Alexander W. Wood

Antagonism of Sphingosine-1-Phosphate Receptors by FTY720 Inhibits Angiogenesis and Tumor Vascularization

FTY720, a potent immunomodulator, becomes phosphorylated in vivo (FTY-P) and interacts with sphingosine-1-phosphate (S1P) receptors. Recent studies showed that FTY-P affects vascular endothelial growth factor (VEGF)-induced vascular permeability, an important aspect of angiogenesis. We show here that FTY720 has antiangiogenic activity, potently abrogating VEGF- and S1P-induced angiogenesis in vivo in growth factor implant and corneal models. FTY720 administration tended to inhibit primary and significantly inhibited metastatic tumor growth in a mouse model of melanoma growth. In combination with a VEGFR tyrosine kinase inhibitor PTK787/ZK222584, FTY720 showed some additional benefit. FTY720 markedly inhibited tumor-associated angiogenesis, and this was accompanied by decreased tumor cell proliferation and increased apoptosis. In transfected HEK293 cells, FTY-P internalized S1P1 receptors, inhibited their recycling to the cell surface, and desensitized S1P receptor function. Both FTY720 and FTY-P apparently failed to impede VEGF-produced increases in mitogen-activated protein kinase activity in human umbilical vascular endothelial cells (HUVEC), and unlike its activity in causing S1PR internalization, FTY-P did not result in a decrease of surface VEGFR2 levels in HUVEC cells. Pretreatment with FTY720 or FTY-P prevented S1P-induced Ca2+ mobilization and migration in vascular endothelial cells. These data show that functional antagonism of vascular S1P receptors by FTY720 potently inhibits angiogenesis; therefore, this may provide a novel therapeutic approach for pathologic conditions with dysregulated angiogenesis.

Metabolism of benzo[a]pyrene VI. Stereoselective metabolism of benzo[a]pyrene and benzo[a]pyrene 7,8-dihydrodiol to diol epoxides

Abstract (±)-7β,8α-Dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-1) and (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-2) are highly mutagenic diol epoxide diastereomers that are formed during metabolism of the carcinogen (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene. Remarkable stereoselectivity has been observed on metabolism of the optically pure (+)- and (−)-enantiomers of the dihydrodiol which are obtained by separation of the diastereomeric diesters with (−)-α-methoxy-α-trifluoromethylphenylacetic acid. The high stereoselectivity in the formation of diol epoxide-1 relative to diol epoxide-2 was observed with liver microsomes from 3-methylcholanthrene-treated rats and with a purified cytochrome P-448-containing monoxygenase system where the (−)-enantiomer produced a diol epoxide-2 to diol epoxide-1 ratio of 6 : 1 and the (+)-enantiomer produced a ratio of 1 : 22. Microsomes from control and phenobarbital-treated rats were less stereospecific in the metabolism of enantiomers of BP 7,8-dihydrodiol. The ratio of diol epoxide-2 to diol epoxide-1 formed from the (−)- and (+)-enantiomers with microsomes from control rats was 2 : 1 and 1 : 6, respectively. Both enantiomers of BP 7,8-dihydrodiol were also metabolized to a phenolic derivative, tentatively identified as 6,7,8-trihydroxy-7,8-dihydrobenzo[a]pyrene, which accounted for ∼30% of the total metabolites formed by microsomes from control and phenobarbital-pretreated rats whereas this metabolite represents ∼5% of the total metabolites with microsomes from 3-methylcholanthrene-treated rats. With benzo[a]pyrene as substrate, liver microsomes produced the 4,5-, 7,8- and 9,10-dihydrodiol with high optical purity (>85%), and diol epoxides were also formed. Most of the optical activity in the BP 7,8-dihydrodiol was due to metabolism by the monoxygenase system rather than by epoxide hydrase, since hydration of (±)-benzo[a]pyrene 7,8-oxide by liver microsomes produced dihydrodiol which was only 8% optically pure. Thus, the stereospecificity of both the monoxygenase system and, to a lesser extent, epoxide hydrase plays important roles in the metabolic activation of benzo[a]pyrene to carcinogens and mutagens.

Broad antitumor and antiangiogenic activities of AG3340, a potent and selective MMP inhibitor undergoing advanced oncology clinical trials.

ABSTRACT: We studied AG3340, a potent metalloproteinase (MMP) inhibitor with pM affinities for inhibiting gelatinases (MMP‐2 and ‐9), MT‐MMP‐1 (MMP‐14), and collagenase‐3 (MMP‐13) in many tumor models. AG3340 produced dose‐dependent pharmacokinetics and was well tolerated after intraperitoneal (i.p.) and oral dosing in mice. Across human tumor models, AG3340 produced profound tumor growth delays when dosing began early or late after tumor implantation, although all established tumor types did not respond to AG3340. A dose‐response relationship was explored in three models: COLO‐320DM colon, MV522 lung, and MDA‐MB‐435 breast. Dose‐dependent inhibitions of tumor growth (over 12.5‐200 mg/kg given twice daily, b.i.d.) were observed in the colon and lung models; and in a third (breast), maximal inhibitions were produced by the lowest dose of AG3340 (50 mg/kg, b.i.d.) that was tested. In another model, AG3340 (100 mg/kg, once daily, i.p.) markedly inhibited U87 glioma growth and increased animal survival. AG3340 also inhibited tumor growth and increased the survival of nude mice bearing androgen‐independent PC‐3 prostatic tumors. In a sixth model, KKLS gastric, AG3340 did not inhibit tumor growth but potentiated the efficacy of Taxol. Importantly, AG3340 markedly decreased tumor angiogenesis (as assessed by CD‐31 staining) and cell proliferation (as assessed by bromodeoxyuridine incorporation), and increased tumor necrosis and apoptosis (as assessed by hematoxylin and eosin and TUNEL staining). These effects were model dependent, but angiogenesis was commonly inhibited. AG3340 had a superior therapeutic index to the cytotoxic agents, carboplatin and Taxol, in the MV522 lung cancer model. In combination, AG3340 enhanced the efficacy of these cytotoxic agents without altering drug tolerance. Additionally, AG3340 decreased the number of murine melanoma (B16‐F10) lesions arising in the lung in an intravenous metastasis model when given in combination with carboplatin or Taxol. These studies directly support the use of AG3340 in front‐line combination chemotherapy in ongoing clinical trials in patients with advanced malignancies of the lung and prostate.

High mutagenicity and toxicity of a diol epoxide derived from benzo[a]pyrene

Abstract (±)-7β,8α-Dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BP 7,8-diol-9,10-epoxide) is a suspected metabolite of benzo[a]pyrene that is highly mutagenic and toxic in several strains of Salmonella typhimurium and in cultured Chinese hamster V79 cells. BP 7,8-diol-9,10-epoxide was approximately 5, 10 and 40 times more mutagenic than benzo[a]pyrene 4,5-oxide (BP 4,5-oxide) in strains TA 98 and TA 100 of S. typhimurium and in V79 cells, respectively. Both compounds were equally mutagenic to strain TA 1538 and non-mutagenic to strain TA 1535 of S. typhimurium . The diol epoxide was toxic to the four bacterial strains at 0.5–2.0 nmole/plate, whereas BP 4,5-oxide was nontoxic at these concentrations. In V79 cells, the diol epoxide was about 60-fold more cytotoxic than BP 4,5-oxide.

Differences in mutagenicity of the optical enantiomers of the diastereomeric benzo[a]pyrene 7,8-diol-9,10-epoxides

Summary Substantial differences in the mutagenic activities of the optically pure (+)- and (−)-enantiomers of the diastereomeric 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrenes were observed in strains TA98 and TA100 of Salmonella typhimurium and Chinese hamster V79 cells. In strains TA98 and TA100 (−)-7β,8α-dihydroxy-9β, 10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene was the most mutagenic compound, inducing from 1.3 to 9.5 times as many mutations as the three other optically active stereoisomers. In Chinese hamster cells (+)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene was the most mutagenic compound inducing from 6 to 18 times as many variant colonies as the three other isomers. These results with known metabolites of the environmental carcinogen benzo[a]pyrene represent the first report of differences in mutagenic activity among optical enantiomers.

Mutagenicity of metabolically activated benzo[a]anthracene 3,4-dihydrodiol: evidence for bay region activation of carcinogenic polycyclic hydrocarbons.

Summary Benzo[a]anthracena and the 5 metabolically possible trans dihydrodiols of benzo[a]anthracene were metabolized, in the presence of S. thyphimurium strain TA100, by a highly purified hepatic microsomal monooxygenase system. The metabolic product(s) of benzo[a]anthracene 3,4-dihydrodiol was nearly 10 times as mutagenic to the bacteria as were the metabolites of benzo[a]anthracene and the other four dihydrodiols. The marked activation of benzo[a]anthracene 3,4-dihydrodiol, presumably to the 3,4-diol-1,2-epoxide is consistent with and supports the hypothesis that bay region epoxides of unsubstituted polycyclic hydrocarbons are ultimately reactive forms of these carcinogenic compounds.

Comparison of the O‐dealkylation of 7‐ethoxycoumarin and the hydroxylation of benzo [a] pyrene in human placenta

A highly sensitive method is described for measuring the in vitro metabolism of 7‐ethoxycoumarin or coumarin to 7‐hydroxycoumarin. This method was used to measure the O‐dealkylation of 7‐ethoxycoumarin and the hydroxylation of coumarin by placentas from 12 nonsmokers or from 12 women who smoked cigarettes. Although an induced level of benzo[a]pyrene (BP) hydroxylase activity was found in each placenta obtained from women who smoked cigarettes during pregnancy, 7‐ethoxycoumarin O‐dealkylase activity was induced (about 2 fold) only in placentas whose BP hydroxylase activity was markedly induced (50 to 100 fold). These results indicate that ethoxycoumarin dealkylase activity parallels BP hydroxylase activity in human placenta only in individuals with markedly induced BP hydroxylase activity. No detectable hydroxylation of coumarin in the 7‐position was observed in placental homogenates from nonsmokers 0., from women who smoked cigarettes.

1α,25-Dihydroxyvitamin D3 inhibits phorbol ester-dependent chemical carcinogenesis in mouse skin

The effect of topical application of 1 alpha, 25-dihydroxyvitamin D3 (1 alpha, 25-(OH)2D3) on the promotional phase of skin tumor formation in mice was evaluated using 7,12-dimethylbenz [a] anthracene as the tumor initiator and 12-0-tetradecanoylphorbol-13-acetate (TPA) as the tumor promoter. Fifteen weeks of twice weekly topical application of 1 alpha, 25-(OH)2D3 1 hour prior to topical treatment with 16 nmol of TPA inhibited tumor formation in a dose-dependent manner. Doses of 0.25-0.50 nmol of the vitamin D3 metabolite inhibited tumor formation approximately 50% and had no significant effect on the survival or weight gain of the mice. These results indicate that in addition to maintaining calcium homeostasis and affecting the growth and differentiation of certain neoplastic cells, 1 alpha, 25-(OH)2D3 can also suppress the formation of chemically induced tumors.

High mutagenicity of metabolically activated chrysene 1,2 dihydrodiol: Evidence for bay region activation of chrysene

Abstract Chrysene and the 3 metabolically possible vicinal trans dihydrodiols of chrysene were tested for mutagenicity towards S. typhimurium strain TA100 in the presence of hepatic microsomes or a highly purified hepatic microsomal monooxygenase system. The products formed during the metabolic activation of chrysene 1,2-dihydrodiol were more than 20 times as mutagenic to the bacteria than the metabolites formed from chrysene, chrysene 3,4-dihydrodiol or chrysene 5,6-dihydrodiol. When the double bond in the 3,4-position of chrysene 1,2-dihydrodiol was saturated, the resulting tetrahydrodiol could not be metabolically activated. These results, which strongly suggest that chrysene 1,2-dihydrodiol is activated by metabolism to either or both of the diastereomeric chrysene 1,2-diol-3,4-epoxides, provide additional support for the bay region theory of polycyclic hydrocarbon carcinogenicity.

Regulation of Tiam1 Nucleotide Exchange Activity by Pleckstrin Domain Binding Ligands

Rho family GTPases play roles in cytoskeletal organization and cellular transformation. Tiam1 is a member of the Dbl family of guanine nucleotide exchange factors that activate Rho family GTPases. These exchange factors have in common a catalytic Dbl homology and adjacent pleckstrin homology domain. Previous structural studies suggest that the pleckstrin domain, a putative phosphoinositide-binding site, may serve a regulatory function. We identified ascorbyl stearate as a compound that binds to the pleckstrin domain of p120 Ras GTPase-activating protein. Furthermore, ascorbyl stearate appears to be a general pleckstrin domain ligand, perhaps by mimicking an endogenous amphiphilic ligand. Tiam1 nucleotide exchange activity was greatly stimulated by ascorbyl stearate. Certain phosphoinositides also stimulated Tiam1 activity but were less potent than ascorbyl stearate. Tiam1 contains an additional N-terminal pleckstrin domain, but only the C-terminal pleckstrin domain was required for activation. Our results suggest that the pleckstrin domains of Dbl-type proteins may not only be involved in subcellular localization but may also directly regulate the nucleotide exchange activity of an associated Dbl homology domain. In addition, this paper introduces ascorbyl stearate as a pleckstrin domain ligand that can modulate the activity of certain pleckstrin domain-containing proteins.