Metastasis accounts for most of deaths caused by cancer. The increasing body of evidence suggests that changes in N-glycosylation of tumor cell proteins such as increased branching, increased sialylation, polysialylation, decreased fucosylation, enhanced formation of Lewis X and sialyl Lewis X antigens are among important factors determining metastatic potential of tumor cell. Most of the adhesion proteins, e.g., integrins, members of immunoglobulin superfamily, and cadherins are heavily N-glycosylated. The other proteins involved in adhesion, like galectins and type-C selectins, recognize N-glycans as a part of their specific ligands. In this review we focus on recent reports concerning the contribution of N-glycosylation of tumor cell adhesion molecules and some selected membrane proteins in the tumor invasion and metastasis.
Sprzężony kwas linolowy (ang. conjugated linoleic acid - CLA) jest terminem zbiorczym obejmującym grupę pozycyjnych i geometrycznych izomerów kwasu linolowego, w których występują sprzężone wiązania podwójne. Spośród tych związków szczególne zainteresowanie budzi aktywność izomerów cis9,trans11 i trans10,cis12. Omawiane izomery występują naturalnie w tłuszczu mleka i mięsa przeżuwaczy, z dominującym udziałem izomeru cis9,trans11 (kwasu żwaczowego). Wyniki licznych prac wskazują na przeciwnowotworowe właściwości izomerów CLA, m.in. na ich zdolność do hamowania wzrostu różnych linii komórek nowotworowych in vitro. Jednocześnie wskazują na odmienne mechanizmy przeciwnowotworowego oddziaływania różnych izomerów CLA. Celem niniejszych badań była ocena indywidualnego wpływu naturalnie występujących izomerów CLA tj. cis9, trans11 i trans10,cis12 na proliferację ustabilizowanych linii komórek nowotworowych piersi. Badania przeprowadzono na dwóch hormonozależnych ludzkich liniach nowotworowych piersi: MCF-7 i T47D (ATCC Collection). Komórki inkubowano z izomerami CLA: cis9,trans11 lub trans10,cis12 w zakresie stężeń 5 - 200 μM i czasie 24 - 120 godzin. W zakresie stężeń 5 - 100 μM nie obserwowano cytotoksycznego wpływu izomerów CLA na komórki w teście LDH (ang. Lactic Dehydrogenase; Roche) i w tym zakresie stężeń badano proliferację komórek. Wpływ izomerów CLA na proliferację komórek określono poprzez ich barwienie fioletem krystalicznym oraz z wykorzystaniem testu ELISA BrdU (5’bromo-2’deoksyurydyna), opartym na wbudowywaniu się BrdU do DNA proliferujących komórek. Z uzyskanych wyników można wnioskować, że obydwa zastosowane izomery CLA działają hamująco na proliferacje komórek linii MCF-7 i T47D szczególnie po czasie dłuższym niż 48 godzin. Komórki linii T47D odpowiadające ~65 % obniżeniem proliferacji wydają się być bardziej wrażliwe na działanie izomerów CLA.
Sprzężony kwas linolowy (ang. conjugated linoleic acid - CLA) jest terminem zbiorczym obejmującym grupę pozycyjnych i geometrycznych izomerów kwasu linolowego, w których występują sprzężone wiązania podwójne. Wyniki licznych prac jednoznacznie wskazują na przeciwnowotworowe właściwości izomerów CLA, m.in. ich zdolność do hamowania wzrostu komórek nowotworowych różnych linii w modelu in vitro. Prowadzone obecnie badania kierują się w stronę modyfikowania zawartości CLA w wielu produktach spożywczych pochodzenia zwierzęcego takich jak: mięso, jaja, masło. Ma to duże znaczenie praktyczne, ponieważ żywność funkcjonalna może zapobiegać i wspomagać leczenie wielu chorób cywilizacyjnych w tym także chorób nowotworowych. Celem przeprowadzonych badań była ocena wpływu lipidów żółtka jaja kurzego wzbogaconego w izomery CLA: cis9,trans11 i trans10,cis12 na proliferację komórek nowotworowych piersi linii MCF-7 (ATCC Collection). Żółtko jaja kurzego zostało wzbogacone w izomery CLA w sposób naturalny na drodze karmienia kur niosek mieszaniną izomerów CLA: cis9,trans11 i trans10,cis12 (4). Komórki inkubowano z hydrolizatem lipidów żółtka jaja kurzego w zakresie stężeń 0,12; 0,36; 0,73 mg/ml i czasie 24 - 72 godziny, po czym mierzono proliferację komórek. W podanym zakresie stężeń nie obserwowano cytotoksycznego wpływu hydrolizatu na komórki w teście LDH (ang. Lactic Dehydrogenase; Roche). Z uzyskanych wyników można wnioskować, że lipidy żółtka jaja z wbudowanymi izomerami CLA bardziej efektywnie hamują proliferację komórek MCF-7 niż lipidy żółtka jaja bez udziału CLA.
Prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) are the markers of human prostatic gland. However, it is still not completely understood if and how, steroid hormones and growth factors affect their expression and metabolism in the respect to the major pathologies of the gland. Appropriate studies were carried out on histopathologically diagnosed benign prostatic hyperplasia — BPH (n = 42) using tissue slices and cells derived from them. They were incubated with steroid hormones: 5-α-dihydrotestosterone (DHT), estradiol (E) and growth factors: epidermal growth factor (EGF), basic fibroblastic growth factor (bFGF) under culture conditions for up to 24 hours. 32P-labelled specific oligonucleotide probes were used to analyze total RNA isolated from each sample for the presence of PAP and PSA mRNAs. DHT, E, bFGF, EGF or both DHT + bFGF and DHT + EGF increased PAP and PSA mRNA levels in a time- and dose-dependent manner. The highest and statistically significant increase (P < 0.001) for PAP mRNA was observed when DHT + bFGF were present in the medium while for PSA mRNA if DHT + EGF were added to the medium. Slow but constant decrease of PAP and PSA mRNA levels was observed in the absence of each of these factors in the incubation medium. The results suggest that early expression of PSA and PAP genes and/or their mRNA stability strongly depend on DHT while differ in their response to EGF and bFGF.
Integrin subunits present on human bladder cells displayed heterogeneous functional specificity in adhesion to extracellular matrix proteins (ECM). The non-malignant cell line (HCV29) showed significantly higher adhesion efficiency to collagen IV, laminin (LN) and fibronectin (FN) than cancer (T24, Hu456) and v-raf transfected (BC3726) cell lines. Specific antibodies to the a2, a5 and β1 integrin sub- units inhibited adhesion of the non-malignant cells, indicating these integrin participation in the adhesion to ECM proteins. In contrast, adhesion of cancer cells was not inhibited by specific antibodies to the β1 integrin subunit. Antibodies to a3 integrin increased adhesion of cancer cells to collagen, LN and FN, but also of the HCV29 line with colagen. It seems that a3 subunit plays a major role in modulation of other integrin receptors especially in cancer cells. Differences in adhesion to ECM proteins between the non-malignant and cancer cell lines in response to Gal and Fuc were not evident, except for the v-raf transfected cell line which showed a distinct about 6-fold increased adhesion to LN on addition of both saccharides. .-Acetylneuraminic acid inhibited adhesion of all cell lines to LN and FN irrespective of their malignancy.
Expression as well as properties of integrins are altered upon transformation. Cell adhesion regulated by integrins is modulated by glycosylation, one of the most frequent biochemical alteration associated with tumorogenesis. Characterisation of carbohydrate moieties of alpha3beta1 integrin on the cultured human bladder carcinoma (T-24, Hu456, HCV 29T) and human normal ureter and bladder epithelium (HCV 29, Hu609) cell lines was carried out after an electrophoresis and blotting, followed by immunochemical identification of alpha3 and beta1 integrin chains and analysis of their carbohydrates moieties using highly specific digoxigenin-labelled lectins. In all the studied cell lines alpha3beta1 integrin was glycosylated although in general each subunit differently. Basic structures recognized in beta1 subunit were tri- or tetraantennary complex type glycans in some cases sialylated (T-24, HCV 29, HCV 29T) and fucosylated (Hu609, HCV 29T). Positive reaction with Phaseolus vulgaris agglutinin and Datura stramonium agglutinin suggesting the presence of beta1-6 branched N-linked oligosaccharides was found in cancerous cell lines (T-24, Hu456) as well as in normal bladder epithelium cells (Hu609). High mannose type glycan was found only in beta1 subunit from Hu456 transitional cell cancer line. On the other hand alpha3 subunit was much less glycosylated except the invasive cancer cell line T-24 where high mannose as well as sialylated tri- or tetraantennary complex type glycans were detected. This observation suggests that changes in glycosylation profile attributed to invasive phenotype are rather associated with alpha3 not beta1 subunit.
Isoelectric focusing of homogenous arylsulfatase B from human placenta pointed to the presence of enzymatically active and inactive forms of high pi (pH 9-8) and of lower pi (pH 6.5-5.5). Glycan chain analysis performed with the use of a Glycan Differentiation Kit showed that basic forms of arylsulfatase B from human placenta contained mostly high mannose/hybrid type glycans, with 6-O-L-fucose bound to the innermost AT-acetylglucosamine residue, whereas acidic forms of the enzyme contained complex type glycans containing fucose and sialic acid. However, the latter forms constitute a small percentage of the total carbohydrate component. Lectin affinity chromatography of the native enzyme confirmed the presence of a core fucose and a sialic acid.
Expression of N-cadherin an adhesion molecule of the cadherin family, in tumor cells is associated with their increased invasive potential. Many studies suggested the role of N-linked oligosaccharides as important factors that contribute to metastasis by influencing tumor cell invasion and adhesion. N-cadherin is a heavily glycosylated protein. We have analysed the carbohydrate profile of this protein synthesized in human melanoma cell lines: WM35 from the primary tumor site and WM239, WM9, and A375 from different metastatic sites. N-cadherin was immunoprecipitated with anti-human N-cadherin polyclonal antibodies. Characterisation of its carbohydrate moieties was carried out by SDS/PAGE electrophoresis and blotting, followed by im- munochemical identification of the N-cadherin polypeptides and analysis of their glycans using highly specific digoxigenin or biotin labelled lectins. The positive reaction of N-cadherin from the WM35 cell line with Galanthus nivalis agglutinin (GNA), Datura stramonium agglutinin (DSA) and Sambucus nigra agglutinin (SNA) indicated the presence of high-mannose type glycans and biantennary complex type oligosac- charides with a2-6 linked sialic acid. N-cadherin from WM239, WM9, and A375 cell lines gave a positive reaction with Phaseolus vulgaris leukoagglutinin (L-PHA) and lotus Tetragonolobus purpureas agglutinin (LTA). This indicated the presence of tri- or tetra-antennary complex type glycans with a-fucose. In addition, N-cadherin from WM9 (lymphomodus metastatic site) and A375 (solid tumor metastatic site) contained complex type chains with 2–3 sialic acid (positive reaction with Maackia amurensis agglutinin — MAA). The results demonstrated that N-glycans of N-cadherin are altered in metastatic melanomas in a way characteristic for invasive tumor cells.
The effect of 5alpha-dihydrotestosterone (DHT) on the level of human prostatic acid phosphatase (hPAP) mRNA was studied using tissue slices from various benign prostatic hyperplastic glands. The absence of DHT in the incubation medium led to a gradual, significant decrease of the hPAP mRNA level. Addition of the hormone induced hPAP mRNA in a time- and dose-dependent manner. The maximal 2-4-fold induction by 10(-9) M DHT was observed after 3-5 h of incubation, and then the hPAP mRNA level was 6-20-fold higher than that in a parallel sample incubated without DHT. The results suggest that DHT is necessary to sustain the expression of hPAP in hyperplastic prostates.