Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 5 uppsatser.

Cervical cancer is reported by the World Health Organisation to be the second most common type of cancer to affect women in poorer socioeconomic countries. Treatment of this pathology remains sub-optimal at advanced stages and continues to be of importance on the research agenda. Previous studies have reported cytotoxic and antiproliferative effects of 2-methoxyestradiol (2-ME) in vitro on a HeLa cervical cancer cell line. These results were promising but use of 2-ME itself is limited due to pharmacodynamic constraints. In an attempt to overcome these, a sulphamoylated analogue of 2-ME, namely 2-ethyl-3-O-sulphamoyl-estra- 1,3,5(10)16-tetraene or compound 19 (C19), was synthesised. In this in vitro study, the induction of a block in mitosis with subsequent culmination of apoptosis and autophagy as types of cells death was investigated after HeLa cells were exposed for 24 hours to a 0.5 μM C19 solution. This was achieved by morphological assessment (fluorescent, Polarization-optical transmitted light differential interference contrast microscopy (PlasDIC) and transmission electron microscopy (TEM)) and flow cytometry (cell cycle progression, cyclin B1 analysis, phosphatidylserine (PS) flip and aggresome formation). Spectrophotometric quantification of the apoptotic initiator and executioner caspases 8 and 3 respectively was done to determine their involvement in the crosstalk between apoptosis and autophagy. Results included the following: (i) PlasDIC microscopy illustrated the appearance of an increased number of cells blocked in metaphase, stress signaling, premature cell shrinkage, hypercondensed chromatin and the presence of apoptotic bodies after C19 exposure. The presence of ghost cells, cell debris and decreased cell density of the treated cells correlated with the autophagy control. (ii) Fluorescence microscopy employing triple staining highlighted an increased lysosomal activity and staining of C19-exposed cells when compared to the control, as well as evidence of apoptotic and metaphase-blocked cells. This is indicative of both the autophagic and apoptotic cell death process. (iii) TEM allowed for examination of the ultrastructure of the intracellular processes, and revealed that apoptotic cells have hallmarks of both autophagy and apoptosis, confirming the results of light microscopy. (iv) Cell cycle analysis demonstrated more cells present in the sub-G1 and G2/M populations, indicating the induction of apoptosis (confirmed with PS fip flow cytometric quantification) and a metaphase block (corroborated by an increased cyclin B1 fluorescence). (v) The increase in autophagosome formation seen on fluorescence- and transmission electron microscopy was confirmed by flow cytometry demonstrating an upregulation of aggresome formation in C19-exposed cells. This investigation demonstrated induction of both types of cells death by this novel compound. (vi) The upregulation of caspases 8 and 3 was demonstrated in the C19-treated cells, indicating apoptosis induction via the extrinsic pathway. (vii) Confocal microscopy demonstrated complete microtubule disintegration in the C19-exposed HeLa cells. Both apoptotic and autophagic cell death mechanisms were induced in C19-treated HeLa cells after spindle abrogation kept the cells in metaphase block. Insight gained into the molecular effect of C19 on HeLa cells may be used as a springboard for in vivo studies, furthering the development of this promising anticancer agent toward clinical application. / Dissertation (MSc)--University of Pretoria, 2012. / Physiology / MSc / Unrestricted

In der vorliegenden Arbeit wird gezeigt, daß der physiologische Östrogenmetabolit 2-Methoxyestradiol (2-ME) eine sehr starke Wachstumshemmung auf Tumorzellen verschiedener solider Tumoren ausübt. Die untersuchten Tumoren sind das hepatozelluläre Karzinom (HCC), das Pankreaskarzinom und das Bronchialkarzinom. Alle drei Tumoren haben eine sehr schlechte Prognose, die sich in den letzten 20 Jahren trotz neuer OP-Techniken und neuer zytostatisch wirkender Substanzen sowie molekularer Therapieansätze nicht wesentlich verbessert hat. Beim HCC und beim Pankreaskarzinom konnten wir durch die Verwendung mehrerer Zelllinien eine Generalisierbarkeit der gefundenen Ergebnisse dokumentieren, denn es kam zu einer dosisabhängigen Wachstumshemmung von bis zu 90% bei allen bis auf eine Pankreaskarzinomzelllinie. Die Wirkung lies sich noch verstärken, indem wir 2-ME beim Pankreaskarzinom mit in der Klinik üblichen Zytostatika wie Gemcitabine, Docetaxel oder einem monoklonalen Antikörper gegen den EGF Rezeptor kombinierten. Wir fanden eine additive Wachstumshemmung in der Kombination mit allen verwendeten Substanzen. Beim Bronchialkarzinom basierten unsere Untersuchungen auf Vorarbeiten, bei denen bereits ein tumorhemmender Effekt durch 2-ME gefunden wurde. Wir kombinierten 2-ME mit Gentherapie. Dazu setzten wir ein Adenovirus, welches das Tumorsuppressorgen p53 exprimiert, ein. Hier konnten wir zeigen, daß das vom Gen des Adenovirus stammende p53 Protein nach systemischer intravenöser Applikation in den Lungenmetastasen exprimiert werden kann. 2-ME stabilisierte dieses p53 Protein, so daß eine ausreichende Menge funktionsfähigen p53 Proteins vorhanden war, um die Tumorzellen zu töten oder im Wachstum zu hemmen. Dies war der erste Bericht über eine erfolgreiche intravenöse Applikation eines adenoviralen Vektors mit einem Tumorsuppressorgen. 2-ME war nicht nur in der Lage, wachstumshemmend auf normale Tumorzellen zu wirken. Wir konnten auch zeigen, daß multiresistente Pankreaskarzinomzellen, die eine bis zu 1000-fach letale Dosis Zytostatika überleben, komplett sensibel gegenüber 2-ME waren. Die Wachstumshemmung durch 2-ME war in diesem Versuch identisch zwischen parentalen sensiblen Zellen und den multiresistenten Zellen. Die IC50 lag hier bei 0,56mM bzw bei 1,65mM je nachdem, ob das mdr-1 Gen exprimiert wurde oder nicht. Diese Werte entsprechen in etwa denen, die bei anderen Tumorzelltypen gefunden wurden. Auch Bronchialkarzinomzellen, die eine Resistenz gegen Cisplatin aufwiesen, zeigten sich ebenfalls komplett sensibel gegenüber 2-ME. Untersuchungen zur Toxizität von 2-ME zeigten, daß Kulturen von normalen humanen Hepatozyten, die von Leberresektaten gewonnen wurden, die Behandlung mit 2-ME überlebten. In parallelen Versuchen mit HCC Zellen zeigte sich eine signifikante Wachstumshemmung der Tumorzellen. Da die normalen Hepatozyten nicht proliferieren, untersuchten wir proliferierende Hepatozyten, indem wir Leberresektionen bei Mäusen durchführten. In der Leberregenerationssphase behandelten wir die Mäuse mit 2-ME. 2-ME hatte keine nachteilige Wirkung auf die Tiere. Nach Beendigung des Versuches waren die resezierten Lebern fast komplett regeneriert. Immunhistochemische Untersuchungen konnten zeigen, daß die Anzahl der apoptotischen Zellen in der regenerierenden Leber in der mit 2-ME behandelten Gruppe nicht zunahm. Durch eine Färbung mit PCNA konnte die Proliferation der Hepatozyten nach Resektion und damit die Regeneration verdeutlicht werden. Hier war die Proliferationsrate unabhängig von der Behandlung mit 2-ME. Als wesentlichen Mechanismus der Wachstumshemmung von Tumorzellen durch 2-ME fanden wir die starke Induktion von Apoptose in allen Zellen, bis auf die relativ resistente Pankreaskarzinomzelllinie PaTu 8988s. Mit mehreren Untersuchungstechniken konnten wir die Apoptose nachweisen. Um die Mechanismen der Apoptoseinduktion zu untersuchen, führten wir Western Blot Untersuchungen durch, die Veränderungen des Expressionsmusters apoptosebezogener Proteine aufzeigen sollten. Wir fanden eine Induktion des p53 Proteins in den HCC Zelllinien, die den Wild-Typ p53 exprimieren. Die Pankreaskarzinomzellen waren alle mutiert für das p53 Gen, so daß hier nach 2-ME Behandlung p53 unabhängige Apoptose vorlag. Messungen des p21 Proteins, einem direkten Effektor von p53, zeigte, daß es parallel zu p53 hochreguliert wurde, was darauf schließen läßt, daß das hochregulierte p53 funktionell aktiv ist. In einer Zelllinie (SK-Hep 1) wurde das stärkste Antiapoptoseprotein bcl-2 herunterreguliert, was eine Förderung der Apoptoseinduktion nach sich zieht. Somit führen mehrere Mechanismen zum apoptotischen Zelltod. Tierversuche an Nacktmäusen zeigten nicht nur, daß 2-ME Tumorzellen töten kann, sondern lassen auch Rückschlüsse für eine klinische Anwendung zu. So konnten wir zeigen, daß subcutane HCC Tumoren zu 55% und Lungenmetastasen von Pankreaskarzinomen und Bronchialkarzinomen um 59, bzw. 55% im Wachstum gehemmt werden konnten. Die Kombination mit dem p53 tragenden Adenovirus verringerte die Tumormasse um weitere 14%. Die gesamte Tumormasse konnte durch diese Kombination um das 336-fache gegenüber der Kontrollgruppe reduziert werden. Den beschriebenen antiangiogenetischen Effekt konnten wir weder beim Pankreaskarzinom, noch beim Bronchialkarzinom nachvollziehen. Die Tiere zeigten keinerlei klinisch apparente Nebenwirkungen wie Durchfall, Gewichtsverlust, Bewegungsarmut oder anderes. Die Kontrollgruppe der Tiere mit Lungenmetastasen vom Pankreaskarzinom hingegen zeigte eine deutliche Tumorkachexie mit 20%igem Gewichtsverlust im Vergleich zur Therapiegruppe. Schlußfolgernd ist 2-ME eine Substanz, die von großem klinischen Interesse ist. Durch die hohe Wirksamkeit in vitro und in vivo bei gleichzeitig sehr geringen Nebenwirkungen wird sie derzeit in der Klinik in Phase I/II Studien getestet. Die orale Gabe macht die Durchführung der Therapie ambulant möglich. Kombinationen mit wirksamen zytostatischen Substanzen scheinen die Wirksamkeit bei Gleichbleiben der Nebenwirkungen noch zu verstärken. Besonders interessant scheint die Therapie bei multiresistenten Tumoren, wie sie beim Tumorrezidiv meist vorliegen. / We here show that the physiological estrogen metabolite 2-Methoxyestradiol (2-ME) has a very strong growth inhibitory effect on cell lines from different solid cancer types. The tumors investigated were hepatocellular carcinome (HCC), pancreatic cancer, and lung cancer. All three tumor types present with a very poor prognosis, which did not improve significantly the last 20 years in spite of new operation techniques, new anticancer drugs, or new molecular approaches. Using several different cell lines of each cancer type we studied, we could confirm a generalized phenomenon of cancer growth inhibition by 2-ME. We found up to 90% growth inhibition in all cell lines with the exception of one pancreatic cancer cell line. This effect could even be increased using combination therapies of 2-ME and Gemcitabine, 5-FU, Taxol or a monoclonal antibody against the EGF receptor. We found an additive growth inhibition when all of these anticancer agents were combined with 2-ME. Our studies on lung cancer were based on previous results, where 2-ME stabilized the p53 protein. We combined 2-ME with gene therapy and used a wild-type p53 expressing adenovirus, which was administered intraveneously. 2-ME was given orally. The p53 gene was expressed in lung colonies. 2-ME stabilized the p53 protein in a quantity that there was enough p53 protein to be able to kill the cancer cells and to inhibit the cancer growth. This was the first report showing that an adenoviral gene transfer using a tumor suppressor gene can have an effect after intraveneous application. We also showed that 2-ME was able to inhibit the growth of multi-resistant pancreatic cancer cells, which were resistant to up to 1000 fold against different anticancer agents. The degree of growth inhibition after 2-ME treatment was identical between normal cancer cells and multi-resistant cancer cells. The IC50 was 0.56µM in mdr-1 gene expressing cells and 1.65µM in mdr-1 gene negative cells. These values are very similar to those seen in normal cancer cells. Lung cancer cells resistant against cisplatin also showed to be sensitive to 2-ME. Toxicitiy studies showed that cultured normal hepatocytes harvested from resected livers survived the treatment with 2-ME. Parallel studies with cancer cells showed strong growth inhibition at the same doses. Since normal hepatocytes do not proliferate in culture, we studied proliferating hepatocytes in vivo after liver resection in mice. After resection, the livers recieve a strong proliferation stimulus, which causes hepatocyte proliferation. During the regeneration, we treated the mice with 2-ME. We found no induction of apoptosis after 2-ME treatment in proliferating hepatocytes. Liver regeneration was not inhibited by 2-ME as shown by immunohistochemistry using PCNA. The major mechanism of growth inhibition due to 2-ME treatment was the induction of apoptosis in all cell lines except one pancreatic cancer cell line (PaTu 8988s). We confirmed the induction of apoptosis with several different methods. To study further mechanisms of induction of apoptosis, we performed western blot analysis for apoptosis related proteins. We found a p53 over-expression in all HCC cells expressing wild-type p53. The pancreatic cancer cells were all mutant for the p53 gene, which suggests the presence of p53 independent mechanisms of apoptosis. The tumor suppressor protein p21, a direct effector protein of p53, was also up-regulated when p53 was up-regulated, which shows that the up-regulated p53 protein is active. In one HCC cell line (SK-Hep1), which is the most sensitive to 2-ME, we found a down-regulation of the strongest anti-apoptosis protein, bcl-2. This effect causes an induction of apoptosis. Thus, different mechanisms lead to an increased induction of apoptosis. Animal experiments on nude mice show significant growth inhibition of different tumors. HCC tumors were implanted subcutaneously and were inhibited by 55%, lung metastases from lung and pancreatic cancer cells were inhibited by 55% and 59%, respectively. The combination of 2-ME and the p53 expressing adenovirus could further inhibit tumor growth by 14%. The total tumor burden could be reduced by 336 fold in this combination therapy compared to the non treated control group. The described antiangiogenetic effect in the literature could not be confirmed in our experiments on pancreatic and lung cancer. The animals did not show any sign of side effects after treatment with 2-ME such as diarrhea, weight loss, hypocinesia, or others. The animals of the control groups with lung metastases from lung or pancreatic cancer showed cachexy due to tumor burden with an average weight loss of 20% compared to the treated animals. In conclusion, 2-ME is a compound of high clinical interest. The strong efficacy in vitro and in vivo on growth inhibition of tumors with no or slight side effects led to the initiation of clinical phase I and II trials. The oral administration allows an outpatient treatment. The combination with other clinically used anti-cancer drugs appears to increase the effect on tumor growth while the side effects don''t increase. Of particular interest will be the treatment of multi-resistant cancers, for example in the case of recurrent cancer.

Oxidative stress (OS) is a frequent complication of various disease conditions such as Alzheimer’s and Parkinson’s disease, atherosclerosis, preeclampsia, rheumatoid arthritis, diabetes including gestational diabetes, etc. OS is defined as an imbalance between the production of reactive species and the ability of an organism to detoxify the reactive intermediates and repair the damage. As a result of OS, the excess of reactive species such as oxygen superoxide (O2-), hydroxyl radical (OH), peroxynitrite (ONOO−), 4-hydroxynonenal (4HNE), etc., have a tendency to react with nearby proteins/nucleic acids/lipids changing their functionality or inactivating them completely. The organism has many ways to protect itself from the harmful effects of oxidants. One strategy employs antioxidants introduced to the body with food. The purpose of this thesis was to investigate the effect of reactive species on the active transport mediated by ABC efflux transporters as well as exploring the possibility of using antioxidants not as interceptors of reactive species but rather as inhibitors of metabolic enzymes and transporters. The BCRP/ABCG2 efflux transporter was selected for the investigation of the effect of reactive anion, ONOO−, generated during OS and the product of OS, 4HNE, formed after a series of chain reactions involving ROS. Experiments conducted with Sf9 membrane vesicles overexpressing BCRP/ABCG2 revealed that both species are capable of inactivating this ABC transporter with IC50 being 31 ± 2.7 μM and 92 ± 1.4 μM for ONOO− and 4HNE, respectively. In presence of 4HNE, Vmax decreased 4-fold and Km remained unchanged, suggesting a noncompetitive inhibition mechanism. However, with addition of 4HNE, positive cooperativity was also observed. With ONOO−, the situation was different: both Vmax and Km changed consistent with mixed type inhibition. Overall, OS-mediated BCRP/ABCG2 inactivation occurred at biologically relevant concentrations of the reactive species. Antioxidants are substances that are known to reduce the amount of ROS/RNS accumulated during OS, but this research considered the use of antioxidants not only as interceptors of ROS/RNS but rather as inhibitors of metabolic enzymes. The effect of the dietary antioxidant, quercetin (Qc), on the metabolism of 2-methoxyestradiol (2Me-E2), a promising potential anticancer agent was investigated. Qc possesses five hydroxyl groups, several of which are targets for UDP-glucuronosyltransferases (UGTs). Thus, the simultaneous presence of Qc and 2Me-E2 could result in decreased glucuronidation of 2Me-E2. Using the LS180 intestinal human colon adenocarcinoma cell line, glucuronidation of 2Me-E2 resulted in formation of only one major glucuronide, 2-Methoxyestradiol-3-glucuronide (2Me-E2-3G). Qc effectively reduced its formation (IC50 = 7.8 ± 0.26 μM) to a minimum level. The decrease in the activity of UGTs increased the intracellular concentration of parent 2Me-E2. Additional increase in cellular concentration of 2Me-E2 was achieved when LS180 cells were pre-incubated with Qc prior the addition of 2Me-E2. Transwell experiments with MDCKII – BCRP cells revealed that BCRP/ABCG2 did not appear to transport 2Me-E2. All in all, the present study showed that OS has a negative impact on active transport mediated by ABC transporters. This, in turn, can affect drug disposition and protection of endogenous organs and tissues. Antioxidants are one of the mechanisms that can effectively reduce the negative impact caused by oxidative species. Nevertheless, this research revealed that they can also be an effective tool to reduce the excessive metabolism of therapeutic drugs. Thus, Qc was found to be a dietary antioxidant that could reduce metabolism of 2Me-E2 and increase it intracellular concentration.

Os melanomas são o tipo mais mortal de câncer de pele, apesar da baixa incidência, 80% das mortes de câncer de pele são devem-se ao melanoma metastático. Novas abordagens farmacológicas e a busca por novos compostos para a terapêutica do melanoma, em aplicações isolados ou em combinação com outros fármacos é imprescindível. Esta busca ocorre principalmente no campo das terapias de alvos específicos, devido à aquisição de resistência tumoral e recidiva. O resveratrol (RES) é um polifenol com atividade anti-oxidante, e seu efeito anti-tumoral foi mostrado pela indução de morte celular, porém o seu estudo não foi aprofundado pela inviabilidade do uso de altas doses in vitro para observação de efeitos celulares. Outro composto, o 2-methoxiestradiol (2ME) é um metabólito do estrógeno cujo efeitos anti-câncer já foi demonstrado em melanoma, porém sem elucidação das vias de sinalização envolvidas. O efeito em células com resistência adquirida também nunca foram testados. Neste estudo ampliamos o painel de linhagens celulares de melanoma humano, e demonstramos que o 2ME induz morte celular, inibe a proliferação destas células sendo que esta inibição está associada a indução de senescência. Pela primeira vez foi observada a inibição de proliferação pelo 2ME em células com a mutação BRAF V600E resistentes ao vemurafenibe (inibidor de BRAF) e duplo resistentes ao vemurafenibe e trametinibe (inibidor de MEK). A inibição de proliferação foi acompanhada pela modulação de p21Cip1, Ciclina B1, pRb, proteínas envolvidas na regulação do ciclo celular. A exposição prolongada ao 2ME inibiu a formação de colônias em todas as linhagens de melanoma (não resistentes e resistentes), mas não teve o mesmo efeito em fibroblastos primários, mostrando efeito seletivo. Em modelo tridimensional de esferóides, foi observado que as linhagens resistentes (Sk-Mel-28R) e duplo resistentes (Sk- Mel-28RT) são mais invasivas que a parental (Sk-Mel-28). Neste modelo, o 2ME foi capaz de inibir a invasão e viabilidade destas células. No modelo de pele reconstituída, na ausência de tratamento, observa-se invasão das células de melanoma pela derme, porém este fenômeno é diminuído quando as peles são tratadas com 2ME. Estes resultados demonstram que o 2ME é um efetivo agente anti-melanoma, independente de sua resistência. / Melanomas are the deadliest type of skin cancer, and in spite of the low incidence, 80% of the skin cancer associated death cases are due to metastatic melanoma. New pharmacological approaches and the search of new compounds for melanoma therapeutics, for monotherapy or combination therapy, are essential. This search occurs mainly in the targeted therapy field because of the melanoma acquisition of resistance to the current treatments. Resveratrol (RES) is a polyphenol with anti-oxidant activity, and its anti-tumor effect has been shown through the induction of cell death. However, the study of this compound has been discontinued in this work due to the impossibility of using high doses in vitro for the observation of cellular effects. Another compound, 2-methoxyestradiol (2ME) is a metabolite from estrogen, and its anti-cancer effects has already been shown in melanoma, but with no elucidation of the signaling pathways involved. Furthermore, the effects in cells with acquired resistance have never been shown. In this study we used a broader panel of human melanoma cell lines and demonstrated that 2ME induces cell death, inhibits proliferation of these cells, and this inhibition is associated with the induction of cell senescence. The inhibition of proliferation caused by 2ME was observed for the first time in BRAF V600E cells that are resistant to Vemurafenib (BRAF inhibitor) and double resistant to Vemurafenib and Trametinib (MEK inhibitor). The proliferation inhibition was related to the modulation of p21Cip1,Cyclin B1 and pRb, which are proteins involved in cell cycle regulation. Long exposure to 2ME in colony formation assay showed the inhibition of colony in all melanoma cell lines (regardless of resistance and mutational status), but not in primary fibroblasts, showing selective effect. In three-dimensional spheroid model, it was observed that the resistant (Sk-Mel- 28R) and double-resistant (Sk-Mel-28RT) cell lines were more invasive than the parental cell line (Sk-Mel-28). In this model, 2ME was able to inhibit cell invasion and cell viability. In the skin reconstruct model, in the absence of treatment, melanoma cell invasion can be observed in the dermis layer. However, after the treatment with 2ME these cell invasion foci are inhibited. Altogether, these effects demonstrate that 2ME is an effective anti-melanoma agent, regardless of resistance.

Os melanomas são o tipo mais mortal de câncer de pele, apesar da baixa incidência, 80% das mortes de câncer de pele são devem-se ao melanoma metastático. Novas abordagens farmacológicas e a busca por novos compostos para a terapêutica do melanoma, em aplicações isolados ou em combinação com outros fármacos é imprescindível. Esta busca ocorre principalmente no campo das terapias de alvos específicos, devido à aquisição de resistência tumoral e recidiva. O resveratrol (RES) é um polifenol com atividade anti-oxidante, e seu efeito anti-tumoral foi mostrado pela indução de morte celular, porém o seu estudo não foi aprofundado pela inviabilidade do uso de altas doses in vitro para observação de efeitos celulares. Outro composto, o 2-methoxiestradiol (2ME) é um metabólito do estrógeno cujo efeitos anti-câncer já foi demonstrado em melanoma, porém sem elucidação das vias de sinalização envolvidas. O efeito em células com resistência adquirida também nunca foram testados. Neste estudo ampliamos o painel de linhagens celulares de melanoma humano, e demonstramos que o 2ME induz morte celular, inibe a proliferação destas células sendo que esta inibição está associada a indução de senescência. Pela primeira vez foi observada a inibição de proliferação pelo 2ME em células com a mutação BRAF V600E resistentes ao vemurafenibe (inibidor de BRAF) e duplo resistentes ao vemurafenibe e trametinibe (inibidor de MEK). A inibição de proliferação foi acompanhada pela modulação de p21Cip1, Ciclina B1, pRb, proteínas envolvidas na regulação do ciclo celular. A exposição prolongada ao 2ME inibiu a formação de colônias em todas as linhagens de melanoma (não resistentes e resistentes), mas não teve o mesmo efeito em fibroblastos primários, mostrando efeito seletivo. Em modelo tridimensional de esferóides, foi observado que as linhagens resistentes (Sk-Mel-28R) e duplo resistentes (Sk- Mel-28RT) são mais invasivas que a parental (Sk-Mel-28). Neste modelo, o 2ME foi capaz de inibir a invasão e viabilidade destas células. No modelo de pele reconstituída, na ausência de tratamento, observa-se invasão das células de melanoma pela derme, porém este fenômeno é diminuído quando as peles são tratadas com 2ME. Estes resultados demonstram que o 2ME é um efetivo agente anti-melanoma, independente de sua resistência. / Melanomas are the deadliest type of skin cancer, and in spite of the low incidence, 80% of the skin cancer associated death cases are due to metastatic melanoma. New pharmacological approaches and the search of new compounds for melanoma therapeutics, for monotherapy or combination therapy, are essential. This search occurs mainly in the targeted therapy field because of the melanoma acquisition of resistance to the current treatments. Resveratrol (RES) is a polyphenol with anti-oxidant activity, and its anti-tumor effect has been shown through the induction of cell death. However, the study of this compound has been discontinued in this work due to the impossibility of using high doses in vitro for the observation of cellular effects. Another compound, 2-methoxyestradiol (2ME) is a metabolite from estrogen, and its anti-cancer effects has already been shown in melanoma, but with no elucidation of the signaling pathways involved. Furthermore, the effects in cells with acquired resistance have never been shown. In this study we used a broader panel of human melanoma cell lines and demonstrated that 2ME induces cell death, inhibits proliferation of these cells, and this inhibition is associated with the induction of cell senescence. The inhibition of proliferation caused by 2ME was observed for the first time in BRAF V600E cells that are resistant to Vemurafenib (BRAF inhibitor) and double resistant to Vemurafenib and Trametinib (MEK inhibitor). The proliferation inhibition was related to the modulation of p21Cip1,Cyclin B1 and pRb, which are proteins involved in cell cycle regulation. Long exposure to 2ME in colony formation assay showed the inhibition of colony in all melanoma cell lines (regardless of resistance and mutational status), but not in primary fibroblasts, showing selective effect. In three-dimensional spheroid model, it was observed that the resistant (Sk-Mel- 28R) and double-resistant (Sk-Mel-28RT) cell lines were more invasive than the parental cell line (Sk-Mel-28). In this model, 2ME was able to inhibit cell invasion and cell viability. In the skin reconstruct model, in the absence of treatment, melanoma cell invasion can be observed in the dermis layer. However, after the treatment with 2ME these cell invasion foci are inhibited. Altogether, these effects demonstrate that 2ME is an effective anti-melanoma agent, regardless of resistance.

2-Methoxyestradiol (2ME) is an endogenous estrogen metabolite with antiproliferative and antiangiogenic properties. 2ME also plays an active role in the induction of apoptosis, especially in cancerous cells. These properties have been confirmed by various in vitro and in vivo studies and render 2ME a potential antitumor agent. The mechanism of action of 2ME, however, is not yet fully elucidated and it is believed that multiple mechanisms are involved that may be dependent on cell type. The aim of this study was to investigate the differential effects of 2ME on cell growth, morphology and spindle formation in the non-tumorigenic MCF-12A breast cell line and the tumorigenic MCF-7 breast cell line. In dose-dependent studies, cell growth was determined spectrophotometrically. Light microscopy was used to investigate the morphological changes induced by 2ME and its effect on spindle formation was investigated by means of indirect immunofluorescence. The estrogen receptor status of the MCF-12A cells was confirmed with immunocytochemistry. In order to investigate the effect of 2ME on the length of the cell cycle, cells were blocked in early S-phase with hydroxyurea, then allowed to continue through the cell cycle and mitotic indices determined at regular time intervals. Checkpoint kinase and Cdc2 kinase assays were used to determine the effect of 2ME on relevant cell cycle kinases. Although 2ME inhibited cell growth in both cell lines, the MCF-7 cells were inhibited from much lower concentrations and growth inhibition was more pronounced than in the MCF-12A cells. Treated MCF-7 cells showed abnormal metaphase cells, membrane blebbing, apoptotic cells and disrupted spindle formation. These observations were either absent, or not as prominent in the MCF-12A cells. Therefore, differential mechanism(s) of growth inhibition are evident between the normal and tumorigenic cells. Although the two cell lines differ in their estrogen receptor status, this could not explain the differential effects, for 2ME has a very low affinity for the estrogen receptor. 2ME had no effect on the length of the cell cycle, but blocked MCF-7 cells in mitosis. There were no significant alterations in the phosphorylation status of Cdc25C after 2ME treatment. However, Cdc2 activity was increased to a greater extend in the MCF-7 cells than in the MCF-12A cells. Therefore, it is suggested that exposure to 2ME disrupts mitotic spindle formation and enhances Cdc2 kinase activity, leading to persistence of the spindle checkpoint and thus prolonged metaphase arrest, which may result in the induction of apoptosis. The tumorigenic MCF-7 cells are especially sensitive to 2ME treatment compared to the normal MCF-12A cells. 2ME shows potential for the treatment of breast cancer. Selecting the concentration of 2ME that has maximum inhibitory effect on tumorigenic, but minimal effect on normal cells is crucial in its possible application as antitumor agent. Furthermore, research concerning the differential action mechanisms of 2ME is essential to create a better understanding regarding the treatment of cancer and may possibly contribute to the development and/or improvement of novel chemotherapeutic agents. / Dissertation (MSc (Physiology))--University of Pretoria, 2008. / Physiology / unrestricted

Paclitaxel is one of the most successful anti-cancer drugs, particularly in the treatment of breast cancer and ovarian cancer. For the investigation of the interaction between paclitaxel and MD-2 protein, and development of new antagonists for lipopolysaccharide, several C10 A-nor-paclitaxel analogs have been synthesized and their biological activities have been evaluated. In order to reduce the myelosuppression effect of the paclitaxel, several C3â ² and C4 paclitaxel analogs have been synthesized and their biological evaluation have been studied. / Master of Science

<p>Prostate cancer is common in the Western society and current treatments are often associated with side effects, therefore improved therapeutic strategies are desired. 2-methoxyestradiol (2-ME), an endogenous metabolite of estradiol-17β inhibits tumor growth <i>in vivo</i> as it prevents angiogenesis. 2-ME has also direct cytotoxic effects on tumor cells. In this study, we have investigated the potential use of PET to record effects 2-ME on prostate cancer cell (PC3) aggregates. The anti-proliferative and pro-apoptotic effects of 2-ME on PC3 cell aggregates <i>in vitro</i> were correlated with the uptake of deoxy-D-glucose, FMAU and choline labeled with ¹⁸F, ¹¹C or ³H. 2-ME clearly reduced growth of PC3 aggregates and induced apoptosis in a dose-dependent manner. However, the PET tracers failed to record the cytotoxicity of 2-ME on PC3 aggregates. </p><p>Further, the signaling events responsible for 2-ME induced prostate cancer cell death were investigated. We found that Smad7, previously implicated in TGF-β-induced responses, is required for 2-ME-induced p38 MAPK activation and subsequent apoptosis in PC-3U cells, as shown by the use of antisense or siRNA techniques and a specific inhibitor of p38 MAPK (SB203580). Interestingly, Smad7 also regulated the expression of the pro-apoptotic Bim protein. </p><p>Shb is a Src Homology 2 domain adapter protein with pro-apoptotic effects. PC3 clones overexpressing Shb exhibited increased rates of apoptosis, both in the presence or absence of 2-ME, as they failed to activate survival mechanisms through ERK and Akt in response to 2-ME. Notably, Shb cells displayed increased activity of the pro-apoptotic kinase c-Abl. Pre-treatment with SB203580 or c-Abl (STI-571) inhibitors completely blocked the apoptotic response to 2-ME. </p><p>In conclusion, Smad7 and Shb appear to be crucial for 2-ME-induced PC3 cell apoptosis via their activation of p38 MAPK and c-Abl. Future therapies exploring these pathways can be envisaged as treatment of prostate cancer.</p>

Prostate cancer is common in the Western society and current treatments are often associated with side effects, therefore improved therapeutic strategies are desired. 2-methoxyestradiol (2-ME), an endogenous metabolite of estradiol-17β inhibits tumor growth in vivo as it prevents angiogenesis. 2-ME has also direct cytotoxic effects on tumor cells. In this study, we have investigated the potential use of PET to record effects 2-ME on prostate cancer cell (PC3) aggregates. The anti-proliferative and pro-apoptotic effects of 2-ME on PC3 cell aggregates in vitro were correlated with the uptake of deoxy-D-glucose, FMAU and choline labeled with 18F, 11C or 3H. 2-ME clearly reduced growth of PC3 aggregates and induced apoptosis in a dose-dependent manner. However, the PET tracers failed to record the cytotoxicity of 2-ME on PC3 aggregates. Further, the signaling events responsible for 2-ME induced prostate cancer cell death were investigated. We found that Smad7, previously implicated in TGF-β-induced responses, is required for 2-ME-induced p38 MAPK activation and subsequent apoptosis in PC-3U cells, as shown by the use of antisense or siRNA techniques and a specific inhibitor of p38 MAPK (SB203580). Interestingly, Smad7 also regulated the expression of the pro-apoptotic Bim protein. Shb is a Src Homology 2 domain adapter protein with pro-apoptotic effects. PC3 clones overexpressing Shb exhibited increased rates of apoptosis, both in the presence or absence of 2-ME, as they failed to activate survival mechanisms through ERK and Akt in response to 2-ME. Notably, Shb cells displayed increased activity of the pro-apoptotic kinase c-Abl. Pre-treatment with SB203580 or c-Abl (STI-571) inhibitors completely blocked the apoptotic response to 2-ME. In conclusion, Smad7 and Shb appear to be crucial for 2-ME-induced PC3 cell apoptosis via their activation of p38 MAPK and c-Abl. Future therapies exploring these pathways can be envisaged as treatment of prostate cancer.