Nous avons récemment découvert et étudié une nouvelle classe d'agents antimicrotubules appelés phényl 4-(2-oxo-3-alkylimidazolidin-1-yl)benzènesulfonates (PAIB-SOs) hautement actifs et sélectifs pour les cellules cancéreuses du sein. Leur mécanisme de sélectivité est basé sur la métabolisation des PAIB-SOs par le CYP1A1 en agents antimitotiques puissants appelés phényl 4-(2-oxo-3-imidazolidin-1-yl)benzènesulfonates (PIB-SOs). L'objectif principal de ma recherche était d'évaluer la période nécessaire à l’induction d’une activité cytotoxique significative de quelques-uns de ces PAIB-SOs prometteurs sur les cellules cancéreuses du sein. Dans un second volet, nous désirions étudier la stabilité métabolique et la cinétique de bioactivation par le CYP1A1 de 8 PAIB-SOs phare en vue d’en sélectionner 4 pour des essais sur des animaux. Premièrement, nos études ont montré que l'activité antiproliférative des PAIB-SOs est concentration et temps dépendante. Un temps de contact des PAIB-SOs étudiés avec les cellules cancéreuses du sein variant de 24 à 36 h est nécessaire pour observer une activité antiproliférative significative. Deuxièmement, nous avons confirmé que tous les PAIB-SOs évalués sont rapidement biotransformés en présence de CYP1A1 en PIB-SOs. Troisièmement, les expériences de stabilité hépatique des PAIB-SOs incubés avec des microsomes soit humains, de souris ou de rats ont montré que leur demi-vie et leur clairance intrinsèque varient selon la structure du PAIB-SO et l’espèce animale étudiée. La stabilité des PAIB-SOs incubés avec des microsomes de souris est plus faible que lorsqu’incubé avec des microsomes humains ou de rats. De plus, la stabilité métabolique de ces PAIB-SOs avec les microsomes humains ou de rats est similaire. Le criblage de notre chimiothèque a permis d’identifier les CEU-829, -938, -934 et -913 comme étant les PAIB-SOs métaboliquement les plus stables et générant les plus basses concentrations de PIB-SOs avec des demi-vies respectives de 22, 55, 31 et 41 minutes dans les microsomes humains, de 43, 52, 23 et 44 minutes dans les microsomes de rat et de 3,7, 20, 12 et 1,6 minutes dans les microsomes de souris. Nos études ont également mis en évidence les CEU-934 et -938 comme des molécules candidates prometteuses pour des études de pharmacocinétique et de pharmacodynamie chez la souris et les CEU-829 et -913 chez les rats. / We recently found and studied a new class of antimicrotubule agents named phenyl 4-(2-oxo-3- alkylimidazolidin-1-yl)benzenesulfonates (PAIB-SOs) that are highly active and selective to breast cancer cells. PAIB-SOs are the first antimicrotuble CYP1A1-dependent prodrugs and their mechanism of selectivity is based on the metabolization in human breast cancer cells of PAIB-SOs by CYP1A1 into potent antimitotics named phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates (PIB-SOs). The main objective of my research was to evaluate the period necessary to trigger an efficient antiproliferative activity on breast cancer cells and to study the metabolic stability and the kinetics of activation by CYP1A1 of 8 promising PAIB-SOs exhibiting high antiproliferative activity and selectivity toward breast cancer cells aiming to select 4 PAIB-SOs for animal studies. We first found that the antiproliferative activities of PAIB-SOs are concentration and timedependent. A contact time of the selected PAIB-SOs with breast cancer cells varying from 24 to 36 h is required to observe a significant antiproliferative activity. We also found that all PAIBSOs are rapidly metabolized in the presence of CYP1A1 into PIB-SOs. Finally, the hepatic stability experiments of PAIB-SOs using human, mouse or rat microsomes showed that the halflife and the intrinsic clearance depend on the structure of the PAIB-SO and the animal species studied. The stability of PAIB-SOs in mouse microsomes was weaker than in rat or human microsomes which were equivalent. Our screening program identified CEU-829, -938, -934 and - 913 as our most stable PAIB-SOs and generating the lowest quantity of PIB-SOs when incubated with human and rodent microsomes. In addition, they respectively exhibited half-lives of 22, 55, 31 and 41 minutes in human microsomes, 43, 52, 23 and 44 minutes in rat microsomes and 3.7, 20, 12 and 1.6 minutes in mouse microsomes. Altogether, our studies identified CEU-934 and - 938 as suitable candidates for further pharmacokinetic and pharmacodynamic evaluation in a mouse model and CEU-829 and -913 in a rat model.

Chan Ho Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 121-138). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.II / ABSTRACT --- p.III / 摘要 --- p.V / ABBREVIATIONS --- p.VI / "TABLE OF CONTENTS, " --- p.VII / LIST OF FIGURES AND TABLES --- p.XI / Chapter CHAPTER 1 --- GENERAL INTRODUCTION --- p.1 / Xenobiotic-metabolizing enzymes --- p.1 / Cytochrome P450 1 family --- p.4 / CYP1A1 --- p.5 / CYP1A2 --- p.5 / CYP1B1 --- p.5 / Transactivation of CYP1 enzymes by Aryl hydrocarbon receptor (AhR) --- p.8 / Implication of PAHs and CYP1 family in breast cancer --- p.10 / Potential role of phytochemicals on cancer prevention --- p.11 / Significance of this project --- p.13 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.14 / Chemicals --- p.14 / Maintenance of cells --- p.14 / Preparation of cell stock --- p.14 / Cell recovery from liquid nitrogen stock --- p.15 / Measurement of cell viability --- p.15 / Preparation of cell lysates (NP-40 cell lysis buffer) --- p.15 / XRE-luciferase gene reporter assay --- p.16 / Manipulation of DNA and RNA --- p.17 / Separation and purification of DNA from agarose gel --- p.17 / Separation of DNA from acrylamide gel --- p.17 / Restriction digestion --- p.18 / Ligation of DNA fragments --- p.18 / Transformation of DH5 a --- p.19 / Small scale plasmid purification from DH5a (mini prep) --- p.19 / Large scale plasmid isolation from DH5a (maxi-prep) --- p.20 / Construction of XRE activated luciferase reporter gene --- p.21 / Measurement of DMBA-DNA adduct formation --- p.21 / Semi-quantitative RT-PCR Assay --- p.22 / ENZYME ACTIVITIES --- p.23 / Isolation of microsomes --- p.23 / EROD activities in intact cells --- p.23 / EROD inhibition assay --- p.24 / Stattstical Analysis --- p.24 / Chapter CHAPTER 3 --- BAICALEIN INHIBITS DMBA-DNA ADDUCT FORMATION BY MODULATING CYP1A1 AND 1B1 ACTIVITIES --- p.26 / Introduction --- p.26 / Results --- p.28 / EROD activities in MCF-7 cells and inhibition assay --- p.28 / Baicalein suppressed DMBA-induced XRE-driven luciferase activities --- p.31 / Baicalein inhibited DMBA-induced CYP1A1 and CYP1B1 mRNA expression --- p.31 / The cytotoxic effect of DMBA was reduced by baicalein --- p.35 / Inhibition of DMBA-DNA adduct formation after baicalein treatment --- p.35 / Discussion --- p.39 / Chapter CHAPTER 4 --- INHIBITION OF DMBA-DNA ADDUCT FORMATION BY (-)-EPIGALLOCATECHIN GALLATE AND THEAFLAVINS --- p.41 / Introduction --- p.41 / Results --- p.45 / Persistence of DMBA-induced DNA adducts --- p.45 / Inhibition of theaflavins and EGCG on human recombinant CYP1A1 and CYP1B1 enzyme activities --- p.48 / EGCG suppressed DMBA-induced EROD activity while thealfavin had no significant effect on this --- p.48 / Kinetic analysis of EGCG on CYP1A1 and CYP1B1 activities --- p.53 / Modulation of DMBA-induced XRE-driven luciferase activities by theaflavins and EGCG --- p.56 / The influence of theaflavins and EGCG on CYP1A1 and CYP1B1 abundance --- p.56 / Discussion --- p.65 / Chapter CHAPTER 5 --- ISOFLAVONES PREVENT DMBA-INDUCED CARCINOGENESIS BY INHIBITING CYP1A1 AND CYP1B1 ACTIVITIES --- p.67 / Introduction --- p.67 / Results --- p.70 / Isoflavones inhibited DMBA-induced EROD activity in MCF-7 cells --- p.70 / Inhibition of MCF-7 microsomal EROD activities by isoflavones --- p.70 / Kinetic analysis of the inhibition of human recombinant CYP1 enzymes by isoflavones --- p.74 / XRE-driven Luciferase activities --- p.83 / Both biochanin A and genistein suppressed DMBA-induced CYP1 mRNA expression --- p.83 / Cytotoxicity of DMBA and isoflavones co-treatment --- p.88 / Isoflavones reduced the binding of activated DMBA to DNA --- p.89 / Discussion --- p.93 / Chapter CHAPTER 6 --- IN VITRO EFFECTS OF BAICALEIN AND THEAFLAVINS ON RAT HEPATIC P450 ACTIVITIES --- p.96 / Introduction --- p.96 / Results --- p.98 / Inhibition of EROD and MROD activities in rat liver microsomes by baicalein --- p.98 / Effects of theaflavins on EROD and MROD activities in rat liver microsomes --- p.102 / Kinetic studies for EROD and MROD activities of theaflavins --- p.104 / DISCUSSION --- p.114 / Chapter CHAPTER 7 --- CONCLUSION --- p.116 / APPENDIX 1 PRIMER LISTS --- p.118 / APPENDIX 2 REAGENTS --- p.119 / BIBLIOGRAPHY --- p.121

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Orientadores: Luis Otavio Zanatta Sarian, Maria Salete Costa Gurgel, Carmem Silvia Passos Lima / Tese (Doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T11:21:21Z (GMT). No. of bitstreams: 1 CardosoFilho_Cassio_D.pdf: 1685855 bytes, checksum: 341644d7cc011e51bf31d1f4881e8878 (MD5) Previous issue date: 2012 / Resumo: Introdução: A terapêutica sistêmica para o câncer de mama envolve o uso do agente antiestrogênico tamoxifeno, fármaco metabolizado pelo fígado no sistema do citocromo P-450 (CYP). Este, por sua vez, é parcialmente codificado pelo gene CYP1A1, e alguns polimorfismos deste gene têm sido associados com interferências na sua eficácia metabólica. Além disso, diferenças interindividuais no CYP explicam parte das variações na resistência ao tamoxifeno e metabolismo dos estrogênios. Dentre esses polimorfismos, o A4889G (M2) e o T6235C (M1) são conhecidos por afetar a ativação da estrona e do estradiol, e por provocar a redução da concentração de metabólitos altamente ativos do tamoxifeno, reduzindo teoricamente o efeito antiestrogênico desta modalidade de hormonioterapia no tecido mamário. Embora plausíveis do ponto de vista biológico, as implicações clínicas dos polimorfismos do CYP1A1, ou seja, as características patológicas dos tumores e um pior prognóstico decorrente do aumento dos estrógenos circulantes e redução dos metabólitos ativos do tamoxifeno, não foram ainda avaliadas. Objetivo: Avaliar a associação entre os polimorfismos M1 e M2 do gene CYP1A1 e as características patológicas e clínicas de mulheres com câncer de mama esporádico, em duas abordagens: 1) determinar as associações entre estes polimorfismos e as características patológicas, clínicas e o padrão de sobrevida global em mulheres com câncer de mama esporádico e 2) determinar as associações entre estespolimorfismos e as caracteríasticas patológicas e o comportamento clínico de tumores de mama com receptores hormonais positivos na vigência do uso de tamoxifeno. Métodos: foram incluídas 741 mulheres com câncer de mama esporádico, 405 das quais com tumores positivos para receptores esteroides e que usaram tamoxifeno como terapia antiestrogênica primária, para as quais os dados referentes a cinco anos de seguimento estavam disponíveis. Foram avaliadas as associações de informações-chave patológicas e clínicas, incluindo a sobrevida geral em cinco anos, com as diferentes combinações de polimorfismos do gene CYP1A1. Resultados: Em mulheres portadoras de ambos os polimorfismos M1 e M2 do CYP1A1, a proporção de tumores grau histológico III (80,3%) foi significativamente menor que nas não-portadoras (89,6%); p ajustado <0,01. O mesmo ocorreu na análise restrita às mulheres com tumores RE+ usando tamoxifeno (76,1% vs. 85,9%; p ajustado= 0,02). Após 60 meses de seguimento, cerca de 75% das mulheres estavam vivas. Não houve diferença significativa na sobrevivência relacionada com o estado do gene CYP1A1. Conclusões: embora associados a tumores de menor grau histológico, não há nenhuma evidência da associação dos polimorfismos do CYP1A1 com prognóstico do câncer da mama / Abstract: Introduction: systemic therapy for breast cancer involves the use of the anti-estrogen agent tamoxifen, which is metabolized by the liver cytochrome P-450 (CYP). This, in turn, is partially encoded by CYP1A1, and some polymorphisms of this gene have been associated with metabolic disturbance at their effectiveness. Moreover, interindividual differences in efficiency of CYP explain part of the variations in resistance to tamoxifen and estrogen metabolism. Among these polymorphisms, the A4889G (M2) and T6235C (M1) are known to affect the activation of estrone and estradiol, and cause the reduction of the concentration of highly active metabolites of tamoxifen, theoretically reducing the anti-estrogenic effect of this form of endocrine therapy in breast tissue. Although plausible from the biological point of view, the clinical implications of polymorphisms of CYP1A1, ie, the pathologic features of tumors and a worse prognosis due to increased circulating estrogens and reduction of active metabolites of tamoxifen have not yet been evaluated. Objectives: To evaluate the association between CYP1A1 A4889G and T6235C gene polymorphisms and clinical and pathological characteristics of women with sporadic breast cancer in two approaches: 1) determine the associations between CYP1A1 A4889G and T6235C gene polymorphisms and the pathological characteristics of the tumors, and the clinical features, including overall survival, of women with sporadic breast cancer and 2) determine the associations between CYP1A1 A4889G and T6235C gene polymorphisms and the pathological characteristics and clinical behavior of estrogen receptor-positive breast tumors in patients using tamoxifen. Methods: We included 741 women with sporadic breast cancer, 405 of whom had tumors positive for steroid receptors and using tamoxifen as primary antiestrogen therapy, for which data on five years of follow-up were available. We evaluated the associations of key pathological and clinical features, including overall survival at five years, with different combinations of the CYP1A1 gene polymorphisms. Results: In women with both polymorphisms of the CYP1A1 gene, the proportion of grade III tumors (80.3%) was significantly lower than in non-carriers (89.6%), adjusted p <0.01. The same was true for women with ER + tumors using tamoxifen (76.1% vs. 85.9%; adjusted p = 0.02). After 60 months of follow up, 75% of the women were alive. There was no significant difference in survival related to the state of the CYP1A1 gene. Conclusions: Although associated with tumors of lower grade, there is no evidence of an association of CYP1A1 polymorphisms with breast cancer prognosis / Doutorado / Oncologia Ginecológica e Mamária / Doutor em Ciências Médicas

Thesis (Ph. D.)--West Virginia University, 2008. / Title from document title page. Document formatted into pages; contains vii, 143 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--West Virginia University, 2003. / Title from document title page. Document formatted into pages; contains x, 123 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 113-123).

The identification of an agent that is selectively activated by a cytochrome P450 (CYP) has the potential for tissue specific dose intensification as a means of significantly improving its therapeutic value. Towards this goal, we disclose evidence for the pathway of activation of a duocarmycin analogue, ICT2700, which targets CYP1A1 for biological activity.

Made available in DSpace on 2016-01-26T12:51:47Z (GMT). No. of bitstreams: 1 glauciamariamfernandes_dissert.pdf: 1922219 bytes, checksum: f00f507bdc9bcdc1daa422a7965c5c41 (MD5) Previous issue date: 2013-12-12 / Fundação de Amparo a Pesquisa do Estado de São Paulo / Introduction: The xenobiotics are exogenous substances to the organism, as N-nitrosamines, heterocyclic amines (HAs) and polycyclic aromatic hydrocarbons (PAHs), can which result in DNA adducts formation. Polymorphisms in genes involved in the metabolism of xenobiotics could contribute to this process and modulate the development of cancer. Objectives: To investigate the CYP1A1*2A (rs4646903), CYP1A1*2C (rs1048943), CYP2E1*5B (rs2031920), CYP1E1*6 (rs6413432), Tyr113His EPHX1 (rs1051740) and His139Arg EPHX1 (rs2234922) polymorphisms related to the metabolism of xenobiotics, the risk of sporadic colorectal (SCRC) cancer, the interaction of these polymorphisms with lifestyle (smoking and drinking) and clinical and histopathological parameters and to evaluate the association of SCRC with socio-demographic factors. Methods: A case-control study was conducted in 641 subjects in the Brazilian population (241 patients with colorectal cancer and 400 controls (individuals without a history of cancer). Real-Time PCR and PCR-RFLP was performed for genotyping. Statistical analysis was performed using the chi-square tet and multiple logistic regression binary. Results: The results showed statistically significant differences between the case and control groups for age greater than 50 years (OR=8.21, 95%CI=5.49-12.28, p<0.01) and male gender (OR=0.50, 95%CI=0.32-0.87, p<0.01) The analysis of polymorphisms revealed an association between the alleles polymorphic CYP2E1*5B (OR=2.84, 95%CI=1.78-4.52, p<0.01, additive model) and CYP2E1*6 (OR=2.78, 95%CI=1.91-4.06, p<0.01, additive model) and the SCRC. Tumor size, lymph node involvement and disease primary site were not associated with polymorphisms. Conclusion: The CYP2E1*5B and CYP2E1*6 polymorphisms are involved in the risk of SCRC and individuals with age &#8805; 50 years are more susceptible to this tumor type, of males are less susceptible. / Introducão: Os xenobióticos são substâncias exógenas ao organismo, tais como as N-nitrosaminas, aminas heterocíclicas (HAs) e hidrocarbonetos policíclicos aromáticos (HPAs), que podem formar adutos de DNA. Polimorfismos em genes envolvidos no metabolismo dos xenobióticos podem contribuir com este processo e, consequentemente, modular o desenvolvimento de câncer. Objetivos: Investigar os polimorfismos CYP1A1*2A (rs 4646903), CYP1A1*2C (rs1048943), CYP2E1*5B (rs 2031920), CYP1E1*6 (rs 6413432), EPHX1 Tyr113His (rs1051740) e EPHX1 His139Arg (rs2234922), relacionados com o metabolismo dos xenobióticos, no risco de câncer de colorretal esporádico (CCRE), a interação desses polimorfismos com os hábitos de vida (tabagismo e etilismo) e parâmetros clínico-histopatológicos e avaliar a associação do CCRE com os fatores sócio-demográficos. Os Métodos: Um estudo caso-controle foi realizado em 641 indivíduos da população brasileira (241 pacientes com câncer de coloretal e 400 controles (indivíduos sem histórico de câncer). As técnicas de PCR em Tempo Real e PCR-RFLP foram realizadas para a genotipagem dos polimorfismos. A análise estatística utilizou os testes de Qui-Quadrado e Regressão Logística Múltipla Binária. Resultados: Os resultados mostraram diferenças estatisticamente significantes entre os grupos caso e controle para idade superior a 50 anos (OR=8,21; IC95%=5,49-12,28, p<0,01) e gênero masculino (OR=0,50; IC95%=0,32-0,87, p<0,01). A análise dos polimorfismos revelou associação entre os alelos polimórficos CYP2E1*5B (OR=2,84; IC95%=1,78-4,52; p<0,01, modelo aditivo) e CYP2E1*6 (OR=2,78; IC95%=1,91-4,06, p<0,01, modelo aditivo) e o CCRE. O tamanho do tumor, envolvimento de linfonodos e sítio primário da doença não foram associados com os polimorfismos. Conclusão: Os polimorfismos CYP2E1*5B e CYP2E1*6 estão envolvidos no risco de CCRE e indivíduos com idade superior ou igual a 50 anos são mais suscetíveis a este tipo tumoral, enquanto aqueles do gênero masculino são menos suscetíveis.

No / We identify cytochrome P450 1A1 (CYP1A1) as a target for tumor-selective drug development in bladder cancer and describe the characterization of ICT2700, designed to be metabolized from a prodrug to a potent cytotoxin selectively by CYP1A1. Elevated CYP1A1 expression was shown in human bladder cancer relative to normal human tissues. RT112 bladder cancer cells, endogenously expressing CYP1A1, were selectively chemosensitive to ICT2700, whereas EJ138 bladder cells that do not express CYP1A1 were significantly less responsive. Introduction of CYP1A1 into EJ138 cells resulted in 75-fold increased chemosensitivity to ICT2700 relative to wild-type EJ138. Negligible chemosensitivity was observed in ICT2700 in EJ138 cells expressing CYP1A2 or with exposure of EJ138 cells to CYP1B1- or CYP3A4-generated metabolites of ICT2700. Chemosensitivity to ICT2700 was also negated in EJ138-CYP1A1 cells by the CYP1 inhibitor alpha-naphthoflavone. Furthermore, ICT2700 did not induce expression of the AhR-regulated CYP1 family, indicating that constitutive CYP1A1 expression is sufficient for activation of ICT2700. Consistent with the selective activity by CYP1A1 was a time and concentration-dependent increase in gamma-H2AX protein expression, indicative of DNA damage, associated with the activation of ICT2700 in RT112 but not EJ138 cells. In mice-bearing CYP1A1-positive and negative isogenic tumors, ICT2700 administration resulted in an antitumor response only in the CYP1A1-expressing tumor model. This antitumor response was associated with detection of the CYP1A1-activated metabolite in tumors but not in the liver. Our findings support the further development of ICT2700 as a tumor-selective treatment for human bladder cancers.