Modification of daunorubicin-GnRH-III bioconjugates with oligoethylene glycol derivatives to improve solubility and bioavailability for targeted cancer chemotherapy.

Daunorubicin-GnRH-III bioconjugates have recently been developed as drug delivery systems with potential applications in targeted cancer chemotherapy. In order to improve their biochemical properties, several strategies have been pursued: (1) incorporation of an enzymatic cleavable spacer between the anticancer drug and the peptide-based targeting moiety, (2) peptide modification by short chain fatty acids, or (3) attachment of two anticancer drugs to the same GnRH-III derivative. Although these modifications led to more potent bioconjugates, a decrease in their solubility was observed. Here we report on the design, synthesis and biochemical characterization of daunorubicin-GnRH-III bioconjugates with increased solubility, which could be achieved by incorporating oligoethylene glycol-based spacers in their structure. First, we have evaluated the effect of an oligoethylene glycol-based spacer on the solubility, enzymatic stability/degradation, cellular uptake, and in vitro cytostatic effect of a bioconjugate containing only one daunorubicin attached through a GFLG tetrapeptide spacer to the GnRH-III targeting moiety. Thereafter, more complex compounds containing two copies of daunorubicin, GFLG spacers as well as Lys(nBu) in position 4 of GnRH-III were synthesized and biochemically characterized. Our results indicated that all synthesized oligoethylene glycol-containing bioconjugates had higher solubility in cell culture medium than the unmodified analogs. They were degraded in the presence of rat liver lysosomal homogenate leading to the formation of small drug containing metabolites. In the case of bioconjugates containing two copies of daunorubicin, the incorporation of oligoethylene glycol-based spacers led to increased in vitro cytostatic effect on MCF-7 human breast cancer cells.

Comparative in vitro biological evaluation of daunorubicin containing GnRH-I and GnRH-II conjugates developed for tumor targeting.

Hormone based drug targeting is a promising tool for selective tumor therapy. In this study, synthesis and systematic comparative biological evaluation of novel drug containing analogs of gonadotropin-releasing hormone GnRH-I and GnRH-II is reported demonstrating their suitability for tumor targeting. The cytotoxic conjugates were prepared by the attachment of the chemotherapeutical agent daunorubicin (Dau) to GnRH analogs directly or through an enzyme-labile spacer with oxime linkage. All conjugates were found to be proteolytically stable under circumstances applied in biological assays. Both GnRH-I and GnRH-II were able to bind similarly to high-affinity GnRH-I receptors on human pituitary and human prostate cancer cells. The in vitro long-term cytotoxic effect of the conjugates was comparable with that of the free drug in human breast and colon cancer cell lines. Furthermore, a concentration-dependent cellular uptake profile was observed. The in vitro apoptotic effect of the compounds was evaluated by flow cytometry analysis using annexin-V. Our results show that both the GnRH-I and the GnRH-II based analogs might be applied for targeted tumor therapy.

Tritium labelling of a cholesterol amphiphile designed for cell membrane anchoring of proteins.

Cell membrane association of proteins can be achieved by the addition of lipid moieties to the polypeptide chain, and such lipid-modified proteins have important biological functions. A class of cell surface proteins contains a complex glycosylphosphatidylinositol (GPI) glycolipid at the C-terminus, and they are accumulated in cholesterol-rich membrane microdomains, that is, lipid rafts. Semisynthetic lipoproteins prepared from recombinant proteins and designed lipids are valuable probes and model systems of the membrane-associated proteins. Because GPI-anchored proteins can be reinserted into the cell membrane with the retention of the biological function, they are appropriate candidates for preparing models via reduction of the structural complexity. A synthetic headgroup was added to the 3ß-hydroxyl group of cholesterol, an essential lipid component of rafts, and the resulting cholesterol derivative was used as a simplified GPI mimetic. In order to quantitate the membrane integrated GPI mimetic after the exogenous addition to live cells, a tritium labelled cholesterol anchor was prepared. The radioactive label was introduced into the headgroup, and the radiolabelled GPI mimetic anchor was obtained with a specific activity of 1.37 TBq/mmol. The headgroup labelled cholesterol derivative was applied to demonstrate the sensitive detection of the cell membrane association of the anchor under in vivo conditions.

Functional properties of cell-free expressed human endothelin A and endothelin B receptors in artificial membrane environments.

The human endothelin receptors are members of the rhodopsin class A of G-protein coupled receptors and key modulators of blood pressure regulation. Their functional in vitro characterization has widely been limited by the availability of high quality samples. We have optimized cell-free expression protocols for the human endothelin A and endothelin B receptors by implementing co-translational association approaches of the synthesized proteins with supplied liposomes or nanodiscs. Efficiency of membrane association and ligand binding properties of the receptors have systematically been studied in correlation to different membrane environments and lipid types. Ligand binding was analyzed by a number of complementary assays including radioassays, surface plasmon resonance and fluorescence measurements. High affinity binding of the peptide ligand ET-1 to both endothelin receptors could be obtained with several conditions and the highest Bmax values were measured in association with nanodiscs. We could further obtain the characteristic differential binding pattern of the two endothelin receptors with a panel of selected agonists and antagonists. Two intrinsic properties of the functionally folded endothelin B receptor, the proteolytic processing based on conformational recognition as well as the formation of SDS-resistant complexes with the peptide ligand ET-1, were observed with samples obtained from several cell-free expression conditions. High affinity and specific binding of ligands could furthermore be obtained with non-purified receptor samples in crude cell-free reaction mixtures, thus providing new perspectives for fast in vitro screening applications.

Cell-free expression of G-protein coupled receptors: new pipelines for challenging targets.

Based on their eminent importance for medical applications, G-protein coupled receptors are currently amongst the most frequently membrane protein targets analyzed by cell-free expression. The cell-free expression approach removes most bottlenecks known from conventional cell-based protein production pipelines and ensures fast access to a selected receptor target. In addition, receptors can be synthesized in presence of a large variety of artificial solubilization environments comprising detergents, lipids, nanodiscs and other amphiphilic compounds. The currently accumulated data based on a variety of analyzed receptors already opens promising perspectives for applications of cell-free synthesized samples in functional characterization and drug screening. Structural evaluation still suffers from high conformational dynamics causing sample instability and might be addressed in future by molecular engineering or immuno-stabilization approaches.

Preparation of semisynthetic lipoproteins with fluorescent cholesterol anchor and their introduction to the cell membrane with minimal disruption of the membrane.

The exogenous introduction of fluorescent lipoproteins into cell membranes is a method for visualizing the cellular traffic of membrane associated proteins, and also for altering the cell surface in a controlled manner. In order to achieve the cell membrane anchoring of proteins and their subsequent fluorescence based detection, a cholesterol derivative was designed. The headgroup of the novel cholesterol anchor contains a fluorescent reporter and a thiol reactive maleimide for protein conjugation. Protein conjugation was demonstrated by the addition of a green fluorescent maleimido anchor to the C-terminus of a Cys extended red fluorescent protein, mCherry. The resulting dual fluorescent cholesteryl lipoprotein was successfully separated from the micellar associates of the surplus fluorescent lipid anchor without denaturing the protein, and the lipoprotein containing only the covalently linked, stoichiometric fluorescent lipid was efficiently delivered to the plasma membrane of live cells. It was demonstrated that the membrane fluorescence could be directly assigned to the protein-anchor conjugate, because no excess of fluorescent lipid species were present during the imaging experiment and the protein and anchor fluorescence colocalized in the cell membrane. Molecular dynamics simulations and subsequent trajectory analysis suggest also the spontaneous and stable membrane association of the cholesterol anchor. Thus, the method could be beneficially applied for studying membrane associated proteins and for preparing mimetics of glycosylphosphatidylinositol (GPI)-anchored proteins to target cholesterol-rich membrane microdomains.

Synthesis, enzymatic stability and in vitro cytostatic effect of Daunorubicin-GnRH-III derivative dimers.

Bioconjugates containing chemotherapeutic agents attached to peptide hormones, such as gonadotropin-releasing hormone (GnRH), are developed as drug delivery systems for targeted cancer chemotherapy. We report here the synthesis and biochemical characterization of disulfide bond-linked dimeric bioconjugates in which daunorubicin was coupled via an oxime linkage to aminooxyacetylated GnRH-III ([Glp-His-Trp-Ser-His-Asp-Trp-Lys(DauAoa-Cys)-Pro-Gly-NH2]2; where Glp is pyroglutamic acid and Aoa is aminooxyacetyl) and its derivatives modified in position four by N-Me-Ser and Lys(Ac). The in vitro stability/degradation of the bioconjugates was determined in human serum, as well as in the presence of rat liver lysosomal homogenate and digestive enzymes. All compounds were stable at least for 24h in human serum and in the presence of pepsin and trypsin, while they were degraded by lysosomal enzymes. The daunorubicin-GnRH-III derivative dimers were partly digested by α-chymotrypsin; however, they had increased stability compared to the corresponding monomers, making them potential candidates for oral administration. The in vitro cytostatic effect of the compounds was determined on MCF-7 human breast cancer cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. All daunorubicin-GnRH-III derivative dimers exerted slightly increased in vitro cytostatic effect (IC50 values in low µM range) than the corresponding monomeric bioconjugates.

Design, synthesis, in vitro stability and cytostatic effect of multifunctional anticancer drug-bioconjugates containing GnRH-III as a targeting moiety.

Bioconjugates containing the GnRH-III hormone decapeptide as a targeting moiety are able to deliver chemotherapeutic agents specifically to cancer cells expressing GnRH receptors, thereby increasing their local efficacy while limiting the peripheral toxicity. However, the number of GnRH receptors on cancer cells is limited and they desensitize under continuous hormone treatment. A possible approach to increase the receptor mediated tumor targeting and consequently the cytostatic effect of the bioconjugates would be the attachment of more than one chemotherapeutic agent to one GnRH-III molecule. Here we report on the design, synthesis and biochemical characterization of multifunctional bioconjugates containing GnRH-III as a targeting moiety and daunorubicin as a chemotherapeutic agent. Two different drug design approaches were pursued. The first one was based on the bifunctional [(4)Lys]-GnRH-III (Glp-His-Trp-Lys-His-Asp-Trp-Lys-Pro-Gly-NH(2)) containing two lysine residues in positions 4 and 8, whose ε-amino groups were used for the coupling of daunorubicin. In the second drug design, the native GnRH-III (Glp-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH(2)) was used as a scaffold; an additional lysine residue was coupled to the ϵ-amino group of (8) Lys in order to generate two free amino groups available for conjugation of daunorubicin. The in vitro stability/degradation of all synthesized compounds was investigated in human serum, as well as in the presence of rat liver lysosomal homogenate. Their cellular uptake was determined on human breast cancer cells and the cytostatic effect was evaluated on human breast, colon and prostate cancer cell lines. Compared with a monofunctional compound, both drug design approaches resulted in multifunctional bioconjugates with increased cytostatic effect.

Acylated mono-, bis- and tris- cinchona-based amines containing ferrocene or organic residues: synthesis, structure and in vitro antitumor activity on selected human cancer cell lines.

A series of novel functionalized mono-, bis- and tris-(S)-{[(2S,4R,8R)-8-ethyl-quinuclidin-2-yl](6-methoxyquinolin-4-yl)}methanamines including ferrocene-containing derivatives was obtained by the reaction of the precursor amine with a variety of acylation agents. Their in vitro antitumor activity was investigated against human leukemia (HL-60), human neuroblastoma (SH-SY5Y), human hepatoma (HepG2) and human breast cancer (MCF-7) cells by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-assay and the 50% inhibitory concentration (IC(50)) values were determined. Our data indicate that the precursor amine has no antitumor activity in vitro, but the bis-methanamines with ureido-, thioureido and amide-type linkers display attractive in vitro cytotoxicity and cytostatic effects on HL-60, HepG2, MCF-7 and SH-SY5Y cells. Besides 1H- and 13C-NMR methods the structures of the new model compounds were also studied by DFT calculations.

Enhanced enzymatic stability and antitumor activity of daunorubicin-GnRH-III bioconjugates modified in position 4.

Here, we report on the synthesis, enzymatic stability, and antitumor activity of novel bioconjugates containing the chemotherapeutic agent daunorubicin attached through an oxime bond to various gonadotropin-releasing hormone-III (GnRH-III) derivatives. In order to increase the enzymatic stability of the bioconjugates (in particular against chymotrypsin), (4)Ser was replaced by N-Me-Ser or Lys(Ac). A compound in which (4)Lys was not acetylated was also prepared, with the aim of investigating the influence of the free ε-amino group on the biochemical properties. The in vitro cytostatic effect of the bioconjugates was determined on MCF-7 human breast, HT-29 human colon, and LNCaP human prostate cancer cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Their stability/degradation (1) in human serum, (2) in the presence of rat liver lysosomal homogenate, and (3) in the presence of digestive enzymes (trypsin, chymotrypsin, and pepsin) was analyzed by liquid chromatography in combination with mass spectrometry. The results showed that (1) all synthesized bioconjugates had in vitro cytostatic effect, (2) they were stable in human serum at least for 24 h, and (3) they were hydrolyzed in the presence of lysosomal homogenate. All compounds were stable in the presence of (1) pepsin and (2) trypsin (except for the (4)Lys containing bioconjugate). In the presence of chymotrypsin, all bioconjugates were digested; the degradation rate strongly depending on their structure. The bioconjugates in which (4)Ser was replaced by N-Me-Ser or Lys(Ac) had the highest enzymatic stability, making them potential candidates for oral administration. In vivo tumor growth inhibitory effect of two selected bioconjugates was evaluated on orthotopically developed C26 murine colon carcinoma bearing mice. The results indicated that the compound containing Lys(Ac) in position 4 had significantly higher antitumor activity than the parent bioconjugate.

A new daunomycin-peptide conjugate: synthesis, characterization and the effect on the protein expression profile of HL-60 cells in vitro.

Daunomycin (Dau) is a DNA-binding antineoplastic agent in the treatment of various types of cancer, such as osteosarcomas and acute myeloid leukemia. One approach to improve its selectivity and to decrease the side effects is the conjugation of Dau with oligopeptide carriers, which might alter the drug uptake and intracellular fate. Here, we report on the synthesis, characterization, and in vitro biological properties of a novel conjugate in which Dau is attached, via an oxime bond, to one of the cancer specific small peptides (LTVSPWY) selected from a random phage peptide library. The in vitro cytostatic effect and cellular uptake of Dau═Aoa-LTVSPWY-NH(2) conjugate were studied on various human cancer cell lines expressing different levels of ErbB2 receptor which could be targeted by the peptide. We found that the new daunomycin-peptide conjugate is highly cytostatic and could be taken up efficiently by the human cancer cells studied. However, the conjugate was less effective than the free drug itself. RP-HPLC data indicate that the conjugate is stable at least for 24 h in the pH 2.5-7.0 range of buffers, as well as in cell culture medium. The conjugate in the presence of rat liver lysosomal homogenate, as indicated by LC-MS analysis, could be degraded. The smallest, Dau-containing metabolite (Dau═Aoa-Leu-OH) identified and prepared expresses DNA-binding ability. In order to get insight on the potential mechanism of action, we compared the protein expression profile of HL-60 human leukemia cells after treatment with the free and peptide conjugated daunomycin. Proteomic analysis suggests that the expression of several proteins has been altered. This includes three proteins, whose expression was lower (tubulin ß chain) or markedly higher (proliferating cell nuclear antigen and protein kinase C inhibitor protein 1) after administration of cells with Dau-conjugate vs free drug.

In vitro degradation and antitumor activity of oxime bond-linked daunorubicin-GnRH-III bioconjugates and DNA-binding properties of daunorubicin-amino acid metabolites.

Bioconjugates with receptor-mediated tumor-targeting functions and carrying cytotoxic agents should enable the specific delivery of chemotherapeutics to malignant tissues, thus increasing their local efficacy while limiting the peripheral toxicity. In the present study, gonadotropin-releasing hormone III (GnRH-III; Glp-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH(2)) was employed as a targeting moiety to which daunorubicin was attached via oxime bond, either directly or by insertion of a GFLG or YRRL tetrapeptide spacer. The in vitro antitumor activity of the bioconjugates was determined on MCF-7 human breast and HT-29 human colon cancer cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Their degradation/stability (1) in human serum, (2) in the presence of cathepsin B and (3) in rat liver lysosomal homogenate was analyzed by liquid chromatography in combination with mass spectrometry. The results show that (1) all synthesized bioconjugates have in vitro antitumor effect, (2) they are stable in human serum at least for 24 h, except for the compound containing an YRRL spacer and (3) they are hydrolyzed by cathepsin B and in the lysosomal homogenate. To investigate the relationship between the in vitro antitumor activity and the structure of the bioconjugates, the smallest metabolites produced in the lysosomal homogenate were synthesized and their binding to DNA was assessed by fluorescence spectroscopy. Our data indicate that the incorporation of a peptide spacer in the structure of oxime bond-linked daunorubicin-GnRH-III bioconjugates is not required for their antitumor activity. Moreover, the antitumor activity is influenced by the structure of the metabolites (daunorubicin-amino acid derivatives) and their DNA-binding properties.

New pemetrexed-peptide conjugates: synthesis, characterization and in vitro cytostatic effect on non-small cell lung carcinoma (NCI-H358) and human leukemia (HL-60) cells.

Pemetrexed (Pem) is a novel antimetabolite type of anticancer drug that demonstrated promising clinical activity in a wide variety of solid tumors, including non-small cell lung carcinoma and malignant pleural mesothelioma. It inhibits enzymes involved in the folate pathway, for which the presence of its free carboxylic groups is necessary. The heteroaromatic ring system of Pem has a modifiable amino group, which opens a possibility to apply a new strategy to conjugate Pem to carrier molecules. Considering this as well as the necessity of untouched carboxylic groups of Pem in the new conjugates, we developed a new synthesis strategy. Here, we describe the synthesis and the characterization of new Pem-peptide conjugates in which cell-penetrating octaarginine or/and lung-targeting H-Ile-Glu-Leu-Leu-Gln-Ala-Arg-NH(2) peptide is attached to the drug by thioether bond. The conjugates characterized by RP-HPLC and MS exhibited cytostatic effect in vitro on non-small cell lung carcinoma as well as on human leukemia cell lines. The IC(50) values of the conjugates were similar, but the conjugates with H-Ile-Glu-Leu-Leu-Gln-Ala-Arg-NH(2) sequence were slightly more effective. Our data show that the in vitro cytostatic effect of the free Pem was essentially maintained after conjugation with cell-penetrating or cell-targeting peptides. Thus, the conjugation strategy reported could lead to the development of a new generation of active Pem conjugates.

Efficient synthesis of an (aminooxy) acetylated-somatostatin derivative using (aminooxy)acetic acid as a 'carbonyl capture' reagent.

Owing to the high chemoselectivity between an aminooxy function and a carbonyl group, oxime ligation is one of the most preferred procedures for the preparation of peptide conjugates. However, the sensitivity of (aminooxy)acetylated peptides to ketones and aldehydes makes their synthesis and storage difficult. In our study, we established the efficient synthesis of an (aminooxy)acetylated-somatostatin derivative in the presence of free (aminooxy)acetic acid, which was used as a 'carbonyl capture' reagent in the final cleavage step. This (aminooxy)acetylated compound was further used for the chemoselective ligation (oxime bond formation) with daunorubicin and 4-fluorobenzaldehyde leading to the formation of conjugates with potential applications in targeted cancer chemotherapy and positron emission tomography.

Synthesis and in vitro antitumor effect of vinblastine derivative-oligoarginine conjugates.

Vinblastine is a widely used anticancer drug with undesired side effects. Its conjugation with carrier molecules could be an efficient strategy to reduce these side effects. Besides this, the conjugate could exhibit increased efficiency against resistant cells, e.g., due to the altered internalization pathway. Oligoarginines, as cell-penetrating peptides, can transport covalently attached compounds into different kinds of cells and enhance the efficiency of those compounds. We report here the coupling of vinblastine through its carboxyl group at position 16 with the N-terminal amino function of L-Trp methyl ester. After hydrolysis of the ester group, 17-desacetylvinblastineTrp was conjugated to the N-terminal amino group of oligoarginine via the C-terminal carboxyl group of the Trp moiety in solution. The antitumor effect of conjugates was studied on sensitive and resistant human leukemia (HL-60) cells in vitro. Our data suggest that all conjugates investigated possess an antiproliferative effect against the studied cells. However, the effect was dependent on the number of Arg residues in the conjugates: Arg8 > Arg6 ≫ Arg4. The conjugate with Arg8 exhibited similar efficicacy as compared with free 17-desacetylvinblastineTrp. The in vitro studies also showed that the tubulin binding ability of vinblastine was essentially preserved even in the octaarginine conjugate. We also observed that two isomers were formed during conjugation. These isomers showed different levels of activity against tubulin polymerization in vitro and in vivo. The 17-desacetylvinblastineTrp-Arg8-1 isomer conjugate possessed high selectivity against the mitotic spindles. HRMS and NMR data suggest that 17-desacetylvinblastineTrp-Arg8-1 and 17-desacetylvinblastineTrp-Arg8-2 are epimers at the tryptophan α carbon atom.

Development of an oxime bond containing daunorubicin-gonadotropin-releasing hormone-III conjugate as a potential anticancer drug.

Here, we report on the synthesis and biological properties of a conjugate in which daunorubicin (Dau) as chemotherapeutic agent was attached through an oxime bond to gonadotropin-releasing hormone-III (GnRH-III) as targeting moiety. In vitro toxicity and the cytostatic effect of the conjugate on MCF-7 human breast and C26 murine colon cancer cell lines were determined, and the results were compared with those obtained for the free daunorubicin, as well as with the doxorubicin containing derivative. In vivo antitumor effect of daunorubicin-GnRH-III was studied on Balb/c female mice transplanted with C26 tumor. Our data indicate that the daunorubicin-GnRH-III conjugate had a lower toxic effect than the free daunorubicin and it was essentially nontoxic up to 15 mg (Dau content)/kg body weight. The treatment of the C26 tumor bearing mice with the conjugate led to tumor growth inhibition and longer survival time in comparison with the controls and with the administration of the free drug. When mice were treated twice with the conjugate (on days 4 and 7 after tumor transplantation), 46% tumor growth inhibition was obtained. In this case, the increase of the median survival time was 38% compared to the controls.

Design, synthesis, and in vitro activity of novel drug delivery systems containing tuftsin derivatives and methotrexate.

During the past decade, biodegradable polymers or oligopeptides recognized by cell-surface receptors have been shown to increase drug specificity, lowering systemic drug toxicity in contrast to small-size fast-acting drugs. The goal of the present study was to develop anticancer bioconjugates based on chemotactic drug targeting (CDT). These constructs are composed of methotrexate (Mtx) attached to a tuftsin-like peptide carrier through an enzyme-labile pentapeptide spacer (GFLGC) and several copies of a chemotactic targeting moiety (H-TKPR, For-TKPR, H-TKPKG, and Ac-TKPKG). Carriers with targeting moieties in the branches were prepared by solid-phase synthesis using mixed Boc and Fmoc strategies. The drug molecule connected to an enzyme-labile spacer was attached to the branched oligopeptide in solution. In vitro chemotaxis, cellular uptake, and cytotoxicity assays were carried out on the MonoMac6 cell line. The most effective conjugates with H-TKPR or Ac-TKPKG targeting moieties in the branches, which have the most advantageous chemotactic properties, can be internalized rapidly, and these conjugates trigger higher toxic effect than the free drug (Mtx). The results suggest that our tuftsin-based drug delivery systems might be potential candidates for targeting cancer chemotherapy.

Different expression of occludin and ZO-1 in primary and metastatic liver tumors.

Tight junction (TJ) components were found to be correlated with carcinogenesis and tumor development. TJs are composed of three main integral membrane proteins; occludin, claudins and JAMs. Alteration of the TJ protein expression may play an important role in the process of cell dissociation, which is among the first steps of tumor invasion and metastasis. Reduced expression of ZO-1 has been reported to be associated with invasion of several tumors. The aim of the present study was to detect differences between occludin and ZO-1 expression in normal liver samples, HCCs and colorectal liver metastases. Expression of occludin and ZO-1 was analysed in 25 surgically removed human hepatocellular carcinomas (HCC) and 25 human colorectal liver metastases. Gene expression levels were measured by real-time RT PCR, protein expression was determined by immunohistochemistry, comparing tumors with the surrounding nontumorous parenchyma and with seven normal liver samples. Occludin and ZO-1 mRNAs showed significant downregulation in HCCs in comparison with normal liver and were also downregulated in the metastases when compared with normal liver. Occludin and ZO-1 proteins were weakly expressed on hepatocytes in normal liver, while strong expression was found on bile canaliculi. In HCCs occludin and ZO-1 did not show immunopositivity on tumor cells, while colorectal metastatic tumors revealed high levels of these molecules. HCCs and metastases are characterized by markedly different protein expression pattern of occludin and ZO-1, which phenomenon might be attributed to the different histogenesis of these tumors.

Cell-penetrating conjugates of pentaglutamylated methotrexate as potential anticancer drugs against resistant tumor cells.

The emerging resistance of tumor cells against methotrexate (MTX) is one of the major limitations of the MTX treatment of tumorous diseases. The disturbance in the polyglutamation which is a main step in the mechanism of methotrexate action is often the reason of the resistance. Delivery of polyglutamylated MTX into cells may evade the mechanisms that are responsible for drug resistance. In this study conjugates of methotrexate and its pentaglutamylated derivatives with cell-penetrating peptides - penetratin and octaarginine - were investigated. The cellular-uptake and in vitro cytostatic activity of conjugates were examined on breast cancer cell cultures (MDA-MB-231 as resistant and MCF-7 as sensitive cell culture). These cell cultures showed very different behaviour towards the conjugates. Although the presence of pentaglutamyl moiety significantly decreased the internalisation of conjugates, some of them were significantly active in vitro. All of the conjugates were able to penetrate in some extent into both cell types, but only the conjugates of penetratin showed in vitro cytostatic activity. The most effective conjugates were the MTX-Glu5-Penetratin(desMet) and MTX-Glu5-GFLG-Penetratin(desMet). The latter was effective on both cell cultures while the former was active only on the resistant tumor cells. Our results suggest that the translocation of polyglutamylated MTX may be a new way to treat sensitive and more importantly resistant tumors. While both penetratin and octaarginine peptides were successfully used to deliver several kinds of cargos earlier in our case the activity of penetratin conjugates was more pronounced.

Cell-free expression of G-protein-coupled receptors.

Cell-free expression has emerged as a new standard for the production of membrane proteins. The reduction of expression complexity in cell-free systems eliminates central bottlenecks and allows the reliable and efficient synthesis of many different types of membrane proteins. Furthermore, the open accessibility of cell-free reactions enables the co-translational solubilization of cell-free expressed membrane proteins in a large variety of supplied additives. Hydrophobic environments can therefore be adjusted according to the requirements of individual membrane protein targets. We present different approaches for the preparative scale cell-free production of G-protein-coupled receptors using the extracts of Escherichia coli cells. We exemplify expression conditions implementing detergents, nanodiscs, or liposomes. The generated protein samples could be directly used for further functional characterization.