Analysis of cyclin B1 and CDK activity during apoptosis induced by camptothecin treatment.

We have studied the role of cyclins and cyclin-dependent kinase (CDK) activity in apoptosis induced by camptothecin (CPT). In this model, 22% of the cells stain for annexin-V at 24 h and then proceed to be 93% positive by 72 h. This time window permits the analysis of cyclins in cells that are committed to apoptosis but not yet dead. We provide evidence that cyclin protein levels and then associated kinase levels increase after CPT treatment. Strikingly, cyclin B1 and cyclin E1 proteins are present at the same time in CPT treated HT29 cells. Although cyclin B1 and E1 CDK complexes are activated in CPT treated cells, only the cyclin B1 complex is required for apoptosis since reduction of cyclin B1 by RNAi or roscovitine treatment reduces the number of annexin-V-stained cells. We have detected poorly organized chromosomes and phosphorylated histone H3 epitopes at the time of maximum cyclin B1/CDK kinase activity in CPT-treated cells, which suggests that these cells enter a mitotic catastrophe. Understanding which CDKs are required for apoptosis may allow us to better adapt CDK inhibitors for use as anti-cancer compounds.

Synthesis and biological evaluation of N-(7-indazolyl)benzenesulfonamide derivatives as potent cell cycle inhibitors.

We herein describe a new synthesis of N-(7-indazolyl)benzenesulfonamide derivatives. These compounds were evaluated for their antiproliferative activities toward L1210 murine leukemia cells. One of them, 4-methoxy-N-(3-chloro-7-indazolyl)benzenesulfonamide, was identified as the most potent with an IC(50) of 0.44 microM.

Synthesis and anticancer activity of new pyrrolocarbazoles and pyrrolo-beta-carbolines.

'Bended' 1, 3 or 'linear' 2 pyrrolidino-fused (aza)carbazoles were prepared and screened towards a few cancer-related targets. Whereas 'bended' derivatives 1 and 3 proved to be weakly toxic, several members of the 'linear' family strongly interact with DNA, especially derivative 28a.

Induction of cyclin E and inhibition of DNA synthesis by the novel acronycine derivative S23906-1 precede the irreversible arrest of tumor cells in S phase leading to apoptosis.

S23906-1 is a diester derivative of 1,2-dihydrobenzo[b]acronycine with an unknown mechanism of action. This cytotoxic compound was 20-fold more potent than acronycine in inhibiting the proliferation of six tumor cell lines. Using a clonogenic assay of cell survival, the HT29 human colon carcinoma cell line was 100-fold more sensitive to S23906-1 than acronycine. Cell cycle analysis, by flow cytometry, showed that S23906-1 induced a partially reversible arrest of HT29 cells in G2+M at 1 microM and below and an irreversible arrest in S phase at 2.5 microM and above. These cell cycle effects were followed by cell death through apoptosis, quantified by annexin-V labeling. Inhibition of DNA synthesis was observed by complete prevention of bromodeoxyuridine (BrdU) incorporation after only 4 h of incubation with 5 microM S23906-1. Interestingly, under the same experimental conditions, a significant increase of cyclin E protein level was observed without any modification of cyclins D1, D2, D3, or A. This overexpressed cyclin E protein was not complexed with Cdk2, as shown by western blotting for Cdk2 in immunoprecipitates of cyclin E. Similar inhibition of BrdU incorporation and elevation of cyclin E protein were observed after treatment with cytosine arabinoside, which reversibly inhibited progression into S phase, but not after DNA damage induced by cisplatin. S23906-1 thus has a novel mechanism of action. A cell line resistant to S23906-1 showed that overexpression of cyclin E was implicated in the novel cytotoxic activity of this compound.

Synthesis, cytotoxic activity, NMR study and stereochemical effects of some new pyrano[3,2-b]thioxanthen-6-ones and pyrano[2,3-c]thioxanthen-7-ones.

Some new substituted pyrano[3,2-b]thioxanthen-6-ones and pyrano[2,3-c]thioxanthen-7-ones were prepared and their cytotoxic activity was evaluated using acronycine as the reference compound. The conformation of the molecules was also investigated in an effort to correlate this parameter with the biological activity.

Cytotoxic activities of novel hexahydroindolizino[8,7-b]indole derivatives prepared by 1,3-dipolar cycloaddition reactions of 3,4-dihydro-beta-carboline ylides.

A series of 1-cyano and 2-cyanohexahydroindolizino[8,7-b]indole derivatives was prepared by 1,3-dipolar cycloaddition of acrylonitrile with ylides derived from 3,4-dihydro-beta-carboline and its 6-methoxy, 6-benzyloxy, 9-methyl and 9-benzyl analogues. The products, together with their reduced 1- or 2-aminomethyl derivatives, were evaluated for cytotoxic activity in L1210 cancer cells. Compounds derived from 6-benzyloxy or 9-benzyl-3,4-dihydro-beta-carboline were found to be the most active, with IC(50)'s in the 2-50 microM range. Of these, two compounds, the 1- and 2-cyano 8-benzyloxyindolizino[8,7-b]indole derivatives 20a and 20c, respectively, were found by cytometric flux analysis to stop cancer cell growth at the G(2)M and 8N (>G(2)M) stage of the cell cycle. These two compounds also showed no loss of cytotoxic activity in K562R cancer cells resistant to doxorubicin.

Design, synthesis and antiproliferative activity of tripentones: a new series of antitubulin agents.

Structure-activity relationship studies of a new series of tripentones (thieno[2,3-b]pyrrolizin-8-ones), led us to prepare several derivatives with antiproliferative activities. The most promising 3-(3-hydroxy-4-methoxyphenyl)thieno[2,3-b]pyrrolizin-8-one 20 (leukemia L1210, IC(50)=15 nM) was shown to be a potent inhibitor of tubulin polymerization.

Synthesis and cytotoxicity of artemisinin derivatives containing cyanoarylmethyl group.

A series of 12alpha-deoxoartemisinyl cyanoarylmethyl dicarboxylates (4a-4o), dicarboxylic acids 12alpha-deoxoartemisinyl ester cyanoarylmethyl amide (5a-5k), and dicarboxylic acids 12alpha-deoxoartemisinyl ester N-methylcyanoarylmethyl amide (6a-6l), showing moderate cytotoxicity against P388 and L1210 cells were prepared. They induced the significant accumulation of L1210 and P388 cells in the G1 phase of the cell cycle. This mechanism of action was quite different from that of the majority of cytotoxic compounds used in the chemotherapy of cancer. Compound 4b possessed better cytotoxicity than the other compounds.

Rebeccamycin analogues from indolo[2,3-c]carbazole.

Glycosylated indolocarbazoles related to the antibiotic rebeccamycin represent an important series of antitumor drugs. In the course of structure-activity relationship studies, we report the synthesis of two new derivatives containing an indolo[2,3-c]carbazole chromophore instead of the conventional indolo[2,3-a]carbazole unit found in the natural metabolites. The N-methylated compound 8 containing one glucose residue behaves as a typical DNA intercalating agent, as judged from circular and electric linear dichroism measurements with purified DNA. In contrast, the bis-glycosylated derivative 7 containing a glucose residue on each indole nitrogen has lost its capacity to form stable complexes with DNA. DNA relaxation experiments reveal that the two drugs 7 and 8 have weak effects on human DNA topoisomerase I. The modified conformation of the indolocarbazole chromophore is detrimental to the stabilization of topoisomerase I-DNA complexes. The lack of potent topoisomerase I inhibition leads to decreased cytotoxicity but, however, we observed that the DNA-intercalating mono-glycosyl derivative 8 is about 5 times more cytoxic than the bis-glycosyl analogue 7. The study suggests that the naturally-occurring indolo[2,3-a]carbazole skeleton should be preserved to maintain the topoisomerase I inhibitory and cytotoxic activities.

Novel antitumor artemisinin derivatives targeting G1 phase of the cell cycle.

Modification of artemisinin structure led us to the discovery of a novel class of antitumor compounds. These artemisinin derivatives containing cyano and aryl groups showed potent antiproliferative effect in vitro against P388 and A549 cells. This activity was reflected in P388 murine leukemia by an accumulation of cells in G1 phase, and induction of apoptosis.

Synthesis of a novel series of cytotoxic bisindole alkaloids.

Original cytotoxic bisindole alkaloids with a 1,2,3,4-tetrahydroquinoline bridge were synthesized by reductive amination with various anilines. The most cytotoxic compounds display a high and dose-dependent cell cycle effect with accumulation in the G1 phase. Influence of substitution of the starting aniline on the reaction and on cytotoxicity of produced dimers was pointed out.

Synthesis and biological activities of indolocarbazoles bearing amino acid residues.

Three indolocarbazole compounds bearing a tripeptide or a lysine group attached to one of the indole nitrogens via a propylamino chain and two rebeccamycin derivatives bearing a lysine residue on the sugar moiety were synthesised with the aim of improving the binding to DNA and the antiproliferative activities. Four tumour cell lines, from murine L1210 leukemia, human HT29 colon carcinoma, A549 non-small cell lung carcinoma and K-562 leukemia, were used to evaluate the cytotoxicity of the drugs. Their effects on the cell cycle of L1210 cells and their antimicrobial properties against two Gram-positive bacteria Bacillus cereus and Streptomyces chartreusis, a Gram-negative bacterium Escherichia coli and a yeast Candida albicans were also investigated.

Synthesis and antiproliferative activity of benzocyclobutacarbazol derivatives. A new class of potential antitumor agents.

Several benzocyclobutacarbazol derivatives were synthesized and evaluated for their potential cytotoxic properties. A number of these compounds exhibited significant antiproliferative activity with concomitant interaction with the cell cycle and represent a new class of potential anticancer agents.

First synthesis and pharmacological evaluation of benzoindolizidine and benzoquinolizidine analogues of alpha- and beta-peltatin.

The benzoindolizidine and quinolizidine analogues of alpha- and beta-peltatin were designed and synthesized by two different synthetic routes involving as the key step the Bischler-Napieralski cyclization of suitably substituted N-acyl-2-arylmethylpyrrolidine and -piperidine derivatives. The in vitro biological activity of these analogues as well as some of their derivatives was subsequently evaluated.

Expeditious synthesis and cytotoxic activity of new cyanoindolo[3,2-c]quinolines and benzimidazo[1,2-c]quinazolines.

Novel 6-cyanoindolo[3,2-c]quinoline and 6-cyanobenzimidazo[1,2-c]quinazoline derivatives have been synthesised by treatment of the appropriate aromatic amines with 4.5-dichloro-1,2,3-dithiazolium chloride 1 (Appel salt). The cytotoxicity and the effect of these compounds on cellular growth were measured.

Synthesis and cytotoxic and antitumor activity of benzo[b]pyrano[3, 2-h]acridin-7-one analogues of acronycine.

Benzo¿bacronycine (6-methoxy-3,3,14-trimethyl-3, 14-dihydro-7H-benzo¿bpyrano¿3,2-hacridin-7-one, 4), an acronycine analogue with an additional aromatic ring linearly fused on the natural alkaloid basic skeleton, was synthesized in three steps, starting from 3-amino-2-naphthalenecarboxylic acid (5). Eight 1, 2-dihydroxy-1,2-dihydrobenzo¿bacronycine esters and diesters (17-24) were obtained by catalytic osmic oxidation, followed by acylation. All these compounds were significantly more cytotoxic than acronycine, when tested against L1210 leukemia cells in vitro. The potency of the cyclic carbonate 24 was in the range of the most active drugs currently used in cancer chemotherapy. Two selected diesters (17 and 24) were evaluated in vivo against P388 leukemia and colon 38 adenocarcinoma implanted in mice. Both compounds were markedly active at doses 16-fold lower than the dose of acronycine itself. Against colon 38 adenocarcinoma, compounds 17 and 24 were highly efficient, inhibiting tumor growth by more than 80%. Diacetate 17 was the most active, inhibiting tumor growth by 96% at 6.25 mg/kg, with two of seven mice being tumor-free on day 43.

Phenotypic and functional changes in regenerated porcine coronary endothelial cells : increased uptake of modified LDL and reduced production of NO.

Porcine coronary arteries with regenerated endothelium exhibit impaired endothelium-dependent relaxations. Experiments were designed to analyze the structural and functional changes occurring in regenerated endothelial cells. Primary cultures from regenerated endothelium contained giant endothelial cells, with an increased number of cells with diameter >14.5 microm, a reduced ability to proliferate, and signs of apoptosis. The uptake of fluorescent acetylated LDL was increased 2-fold in cultures from regenerated endothelium. The increased uptake of acetylated LDL was confirmed ex vivo in injured coronary arteries. In cultures from regenerated endothelium, cGMP production was decreased under basal conditions and during stimulation with serotonin, bradykinin, and A23187. Thus, during regeneration, there is accelerated senescence of endothelial cells accompanied by increased incorporation of modified LDL and reduction of NO production without decrease in endothelial NO synthase expression. These alterations help to explain the altered endothelium-dependent responses 28 days after balloon injury.

Design, synthesis and biological activity of 7-O-(4-O-acetyl-3-iodo-2,3,6-trideoxy-alpha-L-arabino-hexopyranosyl) daunomycinone and 7-O-(3-chloro-2,3,6-trideoxy-4-O-propanoyl-alpha-L-lyxo- hexopyranosyl)daunomycinone.

The synthesis and pharmacological evaluation of 7-O-(4-O-acetyl-3-iodo-2,3,6-trideoxy-alpha-L-arabino-hexopyranosyl) daunomycinone and 7-O-(3-chloro-2,3,6-trideoxy-4-O-propanoyl-alpha-L-lyxo-hexopyrano syl) daunomycinone are described. Their cytotoxic activity was evaluated against normal and resistant cell lines. Both compounds exhibited activity against the adriamycin resistant cell line KB-A1. These results support the hypothesis that the increased lipophilicity of the sugar part of anthracyclines is associated with their ability to overcome multidrug resistance (MDR).

Effects of ceramide on apoptosis, proteoglycan degradation, and matrix metalloproteinase expression in rabbit articular cartilage.

Cartilage loss in osteoarthritis is characterized by matrix degradation and chondrocyte death. The lipid messenger ceramide is implicated in signal transduction of the catabolic cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1), as well as in apoptosis. The aim of this study was to examine the in vitro effects of ceramide on proteoglycan degradation, matrix-metalloproteinase (MMP) expression and activity, and chondrocyte apoptosis in rabbit articular cartilage. Cell-permeant ceramide C(2) stimulated proteoglycan degradation in cartilage explants starting from 3 x 10(-5) M, with 100% increase at the dose of 10(-4) M. This effect was probably due to MMPs since it was blocked by the MMP inhibitor batimastat. Furthermore, in isolated chondrocytes, C(2) stimulated the expression of MMP-1, 3, and 13 at the mRNA level, MMP activity, and MMP-3 production. Ceramide also caused chondrocyte apoptosis at doses ranging from 10(-5) to 10(-4) M. This study supports the hypothesis that ceramide might play a mediatory role in both matrix degradation and apoptosis in processes of cartilage loss such as those observed in osteoarthritis.

Synthesis and in vitro cytotoxic evaluation of N-substituted benzo[5,6]cycloheptal[b]indoles.

A new series of V-substituted benzo[5,6]cyclohepta[b]indole derivatives were synthesised and evaluated for in vitro cytotoxic activities against L1210 murine leukemia and HT29 cell lines. Among them, several compounds showed potent antitumor activity and blocked cell cycle progression of L1210 cells in G2+M phase.