Pavel Jandík

The analysis of S100A9 and S100A8 expression in matched sets of macroscopically normal colon mucosa and colorectal carcinoma: The S100A9 and S100A8 positive cells underlie and invade tumor mass

The expression of calcium‐binding protein S100A9 was investigated in 23 matched sets of colorectal carcinoma and normal colon mucosa using two‐dimensional gel electrophoresis. We found that, from a group of 23 patients, the level of S100A9 protein, in comparison with matched normal colon mucosa, was significantly increased in malignant tissues of 16 patients (70%). Furthermore, an additional protein, identified by matrix‐assisted laser desorption/ionization ‐ mass spectrometry (MALDI‐MS) as S100A8, exhibited an increased expression in the same specimens of malignant tissues as the S100A9 protein. The immunohistological analysis revealed the accumulation of S100A9 positive cells, macrophages and polymorphonuclear leukocytes along the invasive margin of colorectal carcinoma. The S100A8 protein was found to be produced in the same location. The possible participation of both proteins and, especially, its heterodimeric complex calprotectin in colorectal carcinoma regression could be taken into account.

Proteome study of colorectal carcinogenesis.

Development of cancer is a complex process involving multiple changes in gene expression. To unravel these alterations, a proteome approach aimed at the identification of qualitative and quantitative changes in protein composition, including their post‐translational modifications, attracts great attention. Our study was focused on the identification of proteins whose amount is altered in the course of malignant transformation of colon mucosa. Proteins extracted from tissue specimens or cell lysates were separated by two‐dimensional gel electrophoresis (2‐DE). Comparative analyses of 2‐DE protein patterns were done using computerized image analysis. Selected proteins exhibiting statistically significant abundance alterations comparing healthy and diseased tissues were identified by mass spectrometry. Globally, we have found 57 proteins that exhibited either a significant decrease or increase in amount in pathological tissues, and 18 of these were annotated by mass spectrometry. The alterations in the expression of nine proteins were common for both precancerous and neoplastic tissues suggesting their role in colon tumorigenesis. The epithelial origin of all identified spots was checked in two cell lines Caco‐2 and DLD‐1 originating from well‐differentiated and poorly differentiated colon carcinoma, respectively.

Protein abundance alterations in matched sets of macroscopically normal colon mucosa and colorectal carcinoma

Our current results, aimed at the detection of protein abundance alterations that could be associated with the process of colon tumorigenesis, are summarized. The matched sets of macroscopically normal colon mucosa and colorectal carcinoma were examined by a one‐ or two‐dimensional electrophoretic approach and proteins were identified using immunoblotting or mass spectrometry. The following results were observed: The levels of liver fatty acid‐binding protein, actin‐binding protein/smooth muscle protein 22‐alpha and cyclooxygenase 2 were downregulated in colorectal carcinoma compared to normal colon mucosa. Conversely, the expression of a novel variant of heat shock protein70 and several members of the S100 protein family of calcium‐binding proteins (two isoforms of S100A9, S100A8, S100A11 and S100A6) were upregulated in transformed colon mucosa. Despite the variations of the levels of expression of given protein among analyzed samples, all quantitative changes were found to be sta tistically significant (Mann‐Whitney test assuming p ≤ 0.05). We conclude that the proteomic approach is useful for the study of complex biological events underlying the process of colorectal tumorigenesis.

Overexpression of calcium-binding protein calgranulin B in colonic mucosal diseases

Subtractive two-dimensional gel electrophoresis (2-DE) has been used for the study of the protein patterns of the normal colonic mucosa and the specimens collected from patients diagnosed for inflammatory bowel disease (IBD), colonic polyps and colorectal cancer. We found a 13 kDa protein that was detected in five of seven adenomas and in 13 of 15 colorectal carcinomas while it was absent or only slightly expressed in normal colonic mucosa. Furthermore, this protein occurred in all specimens collected from patients suffering from IBD and its quantity reflected the increased severity of inflammation. The combination of microsequencing and mass spectrometry led to the identification of the 13 kDa spot as calgranulin B. Our results indicate that the production of calgranulin B is unregulated in inflammatory, preneoplastic and neoplastic lesions of colonic mucosa.

A Relationship between K-ras Gene Mutations and Some Clinical and Histologic Variables in Patients with Primary Colorectal Carcinoma

Abstract Mutations in the Kirsten ras 2 (K-ras) gene were described as early events in the process of colorectal carcinogenesis. The aim of this study was to find a possible relationship between the presence of K-ras mutation in samples of primary colorectal carcinomas and the clinico-pathological data of the investigated patients. Mutation in codon 12 of the K-ras gene was determined in 18 of 53 colorectal carcinomas (34 %) in our group of patients. The presence of K-ras gene mutations was not related to gender, age of subject at diagnosis, staging or cancer location (p > 0.05). Sixteen of the 42 (38 %) moderately differentiated carcinomas, and two of the eight (25 %) well differentiated carcinomas contained K-ras mutation in codon 12, but none of the three poorly differentiated carcinomas contained the mutation. Moderately differentiated tumours contained an aspartate code GAT (in eight cases), a valine code GTT (in six cases), an alanine code GCT (in one case) and a serine code AGT (in one case) in codon 12. Well differentiated tumours contained only the valine code GTT (two cases). Our results show that the frequency of mutations in the K-ras gene in carcinomas in Central Europe is not different from the frequencies found in other parts of the world. The homogeneous incidence of K-ras mutation does not seem to be related to ethnic factors, dietary habits, or the composition of the diet.

Association between renal tubular cell dysfunction and increased urinary zinc excretion in cancer patients

Although an increase in renal zinc excretion in cancer patients is well documented, the mechanisms involved are still disputed. As recent studies have raised the question of the role of renal tubular cell dysfunction in the elevation of urinary zinc output, we have examined urinary zinc excretion and the excretion of N-acetyl-beta-D-glucosaminidase (NAG), an indicator of tubular cell dysfunction, in 30 patients with cancer, 28 healthy controls and 20 patients with benign non-inflammatory disorders. As expected, urinary zinc excretion was significantly higher in the cancer patients compared with controls and patients with benign disorders (2.64 +/- 3.05 vs. 0.86 +/- 0.36 and 0.89 +/- 0.29 mmol mol creatinine-1, p < 0.001). NAG activity was also elevated (18.9 +/- 20.1 vs. 4.32 +/- 3.33 and 9.99 +/- 9.72 mukat mol creatinine-1, p < 0.001 and p < 0.05, respectively). A significant correlation between urine zinc and NAG was observed in all three groups (rho = 0.73, p < 0.001, rho = 0.55, p < 0.01 and rho = 0.45, p < 0.05, respectively). In conclusion, our data provide additional support for the role of renal tubular cell dysfunction in hyperzincuria in cancer. Urinary zinc measurement may represent an alternative approach to detecting renal tubular dysfunction in human pathology.

Urinary zinc excretion and acute phase response in cancer patients

We investigated urinary zinc and serum levels of C-reactive protein, alpha-1 acid glycoprotein, haptoglobin, transferrin and prealbumin in 55 patients with solid tumors and 20 controls. Urinary zinc, serum C-reactive protein, alpha-1 acid glycoprotein and haptoglobin were significantly higher, and serum prealbumin was significantly lower in cancer patients. A significant positive correlation between urinary zinc and C-reactive protein, alpha-1 acid glycoprotein and haptoglobin, as well as a negative correlation with transferrin and prealbumin were observed. Hyperzincuria in cancer patients appears to be linked to the acute phase response. Our data provide further evidence implicating systemic inflammatory response in increased urinary zinc excretion.