Erika Orbán

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.

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.

Anthracycline-GnRH derivative bioconjugates with different linkages: Synthesis, in vitro drug release and cytostatic effect

To increase the selectivity and consequently to minimize the side effects of chemotherapeutic agents, receptor mediated tumor targeting approaches have been developed. In the present work, various anthracycline-GnRH derivative bioconjugates were synthesized with the aim of investigating the influence of (i) different anthracycline anticancer drugs, (ii) different linkages between the targeting moiety and the anticancer drug, and (iii) different targeting moieties (e.g., GnRH-III and [D-Lys⁶]-GnRH-I) on their in vitro drug release and cytostatic effect. The anthracyclines, daunorubicin or doxorubicin, were attached to the ε-amino group of Lys of GnRH-III or [D-Lys⁶]-GnRH-I through oxime, hydrazone or ester bonds. In another bioconjugate, a self-immolative p-aminobenzyloxycarbonyl spacer was used to link daunorubicin to GnRH-III. The in vitro degradation of the bioconjugates was investigated in the presence of rat liver lysosomal homogenate and cathepsin B. The cellular uptake of the compounds was evaluated by flow cytometry and their in vitro cytostatic effect was determined by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay. The results indicate that on the tested cancer cell lines there is no significant difference in the cellular uptake and in vitro cytostatic effect of bioconjugates containing GnRH-III or [D-Lys⁶]-GnRH-I as a targeting moiety. The bioconjugates containing ester bond, hydrazone bond and the self-immolative spacer exert the highest cytostatic effect, followed by oxime bond-linked compounds.

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.

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. Copyright

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.

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 (IC50) 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 cellular uptake and in vitro antitumor activity of short-chain fatty acid acylated daunorubicin-GnRH-III bioconjugates.

Here we report on the synthesis and biochemical characterization (enzymatic stability, cellular uptake, in vitro antitumor activity, membrane interaction and GnRH-receptor binding affinity) of novel short-chain fatty acid (SCFA) acylated daunorubicin-GnRH-III bioconjugates, which may serve as drug delivery systems for targeted cancer chemotherapy. Ser in position 4 of GnRH-III was replaced by Lys, followed by the acylation of its ε-amino group with various fatty acids. SCFAs are potentially chemoprotective agents by suppressing the growth of cancer cells and therefore may enhance the antitumor activity of the bioconjugates. We found that all synthesized bioconjugates had high cytostatic effect in vitro, were stable in cell culture medium for 6 h and degraded in the presence of rat liver lysosomal homogenate leading to the formation of an oxime bond-linked daunorubicin-Lys as the smallest active metabolite. In the presence of α-chymotrypsin, all compounds were digested, the degradation rate strongly depending on the type of fatty acid. The bioconjugate containing Lys(nBu) in position 4 was taken up most efficiently by the cancer cells and exerted higher in vitro cytostatic effect than the previously developed GnRH-III((4)Lys(Ac), (8)Lys(Dau = Aoa)) or the parent GnRH-III(Dau = Aoa) bioconjugate. Our results could be explained by the increased binding affinity of the newly developed compound containing Lys(nBu) to the GnRH receptors.

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.

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.