Toshiwo Andoh

Catalytic inhibitors of DNA topoisomerase II

Catalytic inhibitors of mammalian DNA topoisomerase II have been found recently in natural and synthetic compounds. These compounds target the enzyme within the cell and inhibit various genetic processes involving the enzyme, such as DNA replication and chromosome dynamics, and thus proved to be good probes for the functional analyses of the enzyme in a variety of eukaryotes from yeast to mammals. Catalytic inhibitors were shown to be antagonists against topoisomerase II poisons. Thus bis(2,6-dioxopiperazines) have a potential to overcome cardiac toxicity caused by potent antitumor anthracycline antibiotics such as doxorubicin and daunorubicin. ICRF-187, a (+)-enantiomer of racemic ICRF-159, has been used in clinics in European countries as cardioprotector. Furthermore, bis(2,6-dioxopiperazines) enhance the efficacy of topoisomerase II poisons by reducing their side effects in preclinical and clinical settings. Bis(2,6-dioxopiperazines) per se among others have antitumor activity, and one of their derivatives, MST-16 or Sobuzoxane, bis(N1-isobutyloxycarbonyloxymethyl-2, 6-dioxopiperazine), has been developed in Japan as an anticancer drug used for malignant lymphomas and adult T-cell leukemia in clinics.

Leptosins isolated from marine fungus Leptoshaeria species inhibit DNA topoisomerases I and/or II and induce apoptosis by inactivation of Akt/protein kinase B

DNA topoisomerases (topo) I and II are molecular targets of several potent anticancer agents. Thus, inhibitors of these enzymes are potential candidates or model compounds for anticancer drugs. Leptosins (Leps) F and C, indole derivatives, were isolated from a marine fungus, Leptoshaeria sp. as cytotoxic substances. In vitro cytotoxic effects of Lep were measured using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide‐based viability assay. Lep F inhibited the activity of topos I and II, whereas Lep C inhibited topo I in vitro. Interestingly both of the compounds were found to be catalytic inhibitors of topo I, as evidenced by the lack of stabilization of reaction intermediate cleavable complex (CC), as camptothecin (CPT) does stabilize. Furthermore, Lep C inhibited the CC stabilization induced by CPT in vitro. In vivo band depletion analysis demonstrated that Lep C likewise appeared not to stabilize CC, and inhibited CC formation by CPT, indicating that Lep C is also a catalytic inhibitor of topo I in vivo. Cell cycle analysis of Lep C‐treated cells showed that Lep C appeared to inhibit the progress of cells from G1 to S phase. Lep C induced apoptosis in RPMI8402 cells, as revealed by the accumulation of cells in sub‐G1 phase, activation of caspase‐3 and the nucleosomal degradation of chromosomal DNA. Furthermore, Leps F and C inhibited the Akt pathway, as demonstrated by dose‐dependent and time‐dependent dephosphorylation of Akt (Ser473). Our study shows that Leps are a group of anticancer chemotherapeutic agents with single or dual catalytic inhibitory activities against topos I and II. (Cancer Sci 2005; 96: 816–824)

DA-Raf1, a competent intrinsic dominant-negative antagonist of the Ras–ERK pathway, is required for myogenic differentiation

Ras activates Raf, leading to the extracellular-regulated kinase (ERK)–mitogen-activated protein kinase pathway, which is involved in a variety of cellular, physiological, and pathological responses. Thus, regulators of this Ras–Raf interaction play crucial roles in these responses. In this study, we report a novel regulator of the Ras–Raf interaction named DA-Raf1. DA-Raf1 is a splicing isoform of A-Raf with a wider tissue distribution than A-Raf. It contains the Ras-binding domain but lacks the kinase domain, which is responsible for activation of the ERK pathway. As inferred from its structure, DA-Raf1 bound to activated Ras as well as M-Ras and interfered with the ERK pathway. The Ras–ERK pathway is essential for the negative regulation of myogenic differentiation induced by growth factors. DA-Raf1 served as a positive regulator of myogenic differentiation by inducing cell cycle arrest, the expression of myogenin and other muscle-specific proteins, and myotube formation. These results imply that DA-Raf1 is the first identified competent, intrinsic, dominant-negative antagonist of the Ras–ERK pathway.

Leptosins M–N1, cytotoxic metabolites from a Leptosphaeria species separated from a marine alga. Structure determination and biological activities

Abstract Leptosins M ( 4 ), M1 ( 5 ) N ( 6 ) and N1 ( 7 ) have been isolated from a strain of Leptosphaeria sp. originally separated from the marine alga Sargassum tortile. Their absolute stereostructures have been elucidated by spectral analyses and some chemical transformations. Their NMR and NOE spectral analyses revealed that 4–7 exist in a single conformer of B type in acetone-d6. All the compounds exhibited significant cytotoxicity against cultured P388 cells. In addition, leptosin M ( 4 ) proved to exhibit significant cytotoxicity against human cancer cell lines, and to inhibit specifically two protein kinases, PTK and CaMKIII, and human topoisomerase II.

Inhibition of DNA topoisomerases I and II, and growth inhibition of human cancer cell lines by a marine microalgal polysaccharide.

We have previously reported purification of an extracellular polysaccharide GA3P, D-galactan sulfate associated with L-(+)-lactic acid, produced by a toxic marine microalga Dinoflagellate Gymnodinium sp. A(3) (GA3), and induction thereby of apoptosis on human myeloid leukemia K562 cells. In the present report, we show that the GA3P is a potent inhibitor of DNA topoisomerase (topo) I and topo II, irrespective of the presence or absence of the lactate group. Dextran sulfate also showed similar level of inhibition of topo I and topo II. We also demonstrated that, unlike camptothecin (CPT) or teniposide (VM-26), the inhibition of topo I or topo II by the polysaccharide does not involve accumulation of DNA-topo I/II cleavable complexes, clearly showing that they are not topo poisons but catalytic inhibitors with dual activity. Furthermore, the polysaccharide, when added to the reaction mixture with CPT or VM-26, inhibited stabilization of cleavable complex induced by the latter compounds. In addition, when added to the reaction mixture after the formation of the cleavable complexes by topo poisons, CPT for topo I and VM-26 for topo II, either GA3P or dextran sulfate diminished the amount of the complexes already accumulated, i.e. reversal of the reaction. These results suggest that the polysaccharides bind to the enzymes with high affinities, and that, as for topo I/II inhibition, the GA3P shares a common mechanism with dextran sulfate. As examined in vitro with a human cancer cell line panel, GA3P exhibited significant cytotoxicity against a variety of cancer cells. These findings show that the polysaccharide GA3P would prove to be a potential anticancer chemotherapeutic agent with dual activity of topo I and topo II catalytic inhibition.

Up‐regulation of Lewis enzyme (Fuc‐TIII) and plasma‐type α1,3Fucosyltransferase (Fuc‐TVI) expression determines the augmented expression of sialyl Lewis x antigen in non‐small cell lung cancer

Sialyl Lewis a and x antigens are well‐known tumor‐associated antigens expressed in many cancer tissues. The expression of the genes encoding 5 α1,3fucosyltransferases, which are able to synthesize the sialyl Lewis antigens, was examined in normal and cancerous lung tissues of patients with non‐small cell lung carcinoma. In all 20 cases examined, the transcripts only for the Lewis gene, encoding the Lewis enzyme (α1,3/4fucosyltransferase, Fuc‐TIII), were abundantly expressed in lung tissue, and interestingly they were markedly up‐regulated in the lung cancer tissues of all 20 cases in comparison with normal lung tissues. Myeloid‐type α1,3fucosyltransferase (Fuc‐TIV) was expressed at an intermediate level but was not up‐regulated in lung cancer tissues. The transcripts for plasma‐type α1,3fucosyltransferase (Fuc‐TVI) gene were detected at a very low level but were apparently up‐regulated in cancer tissues. Fuc‐TVI was found to exhibit stronger relative activity for sialyl Lewis x synthesis (almost 6.4‐fold that of Fuc‐TIII). The amount of sialyl Lewis x antigen on mucins in the lung cancer tissues was found to be determined by both enzymes, the Lewis enzyme (Fuc‐TIII) and Fuc‐TVI. However, the amount of the sialyl Lewis a antigens was not determined by any of the α1,3‐fucosyltransferases, although the expression of sialyl Lewis a antigens definitely required the Lewis enzyme. Int. J. Cancer 83:70–79, 1999.

Isolation of a novel mouse gene, mSVS-1/SUSD2, reversing tumorigenic phenotypes of cancer cells in vitro

We report isolation of a novel tumor‐reversing gene, tentatively named SVS‐1, encoding a protein of 820 amino acids with localization on the plasma membrane as a type I transmembrane protein. The gene was found among those downregulated in the activated oncogene‐v‐K‐ras‐transformed NIH3T3 cells, Ki3T3, with tumorigenic phenotype. SVS‐1 protein harbors several functional domains inherent to adhesion molecules. Histochemical staining of mouse tissues using antibody raised against the protein showed the expression of the protein in restricted regions and cells, for example, strongly positive in apical membranes of epithelial cells in renal tubules and bronchial tubes. The protein inducibly expressed in human fibrosarcoma HT1080 cells and cervical carcinoma HeLa cells was found to be localized primarily on the plasma membrane, as stained with antibodies against FLAG tag in the N‐terminus and against the C‐terminal peptide of the protein. Expression of the protein in cells induced a variety of biological effects on cancer cells: detachment from the substratum and aggregation of cells and growth inhibition in HeLa cells, but no inhibition in non‐tumorigenic mouse NIH3T3 cells. Inhibition of clonogenicity, anchorage‐independent growth, migration and invasion through Matrigel was also observed. Taken together these results suggest that the SVS‐1 gene is a possible tumor‐reversing gene. (Cancer Sci 2007; 98: 900–908)

ICRF-193, a catalytic inhibitor of DNA topoisomerase II, delays the cell cycle progression from metaphase, but not from anaphase to the G1 phase in mammalian cells

We have shown previously that ICRF‐193, a catalytic inhibitor of DNA topoisomerase II (topo II), delays cell cycle progression in HeLa S3 cells. We report here that the delay of the transition in M phase is observed when HeLa S3 cells were treated with ICRF‐193 during metaphase, but not thereafter. ICRF‐193 also delayed the degradation of cyclin B in the transition from M to G1 phase, while in Chinese hamster ovary (CHO) cells the drug did not delay the progression in M phase. Since HeLa S3 and CHO cells are ‘stringent’ and ‘relaxed’ in mitotic control, respectively, it is suggested that under topo II inhibition, the M phase checkpoint operates through an inability for chromosome segregation.

Down-regulation of Bcl-2-interacting protein BAG-1 confers resistance to anti-cancer drugs.

BAG-1 was originally identified as a binding partner of anti-apoptotic factor Bcl-2 [Takayama et al., Cell 80 (1995) 279-284]. Exogenous expression of BAG-1 was reported to confer cells resistance to several stresses [Chen et al., Oncogene 21 (2002) 7050]. We have obtained human cervical cancer HeLa cells with down-regulated BAG-1 levels by using a highly specific and efficient RNA interference approach. Surprisingly, cells with down-regulated BAG-1 exhibited significantly lower sensitivity against several anti-cancer drugs than parental cells expressing normal levels of the protein. Furthermore, growth rate of the cells was reduced when BAG-1 was down-regulated. Activity of ERK pathway appeared to be decreased in BAG-1 down-regulated cells, as shown by the reduced phosphorylation of ERK1/2 proteins. Taken together resistance against anti-cancer drugs acquired by BAG-1 down-regulated cells may well be accounted for by the retardation of cell cycle progression, implicating the importance of BAG-1 in cell growth regulation.

Signal transduction pathways leading to cell cycle arrest and apoptosis induced by DNA topoisomerase poisons

SummaryIn this overview, I have summarized the important pathways of stress-induced signal transduction: stabilization and activation of p53 playing a central role in stress-induced cell cycle checkpoint and apoptosis, and activation of ASK1-JNK/p38 pathway often induced by a variety of stress stimuli, which appears to be essentially required for apoptosis to follow.