Biomedical subjects

Joachim Gullbo

Publications and source records attributed to Joachim Gullbo.

17 recordsLinked to original sources

PubMed2006

Pharmacological profiling of disulfiram using human tumor cell lines and human tumor cells from patients.

The thiocarbamate drug disulfiram has been used for decades in the treatment of alcohol abuse. Disulfiram induces apoptosis in a number of tumor cell lines and was recently by us proposed to act as a 26S proteasome inhibitor. In this work we characterized disulfiram in vitro with regard to tumor-type specificity, possible mechanisms of action and drug resistance and cell death in human tumor cell lines and in 78 samples of tumor cells from patients using the fluorometric microculture cytotoxicity assay and the automated fluorescence-imaging microscope ArrayScan((R)). Disulfiram induced cytotoxicity in a biphasic pattern in both cell lines and patient tumor cells. Disulfiram induced apoptosis as measured by cell membrane permeability, nuclear fragmentation/condensation and caspase-3/7 activation using high content screening assays. For many of the cell lines tested disulfiram was active in sub-micromolar concentrations. When comparing the logIC(50) patterns with other cytotoxic agents, disulfiram showed low correlation (R<0.5) with all drugs except lactacystin (R=0.69), a known proteasome inhibitor, indicating that the two substances may share mechanistic pathways. Disulfiram was more active in hematological than in solid tumor samples, but substantial activity was observed in carcinomas of the ovary and the breast and in non-small cell lung cancer. Disulfiram also displayed higher cytotoxic effect in cells from chronic lymphocytic leukemia than in normal lymphocytes (p<0.05), which may indicate some tumor selectivity. These results together with large clinical experience and relatively mild side effects encourage clinical studies of disulfiram as an anti-cancer agent.

Cell Line, Tumor↗

PubMed2006

Inhibition of proteasome activity, nuclear factor-KappaB translocation and cell survival by the antialcoholism drug disulfiram.

The proteasome pathway is an important target for anticancer drug development. Here, we identify the antialcoholism drug disulfiram and its analogue pyrrolidine dithiocarbamate (PDTC) as inhibitors of the 26S proteasome activity in a cell-based screening assay. As expected for proteasome inhibitors, these compounds also inhibited TNF-alpha-induced nuclear factor-KappaB (NF-KappaB) translocation and were cytotoxic. Disulfiram was more cytotoxic against chronic lymphocytic leukemia cells compared to peripheral blood mononuclear cells (PBMC) at clinically achievable concentrations. Proteasome and NF-KappaB inhibition were achieved with a potency in the same range as that of the clinically used proteasome inhibitor bortezomib. Disulfiram was also able to induce accumulation of p27(Kip1) and to prolong the half-life of c-Myc, both targets for proteasome-dependent degradation. It is concluded that the previously observed antitumoral and NF-KappaB inhibiting activity of disulfiram and PDTC could be attributed to their inhibition of the 26S proteasome.

Alcohol Deterrents↗

PubMed2006

Phenotype-based screening of mechanistically annotated compounds in combination with gene expression and pathway analysis identifies candidate drug targets in a human squamous carcinoma cell model.

The squamous cell carcinoma HeLa cell line and an epithelial cell line hTERT-RPE with a nonmalignant phenotype were interrogated for HeLa cell selectivity in response to 1267 annotated compounds representing 56 pharmacological classes. Selective cytotoxic activity was observed for 14 of these compounds dominated by cyclic adenosine monophosphate (cAMP) selective phosphodiesterase (PDE) inhibitors, which tended to span a representation of the chemical descriptor space of the library. The PDE inhibitors induced delayed cell death with features compatible with classical apoptosis. The PDE inhibitors were largely inactive when tested against a cell line panel consisting of hematological and nonsquamous epithelial phenotypes. In a genome-wide DNA microarray analysis, PDE3A and PDE2A were found to be significantly increased in HeLa cells compared to the other cell lines. The pathway analysis software PathwayAssist was subsequently used to extract a list of proteins and small molecules retrieved from Medline abstracts associated with the hit compounds. The resulting list consisted of major parts of the cAMP-protein kinase A pathway linking to ERK, P38, and AKT. This molecular network may provide a basis for further exploitation of novel candidate targets for the treatment of squamous cell carcinoma.

Carcinoma, Squamous Cell↗

PubMed2005

Screening for apoptosis--classical and emerging techniques.

There has been a rapid development of cell-based assays and screening methods to identify promising apoptosis-inducing drug candidates for the treatment of cancer. Distinguishing between the complex processes involved in apoptosis and other forms of cell death requires information on both biochemical and morphological processes in the cell. Traditionally, many assays have been limited to measuring, for example, caspase activity using fluorogenic substrates. However, these screening assays provide only limited information on the complex processes involved in apoptosis. In this review we describe some of the available apoptosis assays amenable to high-throughput screening. In particular, image-based high-content screening assays to evaluate multiple biochemical and morphological parameters in apoptotic cells are described. Through combining the imaging of cells in microtiter plates with powerful image analysis algorithms, one can acquire deeper knowledge on multiple biochemical or morphological pathways at the single-cell level at an early stage in the development of novel anti-cancer drugs.

Animals↗

PubMed2004

Cytotoxic cyclotides from Viola tricolor.

A crude fraction of Viola tricolor rich in small lipophilic proteins was prepared and subjected to fractionation guided by bioactivity, using RP-HPLC and a fluorometric cytotoxicity assay. Two human cancer cell lines, U-937 GTB (lymphoma) and RPMI-8226/s (myeloma), were used in this study. The most potent compounds isolated, that is, the compounds showing the lowest IC(50) values, were shown to be three small proteins: vitri A (IC(50) = 0.6 microM and IC(50) = 1 microM, respectively), varv A (IC(50) = 6 microM and IC(50) = 3 microM, respectively), and varv E (IC(50) = 4 microM in both cell lines). Their sequences, determined by automated Edman degradation, quantitative amino acid analysis, and mass spectrometry, were cyclo-GESCVWIPCITSAIGCSCKSKVCYRNGIPC (vitri A), cyclo-GETCVGGTCNTPGCSCSWPVCTRNGLPVC (varv A), and cyclo-GETCVGGTCNTPGCSCSWPVCTRNGLPIC (varv E), of which vitri A is described for the first time. Each forms a head-to-tail cyclic backbone, with six cysteine residues being involved in three disulfide bonds, characteristic of the family of small proteins called the cyclotides. This is the first report on cyclotides from the species V. tricolor and the first report on the sequence of the cytotoxic cyclotide vitri A.

Amino Acid Sequence↗

PubMed2004

Antitumor efficacy and acute toxicity of the novel dipeptide melphalanyl-p-L-fluorophenylalanine ethyl ester (J1) in vivo.

The novel alkylating dipeptide melphalanyl-p-L-fluorophenylalanine ethyl ester (J1) was evaluated for acute toxicity and antitumor activity in mice, with melphalan as a reference. To determine a safe and tolerable dose for efficacy studies the acute toxicity following intravenous injection in the tail vein was monitored using a 14-day schedule with up to four doses. The highest tested dose, 25 micromoles/kg, was considered close to this level, with minor effects on body weight gain but significant effects on hematological parameters. Melphalan and J1 appeared equitoxic with no statistically significant differences. Subsequently a mouse hollow fiber model was employed with subcutaneous implantation of fibers containing human tumor cells. Three different human tumor cell lines as well as two samples of primary human tumor cells (ovarian carcinoma and chronic lymphatic leukemia) were used as tumor models. At the dose level tested there was a marked and statistically significant decrease in both T-cell leukemia CCRF-CEM and small cell lung cancer NCI-H69 tumor cell growth and viability in response to J1 as compared with both placebo and melphalan treated groups. In primary ovarian carcinoma cells only J1 treatment resulted in significant tumor regression (net cell kill). In summary the results indicate that, despite an expected short half time in the blood circulation, the promising in vitro data from the previous studies of J1 seems translatable into the in vivo situation. At equal doses of alkylating units J1, compared to melphalan, was more active in the mouse hollow-fiber model, but showed similar general toxicity.

Animals↗

PubMed2004

Development and characterization of two human tumor sublines expressing high-grade resistance to the cyanoguanidine CHS 828.

The cyanoguanidine CHS 828 has shown promising antitumor properties and is currently in early clinical trials, although the mechanism of action still is largely unknown. In this study, resistant sublines of the histiocytic lymphoma cell line U-937 GTB and the myeloma line RPMI 8226 were developed by culturing under gradually increasing concentrations of CHS 828 until reaching 25 times the parental line EC50s. The new phenotypes demonstrate more than 400-fold resistance to CHS 828 and cross-resistance to six cyanoguanidine analogs, but no resistance to nine standard drugs of different mechanistic classes or to the cytotoxic guanidines m-iodobenzylguanidine and methylglyoxal-bis(guanylhydrazone). The resistant phenotypes were stable for several months even if cultivated in drug-free medium and no difference in proliferation, ultrastructural or morphologic appearance in the sublines could be detected. Neither was decreased accumulation of tritium-labeled CHS 828 observed. Furthermore, the new U-937 phenotype was not accompanied by changes in differentiation or an altered cell-cycle distribution. In the myeloma cell line, esterase activity was shown to be moderately enhanced. Two-dimensional protein electrophoresis was undertaken to unmask possible resistance-mediating proteins and/or the target molecule(s) for CHS 828. In the myeloma cell line, lambda light chain immunoglobulin (down-regulated) and a fatty acid-binding protein (up-regulated) were identified. The findings presented here indicate that development of specific cellular alterations is responsible for the gained CHS 828 resistance.

Antineoplastic Agents↗

PubMed2003

Activity of hydrolytic enzymes in tumour cells is a determinant for anti-tumour efficacy of the melphalan containing prodrug J1.

Recently, we presented a series of melphalan containing di- and tripeptides with high cytotoxic activity and J1 (l-melphalanyl-p-l-fluorophenylalanine ethyl ester) was identified as one of the most interesting compounds. It was speculated that the increased activity compared to melphalan itself, demonstrated both in vitro and in vivo, resided in increased transport over the tumour cell membrane and/or hydrolytic cleavage and liberation of melphalan inside the cells. Indeed, overexpression of hydrolytic enzymes like peptidases, esterases and proteases has been described in several types of human malignancies, thus providing a target for selective chemotherapy. In this work, the details of the increased activity was further investigated and potential tumour selectivity is discussed. The intracellular delivery of melphalan is investigated in detail using peptidase resistant dipeptide derivatives, by enzyme inhibitors and probes for enzymatic activity and by studying the time dependency of drug effect as well as intracellular drug concentrations (cellular pharmacokinetics). The results show that the activity of the dipeptide mustards is highly dependent on intracellular hydrolysis, which result in rapid intracellular release of the alkylating unit (i.e. free melphalan) in cells with high enzymatic activity. The maximum intracellular melphalan concentration following J1 exposure was reached already after 15 min, thereafter declining with a half-life of approximately 1 h. This rapid intracellular loading resulted in less reduction of activity for J1 than for melphalan and six other standard drugs when human tumour cell lines were exposed to the drugs for a limited time (simulating short half-life in vivo). Peptidase inhibitors inhibited the activity and intracellular release of melphalan, and dipeptide derivatives designed to resist the action of peptidases was less active than the corresponding normal dipeptide.

Antineoplastic Agents, Alkylating↗

PubMed2003

Antitumor activity of the alkylating oligopeptides J1 (L-melphalanyl-p-L-fluorophenylalanine ethyl ester) and P2 (L-prolyl-m-L-sarcolysyl-p-L-fluorophenylalanine ethyl ester): comparison with melphalan.

Peptichemio, a mixture of six short oligopeptides all comprising the alkylating amino acid m-L-sarcolysin, has shown clinical activity in several malignancies. Previous studies have suggested that activity mainly resides in one of the peptides, P2 (L-prolyl-m-L-sarcolysyl-p-L-fluorophenylalanine ethyl ester). In the present study the in vitro activity of P2 was further investigated and compared to melphalan and the novel alkylating dipeptide J1 (L-melphalanyl-p-L-fluorophenylalanine ethyl ester), which is structurally related to P2 and melphalan. Cytotoxic activity was studied using patient tumor cells in a non-clonogenic cytotoxicity assay, whereas cellular response, and kinetics thereof, were studied in the lymphoma cell line U-937 GTB. Cellular metabolism was studied using microphysiometry, kinetic effects on macromolecular synthesis by radiolabeled substrate incorporation and, finally, the microculture kinetic assay of apoptosis was used to monitor morphologic changes following drug exposure. The assays compared P2 favorably with melphalan. Interestingly J1 was even more cytotoxic, and produced more pronounced effects in the kinetic assays for macromolecular synthesis, metabolic activity and apoptosis. The results indicate that the delivery properties of J1 are improved compared to those of melphalan and P2.

Antineoplastic Agents, Alkylating↗

PubMed2003

Structure-activity relationship for alkylating dipeptide nitrogen mustard derivatives.

The strategy of using small peptides for effective targeting of tumor cells in chemotherapy has proven beneficial. Recently we showed that J1 (L-melphalanyl-p-L-fluorophenylalanine ethyl ester), an alkylating nitrogen mustard-containing dipeptide, exhibited strong cytotoxic activity in fresh human tumor samples in addition to rapid and pronounced inhibition of macromolecular syntheses and cellular respiration in the human tumor lymphoma cell line U-937 GTB. In this study, an additional series of 17 nitrogen mustard-containing dipeptides has been synthesized and analyzed for cytotoxic activity in a panel of 10 human tumor cell lines. The results were compared to the single amino acid mustard derivative melphalan and its ethyl and isopropyl esters. Also P2 (L-prolyl-m-L-sarcolysyl-p-L-fluorophenylalanine ethyl ester), a tripeptide that previously has shown impressive effects in human tumor cells, was used as reference. The tested compounds displayed various activities in the different cell lines but also showed a high correlation, indicating a similar mechanism of action. Factors like amino acid composition, amino acid sequence, modifications of the C- and N-termini, and to a minor extent the lipophilicity of the dipeptide derivatives appear to influence the in vitro activity. The results indicate that the activity of these compounds not only relies on their chemical reactivity, but also on active biological interactions such as transport across membranes and/or enzymatic liberation of reactive molecular entities.

Alkylation↗

PubMed2003

Antitumor activity of the novel melphalan containing tripeptide J3 (L-prolyl-L-melphalanyl-p-L-fluorophenylalanine ethyl ester): comparison with its m-L-sarcolysin analogue P2.

Peptichemio (PTC), a mixture of six oligopeptides all containing m-L-sarcolysin, has previously shown impressive results in clinical trials. The tripeptide P2 (L-prolyl-m-L-sarcolysyl-p-L-fluorophenylalanine ethyl ester) has been suggested as the main contributor to PTC activity. In contrast to its analogue melphalan, m-L-sarcolysin never reached clinical use. To allow a direct comparison, the corresponding melphalan containing tripeptide J3 (L-prolyl-L-melphalanyl-p-L-fluorophenylalanine ethyl ester) was synthesized and its activity was compared with that of P2; the activities of melphalan and m-L-sarcolysin were studied in parallel. Cytotoxic activity in human tumor cell lines and some fresh human tumor specimens were analyzed as well as effects on cellular metabolism, macromolecular synthesis, and preliminary evaluation of the cell death characteristics. The results show that melphalan and m-L-sarcolysin display similar activity in these systems and that the tripeptides were more active than their parent monomers. Surprisingly however, the melphalan containing tripeptide J3 demonstrated a significantly more rapid and stronger activity than the m-L-sarcolysin analogue P2. Finally, the in vivo toxicity and activity of melphalan and J3 were investigated in mice bearing human leukemia cells in s.c. fibers. The in vitro results seem translatable into the in vivo situation, demonstrating better antileukemic effect of J3 but similar side effects as melphalan.

Antineoplastic Agents, Alkylating↗

PubMed2002

Ligatoxin B, a new cytotoxic protein with a novel helix-turn-helix DNA-binding domain from the mistletoe Phoradendron liga.

A new basic protein, designated ligatoxin B, containing 46 amino acid residues has been isolated from the mistletoe Phoradendron liga (Gill.) Eichl. (Viscaceae). The protein's primary structure, determined unambiguously using a combination of automated Edman degradation, trypsin enzymic digestion, and tandem MS analysis, was 1-KSCCPSTTAR-NIYNTCRLTG-ASRSVCASLS-GCKIISGSTC-DSGWNH-46. Ligatoxin B exhibited in vitro cytotoxic activities on the human lymphoma cell line U-937-GTB and the primary multidrug-resistant renal adenocarcinoma cell line ACHN, with IC50 values of 1.8 microM and 3.2 microM respectively. Sequence alignment with other thionins identified a new member of the class 3 thionins, ligatoxin B, which is similar to the earlier described ligatoxin A. As predicted by the method of homology modelling, ligatoxin B shares a three-dimensional structure with the viscotoxins and purothionins and so may have the same mode of cytotoxic action. The novel similarities observed by structural comparison of the helix-turn-helix (HTH) motifs of the thionins, including ligatoxin B, and the HTH DNA-binding proteins, led us to propose the working hypothesis that thionins represent a new group of DNA-binding proteins. This working hypothesis could be useful in further dissecting the molecular mechanisms of thionin cytotoxicity and of thionin opposition to multidrug resistance, and useful in clarifying the physiological function of thionins in plants.

Amino Acid Sequence↗

PubMed2002

Modulation of pyridyl cyanoguanidine (CHS 828) induced cytotoxicity by 3-aminobenzamide in U-937 GTB cells.

The role of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) and the ADP-ribosylation inhibitor 3-aminobenzamide (3-ABA) in the cytotoxicity induced by the novel antitumoral cyanoguanidine CHS 828 was investigated in the human lymphoma cell line U-937 GTB. Exposing cells to CHS 828 and 3-ABA in combination resulted in a 100-fold higher IC(50) compared to exposure to CHS 828 alone. CHS 828 did not activate PARP, measured as PARP-activity and formation of poly(ADP-ribose). The ATP-levels and levels of extracellular acidification rate of cells exposed to CHS 828 in combination with 3-ABA were maintained for a longer period than for cells exposed to CHS 828 alone. To characterize the mode of cell death, caspase-3 activity and gross morphology were assessed. 3-ABA increased and delayed the caspase-3 activity in cells exposed to CHS 828. Cells exposed to high concentrations of CHS 828 showed a necrotic morphology, while high concentrations of CHS 828 in combination with 3-ABA switched the mode of cell death, generating an apoptotic morphology. The results indicate that the cytotoxicity and morphology induced by CHS 828 is not due to PARP activation but can be modulated by the ADP-ribosylation inhibitor 3-ABA.

Antineoplastic Agents↗

PubMed2002

Selective cytotoxicity evaluation in anticancer drug screening of fractionated plant extracts.

Chosen to reflect biodiversity in a phylogenetic sense, 100 fractionated plant extracts were screened in vitro for cytotoxicity following extraction and fractionation (polypeptide isolation). Of these 100 extracts, 30 were selected and then characterized preliminarily for antitumor potency and mode of action by testing them on two cell lines and primary cultures of human tumor cells. On the basis of cytotoxicity potency, 10 of the extracts were further characterized for anticancer activity in 10 human tumor cell lines. This final testing resulted in seven potential lead plants with superior evidence of antitumor potential: Colchicum autumnale L. (Colchicaceae), Digitalis lanata Ehrh. and Digitalis purpurea L. (Plantaginaceae), Helleborus cyclophyllus Boiss. (Ranunculaceae), Menyanthes trifoliata L. (Menyanthaceae), and Viola arvensis Murr. and Viola patrinii Ging. (Violaceae). Within a database of antitumor compounds, the activity profiles of the extracts from these seven plants were compared, by correlation analysis, with those of more than 100 other compounds, including 39 standard drugs from different classes of cytotoxic mechanisms. The activity profiles of six of these candidates were uncorrelated with those of the standard drugs, possibly indicating new pathways of drug-mediated cell death.

Antineoplastic Agents, Phytogenic↗

PubMed2002

Cyclotides: a novel type of cytotoxic agents.

Cytotoxic activities of three naturally occurring macrocyclic peptides (cyclotides) isolated from the two violets, Viola arvensis Murr. and Viola odorata L., were investigated. A nonclonogenic fluorometric microculture assay was used to examine cytotoxicity in a panel of 10 human tumor cell lines representing defined types of cytotoxic drug resistance. Additionally, primary cultures of tumor cells from patients, and for comparison normal lymphocytes, were used to quantify cytotoxic activity. All three cyclotides, varv A, varv F, and cycloviolacin 02, exhibited strong cytotoxic activities, which varied in a dose-dependent manner. Cycloviolacin 02 was the most potent in all cell lines (IC50 0.1-0.3 microM), followed by varv A (IC50 2.7-6.35 microM) and varv F (IC50 2.6-7.4 microM), respectively. Activity profiles of the cyclotides differed significantly from those of antitumor drugs in clinical use, which may indicate a new mode of action. This, together with the exceptional chemical and biological stability of cyclotides, makes them interesting in particular for their potential as pharmacological tools and possibly as leads to antitumor agents.

Amino Acid Sequence↗

PubMedDate not supplied

Cytotoxic activity of a new lipid formulation of doxorubicin in cell lines and primary tumor cells.

BACKGROUND: Liposomal formulations of the anthracyclines are being developed to circumvent toxicity and prolong effect. The current study investigates the in vitro activity of a novel doxorubicin micelle formulation, containing a vehicle designed to release pharmacologically active subcomponents. MATERIALS AND METHODS: The cytotoxicity of doxorubicin formulated in a vehicle containing C4 (N-docosahexaenoyl-O-phospho-2-aminoethanol) and C11 (N-all trans-retinoyl-O-phospho-L-tyrosine) was measured in a panel of human tumor cell lines, 19 primary cultures of human tumor cells and 5 lymphocyte preparations. RESULTS AND CONCLUSION: At the tested ratio between doxorubicin and C4/C11 (1:50), C4/C11 contributed significantly to the in vitro toxicity. However, the molar EC50-values were lower for doxorubicin than for C4/C11. Synergistic interactions between doxorubicin and C4/C11 were evident in a majority of the cell types studied. C4/C11 increased the cellular load of the fluorescent Pgp substrate calcein. To further investigate the possible benefits of the new formulation, in vivo studies are ongoing.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMedDate not supplied

p53 status and its in vitro relationship to radiosensitivity and chemosensitivity in lung cancer.

The following study was designed to investigate if mutations within the p53 gene are associated with radiation responsiveness or response to different cytotoxic drugs. Nine human lung cancer cell lines were examined (four SCLC and five NSCLC cell lines). cDNA-based sequencing of the entire p53 gene was performed. All cell lines were characterised with respect to drug-sensitivity towards eight cytotoxic drugs using the FMCA method and data from the clonogenic assay were studied to obtain information concerning radioresponsiveness. All the cell lines expressed mutations; six were missense mutations and three were deletions. A statistically significant increase in radiosensitivity was found for mutations in exon 7 (p = 0.019), compared with the other mutations localised within different exons of p53. Further statistical analyses using Fisher's two-tailed exact test confirmed that mutations in exon 7 were significantly associated with radiosensitivity, p = 0.047. No correlation concerning mutations in separate exons and response towards different chemotherapeutic agents could be found. Our results indicate that p53 mutations in exon 7 might be associated with increased radiation sensitivity in these human lung cancer cell lines, but our data should be interpreted with caution since several other explanations might exist regarding what determines the response towards radiation.

Carcinoma, Non-Small-Cell Lung↗