O câncer é a segunda maior causa de morte no Brasil, atrás apenas de doenças cardíacas. Por isto, é evidente que grandes recursos sejam direcionados para a pesquisa no descobrimento de novas opções com a finalidade de erradicar esta doença. Dentre estas opções, a quimioprevenção do câncer tem chamado a atenção já que, mesmo com os imensos avanços no conhecimento sobre os mecanismos da carcinogênese e conseqüente desenvolvimento de novas drogas, os dados estatísticos de mortalidade não se tornaram menores. Somando-se a estes fatos, deve ser considerado que no Brasil o tratamento padrão do câncer não chega a todas as pessoas por ser extremamente caro. Desta forma, a quimioprevenção do câncer com fatores presentes na dieta ou oriundos de fontes consideradas baratas como fitoterápicos, deve ser apreciada. Assim, este trabalho teve como objetivo avaliar os efeitos quimiopreventivos e antineoplásicos de uma planta brasileira, o guaraná (Paullinia cupana Mart var. sorbilis). Foram utilizados alguns experimentos em camundongos como indução genotóxica em fígado pela Dietilnitrosamina (DEN); carcinogênese pulmonar induzida pela 4-(metilnitrosamino)-1-(3-piridil)-1-butanona (NNK), uma nitrosamina presente no tabaco; tumor ascítico de Ehrlich; disseminação hematógena de melanoma B16/f10; e cultivo de células tumorais e não tumorais. Além disso, caracterizou-se o papel da Conexin43 na carcinogênese pulmonar induzida pelo NNK e os efeitos do guaraná sobre o receptor CAR e sua ação quando da administração do ligante do CAR, 1,4-bis[2-(3,5-dichloropiridiloxi)]benzeno (TCPOBOP). Pudemos observar efeitos quimiopreventivos e antineoplásicos do guaraná dependendo do modelo utilizado, demonstrando que seu modo de ação principal é a redução da proliferação celular. Além disso, observamos que os tumores de pulmão dos animais tratados com a planta apresentavam menor tamanho, menor grau maligno, menor índice de proliferação celular e menor ativação do fator de transcrição CREB. Observamos também que a Conexina43 (Cx43) tem importante papel na carcinogênese pulmonar induzida pelo NNK, atuando como supressor tumoral e em fases tardias possivelmente tendo papel inverso, ou seja, como um oncogene. Caracterizamos os efeitos do guaraná sobre a ativação do receptor CAR e demonstramos que, por si só, o guaraná induz a expressão do CAR, além de alterar a expressão de alguns de seus transcritos como a CYP2B10 e CYP3A11. Ao analisarmos os efeitos de extratos de guaraná sobre células de tumor de pulmão (E9) in vitro, verificamos o mesmo efeito antiproliferativo, diminuindo a expressão do PCNA e da Conexina43 de maneira dose-dependente, além de verificar um aumento da expressão do receptor CAR. Ao fim propomos uma hipótese de mecanismo de ação baseando-se nas alterações encontradas oriundas da administração do guaraná. Concluímos que o guaraná apresenta componentes com ação antitumoral em camundongos, tendo efeito quimiopreventivo ou antineoplásico dependendo do modelo utilizado. / Cancer is the second biggest cause of deaths in Brazil, only behind of cardiac diseases. As a result, it is evident that great resources for research will be directed towards the discovery of new options to eradicate this disease. Among these options, cancer chemoprevention has calling for attention since the huge advances in the knowledge of carcinogenesis and development of new drugs did not decrease statistical data on mortality due to cancer. In addition, it must be considered that in Brazil, cancer therapy is not available for all given that it is too expensive. Therefore, cancer chemoprevention with dietary factors or from medicinal plants has got to be treasured. Following these lines, the aim of this work was to evaluate the chemopreventive and antineoplastic effects of a Brazilian plant, Paullinia cupana Mart var. sorbilis, most known as guarana. It was used several experiments in mice and cell culture essays as: protection against DEN-induced DNA damage; NNK-induced lung carcinogenesis; Ehrlich Ascitic Tumor; metastasis of B16/f10 melanoma cells; and cell culture of a tumorigenic and a non-tumorigenic cell lines. Additionally, it was characterized the role of Connexin43 in the NNK-induced lung carcinogenesis and the effects of guarana on the CAR receptor before and after the administration of TCPOBOP. We note a chemopreventive or antineoplastic effect of guarana depending on the model employed and showed that the mode of action responsible for these effects was reduced cell proliferation. Also, the lung tumors of guarana-treated animals were smaller, less aggressive, with decreased cell proliferation and CREB activation. On the other hand, we observed that Connexin43 have an important role on NNK-induced lung carcinogenesis because it may act as a tumor suppressor and in advanced stages as an oncogene. The effects of guarana on the CAR activation were characterized and we showed that guarana induces CAR mRNA expression, altering the levels of its transcripts as CYP2B10 and CYP3A11. We also examined the effects of guarana extracts on a lung tumor cell culture (E9 cells) and demonstrated the same antiproliferative effect observed previously, by decreased PCNA and Connexin43 proteins in a dose-dependent manner along with an increase in CAR protein. At last we hypothesized a mechanism of action for guarana effects basing in our findings. We concluded that guarana presents substances that have antitumoral effects in mice, enclosing a chemopreventive or antineoplastic effect depending on the model studied.

published_or_final_version / Anatomy / Master / Master of Philosophy

Thesis (Ph.D. in Toxicology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 156-176). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Ο καρκίνος της ουροδόχου κύστης αποτελεί την 4η κατά συχνότητα μορφή καρκίνου στους άνδρες στο Δυτικό κόσμο, ακολουθώντας τον καρκίνο του προστάτη, του πνεύμονα και του κόλου. Η επιφανειακή μορφή του καρκίνου της κύστεως έχει το ιδιαίτερο χαρακτηριστικό των πολύ συχνών υποτροπών, οι οποίες ευθύνονται για τη μεγάλη νοσηρότητα της νόσου. Οι πολύ συχνές υποτροπές έχουν, όπως εύκολα γίνεται αντιληπτό, τεράστιο κοινωνικοοικονομικό κόστος, εφ’ όσον ένα σημαντικό τμήμα των πασχόντων αποτελεί μέρος του οικονομικά ενεργού πληθυσμού. Η καρκινογένεση δεν είναι μια απλή διαδικασία, αλλά μια αλληλουχία αλλαγών οι οποίες αφορούν τους κυτταρικούς μηχανισμούς αύξησης, διαφοροποίησης και απόπτωσης και οδηγούν στη μετατροπή ενός φυσιολογικού κυττάρου σε νεοπλασματικό. Ως «Χημειοπρόληψη» ορίζεται η χρήση ειδικών φυσικών ή συνθετικών χημικών ουσιών που μπορούν να παρέμβουν σε κάποια από τα μοριακά αυτά γεγονότα και να προλάβουν, καταστείλουν ή αναστρέψουν, την εξέλιξη προκαρκινικών βλαβών σε διηθητικό καρκίνο. Η διαφορά στην επίπτωση του νεοπλάσματος και τα διαφορετικά κλινικοπαθολογοανατομικά χαρακτηριστικά του καρκίνου της ουροδόχου κύστεως ανάμεσα στα δύο φύλα, υποδεικνύουν ένα σημαντικό ρόλο των ορμονών του φύλου στην παθογένεια του νεοπλάσματος. Υποθέσεις μόνο γίνονται για τη σημασία των οιστρογόνων στην ανάπτυξη καρκίνου της ουροδόχου κύστεως, αφού ακόμα ο ρόλος τους δεν έχει αποσαφηνιστεί. Τα οιστρογόνα ασκούν τη δράση τους μέσω των υποδοχέων τους (ERα, ERβ) οι οποίοι αποτελούν μέλη μιας υπεροικογένειας μεταγραφικών παραγόντων, των πυρηνικών υποδοχέων. Οι πυρηνικοί υποδοχείς ρυθμίζουν τη γονιδιακή έκφραση μέσω θετικής ή αρνητικής παρεμβάσεως στη δράση άλλων μεταγραφικών παραγόντων, όπως του NFκB, με τη βοήθεια ενός μηχανισμού που ονομάζεται cross-talk. Αν και η φλεγμονή με την καρκινογένεση έχουν αναγνωρισμένη σχέση από παλιά, συνδέθηκαν άμεσα με την παρατήρηση ότι υπερέκφραση του γονιδίου για το ένζυμο κυκλοοξυγενάση-2 (COX-2), αποτελεί πρώιμο γεγονός της καρκινογένεσης (Greten et al, 2004). Η COX-2, όπως επίσης και ο μεταγραφικός παράγοντας NFκB, που σχετίζεται με τη φλεγμονή, έχει διαπιστωθεί ότι συμμετέχουν στις διαδικασίες της καρκινογένεσης.. Η σύνδεση των οιστρογόνων στους οιστρογόνικους υποδοχείς επάγει τη δέσμευση συν-ρυθμιστικών παραγόντων, οι οποίοι διακρίνονται σε δύο μεγάλες κατηγορίες τους συν-ενεργοποιητές (coactivators), όπως p300 και τους συν-καταστολείς (corepressors), όπως NCoR. Κατά την παρούσα εργασία μελετήθηκε η μεμονωμένη όσο και η συνδυαστική έκφραση των πέντε παραπάνω μορίων στο φυσιολογικό επιθήλιο και στα καρκινώματα διαφόρων Grade, σε ιστικά δείγματα από 140 ασθενείς που υποβλήθηκαν σε διαγνωστική βιοψία διουρηθρική εκτομή νεοπλάσματος κύστεως ή ριζική κυστεκομή. Η μέθοδος που εφαρμόστηκε ήταν η ανοϊστοχημεία σε τομές παραφίνης, η οποία λόγω του μορφολογικού της χαρακτήρα επέτρεψε τη λήψη δεδομένων για τη σχετική εντόπιση των μορίων στους ενδοκυττάριους χώρους, τις ενδοεπιθηλιακές στιβάδες, τις φυσιολογικές ή παθολογικές ιστολογικές βαθμίδες και το επιθηλιακό ή μεσεγχυματικό διαμέρισμα. Ο παράγοντας NFκB (υπομονάδα p65) εμφάνισε μεικτή υποκυττάρια εντόπιση. Στην παρούσα ανοσοϊστοχημική μελέτη το επίπεδο της έκφρασης του NFκB στον πυρήνα των καρκινικών κυττάρων παρουσίαζε μια στατιστικά σημαντική συνολική αύξηση στα τρία επίπεδα διαφοροποίησης των καρκινωμάτων. Τα καρκινώματα χαμηλής διαφοροποίησης παρουσίαζαν ισχυρότερη ανοσοθετικότητα του NFκB από τα μετρίας και καλής διαφοροποίησης. Η αύξηση της πυρηνικής εντόπισης του NFκB συνδυάζεται με ταυτόχρονη ελάττωση της κυτταροπλασματικής, γεγονός που επιβεβαιώνει τη βιολογική δράση του. Ο πυρηνικός υποδοχέας ERβ, που εντοπίζεται στο πυρήνα των καλώς διαφοροποιημένων καρκινικών κυττάρων, είναι στατιστικά σημαντικά αυξημένος σε σχέση με λιγότερο διαφοροποιημένα νεοπλασματικά κύτταρα. Στην παρούσα ανοσοϊστοχημική μελέτη τα κύτταρα του φυσιολογικού επιθηλίου της ουροδόχου κύστης, εκφράζουν έντονα τον πυρηνικό υποδοχέα και κατά την πρόοδο της καρκινογένεσης η έκφραση του ελαττώνεται, παράλληλα με την απώλεια της διαφοροποίησης των καρκινικών κυττάρων. Στην εξέλιξη της καρκινογένεσης, η COX-2 επάγεται σταθερά, σύμφωνα με τα αποτελέσματα της παρούσας εργασίας, με διαδοχικές αυξήσεις που συνοδεύουν όλα τα στάδια της προοδευτικής αποδιαφοροποίησης των κυττάρων. Η πυρηνική έκφραση του p300 αυξάνεται σταδιακά καθώς τα καρκινώματα αποκτούν χαρακτήρες αποδιαφοροποίησης, συσχέτιση στατιστικώς σημαντική. Η πυρηνική έκφραση του NCoR ελαττώνεται σταδιακά καθώς τα καρκινώματα αποκτούν χαρακτήρες αποδιαφοροποίησης, συσχέτιση στατιστικώς σημαντική, σύμφωνα με τα ευρήματα της παρούσας μελέτης. Στις υπόλοιπες συσχετίσεις μελετήθηκε η συν-έκφραση πλέον των παραγόντων σε κάθε ασθενή, με σκοπό την εξαγωγή συμπερασμάτων για ενδεχόμενη αλληλεπίδραση τους. Αναλυτικότερα, παρατηρήθηκε στα καρκινώματα της ουροδόχου κύστεως ισχυρή θετική συσχέτιση του NFκB με την έκφραση της COX-2, υποδηλώνοντας τον υποστηρικτικό ρόλο των δύο αυτών παραγόντων στην πρόοδο της καρκινογένεσης. Από τη συσχέτιση NFκB και ERβ προέκυψε κατασταλτική επίδραση του πρώτου στην ογκοανασταλτική δράση του δεύτερου, υποδηλώνοντας σχέση ανταγωνισμού στη δέσμευση συνπαραγόντων και κατάληψης ίδιων περιοχών στους υποκινητές γονιδίων, ενώ δεν αναδείχθηκε συνομιλία ανάμεσα στον ERβ και COX-2. Τέλος αποκαλύφθηκε συνεργική δράση του NFκB με τον p300 στην καρκινογένεση, με τον ERβ και NCoR να χάνουν την ικανότητα πρόκλησης κυτταρικής διαφοροποίησης και άρα την προστατευτική επίδρασή τους. Η έκφραση του ERβ συσχετίστηκε με την ιστοπαθολογική βαρύτητα, ανά βαθμό έκφρασης των συνρυθμιστών p300 και NCoR, ώστε να διευκρινιστεί εάν η συγκέντρωση τους στα κύτταρα είναι καθοριστικός παράγοντας για την επίδραση του πυρηνικού υποδοχέα στον ιστολογικό φαινότυπο. Η επεξεργασία των δεδομένων φανερώνει αρνητική συσχέτιση της προόδου της καρκινογένεσης με την πυρηνική έκφραση του ERβ, αλλά μόνο όταν τα κύτταρα υπερεκφράζουν παράλληλα τον p300. Η απώλεια της έκφρασης του NCoR αναστέλλει την ενεργοποίηση του πυρηνικού υποδοχέα ERβ, γεγονός που διαπιστώθηκε και στην παρούσα μελέτη κατά τη διάρκεια της απώλεια διαφοροποίησης των καρκινικών κυττάρων της ουροδόχου κύστης. Επομένως, το τελικό αποτέλεσμα της δράσης του ERβ εξαρτάται από τον ανταγωνισμό των ενεργοποιητών και καταστολέων για τις ίδιες θέσεις σύνδεσης στο μόριο του ERβ. Συνολικά, η χρήση αγωνιστών των ERβ και NCoR με παράλληλη αναστολή των NFκB, COX2 και NCoR, θα είχε πιθανότατα ευνοϊκό αποτέλεσμα στην αναστροφή της καρκινογένεσης στην ουροδόχο κύστη. Ειδικές παράμετροι του χημειοπροληπτικού σχήματος, θα ήταν ωφέλιμο να τροποποιούνται ύστερα από εξατομικευμένη αξιολόγηση του δικτύου των πέντε παραγόντων. / Backround Bladder cancer is the forth most common malignancy among men in the Western World, following prostate, lung, and colon cancer. However, due to the highly recurrent nature of the disease, bladder cancer is the most prevalent and the most expensive per patient treated. Carcinogenesis is a complicated multistage process that gradually deprives normal cells of their natural phenotype, resulting in tissue disturbance, from which tumors finally emerge. During its lengthy course it is accompanied by an evenly prolonged inflammatory response. Chemoprevention pursues the arrest of both processes, by means of pharmacological targetting key molecules, involved in cell growth, differentiation and apoptosis, as well as in chronic inflammation. Nuclear Hormone Receptors are appropriate targets, as they are induced by ligand binding to mediate gene transcription. Epidemiological and molecular data support the possible role of ERβ and NFκB between the two collateral processes, providing evidence for target-specific chemopreventive strategies. ERβ promotes cellular differentiation and restriction of inflammation. Nuclear receptor coregulators provide a great level of sophistication in the dynamic process of transcriptional regulation. The transcriptional coactivator p300 is a ubiquitous nuclear protein and transcriptional cofactor with intrinsic acetyltransferase activity. NCoR is a protein that contain distinct functional domains responsible for interaction with NRs, and activation of HDAC proteins, ultimately resulting in targeted repression of transcription The inducible transcription factor NF-κB, immediately after being released from a cytoplasmic inhibitor, translocates into nucleus, where it enhances transcription of anti-apoptotic and pro-inflammatory genes. COX-2, an enzyme often induced in neoplastic conditions, perpetuates the chronic inflammatory state in the epithelium and its microenvironment, by means of prostaglandin synthesis. Elucidation of the molecular networks implicated in estrogen signaling is very important in view of the potential use of selective estrogen receptor modulators in chemoprevention and targeted anticancer therapy. Materials and Methods. In our retrospective study we included 111 consecutive patients (74 males and 37 females), aged 23-90 years (mean 70±10) diagnosed with TCC of the bladder by either biopsies, transurethral resection of bladder tumor, or radical cystectomies, between 2000 and 2002 from the Urological Department of Urology of University Hospital of Patras, Greece. None of the patients had received any preoperative intravesical therapy. Bladder tumors were graded and staged according to the World Health Organization (WHO) grading. Paraffin section immunohistochemistry was utilized and relative expression was estimated in intracellular compartments, intraepithelial layers, and histologic categories. NF-κB(p65 subunit) demonstrated mixed subcellular presence, COX2 cytoplasmic whereas ERβ, p300 and NCoR staining patterns were nuclear. NF-κB and COX-2, were constantly upregulated as tumorigenesis progressed. Results NF-κB, COX-2 and p300 expression correlated positively with progression of carcinogenesis, suggesting a potential involvement in bladder tumorigenesis. On the contrary, ERβ and NCoR were severely diminished in cancer, compared to normal epithelium, and they were affected by tumor Grade. The remaining correlations are based on coexpression analysis of the aforementioned factors, individually for each patient, to permit judgement of molecular interactions. In detail, an inverse staining between ERβ and nuclear p65 immunoreactivity was observed and we could suggest that there is a reciprocal transactivation between ERβ and activated NFκB. COX-2 was positively associated in bladder carcinomas with NFκB, a finding which may denote the nuclear factor contribution to the enzyme induction. A correlation has been established, when correlating the expression of ERβ with the coregulators, positive with NCoR and negative with p300, indicating a potential role of these key molecules in bladder carcinogenesis. Furthermore, p300 and NCoR, may not be strictly segregated and in bladder cancer cells interact directly, since, according to biochemical purification studies, p300 is capable of directed negative interaction with NCoR. Conclusions The inhibition of ERβ in combination with the antiapoptotic properties of NFκB may contribute to the pathogenesis of TCC. Selective ERβ and NCoR agonist and agents-inhibitors of NFκB, COX2 and p300 may represent a possible new treatment strategy, by virtue of their role in bladder carcinogenesis. Subtle variations in the chemopreventive regimen, based on personalized molecular profiling, would hopefully achieve a patient-tailored therapeutic approach.

Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2016-10-19T10:06:31Z No. of bitstreams: 2 Tese - Cristiene Costa Carneiro - 2016.pdf: 2763720 bytes, checksum: 5287cb8e3862369e99646711609afd00 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2016-10-19T16:08:06Z (GMT) No. of bitstreams: 2 Tese - Cristiene Costa Carneiro - 2016.pdf: 2763720 bytes, checksum: 5287cb8e3862369e99646711609afd00 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-10-19T16:08:06Z (GMT). No. of bitstreams: 2 Tese - Cristiene Costa Carneiro - 2016.pdf: 2763720 bytes, checksum: 5287cb8e3862369e99646711609afd00 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-09-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Punicalagin and gemin D are ellagitannins found in some species of plants of medical importance such as Punica granatum and Geun japonicun. For this study, punicalagin and gemin D were isolated, respectively, from the leaves of Lafoensia pacari and Eugenia uniflora, two species of Brazilian medicinal plants with several biological activities, such as antitumoral, antioxidant and healing of wounds. In the present study, we evaluated the genotoxic, cytotoxic, antigenotoxic and anticytotoxic effects of gemin D using the Ames test in Salmonella typhimurium, the micronucleus (MN) test and comet assay in mice. With the punicalagin ellagitannin, we assessed the same effects mentioned above using the comet and MN tests in mice, and we also evaluated the angiogenic and antiangiogenic activities of this ellagitannin by the chick chorioallantoic membrane (CAM) angiogenic assay. The results obtained with gemin D showed that this tannin did not present genotoxic effect by the Ames and MN tests, however, in the comet assay, the highest dose of gemin D (100 mg/kg) induced increase of breaks in DNA in comparison to the negative control (p < 0.05). In the antigenotoxicity, gemin D protected DNA against the harmful action of 4-nitroquinoline-1-oxide and sodium azide by the Ames test, and also against cyclophosphamide (CPA) in pre- and co-treatment by MN and comet tests in mice, but it did not protect DNA in the post-treatment. The results obtained with punicalagin showed that this tannin exhibited no genotoxic effect by MN test and comet assay in mice. Only the highest dose of punicalagin (50 mg/kg) exhibited significant cytotoxic effect by MN test, and in the co-treatment with CPA, this cytotoxicity was enhanced. Co-treatment, pre-treatment and post-treatment of punicalagin with CPA led to a significant reduction in the number of DNA breaks and in the frequency of CPA-induced MN, indicating antigenotoxic effect. Using the CAM model, punicalagin exhibited angiogenic activity in all concentrations, mainly at the lowest concentration (12.5 µg/µL). Therefore, gemin D and punicalagin exhibited relevant antigenotoxic and cytotoxic effects, which indicate that they may be probables candidates for chemoprevention or for the development of new cancer therapies. In addition, the angiogenic activity presented by punicalagin in this study could contribute for the processes of tissue repairing and wound healing. / Punicalagin e gemin D são elagitaninos encontrados em algumas espécies de plantas de importância médica, tais como Punica granatum e Pelargonium sidoides. Para o presente estudo, punicalagin e gemin D foram isolados, respectivamente, das folhas de Lafoensia pacari e Eugenia uniflora, duas espécies de plantas medicinais brasileiras com diversas atividades biológicas, tais como, antitumoral, antioxidante e cicatrizante de feridas. No presente estudo, nós avaliamos os seguintes efeitos: genotóxico, citotóxico, antigenotóxico e anticitotóxico de gemin D utilizando o teste de Ames em Salmonella typhimurium, o teste do micronúcleo (MN) e o ensaio cometa em camundongos. Com o elagitanino punicalagin, nós investigamos os mesmos efeitos citados anteriormente utilizando os testes cometa e MN em camundongos, e também avaliamos a atividade angiogênica e antiangiogênica desse tanino utilizando o ensaio em membrana corioalantóide (MCA) do ovo embrionado de galinha. Os resultados obtidos com gemin D mostraram que esse tanino não apresentou efeito genotóxico pelos testes de Ames e MN, porém, no ensaio cometa, a maior dose de gemin D (100 mg/kg) induziu aumento de quebras no DNA em comparação com o controle negativo (p < 0.05). Na avaliação antigenotóxica, gemin D protegeu o DNA contra as ações lesivas de 4-nitroquinolina-1-óxido e azida sódica pelo teste de Ames, e também contra ciclofosfamida (CIF) no pré- e co-tratamento pelos testes cometa e MN em camundongos, entretanto, ele não protegeu o DNA no pós-tratamento. Os resultados obtidos com punicalagin mostraram que esse elagitanino não exibiu efeito genotóxico pelos testes cometa e MN em camundongos. Apenas a maior dose de punicalagin (50 mg/kg) exibiu efeito citotóxico significativo pelo teste do MN, e no co-tratamento com CIF, essa citotoxicidade foi maior do que a apresentada pelo controle positivo (CIF). O co-, pré e pós-tratamento de punicalagin com CIF em camundongos levou a uma redução significativa no número de quebras no DNA e na frequência de MN induzida por CIF, indicando efeito antigenotóxico. Utilizando o modelo MCA, punicalagin exibiu atividade angiogênica em todas as concentrações testadas, especialmente a menor concentração (12.5 µg/µL). Assim, gemin D e punicalagin demonstraram relevantes efeitos antigenotóxico e citotóxico, indicando que eles podem ser prováveis candidatos para quimioprevenção ou desenvolvimento de novas terapias para o câncer. Além disso, a atividade angiogênica apresentada por punicalagin nesse estudo poderia contribuir em processos de reparação tecidual e cicatrização de feridas, como por exemplo no tratamento de úlceras gástricas e queimaduras.

Foram avaliados as atividades quimiopreventivas da &#946;-ionona (BI) e do geraniol (GR) durante a etapa de promoção da hepatocarcinogênese. Ratos Wistar foram submetidos ao modelo de hepatocarcinogênese do \"Hepatócito Resistente\" e receberam durante 5 semanas consecutivas: 16 mg/100 g p.c. de BI, 25 mg/100 g p.c. de GR, 16 mg/100 g p.c. de BI + 25 mg/100 g p.c. de GR ou somente 25 ml/100 g p.c. de óleo de milho (grupo OM, controle). Em comparação ao grupo OM, o grupo BI apresentou menor multiplicidade de nódulos hepáticos e os grupos BI, GR e GR+BI apresentaram menor número de lesões pré-neoplásicas (LPN) persistentes e maior porcentagem de LPN em remodelação. Comparados ao grupo OM, o grupo BI apresentou menor área de LPN em remodelação e menor porcentagem da área do corte histológico ocupado por LPN persistentes enquanto o grupo GR apresentou menor porcentagem da área do corte histológico ocupado por LPN em remodelação. O tratamento com BI tendeu a reduzir o colesterol plasmático e inibir a proliferação celular em LPN. O grupo GR apresentou maior número de corpúsculos apoptóticos em LPN persistentes em comparação ao grupo OM. Comparados ao grupo OM, os grupos BI e GR+BI apresentaram menor porcentagem de LPN hepáticas em remodelação positivas para a proteína p53 citoplasmática. Já o grupo GR apresentou maior porcentagem de LPN hepáticas persistentes positivas para p53. Os níveis da proteína RhoA nas membranas celulares hepáticas foram menores no grupo GR quando comparados aos do grupo OM. Assim, BI e GR são quimiopreventivos supressores promissores da hepatocarcinogênese. / Chemopreventive activities of &#946;-ionone (BI) and geraniol (GR) were evaluated during promotion phase of hepatocarcinogenesis. Wistar rats received GR (25 mg/100 g body weight), BI (16 mg/100 g body weight), GR (25 mg/100 g body weight) +BI (16 mg/100 g body weight) or corn oil (CO, control). Multiplicity of hepatic nodules were smaller in BI group, compared to CO group. BI, GR and GR+BI groups presented decreased number of persistent preneoplastic lesions (PNL) and increased % of remodeling PNL. BI group presented decreased % liver section area occupied by persistent and remodeling PNL. GR group presented decreased % liver section area occupied by remodeling PNL. BI group tended to present reduced cholesterol concentration and inhibited cell proliferation. GR group increased number of apoptotic bodies in persistent PNL. BI and GR+BI groups reduced the % of remodeling and increased the % of persistent p53 positive PNL. RhoA protein in cellular membranes was reduced in GR group. Thus, BI and GR could be considered as chemopreventive agents against hepatocarcinogenesis.

O câncer é a segunda maior causa de morte no Brasil, atrás apenas de doenças cardíacas. Por isto, é evidente que grandes recursos sejam direcionados para a pesquisa no descobrimento de novas opções com a finalidade de erradicar esta doença. Dentre estas opções, a quimioprevenção do câncer tem chamado a atenção já que, mesmo com os imensos avanços no conhecimento sobre os mecanismos da carcinogênese e conseqüente desenvolvimento de novas drogas, os dados estatísticos de mortalidade não se tornaram menores. Somando-se a estes fatos, deve ser considerado que no Brasil o tratamento padrão do câncer não chega a todas as pessoas por ser extremamente caro. Desta forma, a quimioprevenção do câncer com fatores presentes na dieta ou oriundos de fontes consideradas baratas como fitoterápicos, deve ser apreciada. Assim, este trabalho teve como objetivo avaliar os efeitos quimiopreventivos e antineoplásicos de uma planta brasileira, o guaraná (Paullinia cupana Mart var. sorbilis). Foram utilizados alguns experimentos em camundongos como indução genotóxica em fígado pela Dietilnitrosamina (DEN); carcinogênese pulmonar induzida pela 4-(metilnitrosamino)-1-(3-piridil)-1-butanona (NNK), uma nitrosamina presente no tabaco; tumor ascítico de Ehrlich; disseminação hematógena de melanoma B16/f10; e cultivo de células tumorais e não tumorais. Além disso, caracterizou-se o papel da Conexin43 na carcinogênese pulmonar induzida pelo NNK e os efeitos do guaraná sobre o receptor CAR e sua ação quando da administração do ligante do CAR, 1,4-bis[2-(3,5-dichloropiridiloxi)]benzeno (TCPOBOP). Pudemos observar efeitos quimiopreventivos e antineoplásicos do guaraná dependendo do modelo utilizado, demonstrando que seu modo de ação principal é a redução da proliferação celular. Além disso, observamos que os tumores de pulmão dos animais tratados com a planta apresentavam menor tamanho, menor grau maligno, menor índice de proliferação celular e menor ativação do fator de transcrição CREB. Observamos também que a Conexina43 (Cx43) tem importante papel na carcinogênese pulmonar induzida pelo NNK, atuando como supressor tumoral e em fases tardias possivelmente tendo papel inverso, ou seja, como um oncogene. Caracterizamos os efeitos do guaraná sobre a ativação do receptor CAR e demonstramos que, por si só, o guaraná induz a expressão do CAR, além de alterar a expressão de alguns de seus transcritos como a CYP2B10 e CYP3A11. Ao analisarmos os efeitos de extratos de guaraná sobre células de tumor de pulmão (E9) in vitro, verificamos o mesmo efeito antiproliferativo, diminuindo a expressão do PCNA e da Conexina43 de maneira dose-dependente, além de verificar um aumento da expressão do receptor CAR. Ao fim propomos uma hipótese de mecanismo de ação baseando-se nas alterações encontradas oriundas da administração do guaraná. Concluímos que o guaraná apresenta componentes com ação antitumoral em camundongos, tendo efeito quimiopreventivo ou antineoplásico dependendo do modelo utilizado. / Cancer is the second biggest cause of deaths in Brazil, only behind of cardiac diseases. As a result, it is evident that great resources for research will be directed towards the discovery of new options to eradicate this disease. Among these options, cancer chemoprevention has calling for attention since the huge advances in the knowledge of carcinogenesis and development of new drugs did not decrease statistical data on mortality due to cancer. In addition, it must be considered that in Brazil, cancer therapy is not available for all given that it is too expensive. Therefore, cancer chemoprevention with dietary factors or from medicinal plants has got to be treasured. Following these lines, the aim of this work was to evaluate the chemopreventive and antineoplastic effects of a Brazilian plant, Paullinia cupana Mart var. sorbilis, most known as guarana. It was used several experiments in mice and cell culture essays as: protection against DEN-induced DNA damage; NNK-induced lung carcinogenesis; Ehrlich Ascitic Tumor; metastasis of B16/f10 melanoma cells; and cell culture of a tumorigenic and a non-tumorigenic cell lines. Additionally, it was characterized the role of Connexin43 in the NNK-induced lung carcinogenesis and the effects of guarana on the CAR receptor before and after the administration of TCPOBOP. We note a chemopreventive or antineoplastic effect of guarana depending on the model employed and showed that the mode of action responsible for these effects was reduced cell proliferation. Also, the lung tumors of guarana-treated animals were smaller, less aggressive, with decreased cell proliferation and CREB activation. On the other hand, we observed that Connexin43 have an important role on NNK-induced lung carcinogenesis because it may act as a tumor suppressor and in advanced stages as an oncogene. The effects of guarana on the CAR activation were characterized and we showed that guarana induces CAR mRNA expression, altering the levels of its transcripts as CYP2B10 and CYP3A11. We also examined the effects of guarana extracts on a lung tumor cell culture (E9 cells) and demonstrated the same antiproliferative effect observed previously, by decreased PCNA and Connexin43 proteins in a dose-dependent manner along with an increase in CAR protein. At last we hypothesized a mechanism of action for guarana effects basing in our findings. We concluded that guarana presents substances that have antitumoral effects in mice, enclosing a chemopreventive or antineoplastic effect depending on the model studied.

O isoprenóide geraniol (GO) apresentou atividade quimiopreventiva quando administrado continuamente durante as fases de iniciação e pós-iniciação em modelo de carcinogênese experimental de cólon por meio da redução do número de focos de criptas aberrantes (FCAs) totais FCAs &#8805; 4 criptas e aumento de apoptose no cólon distal. Dessa forma, optou-se por avaliar os eventuais efeitos de três doses de GO (GO1: 25mg/100g de peso corpóreo [p.c.], G02: 50 mg/100g de p.c. e G03: 100 mg/100g de p.c.) em categorias de lesões pré-neoplásicas (LPNs) induzidas por dimetilhidrazina (DMH) durante a fase de pós-iniciação tardia de modelo de carcinogênese experimental de cólon, caracterizada por apresentar lesões mais avançadas e com alto grau de alterações celulares morfológicas, bioquímicas e moleculares denominadas de displasia. Para isso, analisamos diferentes biomarcadores como: FCAs totais e FCAs < ou &#8805; 4 criptas em cólons corados com azul de metileno; focos depletados ou positivos de mucina (FPMs ou FDMs) em cólons corados com azul de toluidina; FCAs convencionais ou displásicos por meio de análise histopatológica em cortes corados com hematoxilina e eosina (HE) e focos positivos ou negativos para beta-catenina (FPBCs ou FNBCs) citoplasmática e/ou nuclear por meio de imunoistoquímica. Além disso, células apoptóticas foram identificadas utilizando-se critérios morfológicos clássicos em FCAs &#8805; 4 no cólon distaI e a expressão de genes envolvidos na carcinogênese de cólon foi avaliada por meio de RT-PCR: HMGCoA-redutase na mucosa colônica e K-Ras e c-myc em FCAs microdissecados. Em relação ao grupo controle, foi possível observar que o grupo tratado com a maior dose de GO (G03) reduziu a freqüência de FCAs &#8805; 4 criptas e FDMs, além de aumentar a apoptose em FCAs &#8805; 4 displásicos no cólon distaI (p &#8804; 0,05). Já, em relação aos outros biomarcadores e às expressões de HMGCoA-redutase, K-Ras e c-myc não observamos diferenças estatísticas entre os tratamentos (p > 0,05). A partir desses resultados, podemos concluir que a dose de 100 mg/100 g de p.c. de GO mostrou ser mais interessante do ponto de vista quimiopreventivo com efeitos observados principalmente no cólon distaI, onde há maiores relatos de incidência de adenocarcinomas colônicos, tanto em animais quanto em humanos. Assim, a indução da morte celular programada em FCAs &#8805; 4 preferencialmente displásicos poderia representar um mecanismo importante de atuação de G03 na redução da freqüência de FCAs &#8805; 4 criptas e de FDMs (também utilizado como marcador de displasia) durante a fase de pós-iniciação tardia de modelo de carcinogênese experimental de cólon. / The isoprenoid geraniol (GO) showed chemopreventive activity when administered continuously during the initiation and post-initiation phases in an experimental model of colon carcinogenesis by reducing the number of total aberrant crypt foci (ACF) and ACFs &#8805; 4 crypts, as well as increasing apoptosis in the distal colon. We therefore chose to evaluate the effects of three different doses of GO (GO1: 25 mg/100 g body weight [b.w.], GO2: 50 mg/100 g b.w. and GO3: 100 mg/100 g of b.w.) on preneoplastic lesions (PNLs) induced by dimethylhydrazine (DMH) during late post-initiation in an experimental model of colon carcinogenesis that is characterized by more advanced lesions and a higher degree of cellular alterations morphological, biochemical and molecular (dysplasia) than previous models. For this study, we analyzed the following biomarkers: total ACFs, ACFs < 4 crypts, and ACFs &#8805; 4 erypts in colons stained with methylene blue; mucin-depleted or mucin-positive foci (MDFs or MPFs) in colons stained with toluidine blue; ACFs, through conventional or dysplastie histopathological analysis of sections stained with hematoxylin and eosin (HE); and cytoplasmic vs. nuclear foci reactivity for beta-catenin (foci positive for beta-eatenin (FPBC) or foci negative for beta catenin (FNBC)) using immunohistochemistry. Additionally, apoptotic cells were identified using classical morphologic criteria in ACFs &#8805; 4 crypts in the distal colon, and the expression of several genes involved in colon carcinogenesis was assessed by RT-PCR, including HMG-CoA reductase in the colonic mucosa and K-Ras and c-myc in microdissected ACFs. Relative to the control group, we observed that the group receiving the highest dose of GO (GO3 group) had a reduced frequency of both ACFs &#8805; 4 crypts and MDFs and that apoptosis increased in dysplastic ACFs &#8805; 4 crypts in the distal colon (p < 0, 05). Expression of HMG-CoA reductase, K-Ras and c-myc did not differ between treatments (p > 0, 05). Based on these results, we conclude that the 100 mg/100 g b.w. dose of GO is the most promising, as it shows evidence of chemopreventive effects mainly in the distal colon, which is a region that is reported to have a higher incidence of colonic adenocarcinomas, both in animaIs and in humans. lnduction of programmed cell death by GO3 in ACFs &#8805; 4 specifically dysplastic could represent an important mechanism of action in reducing the frequency of both ACFs &#8805; 4 crypts and MDFs during late post-initiation in this experimental model of colon carcinogenesis.

A ingestão de folato é inversamente associada com o risco de diversos cânceres. Apesar da deficiência dessa vitamina ser classicamente considerada fator de risco para câncer de fígado, não existem estudos avaliando o efeito da suplementação com ácido fólico (AF) durante as etapas iniciais da hepatocarcinogênese. Assim, o objetivo do presente estudo foi avaliar o efeito da suplementação com AF continuamente durante as etapas de iniciação e seleção/promoção da hepatocarcinogênese em ratos. Os animais receberam diariamente 0,08 mg (grupo AF8) ou 0,16 mg (grupo AF16) de AF/100 g de peso corpóreo ou água (grupo controle [GC]). Após duas semanas de tratamento, todos os animais foram submetidos ao modelo de hepatocarcinogênese do &#8220;Hepatócito Resistente&#8221; (iniciação com dietilnitrosamina, seleção/promoção com 2-acetilaminofluoreno e hepatectomia parcial a 70%). A eutanásia dos animais ocorreu após 8 semanas de tratamento. Quando comparado ao GC, o grupo AF16, mas não o AF8, apresentou menores nódulos macroscópicos (p<0,05), menor (p<0,05) número de lesões pré-neoplásicas (LPN) persistentes, maior (p<0,05) número de LPN em remodelação, menor (p<0,05) proliferação celular nas LPN persistentes, menos (p<0,05) danos no DNA hepático e tendência (p<0,10) a apresentar menor expressão de c-myc em LPN microdissecadas. Não foram observadas diferenças significativas (p>0,05) entre os grupos experimentais com relação à indução de apoptose nas LPN persistentes e em remodelação bem como no padrão de metilação global do DNA em LPN microdissecadas. Em resumo, a suplementação com AF durante as etapas iniciais da hepatocarcinogênese resultou em atividade quimiopreventiva de forma dose-efeito. Alteração no fenótipo das LPN, inibição de danos no DNA hepático e da expressão de c-myc representam relevantes efeitos celulares e moleculares dessa vitamina. / Dietary intake of folate is inversely associated with the risk of several malignancies. Although folate deficiency is associated with liver cancer, there is no data on folic acid (FA) supplementation during hepatocarcinogenesis. The aim of the present study was to evaluate the effect of FA supplementation during early hepatocarcinogenesis. Rats receiving daily 0.08 mg (FA8 group) or 0.16 mg (FA16 group) of FA/100 g body weight or water (CO group, controls) were used. After a 2 week-treatment, all animals were submitted to the resistant hepatocyte model of hepatocarcinogenesis (initiation with diethylnitrosamine, selection/promotion with 2-acetylaminofluorene and partial hepatectomy). All animals were euthanized after 8 weeks of treatment. When compared to CO group, FA16 group, but not FA8 group, presented: smaller (p < 0.05) macroscopic nodules; reduced (p < 0.05) number of persistent and increased (p < 0.05) number of remodeling preneoplastic lesions (PNL); reduced (p < 0.05) cell proliferation in persistent PNL; decreased (p < 0.05) hepatic DNA damage; and a tendency (p < 0.10) of decreased c-myc expression in microdissected PNL. No differences (p > 0.05) were observed between CO, FA8 and FA16 groups regarding apoptosis in both persistent and remodeling PNL, and global DNA methylation pattern in microdissected PNL. In conclusion, FA supplementation during early hepatocarcinogenesis resulted in a dose-response chemopreventive activity. Reversion of PNL phenotype and inhibition of DNA damage and of c-myc expression represent relevant FA cellular and molecular effects.

A &#946;-ionona (BI) é um isoprenóide que apresenta atividade quimiopreventiva durante a fase de promoção da hepatocarcinogênese. O presente trabalho teve como objetivo avaliar a expressão de genes modulados pela BI envolvidos na quimioprevenção durante a fase de promoção da hepatocarcinogênese induzida pelo modelo do \"Hepatócito Resistente\" (RH). Ratos Wistar machos foram submetidos ao modelo do RH e tratados durante 4 semanas consecutivas com BI (16 mg/100 g de p.c.) ou óleo de milho (OM) (0,25 ml/100 g de p.c.; grupo controle). O perfil da expressão de 1.176 genes foi analisado por macroarray no fígado dos grupos BI, OM e de ratos considerados normais (grupo N). A expressão gênica foi considerada aumentada, quando a razão de expressão foi &#8805; 1,5 ou diminuída, quando &#8804; 0,5. Aplicou-se análise hierárquica de clustering e classificação ontológica dos genes diferencialmente expressos. A expressão gênica foi validada por RT-PCR do tipo \"duplex\", utilizando-se tecido hepático microdissecado de: lesões pré-neoplásicas persistentes (pLPN) ou em remodelação (rLPN) e de regiões ao redor das LPN (surrounding). Um total de 133 e 32 genes foi considerado diferencialmente expresso entre os grupos OM (em relação ao N) e BI (em relação ao OM), respectivamente. Trinta e sete por cento dos genes diferencialmente expressos no grupo BI vs OM referiam-se a receptores celulares. Destes, 4 genes codificantes para receptores nucleares foram identificados como possíveis alvos da BI na quimioprevenção da hepatocarcinogênese: RXR&#945; (receptor X de retinóide &#945;), RAR&#946; (receptor de ácido retinóico &#946;), COUP-TFI (chicken ovalbumin upstream promoter-transcription factor I) e Nur77 (nuclear receptor 77). Em comparação ao grupo OM, a expressão de RXR&#945; e RAR&#946; foi maior (p<0,05) especificamente em pLPN e rLPN do grupo &#946;I, respectivamente. Em comparação ao grupo N, Nur77 apresentou maior (p<0,05) expressão no surrounding e nas rLPN do grupo OM. Por outro lado, a expressão de Nur77 em rLPN foi menor (p<0,05) no grupo BI do que no OM. Comparada ao grupo N, a expressão de COUP-TFI foi maior (p<0,05) no grupo OM, tanto no surrounding das LPN como nas pLPN e rLPN. Em comparação ao grupo OM, a expressão de COUP-TFI foi menor (p<0,05) no grupo BI, especificamente nas pLPN e nas rLPN. Os resultados sugerem que os receptores nucleares RXR&#945;, RAR&#946;, Nur77 e COUP-TFI representam alvos moleculares da BI relevantes para a quimioprevenção da hepatocarcinogênese em ratos. / &#946;-ionone (BI) is an isoprenoid which has chemopreventive activity during the promotion phase of hepatocarcinogenesis. This study aimed to evaluate the expression of genes modulated by BI involved in chemoprevention during the promotion phase of hepatocarcinogenesis induced model of \"Resistant Hepatocyte (RH). Male Wistar rats were submitted to the RH model and treated for 4 consecutive weeks with BI (16 mg/100 g bw) or corn oil (CO) (0.25 ml/100 g bw, control group). The expression profile of 1,176 genes was analyzed by macroarray in the liver of groups BI, CO and normal rats (group N). Gene expression was considered increased when the expression ratio was 1.5 or decreased when 0.5. Hierarchical clustering analysis and ontological classification of differentially expressed genes were applied. Gene expression was validated by RT-PCR \"duplex\", using microdissected hepatic tissue from: persistent pre-neoplastic lesions (pPNL) or remodeling pre-neoplastic lesions (rPNL) and regions around the PNL (surrounding). A total of 133 genes and 32 were considered differentially expressed between the two groups (CO to N) and BI (relative to CO), respectively. 37% of differentially expressed genes in group BI vs CO were related to cell receptors. Of these, four genes encoding for nuclear receptors have been identified as possible targets of BI in the chemoprevention of hepatocarcinogenesis: RXR (retinoid X receptor &#945;), RAR&#946; (retinoic acid receptor &#946;), COUP-TFI (chicken ovalbumin upstream promoter-transcription factor I) and Nur77 (nuclear receptor 77). Compared to CO group, the expression of RXR&#945; and RAR&#946; was higher (p <0.05) specifically in pPNL and rPNL of BI group, respectively. Compared to the group N, Nur77 showed higher (p <0.05) expression in the surrounding and rPNL of CO group. The expression of Nur77 in rPNL was lower (p <0.05) in BI than the CO group. Compared to N group, the expression of COUP-TFI was higher (p <0.05) in CO group (surrounding, pPNL and rPNL). Compared to CO group, the expression of COUP-TFI was lower (p <0.05) in BI group, specifically in the pPNL and rPNL. The results suggest that the nuclear receptors RXR&#945;, RAR&#946;, Nur77 and COUP-TFI represent relevant molecular targets of BI in the chemoprevention of hepatocarcinogenesis in rats.