Gene profiling of clinical routine biopsies and prediction of survival in non-small cell lung cancer.

RATIONALE: Global gene expression analysis provides a comprehensive molecular characterization of non-small cell lung cancer (NSCLC). OBJECTIVES: To evaluate the feasibility of integrating expression profiling into routine clinical work-up by including both surgical and minute bronchoscopic biopsies and to develop a robust prognostic gene expression signature. METHODS: Tissue samples from 41 chemotherapy-naive patients with NSCLC and 15 control patients with inflammatory lung diseases were obtained during routine clinical work-up and gene expression profiles were gained using an oligonucleotide array platform (NovaChip; 34'207 transcripts). Gene expression signatures were analyzed for correlation with histological and clinical parameters and validated on independent published data sets and immunohistochemistry. MEASUREMENTS AND MAIN RESULTS: Diagnostic signatures for adenocarcinoma and squamous cell carcinoma reached a sensitivity of 80%/80% and a specificity of 83%/94%, respectively, dependent on the proportion of tumor cells. Sixty-seven of the 100 most discriminating genes were validated with independent observations from the literature. A 13-gene metagene refined on four external data sets was built and validated on an independent data set. The metagene was a strong predictor of survival in our data set (hazard ratio = 7.7, 95% CI [2.8-21.2]) and in the independent data set (hazard ratio = 1.6, 95% CI [1.2-2.2]) and in both cases independent of the International Union against Cancer staging. Vascular endothelial growth factor-beta, one of the key prognostic genes, was further validated by immunohistochemistry on 508 independent tumor samples. CONCLUSIONS: Integration of functional genomics from small bronchoscopic biopsies allows molecular tumor classification and prediction of survival in NSCLC and might become a powerful adjunct for the daily clinical practice.

DNA binding to guide the development of tetrahydroindeno[1,2-b]pyrido[4,3,2-de]quinoline derivatives as cytotoxic agents.

The tetrahydroindeno[1,2-b]pyrido[4,3,2-de]quinoline chromophore was initially designed as a DNA intercalating unit because of its planar structure. Unexpectedly, one molecule (15d) bearing two N-methylpiperazine chains on both sides of this condensed pentacyclic skeleton fits into the minor groove of DNA and preferentially recognizes AT-rich sequences. The monosubstituted compound 16d was identified as a potent cytotoxic DNA intercalator, whereas the disubstituted analogue 15d represents a new structural motif for the development of DNA sequence-reading small molecules.

Topoisomerase I-mediated DNA cleavage as a guide to the development of antitumor agents derived from the marine alkaloid lamellarin D: triester derivatives incorporating amino acid residues.

The marine alkaloid lamellarin D (LAM-D) has been recently characterized as a potent poison of human topoisomerase I endowed with remarkable cytotoxic activities against tumor cells. We report here the first structure-activity relationship study in the LAM-D series. Two groups of triester compounds incorporating various substituents on the three phenolic OH at positions 8, 14 and 20 of 6H-[1]benzopyrano[4',3':4,5]pyrrolo[2,1-a]isoquinolin-6-one pentacyclic planar chromophore typical of the parent alkaloid were tested as topoisomerase I inhibitors. The non-amino compounds in group A showed no activity against topoisomerase I and were essentially non cytotoxic. In sharp contrast, compounds in group B incorporating amino acid residues strongly promoted DNA cleavage by human topoisomerase I. LAM-D derivatives tri-substituted with leucine, valine, proline, phenylalanine or alanine residues, or a related amino side chain, stabilize topoisomerase I-DNA complexes. The DNA cleavage sites detected at T downward arrow G or C downward arrow G dinucleotides with these molecules were identical to that of LAM-D but slightly different from those seen with camptothecin which stimulates topoisomerase I-mediated cleavage at T downward arrow G only. In the DNA relaxation and cleavage assays, the corresponding Boc-protected compounds and the analogues of the non-planar LAM-501 derivative lacking the 5-6 double bond in the quinoline B-ring showed no effect on topoisomerase I and were considerably less cytotoxic than the corresponding cationic compounds in the LAM-D series. The presence of positive charges on the molecules enhances DNA interaction but melting temperature studies indicate that DNA binding is not correlated with topoisomerase I inhibition or cytotoxicity. Cell growth inhibition by the 41 lamellarin derivatives was evaluated with a panel of tumor cells lines. With prostate (DU-145 and LN-CaP), ovarian (IGROV and IGROV-ET resistant to ecteinascidin-743) and colon (LoVo and LoVo-Dox cells resistant to doxorubicin) cancer cells (but not with HT29 colon carcinoma cells), the most cytotoxic compounds correspond to the most potent topoisomerase I poisons. The observed correlation between cytotoxicity and topoisomerase I inhibition strongly suggests that topoisomerase I-mediated DNA cleavage assays can be used as a guide to the development of superior analogues in this series. LAM-D is the lead compound of a new promising family of antitumor agents targeting topoisomerase I and the amino acid derivatives appear to be excellent candidates for a preclinical development.

Lamellarin D: a novel potent inhibitor of topoisomerase I.

We report the identification and characterization of a novel potent inhibitor of DNA topoisomerase I: lamellarin D (LAM-D), initially isolated from a marine mollusk, Lamellaria sp., and subsequently identified from various ascidians. This alkaloid, which displays potent cytotoxic activities against multidrug-resistant tumor cell lines and is highly cytotoxic to prostate cancer cells, bears a 6H-[1]benzopyrano[4',3':4,5]pyrrolo[2,1-a]isoquinolin-one pentacyclic planar chromophore, whereas its synthetic 5,6-dehydro analogue, LAM-501, has a significantly tilted structure. DNA binding measurements by absorbance, fluorescence, and electric linear dichroism spectroscopy show that LAM-D is a weak DNA binder that intercalates between bp of the double helix. In contrast, the nonplanar analogue LAM-501 did not bind to DNA and failed to inhibit topoisomerase I. DNA intercalation may be required for the stabilization of topoisomerase I-DNA complexes by LAM-D. In the DNA relaxation assay, LAM-D strongly promoted the conversion of supercoiled DNA into nicked DNA in the presence of topoisomerase I. The marine product was approximately 5 times less efficient than camptothecin (CPT) at stabilizing topoisomerase I-DNA complexes, but interestingly, the two drugs exhibited slightly distinct sequence specificity profiles. Topoisomerase I-mediated DNA cleavage in the presence of LAM-D occurred at some sites common to CPT, but a few specific sites identified with CPT but not with LAM-D or conversely unique sites cleaved by LAM-D but not by CPT were detected. The distinct specificity profiles suggest that LAM-D and CPT interact differently with the topoisomerase I-DNA interface. A molecular modeling analysis provided structural information on the orientation of LAM-D within the topoisomerase I-DNA covalent complex. The marine alkaloid did not induce DNA cleavage by topoisomerase II. Immunoblotting experiments revealed that endogenous topoisomerase I was efficiently trapped on DNA by LAM-D in P388 and CEM leukemia cells. P388/CPT5 and CEM/C2 cell lines, both resistant to CPT and expressing a mutated top1 gene, were cross-resistant to LAM-D. Collectively, the results identify LAM-D as a novel lead candidate for the development of topoisomerase I-targeted antitumor agents.

Indolizino[1,2-b]quinolines derived from A-D rings of camptothecin: synthesis and DNA interaction.

Camptothecin consists of a lactone E-ring adjacent to a tetracyclic A-D ring planar chromophore which are essential for topoisomerase I inhibition and DNA interaction, respectively. The A-D ring system can be exploited to develop DNA-binding molecules. Indolizino[1,2-b]quinoline derivatives substituted with a piperidinoethyloxy side chain on the A-ring and an aminomethyl function on the D one were synthesized and their DNA-binding properties and in vitro cytotoxicity investigated.

Indolocarbazole glycosides in inactive conformations.

Indolocarbazole glycosides related to rebeccamycin represent a promising category of antitumor agents targeting DNA and topoisomerase I. These drugs prefer to adopt a closed conformation with an intramolecular hydrogen bond between the indole NH group and the pyranose oxygen atom. Three pairs of indolocarbazole monoglycosides bearing an NH or an N-methyl indole moiety were synthesized and their biological properties investigated at the molecular and cellular level. Replacing the indole NH proton with a methyl group reduces DNA interaction and abolishes activity against DNA topoisomerase I. Surface plasmon resonance studies performed with a pair of water-soluble indolocarbazole glycosides and two hairpin oligonucleotides containing an [AT]4 or a [CG]4 sequence indicate that both the NH and the N-methyl derivative maintain a relatively high affinity for DNA (Keq = 2 - 6 x 10(5) M(-1)) but the incorporation of the methyl group restricts access to the DNA. The number of ligand binding sites (n) on the oligonucleotides is about twice as high for the NH compound compared to its N-methyl analogue. Modeling and 1H NMR studies demonstrate that addition of the N-methyl group drives a radical change in conformation in which the orientation of the aglycone relative to the beta-glucoside is reversed. The loss of the closed conformation by the N-methyl derivatives perturbs thir ability to access DNA binding sites and prevents the drug from inhibiting topoisomerase I. As a consequence, the NH compounds exhibit potent cytotoxicity against CEM leukemia cells with an IC50 value in the 1 microM range, whereas the N-methyl analogues are 10 to 100 times less cytotoxic. These studies offer circumstantial evidence supporting the importance of the closed conformation in the interaction of indolocarbazole glycosides with their molecular targets, DNA and topoisomerase I.

Highly cytotoxic benzo[c]pyrido[2,3,4-kl]acridines.

Several benzo[c]pyrido[2,3,4-kl]acridines bearing different substituents on the A and E rings were synthesized and evaluated for their capacity to bind to DNA and to inhibit DNA topoisomerases. Potent cytotoxic compounds were discovered but no strict correlation with their DNA binding affinity and effects on topoisomerases were observed. DNA is one but not the unique target of these compounds.

Formaldehyde-induced DNA cross-link of indolizino[1,2-b]quinolines derived from the A-D rings of camptothecin.

Camptothecin consists of a lactone E ring adjacent to tetracyclic A-D rings of a planar chromophore, which are essential for topoisomerase I inhibition and DNA interaction. The A-D rings can be exploited to develop DNA-sequence-reading molecules. Indolizino[1,2-b]quinoline derivatives substituted with a piperidinoethyloxy side chain and an aminomethyl function on rings A and D, respectively, were synthesized, and their DNA binding and formaldehyde-mediated bonding properties were investigated.

DNA binding and topoisomerase I poisoning activities of novel disaccharide indolocarbazoles.

The antibiotics AT2433-A1 and AT2433-B1 are two indolocarbazole diglycosides related to the antitumor drug rebeccamycin known to stabilize topoisomerase I-DNA complexes. This structural analogy prompted us to explore the binding of four indolocarbazole diglycosides with DNA and their capacity to interfere with the DNA cleavage-reunion reaction catalyzed by topoisomerase I. The molecular basis of the drug interaction with double-stranded DNA and with purified chromatin, with particular emphasis on the role of the carbohydrate moiety, was investigated by means of complementary spectroscopic techniques, including surface plasmon resonance and electric linear dichroism. We compared the DNA binding properties, sequence recognition, and effects on topoisomerase I-mediated DNA relaxation and cleavage of AT2433-A1 bearing a 2,4-dideoxy-4-methylamino-L-xylose residue, its dechlorinated analog AT2433-B1, the diastereoisomer iso-AT2433-B1 with an inverted aminosugar residue, and compounds 5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione, 12-beta-D-glucopyranosyl-12,13-dihydro-6-methyl (JDC-108) and 5H-indolo[2,3-a]pyrrolo[3, 4-c]carbazole-5,7(6H)-dione, 12-(6-O-alpha-D-galacto-pyranosyl-beta-D-glucopyranosyl)-12,13-dihydro-6-methyl (JDC-277) with an uncharged mono- and disaccharide, respectively. The two antibiotics AT2433-A1 and AT2433-B1 proved to be highly cytotoxic to leukemia cells and this may be a consequence of their tight intercalative binding to DNA, preferentially into GC-rich sequences as inferred from DNase I footprinting studies and surface plasmon resonance measurements. Like the diastereoisomer iso-AT2433-B1, they have no inhibitory effect on topoisomerase I, in contrast to the uncharged diglycoside JDC-277, which stimulates DNA cleavage by the enzyme mainly at TG sites, as observed with camptothecin. Cytotoxicity measurements with CEM and CEM/C2 human leukemia cell lines sensitive and resistant to camptothecin, respectively, also suggested that topoisomerase I contributes, at least partially, to the mechanism of action of the neutral diglycoside JDC-277 but not to that of the cationic AT2433 compounds. Together, the results indicate that sequence-selective DNA interaction and topoisomerase I inhibition is controlled to a large extent by the stereochemistry of the diglycoside moiety.

Distribution of furamidine analogues in tumor cells: influence of the number of positive charges.

Fluorescence microscopy has been used to study the cellular distribution properties of a series of DNA binding cationic compounds related to the potent antiparasitic drug furamidine (DB75). The compounds tested bear a diphenylfuran or a phenylfuranbenzimidazole unfused aromatic core substituted with one or two amidine or imidazoline groups. The synthesis of five new compounds is reported. The B16 melanoma cell line was used to compare the capacities of mono-, bis-, and tetracations to enter the cell and nuclei. The high-resolution fluorescence pictures show that in the furamidine series, the compounds with two or four positive charges selectively accumulate in the cell nuclei whereas, in most cases, those bearing only one positive charge show reduced cell uptake capacities. One of the monocationic compounds, DB607, distributes in the cytoplasm, possibly in mitochondria, with no distinct nuclear accumulation. In sharp contrast, furamidine and benzimidazole analogues, including the drug DB293 that forms DNA minor groove dimers, efficiently accumulate in the cell nuclei and the intranuclear distribution of these DNA minor groove binders is significantly different from that seen with the DNA intercalating drug propidium iodide. The results suggest that the presence of two amidine terminal groups plays a role in facilitating nuclear accumulation into cells, probably as a result of nucleic acid binding. The determination of DNA melting temperature increases on addition of these compounds supports the importance of DNA binding in nuclear uptake.

Dimers from dechlorinated rebeccamycin: synthesis, interaction with DNA, and antiproliferative activities.

In the course of structure-activity relationships on rebeccamycin analogues, two dimers of dechlorinated rebeccamycin were synthesised with the aim to improve the interaction with DNA and in vitro antiproliferative activities. The synthesis of two dimeric compounds obtained by joining two molecules of dechlorinated rebeccamycin via the imide nitrogen is described. Melting temperature and DNase I footprinting studies were performed to investigate their interaction with DNA. Four tumour cell lines, murine L1210 leukaemia, human HT29 colon carcinoma, A549 non-small cell lung carcinoma and K-562 leukaemia, were used to evaluate the cytotoxicity of the drugs. Their effects on the cell cycle of L1210 cells were also investigated.

DNA sequence recognition by the indolocarbazole antitumor antibiotic AT2433-B1 and its diastereoisomer.

The antibiotic AT2433-B1 belongs to a therapeutically important class of antitumor agents. This natural product contains an indolocarbazole aglycone connected to a unique disaccharide consisting of a methoxyglucose and an amino sugar subunit, 2,4-dideoxy-4-methylamino-L-xylose. The configuration of the amino sugar distinguishes AT2433-B1 from its diastereoisomer iso-AT2433-B1. Here we have investigated the interaction of these two disaccharide indolocarbazole derivatives with different DNA sequences by means of DNase I footprinting and surface plasmon resonance (SPR). Accurate binding measurements performed at 4 and 25 degrees C using the BIAcore SPR method revealed that AT2433-B1 binds considerably more tightly to a hairpin oligomer containing a [CG](4) block than to an oligomer with a central [AT](4) tract. The kinetic analysis shows that the antibiotic dissociates much more slowly from the GC sequence compared to the AT one. Preferential binding of AT2433-B1 to GC-rich sequences in DNA was independently confirmed by DNase I footprinting experiments performed with a 117 bp DNA restriction fragment. The specific binding sequence 5'-AACGCCAG identified from the footprints was then converted into a biotin-labeled DNA hairpin duplex and compound interactions with this specific sequence were characterized by high resolution BIAcore SPR experiments. Such a combined approach provided a detailed understanding of the molecular basis of DNA recognition. The discovery that the glycosyl antibiotic AT2433-B1 preferentially recognizes defined sequences offers novel opportunities for the future design of sequence-specific DNA-reading small molecules.

DNA targeting of two new antitumour rebeccamycin derivatives.

In the course of a medicinal chemistry program aimed at discovering novel tumour-active rebeccamycin derivatives targeting DNA and/or topoisomerase I, a series of analogues with the sugar residue linked to the two indole nitrogens was recently developed. Two promising drug candidates in this staurosporine-rebeccamycin hybrid series were selected for a DNA-binding study reported here. The DNA interaction of the cationic indolocarbazole glycosides MP059 bearing a N,N-diethylaminoethyl side chain and MP072 containing a sugar bearing an amino group was compared with that of the uncharged analogue MP024. The results show that the addition of a cationic substituent, either directly on the indolocarbazole chromophore or on the carbohydrate residue, significantly reinforces the interaction of the drugs with nucleic acids. The two cationic molecules MP059 and MP072 recognise preferentially sequences containing GpT.ApC and TpG.CpA steps but they do not inhibit topoisomerase I, in contrast to the parent uncharged derivative MP024 which stimulates DNA single strand breaks by topoisomerase I. The cytotoxic activity of the indolocarbazole derivatives bearing positively charged groups is one order of magnitude higher than that of the neutral compound MP024. The high cytotoxic potential can be attributed to the enhanced DNA binding and sequence recognition capacity of the cationic compounds. The study provides useful information for further structure-activity relationship studies in the indolocarbazole series.