P388 Murine Leukemia Cells Research Articles

Cytotoxicity and cell cycle effects of the plant alkaloids cryptolepine and neocryptolepine: relation to drug-induced apoptosis

Cryptolepine and neocryptolepine are two indoloquinoline derivatives isolated from the roots of the african plant Cryptolepis sanguinolenta. These two alkaloids, which only differ by the respective orientation of their indole and quinoline rings, display potent cytotoxic activities against tumour cells and present antibacterial and antiparasitic properties. Our previous molecular studies indicated that these two natural products intercalate into DNA and interfere with the catalytic activity of human topoisomerase II. Here we have extended the study of their mechanism of action at the cellular level. Murine and human leukemia cells were used to evaluate the cytotoxicity of the drugs and their effects on the cell cycle were measured by flow cytometry. Cryptolepine, and to a lesser extent neocryptolepine, provoke a massive accumulation of P388 murine leukemia cells in the G2/M phase. With HL-60 human leukemia cells, the treatment with cryptolepine leads to the appearance of a hypo-diploid DNA content peak (sub-G1) characteristic of the apoptotic cell population. With both P388 and HL-60 cells, cryptolepine proved about four times more toxic than its isomer. But the use of the HL-60/MX2 cell line resistant to the anticancer drug mitoxantrone suggests that topoisomerase II may not represent the essential cellular target for the alkaloids, which are both only two times less toxic to the resistant HL-60/MX2 cells compared to the parental cells. The capacity of the drugs to induce apoptosis of HL-60 human leukemia cells was examined by complementary biochemical techniques. Western blotting analysis revealed that cryptolepine, but not neocryptolepine, induces cleavage of poly(ADP-ribose) polymerase but both alkaloids induce the release of cytochrome c from the mitochondria. The cleavage of poly(ADP-ribose) polymerase observed with cryptolepine correlates with the appearance of a marked sub-G1 peak in the cell cycle experiments. The proteolytic activity of Asp-Glu-Val-Asp- or Ile-Glu-Thr-Asp-caspases was found to be enhanced much more strongly with cryptolepine than with its isomer, as expected from their different cytotoxic potential. Despite the activation of the caspase cascade, we did not detect internucleosomal cleavage of DNA in the HL-60 cells treated with the alkaloids. Altogether, the results shed light on the mechanism of action of these two plant alkaloids.

Classification of multidrug-resistance reversal agents using structure-based descriptors and linear discriminant analysis.

Linear discriminant analysis is used to generate models to classify multidrug-resistance reversal agents based on activity. Models are generated and evaluated using multidrug-resistance reversal activity values for 609 compounds measured using adriamycin-resistant P388 murine leukemia cells. Structure-based descriptors numerically encode molecular features which are used in model formation. Two types of models are generated: one type to classify compounds as inactive, moderately active, and active (three-class problem) and one type to classify compounds as inactive or active without considering the moderately active class (two-class problem). Two activity distributions are considered, where the separation between inactive and active compounds is different. When the separation between inactive and active classes is small, a model based on nine topological descriptors is developed that produces a classification rate of 83.1% correct for an external prediction set. Larger separation between active and inactive classes raises the prediction set classification rate to 92.0% correct using a model with six topological descriptors. Models are further validated through Monte Carlo experiments in which models are generated after class labels have been scrambled. The classification rates achieved demonstrate that the models developed could serve as a screening mechanism to identify potentially useful MDRR agents from large libraries of compounds.

ChemInform Abstract: A Focused Compound Library of Novel N-(7-Indolyl)benzenesulfonamides for the Discovery of Potent Cell Cycle Inhibitors.

A series of compounds containing an N-(7-indolyl)benzenesulfonamide pharmacophore was synthesized and evaluated as a potential antitumor agent. Cell cycle analysis with P388 murine leukemia cells revealed that there were two different classes of potent cell cycle inhibitors; one disrupted mitosis and the other caused G1 accumulation. Herein described is the SAR summary of the substituent patterns on this pharmacophore template.

Microcyclamide, a cytotoxic cyclic hexapeptide from the cyanobacterium Microcystis aeruginosa.

Microcyclamide (1), a cytotoxic cyclic hexapeptide, was isolated from the cultured cyanobacterium Microcystis aeruginosa (NIES-298). Its structure was elucidated to be 1 on the basis of two-dimensional (1)H-(1)H, (1)H-(13)C, and (1)H-(15)N NMR correlation experiments and the HRFABMS measurement. The absolute stereochemistry of the asymmetric centers was determined by the Marfey's method. This peptide showed a moderate cytotoxicity against P388 murine leukemia cells.

Comparative QSAR studies on substituted bis-(acridines) and bis-(phenazines)-carboxamides: a new class of anticancer agents

Quantitative structure–activity relationships have been formulated for two sets of DNA binding topoisomerase agents (bis-acridines and bis-phenazines) acting on murine P388 leukemia cells, murine Lewis lung carcinoma (LLC) cells and human Jurkat leukemia wild-type (JLC) cells. For the acridines, all three QSARs (1–3) show only a (small negative) hydrophobic effect. In sharp contrast, the phenazines in all three studies (4–6) show a strong hydrophobic effect, with the optimum ClogP being near 7.3 for all examples. This suggests that, despite the structural similarity of the compounds, different modes of enzyme and/or DNA binding may be involved.

Antineoplastic agents 440. Asymmetric synthesis and evaluation of the combretastatin A-1 SAR probes (1S,2S)- and (1R,2R)-1, 2-dihydroxy- 1-(2',3'-dihydroxy-4'-methoxyphenyl)-2-(3' ',4' ',5' '-trimethoxyphenyl)-ethane.

The synthetic (E)-isomer (3b) of natural combretastatin A-1 (1a) isolated from the African bushwillow Combretum caffrum was the focus of chiral hydroxylation (Sharpless) reactions as part of a structure-activity relationship study. The resulting (R,R)- and (S,S, )-diols (6 and 7) and synthetic intermediates were evaluated against a series of cancer cell lines, microorganisms, and tubulin. Chiral diols 6 and 7 showed increased activity against the P-388 murine lymphocytic leukemia cell line with ED(50) values of 3.9 and 2.9 microg/mL, respectively, when compared to the precursor (E)-stilbene 3b. In contrast, (E)-stilbene 3b exhibited more potent antibiotic activity than the chiral diols (6 and 7). Both diols, (R,R)-6 and (S, S)-7, displayed less cancer cell growth inhibition and less antibiotic activity than did natural combretastatin A-1 (1a) (P-388 ED(50) 0.25 microg/mL).

A focused compound library of novel N-(7-indolyl)benzenesulfonamides for the discovery of potent cell cycle inhibitors

A series of compounds containing an N-(7-indolyl)benzenesulfonamide pharmacophore was synthesized and evaluated as a potential antitumor agent. Cell cycle analysis with P388 murine leukemia cells revealed that there were two different classes of potent cell cycle inhibitors; one disrupted mitosis and the other caused G1 accumulation. Herein described is the SAR summary of the substituent patterns on this pharmacophore template.

Hachijodines A-G: seven new cytotoxic 3-alkylpyridine alkaloids from two marine sponges of the genera Xestospongia and amphimedon.

Seven cytotoxic 3-alkylpyridine alkaloids, hachijodines A-G, have been isolated from two marine sponges of the genera Xestospongia and Amphimedon. Their structures were determined on the basis of spectral data. These alkaloids are moderately cytotoxic against P388 murine leukemia cells with IC(50) values of 1.0-2.3 microg/mL.

Amaminols A and B, new bicyclic amino alcohols from an unidentified tunicate of the family Polyclinidae

Two new cytotoxic amino alcohols, amaminols A and B, have been isolated from an unidentified tunicate of the family Polyclinidae. Their structures including stereochemistry were elucidated by spectroscopic and chemical methods. Both compounds were cytotoxic against P388 murine leukemia cells.

Mechanism of action of aragusterol a (YTA0040), a potent anti-tumor marine steroid targeting the G(1) phase of the cell cycle.

Aragusterol A (YTA0040), isolated from the Okinawan marine sponge of the genus Xestospongia, is a potent anti-tumor marine steroid that possesses a unique structural component. This compound showed broad-spectrum anti-proliferative activity against a panel of 14 human cancer cell lines (IC(50) = 0.01-1.6 microM). P-glycoprotein-mediated, multidrug-resistant cells showed cross-resistance to YTA0040 cells, whereas cisplatin-resistant non-small-cell lung-cancer (NSCLC) sublines showed a collateral sensitivity to YTA0040. In transplantable murine tumor models, YTA0040 displayed a broad spectrum and high degree of anti-tumor activity when administered i.p. or p.o. (life span T/C = 135-234%). In P388 murine leukemia cells, YTA0040 caused dose- and time-dependent suppression of nucleic acid and protein synthesis, with protein synthesis being more potently and rapidly inhibited than nucleic acid synthesis. Flow-cytometric analysis revealed that YTA0040 blocked the entry of human NSCLC-derived A549 cells into S phase, leading to arrest in the G(1) phase of the cell cycle. Western blot analysis demonstrated that YTA0040 caused a dose-dependent decrease in the levels of expression of hyperphosphorylated pRb and cyclin A in A549 cells. The level of p53 protein expression was decreased by YTA0040 treatment. A higher concentration of YTA0040 down-regulated the levels of expression of CDK2, CDK4, cyclin D1 and cyclin E. These findings indicated that YTA0040 arrested human NSCLC cells in late G(1) phase of the cell cycle through inhibition of pRb phosphorylation. Inhibition of pRb phosphorylation by YTA0040 resulted from down-regulation of levels of expression of the CDKs and cyclins involved in the G(1)/S transition and not from induction of p53 and/or the CDK inhibitor p21.

Theopederins F-J: Five new antifungal and cytotoxic metabolites from the marine sponge, theonella swinhoei

Five new bioactive metabolites, theopederins F-J, have been isolated from the marine sponge, Theonella swinhoei. Their structures are derivatives of theopederins on the basis of spectral data. Theopederin F was antifungal against Saccharomyces cerevisiae at 1 μg/disk and cytotoxic against P388 murine leukemia cells with an IC 50 value of 0.15 ng/mL.

Discovery of novel antitumor sulfonamides targeting G1 phase of the cell cycle.

Described herein is the discovery of a novel series of antitumor sulfonamides targeting G1 phase of the cell cycle. Cell cycle control in G1 phase has attracted considerable attention in recent cancer research, because many of the important proteins involved in G1 progression or G1/S transition have been found to play a crucial role in proliferation, differentiation, transformation, and programmed cell death (apoptosis). We previously reported our first antitumor sulfonamide E7010 as a novel tubulin polymerization inhibitor. Interestingly enough, continuous research on structurally related compounds led us to the finding of another class of antitumor sulfonamides that block cell cycle progression of P388 murine leukemia cells in G1 phase, but not in M phase. Of the compounds examined, N-(3-chloro-7-indolyl)-1,4-benzenedisulfonamide (E7070) showed significant antitumor activity against HCT116 human colon carcinoma both in vitro (IC(50) 0.11 microg/mL in cell proliferation assay) and in vivo (not only growth suppression but also a marked reduction of tumor size in nude mice). Because of its promising efficacy against human tumor xenografts and its unique mode of action, E7070 is currently undergoing phase I clinical trials in European countries.

Uptake and intracellular distribution of amrubicin, a novel 9-amino-anthracycline, and its active metabolite amrubicinol in P388 murine leukemia cells.

Amrubicin, a 9‐aminoanthracycline anti‐cancer drug, and its C‐13 hydroxyl metabolite amrubicinol, were examined for growth‐inhibitory activity as well as cellular uptake and distribution in P388 murine leukemia cells and doxorubicin‐resistant P388 cells. Also discussed are the differences in the mechanisms of action among amrubicin, amrubicinol and doxorubicin in terms of their cellular pharmacokinetic character. In P388 cells, amrubicinol was about 80 times as potent as amrubicin, and about 2 times more potent than doxorubicin in a 1‐h drug exposure growth‐inhibition test. A clear cross‐resistance was observed to both amrubicin and amrubicinol in doxorubicin‐resistant P388 cells, though the resistance index was lower for amrubicin. The intracellular concentration of amrubicinol was about 6 times and 2 times higher than those of amrubicin and doxorubicin, respectively. Compared to doxorubicin, amrubicin and amrubicinol were released rapidly after removal of the drugs from the medium. A clear correlation was found between the growth‐inhibiting activity and the cellular level of amrubicin and amrubicinol in P388 cells. About 10 to 20% of amrubicin or amrubicinol taken up by the cells was detected in the cell nuclear fraction, whereas 70 to 80% of doxorubicin was localized in this fraction. These results suggest that amrubicin and amrubicinol exert cytotoxic activity via a different mechanism from that of doxorubicin.

Rubrosides A−H, New Bioactive Tetramic Acid Glycosides from the Marine Sponge Siliquariaspongia japonica1

Eight new tetramic acid glycosides named rubrosides A−H have been isolated from the marine sponge Siliquariaspongia japonica. Their structures were elucidated on the basis of spectral data as tetramic acid glycosides containing polyenes terminating in a 4-chloro-2-methyltetrahydrofuran ring. The absolute stereochemistry of the furan functionality in the two major metabolites, rubrosides D and F, was determined by the NMR method using chiral anisotropic reagents for tetrahydro-2-furoic acid derived by RuO4 oxidation. The absolute stereochemistry of tetramic acid and of the sugar moieties in all rubrosides was deduced by chiral GC analysis of chemical degradation products. The rubrosides induced numerous large intracellular vacuoles in 3Y1 rat fibroblasts at concentrations of 0.5−1.0 μg/mL, and rubrosides A, C, D, and E were cytotoxic against P388 murine leukemia cells with IC50 values of 0.046−0.21 μg/mL. Most rubrosides show antifungal activity against Aspergillus fumigatus and Candida albicans.

Stereocontrolled syntheses of novel styryl lactones, (+)-goniodiol, (+)-goniotriol, (+)-8-acetylgoniotriol, (+)-goniofufurone, (+)-9-deoxygoniopypyrone, (+)-goniopypyrone, and (+)-altholactone from common intermediates and cytotoxicity of their congeners

Concise syntheses of (+)-goniodiol, (+)-goniotriol, (+)-8-acetylgoniotriol, (+)-goniofufurone, (+)-9-deoxygoniopypyrone, (+)-goniopypyrone, and (+)-altholactone and their congeners from chiral lactonic aldehydes 27 and 36 as common intermediates are described. The key features in the syntheses are based on the in situ generation of unstable aldehydes 27 and 36 followed by their chemoselective reaction with triisopropoxyphenyltitanium to afford both diastereomers 28, 29 and 37, 38 at the C-8 positions. The cytotoxicity of styryl lactone congeners against P388 murine leukemia cells was examined.

The synthesis and P388 cytotoxicity of mycalazol 11 and related 5-acyl-2-hydroxymethylpyrroles

We report a general method for the synthesis of mycalazol 11 and some related 5-acyl-2-hydroxymethylpyrroles using a Stille coupling of 5-(tri- n-butylstannyl)pyrrole-2-carboxaldehyde with an acid chloride as the key step. The newly prepared 5-acyl-2-hydroxymethylpyrroles 5–7, together with the 5-carboxamido-2-hydroxymethylpyrrole 10, have been assayed for in vitro cytotoxicity against the P388 murine leukemia cell line.

Photodynamic therapy: a mitochondrial inducer of apoptosis.

Photodamage to the mitochondria of murine leukemia P388 cells resulted in immediate loss of the mitochondrial membrane potential together with the release of cytochrome c into the cytosol. This was followed by a rapid activation of caspase 3-like proteases, as indicated by a marked rise in DEVDase activity. There was no significant effect on WEHDase or VEIDase activities, suggesting that only the late-stage caspases had been effected. The apoptotic response to mitochondrial photodamage was abolished by the broad-spectrum caspase inhibitor zVAD-fmk, but this did not prevent loss of viability after mitochondrial photodamage. These studies indicate that the release of cytochrome c from photodamaged mitochondria is sufficient to directly initiate a caspase-dependent apoptotic response.

Characterization of cytotoxicity induced by sphingolipids in multidrug-resistant leukemia cells

Certain sphingolipids (SLs) exert fundamental roles in differentiative, growth-inhibitory and apoptotic pathways induced by a number of agents in leukemia cells. Multidrug-resistance (MDR) is a major cause of therapeutic failure in leukemia. SLs are among the diverse substrates for the MDR p-170 glycoprotein drug-efflux pump. We tested the hypothesis that expression of MDR would thereby block the cytotoxicity induced by the SLs sphingosine, sphinganine and N-hexanoyl-sphingosine. An MDR-expressing subline of murine P388 leukemia cells demonstrated an ED 50 value ≥2 log 10 higher than the parental line in response to doxorubicin. In contrast, the ED 50 values for each of the SLs were only approximately 1.5 to two-fold higher in the MDR line than in the parental; induction of DNA damage by SLs was comparable or actually greater in MDR compared to parental cells. Therefore, expression of MDR does not significantly affect the cytotoxic function of these SLs, nor do these SLs likely contribute to MDR.

Characterization of multidrug resistance and monitoring of tumor response by combined 31P and 1H nuclear magnetic resonance spectroscopic analysis.

A combined 31P and 1H nuclear magnetic resonance (NMR) spectroscopic study was carried out in drug-sensitive and adriamycin- and mitoxantrone-resistant P388 murine leukemic cells. Typical spectral changes characteristic of multidrug resistance were observed in resistant cells compared to drug-sensitive cells. Quantitative comparison of phosphate metabolites ATP, phosphocreatine, phosphomonoesters and phosphodiesters revealed a significant alteration in the metabolism of resistant cells. The elevated levels of energy metabolites supported the energy-dependent process of drug efflux in resistant cells. An increased rate of glycolysis in resistant cells as indicated by the elevated lactate level further supported this. The near total loss of energy metabolites and marked decrease in phospholipid metabolites in sensitive cells upon treatment with drug compared to unaltered metabolite levels in resistant cells suggested that the spectral changes can reveal the subtle differences in tumor response between drug-sensitive and -resistant cells. The results substantiate the potential of this non-invasive method to characterize the multidrug resistance phenotype and monitor tumor response.

Thiomycalolides: new cytotoxic trisoxazole-containing macrolides isolated from a marine sponge Mycale sp.

Two new cytotoxic trisoxazole macrolides, thiomycalolides A (2) and B (3), have been isolated from a marine sponge Mycale sp. The structures were determined to be glutathione adducts of mycalolides A and B by interpretation of spectral data and chemical transformation. Thiomycalolides A and B are highly cytotoxic against P388 murine leukemia cells.