Cellular uptake and interaction with purified membranes of rebeccamycin derivatives

Rebeccamycin is an antitumor antibiotic possessing a DNA-intercalating indolocarbazole chromophore linked to a glycosyl residue. The carbohydrate moiety of rebeccamycin and related synthetic analogues, such as the potent antitumor drug NB-506 (6-N-formylamino-12,13-dihydro-1, 11-dihydroxy-13-(beta-D-glucopyranosyl)-5H-indolo[2,3-a]pyrrolo- [3,4-c]carbazole-5,7-(6H)-dione), is a key element for both DNA-binding and inhibition of DNA topoisomerase I. In this study, we have investigated the cellular uptake of rebeccamycin derivatives and their interaction with purified membranes. The transport of radiolabeled [3H]dechlorinated rebeccamycin was studied using the human leukemia HL60 and melanoma B16 cell lines as well as two murine leukemia cell lines sensitive (P388) or resistant (P388CPT5) to camptothecin. In all cases, the uptake is rapid but limited to about 6% of the drug molecules. In HL60 cells, the uptake entered a steady-state phase of intracellular accumulation of about 0.26+/-0.05 pmol/10(6) cells, which persisted to at least 90 min. The efflux of exchangeable radiolabeled molecules was relatively weak. Fluorescence studies were performed to compare the interaction of a rebeccamycin derivative and its aglycone with membranes purified from HL60 cells. The glycosylated drug molecules bound to the cell membranes can be extracted upon washing with buffer or by adding an excess of DNA. In contrast, the indolocarbazole drug lacking the carbohydrate domain remains tightly bound to the membranes with very little or no exchange upon the addition of DNA. The membrane transport and binding properties of indolocarbazole drugs related to rebeccamycin are reminiscent to those of other DNA-intercalating antitumor agents. The uptake most likely occurs via a passive diffusion through the plasma membranes and the glycosyl residue of the drug plays an essential role for the translocation of the drug from the membranes to the internal cell components, such as DNA.     

Embryology and anatomy of the anorectum. Basis of surgery

The rectum is a pelvic organ, complex in its morphology and its topographic relationships. Its double embryologic origin explains the two types of tumors that develop in the rectum: (1) lieberkühnian adenocarcinoma in the pelvic rectum and (2) squamous epithelioma in the anal canal. Its venous and lymphatic supply, intensively developed, realizes early pathway of tumoral dissemination. The pelvic relationships of the rectum and anus explain the technical difficulty of rectal surgery, especially when subperitoneal resection and anastomosis are concerned. Imaging of this area permits an early diagnosis of rectal tumors and allows a less invasive surgery with a carcinologic precision.     

Formaldehyde-induced alkylation of a 2'-aminoglucose rebeccamycin derivative to both A.T and G.C base pairs in DNA

Rebeccamycin derivatives represent a promising class of antitumor agents. In this series, two glycosylated indolocarbazoles, NB-506 and NSC-655649, are currently undergoing clinical trials. Their anticancer activities are associated with their capacities to interact with DNA and to inhibit DNA topoisomerases. Previous studies revealed that the planar indolocarbazole chromophore can intercalate into DNA, locating the appended carbohydrate residue in one of the two helical grooves, probably the minor groove as is the case with the anthracyclines and other DNA-binding antibiotics. The sugar residue contributes significantly to the DNA binding free energy of NB-506. However, the exact positioning of the glycosyl residue of rebeccamycin derivatives in the drug-DNA complex remains poorly understood. To better understand how glycosylated indolocarbazoles interact with DNA, we investigated the interaction of a rebeccamycin derivative (85) bearing a 2'-amino group on the sugar residue. We show that the presence of the 2'-amino function permits the formation of covalent drug-DNA complexes in the presence of formaldehyde. Complementary biochemical and spectroscopic measurements attest that 85 reacts covalently with the 2-amino group of guanines exposed in the minor groove of the double helix, as is the case with daunomycin. In contrast to daunomycin, 85 also forms cross-links with an oligonucleotide containing only A.T base pairs. The covalent binding to A.T base pairs was detected using a gel mobility shift assay and was independently confirmed by thermal denaturation studies and by fluorescence measurements using a series of synthetic polynucleotides. The HCHO-mediated alkylation reaction of the drug with A.T base pairs apparently involves the 6-amino group of adenines exposed in the major groove whereas the covalent attachment to G.C base pairs implicates the 2-amino group of guanines situated in the opposite minor groove. Therefore, the results suggest that either the drug is able to switch grooves in response to sequence or it can simultaneously bind to both the minor and major grooves of the double helix. This study will help to guide the rational design of new DNA-binding antitumor indolocarbazole drugs and also provides a general experimental approach for probing minor versus major groove interactions between small molecules and DNA.     

Pim-selective inhibitor DHPCC-9 reveals Pim kinases as potent stimulators of cancer cell migration and invasion

Background  

Pim family kinases are small constitutively active serine/threonine-specific kinases, elevated levels of which have been detected in human hematopoietic malignancies as well as in solid tumours. While we and others have previously shown that the oncogenic Pim kinases stimulate survival of hematopoietic cells, we now examined their putative role in regulating motility of adherent cancer cells. For this purpose, we inhibited Pim kinase activity using a small molecule compound, 1,10-dihydropyrrolo[2,3-a]carbazole-3-carbaldehyde (DHPCC-9), which we had recently identified as a potent and selective inhibitor for all Pim family members.     

Risk factors of failure and immediate complication of subclavian vein catheterization in critically ill patients

OBJECTIVE: To identify the risk factors of failure and immediate complication of subclavian vein catheterization (SVC). DESIGN: Prospective observational study. SETTING: Surgical critical care unit of a tertiary university hospital. PATIENTS: Critically ill patients requiring a first SVC. INTERVENTION: Subclavian vein catheterization was attempted in 707 patients without histories of surgery or radiotherapy in the subclavian area. Failed catheterizations, arterial punctures, pneumothoraces and misplacements of the catheter tip were recorded. Risk factors of failure and immediate complication were isolated among patients' characteristics, procedure parameters (side and number of venipunctures) and the operator's experience using a univariate +/- multivariate analysis. MEASUREMENTS AND MAIN RESULTS: Five hundred sixty-two SVCs (79.5%) were achieved without adverse events. Among the remaining 145 catheterizations, 67 (9.5%) failures, 55 (7.8%) arterial punctures, 22 (3.1%) pneumothoraces and 30 (4.2%) misplacements of the catheter tip occurred. More than one venipuncture was the only risk factor of failed catheterization [2 venipunctures, odds ratio =7.4 (2.1-26); >2 venipunctures, odds ratio =49.1 (16.8-144.1)]. More than one venipuncture and age 77 years or more were predictive of the occurrence of immediate complications [2 venipunctures, odds ratio =3.6 (1.8-7.0); >2 venipunctures, odds ratio =14 (7.7-25.3); age >or=77, odds ratio =1.8 (1.0-3.1)]. The operator's training was not predictive of failed catheterization or immediate complication. CONCLUSION: For SVC, more than one venipuncture is predictive of failed catheterization and immediate complication. Age 77 years or more was predictive of immediate complications.     

Peculiar acute toxic colitis after ingestion of colocynth: a clinicopathological study of three cases.

We report three examples of toxic acute colitis which occurred after ingestion of colocynth (Citrullus colocynthis) for ritual purposes. The prominent clinical feature was dysenteric diarrhoea; colonoscopic changes included congestion and hyperaemia of the mucosa with abundant exudates but no ulceration or pseudopolyp formation. A causal relationship between colonic injury and the intake of colocynth was supported by the following features: (1) the pharmacology of the colocynth extract ingested; (2) the temporal relationship between colocynth intake and clinical onset (eight to 12 h); (3) the rapid recovery within three to six days, with normal endoscopy at day 14; (4) the absence of other possible causes for the observed patterns, except in one case, in which a concomitant intestinal infection with Clostridium perfringens Type A was discovered; (5) the specific pathological features. Colonic biopsies taken 27, 44, and 72 h after colocynth intake showed: erosions with fibrino-purulent exudate, early fibrosis of the lamina propria, hyaline thickening of the superficial epithelial basal membrane. These pathological features completely disappeared within 14 days in all three cases.     

Semi-synthesis of A23187 (calcimycin) analogs. III. Modification of benzoxazole ring substituents, ionophorous properties in an organic phase

Ten semi-synthetic analogs of A23187 (calcimycin), with only the benzoxazole ring substituents modified together with the ionophore X14885A were studied with regard to their calcium and magnesium carrier properties through an organic phase (toluene - butanol, 70:30). The results indicate that the carboxylic group and the oxazolic nitrogen, maintained in the ortho position are essential for the ionophorous properties. Further, the introduction of a substituent in place of the NHCH3 group, producing steric hindrance of the carboxylic group leads to a destabilization of the 2:1 associations with cations.     

Synthesis, in vitro antiproliferative activities, and Chk1 inhibitory properties of indolylpyrazolones and indolylpyridazinedione

The synthesis of 5-indolylpyrazol-3-one, 4-indolylpyrazol-3-one and 4-indolyl-pyridazin-3,6-dione is reported. Their Chk1 inhibitory properties have been evaluated and their in vitro antiproliferative activities toward three tumor cell lines: murine leukemia L1210, human colon carcinoma HT29 and HCT116 have been determined. 4-Indolyl-pyridazin-3,6-dione is inactive against Chk1 and exhibits weak cytotoxicities toward the tumor cell lines tested. The IC(50) values toward Chk1 of the two indolylpyrazolones are identical and are in the micromolar range, but the cytotoxicities of 4-indolylpyrazol-3-one are significantly stronger than those of 5-indolylpyrazol-3-one. Since 4-indolylpyrazol-3-one and 5-indolylpyrazol-3-one can present several conformers and tautomeric forms, molecular modelling in the ATP binding site of Chk1 has been carried out to investigate which form could induce the best stabilization in the active site of the enzyme. To get an insight into the kinase selectivity of these compounds, their inhibitory activities toward Src kinase were evaluated.     

Synthesis and antiproliferative activities of diversely substituted glycosyl-isoindigo derivatives

In the course of structure-activity relationship studies, diversely substituted 1-(beta-D-glucopyranosyl)-isoindigo derivatives were prepared from commercially available indolines. Their antiproliferative activities were evaluated toward a panel of human solid cancer cell lines (PC 3, DLD-1, MCF-7, M4Beu, A549, PA 1), a murine cell line (L929) and a human fibroblast primary culture to get an insight into the substitution pattern required for the best biological potencies.     

Synthesis and antiproliferative activities of indolin-2-one derivatives bearing amino acid moieties

A convenient synthesis of indolin-2-ones substituted in the 3 position by an aminomethylene group bearing different amino acid moieties is described. Their antiproliferative activities were evaluated toward a panel of human solid tumor cell lines (PC 3, DLD-1, MCF-7, M4 Beu, A549, PA 1) and healthy cell lines (a murine fibroblast L929 and a human fibroblast primary culture).