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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.

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

Die Umwandlung in wasserlösliche Verbindungen, die renal ausgeschieden werden können, ist ein grundlegendes Prinzip im Abbau von Fremdstoffen. Hierbei unterscheidet man Phase-I- und Phase-II-Reaktionen. Die Aktivität vieler Phase-I- und Phase-II-Enzyme ist genetisch beeinflusst und kann starke interindividuelle Unterschiede im Metabolismus von Fremdstoffen verursachen und dadurch das Krebsrisiko und das Risiko für Arzneimittelnebenwirkungen beeinflussen. Die Häufigkeitsverteilungen der Allele der Gene, die Phase-I- und Phase-II-Enzyme kodieren, zeigen eine große interethnische Varianz. Die Polymorphismen dieser Enzyme wurden bisher jedoch noch nicht in der größten slawischen Volksgruppe, der russischen, untersucht. An der vorliegenden Studie nahm eine Gruppe von 325 Personen russischer Abstammung teil - gesunde Probanden bzw. Patienten, die nicht an einer malignen Erkrankung litten. Die Polymorphismen von zwei Enzymen der Phase I, CYP1A1 und CYP2D6, und von einem Enzym der Phase II, NAT2, wurden mittels PCR-RFLP-Genotypisierung und Real-time-PCR-Verfahren komplett untersucht. Die Häufigkeit der CYP1A1 Allele mit hoher Aktivität, CYP1A1*2A und CYP1A1*2B, betrug 4,6% (3,1%-6,5%) bzw. 5,1% (3,5%-7,1%). Die Häufigkeiten der genetischen Varianten von CYP1A1 waren: m1 (3801T>C) - 9,8% (95% Vertrauensbereich, 7,7%-12,4%), m2 (2455A>G) - 5,0% (95% VB, 3,5%-7,1%), m4 (2453C>A) - 2,5% (1,4%-4,0%), m5 (-4335G>A) - 25,8% (22,5%-29,4%), m6 (-3219C>T) - 6,0% (4,3%-8,1%), und m7 (-3229G>A) - 2,9% (1,8%-4,5%). Die Mutation m3, die bisher nur bei Afrikaner gefunden wurde, konnten wir nicht nachweisen. 5,9% (3,5%-9,2%) aller Probanden waren CYP2D6 Langsam-Metabolisierer und 3,4% (1,7%-6,3%) wurden als Ultraschnell-Metabolisierer identifiziert (CYP2D6*1x1/*1). Bei der Genotypisierung von acht verschiedenen Punktmutationen im NAT2-Gen ergab sich für 59,7% (54,1%-65,1%) der Studienteilnehmer ein Genotyp, der mit einer Langsam-Acetylierer-Status einhergeht. 34,7% (29,6%-40,2%) der Probanden hatten ein und 5,6% (3,3%-8,6%) zwei für die Schnellacetylierung kodierende Allele. Die Allelverteilung der für die wichtigsten Enzyme im Arzneimittelstoffwechsel kodierenden Gene ist bei Russen ähnlich wie bei anderen Kaukasiern. Es kann deshalb erwartet werden, dass die genetisch-bedingten Unterschiede in der Wirksamkeit und im Auftreten von Arzneimittelnebenwirkungen in der russischen Bevölkerung vergleichbar sind mit denen in anderen europäischen Populationen. / The basic principle of drug and xenobiotic metabolism in the body is to make them more water soluble and thus more readily excreted in the urine. Genetic polymorphisms of phases I and II xenobiotic transformation reactions are known to contribute considerably to interindividual variations in the metabolism of numerous drugs and xenobiotics and to associate with altered risk of adverse drug reactions and some cancers. The frequency of functionally important mutations and alleles of genes coding for xenobiotic metabolizing enzymes shows a wide ethnic variation. However, little is known of the frequency distribution of the major allelic variants in the Russian population. In this study we investigated 325 individuals of Russian origin, who were healthy volunteers or patients without malignant diseases. Our study included the complete investigation of two enzymes of phase I, CYP1A1 and CYP2D6, and one phase II enzyme, NAT2, using PCR-RFLP genotyping and LightCycler method. The frequencies of the CYP1A1 high-activity alleles, CYP1A1*2A and CYP1A1*2B, were 4.6% (3.1%-6.5%) and 5.1% (3.5%-7.1%), respectively. The mutations m1 (3801T>C), m2 (2455A>G), m4 (2453C>A), m5 (-4335G>A), m6 (-3219C>T), and m7 (-3229G>A) of CYP1A1 occurred in 9.8% (95% confidence interval, 7.7%-12.4%), 5.0% (95% C. I., 3.5%-7.1%), 2.5% (1.4%-4.0%), 25.8% (22.5%-29.4%), 6.0% (4.3%-8.1%), and 2.9% (1.8%-4.5%) of alleles, respectively. We did not find the m3 mutation, which has only been detected in Africans up to now. 5.9% (3.5%-9.2%) of all subjects were CYP2D6 poor metabolizers, whereas 3.4% (1.7%-6.3%) were identified as ultra-rapid metabolizers (CYP2D6*1x1/*1). Genotyping eight different single nucleotide polymorphisms in the NAT2 gene provided a genotype associated with slow acetylation in 59.7% (54.1%-65.1%) of individuals, 34.7% (29.6%-40.2%) of participants carried at least one allele encoding rapid acetylation, and 5.6% (3.3%-8.6%) were homozygous for the rapid-acetylation allele (wild-type allele *4 or mutant allele *12A). The overview of allele distribution of the important drug and xenobiotic metabolizing enzymes among Russians shows that the allele frequency is similar to that of other Caucasians. Therefore it may be expected that drug side effects and efficacy problems due to an individual's genetic background are similar compared to those in other European populations.

Made available in DSpace on 2016-08-10T10:38:42Z (GMT). No. of bitstreams: 1 KLEBER SANTIAGO FREITAS E SILVA.pdf: 1080005 bytes, checksum: 9dfcb821b9cd1574718e2442cabc533b (MD5) Previous issue date: 2013-02-18 / In healthy women, a great number of intra and extracellular controls prevent the attachment and proliferation of ectopic endometrial cells. In endometriosis, abnormalities in those controls can lead to the survival of endometrial cells and consequently their attachment to the peritoneal cavity and disease progression. Endometrial cells with genetic polymorphisms respond to local signals, and they proliferate instead of undergoing apoptosis. The products of these abnormal cells stimulate the invasion of tissues and induce an inflammatory response. The disease has a complex trait and it is related to several factors such as genetic, immunological and environmental. This study examined six polymorphisms presented by six different genes (p53; Estrogen Receptor &#946;; Progesterone receptor; GSTM1; GSTT1; CYP1A1). We obtained the polymorphic genotype frequencies from the same 50 patients for all genes and we analyzed them using the Fisher's Exact Test or G Test. First we analyzed the genes in a group of two and subsequently in a group of three. We found significant association between polymorphisms in six pairs of genes (p53-Er&#946; with frequency 5.9 times higher in the experimental group; p53-GSTM1, 2.39 times higher; p53-CYP1A1 with 65.5% of the patients with the polymorphism; ER&#946;-PROGINS 3.0 times higher in the experimental group; GSTM1-PROGINS and GSTT1-CYP1A1 both with 31.25% of the patients with the polymorphism). Positive results were found in 15 situations when genes were analyzed in a group of three; the most significant result corresponding to the polymorphisms of the genes p53, Er&#946; e GSTM1 with 20% of the patients carrying these polymorphisms; PROGINS, Er&#946; e GSTM1 with 18% and p53, Er&#946; e PROGINS with 12%. The results support the idea that the presence of polymorphisms in more than one endometriosis-related gene can lead to the onset of the disease and its progression. Studies should aim at these genes in order to understand the relationship among them more clearly and the possibility of developing new diagnostic techniques based on molecular markers of these genes. / Controles intra e extracelulares impedem a implantação e a proliferação de células endometriais ectópicas nas mulheres saudáveis. Anormalidades em quaisquer desses controles levam à sobrevida dessas células, implantação e a consequente progressão da Endometriose. Células endometriais com polimorfismos genéticos respondem a sinais locais e se proliferam não sofrendo apoptose. Os produtos dessas células anormais estimulam a invasão de tecidos e induzem respostas inflamatórias. A doença é complexa e relacionada a fatores como o genético, o imunológico e o ambiental. Este trabalho analisou seis polimorfismos de seis diferentes genes (p53; Receptor &#946; de estrógeno; Receptor de progesterona; GSTM1; GSTT1; CYP1A1). As frequências dos genótipos polimórficos foram obtidas das mesmas 50 pacientes para todos os genes e analisadas pelo Teste Exato de Fisher ou Teste G. Os genes foram analisados dois a dois e posteriormente três a três. Resultados significativos foram encontrados para seis pares de genes (p53- RE&#946; com frequência de polimorfismo 5,9 vezes maior no grupo endometriose; p53-GSTM1 com frequência 2,39 vezes maior; p53-CYP1A1 com 65,5% das pacientes com endometriose apresentando os polimorfismos; RE&#946;-PROGINS com frequência 3,0 vezes maior; GSTM1-PROGINS e GSTT1-CYP1A1, ambos com 31,25% das pacientes do grupo endometriose apresentando os polimorfismos). Em 15 situações quando os genes foram analisados três a três o p foi menor que 0,05. Os polimorfismos de maior frequência foram dos genes p53, RE&#946; e GSTM1 com 20% das pacientes com endometriose apresentando esses polimorfismos; PROGINS, RE&#946; e GSTM1 com 18% e p53, RE&#946; e PROGINS com 12%. Esses resultados corroboram a ideia de que a presença de polimorfismos em mais de um gene relacionado à endometriose pode levar ao aparecimento e deselvolvimento da doença. Estudos devem ser direcionados a esses genes na tentativa de compreender mellhor a relação entre eles e o possível desenvolvimento de novas técnicas de diagnóstico baseada nos marcadores moleculares desses mesmos genes.

Die vorliegende Studie präsentiert eine Analyse der Integration der AhR und Wnt/beta-catenin Signalwege in den CYP1A1 Promotor sowie den regulatorischen Einfluss der Promotorlogik auf die Genexpression. Experimentell wurde diese Analyse mithilfe 29 mutagener Reporterkonstrukte des humanen CYP1A1 Promotors durchgeführt. Ein mathematisches Modell, welches eine Repräsentation des Crosstalks der Signaltransduktionswege mit einer statistisch mechanischen Beschreibung der kombinatorischen Promotorbelegung kombiniert, komplementierte den experimentellen Ansatz. Unter zusätzlicher Zuhilfenahme von gut kontrollierbaren synthetischen Promotorkonstrukten fand ich heraus, dass nur jenes Dioxin-responsive Element das sich am nächsten am Transkriptionsstartpunkt befindet, die Promotorbelegung an die RNA Polymerase kommuniziert. Außerdem beobachtete ich, dass Transkriptionsfaktoren alleine mit Transkriptionsfaktoren interagieren die mit benachbarten Bindestellen assoziieren, d.h. Interaktionen überbrücken keine größeren Entfernungen. Der Modellierungsansatz ermöglichte zudem die erfolgreiche Vorhersage einer UND-Gatter-ähnlichen Integration der beiden Signalwege in den Promotor. Für die genomische Architektur des CYP1A1 Promotors konnte ich die Signifikanz der Zielbindestelle des Wnt/beta-catenin Signalwegs innerhalb des cis-regulatorischen Region demonstrieren. Mithilfe des Modells fand ich heraus, dass diese Bindestelle am stärksten und vielfältigsten mit den restlichen Transkriptionsfaktoren interagiert. Zusätzliche konnte, im Vergleich zu dem alles-oder-nichts UND-Gatter der synthetischen Konstrukte, eine sehr viel graduellere Antwort auf die Integration der beiden Signalwege aufgezeigt werden. Abschließend wurde das physiologisch zu beobachtende Expressionsmuster von dem Modell vorhergesagt und experimentell validiert. / The study at hand presents an analysis of the integration of the AhR and the Wnt/beta-catenin signaling pathways into the CYP1A1 promoter as well as the regulatory influence of the promoter logic on gene expression. Experimentally, this analysis was conducted with the help of 29 mutant constructs of the human CYP1A1 promoter. I complemented this experimental approach with a set of mathematical models that combined a representation of the signaling crosstalk with a statistical mechanics description of the combinatorial promoter occupancy. With the help of well controllable synthetic promoter constructs I found that only the dioxin responsive element closest to the transcription start site communicates the promoter occupancy to the RNA polymerase. Furthermore, transcription factors only interact with transcription factors that associate with nearby binding sites, i.e., no long-distance binding was observed. The modeling approach subsequently enabled the successful prediction of an AND-gate-like integration of the two signaling pathways into the promoter. For the genomic architecture of the CYP1A1 promoter, I could demonstrate the importance of the Wnt/beta-catenin pathway target binding site within the cis-regulatory region. The model uncovered that this binding site is the strongest and most promiscuous interaction partner of the remaining transcription factors. In addition, a less switch-like response to the integration of the two signaling pathways as compared to the all-or-none AND-gate within the synthetic constructs could be demonstrated. And lastly, the physiological expression pattern in liver lobules could be successfully predicted by the model and experimentally verified.

5 Abstract Cytochrome P450 1A1 (CYP1A1) is one of the major isoforms of the cytochrome P450 superfamily. It is primarily an extrahepatic enzyme which is responsible for oxidation of many polycyclic aromatic hydrocarbons and other xenobiotics. Besides of the role in detoxification metabolism CYP1A1 is the one most important isoform involved in activation of procarcinogens. The main aim of this project was preparation of two modifications of the rat CYP1A1 gene with codon optimization for expression in E. coli by gene synthesis. One was wild type (wt1A1) and the other was with modified N-terminal anchor (mod1A1) - for both modifications with or without His Tag at the C-end of CYP1A1. Furthermore, an aim was to compare their level of expression in different strains of E. coli and try to purify and assess enzymatic activity of the gene's products. From pre-prepared oligonucleotides 2 "syntons" (parts of gene) were synthetized and separately inserted into pUC19. After verified sequence of the "syntons" they were cleaved from pUC19 and inserted together into pET-22b. These vectors were prepared for transformation of 3 strains of E. coli (BL-21 (DE3) GOLD, RIL a RIPL). For production of proteins many conditions were tested: temperature (18, 22, 24, 27 a 37 řC), time of production (untill 48 hours), concentration...

Most members of the PAS (PER-ARNT-SIM) protein family are transcription factors, mediating development and adaptive responses to the environment, such as circadian rhythms and toxin responses. Because the PAS domain mediates protein-protein interactions and functional cross-talk between distinct biological processes, we hypothesized that PAS genes in the circadian clockworks, namely Per1 and Per2, may be involved in development and toxin responses, which are modulated by other PAS members. To explore the possible role of clock genes in development, we examined mammary epithelial cells in vitro and the mouse mammary gland in vivo for evidences of changes in clock gene expression during different stages of development and differentiation. Our results showed that Per1 and Bmal1 expression were up-regulated in differentiated HC-11 cells, whereas Per2 mRNA levels were higher in undifferentiated cells. A similar differentiation-dependent profile of clock gene expression was observed in mouse mammary glands; Per1 and Bmal1 mRNA levels were elevated in late pregnant and lactating mammary tissues, whereas Per2 expression was higher in proliferating virgin and early pregnant glands. These data suggest that circadian clock genes may play a role in mouse mammary gland development. To examine clock gene function in toxin responses, we evaluated whether disruption or inhibition of Per1 and/or Per2 alters toxin-induced activity of the AhR signaling pathway in the mouse mammary gland and liver. We assessed the activation of the AhR signaling pathway in response to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a prototypical AhR agonist, by analyzing the mRNA abundance of its two target genes, cytochrome P450, subfamily I, polypeptide 1 (Cyp1A1) and Cyp1B1. Our results showed that the targeted disruption of Per1, but not Per2, significantly increases the TCDD-induced p450 expression in the mammary gland and liver in vivo. Similar changes in TCDD-mediated p450 expression were observed in vitro using mammary primary cultures of mammary cells derived from from Per1ldc, Per2ldc and Per1ldc/Per2ldc mutant mice and Hepa1c1c7 cells subjected to siRNA-mediated inhibition of Per1 or Per2. These discoveries suggest that the clock gene Per1 may modulate toxin responses perhaps by functioning as a negative regulator for TCDD-mediated activation of the AhR signaling pathway.

Salvia genus is a widely cultivated genus and used in medicine for various purposes as having antimicrobial, antioxidant, anticarcinogen and anti-inflammatory features. In this study the aim was to investigate phenolic composition of Salvia absconditiflora and understand the possible effects of those constituents in cancer related drug metabolizing enzymes. Salvia absconditiflora showed 80,43 % Radical Scavenging Activity against DPPH radical. Total flavonoid content was found as one third of total phenolic content. Presence of important phenolic acids and flavonoids such as caffeic acid, luteolin, coumaric acid are validated with LC-MS/MS analysis. Cytotoxicity of Salvia absconditiflora treatment on MCF-7 and MDA-MB-231 breast cancer cell lines were investigated through XTT and TBE assays both dose and time dependent manner. Cell proliferation was inhibited 50 % by different IC50 values calculated in different assays and different time intervals. This suggests that two breast cancer cell lines response in a different way to cytotoxic treatments. Cancer related drug metabolizing enzyme gene modulations were investigated with qRT-PCR. CYP1A1 and CYP1B1 were upregulated in MCF-7 but down-regulated in MDA-MB-231 cells in response to Salvia absconditiflora treatment.

Salvia genus is a widely cultivated genus and used in medicine for various purposes as having antimicrobial, antioxidant, anticarcinogen and anti-inflammatory features. In this study the aim was to investigate phenolic composition of Salvia absconditiflora and understand the possible effects of those constituents in cancer related drug metabolizing enzymes. Salvia absconditiflora showed 80,43 % Radical Scavenging Activity against DPPH radical. Total flavonoid content was found as one third of total phenolic content. Presence of important phenolic acids and flavonoids such as caffeic acid, luteolin, coumaric acid are validated with LC-MS/MS analysis. Cytotoxicity of Salvia absconditiflora treatment on MCF-7 and MDA-MB-231 breast cancer cell lines were investigated through XTT and TBE assays both dose and time dependent manner. Cell proliferation was inhibited 50 % by different IC50 values calculated in different assays and different time intervals. This suggests that two breast cancer cell lines response in a different way to cytotoxic treatments. Cancer related drug metabolizing enzyme gene modulations were investigated with qRT-PCR. CYP1A1 and CYP1B1 were up-regulated in MCF-7 but down-regulated in MDA-MB-231 cells in response to Salvia absconditiflora treatment.