Coexpression of IL-6 and TNF-α: prognostic significance on breast cancer outcome.

Presented study was conducted to investigate the prognostic significance of the coexpression of serum interleukin-6 (IL-6) and tumor necrosis factor-a (TNF-a) in breast cancer, by correlating their presence with clinicopathological characteristics indicative of tumor progression and the overall survival of breast cancer patients. One hundred twelve consecutive patients with primary breast cancer were prospectively included and evaluated. Serum concentrations of IL-6 and TNF-a were measured by quantitative sandwich enzyme immunoassay (ELISA). Median split was used to subdivide patients with low or high IL-6 and TNF-a levels. A positive association between the expression of the two cytokines was found. The coexpression of high IL-6 and TNF-α was independently associated with extended lymph node (>3) involvement (aOR, 7.8) and lymphovascular invasion (aOR, 14.1), increasing the prognostic significance of each cytokine separately; it also provided additional prognostic information regarding survival, defining a high-risk subgroup of patients with significantly shorter survival and higher risk of death compared to patients with both cytokines low (aHR, 4.45) and patients with only one cytokine high (aHR, 3.63). Our findings suggest that the coexpression of these two cytokines could be used clinically as a useful tumor marker for the extension and the outcome of the disease.

Long-term persistence of acquired resistance to 5-fluorouracil in the colon cancer cell line SW620.

Treatment resistance to antineoplastic drugs represents a major clinical problem. Here, we investigated the long-term stability of acquired resistance to 5-fluorouracil (FU) in an in vitro colon cancer model, using four sub-clones characterised by increasing FU-resistance derived from the cell line SW620. The resistance phenotype was preserved after FU withdrawal for 15weeks (~100 cell divisions) independent of the established level of drug resistance and of epigenetic silencing. Remarkably, resistant clones tolerated serum deprivation, adopted a CD133(+) CD44(-) phenotype, and further exhibited loss of membrane-bound E-cadherin together with predominant nuclear ß-catenin localisation. Thus, we provide evidence for a long-term memory of acquired drug resistance, driven by multiple cellular strategies (epithelial-mesenchymal transition and selective propagation of CD133(+) cells). These resistance phenomena, in turn, accentuate the malignant phenotype.

Evaluation of predictive and prognostic significance of serum TGF-beta1 levels in breast cancer according to HER-2 codon 655 polymorphism.

The present study was conducted to clarify the predictive and prognostic significance of serum TGF-I(2)1 in breast cancer in relation to Ile655Val single nucleotide polymorphism (SNP) of human epidermal growth factor receptor-2 (HER-2). In a case-control study, 56 consecutive patients with primary breast cancer were prospectively included and evaluated. The control group consisted of 45 healthy women. Serum concentrations of TGF-I(2)1 were measured by quantitative sandwich enzyme immunoassay (ELISA). HER-2 SNP was genotyped using PCR-RFLP method. Serum levels of TGF-I(2)1 were significantly increased in breast cancer patients compared to healthy controls (p<0.001). For the evaluation of the diagnostic significance of serum TGF-I(2)1 the area under the receiver operating characteristic (ROC) curve (AUC) was 0.804, while the optimal cut-off point of 30.86 ng/ml was determined to classify breast cancer patients, which yielded sensitivity of 77%, specificity of 78% and accuracy of 77%. Significantly elevated serum TGF-I(2)1 levels were associated with advanced stages (p=0.023), positive lymph nodes (p=0.019) and postmenopausal status (p=0.031). A marginal trend towards higher TGF-I(2)1 levels was found among patients with Val-containing genotypes compared to homozygous Ile-Ile (p=0.094). In multivariate analysis lymph node metastases (p=0.009) remained the only significant independent determinant of high TGF-I(2)1 levels. With regard to prognostic significance for advanced stages (AUC, 0.704) and lymph node metastasis (AUC, 0.683), when the optimal cut-off value was set at 65.15 pg/ml, the sensitivity was 86% and 67%, the specificity was 60% and 62% and accuracy was 66% and 64%, respectively. Survival was shorter in patients with increased serum TGF-I(2)1 (36 months vs 46 months, p=0.022). Multivariate analysis demonstrated a marginal prognostic significance of serum TGF-I(2)1 for survival (p=0.072). The combination of high TGF-I(2)1 and Val-Val genotype predicts a worse prognosis than high serum TGF-I(2)1 alone. Our findings suggest that serum TGF-I(2)1 is involved in tumor malignancy and lymph node metastasis and could be used clinically as a useful tumor marker for evaluation, the extension and the outcome of the disease. They also provide clinical evidence for a significant association between HER-2 Ile655Val SNP and serum TGF-I(2)1, resulting to more aggressive phenotype of the tumor and poor prognosis.

The influence of serum HER-2 levels and HER-2 codon 655 polymorphism on breast cancer outcome.

HER-2 (human epidermal growth factor receptor-2) proto-oncogene is a member of the EGFR family and plays an important role in the regulation of cell growth, differentiation and survival and is involved in the regulation of normal breast growth and development. Alterations of HER-2 have been associated with carcinogenesis and poor prognosis of breast cancer. The present case-control study was conducted to clarify the predictive and prognostic significance of serum HER-2 protein in breast cancer patients in relation to Ile655Val single nucleotide polymorphism (SNP) of this gene. Fifty-six consecutive patients with primary breast cancer and 45 healthy women were prospectively included and evaluated. Serum levels of HER-2 were significantly increased in breast cancer patients compared to healthy controls (p=0.035). The optimal cut-off point of 1.98 ng/ml, which was determined to classify breast cancer patients, yielded sensitivity of 54%, specificity of 73% and accuracy of 62%. Significantly elevated serum HER-2 levels were associated with lymphovascular invasion (p=0.022), poor differentiation (p=0.011), advanced clinical stages (p=0.001), lymph node metastasis (p=0.011), higher number of positive lymph nodes (p=0.007) and the immunohistochemical overexpression of HER-2 protein (p=0.016). Regarding to HER-2 Ile655Val SNP, Ile-Val and Val-Val genotypes exhibited highly significant serum HER-2 elevation compared to homozygous Ile-Ile (both p<0.001). In multivariate analysis advanced stages (p=0.003) and Val-containing genotypes (p=0.009) remained the two significant independent determinants of high HER-2 levels. Survival analysis demonstrated an independent prognostic significance of homozygous Val-Val genotype for reduced survival (p=0.045), but not of serum HER-2 (p=0.181). Our findings confirm that serum HER-2 could be used clinically as a useful tumor marker for the diagnosis and the progression of breast cancer. Furthermore, they provide clinical evidence that HER-2 Ile655Val SNP does affect serum HER-2 levels and it can be regarded as a predictive biomarker for breast cancer patients with poor prognosis.

Cell signaling in cancer.

The pathways that control cell differentiation and growth are almost always compromised in cancer. Although in the last years there have been major advances in understanding these changes and how they contribute to tumor initiation and growth, the task is far from complete. In this review we discuss some of the factors that are found in major key nodes of the signaling pathways. Included among them are the nuclear factor kappaB (NF-kappaB) that has a quite central role in inflammation, and the mammalian target of rapamycin (mTOR) kinase. Also an eminent role is played by the EGF (epidermal growth factor) and the Snail/Slug family of repressors. Since the expansion of tumor cells depends heavily on nutrient and oxygen supply, it requires the growth of new blood vasculature which is directed by the hypoxia inducible factor (HIF).

Allelic imbalance of HER-2 codon 655 polymorphism among different religious/ethnic populations of northern Greece and its association with the development and the malignant phenotype of breast cancer.

Alterations of c-erbB-2 (neu or HER-2) proto-oncogene have been associated with carcinogenesis and poor prognosis of breast cancer. A single nucleotide polymorphism (SNP) at codon 655 resulting in a G to A transition (Ile655Val) in the transmembrane domain-coding region of this gene has been associated with an increased risk of breast cancer. This study aims to determine the prevalence of the HER-2 genotype and its association with breast cancer in the Greek Christian and Greek Muslim population of Thrace, Greece. In this case-control study, we genotyped 56 patients (43 Christians and 13 Muslims) with primary breast cancer and 45 healthy women (32 Christians and 13 Muslims) for the Ile655Val polymorphism, with the PCR-RFLP method. The Val allele and the Val-containing genotypes were significantly more frequent in Muslims than in Christians (p=0.020 and p=0.008, respectively). Among the Greek Christian population, a 5-fold and a 3.1-fold increase in risk of breast cancer was associated with the Val-Val genotype and the Ile-Val or Val-Val genotypes (95% CI, 1.3-18.4; p=0.017 and aOR, 3.1; 95% CI, 1.2-8.3; p=0.025; respectively) compared to homozygous Ile-Ile. No significant association was found in the Muslim population. Among the entire cohort, the Val allele confers a modest increase in breast cancer risk (OR, 2.6; 95% CI, 0.9-7.6; p=0.076, for Val-Val and OR, 2.2; 95% CI, 0.9-5.2; p=0.079 for Ile-Val or Val-Val). This effect was even more pronounced in younger women. Among breast cancer patients, invasive carcinomas, low differentiation tumors, advanced stages, positive lymph nodes, high number of lymph nodes and HER-2 overexpression were more frequent in patients with allele Val than those with allele Ile. Our study proposes the allelic imbalance of Ile655Val polymorphism between Greek Christian and Greek Muslim populations of Thrace contributes to the inconsistent association between this SNP and breast cancer risk across these two different ethnic groups. The association of the HER-2 genotype with clinicopathologic characteristics and HER-2 expression may indicate its possible implication on the more aggressive phenotype.

Circulating matrix metalloproteinases and their inhibitors in inguinal hernia and abdominal aortic aneurysm.

AIM: There is evidence supporting the role of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) in aortic and abdominal wall connective tissue degeneration, resulting in aneurysm and hernia formation. Furthermore, clinical association studies have demonstrated increased prevalence of abdominal wall hernias in patients with aortic aneurysms. Our objective was to estimate the levels of MMPs and TIMPs in the blood of patients with aortic aneurysm and inguinal hernia, in order to investigate whether there is potential pathogenic linkage of impaired collagen metabolism. METHODS: Plasma concentrations of MMP-9, MMP-2, TIMP-1 and TIMP-2 were quantified using ELISA in 33 male patients with abdominal aortic aneurysm and 91 male patients with inguinal hernia. They were consecutive patients undergoing repair during the study period. The same substances were measured in 35 healthy male controls. RESULTS: MMP-9 and MMP-2 concentrations were lower in the plasma of patients with inguinal hernia and abdominal aortic aneurysm than controls, with hernia patients having the lowest circulating levels. The levels of TIMP-2 were significantly elevated in patients with inguinal hernia and significantly reduced in patients with aortic aneurysm, whereas opposite correlations were found for circulating TIMP-1. CONCLUSION: Different patterns of circulating MMP and TIMP levels were found in patients with aneurysm and hernia compared with controls. Underlying pathogenic processes implicating MMPs and TIMPs in connective tissue metabolism are expressed by differing plasma levels in the two disease states. Further research including combined plasma and tissue analyses is required to further investigate potential common pathogenesis of these diseases.

Effect of HbS in the determination of HbA2 with the Menarini HA-8160 analyzer and comparison with other instruments.

It is known that the presence of hemoglobin S (HbS) affects the determination of hemoglobin A(2) (HbA(2)) levels in clinical samples. We quantitated this effect using the Menarini HA-8160 analyzer and compared with other instruments (HELENA beta-thal quik column, TOSOH HLC-723G7 and BIORAD Variant II) using the HELENA SAS-MX alkaline gel electrophoresis kit as the reference method. The %HbA(2) values from the HA-8160 analyzer and the alkaline gel electrophoresis show a good linear correlation in the absence of HbS. A strong positive bias in the %HbA(2) values from the HA-8160 is apparent in the presence of HbS in the samples, when compared with the alkaline electrophoresis. The analytical imprecision and bias of the three HPLC instruments are comparable both in the presence and absence of HbS. The manual column method shows a lower bias in the absence of HbS but is more affected when HbS is present in the samples.

Gelatinases [matrix metalloproteinase-2 (MMP-2) and MMP-9] induce carotid plaque instability but their systemic levels are not predictive of local events.

Matrix metalloproteinases (MMPs) appear to play a central role in atherosclerotic plaque remodeling; however, the relationship of increased MMP levels in inducing carotid plaque instability remains controversial. We investigated whether gelatinases (MMP-2 and MMP-9) are implicated in carotid intraplaque hemorrhage and whether their serum levels may predict local carotid events. Nineteen carotid specimens obtained by endarterectomy of 18 patients were studied. The presence of gross intraplaque hemorrhage was recorded before plaque removal and quantification of MMP-2, MMP-9, and tissue inhibitor of metalloproteinase-1 (TIMP-1) in extracts from (1) the more stenotic area of the plaque, (2) the periphery of the plaque, and (3) serum was performed by enzyme-linked immunosorbent assay. MMP-9 levels measured in extracts from the most stenotic area were significantly higher in patients with intraplaque hemorrhage (p = 0.007); however, serum levels showed no difference, while those taken from the periphery of the lesion were also increased but did not reach a statistically significant level (p = 0.06). An increase in MMP-2 values was observed in the periphery of the lesion (p = 0.04) in patients with intraplaque hemorrhage. TIMP-1 levels showed no difference between the two groups regardless of the presence or absence of intraplaque hemorrhage. No significant differences in MMP levels were observed between symptomatic and asymptomatic patients. Increased levels of MMPs, particularly MMP-9, have been implicated in carotid intraplaque hemorrhage without their serum levels being predictive of local events.

Partial purification and properties of (Na+ + K+)-ATPase from Potamon potamios.

1. The tissue distribution of the (Na+ + K+)-ATPase in the freshwater/land crab Potamon Potamios was studied. 2. Gills were found to display the highest total activity in the whole animal (47%) but the highest specific activity was detected in the heart (15.15 mumol Pi/mg protein/min). 3. All other organs tested were found to have low enzyme activity. 4. The freshwater/land crab ATPase enzyme was inhibited by ouabain with a Ki of 0.5 mM.Km values for ATP, Mg2+ and K+ were 1.4, 4.0 and 1.2 mM respectively. The enzyme also showed a break in the Arrhenius plot at 23 degrees C. 5. A purification method of microsomal ATPase is described involving ultracentrifugation and electrofocusing.

Sp1 plays a critical role in the transcriptional activation of the human cyclin-dependent kinase inhibitor p21(WAF1/Cip1) gene by the p53 tumor suppressor protein.

In the present study we present evidence for the critical role of Sp1 in the mechanism of transactivation of the human cell cycle inhibitor p21(WAF1/Cip1) (p21) gene promoter by the tumor suppressor p53 protein. We found that the distal p53-binding site of the p21 promoter acts as an enhancer on the homologous or heterologous promoters in hepatoma HepG2 cells. In transfection experiments, p53 transactivated the p21 promoter in HaCaT cells that express Sp1 but have a mutated p53 form. In contrast, p53 could not transactivate the p21 promoter in the Drosophila embryo-derived Schneider's SL2 cells that lack endogenous Sp1 or related factors. Cotransfection of SL2 cells with p53 and Sp1 resulted in a synergistic transactivation of the p21 promoter. Synergistic transactivation was greatly decreased in SL2 cells and HaCaT cells by mutations in either the p53-binding site or in the -82/-77 Sp1-binding site indicating functional cooperation between Sp1 and p53 in the transactivation of the p21 promoter. Synergistic transactivation was also decreased by mutations in the transactivation domain of p53. Physical interactions between Sp1 and p53 proteins were established by glutathione S-transferase pull-down and coimmunoprecipitation assays. By using deletion mutants we found that the DNA binding domain of Sp1 is required for its physical interaction with p53. In conclusion, Sp1 must play a critical role in regulating important biological processes controlled by p53 via p21 gene activation such as DNA repair, cell growth, differentiation, and apoptosis.