Carbamoylating activity associated with the activation of the antitumor agent laromustine inhibits angiogenesis by inducing ASK1-dependent endothelial cell death.

The anticancer agent 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine (laromustine), upon decomposition in situ, yields methyl isocyanate and the chloroethylating species 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE). 90CE has been shown to kill tumor cells via a proposed mechanism that involves interstrand DNA cross-linking. However, the role of methyl isocyanate in the antineoplastic function of laromustine has not been delineated. Herein, we show that 1,2-bis(methylsulfonyl)-1-[(methylamino)carbonyl]hydrazine (101MDCE), an analog of laromustine that generates only methyl isocyanate, activates ASK1-JNK/p38 signaling in endothelial cells (EC). We have previously shown that ASK1 forms a complex with reduced thioredoxin (Trx1) in resting EC, and that the Cys residues in ASK1 and Trx1 are critical for their interaction. 101MDCE dissociated ASK1 from Trx1, but not from the phosphoserine-binding inhibitor 14-3-3, in whole cells and in cell lysates, consistent with the known ability of methyl isocyanate to carbamoylate free thiol groups of proteins. 101MDCE had no effect on the kinase activity of purified ASK1, JNK, or the catalytic activity of Trx1. However, 101MDCE, but not 90CE, significantly decreased the activity of Trx reductase-1 (TrxR1). We conclude that methyl isocyanate induces dissociation of ASK1 from Trx1 either directly by carbamoylating the critical Cys groups in the ASK1-Trx1 complex or indirectly by inhibiting TrxR1. Furthermore, 101MDCE (but not 90CE) induced EC death through a non-apoptotic (necroptotic) pathway leading to inhibition of angiogenesis in vitro. Our study has identified methyl isocyanates may contribute to the anticancer activity in part by interfering with tumor angiogenesis.

Thioredoxin reductase is inhibited by the carbamoylating activity of the anticancer sulfonylhydrazine drug laromustine.

The thioredoxin system facilitates proliferative processes in cells and is upregulated in many cancers. The activities of both thioredoxin (Trx) and its reductase (TrxR) are mediated by oxidation/reduction reactions among cysteine residues. A common target in preclinical anticancer research, TrxR is reported here to be significantly inhibited by the anticancer agent laromustine. This agent, which has been in clinical trials for acute myelogenous leukemia and glioblastoma multiforme, is understood to be cytotoxic principally via interstrand DNA crosslinking that originates from a 2-chloroethylating species generated upon activation in situ. The spontaneous decomposition of laromustine also yields methyl isocyanate, which readily carbamoylates thiols and primary amines. Purified rat liver TrxR was inhibited by laromustine with a clinically relevant IC(50) value of 4.65 µM. A derivative of laromustine that lacks carbamoylating activity did not appreciably inhibit TrxR while another derivative, lacking only the 2-chloroethylating activity, retained its inhibitory potency. Furthermore, in assays measuring TrxR activity in murine cell lysates, a similar pattern of inhibition among these compounds was observed. These data contrast with previous studies demonstrating that glutathione reductase, another enzyme that relies on cysteine-mediated redox chemistry, was not inhibited by methylcarbamoylating agents when measured in cell lysates. Mass spectrometry of laromustine-treated enzyme revealed significant carbamoylation of TrxR, albeit not on known catalytically active residues. However, there was no evidence of 2-chloroethylation anywhere on the protein. The inhibition of TrxR is likely to contribute to the cytotoxic, anticancer mechanism of action for laromustine.

Inhibition of all-trans retinoic acid-induced granulocytic differentiation of WEHI-3B D+ cells by forced expression of SCL (TAL1) and GATA-1.

All-trans retinoic acid (ATRA) induces granulocytic maturation of WEHI-3B D+ leukemia cells and LiCl enhances this maturation, while WEHI-3B D- cells are non-responsive to ATRA. Transfection of SCL, expressed in D- but absent in D+ cells, into D+ cells, caused resistance to ATRA, while transfection of GATA-1 into D+ cells produced resistance to the combination of ATRA and LiCl. SCL expression in D+ cells did not induce the expression of c-Kit, a putative target gene for SCL. LiCl, known to inhibit some kinases by displacing Mg2+, did not affect tyrosine kinase activity of the cytoplasmic domain of c-Kit.

Inhibition of human DNA polymerase beta activity by the anticancer prodrug Cloretazine.

The antineoplastic prodrug Cloretazine exerts its cytotoxicity via a synergism between 2-chloroethylating and carbamoylating activities that are cogenerated upon activation in situ. Cloretazine is reported here to inhibit the nucleotidyl-transferase activity of purified human DNA polymerase beta (Pol beta), a principal enzyme of DNA base excision repair (BER). The 2-chloroethylating activity of Cloretazine alkylates DNA at the O(6) position of guanine bases resulting in 2-chloroethoxyguanine monoadducts, which further react to form cytotoxic interstrand DNA crosslinks. Alkylated DNA is often repaired via BER in vivo. Inhibition of the polymerase activity of Pol beta may account for some of the synergism between Cloretazine's two reactive subspecies in cytotoxicity assays. This inhibition was only observed using agents with carbamoylating activity. Furthermore, while therapeutically relevant concentrations of Cloretazine inhibited the polymerase activity of Pol beta, the enzyme's lyase activity, which may also participate in BER, was not significantly inhibited.

Disruption of cAMP and prostaglandin E2 transport by multidrug resistance protein 4 deficiency alters cAMP-mediated signaling and nociceptive response.

Multidrug resistance protein 4 (MRP4; ABCC4) is a member of the MRP/ATP-binding cassette family serving as a transmembrane transporter involved in energy-dependent efflux of anticancer/antiviral nucleotide agents and of physiological substrates, including cyclic nucleotides and prostaglandins (PGs). Phenotypic consequences of mrp4 deficiency were investigated using mrp4-knockout mice and derived immortalized mouse embryonic fibroblast (MEF) cells. Mrp4 deficiency caused decreased extracellular and increased intracellular levels of cAMP in MEF cells under normal and forskolin-stimulated conditions. Mrp4 deficiency and RNA interference-mediated mrp4 knockdown led to a pronounced reduction in extracellular PGE(2) but with no accumulation of intracellular PGE(2) in MEF cells. This result was consistent with attenuated cAMP-dependent protein kinase activity and reduced cyclooxygenase-2 (Cox-2) expression in mrp4-deficient MEF cells, suggesting that PG synthesis is restrained along with a lack of PG transport caused by mrp4 deficiency. Mice lacking mrp4 exhibited no outward phenotypes but had a decrease in plasma PGE metabolites and an increase in inflammatory pain threshold compared with wild-type mice. Collectively, these findings imply that mrp4 mediates the efflux of PGE(2) and concomitantly modulates cAMP mediated signaling for balanced PG synthesis in MEF cells. Abrogation of mrp4 affects the regulation of peripheral PG levels and consequently alters inflammatory nociceptive responses in vivo.

Differential inhibition of cellular glutathione reductase activity by isocyanates generated from the antitumor prodrugs Cloretazine and BCNU.

The antitumor, DNA-alkylating agent 1,3-bis[2-chloroethyl]-2-nitrosourea (BCNU; Carmustine), which generates 2-chloroethyl isocyanate upon decomposition in situ, inhibits cellular glutathione reductase (GR; EC 1.8.1.7) activity by up to 90% at pharmacological doses. GR is susceptible to attack from exogenous electrophiles, particularly carbamoylation from alkyl isocyanates, rendering the enzyme unable to catalyze the reduction of oxidized glutathione. Evidence implicates inhibition of GR as a cause of the pulmonary toxicity often seen in high-dose BCNU-treated animals and human cancer patients. Herein we demonstrate that the prodrug Cloretazine (1,2-bis[methylsulfonyl]-1-[2-chloroethyl]-2-[(methylamino)carbonyl]hydrazine; VNP40101M), which yields methyl isocyanate and chloroethylating species upon activation, did not produce similar inhibition of cellular GR activity, despite BCNU and Cloretazine being equally potent inhibitors of purified human GR (IC(50) values of 55.5 microM and 54.6 microM, respectively). Human erythrocytes, following exposure to 50 microM BCNU for 1h at 37 degrees C, had an 84% decrease in GR activity, whereas 50 microM Cloretazine caused less than 1% inhibition under the same conditions. Similar results were found using L1210 murine leukemia cells. The disparity between these compounds remained when cells were lysed prior to drug exposure and were partially recapitulated using purified enzyme when 1mM reduced glutathione was included during the drug exposure. The superior antineoplastic potential of Cloretazine compared to BCNU in animal models could be attributed in part to the contribution of the methyl isocyanate, which is synergistic with the co-generated cytotoxic alkylating species, while at the same time unable to significantly inhibit cellular GR.

Increasing the kinase specificity of k252a by protein surface recognition.

[reaction: see text] Here we describe a miniature protein (1) that presents the cAMP-dependent protein kinase (PKA) recognition epitope found within the heat-stable Protein Kinase Inhibitor protein (PKI) and a miniature protein conjugate (1-K252a) in which 1 is joined covalently to the high-affinity but nonselective kinase inhibitor K252a. Miniature protein 1 recognizes PKA with an affinity that rivals that of PKI and, in the context of 1-K252a, leads to a dramatic increase in kinase specificity.