Ferenc Timár

The antiproliferative action of a melphalan hexapeptide with collagenase-cleavable site

Purpose: The objective of the present study was to examine the relevance of collagenase in the antitumor action of a melphalan peptide (MHP) with a collagenase-cleavable sequence. The question was addressed as to whether collagenase may act as an activator or a target in the antiproliferative mechanism of MHP. Methods: Melphalan was inserted into peptides representing the sequence Pro-Gln-Gly-Ile-Ala.Gly of the collagenase-cleavable site in collagens. Changes in growth and collagenase IV activities of HT-1080, HT-29, HT-168, and MCF-7 cell cultures were investigated. Results: The present investigations provide data indicating that Pro-Gln-Gly-Ile-Mel-Gly (melphalan hexapeptide, MHP) is a substrate for both bacterial and 72-kDa type IV collagenases and that in this way it can generate Ile-Mel-Gly (melphalan tripeptide, MTP) of higher cytotoxic potency. Indeed, the formation of MTP was detected in the conditioned medium of HT-1080, a collagenase IV-producing human fibrosarcoma. In a comparison of equimolar concentrations of melphalan and its two peptide derivatives (MHP and MTP), superior antiproliferative action of MTP was seen in HT-29, HT-1080, and HT-168 tumor cell cultures. However, the relatively modest cytostatic actions of MHP were increased when bacterial collagenase was added to the cell cultures. After melphalan treatment, reduced levels of both 92 and 72-kDa type IV collagenases were seen in the HT-1080 cell cultures. However, the reduction of collagenase activity and the cell counts did not run parallel in the MTP- or MHP-treated cultures; indeed, collagenase activity related to cell numbers showed an elevated level. Conclusions: As the conversion of MHP to the more toxic MTP was detected in the presence of collagenases, it is possible that collagenase-directed activation of prodrugs may be a promising approach for the development of more selective cytostatic drugs against malignant tumors with high collagenase activities.

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 descrease 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.

Role of the Basement Membrane in Tumor Cell Dormancy and Cytotoxic Resistance

Objectives and Methods: Tumor dormancy and resistance to cytotoxic agents are key limiting events in the treatment of malignant diseases. To determine whether both are influenced by the extracellular milieu in which tumors reside, HT1080 human fibrosarcoma, MCF-7 breast carcinoma and OSCORT osteosarcoma cell proliferation, viability, apoptosis and cytoreductive-treatment-induced death were investigated in the presence or absence of extracellular matrix (ECM). Results: ECM-adherent, but not plastic-adherent HT1080 cells formed a multicellular network accompanied by reduced proliferation and lowered DNA synthetic capacity. The number of cells in S-phase was dramatically reduced. Viable cells entered a state of dormancy reminiscent of that observed in the step of metastasis after extravasation, i.e. prior to the initiation of progressive growth. Such ECM-induced dormancy could be reversed by plating cells on plastic, but only after a 48-hour lag period. No difference was indicated in clonogenicity of HT1080 cells originated from plastic or ECM gel. However, the cells released from ECM gel showed significantly reduced migration ability. The resistance of anchored cells against cytotoxic damage was increased by ECM gel. Examination of cytoreductive treatment revealed that ECM adherence at the time of injury is partially protective, a property which was also moderately apparent when injured cells were transferred to the basement membrane. Conclusions: Taken together, these results suggest that the ECM plays a key role in tumor dormancy and cytotoxic resistance, both explorable at the molecular level using our in vitro model system.

A Facile Synthesis of C2-Substituted Pyrrolo[2,3-f]quinolines with Cytotoxic Activity

An expeditious four-step synthesis of the 1H-pyrrolo[2,3-f]quinoline-2- carboxamides (5a-h) is described. Readily available 6-quinolinecarboxaldehyde is converted to the parent acid (6) by nucleophilic attack of the azido-ester (9) and intramolecular cyclization of (10) followed by hydrolysis of the methyl ester (11). The cytotoxicity of the target molecules (5a-h) was evaluated in four tumour cell lines in vitro. One compound (5d) showed sufficient activity (IC50 = 10.2 ?M) in the human non-small cell lung cell line NSCLC-N16-L16 to be worthy of further study.

The effect of D-penicillamine on CCl4-induced experimental liver cirrhosis.

The effect of D-penicillamine (Pe) on liver fibrosis-cirrhosis induced by chronic CCl4 and phenobarbital (Pb) administration in Fischer 344 male rats was studied. Morphometric analysis did not reveal a decrease in the amount of connective tissue fibers after Pe-treatment. Compared to the CCl4 and Pb-treated control groups, Pe had no significant effect on the concentrations of hydroxyproline, a parameter of collagen degradation, either; however, it increased the glycosaminoglycan concentrations. Lymphocyte stimulation by Con-A in the Pe-treated groups did not differ from that of the CCl4 and Pb-treated ones. According to our studies, Pe-treatment was ineffective in rats with liver fibrosis-cirrhosis induced by CCl4 and Pb administration. It seems that Pe can be effective only in the cirrhosis types accompanied by a considerable copper accumulation due to suppression of the toxic effects of copper.

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.

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.

Modification of the inhibitory effects of CCl4, on phospholipid and protein biosynthesis by prostacyclin

CCl4 induced cellular injury and its modification by prostacyclin (PGI2) was studied in cultured rat hepatocytes. Biosynthesis of both intracellular and serum proteins and that of phospholipids decreased upon CCl4 treatments (IC50 7.0, 2.5 and 3.2 mM, respectively). After 1 hr exposure of the cells to CCl4, the reductions in the biosynthesis increased further with time. PGI2 treatments (10(-5)-10(-9) M) of the hepatocytes subsequent to CCl4 poisoning resulted in partial recovery from the cell injury evaluated at the fifth hour of the experiment. Optimal effects of PGI2 were found at a concentration of 10(-7)-10(-8) M, while higher and lower concentrations offered less protection. Upon the addition of CCl4 a higher catabolic rate of PIP2 and an increased formation of inositol phosphates were observed. This alteration was shown to precede the defects in the labelling of the major phospholipid components. Furthermore, these changes were circumvented in the presence of PGI2. Thus, PIP2 metabolism appears to be a critical process in the mechanism of this type of cellular injury and its protection by PGI2.

Membrane Properties of Lewis Lung Tumor Cells with “Low” and “High” Metastatic Capacity: Anti-Metastatic Effect of a Glycosaminoglycan Biosynthesis Blocking Agent 5-Hexyl-2′-Deoxyuridine (HUdR)

The main cause of a cancer patient’s death is the development of tumor metastases. Therefore, understanding of biological events of metastasization has a great clinical impact (1). A number of important steps in this process are mediated by the surface characteristics of tumor cells, particularly by surface glycoconjugate molecules (2,3,4).

Establishment and characterization of a new transplantable pancreatic cancer xenograft (PZX-5) in immunosuppressed mice

SummaryConclusionA new, stable, transplantable human pancreatic cancer xenograft (PZX-5) model has been established in CBA immunosuppressed mice.BackgroundNumerous human pancreatic carcinomas have been successfully transplanted into athymic nude mice. However, artificially immunosuppressed animals have rarely been used as recipients. Because this model system proved to be reliable for hosting many human malignancies at our institute, successive xenotransplantations of a ductal adenocarcinoma have been carried out.MethodImmunosuppression of CBA/CA mice was achieved by thymectomy, whole-body irradiation and bone-marrow reconstruction. Tumor fragments were subcutaneously implanted from a well/moderately differentiated ductal pancreatic adenocarcinoma and serially transplanted for more than 20 mo. The xenografted tumors were characterized using morphological, immunohistochemical, biochemical, and flow cytometric methods.ResultsDuring the serial transplantations, the neoplasm maintained its original morphological-pathobiological characteristics. It produced a large amount of mucin and expressed carcinoembryonic antigen (CEA). Neither the mitotic activity nor the degree of differentiation was altered, and CEA was permanently detected. Flow cytometric DNA analysis revealed an aneuploid pattern (DNA index 1.45±0.03), which has remained within the same range during xenograftings. The doubling time in an in vitro system proved to be 18 h. The human character has been well preserved even 9 mo posttransplantation, as was evidenced by LDH-isoenzyme electrophoresis. The results indicate that the thymectomized—whole-body irradiated—bone-marrow reconstructed immunosuppressed mice are also appropriate hosts for pancreatic cancer xenografts.