (mint például az idősek körében gyakrabban előforduló sporadikus vastagbélrák) kialakulásával való kapcsolatának feltérképezése új és ígéretes területe a molekuláris gasztroenterológiának. Ismert tény, hogy az emésztőrendszerben az öregedés során kialakuló molekuláris változások egy része (például a DNS-metiláció, a telomerrövidülés) a vastagbélrák bizonyos típusaiban is azonosítható. A sporadikus vastagbélrák epidemiológiai és molekuláris tulajdonságait ismerve továbbra is nyitott kérdés, hogy pontosan milyen molekuláris mechanizmusok, genetikai vagy génexpressziós szintű, illetve epigenetikai változások magyarázzák, hogy 45–50 éves kor felett a sporadikus vastagbélrákos megbetegedések száma ugrásszerűen megnő, és ezek vajon hogyan hozhatók összefüggésbe az öregedés mechanizmusával. Hosszú távon a megelőzés és a korszerű kezelés szempontjából is kiemelkedő jelentőségű lehet a megismert folyamatok célzott befolyásolása. Dolgozatunk az öregedés során a vastagbélben kialakuló mikroszkópos és molekuláris változásokat foglalja össze, és ezek lehetséges szerepét mutatja be az időskori sporadikus vastagbélrák kialakulásában.

A recently published transcript set is suitable for gene expression-based discrimination of normal colonic and colorectal cancer (CRC) biopsy samples. Our aim was to test the discriminatory power of the CRC-specific transcript set on independent biopsies and on formalin-fixed, paraffin-embedded (FFPE) tissue samples. Total RNA isolations were performed with the automated MagNA Pure 96 Cellular RNA Large Volume Kit (Roche) from fresh frozen biopsies stored in RNALater (CRC (n = 15) and healthy colonic (n = 15)), furthermore from FFPE specimens including CRC (n = 15) and normal adjacent tissue (NAT) (n = 15) specimens next to the tumor. After quality and quantity measurements, gene expression analysis of a colorectal cancer-specific marker set with 11 genes (CA7, COL12A1, CXCL1, CXCL2, CHI3L1, GREM1, IL1B, IL1RN, IL8, MMP3, SLC5A7) was performed with array real-time PCR using Transcriptor First Strand cDNA Synthesis Kit (Roche) and RealTime ready assays on LightCycler®480 System (Roche). In situ hybridization for two selected transcripts (CA7, CXCL1) was performed on NAT (n = 3), adenoma (n = 3) and CRC (n = 3) FFPE samples. Although analytical parameters of automatically isolated RNA samples showed differences between fresh frozen biopsy and FFPE samples, both quantity and the quality enabled their application in gene expression analyses. CRC and normal fresh frozen biopsy samples could be distinguished with 93.3 % sensitivity and 86.7 % specificity and FFPE samples with 96.7 and 70.0 %, respectively. In situ hybridization could confirm the upregulation of CXCL1 and downregulation of CA7 in colorectal adenomas and tumors compared to healthy controls. According to our results, gene expression analysis of the analyzed colorectal cancer-specific marker set can also be performed from FFPE tissue material. With the addition of an automated workflow, this marker set may enhance the objective classification of colorectal neoplasias in the routine procedure in the future.