MST-16, a novel bis-dioxopiperazine anticancer agent, ameliorates doxorubicin-induced acute toxicity while maintaining antitumor efficacy.
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MST-16 [4,4-1,2-(ethanediyl)bis(1-isobutoxycarbonyl-oxy-methyl-2,6-pipera zinedione)], recently approved as an oral anticancer drug for clinical use in Japan, was evaluated as a chemotherapeutic agent in combination with doxorubicin (DOX) in vitro and in vivo. Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and murine Colon 26 and human KATO III adenocarcinoma cells were used. The combination index derived from these cytotoxic values indicated a synergistic interaction between DOX and MST-16 or its active metabolite, ICRF-154 (1,1'-ethylenedi-3,5-dioxopiperazine). A maximal tolerated dose of DOX administered to female BALB/c mice bearing a solid Colon 26 tumor resulted in severe body weight loss and diarrhea, but a limited tumor growth delay (1.8 days). However, when combined with an oral dose of MST-16, DOX-induced body weight loss and diarrhea were significantly ameliorated, and an additive tumor growth delay (8.7 days) was obtained. The LD50 of DOX administered i.p. to control female BALB/c mice increased more than 1.5-fold when combined with MST-16. Thus, MST-16 ameliorates DOX-induced acute toxicity while maintaining antitumor efficacy. These results indicate that MST-16 may be effective chemotherapy for cancer patients when combined with DOX. (+info)
L-2-[(13)C]oxothiazolidine-4-carboxylic acid: a probe for precursor mobilization for glutathione synthesis.
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L-2-oxothiazolidine-4-carboxylic acid (OTZ), a 5-oxoproline analog, is metabolized by 5-oxoprolinase and converted to cysteine, the rate-limiting amino acid for GSH synthesis, with the release of CO(2). [(13)C]OTZ (1.5 mg/kg) was used in 12 healthy men and women (ages 23-73 yr) to indirectly assess precursor mobilization for GSH synthesis when stores were reduced by 2 g acetaminophen. Expired breath samples were analyzed for (13)CO(2), and results were analyzed using noncompartmental and two-compartment open minimal models. Results show an increase in (13)C excretion (higher OTZ hydrolysis) when GSH stores were reduced and 5-oxoprolinase substrate utilization patterns, consequently, were altered (P < 0. 01). A metabolic rate index (MRI) of the OTZ probe was found to be significantly higher after reduction of GSH content by acetaminophen (P < 0.05). The difference in adaptive capacity (difference between control and postacetaminophen metabolic rate indexes) was two times as large in the young than the old subjects (P < 0.01). These data support the use of [(13)C]OTZ as a probe to identify individuals who may be at risk for low GSH stores or who have an impaired capacity to synthesize GSH. (+info)
IL-4 increases surfactant and regulates metabolism in vivo.
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Mice that express interleukin (IL)-4 in Clara cells (CCSP-IL-4) develop chronic airway inflammation and an alveolar proteinosis-like syndrome. To identify the role of IL-4 in surfactant homeostasis, we measured lipid and protein metabolism in the lungs of CCSP-IL-4 mice in vivo. Alveolar saturated phosphatidylcholine (Sat PC) pools were increased 6.5-fold and lung tissue Sat PC pools were increased 4. 8-fold in the IL-4 transgenic mice. Whereas surfactant protein (SP) A was increased proportionately to Sat PC, SP-D was increased approximately 90-fold in the IL-4 mice compared with wild-type mice and was associated with 2.8-fold increase in SP-D mRNA. The incorporation of palmitate and choline into Sat PC was increased about twofold in CCSP-IL-4 mice. Although trace doses of radiolabeled Sat PC were cleared from the air spaces and lungs of CCSP-IL-4 mice more slowly than in wild-type mice, net clearance of Sat PC from the lungs of CCSP-IL-4 mice was sixfold higher in the IL-4 mice than in wild-type mice because of the larger Sat PC pool sizes. Expression of IL-4 in Clara cells increased surfactant lipid synthesis and clearance, establishing a new equilibrium with increased surfactant pools and an alveolar proteinosis associated with a selective increase in SP-D protein, demonstrating a previously unexpected effect of IL-4 in pulmonary surfactant homeostasis. (+info)
Cell line dependence of Bcl-2-induced alteration of glutathione handling.
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Bcl-2 has been associated with both oxidative and antioxidative effects in vivo. Moreover, despite evidence that Bcl-2 is antiapoptotic by virtue of its effect on reactive oxygen species and their scavengers, Bcl-2 exerts its antiapoptotic effects even under anaerobic conditions. The reasons for the variable relationship between Bcl-2 and reactive oxygen species are not clear. The present studies demonstrate that the impact of Bcl-2 on glutathione (GSH) metabolism is cell line-dependent. Bcl-2 overproduction in PC12 cells is associated with increased functional thiol reserves, increased reductive activation of chemotherapeutic prodrugs, and GSH accumulation after treatment with N-acetylcysteine. In contrast, Bcl-2-overproducing MCF-7 breast cancer cells demonstrate neither altered GSH handling nor potentiation of chemotherapeutic prodrug reduction. These findings indicate that the effects of Bcl-2 on GSH handling are millieu-dependent. This could account for the variable effects of Bcl-2 in in vivo systems. Furthermore, since our previous studies have demonstrated that reduction-dependent prodrugs may be useful chemotherapeutic agents against tumors that demonstrate altered GSH handling, screening in vitro for alteration of GSH handling may predict responsiveness of such tumors to these reduction-dependent agents. (+info)
Human phenol sulfotransferases hP-PST and hM-PST activate propane 2-nitronate to a genotoxicant.
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The industrial solvent 2-nitropropane (2-NP) is a genotoxic hepatocarcinogen in rats. The genotoxicity of the compound in rats has been attributed to sulfotransferase-mediated formation of DNA-reactive nitrenium ions from the anionic form of 2-NP, propane 2-nitronate (P2N). Whether human sulfotransferases are capable of activating P2N is unknown. In the present study we have addressed this question by investigating the genotoxicity of P2N in various V79-derived cell lines engineered for expression of individual forms of human sulfotransferases, the phenol-sulfating and the monoamine-sulfating phenol sulfotransferases (hP-PST and hM-PST) and the human hydroxysteroid sulfotransferase (hHST). Genotoxicity was assessed by measuring the induction of DNA repair synthesis and by analyzing the formation of DNA modifications. P2N induced repair synthesis in V79-hP-PST and V79-hM-PST cells, whereas induction of repair synthesis in V79-hHST cells was negligible. P2N also resulted in the formation of 8-aminodeoxyguanosine and increased the level of 8-oxodeoxyguanosine in V79-hP-PST cells, but not in the parental V79-MZ cells, which do not show any sulfotransferase activity. Acetone oxime, the tautomeric form of the first reduction product of 2-NP, 2-nitrosopropane, was inactive in all cell lines. The results show that the human phenol sulfotransferases P-PST and M-PST are capable of metabolically activating P2N (P-PST >> M-PST) and that the underlying mechanism is apparently identical to that resulting in the activation of P2N in rat liver, where 2-NP causes carcinomas. These results support the notion that 2-NP should be regarded as a potential human carcinogen. (+info)
Prostate-specific antigen promoter/enhancer driven gene therapy for prostate cancer: construction and testing of a tissue-specific adenovirus vector.
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A range of luciferase reporter vectors was constructed, incorporating 5'-flanking sequences from the prostate-specific antigen (PSA), human glandular kallikrein 2 (hKLK2), and cytomegalovirus (CMV) promoters for expression control. Tissue specificity was evaluated in the PSA-positive line LNCaP and PSA-negative cells from different tissues of origin (CoLo320, DG75, EJ, A2780, and Jurkat). The minimal 628-bp PSA and hKLK2 promoters showed only low-level expression in either PSA-positive or PSA-negative cells and showed no increase with the addition of androgen. Tandem duplication of the PSA promoter slightly increased expression in PSA-positive LNCaP cells. The addition of CMV enhancer sequences upstream of a single PSA or hKLK2 promoter substantially but nonspecifically increased luciferase expression in all cell lines tested. However, placing a 1455-bp PSA enhancer sequence upstream of either the PSA or hKLK2 promoters increased expression 20-fold in the PSA-positive cell line LNCaP but not in the PSA-negative lines. Tandem duplication of the PSA enhancer increased expression to approximately 50-fold higher than either promoter alone while retaining tissue-specific control. The level of expression was reduced by the addition of a third copy of the PSA enhancer. Expression from all enhancer constructs was increased 100-fold above basal levels when induced with the androgen dihydrotestosterone, with the PSA-based constructs consistently exhibiting roughly twice the level of expression of the hKLK2-based constructs at all androgen concentrations. Adenovirus vectors were produced in which either enhanced green fluorescent protein or nitroreductase could be expressed from the optimized PSA double enhancer-promoter construct and evaluated in LNCaP cells and the bladder-derived line EJ. Control vectors with the CMV promoter gave good levels of expression in both cell lines, whereas the PSA constructs only produced detectable levels of protein in the LNCaP cells as assessed by fluorescence of enhanced green fluorescent protein or by Western blotting of nitroreductase. LNCaP but not EJ cells were selectively sensitized to the prodrug CB1954 following infection with Ad-PSA(EEP)-NR. The PSA-based nitroreductase virus produced comparable amounts of nitroreductase and sensitization to CB1954 approaching that of the CMV-driven virus. Plasmid and adenovirus constructs combining PSA enhancer and promoter sequences demonstrate selective expression of linked genes in PSA-positive cells. The expression is induced by androgen and gives therapeutically relevant levels of effector proteins. (+info)
The lipophilic properties of angiotensin I-converting enzyme inhibitors do not influence their diffusion through cultured endothelium.
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The background for these investigations was the discovery that formation of angiotensin II by the renin angiotensin system can take place in extravascular tissues (e.g., cardiomyocytes and neurons) and within single cells. Consequently, the question arose about whether such tissue-based systems might be differentially influenced by angiotensin I-converting enzyme (ACE) inhibitors with distinct physicochemical properties. Therefore, the aim of this study was to investigate how the membrane penetration of various ACE inhibitors depends on their lipophilia. All diacid forms of ACE inhibitors are dissociated at a pH of 7.4 and scarcely extractable into octanol (extraction coefficient < 10%). In contrast, the extraction coefficients of the parent substances showed marked differences in the following order of increasing lipophilia: enalapril = perindopril < captopril = ceranapril < ramipril < quinapril < HOE288 = zofenopril < fosinopril < HOE065. For selected substances, the kinetics of diffusion through a monolayer of cultured bovine aortic endothelium were determined. The diffusion rates (expressed as half lives) of captopril (59.6 min), enalapril (53.4 min), enalaprilat (50.8 min), ramipril (56.9 min) and ramiprilat (51.1 min) are similar indicating: 1) that penetration is independent on lipophilia and 2) that endothelium constitutes no specific barrier for the passage of ACE inhibitors into the vessel wall. (+info)
Expression of endogenously activated secreted or cell surface carboxypeptidase A sensitizes tumor cells to methotrexate-alpha-peptide prodrugs.
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Methotrexate (MTX) is one of the most commonly used agents in the treatment of solid malignancies; however, the toxicities of MTX to bone marrow and gastrointestinal tract complicate this therapy. We, therefore, propose a gene-dependent enzyme prodrug therapy to limit these toxicities by localizing the production of MTX to the site of the tumor. The combination of MTX-alpha-peptide prodrugs, which cannot be internalized by the cellular reduced folate carrier, with carboxypeptidase A (CPA), which can remove the blocking peptide, has been demonstrated previously in vitro using antibody-dependent enzyme prodrug therapy. CPA is normally synthesized as a zymogen that is inactive without proteolytic removal of its propeptide by trypsin. Therefore, to adapt this system to gene-dependent enzyme prodrug therapy, a mutant form of CPA was engineered, CPA(ST3), that does not require trypsin-dependent zymogen cleavage but is instead activated by ubiquitously expressed intracellular propeptidases. Purification, peptide sequencing, and kinetic analysis indicated that mature CPA(ST3) is structurally and functionally similar to the trypsin-activated, wild-type enzyme. In addition, CPA(ST3)-expressing tumors cells were sensitized to MTX prodrugs in a dose- and time-dependent manner. To limit diffusion of CPA, a cell surface localized form was generated by constructing a fusion protein between CPA(ST3) and the phosphatidylinositol linkage domain from decay accelerating factor. SDS-PAGE and flow cytometric analysis of infected tumor cells indicated that CPA(DAF) was cell surface localized. Finally, after retroviral transduction, this enzyme/prodrug strategy exhibited a potent bystander effect, even when <10% of the cells were transduced, because extracellular production of MTX sensitized both transduced and nontransduced cells. (+info)