Differential inhibition of single and cluster type tumor cell migration.

For the control of tumor metastasis it is important to identify chemical compounds with antimigratory potency. Agents acting against single cell and cluster type migration are necessary for successful antimetastatic therapy. In the present study, the migration of HT-1080 fibrosarcoma cells and OSCORT osteosarcoma cells was compared in a Boyden chamber and in an extracellular matrix (ECM)-based three-dimensional cell culture (3-DCC) model system. The Boyden chamber offers a model of single tumor cell migration, whereas the 3-DCC model system demonstrates invasive growth in the form of a cluster. Since PD98059 (MEK inhibitor) exclusively reduced migration in the 3-DCC model, it may be plausible that the ERK/MAPK signaling pathway is essential for cluster type migration. Interestingly, single cell migration was stimulated upon blocking phosphatidylinositol 3-kinase (PI3K) and also p38-MAPK by treatment with LY294002 and SB203580 respectively. A remarkable reduction of single cell migration was observed following treatment with okadaic acid, a phosphatase 1 (PP1) and 2A (PP2A) inhibitor, which was rather intriguing. This study provided evidence that certain cytotoxic/cytostatic agents at appropriate concentrations were able to preferentially inhibit certain types of migration relative to cell proliferation. Single cell migration was selectively inhibited by taxol at very low subtoxic concentration, whereas 5-hexyl-2'-deoxyuridine (HUdR) exclusively inhibited the cluster type of migration. The borrelidin compound was able to inhibit both types of tumor cell migration, but single tumor cell migration was much less affected. It is interesting that migration was more reduced than proliferation by borrelidin, especially at the advanced growth stage. Taxol is recommended as an agent acting against single cell migration, as well as HUdR and borrelidin as leading compounds for developing antimetastatic drugs against cluster type migration.

Extracellular matrix induces doxorubicin-resistance in human osteosarcoma cells by suppression of p53 function.

BACKGROUND: Osteosarcoma is the most common primary malignant bone tumor in childhood and adolescence. The several chemotherapy-resistant cases of osteosarcoma are at a higher risk of relapse and adverse outcome. OBJECTIVE: The aim of the current study was to determine the role of extracellular matrix in the resistance developed against chemotherapeutic treatments of human osteosarcoma cells. MATERIALS AND METHODS: A cell line, named OSCORT was established from the biopsy of a 17-year-old male patient with primary osteosarcoma. Cell proliferation, apoptosis and quantification of DNA damage after treatments with doxorubicin were investigated in classical and three-dimensional cell culture systems using an extracellular matrix gel. The experimental results were related to the clinical observations of the case. RESULTS: The cells cultured in extracellular matrix gel have shown resistance to doxorubicin similar to that seen in the clinical case, as demonstrated by their proliferation, apoptosis and doxorubicin-induced DNA damage characteristics. Among the extracellular matrix components, the heparan sulfate proteoglycan and-to a lesser extent-fibronectin were involved in the doxorubicin resistance. Laminin and nidogen did not decrease the cytoreductive effect of doxorubicin, while collagen IV even increased it. The extracellular matrix gel decreased the protein levels of p53 and abrogated its cell nuclear translocalization. The most frequent known mutations in the p53 gene were not found in OSCORT cells. CONCLUSION: The current study provides experimental evidence for an epigenetical, extracellular matrix-induced loss of p53 function, which lead to a potent chemotherapy resistance showing accordance with the clinical experience.

[Study of drugs against neoplastic metastasis]. / A tumorok áttétképzését korlátozó hatóanyagok kutatása.

Further progress in the therapy of malignant diseases is expected from the introduction of potent antimetastatic drugs. Surveying of the complex and multi-step behavior of the metastatic process, compounds showing inhibitory action against tumor cell migration may be ranked among the promising antimetastatic agents. Our present study indicate, however, that the antimigratory actions of certain antitumor drugs (doxorubicin, taxol), and inhibitors of signal transduction (PD-98059, LY-294002, SB-203580) are highly dependent on the assay applied (Boyden-chamber, 3D ECM cell culture). It has been proposed that agents interrupting cell-extracellular matrix contacts (hexyldeoxyuridine, borrelidin) and others interfering with the regulatory mechanism of gene translation (rapamycin, ribavirin) could be regarded as leading compounds in the antimetastatic drug development process. Nevertheless, for introducing diagnostically based targeted therapy the forthcoming tasks must include the further elucidation of the molecular mechanisms implicated in the amoeboid and cluster type of cell migration.

[Role of syndecan-1 proteoglycan in the invasiveness of HT-1080 fibrosarcoma]. / A syndecan-1 proteoglikán hatása a HT-1080 fibroszarkóma invazivitására.

Syndecan-1 is a transmembrane heparan sulfate proteoglycan which plays pivotal role in cell-cell and cell-extracellular matrix interactions. However, its implication in the establishment of malignant phenotype is still controversial. Its expression indicates differentiated phenotype in certain tumors, while it confers invasive nature for others. For the better understanding of the role of syndecan-1 in cancer we transfected HT-1080 fibrosarcoma cell line with the full and a truncated construct of syndecan-1 and established stable cell lines with them. We studied the in vitro and in vivo growth capacity and metastatic potential of the transfectants in comparison with the cell line bearing only the EGFP expression vector. Our results showed that the growth rate of syndecan transfectants increased and they developed more lung metastases than the control cells. As local growth of the full transfectant was faster than that of the 78sig we presume that the full protein and maybe the shedding is needed for the local development of the tumor, but the intracellular and transmembrane domain is sufficient to promote metastasis formation.

Repopulation of osteosarcoma cells after treatment with doxorubicin in the presence of extracellular matrix biopolymers.

PURPOSE: To elucidate the role of extracellular matrix (ECM) in repopulation capacity of osteosarcoma cells after doxorubicin treatment. METHODS: OSCORT cells established in our laboratory from a human osteosarcoma, were treated with doxorubicin in monolayer for 4 h, then cells were further incubated either in monolayer or in ECM-containing three-dimensional cell-culture (3-DCC), apoptosis induction and changes in cell number were measured. Alkaline comet assay was applied to estimate DNA damage, immunoblot technique and immunocytochemistry were used to investigate p53 protein synthesis, and the repopulating capacity in monolayer culture and in ECM-based 3-DCC, after doxorubicin treatment was measured. In addition to OSCORT culture five other human cell lines (HT-1080, PC-3, MDA-MB231, A-431 and ZR-75-1) were used to compare the antimigratory and antiproliferative effects of doxorubicin. RESULTS: The apoptotic index, the extent of DNA damage and the representation of p53 were much lower in the OSCORT cell cultures if the cells were exposed to ECM after treatment with doxorubicin. The doxorubicin-treated OSCORT cells transferred from the monolayer culture were not able to proliferate at all, at the same time, the cytoprotection provided by ECM prevailed upon transferring the cells into plastic dish, and resulted in potent repopulation capacity of the cells. CONCLUSIONS: Present data indicate that ECM contributes to failure in therapy of human osteosarcoma in clinical situation. Overall, the application of ECM-based 3-DCC could be suggested as an appropriate model system for the better understanding of antitumor drug action and hereby to set the stage for promising novel pharmacological approaches in cancer therapy.

Invasive growth and topoisomerase-switch induced by tumorous extracellular matrix in osteosarcoma cell culture.

Osteosarcoma cells are capable of extracellular matrix (ECM) synthesis. The ability of ECM to trigger the proliferation of a novel osteosarcoma cell line (OSCORT) was tested in this study in relation to a known tumor ECM, isolated from Engelbreth-Holm-Swarm (EHS) sarcoma (EHS-ECM). OSCORT was grown in monolayer, in EHS-ECM and in ECM deposited by the cells (OSCORT-ECM). Both EHS-ECM and OSCORT-ECM increased the proliferation and migration of OSCORT cells. Among the ECM biopolymers, heparan sulfate proteoglycan (HSPG) and fibronectin enhanced invasive growth, collagen type IV reduced it, while laminin had no effect. Among the ECM components HSPG and collagen IV increased both the synthesis and activation of collagenase type IV, and all the ECM components substantially increased beta1 integrin levels in the cells. The majority of ECM biopolymers decreased the level of topoisomerase I (except laminin) and elevated topoisomerase II (except fibronectin) in OSCORT. The switch in the ratio between the activities of topoisomerases I and II was mainly due to HSPG. The HSPG synthesized by OSCORT cells is described as agrin, which is a novel finding. The present study showed that HSPG (agrin) showed the most remarkable stimulatory action on the growth and migration of OSCORT cells. HSPG-induced topoisomerase II-induction deserves further experimentation, to discover its relevance to tumor progression.

Antiproliferative and antimigratory effects of doxorubicin in human osteosarcoma cells exposed to extracellular matrix.

Osteosarcoma cells are involved in the remodeling of the extracellular matrix (ECM) that affects their growth, invasive and metastatic activities. The tumour ECM provided effective protection against chemotherapy agents in several previously studied malignancies. The current study examined the effects of doxorubicin on cells that were migrated into a 3-dimensional extracellular matrix gel (ECM-gel) in comparison with its effects on cells remaining in the monolayer compartment. A human osteosarcoma cell line (OSCORT) was treated with doxorubicin in monolayer culture for 4 or 24 hours, and then overlaid by ECM-gel for 24 hours. Tumour cells remaining in the monolayer were separated from the cells migrated into ECM-gel, and both of them were characterized. OSCORT cells migrated into ECM-gel showed elevated levels and activity of topoisomerase II, increased protein expression of beta1 integrin and matrix metalloproteinase-9 activity. Doxorubicin treatment for 4 hours resulted in increased cytotoxicity in the monolayer compartment relative to the cells migrated into ECM-gel, whereas 24-hour treatment at a low concentration (0.01 microg/ml) showed an antimigratory effect. Different antiproliferative and antimigratory effects of doxorubicin treatment schedules warrant short-term, high-dose treatment for targeting the tumour growth, and long-term, low-dose treatment for targeting the invasion of osteosarcoma.

Selective suppression of an endothelin and platelet-derived growth factor containing vesicular system in endothelium of rat saphenous vein by long-term orthostasis.

Electron-dense vesicles were observed in rat vascular endothelium. The purpose of this study was to characterize their content(s), venous-arterial distribution and response to chronic orthostatic stress in extremity vessels. Saphenous and brachial vessels - saphenous vein (SV), saphenous artery (SA), brachial vein, brachial artery - were prepared for electron microscopy to quantitate the vesicle area within the endothelium following immunohistochemical and immunocytochemical identification. The effect of long-term orthostasis was assessed by exposure to head-up tilt for 2 weeks. The vesicular area in relation to the total cross-sectional area of the endothelial cells in the SV and SA of normal and confined control groups was 3.88 +/- 0.38 versus 0.89 +/- 0.06% (p < 0.05) and 4.92 +/- 0.25 versus 1.09 +/- 0.47% (p < 0.05), respectively. Head-up tilt suppressed the vesicle content of the SV to 2.26 +/- 0.39% (p < 0.05), but it remained low in the SA (1.29 +/- 0.45%), brachial vein (0.45 +/- 0.12%) and brachial artery (0.59 +/- 0.17%). Endothelin and platelet-derived growth factor, but not acidic phosphatase activity or lipid content, could be identified in the vesicles. Plasma endothelin levels were unchanged. We conclude that dense vesicles in the endothelium of extremity vessels are not cell degradation products. They may represent a vesicular secretory or storage system for endothelin and platelet-derived growth factor which participates in regional vascular adaptation to long-term orthostatic load.

[Potentiation of 5-fluorouracil efficacy. Molecular mechanisms playing a role in the cytotoxic action of 5-fluorouracil and 5-ethyl-2'-deoxyuridine (EUdR) combination]. / Az 5-fluorouracil (5-FU) hatékonyságának fokozása. Az 5-FU és 5-etil-2'-dezoxiuridin (EUdR) kombináció hatásában szerepet játszó molekuláris tényezôk.

UNLABELLED: Pharmacologic modulation of 5-fluorouracil (5-FU) metabolism provides a possibility for the enhancement of its clinical efficacy. AIM: The purpose of the present work was to study the effect of 5-ethyl-2'-deoxyuridine (EUdR), a potent 5-FU modulator, on different molecular mechanisms, influenced by 5-FU itself, and to obtain further data about the mode of action of the combination. MATERIALS AND METHODS: SW620 cell line was used for the experiments. Cytotoxicity was studied by MTT test, cell kinetic changes by FACStar flow cytometer, apoptosis by fluorescent microscope after staining the cells with acridine orange and ethydium bromide, DNA fragmentation by PAGE electrophoresis after RNase and proteinase-K digestion, thymidine incorporation with 3H-thymidine, p53 and PCNA protein expression by Western blotting. RESULTS: The cytotoxicity of 5-FU was potentiated dose dependently by EUdR. One hundred muM concentration of EUdR resulted in a 40% decrease of the IC50 value of 5-FU. Cell cycle arrest in the G2/M transition phase was most pronounced after combined treatment with 5-FU+EUdR. EUdR potentiated the incorporation of 3Hthymidine into DNA. In addition to the increase of apoptosis rate, the expression of p53 protein, caused by 5-FU was further potentiated by UdR. CONCLUSION: This study demonstrated a potential novel approach to increase the efficacy of 5-FU by EUdR, which incorporated two complementary molecular actions, the selective modulation of TS inhibition and potentiation of the p53 protein expression, consequently leading to an increase in the apoptotic rate.

Vacuole formation in the endothelium of rat extremity vessels depends on fixation techniques and vessel type.

Applying immersion fixation for electron microscopy, huge clear endothelial membrane-bound vacuoles of 0.1-3 microm diameter were noted in the extremity veins of Sprague-Dawley rats. Histological and electron microscopic histochemical methods were applied to determine whether they were the product of programmed cell death or any other kind of cell damage. Image analyzer was used to measure the total area of the vacuoles in the endothelium cells. Neither lipid content nor acidic phosphatase activity could be identified in the vacuoles. In saphenous and brachial veins, the vacuoles occupied 20.6 +/- 2.21% and 18 +/- 2.45% of the endothelium, respectively. Venous endothelium of two different strains of rat also contained the vacuoles. No such structures appeared in extremity arteries. Long-term tilting did not influence vacuolization. Using in vivo whole body fixation, only pinocytotic and dense microvesicles, but no vacuoles were noted. In conclusion, the clear vacuolar structures represent neither lipid inclusions nor secondary lysosomes. The method of tissue fixation is critical when venous endothelial vesicles are investigated. It is presumed that the vacuoles originated from intra- or intercellular microstructures, and that in case of the collapsible vein segments, their size is increased under the pathological-hypoxic and low-pressure-conditions of in vitro fixation.

Adhesion dynamics and cytoskeletal structure of gliding human fibrosarcoma cells: a hypothetical model of cell migration.

During motility of fibroblast type cells on planar surfaces, adhesions are formed at the anterior of the protruding lamella, which remain stationary relative to the substrate and undergo a maturation process as the cell passes over them. Through these adhesions force is exerted, the orientation of which is parallel to the direction of the movement. Here we show that, during gliding-type motility of human tumor cells, characterized by a semicircular shape, adhesions were found at the outer rim of the cells, along the semicircle. Time-lapse microscopy of GFP-vinculin-expressing cells showed that these adhesions were constantly renewed at the cell edge and followed a curved trajectory according to the graded radial extension model. Eventually, the adhesions reached the long axis of the cell where they were retracted into the cell body. Actin cables formed arcs, with the concave face at the anterior of the lamella found to be oriented in the direction of movement. Since adhesions moved backward with respect to the cell, actin cables connected to these adhesions must continuously grow, reaching maximal size at the long axis of the cell. Contraction of the arcs is responsible for the forward movement of the cell body.

In vitro antiangiogenic effect of sandostatin (octreotide) on the proliferation of the placental vessels.

BACKGROUND: The somatostatin analog octreotide (Sandostatin) exerts its antineoplastic effect through different mechanisms. There are several in vitro and in vivo studies available demonstrating an antiangiogenic activity, however, some other observations failed to reinforce these results. We investigated the potential angio-inhibiting activity of this drug in an in vitro system using human placental fragments. MATERIALS AND METHODS: In an in vitro angiogenesis assay, small pieces of human placenta were embedded in a fibrin gel and the effect of 1, 10 and 100 micrograms/ml Sandostatin was assessed on the microvessel formation. The results were expressed as microvessel counts per mm perimeter. RESULTS: At a dose of 1 microgram/mL octreotide did not influence the microvessel outgrowth, however, a 10 micrograms/ml concentration resulted in a 61% inhibition, while in the samples treated with 100 micrograms/ml Sandostatin only occasional capillary tubes formed. CONCLUSION: Sandostatin effectively inhibited the outgrowth of the placental vessels in a dose-dependent manner. In the mechanism of action of this drug the antiangiogenic effect should also be taken into account.

Modification of Acute and Chronic Liver Damage by Thiazolidine Compounds.

As high sulfhydril levels were shown to reduce the action of agents causing tissueinjury, increasing glutathion concentrations may have cytoprotective potential. In this study the hepatoprotective effects of several derivatives of 4carboxy5,5dimethyl thiazolidine, a modulator of glutathion metabolism were studied in rat liver damaged with CCl4. It was found that 4(S) carboxy 5,5dimethyl2 (5'nitro2furyl) thiazolidine (dimethylthiazolidinenitrofuran: DTNF) had the most significant hepatoprotective action; therefore it was subjected to detailed investigation in various models for acute and chronic liver injury. This compound was shown to ameliorate allylalcohol induced liver injury in rats, galactosamine induced hepatitis of mice and CCl4 induced chronic liver damage in rats. Our study on protein synthesis in primary hepatocyte suspension culture showed that cell injury induced by CCl4 could be reduced in the presence of this thiazolidine compound.