Marzanna Cechowska-Pasko

Enhanced prolidase activity and decreased collagen content in breast cancer tissue

Adherent interactions between integrins and extracellular matrix (ECM) proteins play an important role in tumorigenicity and invasiveness. The major component of ECM is collagen that plays a central role in the interaction with integrins. The expression of certain collagenases (gelatinases) by tumour cells is one of the characteristic features of the so‐called metastatic phenotype, presumably by breaking down ECM barriers as well by altering the ECM–cell interaction. Although extracellular collagenases initiate the breakdown of collagen, the final step of collagen degradation is catalysed by intracellular prolidase. Collagen deposition, gelatinolytic and prolidase activities, expression of β1‐integrin receptor and their possible relationships were studied in seven operable breast cancer cases. In breast cancer tissue, we have found significant decrease in the amount of collagen. The decrease in collagen deposition in breast cancer tissue was accompanied by increase in the tissue gelatinolytic and prolidase activities. Simultaneously, a slight decrease in the expression of β1‐integrin receptor in breast cancer tissue was observed. These results suggest that alteration in collagen metabolism in breast cancer tissue may reflect tissue remodelling, characteristic for invasive phenotype of cancer cells. Increased gelatinolytic and prolidase activities in breast cancer tissue may enhance stromal matrix degradation and thus may promote metastatic dissemination. On the basis of the data, it seems that compounds endowed with gelatinolytic and prolidase inhibitory activities may be considered as a potential drug candidates for breast cancer therapy.

Decrease in the glycosaminoglycan content in the skin of diabetic rats. The role of IGF-I, IGF-binding proteins and proteolytic activity

The results of our previous studies demonstrated that acute streptozotocin-induced diabetes in rats evoked a decrease in skin collagen content with little effect on glycosaminoglycans (GAG) content. In our present study we employed the model of chronic diabetes in order to check its effect on skin GAG content.It was found that the skin of diabetic rats showed a significant decrease in almost all the investigated GAGs by 50-70%. The decrease in heparan sulfate content was slight and statistically insignificant. We sought to determine whether the insulin-like growth factor-I (IGF-1) and IGF-binding proteins (IGF-BPs) levels are altered in animals with experimental diabetes and might contribute to the decrease in tissue GAG content. Circulating IGF-I level was found to be reduced in animals with diabetes and significant changes in serum IGF-BPs were observed. The amount of high molecular weight binding proteins (HMW-BPs) was decreased and the content of low molecular weight binding proteins (LMW-BPs), known as IGF-I inactivating substances, markedly increased. Furthermore, diabetic rats demonstrated an increase of skin proteolytic activity. We conclude that the decrease of GAG content in the skin of diabetic rats is a result of three co-existing phenomena: decreased circulating IGFI level, increased plasma content of LMW BPs and increased proteolytic activity of the skin.

The effect of hypoxia on the expression of 150 kDa oxygen-regulated protein (ORP 150) in HeLa cells.

Correct protein folding is an important factor, for the translocation of newly synthesised proteins to specific subcellular compartments, extracellular matrix or to biological fluids. This process is regulated by a group of specific proteins, referred to as chaperones. Many stress conditions, such as oxygen or glucose deprivation, slow down the folding process and cause accumulation of unfolded/misfolded proteins in the cell. Molecular chaperones are induced in these conditions; with some named as oxygen-regulated proteins (ORPs). These bind to unfolded / misfolded proteins to facilitate correct assembly. ORP 150 is the subject of this study. Hypoxia results in an enhancement of ORP 150 expression in several tumour cell lines cultured in vitro. HeLa cells grown in hypoxic conditions (despite an intensive expression of ORP 150) demonstrate higher rates of apoptosis in comparison to those cultured in normoxic conditions. Furthermore, the inhibition of ORP 150 synthesis by transfection of these cells with a specific siRNA resulted in an intensification of apoptosis, as indicated by specific markers of this process; the enhancement of poly ADP-ribose protein cleavage and the increase in Bim protein expression. We conclude from our study that the increase in ORP 150 synthesis protects the cells against the proapoptotic effect of hypoxia.

Decreased biosynthesis of glycosaminoglycans in the skin of rats with chronic diabetes mellitus

The skin of rats with experimental (streptozotocin-induced) chronic diabetes mellitus exhibits significant decrease in glycosaminoglycans (GAGs) content. In the present study we asked the question whether the decrease in GAGs content is a result of decline in GAG biosynthesis or an increase in their degradation. We demonstrated by a pulse-labeling experiments that diabetes results in a decrease of [14C]-glucosamine incorporation into both hyaluronic acid and sulphated GAGs. During the chase period, there was no significant degradation of the pulse-labeled GAGs, suggesting that the reduction of GAGs content in the skin of diabetic rats is a result of decrease in GAG biosynthesis. Especially the biosynthesis of sulphated GAGs is deeply reduced. This phenomenon may be one of the factors which impairs the wound healing in diabetic subjects.

Glucose-depleted medium reduces the collagen content of human skin fibroblast cultures.

Glucose deprivation appeared to be a factor which induces oxygen regulated protein (ORP) 150 expression in the human skin fibroblasts cultures. Whereas glucose deprivation resulted in a slight (statistically insignificant) decrease of protein content in these cultures, a marked decrease of collagen content was observed, resulting in a distinct reduction of hydroxyproline: protein ratio. Furthermore, the appearance of ORP150 in glucose-deprived cultures coexisted with an increase of gelatinolytic activity and slight reduction in the expression of insulin-like growth factor-I (IGF-I) receptor. Since IGF-I is a main stimulator of collagen synthesis, the reduction in the expression of IGF-I receptor may result in a decrease of collagen synthesis. It is suggested that ORP 150 is a chaperon, which protects intracellular proteins against proteolytic effects exerted by hypoxia or glucose shortage. Since the total amount of protein in fibroblast cultures did not change much, it appears that collagen (in contrast to other proteins) was not efficiently protected. The decrease in collagen synthesis and the enhancement of collagen degradation by gelatinases may result in distinct reduction of collagen content in glucose-deprived fibroblast cultures.

Age-dependent changes in glycosaminoglycan content in the skin of fasted rats.A possible mechanism

It is well recognized that during fasting or aging of animals there is a decreased content of several extracellular matrix components in the skin, including glycosaminoglycans (GAGs) and decrease in biosynthesis of these macromolecules. The mechanism for the phenomena is not known. We considered skin and blood lactate as a potential candidate to control GAG metabolism in tissues. Energetic metabolism, reflected by NAD/NADH and lactate/pyruvate ratios is changed during aging or fasting and lactate inhibits at least some GAGs biosynthesis. Therefore we have compared the level of lactate and the ratios of lactate to pyruvate in the blood and skin of fasted young and fasted adult rats and correlated them with the content of skin glycosaminoglycans. It has been found that the skin of adult rats contains about 60% of GAGs found in the skin of young animals. Fasting of both groups of animals resulted in further decrease in skin GAG content. GAG content in the skin of fasted young animals was decreased by 30% while in fasted adult rats no significant differences were observed, compared to fed animals. Lactate concentration was found to be increased over 2-fold in the skin of young fasted rats, compared to young controls. The lactate concentration in adult animals was not changed during fasting, although in both cases the lactate levels were almost 3-fold higher than in young control rats. In blood, lactate concentration increased by 40% during fasting of young animals while it decreased by about 40% during fasting of adult rats. Although no differences were found in blood lactate level between young and adult rats, the ratio of lactate/pyruvate was decreased by over 2 fold in adult rats. The relative differences in mean GAG content in the skin of all experimental groups of animals were related to the similar differences in blood glucose and lactate/pyruvate ratio. Therefore not only skin lactate but also blood lactate concentrations may reflect the extent of skin GAG biosynthesis. We have noticed that increase in the ratio of skin lactate/pyruvate concentration and decrease of the ratio in the blood is accompanied by decrease in the skin GAG content. We suggest that the phenomenon may result from utilization of lactate into glucose in the Cori cycle which regulate glucose availability for GAG biosynthesis. Therefore it can be suggested that lactate may participate in inhibition of skin GAG biosynthesis and the extent of the inhibition is reflected by the ratio of lactate/pyruvate concentrations both in the skin and blood.

Hypoxia Enhances the Senescence Effect of Bortezomib—The Proteasome Inhibitor—On Human Skin Fibroblasts

The 26S proteasome inhibitor, bortezomib, selectively induces apoptosis in some cancer cells. However, the nature of its selectivity remains unknown. The study presented here provides novel information on cellular effects of bortezomib in normal fibroblasts. We have found that in normoxic conditions the percent of apoptotic cells did not change significantly, independently on incubation time and examined concentration of bortezomib (25 nmol/L or 50 nmol/L). In hypoxic conditions we did not observe any effect of bortezomib on apoptosis of fibroblasts incubated for 24 h and 48 h in comparison to control. Only in the case of fibroblasts incubated for 12 hours in hypoxia significant increase in apoptosis, dependent on concentration of bortezomib, was observed. Our study has shown that bortezomib causes a time-dependent increase in senescence of normal fibroblasts, especially of these incubated in hypoxic conditions. Moreover, we demonstrated that oxygen regulated protein 150 (ORP150) expression was induced in fibroblasts in hypoxia conditions only, suggesting that this protein may play an important role in the cytoprotective response to environmental stress.

Metronidazole Decreases Viability of DLD-1 Colorectal Cancer Cell Line

The aim of our study was to evaluate the impact of metronidazole (MTZ) on DLD-1 colorectal cancer cell (CRC) line. Toxicity of MTZ was determined by MTT test. Cells were incubated with MTZ used in different concentrations for 24, 48, and 72 hours. The effect of MTZ on DNA synthesis was measured as [3H]-thymidine incorporation. The morphological changes in human DLD-1 cell line were defined by transmission electron microscope OPTON 900. The influence of MTZ on the apoptosis of DLD-1 cell lines was detected by flow cytometry and fluorescence microscopy, while cell concentration, volume, and diameter were displayed by Scepter Cell Counter from Millipore. Our results show that cell viability was diminished in all experimental groups in comparison with the control, and the differences were statistically significant. We did not find any significant differences in [3H]-thymidine incorporation in all experimental groups and times of observation. Cytofluorimetric assays demonstrated a statistically significant increase of apoptotic rate in MTZ concentrations 10 and 50 μg/mL after 24 hours; 0.1, 10, 50, and 250 μg/mL after 48 hours; and in all concentrations after 72 hours compared with control groups. In the ultrastructural studies, necrotic or apoptotic cells were occasionally seen. In conclusion, MTZ affects human CRC cell line viability. The reduction of cell viability was consistent with the apoptotic test.

Glycosaminoglycan-degrading enzymes in the skin of fasted rats

During fasting of animals, there is decreased content of skin glycosaminoglycans (GAGs) accompanied by decrease in their biosynthesis. Since tissue GAG content depends on both synthesis and degradation of these molecules, we asked whether fasting affects the activity of several tissue glycosidases. Therefore we measured the activity of skin neutral and acidic endoglycosidases, some exoglycosidases: beta-N-acetylhexosaminidase [EC 3.2.1.30], beta-galactosidase [EC 2.1.23], beta-glucuronidase [EC 3.2.1.31], alpha-iduronidase [EC 3.2.1.76], and two sulfatases: arylsulfatase B [EC 3.1.6.1] and 6-sulfatase [EC 3.1.6.14] in the skin of control and fasted rats. Although fasting was accompanied by distinct decrease in the activity of most neutral endoglycosidases, no characteristic changes in the activity of exoglycosidases were found. In contrast, we found that fasting is associated with increase in the activity of acidic endoglycosidases (of lysosomal origin) which degraded hyaluronic acid, chondroitin-4-sulfate, chondroitin-6-sulfate and heparin. The same GAGs were decreased in the skin of fasted rats. Our data suggest that the phenomenon is a result of increased intracellular degradation of these molecules. Therefore, not only decreased biosynthesis of GAGs during fasting, but also increased their intracellular degradation may contribute to decrease in GAG skin content.

Differential effect of fasting on IGF-BPs in serum of young and adult rats and its implication to impaired skin GAG content

During fasting or aging of animals there is a decreased content of skin glycosaminoglycans (GAGs). It has been found that the skin of adult rats contains about 60% of GAGs found in the skin of young animals. Fasting of both groups of animals (young and adult) resulted in decrease of GAG content. However, GAG content in the skin of fasted young rats decreased by 30% and in fasted adult rats by 15% only, compared to fed animals, respectively. The mechanism for the phenomena is not known. We considered insulin-like growth factor-I (IGF-I) as a potential candidate involved in regulation of GAG biosynthesis in both experimental models of animals. Adult rat sera were found to contain about 75% of IGF-I recovered from young rat sera. Fasting of both groups of animals resulted in dramatic decrease in serum IGF-I levels to about 50% of initial values. Since IGF-I activity and IGF-I serum half-life depends on the level of specific IGF-binding proteins (IGFBPs) we determined (i) relationship between main groups of IGFBPs, namely high molecular weight binding proteins (HMWBPs) and low molecular weight binding proteins (LMWBPs) and (ii) the amounts of IGF-I bound to respective proteins in the sera of all experimental animals. Control young rat serum was found to contain about 90% of HMWBPs and about 10% of LMWBPs as determined by ligand binding assay. In contrast, control adult rat serum contained about 60% of HMWBPs and about 40% of LMWBPs. Fasting of both groups of animals resulted in significant increase in serum levels of LMWBPs. Control young rat serum was found to contain about 8% IGF-I bound to LMWBPs while serum of control adult rats contained 18% IGF-I bound to these proteins. In sera of fasted young animals however, about 75% of the bound IGF-I was recovered from LMWBPs (about 60% of total serum IGF-I) while in sera of fasted adult animals only about 56% of the bound IGF-I was recovered from LMWBPs (about 50% of total serum IGF-I). Evidence was provided that during fasting of both groups of animals there is a significant decrease in serum BP-3 and dramatic increase in serum BP-1 concentrations, compared to respective controls. However, the concentration of BP-1 in serum of fasted young rats was increased by about 60 fold while in serum of fasted adult rats only by about 10 fold, compared to respective control animals. Negative correlation between skin GAG content and LMWBPs derived IGF-I during fasting of young (r = - 0.943, p < 0.001) and adult ( r = - 0.571, p < 0.01) rats was found.The data presented suggest that the effects of aging and fasting on decreased skin GAG content may be due to induction of LMWBPs that are known to (i) inhibit IGF-I dependent function and (ii) increase clearance of IGF-I from circulation. However, the effects of fasting are distinct in respect to young and adult rats suggesting that mechanisms involved in regulation of IGF-I bioactivity during aging are more complex that during fasting.