Biomedical subjects

P Poucková

Publications and source records attributed to P Poucková.

At least 19 recordsLinked to original sources

PubMed2006

Proteinase-activated receptor-2 expression in breast cancer and the role of trypsin on growth and metabolism of breast cancer cell line MDA MB-231.

Proteinase-activated receptor-2 (PAR-2) is a ubiquitous surface molecule participating in many biological processes. It belongs to the family of G protein-coupled receptors activated by the site-specific proteolysis of trypsin and similar proteases. Altered function of PAR-2 has been described in different malignant tumors. In the present study, we investigated the expression of PAR-2 in breast cancer surgical specimens and the role of trypsin in breast cancer cell line MDA MB-231 proliferation and metabolism. A total of 40 surgical samples of infiltrative ductal breast cancer and breast cancer cell line were included in this study. We analyzed PAR-2 expression by immunohistochemistry, RT-PCR and western blot. Activation of PAR-2 on cell line MDA MB-231 was measured using calcium mobilization assay determined by flow cytometry. MTT cell metabolism assay and cell count analysis were used to assess the trypsin influence on breast cancer cell line MDA MB-231 proliferation. Immunohistochemical examination showed the expression of PAR-2 in all samples of breast cancer surgical specimens and high levels of cell lines which was confirmed by RT-PCR and western blot. Calcium mobilization assay corroborated the activation of PAR-2 on cell line MDA MB-231 either by trypsin or by an agonistic peptide. Cell metabolism assay and cell count analysis showed significant differences of proliferative activity of breast cancer cells dependent on the presence or absence of trypsin and serum in the culture medium. PAR-2 is expressed by high levels in infiltrative ductal breast cancer tissue specimens. PAR-2 is also strongly expressed in studied breast cancer cell lines. PAR-2 is activated by trypsin and also by agonistic peptide in the model of breast cancer cell line MDA MB-231. Activation of PAR-2 in vitro influences proliferative and metabolic activity of breast cancer cell line MDA MB-231. The action of trypsin is modified by the presence of serum which is a potential source of protease inhibitors.

Blotting, Western↗

PubMed2006

Mung bean sprout (Phaseolus aureus) nuclease and its biological and antitumor effects.

Bovine seminal ribonuclease (BS RNase), a dimeric homolog of bovine pancreatic ribonuclease (RNase A), is known to display special biological activities namely cytotoxicity for human tumor cells. Because some plant ribonucleases have a similar mass weight and structure as the animal ribonuclease, effects of a commercial product of Mung bean (Phaseolus aureus) nuclease (PhA) were studied on proliferation of ML-2 human tumor cells, as well as it's aspermatogenic, embryotoxic, immunogenic, and immunosuppressive activity, and therapeutic efficiency in athymic mice bearing human melanoma tumor. Concerning the antiproliferative activity, PhA nuclease was almost non-effective in vitro on ML-2 cells and also immunosuppressive activity on human lymphocyte in mixed culture was very low compared to that of BS RNase. However, significant antitumor activity was detected on human melanoma tumor after intratumoral or intraperitoneal administration into the mice. Furthermore conjugate of PhA nuclease with polyethylene glycol (PEG) injected seven times at the dose of 10 microg intraperitoneally showed identical antitumor activity as that of bovine seminal ribonuclease (BS RNase) injected by the same way at ten times higher dose. Both PhA and BS RNases exerted strong aspermatogenic effect on the width of spermatogenic layers while RNase A administration at ten times higher concentration was ineffective. PhA nuclease when compared by means of antibody cross reaction with RNase A, BS RNase and wheat leaf neutral RNase (WLN-RNase) was found to be immunologically similar to RNase A and WLN-RNase, meanwhile BS RNase showed much higher antigenicity in comparison with them.

Animals↗

PubMed2004

Polymer-conjugated bovine pancreatic and seminal ribonucleases inhibit growth of human tumors in nude mice.

The hydrophilic poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) was used for RNase A or BS-RNase modification to prevent their degradation in bloodstream or fast elimination. Two PHPMA chains (classic and star-like) were synthesized and their conjugates with both enzymes were tested on the CD-1 nude mice bearing various human tumors. These RNase conjugates injected intravenously or intraperitoneally into the mice bearing melanoma, neuroblastoma or ovarian tumor caused significant reduction of transplanted tumors following ten daily doses of 2.5 and/or 1 mg/kg, respectively, while free RNase A or BS-RNase injected in doses of 10 mg/kg exerted only negligible antitumor activity. Histological examination confirmed potent cytotoxic effect of RNase A conjugates in ovarian tumor. Despite the antitumor activity observed in vivo, the in vitro cytotoxic activity of RNase A conjugates was not pronounced and did not differ from that caused by the free RNase A. The in vitro experiments with 125I-labeled preparations demonstrated that polymer conjugates were internalized by tumor cells very poorly in contrast to the dose-dependent internalization of the wild enzyme preparation. Surprisingly, mice injected with EL-4 leukemic cells, which were preincubated for 4 h with BS-RNase conjugates, exerted significantly prolonged survival compared with the control non-treated mice. It may be supposed that both BS-RNase and RNase A conjugates with PHPMA act after administration in vivo by a mechanism different from that or those occurring under in vitro conditions because in vivo they exert an antitumor action, whereas in vitro, they are ineffective. The experiments proved that RNase A, when conjugated to PHPMA, produced identical aspermatogenic and antitumor effects as BS-RNase conjugated to this polymer and that this preparation may be regarded as a potential anticancer drug.

Animals↗

PubMed2002

Poly[N-(2-hydroxypropyl)methacrylamide] conjugates of bovine pancreatic ribonuclease (RNase A) inhibit growth of human melanoma in nude mice.

Recently hydrophilic poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) was used for BS-RNase modification to prevent its degradation in bloodstream or fast elimination. Polymer-conjugated BS-RNase preparations proved to be cytotoxic after intravenous or intraperitoneal application, whereas native BS-RNase was ineffective. Here RNase A unimer was conjugated with two HPMA polymers (classic and star) and their antitumor effects both in vitro and in vivo were compared with those of BS-RNase polymers. Surprisingly, the antitumor effect of RNase A conjugates was also pronounced. The RNase A conjugates (classic and star) injected intravenously to mice bearing melanoma tumor caused a significant reduction in tumor volume following ten doses of 5 and 1 mg/kg, respectively. Despite the antitumor activity observed in vivo, the in vitro tested cytotoxic activity of RNase A did not differ from that caused by native RNase A while native BS-RNase (50 microg/ml) totally inhibited DNA synthesis in treated cells. The experiments with 125I-labeled preparations demonstrated concentration-dependent internalization of native BS-RNase by tumor cells within an hour, whereas the polymer conjugate (S-BS) was not internalized. On the contrary, the in vivo experiments showed that whereas 40% of S-BS conjugate persisted in bloodstream for 24h after administration, 98% of the native BS-RNase was already eliminated. Improved antitumor activities of PHPMA-modified RNases in vivo might be ascribed to their prolonged retention in bloodstream, better proteolytic stability and resistance to the action of the ribonuclease inhibitor.

Animals↗

PubMed2002

Proteinase-activated receptor-2 expression on cerebral neurones after radiation damage: immunohistochemical observation in Wistar rats.

Radiation damage results in blood-brain barrier damage followed by blood plasma transfer into the neuropil. The transferred liquid contains high amounts of biologically active substances/proteinases including factor Xa and a free pool of serum trypsin, which is not bound to antiproteases (alpha1 AT, alpha2-macroglobulin). The aim of this study was to follow up expression of proteinase-activated receptor-2 (PAR-2) in the brains of Wistar rats after single exposure to radiation at 26 Gy (60Co, 23 min, 15 sec). After irradiation, the animals were sacrificed on days 10, 20, 30 and 40. Control rat brains served as negative control. Coronal sections of caudal diencephalons were investigated using histology and immunohistochemistry. Polyclonal goat specified antibody against the NH-end of murine and rat PAR-2. Significant PAR-2 membrane positivity of scattered swollen neurons in deeper cortical layers was found in irradiated animals compared with controls. Although this membrane positivity was noticed in all irradiated animals, the most prominent occurred on day 30. Diffuse cytoplasmic positivity was also demonstrated on shrunken neurons in the cortex and hippocampus. Increased cytoplasmic and polarized membrane positivity was also noticed on the neurons of hypothalamic nuclei The causal relationship between blood-brain barrier damage, PAR-2 activation and neurodegeneration has not yet been verified. However, the present findings indicate that PAR-2 mediates a certain cellular response. It remains to be demonstrated whether this is a response to higher concentrations of factor Xa, a free pool of trypsin or other unknown possible proteinases in brain tissue; whether changes in PAR-2 expression are consequences of direct radiation damage to neuronal cells; whether this reaction is protective; and whether primary PAR-2 activation results in neuronal damage.

Animals↗

PubMed2001

Mixture of trypsin, chymotrypsin and papain reduces formation of metastases and extends survival time of C57Bl6 mice with syngeneic melanoma B16.

PURPOSE: The aim of the present study was to investigate the effect of a mixture of proteolytic enzymes (comprising trypsin, chymotrypsin and papain) on the metastatic model of syngeneic melanoma B16. METHODS: 140 C57B16 mice were divided into two control and two "treated" groups. Control groups received saline rectally, twice a day starting 24 h after intracutaneous transplantation (C1) or from the time point of the primary B16 melanoma extirpation (C2), respectively. "Treated" groups were rectally administered a mixture of 0.2 mg trypsin, 0.5 mg papain, and 0.2 mg chymotrypsin twice daily starting 24 h after transplantation (E1) or after extirpation of the tumor (E2), respectively. Survival of mice and B16 melanoma generalization were observed for a period of 100 days. Immunological evaluation of B16 melanoma cells in the ascites was accomplished. CD44, CD54 and CD106 cells were measured by flow cytometry. RESULTS: Administration of proteolytic enzymes to mice inhibited the growth of primary tumors, and tumor recurrences were less numerous. Importantly, metastasis was considerably curtailed both in the vicinity of the primary tumor and at distant locales. These findings correlated with a decreased expression of CD44 and CD54 molecules in tumors exposed to proteolytic enzymes in vivo. CONCLUSIONS: Our data suggest that serine and cysteine proteinases suppress B16 melanoma, and restrict its metastatic dissemination in C57B16 mice.

Administration, Rectal↗

PubMed2001

Expression of proteinase-activated receptor 2 during taurocholate-induced acute pancreatic lesion development in Wistar rats.

BACKGROUND: Proteinase-activated receptor 2 (PAR-2) is a G-protein coupled transmembrane receptor activated by trypsin by site-specific cleavage. Its presence on pancreatic structures was demonstrated in the past. PAR-2 physiologically involves in duct/acinary cells secretion, arterial tonus regulation or capillary liquid turnover. During development of acute pancreatitis/acute pancreatic lesion (APL) these mentioned structures are influenced by very high concentration of trypsin due to its increased basolateral secretion into the interstitium. The aim of our study as presented was to investigate whether PAR-2 is also involved in APL following changes of PAR-2 expression. METHODS: APL was investigated in Wistar rats after injection of 0.1 mL taurocholate into the ductus choledochus. Anatomy, histology, reverse transcriptase polymerase chain reaction (RT PCR) as well as immunohistochemistry and Western-blot analysis of pancreatic tissue were performed using antibody mapping of the new NH2 terminal of PAR-2 after trypsin cleavage. Results from control rats and d 1 or d 4 rats after taurocholate injection were compared. RESULTS: Much higher positivity on acinary/duct cells was observed in APL induced animals than in controls. Similar findings were noticed on arterial smooth muscle cells. Surprisingly, parallel to the exocrine pancreas and vessel findings, enhanced Langerhans' islet cell positivity was observed in experimental animals. CONCLUSIONS: Based on these results, we have demonstrated that during APL development PAR-2 expression increases. This effect is caused by conformational changes after PAR-2 activation, and the new NH2 terminal of activated receptor presentation. We suggest that PAR-2 physiological functions are enhanced during APL development.

Acute Disease↗

PubMed2001

Production of TGF-beta1 in lungs after low-dose whole-body radiation exposure in fibrosing (C57BL/6) and non-fibrosing (C3H/J) mouse strain.

The aim of the presented study was to observe acute and subacute discrete TGF-beta1 production after a low-dose whole-body radiation stimulus, known to induce thrombocytopenia. TGF-beta1 mRNA production and the number of thrombocytes was followed up in two mouse strains with different tendencies to the origination of fibroses. Mice of the C57BL/6 and C3H/J strains were exposed to a whole-body dose of 7 Gy. Non-irradiated mice of both strains were used as negative controls. The relative number of thrombocytes recorded in lung capillaries was significantly lower in both strains on day 9 after irradiation in comparison with controls. This finding was in accordance with a decrease in the number of thrombocytes in the peripheral blood in irradiated animals of both strains. On day 56 relative platelet counts reached physiological numbers in comparison to controls. On the other hand, TGF-beta1 mRNA production was higher in the C57BL/6 strain (on day 9) contrary to minimal production in the C3H/J strain (on day 9) or no production in both groups on day 56 and in controls. Thus, TGF-beta1 production without increased thrombocyte trapping in lung vessels in acute stage suggests that an additional mechanism is involved in low-dose radiation-induced cytokine synthesis in lung tissue besides the release of growth factors from thrombocytes.

Animals↗

PubMed2001

Polymer conjugated bovine seminal ribonuclease inhibits growth of solid tumors and development of metastases in mice.

Bovine seminal ribonuclease (BS-RNase) exerts a potent cytotoxic activity when administered intratumorally (i.t.) to the nude mice bearing human tumors. The ineffective treatment with intravenous (i.v.) or intraperitoneal (i.p.) administration led us to the synthesis of polymeric conjugates with BS-RNase to prevent it from degradation in the blood vessel. Hydrophilic poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) was used for BS-RNase modification and a PHPMA-BS-RNase conjugates were prepared. Classic conjugate (P-BS) with BS-RNase bound to the polymer by its oligopeptide site chains was prepared by aminolytic reaction of the polymer precursor bearing reactive ester groups situated in the side chains of polymer, while star-like conjugate (S-BS) was synthesized by the reaction of PHPMA containing end-chain reactive group with BS-RNase in aqueous buffer solution at pH 8. In contrast to the total ineffectiveness of free BS-RNase administered i.v. at a daily dose 10 mg/kg, application of P-BS and S-BS conjugates at doses 2 mg/kg and 0.5 mg/kg caused significant inhibition of the growth of human melanoma in nude mice. On the base of these results the effect of i.v. administered S-BS on the metastatic process and the survival of C57Bl/6 inbred mice inoculated with B16 melanoma cells was investigated. Sixty per cent of mice treated with S-BS (0.5 mg/kg/day) survived 100 days without metastatic foci when the experiment terminated. The average survival time of the treated groups was 75.5 days compared to 32.7 days in the control group. BS-RNase conjugated to water soluble polymers appears to be the first BS RNase preparation which exerts anticancer and antimetastatic activity following its intravenous administration.

Animals↗

PubMed2000

Butylaminiperindopril decreases transforming growth factor-beta 1 messenger RNA production in lungs of C57Bl6 mice after low-dose whole-body irradiation.

Transforming growth factor (TGF)-beta is believed to play a key role in the development of many autoimmune and malignant diseases, such as radiation and drug-induced organ disease. The aim of the present study was to determine messenger RNA (mRNA) production of TGF-beta 1 in the lungs of C57Bl6 mice after low-dose whole-body irradiation. Control (irradiated) and irradiated angiotensin-converting enzyme (ACE) inhibitor-treated animals were simultaneously examined. The ACE inhibitor group received butylaminiperindopril for 9 days after irradiation (7 Gy) at a daily dose of 0.1 mg/kg per rectum. On day 9 all mice were sacrificed and the production of mRNA TGF-beta 1 in lung tissue was determined semiquantitatively using reverse transcriptase polymerase chain reaction. In butylaminiperindopril-treated mice, a decrease in transcript of TGF-beta 1 (to 59% in comparison with controls) was observed.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed1998

Antitumor action of bovine seminal ribonuclease.

Unlike the bovine pancreatic ribonuclease (RNAase A), bovine seminal ribonuclease (BS RNAase) displays various biological activities including antitumor cytotoxicity. To learn more about its antitumor activity, we investigated BS RNAase effect on athymic nude mice bearing various tumors. BS RNAase (250 micrograms per mouse per day) was administered to the mice with prostate carcinoma for three weeks by three different routes (intraperitoneally--i.p., subcutaneously--s.c., and intratumorally-i.t.). Administration i.p. was ineffective, while s.c. administration reduced significantly size of tumors and i.t. administration abolished half of the tumors in treated mice. The i.t. administration of BS RNase to nude mice bearing melanoma showed even better results. Eighty % of mice were without tumors and in the other mice the tumors were significantly diminished. The best antitumor effect was obtained in case of seminoma. All mice bearing this tumor were cured after ten doses of BS RNAase.

Animals↗

PubMed1998

Proteinases reduce metastatic dissemination and increase survival time in C57Bl6 mice with the Lewis lung carcinoma.

The effect of combined proteolytic enzymes, administered by the rectal route, on the metastatic process and the time of survival in C57Bl6 mice with the Lewis lung carcinoma inoculated subcutaneously was investigated. In the control group, which received no enzyme treatment, 90% of animals died of the metastatic spread of cancer by day 18 after primary tumor extirpation. In Group A, which received the multi-enzyme solution from the time of primary tumor extirpation, 30% of mice died of disseminated cancer by day 25. In Group B, which was treated with the enzymes from 6 days before primary tumor extirpation, only 10% of animals showed the metastatic process by day 15. In Group C, which received the enzymes from 24 hours after intracutaneous tumor inoculation, no metastatic dissemination was discernible. In these three groups, the enzyme treatment was carried out throughout the study. None of the control animals survived for 100 days when the study was ended. The treated groups A, B and C showed survival rate 60%, 90% and 100% of animals, respectively, by 100 days.

Administration, Rectal↗

PubMed1998

Polyenzyme preparation Wobe-Mugos inhibits growth of solid tumors and development of experimental metastases in mice.

Long-term rectal administration of enzyme mixture containing papain, trypsin and chymotrypsin in the same ratio as the preparation Wobe-Mugos E (Mucos Pharma, Germany) was evaluated for their antitumor effects in C57Bl6 inbred mice inoculated with Bl6 melanoma cells. 30% of animals in the test group (3 pcs) have been cured of cancer. In the rest of animals (70%) the survival time was prolonged by 58.3% compared to the control group (from average survival time of 24 days in control group to 38 days in the test group). Based on histological and immunohistochemical evaluation a faster process of metastasizing was found in control group than in the group treated with the polyenzyme preparation. In the case of melanoma Bl6 an antimetastatic effect of the preparation was thus proved.

Animals↗

PubMed1998

Antitumor action of bovine seminal ribonuclease. Cytostatic effect on human melanoma and mouse seminoma.

This paper reports on the antitumor activity of BS RNase on human melanoma and mouse seminoma. Human melanoma cells established in culture were extremely susceptible to BS RNase, administered in concentrations ranging from 1-100 microg/ml. Concentrations of BS RNase over 10 microg/ml caused complete inhibition of cell growth. Bovine pancreatic ribonuclease (RNase A), a prototype of the ribonuclease superfamily, did not exert any effect under these conditions. Based on our previous results, athymic mice bearing human melanoma or mouse seminoma were treated with intratumoral administration of BS RNase (12.5 mg/kg b.w.). This dose was injected for five consecutive days excluding weekends. The intratumoral administration of BS RNase to nude mice bearing human melanoma showed a significant antitumor effect. There were no tumors seen in eighty percent of mice treated for three weeks, and tumors in the other mice diminished significantly. After some delay the tumors started to regrow. Prolonging of the treatment to five weeks had a similar effect. The effect of BS RNase on mouse seminoma was well pronounced. Five to seven doses of BS RNase were sufficient to eliminate tumors in all treated mice. However, as in the previous experiment, the growth of tumor tissue later reappeared.

Animals↗

PubMed1997

Distribution and photodynamic effect of zinc phthalocyanine disulfonate in nude mice bearing mammary carcinoma.

The distribution study of zinc phthalocyanine disulfonate (ZnPcS2) in nude mice bearing mammary carcinoma (T50/80) revealed a rapid uptake of the dye by tumor. In experimental photodynamic therapy (PDT), the tumors were exposed to laser radiation (670 nm, 100 mW/cm2, 150 J/ /cm2) after intravenous administration of ZnPcS2 in saline. The results showed the maximum tumor destruction to be achieved for the time interval between injection of the drug (2 mg/kg) and exposure to laser light of 5 min, while a significantly less damage was observed when the time interval was 24 h (p < 0.0001). The degree of damage produced by the treatment was monitored in vivo by means of noninvasive NMR-imaging and subsequently confirmed histologically.

Animals↗

PubMed1996

Antitumor action of bovine seminal ribonuclease.

Unlike the bovine pancreatic ribonuclease (RNaseA), bovine seminal ribonuclease (BS RNase) displays various biological activities, including antitumor activity, immunosuppressivity, spermatogenicity and embryotoxicity. To learn more about its antitumor effect we tested BS RNase on the growth of 16 cell lines derived from patients with various hematological malignancies. The cells of lymphoid origin were generally more susceptible to BS RNase, administered in the range of concentrations from 2 to 100 micrograms/ml, than the myeloid ones. RNaseA used at the same concentrations did not exert any inhibitory effect. The inhibitory effect of BS RNase persisted in cultured cells after three times wash in complete medium and cell recultivation in fresh medium free of BS RNase. Four cell lines were very little sensitive (KG-1 and U-937) or resistant (JOK and NAMALWA) to BS RNase regardless of their origin. The in vivo antitumor effect of BS RNase was tested on human prostate carcinoma transplanted to athymic nude mice. The daily dose of BS RNase (0.25 mg/20 g) was administered for three weeks except weekends (15 doses) by three different ways (intraperitoneally-i.p., subcutaneously-s.c. and intratumorally-i.t.). Whereas i.p. administration was ineffective, s.c. administration significantly reduced size of the tumors and i.t. administration abolished half of the tumors in treated mice. The average weight of treated mice decreased during the experiment by 10-15%.

Animals↗

PubMed1995

Meso-tetra-(4-sulfonatophenyl)-porphine of low neurotoxicity.

The acute toxicity of specially purified meso-tetra-(4-sulfonatophenyl)-porphine (TPPS4) was measured in mice DBA/2 and Wistar rats after i.v. administration of the dye dissolved in saline solution. LD50 of our preparation was 352.5 mg/kg for mice and 574.9 mg/kg for rats. Motor nerve conduction velocity was measured in rats and rabbits in ether anesthesia N. ischiadicus and n. tibialis were stimulated by surface electrodes and M response was registered. No signs of neurotoxicity were found, even after application of 150 mg/kg b.w. of our TPPS4. Histologic evaluation was performed by routine method and standard electron microscopic procedure. Only minimal and sporadic axonal changes were found. The clinical application of low toxic TPPS4 was reconsidered.

Animals↗

PubMed1992

[Relation between lipid-bound sialic acid in blood serum and plasma andin human tumors].

Athymic mice with transplanted osteosarcoma and carcinoma of the rectum were found to have increased blood plasma levels of lipid-bound sialic acid (LSA). To verify the applicability of the method of LSA determination, patients with cancer of the mammary gland, rectum, and colon were examined for their LSA level. The serum LSA level was significantly increased in patients with cancer of the mammary gland and rectum, compared to levels determined in the serum of healthy volunteers. The serum LSA level elevation was even more pronounced in patients with carcinoma of the colon. In patients with carcinoma of the colon who were in remission at the time of blood collection the serum LSA level was found to be reduced to control values.

Animals↗