Giovanna Bises

25-Hydroxyvitamin D3-1α-hydroxylase Expression in Normal and Malignant Human Colon

1,25-dihydroxyvitamin D3 has anti-mitotic, pro-differentiating, and pro-apoptotic activity in tumor cells. We demonstrated that the secosteroid can be synthesized and degraded not only in the kidney but also extrarenally in intestinal cells. Evaluation of 1,25-dihydroxyvitamin D3-synthesizing CYP27B1 hydroxylase mRNA (real-time PCR) and protein (immunoblotting, immunofluorescence) showed enhanced expression in high- to medium-differentiated human colon tumors compared with tumor-adjacent normal mucosa or with colon mucosa from non-cancer patients. In high-grade undifferentiated tumor areas expression was lost. Many cells co-expressed CYP27B1 and the vitamin D receptor. We suggest that autocrine/paracrine antimitotic activity of 1,25-dihydroxyvitamin D3 could prevent intestinal tumor formation and progression. (J Histochem Cytochem 52:985–989, 2004)

The Vitamin D endocrine system of the gut—Its possible role in colorectal cancer prevention

While Vitamin D insufficiency in the US and European population is rising, epidemiological studies suggest an inverse correlation between low serum levels of 25-hydroxyvitamin D(3) (25-OH-D(3)) and colorectal cancer incidence. The antimitotic, prodifferentiating and proapoptotic active metabolite 1alpha,25-dihydroxyvitamin D(3) (1,25-(OH)(2)-D(3)) is synthesized also by colonocytes, since these possess Vitamin D synthesizing (CYP27B1) and catabolic (CYP24) hydroxylases similar to the kidney. Early during colon tumor progression, expression of CYP27B1 and of the Vitamin D receptor increases, suggesting an autocrine/paracrine growth control in colon tissue as a physiological restriction against tumor progression. However, in human adenocarcinomas expression of the catabolic CYP24 is also enhanced when compared with adjacent normal mucosa. Therefore, to maintain colonic accumulation of 1,25-(OH)(2)-D(3) its catabolism needs to be restricted. Our studies in mice show that low nutritional calcium causes hyperproliferation of colon crypts and significant elevation of CYP24 expression, which can be completely abrogated by soy feeding. We suggest that phytoestrogens in soy, known to be estrogen receptor modulators, are responsible for decreased CYP24 expression. These results and our observation that 17beta-estradiol can elevate CYP27B1 expression in rectal tissue of postmenopausal women, may underlie the observed protective effect of estrogens against colorectal cancer in females.

Gender-Specific Modulation of Markers for Premalignancy by Nutritional Soy and Calcium in the Mouse Colon

Sporadic colorectal cancer develops as a multistep process during decades of latency. Multiple factors, in particular nutrition, influence progression. Both nutritional calcium and soy are known to reduce sporadic cancer incidence. Soy contains high levels of phytoestrogens. Among them genistein is recognized as an antioxidant and cell-cycle inhibitor. However, timing and length of consumption of genistein as well as gender- and colon site-specific activity may result in beneficial or detrimental effects. We therefore evaluated the effect in mice of a basic AIN76A diet containing 20% soy as main protein source fed for 1 or 2 generations. In another set of animals, normal calcium levels (0.5%) were replaced by low calcium (0.04%) with or without supplementation of genistein (0.04%). Expression of the vitamin D receptor, cyclooxygenase (COX)-2, proapoptotic Bak and antiapoptotic Bcl-2 protein, as well as estrogen receptor (ER)-alpha and ER-beta mRNA were evaluated. Results were identical whether soy was fed for 1 or 2 generations. Soy decreased Bak and increased COX-2 and ER-alpha expression site-specifically in female mice. Vitamin D receptor protein was reduced only in males. In animals fed 0.04% dietary calcium, COX-2 protein was increased mainly in females, but supplementation of genistein to the diet lowered COX-2 expression significantly in both genders. Our results suggest that genistein counteracts the induction of a marker of colonic premalignancy by low nutritional calcium in both genders. However, soy itself enhances COX-2 and reduces Bak, but only in females. This suggests detrimental activity of an unknown component of soy triggered by a high-estrogen background.

Clone-specific expression, transcriptional regulation, and action of interleukin-6 in human colon carcinoma cells

BackgroundMany cancer cells produce interleukin-6 (IL-6), a cytokine that plays a role in growth stimulation, metastasis, and angiogenesis of secondary tumours in a variety of malignancies, including colorectal cancer. Effectiveness of IL-6 in this respect may depend on the quantity of basal and inducible IL-6 expressed as the tumour progresses through stages of malignancy. We therefore have evaluated the effect of IL-6 modulators, i.e. IL-1β, prostaglandin E2, 17β-estradiol, and 1,25-dihydroxyvitamin D3, on expression and synthesis of the cytokine at different stages of tumour progression.MethodsWe utilized cultures of the human colon carcinoma cell clones Caco-2/AQ, COGA-1A and COGA-13, all of which expressed differentiation and proliferation markers typical of distinct stages of tumour progression. IL-6 mRNA and protein levels were assayed by RT-PCR and ELISA, respectively. DNA sequencing was utilized to detect polymorphisms in the IL-6 gene promoter.ResultsIL-6 mRNA and protein concentrations were low in well and moderately differentiated Caco-2/AQ and COGA-1A cells, but were high in poorly differentiated COGA-13 cells. Addition of IL-1β (5 ng/ml) to a COGA-13 culture raised IL-6 production approximately thousandfold via a prostaglandin-independent mechanism. Addition of 17β-estradiol (10-7 M) reduced basal IL-6 production by one-third, but IL-1β-inducible IL-6 was unaffected. Search for polymorphisms in the IL-6 promoter revealed the presence of a single haplotype, i.e., -597A/-572G/-174C, in COGA-13 cells, which is associated with a high degree of transcriptional activity of the IL-6 gene. IL-6 blocked differentiation only in Caco-2/AQ cells and stimulated mitosis through up-regulation of c-myc proto-oncogene expression. These effects were inhibited by 10-8 M 1,25-dihydroxyvitamin D3.ConclusionIn human colon carcinoma cells derived from well and moderately differentiated tumours, IL-6 expression is low and only marginally affected, if at all, by PGE2, 1,25-dihydroxyvitamin D3, and 17β-estradiol. However, IL-6 is highly abundant in undifferentiated tumour cells and is effectively stimulated by IL-1β. In case of overexpression of an IL-6 gene variant with extreme sensitivity to IL-1β, massive release of the cytokine from undifferentiated tumour cells may accelerate progression towards malignancy by paracrine action on more differentiated tumour cells with a still functioning proliferative IL-6 signalling pathway.

Quantitative Image Analysis of Epithelial and Stromal Area in Histological Sections of Colorectal Cancer: An Emerging Diagnostic Tool

In colorectal cancer (CRC), an increase in the stromal (S) area with the reduction of the epithelial (E) parts has been suggested as an indication of tumor progression. Therefore, an automated image method capable of discriminating E and S areas would allow an improved diagnosis. Immunofluorescence staining was performed on paraffin-embedded sections from colorectal tumors (16 samples from patients with liver metastasis and 18 without). Noncancerous tumor adjacent mucosa (n = 5) and normal mucosa (n = 4) were taken as controls. Epithelial cells were identified by an anti-keratin 8 (K8) antibody. Large tissue areas (5–63 mm2/slide) including tumor center, tumor front, and adjacent mucosa were scanned using an automated microscopy system (TissueFAXS). With our newly developed algorithms, we showed that there is more K8-immunoreactive E in the tumor center than in tumor adjacent and normal mucosa. Comparing patients with and without metastasis, the E/S ratio decreased by 20% in the tumor center and by 40% at tumor front in metastatic samples. The reduction of E might be due to a more aggressive phenotype in metastasis patients. The novel software allowed a detailed morphometric analysis of cancer tissue compartments as tools for objective quantitative measurements, reduced analysis time, and increased reproducibility of the data.

Segmentation of cell nuclei within complex configurations in images with colon sections

This paper addresses the development of an automatic segmentation technique for detecting cell nuclei. The technique uses a new approach for segmenting nuclei in images taken from tissues with colon carcinoma. The segmentation problems encountered in these images and solved by the proposed technique are related to the non-uniform illumination on the background, out-of-focus nuclei, the physical structure of cells in the tissue section, the activity status of the cell and the clustered cell nuclei. First, the region growing method is used for accurate background detection. The separation regions between grouped cell nuclei are detected using the cross-correlation method and validated based on their link with the background. Then, the nuclei boundaries are identified by applying the watershed algorithm on the complemented distance transform of the binary image containing the selected separation lines.

Automated REcognition of tissue-associated erythrocytes (ARETE)-a new tool in tissue cytometry.

Automated microscopic image analysis of immunofluorescence‐stained targets on tissue sections is challenged by autofluorescent elements such as erythrocytes, which might interfere with target segmentation and quantification. Therefore, we developed an automated system (Automated REcognition of Tissue‐associated Erythrocytes; ARETE) for in silico exclusion of erythrocytes. To detect erythrocytes in transmission images, a cascade of boosted decision trees of Haar‐like features was trained on 8,640/4,000 areas (15 × 15 pixels) with/without erythrocytes from images of placental sections (4 µm). Ground truth data were generated on 28 transmission images. At least two human experts labelled the area covered by erythrocytes. For validation, output masks of human experts and ARETE were compared pixel‐wise against a mask obtained from majority voting of human experts. F1 score, specificity, and Cohens κ coefficients were calculated. To study the influence of erythrocyte‐derived autofluorescence, we investigated the expression levels of a protein (receptor for advanced glycated end products; RAGE) in placenta and number of Ki‐67‐positive/cytokeratin 8‐positive epithelial cells in colon sections. ARETE exhibited high sensitivity (99.87%) and specificity (99.81%) on a training‐subset and processed transmission images (1,392 × 1,024 pixels) within 4 sec. ARETE and human experts F1‐scores were 0.55 versus 0.76, specificities 0.85 versus 0.92 and Cohens κ coefficients 0.41 versus 0.68. A ranking of Cohens κ coefficient by the scale of Fleiss certified “good agreement” between ARETE and the human experts. Applying ARETE, we demonstrated 4–14% false‐positive RAGE‐expression in placenta, and 18% falsely detected proliferative epithelial cells in colon, caused by erythrocyte‐autofluorescence. ARETE is a fast system for in silico reduction of erythrocytes, which improves automated image analysis in research and diagnostic pathology.

Abstract 1874: Impact of vitamin D and calcium on PGE2 metabolism in colorectal cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Aberrant expression of cyclooxygenase 2 (COX2), the enzyme responsible for synthesizing prostaglandin E2 (PGE2), is one of the early events in colorectal cancer (CRC) development. Repression of COX2 results in the inhibition of prostaglandin synthesis which can delay colon tumorigenesis. It has been shown previously that in cancer cells 1α,25-dihydroxyvitamin D3 (1,25D3) downregulates the expression of COX2 and increasing that of the PGE2 catabolizing enzyme 15-prostaglandin-dehydrogenase (15-PGDH). Due to the differentiation-promoting, pro-apoptotic and anti-proliferative effects calcium and 1,25D3 have become factors of great interest for the chemoprevention of CRC. In order to study the effect of calcium on the PGE2 pathway, we treated the colon cancer cell line Caco-2 with low calcium (0.025 mM) for 48 hours. This led to an increase in COX2 expression while decreasing that of 15-PGDH. Similar effect was seen in vivo: in mice low calcium (0.04%) diet elevated COX2 expression in the colon when compared with the control diet (0.5%). Interestingly, expression of the vitamin D catabolizing enzyme, CYP24A1 was also augmented, in parallel with an increase of crypt cell proliferation. However, expression of the 1,25D3 synthesizing CYP27B1 was not affected. These results suggested that low dietary calcium might reduce 1,25D3 levels in the colon, thereby lowering the impact of 1,25D3 on the COX2-PGE2 pathway. To investigate whether this expression profile is prevalent also in the human colon, we screened over 200 tissue specimens from CRC patients by QRT-PCR. In colon tumors, the mRNA ratio of 15-PGDH and COX2 was significantly downregulated (p<0.0001). This unbalance implicates an elevated PGE2 production. In these tumor samples the CYP24A1/CYP27B1 mRNA ratio was also significantly increased (p<0.05) as compared with the adjacent mucosa. Additionally, CYP24A1 staining confirmed elevated levels of the enzyme in adenocarcinomas. Thus, in these colorectal tumors local 1,25D3 levels were most probably insufficient to inhibit PGE2 accumulation due to high CYP24A1 expression. Low dietary calcium is a promoter of CYP24A1 expression. Our data represent one of the possible molecular mechanisms for the synergistic action of calcium and vitamin D levels in CRC chemoprevention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1874.