Implication of alpha5beta1 integrin in invasion of drug-resistant MCF-7/ADR breast carcinoma cells: a role for MMP-2 collagenase.

Expression of alpha5beta1 integrin in the drug-resistant MCF-7/ADR breast carcinoma cells was inhibited by treatment of these cells with alpha5-specific siRNA. The decrease of alpha5beta1 expression resulted in a sharp decrease of expression of MMP-2 collagenase and inhibition of invasion activity of these cells in vitro. Similar decrease of invasion was also observed during inhibition of MMP-2 expression by treatment of these cells with MMP-2-specific siRNA. Inhibition of alpha5beta1 expression was also accompanied by significant decrease in cell content of active (phosphorylated) forms of signal protein kinases Akt and Erk1/2. Inhibition of activity of these kinases by treatment of cells with PI-3K/Akt-specific inhibitor LY294002 or Erk-specific inhibitor PD98059 resulted in inhibition of MMP-2 expression and the decrease of invasion in vitro. These data suggest that alpha5beta1 controls invasion ability of these cells by regulating expression of MMP-2, which involves PI-3K and Erk1/2 protein kinase signaling.

The role of beta1 integrin subfamily in anchorage-dependent apoptosis of breast carcinoma cells differing in multidrug resistance.

Integrin expression was investigated in MCF-7 human breast adenocarcinoma line and in the MCF-7Dox line, which was selected from MCF-7 by a resistance to multiple antitumor drugs (MDR). We have shown that acquisition of MDR was accompanied by a drastically reduced expression of some integrins of the beta1-subfamily (alpha2beta1, alpha3beta1, alpha6beta1) and of alpha vbeta5 intergin in the adenocarcinoma cells. In contrast, expression of alpha5beta1 integrin was markedly increased in the MDR cells. Along with multiple antitumor drug resistance, MCF-7Dox cells demonstrate elevated resistance to anchorage-dependent apoptosis (anoikis) and enhanced in vitro invasive activity. To elucidate the implication of beta1-integrins in the above phenotypic modifications, the effect of beta1-integrin signaling was assayed. Stimulation of beta1-mediated signaling was accomplished by treating of the cells with antibodies to the beta1-subunit common for members of the beta1-subfamily. These data show that activation of beta1-integrin signaling markedly upregulated anoikis of the adenocarcinoma cells.

Monomeric and multimeric blockers of selectins: comparison of in vitro and in vivo activity.

The potency of the oligosaccharides SiaLe(x), SiaLe(a), HSO(3)Le(x), and HSO(3)Le(a), their conjugates with polyacrylamide (PAA, 40 kD), and other monomeric and polymeric selectin inhibitors has been compared with that of the polysaccharide fucoidan. The following assay systems were used: 1) a 96-well assay based either on the use of recombinant E-, P-, and L-selectins or an analogous assay with natural P-selectin isolated from human platelets; 2) a platelet-based P-selectin cell assay; and 3) a rat model of peritoneal inflammation. IC(50) values for the neoglycoconjugate SiaLe(a)-PAA were 6, 40, and 85 microM for recombinant E-, P-, and L-selectins, respectively; all monomeric inhibitors were about two orders of magnitude weaker. PAA-conjugates, containing as a ligand tyrosine-O-sulfate (sTyr) in addition to one of the sialylated oligosaccharides, were the most potent synthetic blockers in vitro. Compared with fucoidan, the most potent known P- and L-selectin blocker, the bi-ligand glycoconjugate HSO(3)Le(a)-PAA-sTyr displayed similar inhibitory activity in vitro towards L-selectin and about ten times lower activity towards P-selectin. All of the tested synthetic polymers displayed a similar ability to inhibit neutrophil extravasation in the peritonitis model (in vivo) at 10 mg/kg. The data provide evidence that monomeric SiaLe(x) is considerably more effective as a selectin blocker in vivo than in vitro, whereas the opposite is true for fucoidan and the bi-ligand neoglycoconjugate HSO(3)Le(a)-PAA-sTyr.

Uncharged P-selectin blockers.

The blocking potency of P- and L-selectin was studied for certain small molecule mannosides and their polyacrylamide (PAA, 30 kDa) conjugates in comparison to SiaLe(x) and fucoidan. Two experimental systems were used: (1) solid phase static assay based on recombinant selectins, and (2) P-selectin dependent rat peritoneal inflammation. betaMan-SC6H4NO2- p was four times more potent P-selectin inhibitor as compared to SiaLe(x). Docking of this molecule onto the P-selectin carbohydrate-binding site demonstrated that a nitro group enabled an electrostatic interaction with residue Lys 84, while the phenyl ring and the CH2 at C-6 contacted the CH2 groups of the same Lys residue. In vivo, betaMan-SC6H4NO2- p blocked experimental inflammation better than SiaLe(x), but significantly lower than fucoidan. In vitro Man-polyacrylic acid conjugates appeared to be very potent inhibitors comparable to fucoidan, uncharged Man-PAA proved rather active, comparable to SiaLe(x)-PAA both in vitro, and in vivo, whereas mannan did not display any P-selectin blocking effect.

Fucoidan inhibits leukocyte recruitment in a model peritoneal inflammation in rat and blocks interaction of P-selectin with its carbohydrate ligand.

Neutrophil recruitment into systemic inflammatory sites in vivo is thought to be initiated by selectin-mediated endothelial adherence. The effect of fucoidan (natural sulfated polymer of L-fucose) on the selectin dependent PMN migration into rat peritoneum following the induction of inflammation by peptone injection was studied. Peritonitis was characterized by an increase in the total cell number (from 45.3 x 10(6) to 91.6 x 10(6)/rat), and by highly elevated PMN content (from 0.2% to 58%) in the rat peritoneal cavity 3 h after peptone injection. Intravenous administration of fucoidan was found to reduce, in a dose-dependent manner, neutrophil migration into peritoneum. Fucoidan in a dose as low as 0.8 mg per rat caused 96.8% reduction of neutrophil extravasation. The inhibitory effect of fucoidan was also dependent on the time intervals between the peptone and fucoidan injections. The maximal inhibitory effect of fucoidan was observed within the first 15 min after the induction of peritonitis and it was maintained at a level of 80% during 1.5 h. Administration of fucoidan 2.5 h after peptone injection had practically no effect on PMN extravasation. Since P-selectin is known to play a key role at the earlier stages of PMN extravasation, it was suggested that the inhibitory effect of fucoidan was mostly due to its interaction with P-selectin. The in vitro experiments demonstrated the high affinity of fucoidan for both isolated P-selectin and P-selectin in plasma membranes of activated platelets.

Lysosomal glycosidases in different populations of human thymocytes.

Lysosomal glycosidase activities were studied in human thymocyte fractions obtained by two methods: (A) fractionation in Percoll density gradient and (B) separation from the cells forming rosettes with sheep erythrocytes (E-RFC). (A) affords fraction L, enriched with immature and endogenously activated thymocytes, and fraction H containing mature thymocytes. By use of (B), fraction E-RFC--enriched with non-activated immature thymocytes--was obtained. Comparative study of E-RFC and H revealed diverse alterations in activities of glycosidases during thymocytes maturation, specifically decreases in alpha-L-fucosidase and alpha-D-mannosidase and an increase in beta-D-galactosidase. Comparing E-RFC and L demonstrates increases in activities of studied glycosidases following endogenous activation of thymocytes.

Isoform patterns of lysosomal glycosidases in phenotypically different leukemic lymphoid cells.

Isoform patterns of six lysosomal glycosidases were studied in leukemic lymphoid cells phenotypically related to B and T cells at distinct stages of differentiation. In all types of cells, the activity of glycosidases under study was expressed in two major isoforms. No correlation was observed between isoform patterns and cell phenotypes. The beta-hexosaminidase isoform ratios for phenotypically related leukemic lymphoid cells but isolated from different sources (blood and spleen) differed. It was suggested that cell localization affects isoform expression. An anomalous alpha-mannosidase was detected in lymphoid cells from lymph nodes, while it was lacking in the phenotypically related blood lymphoid cells from the same patients. Isoform I of beta-hexosaminidase was recorded in lymphoid cells of patients with anemias.

Acidic alpha-D-mannosidase in phenotypically different leukemic lymphoid cells.

The activity of acid alpha-mannosidase in phenotypically characterized lymphoid cells, isolated from peripheral blood, spleen and lymph nodes of patients with various lymphoproliferative disorders has been studied. Cells with different immunophenotypes were shown to have different alpha-mannosidase activity levels. The lowest alpha-mannosidase activity was observed in cells phenotypically corresponding to early B cells obtained from B-CLL patients. The highest activity was determined in cells with phenotypes of activated, CD11c-expressing B cells from B-NHL and HCL patients. There were considerable differences in alpha-mannosidase activity between peripheral blood and spleen lymphoid cells of B-NHL patients with spleen damage. The data obtained may be used in classification, primary diagnosis and staging of hematopoietic malignancies.

Activity of some proteinases and glycosidases in human leukemic lymphoid cells at various stages of differentiation.

Activities of some glycosidases and proteinases in human leukemic lymphoid cells at various stages of differentiation have been compared. It was found that cells with different immunological phenotypes gave different enzymic spectra. Glycosidases and proteinases in lymphoid cell precursors had higher activity level than the enzymes in mature T- and B- cells. In cells of B- lineage, all activities were lower than in common precursor of lymphoid cells. In T-cells at the earlier stages of thymic differentiation, activities of all proteinases and most of glycosidases were higher than in common precursor cells whereas in mature T-helpers and T-suppressors the activities were markedly lower. Most of hydrolases in mature T-cells were twice more active than the enzymes in mature B-cells. The opposite-directional changes in activities of some hydrolases at the earlier stages of differentiation of lymphoid cells along B- or T- cells pathways are suggested.

Studies on some biologically active dextrans.

The relationship between the structures of six native dextrans and their effects on nonspecific resistance to infection (n.s.r.i.) in mice and also anticomplementary activity has been studied. The data obtained showed that the n.s.r.i. activity of dextrans generally increased with increase of extent of branching, but no direct correlation between these two factors was found. Data on exodextranase-catalyzed hydrolysis of dextrans suggest that the length of the outer chains may be important for the n.s.r.i. activity of the dextrans. Dextrans characterized by a significant extent of branching were anticomplementary, but no relationship between extent of branching and anticomplementary activity was observed.