Anti-tumor drug candidate 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole induces single-strand breaks and DNA-protein cross-links in sensitive MCF-7 breast cancer cells

Purpose: The fluorinated benzothiazole analogue 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203, NSC 703786) exhibits selective and potent anticancer activity, and its lysylamide prodrug (Phortress, NSC 710305) recently entered Phase I clinical trials in the United Kingdom. Only cancer cells sensitive to the anti-proliferative effects of 5F 203 deplete this drug candidate from nutrient media. 5F 203 induces cell cycle arrest, cytochrome P450 1A1 (CYP 1A1) mRNA and protein expression, and is metabolized into reactive electrophilic species that can covalently bind to DNA and form adducts in sensitive (i.e., MCF-7) but not in resistant (i.e., MDA-MB-435) breast cancer cells. Methods: In this present study, we investigated additional anticancer effects of 5F 203 in MCF-7 cells. In addition, we sought to determine if cells deficient in the xeroderma pigmentosum D gene, a gene critical in DNA repair, would show greater sensitivity to the cytotoxic effects of 5F 203 than those complemented with XPD. Results: Alkaline Elution revealed that 5F 203 induced single-strand breaks and DNA-protein cross-links in sensitive MCF-7 cells. In contrast, we detected no double-strand breaks or protein-associated strand breaks typically associated with topoisomerase I (top1) or topoisomerase II (top2) inhibition. In addition, 5F 203 was unable to trap top1- or top2-DNA cleavage complexes in MCF-7 cells. 5F 203 induced cell cycle arrest in MCF-7 cells following DNA damage after brief exposures. Cells deficient in the nucleotide excision repair xeroderma pigmentosum group D (XPD) gene displayed sensitivity to 5F 203 while cells complemented with XPD displayed resistance to 5F 203. Conclusion: These data suggest that the anti-cancer activity of 5F 203 depends upon targets other than top1 or top2 and on the ability of this benzothiazole to form single-strand breaks and DNA-protein cross-links in cancer cells.     

A systematic study of nitrated indenoisoquinolines reveals a potent topoisomerase I inhibitor

The biological activity of indenoisoquinoline topoisomerase I inhibitors is significantly enhanced by nitration of the isoquinoline ring. In the present study, nitrated analogues were synthesized with the indenone ring substituted with methoxy groups to further explore a previously identified structure-activity relationship between the nitrated isoquinoline ring and a methylenedioxy-substituted indenone ring. The results indicate that a single methoxy group at the 9-position of an indenoisoquinoline affords superior biological activity. Hypothetical binding models have been developed to rationalize these results, and they indicate that pi-stacking between the indenoisoquinolines and the DNA base pairs, as visualized by electrostatic complementarity, is important for the intercalation and biological activity of the indenoisoquinoline analogues. Collectively, the analysis of methoxy groups on the indenone ring also illustrates a strict steric requirement for substituents extending toward the nonscissile DNA backbone and emphasizes a need for planarity to afford potent biological activity.     

Oral glucosamine for 6 weeks at standard doses does not cause or worsen insulin resistance or endothelial dysfunction in lean or obese subjects

Glucosamine is a popular nutritional supplement used to treat osteoarthritis. Intravenous administration of glucosamine causes insulin resistance and endothelial dysfunction. However, rigorous clinical studies evaluating the safety of oral glucosamine with respect to metabolic and cardiovascular pathophysiology are lacking. Therefore, we conducted a randomized, placebo-controlled, double-blind, crossover trial of oral glucosamine at standard doses (500 mg p.o. t.i.d.) in lean (n = 20) and obese (n = 20) subjects. Glucosamine or placebo treatment for 6 weeks was followed by a 1-week washout and crossover to the other arm. At baseline, and after each treatment period, insulin sensitivity was assessed by hyperinsulinemic-isoglycemic glucose clamp (SI(Clamp)) and endothelial function evaluated by brachial artery blood flow (BAF; Doppler ultrasound) and forearm skeletal muscle microvascular recruitment (ultrasound with microbubble contrast) before and during steady-state hyperinsulinemia. Plasma glucosamine pharmacokinetics after oral dosing were determined in each subject using a high-performance liquid chromatography method. As expected, at baseline, obese subjects had insulin resistance and endothelial dysfunction when compared with lean subjects (SI(Clamp) [median {25th-75th percentile}] = 4.3 [2.9-5.3] vs. 7.3 [5.7-11.3], P < 0.0001; insulin-stimulated changes in BAF [% over basal] = 12 [-6 to 84] vs. 39 [2-108], P < 0.04). When compared with placebo, glucosamine did not cause insulin resistance or endothelial dysfunction in lean subjects or significantly worsen these findings in obese subjects. The half-life of plasma glucosamine after oral dosing was approximately 150 min, with no significant changes in steady-state glucosamine levels detectable after 6 weeks of therapy. We conclude that oral glucosamine at standard doses for 6 weeks does not cause or significantly worsen insulin resistance or endothelial dysfunction in lean or obese subjects.     

A novel polypyrimidine antitumor agent FdUMP[10] induces thymineless death with topoisomerase I-DNA complexes

FdUMP[10], a 10mer of 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP), the thymidylate synthase inhibitory metabolite of 5-fluorouracil (FU), is most closely correlated with the DNA topoisomerase I (Top1) inhibitor camptothecin in the National Cancer Institute COMPARE analysis, but not with FU. FdUMP[10] exhibits more potent antiproliferative activity than FdUMP or 5-fluoro-2'-deoxyuridine (FdU) and is markedly more active than FU. Camptothecin-resistant P388/CPT45 cells lacking Top1 are cross-resistant to FdUMP[10] as well as to FdUMP, FdU, and the thymidylate synthase inhibitor raltitrexed (Tomudex). FdUMP[10] induces DNA single-strand breaks and cellular Top1-DNA complexes. Such complexes are also observed in response to FdUMP, FdU, raltitrexed, and FU. The FdUMP[10]-induced Top1-DNA complexes are not inhibited by the caspase inhibitor z-VAD-fmk and form independently of apoptotic DNA fragmentation, indicating that they do not correspond to apoptotic Top1-DNA complexes. In biochemical assay, Top1 is directly trapped at uracil and FdU misincorporation sites. We propose that FdUMP[10] damages DNA by trapping Top1 at uracil and FdU misincorporation sites resulting from thymidylate synthase inhibition and thymine depletion.     

DNA-protein cross-links and replication-dependent histone H2AX phosphorylation induced by aminoflavone (NSC 686288), a novel anticancer agent active against human breast cancer cells

Aminoflavone (5-amino-2,3-fluorophenyl)-6,8-difluoro-7-methyl-4H-1-benzopyran-4-one) (NSC 686288) is a candidate for possible advancement to phase I clinical trial. Aminoflavone has a unique activity profile in the NCI 60 cell lines (COMPARE analysis; http://www.dtp.nci.nih.gov/docs/dtp_search.html), and exhibits potent cellular and animal antitumor activity. To elucidate the mechanism of action of aminoflavone, we studied DNA damage in MCF-7 cells. Aminoflavone induced DNA-protein cross-links (DPC) and DNA single-strand breaks (SSB). Aminoflavone induced high levels of DPC and much lower level of SSB than camptothecin, which induces equal levels of DPC and SSB due to the trapping topoisomerase I-DNA complexes. Accordingly, neither topoisomerase I nor topoisomerase II were detectable in the aminoflavone-induced DPC. Aminoflavone also induced dose- and time-dependent histone H2AX phosphorylation (gamma-H2AX). Gamma-H2AX foci occurred with DPC formation, and like DPC, persisted after aminoflavone removal. Aphidicolin prevented gamma-H2AX formation, suggesting that gamma-H2AX foci correspond to replication-associated DNA double-strand breaks. Accordingly, no gamma-H2AX foci were found in proliferating cell nuclear antigen-negative or in mitotic cells. Bromodeoxyuridine incorporation and fluorescence-activated cell sorting analyses showed DNA synthesis inhibition uniformly throughout the S phase after exposure to aminoflavone. Aminoflavone also induced RPA2 and p53 phosphorylation, and induced p21(Waf1/Cip1) and MDM2, demonstrating S-phase checkpoint activation. These studies suggest that aminoflavone produces replication-dependent DNA lesions and S-phase checkpoint activation following DPC formation. Gamma-H2AX may be a useful clinical marker for monitoring the efficacy of aminoflavone in tumor therapies.     

4-Amino-5-benzoyl-2-(4-methoxyphenylamino)thiazole (DAT1): a cytotoxic agent towards cancer cells and a probe for tubulin-microtubule system

Microtubule binding drugs are of special interest as they have important roles in the modulation of cellular functions and many of them act as anticancer agents. 4-Amino-5-benzoyl-2-(4-methoxyphenylamino)thiazole (DAT1) was identified as one of the active compounds from a series of diaminoketothiazoles in a cell-based screening assay to discover cytotoxic compounds. DAT1 shows cytotoxicity with GI(50) values ranging from 0.05 to 1 microM in different malignant cell lines with an average value of 0.35 microM. It blocks mitosis in the prometaphase and metaphase stages. In HeLa cells, DAT1 blocks the spindle function by disturbing spindle microtubule and chromosome organization. The drug also inhibits assembly of brain microtubules and binds tubulin specifically at a single site with induction of fluorescence. The dissociation constant of DAT1 binding to tubulin was determined as 2.9+/-1 microM at 24 degrees C. The binding site of DAT1 on tubulin overlaps with that of the conventional colchicine-binding site. DAT1 can thus be considered as a lead compound of a new class of small molecules and this study can be used as a step to develop potent antimitotic agents for the control of cytoskeletal functions and cell proliferation. It would also be an interesting probe for the structure-function studies of tubulin-microtubule system.     

Rapid and accurate determination of tissue optical properties using least-squares support vector machines

Diffuse reflectance spectroscopy (DRS) has been extensively applied for the characterization of biological tissue, especially for dysplasia and cancer detection, by determination of the tissue optical properties. A major challenge in performing routine clinical diagnosis lies in the extraction of the relevant parameters, especially at high absorption levels typically observed in cancerous tissue. Here, we present a new least-squares support vector machine (LS-SVM) based regression algorithm for rapid and accurate determination of the absorption and scattering properties. Using physical tissue models, we demonstrate that the proposed method can be implemented more than two orders of magnitude faster than the state-of-the-art approaches while providing better prediction accuracy. Our results show that the proposed regression method has great potential for clinical applications including in tissue scanners for cancer margin assessment, where rapid quantification of optical properties is critical to the performance.