Class of 2006 Abstract / Doxorubicin is a member of the anthracycline antibiotic class of chemotherapeutic agents and is among the most effective chemotherapies in the treatment of breast cancer1. However, the efficacy of the drug is limited by its ability to cause a heart failure. In younger women with high – risk breast cancers, doxorubicin improves their chances of survival from the breast cancer, but the risk of heart failure, should they survive, will shorten their lives substantially and reduce their quality of life. Periodic monitoring of left ventricular ejection fraction (LVEF) during treatment with doxorubicin has been shown to reduce morbidity and mortality from doxorubicin – induced heart failure19 and for the past 20 years, the Schwartz guidelines have provided an algorithm for the frequency of LVEF monitoring19. According to this guideline, women in their 30’s who are otherwise healthy do not receive LVEF monitoring until they have received a threshold dose of 250-300mg/m2. We have conducted a decision – analysis cost – effectiveness study of heart failure prevention by LVEF monitoring with equilibrium radionuclide angiography (ERNA) either in adherence to the Schwartz guidelines or before each dose of doxorubicin (serial monitoring). Our findings show that, both in terms of quality of life and cost outcomes, ERNA monitoring of LVEF according to the Schwartz guidelines is the dominant strategy. Monte Carlo analysis of 5000 patients showed that the Schwartz guidelines have an average cost- effectiveness of $110/QALY, versus $390/QALY for serial monitoring. Sensitivity analysis of uncertain variables invariably showed that Schwartz guidelines are dominant.

A resistência à quimioterapia, a qual se constitui em um dos maiores desafios a serem superados durante o tratamento do cancer de mama, pode ser modulada por várias cascatas de sinalização cellular que levam ao aumento da sobrevivência celular. A via PI3K/Akt/mTOR é uma importante reguladora desse processo, visto que ela pode favorecer o crescimento tumoral após sua ativação por diversos receptores de fatores de crescimento, incluindo os receptores HER-2. Assim, nossa proposta foi investigar se a inibição dessa via poderia aumentar a sensibilidade de linhagens epiteliais mamárias humanas ao agente quimioterápico doxorrubicina. Também decidimos analisar o perfil de expressão gênica gerado por essa abordagem com o objetivo de compreender os mecanismos moleculares associados à resposta biológica. A rapamicina (um inibidor de mTOR) e a doxorrubicina foram utilizadas individualmente ou em combinação no tratamento de três linhagens epiteliais mamárias humanas: HB4a, C5.2 e SKBR3. A linhagem HB4a, uma linhagem derivada de epitélio mamário normal, expressa níveis basais do receptor HER-2. As outras duas linhagens expressam altos níveis desse receptor: a linhagem SKBR3 (derivada de adenocarcinoma mamário), expressa constitutivamente esse receptor e a linhagem C5.2 foi derivada da linhagem HB4a pela transfecção com o cDNA full-lenght do gene HER-2. A células foram tratadas com 20 nM de rapamicina associada ou não a doses de 30 nM a 1000 nM de doxorrubicina por 24, 48 e 72 horas. Após 24 horas, os resultados de citometria de fluxo mostraram que, enquanto as linhagens SKBR3 e C5.2 apresentaram indução de parada na fase G0/G1 do ciclo celular, a linhagem HB4a não demonstrou alterações significativas na distribuição do ciclo celular pelo tratamento com rapamicina. Esses resultados sugeriram que as linhagens SKBR3 e C5.2 foram mais sensíveis à rapamicina quando comparadas à linhagem HB4a. A doxorrubicina, por sua vez, induziu parada na fase S-G2/M do ciclo celular, a qual foi maior na linhagem HB4a comparada com as demais. De forma similar ao tratamento com doxorrubicina sozinha, o tratamento combinado também induziu parada em S-G2/M nas linhagens HB4a e C5.2. Assim, os efeitos da doxorrubicina foram predominantes aos da rapamicina na distribuição do ciclo celular no tratamento combinado nessas linhagens. Por outro lado, a linhagem SKBR3 mostrou um efeito proeminente da rapamicina, visto que sua associação com doxorrubicina induziu parada na fase G0/G1 do ciclo celular após exposição a 30 nM de doxorrubicina. Os ensaios de viabilidade celular mostraram que o tratamento combinado foi 20% mais eficiente na inibição do crescimento celular quando comparado à doxorrubicina sozinha nas três linhagens celulares, o que foi associado a uma redução de 2 vezes no IC50 da doxorrubicina. Para cada linhagem, os dados de microarray foram analisados usando o SAM (FDR 5%) para comparar os tratamento com doxorrubicina, rapamicina e o tratamento combinado aos seus respectivos controles. Considerando apenas as linhagens C5.2 e SKBR3, a anotação funcional dos genes modulados pelo tratamento combinado mostrou que os genes relacionados ao tratamento combinado estavam relacionados ao rearranjo de RNAs (splicing). Além disso, genes exclusivamente modulados pelo tratamento combinado mostrou funções associadas ao transporte intracellular enquanto que a resposta mediada por HER-2 no tratamento combinado incluiu alterçãoes na via do ubiquitina-proteossomo, glicólise e cadeia trasnportadora de elétrons. Em conjunto, nossos resultados pareceram afetar vias moleculares importantes relacionadas à sobrevivência tumoral em linhagens caracterizadas pela superexpressão de HER-2, o que foi associado a uma supressão de 2 vezes no IC50 da doxorrubicina. Esses dados ressaltam a relevância clínica da terapia combinada no contexto do HER-2, visto que essa associação pode ser capaz de reduzir a toxicidade dos pacientes submetidos à quimioterapia / Chemotherapy resistance, which is the major challenge to be overcome during breast cancer treatment, can be modulated by several signaling cascades that lead to increased cell survival. The PI3K/Akt/mTOR pathway is an important regulator of this process, as it can favour the tumor growth after its activation by several growth factor receptors, including HER-2 receptors. Thus, we proposed to investigate whether the inhibition of this pathway could improve the sensitivity of human HER-2-overexpressing breast epithelial cell lines to the chemotherapeutic agent doxorubicin. We also decided to analyze the gene expression profile generated by this approach to better understand the molecular mechanisms associated with the biological response. Rapamycin (a mTOR inhibitor) and doxorubicin were used individually or in combination on three human breast epithelial cell lines: HB4a, C5.2 e SKBR3. The HB4a cell, a normal mammary epithelial cell line, expresses basal levels of HER-2. The two other cell lines express high levels of this receptor: the SKBR3 cell (derived from a mammary adenocarcinoma) expresses constitutively this receptor.and the C5.2 cell was derived from HB4a cells transfected with the cDNA-full length of HER-2. The cells were treated with 20 M of rapamycin with or without 30nM to 1000nM of doxorubicin for 24, 48 and 72 hours. After 24 hours, the flow citometry results showed that while SKBR3 and C5.2 were arrested in G0/G1 phase, HB4a demonstrated no alterations in the cell cycle distribution after rapamycin treatment. These results showed that SKBR3 and C5.2 cells were more sensitive to rapamycin compared to HB4a. Doxorubicin, in turn, led the cells to a S-G2/M arrest which was greater in HB4a cells compared to SKBR3 and C5.2. Similarly to doxorubicin alone, the combined treatment also caused an increase of S-G2/M arrest in HB4a and C5.2 cells. So, doxorubicin effects were predominant to the rapamycin effects on the cell cycle distribution of the combined treatment in these cell lines. Otherwise, SKBR3 showed a predominant effect of rapamycin since its association with doxorubicin caused a G0/G1 arrest after exposure to 30 nM of doxorubicin. The viability assays showed that the combined treatment was 20% more effective in inhibiting cell growth than doxorubicin alone in the three cell lines, which was associated with a reduction in the IC50 of doxorubicin by 2-fold. For each cell line, microarray data were analyzed using SAM (FDR 5%) to compare doxorubicin, rapamycin and the combined treatment to the respective untreated cells. Considering only C5.2 and SKBR3 cells, Gene Ontology (GO) annotations of the genes modulated by the combined treatment showed altered genes related to RNA splicing. Moreover, genes exclusively modulated by the combined treatment showed altered functions related to intracellular trafficking whereas HER-2-mediated response to the combined treatment included changes in the ubiquitin-proteasome pathway, glycolysis and electron transport chain. Altogether, our results seemed to affect important molecular pathways related to tumor survival in HER-2- overexpressing cell lines, which was associated to a 2-fold suppression in the IC50 of doxorubicin. These findings highlight the clinical relevance of the rapamycin/doxorubicin combination therapy in HER-2 context, since this association could be able to decrease toxicity of patients undergoing chemotherapy

O objetivo deste trabalho foi estudar a permeação iontoforética da Doxorrubicina (DOX) em formulações semi-sólidas e testar a citotoxicidade destas formulações em cultura de células de melanoma com e sem a aplicação de corrente elétrica de baixa intensidade. O estudo de liberação da DOX das formulações (gel de Hidroxietilcelulose ? HEC, gel de quitosana e solução aquosa) mostrou que o gel de quitosana possuiu uma velocidade de liberação quase três vezes maior do que nas demais formulações. Os estudos de permeação passiva mostraram que o fármaco não atravessa a pele em quantidades detectáveis. No entanto, a iontoforese contribui significativamente não só na permeação da DOX, mas também na sua retenção cutânea. O gel de HEC foi o que levou a uma maior retenção cutânea do fármaco em comparação com as demais formulações. Nos estudos de citotoxicidade realizados em células de melanoma de camundongos, verificou-se que as formulações contendo DOX possuíram citotoxicidade maior comparadas ao controle (solução de DOX). Isso significa que os componentes de cada formulação contribuem no poder de citotoxicidade contra as células de melanoma. A solução de monoleína 5% em propilenoglicol apresentou maior atividade citotóxica dentre todas as formulações estudadas. Seus componentes, monoleína e propilenoglicol contribuem sinergicamente para sua atividade citotóxica, a qual é de aproximadamente 90% quando a concentração de DOX é de 20 ng/mL. Enquanto que em solução de DOX sua citotoxicidade é de aproximadamente 34% na mesma concentração. Foi feita a padronização dos estudos de aplicação de corrente elétrica em cultura de células, quanto a placa de cultura, número de células, volume do meio de cultura e ponte salina (utilizada para a passagem da corrente para o meio de cultura). A aplicação de 0,1 a 0,5 mA/cm2 de corrente elétrica não causou morte significativa para as células de melanoma quando aplicada por um período de 10 a 60 minutos. A citotoxicidade das formulações com e sem aplicação de corrente elétrica por 10 minutos não apresentaram diferença significativa. Porém, a aplicação de 20 minutos de corrente elétrica aumentou significativamente a citotoxicidade da DOX em solução aquosa. Conclui-se, resumidamente, que a aplicação de corrente elétrica de baixa intensidade aumentou a penetração da DOX através da pele e auxiliou a entrada do fármaco nas células tumorais, quando esta é dissolvida em solução aquosa. / The aim of this work was to study the iontophoretic delivery of Doxorubicin (DOX) dispersed in semi-solid formulations and test its citotoxicity activity on melanoma cell lines, with or without the application of a low intensity electrical current. The release study of DOX from the formulations (hydroxyethylcellulose ? HEC, chitosan gel and aqueous solution) showed that chitosan gel increased almost 3 times the diffusion coefficient of the drug when compared to a water solution. Passive permeation studies showed that the drug does not cross the skin in detected amounts. However, iontophoresis of DOX increased significantly not only the permeation but also the skin retention of the drug. HEC gel improved DOX skin retention when compared to other formulations. Cytotoxicity studies, performed in rat melanoma cell culture indicated that formulations containing DOX have high citotoxicity compared to the control (DOX solution). These results means that the components of the formulations probably contribute to melanoma cells death. Monoolein 5% solution in propileneglicol showed high citotoxicity compared to the other formulations. Its components act sinergically and produce a great citotoxicity: approximately 90% when the concentration of DOX is 20 ng/mL, whereas in DOX solution its citotoxicity is approximately 34% on this concentration. Standardization of the electrical current studies has been made as matter as the culture plate, number of cells, volume of culture medium and agar bridge (used to pass electrical current for the culture medium). The application of 0,1 to 0,5 mA/cm2 of electrical current during 10 to 60 minutes did not kill melanoma cell lines significantly. The cytotoxicity of DOX incorporated in water and semi-solid formulations are not statistical different in the presence or not of an electrical current for 10 minutes. However, 20 minutes of an electrical current raised significantly the citotoxicity effects of DOX in aqueous solution. In summary, the application of low intensity electrical current increases the penetration of DOX through the skin and helps the drug to enter into the tumor cells, when dispersed in water solution

A resistência à quimioterapia, a qual se constitui em um dos maiores desafios a serem superados durante o tratamento do cancer de mama, pode ser modulada por várias cascatas de sinalização cellular que levam ao aumento da sobrevivência celular. A via PI3K/Akt/mTOR é uma importante reguladora desse processo, visto que ela pode favorecer o crescimento tumoral após sua ativação por diversos receptores de fatores de crescimento, incluindo os receptores HER-2. Assim, nossa proposta foi investigar se a inibição dessa via poderia aumentar a sensibilidade de linhagens epiteliais mamárias humanas ao agente quimioterápico doxorrubicina. Também decidimos analisar o perfil de expressão gênica gerado por essa abordagem com o objetivo de compreender os mecanismos moleculares associados à resposta biológica. A rapamicina (um inibidor de mTOR) e a doxorrubicina foram utilizadas individualmente ou em combinação no tratamento de três linhagens epiteliais mamárias humanas: HB4a, C5.2 e SKBR3. A linhagem HB4a, uma linhagem derivada de epitélio mamário normal, expressa níveis basais do receptor HER-2. As outras duas linhagens expressam altos níveis desse receptor: a linhagem SKBR3 (derivada de adenocarcinoma mamário), expressa constitutivamente esse receptor e a linhagem C5.2 foi derivada da linhagem HB4a pela transfecção com o cDNA full-lenght do gene HER-2. A células foram tratadas com 20 nM de rapamicina associada ou não a doses de 30 nM a 1000 nM de doxorrubicina por 24, 48 e 72 horas. Após 24 horas, os resultados de citometria de fluxo mostraram que, enquanto as linhagens SKBR3 e C5.2 apresentaram indução de parada na fase G0/G1 do ciclo celular, a linhagem HB4a não demonstrou alterações significativas na distribuição do ciclo celular pelo tratamento com rapamicina. Esses resultados sugeriram que as linhagens SKBR3 e C5.2 foram mais sensíveis à rapamicina quando comparadas à linhagem HB4a. A doxorrubicina, por sua vez, induziu parada na fase S-G2/M do ciclo celular, a qual foi maior na linhagem HB4a comparada com as demais. De forma similar ao tratamento com doxorrubicina sozinha, o tratamento combinado também induziu parada em S-G2/M nas linhagens HB4a e C5.2. Assim, os efeitos da doxorrubicina foram predominantes aos da rapamicina na distribuição do ciclo celular no tratamento combinado nessas linhagens. Por outro lado, a linhagem SKBR3 mostrou um efeito proeminente da rapamicina, visto que sua associação com doxorrubicina induziu parada na fase G0/G1 do ciclo celular após exposição a 30 nM de doxorrubicina. Os ensaios de viabilidade celular mostraram que o tratamento combinado foi 20% mais eficiente na inibição do crescimento celular quando comparado à doxorrubicina sozinha nas três linhagens celulares, o que foi associado a uma redução de 2 vezes no IC50 da doxorrubicina. Para cada linhagem, os dados de microarray foram analisados usando o SAM (FDR 5%) para comparar os tratamento com doxorrubicina, rapamicina e o tratamento combinado aos seus respectivos controles. Considerando apenas as linhagens C5.2 e SKBR3, a anotação funcional dos genes modulados pelo tratamento combinado mostrou que os genes relacionados ao tratamento combinado estavam relacionados ao rearranjo de RNAs (splicing). Além disso, genes exclusivamente modulados pelo tratamento combinado mostrou funções associadas ao transporte intracellular enquanto que a resposta mediada por HER-2 no tratamento combinado incluiu alterçãoes na via do ubiquitina-proteossomo, glicólise e cadeia trasnportadora de elétrons. Em conjunto, nossos resultados pareceram afetar vias moleculares importantes relacionadas à sobrevivência tumoral em linhagens caracterizadas pela superexpressão de HER-2, o que foi associado a uma supressão de 2 vezes no IC50 da doxorrubicina. Esses dados ressaltam a relevância clínica da terapia combinada no contexto do HER-2, visto que essa associação pode ser capaz de reduzir a toxicidade dos pacientes submetidos à quimioterapia / Chemotherapy resistance, which is the major challenge to be overcome during breast cancer treatment, can be modulated by several signaling cascades that lead to increased cell survival. The PI3K/Akt/mTOR pathway is an important regulator of this process, as it can favour the tumor growth after its activation by several growth factor receptors, including HER-2 receptors. Thus, we proposed to investigate whether the inhibition of this pathway could improve the sensitivity of human HER-2-overexpressing breast epithelial cell lines to the chemotherapeutic agent doxorubicin. We also decided to analyze the gene expression profile generated by this approach to better understand the molecular mechanisms associated with the biological response. Rapamycin (a mTOR inhibitor) and doxorubicin were used individually or in combination on three human breast epithelial cell lines: HB4a, C5.2 e SKBR3. The HB4a cell, a normal mammary epithelial cell line, expresses basal levels of HER-2. The two other cell lines express high levels of this receptor: the SKBR3 cell (derived from a mammary adenocarcinoma) expresses constitutively this receptor.and the C5.2 cell was derived from HB4a cells transfected with the cDNA-full length of HER-2. The cells were treated with 20 M of rapamycin with or without 30nM to 1000nM of doxorubicin for 24, 48 and 72 hours. After 24 hours, the flow citometry results showed that while SKBR3 and C5.2 were arrested in G0/G1 phase, HB4a demonstrated no alterations in the cell cycle distribution after rapamycin treatment. These results showed that SKBR3 and C5.2 cells were more sensitive to rapamycin compared to HB4a. Doxorubicin, in turn, led the cells to a S-G2/M arrest which was greater in HB4a cells compared to SKBR3 and C5.2. Similarly to doxorubicin alone, the combined treatment also caused an increase of S-G2/M arrest in HB4a and C5.2 cells. So, doxorubicin effects were predominant to the rapamycin effects on the cell cycle distribution of the combined treatment in these cell lines. Otherwise, SKBR3 showed a predominant effect of rapamycin since its association with doxorubicin caused a G0/G1 arrest after exposure to 30 nM of doxorubicin. The viability assays showed that the combined treatment was 20% more effective in inhibiting cell growth than doxorubicin alone in the three cell lines, which was associated with a reduction in the IC50 of doxorubicin by 2-fold. For each cell line, microarray data were analyzed using SAM (FDR 5%) to compare doxorubicin, rapamycin and the combined treatment to the respective untreated cells. Considering only C5.2 and SKBR3 cells, Gene Ontology (GO) annotations of the genes modulated by the combined treatment showed altered genes related to RNA splicing. Moreover, genes exclusively modulated by the combined treatment showed altered functions related to intracellular trafficking whereas HER-2-mediated response to the combined treatment included changes in the ubiquitin-proteasome pathway, glycolysis and electron transport chain. Altogether, our results seemed to affect important molecular pathways related to tumor survival in HER-2- overexpressing cell lines, which was associated to a 2-fold suppression in the IC50 of doxorubicin. These findings highlight the clinical relevance of the rapamycin/doxorubicin combination therapy in HER-2 context, since this association could be able to decrease toxicity of patients undergoing chemotherapy

Cognitive decline is increasingly recognized as a side effect of chemotherapy. However, cognitive decline doesn't occur in all patients receiving chemotherapy, and there is variability in the cognitive domains affected (Ahles; JCO,Oct 20, 2012:3675-3686). Safety pharmacogenomics, i.e. using genetic variations to predict response/toxicity, offers an exciting approach to identify the subset of patients most likely to suffer from cognitive decline post chemotherapy. Consequently specific therapeutic interventions can be developed to target this group of patients, and/or alternate chemotherapeutic regimens can be used to limit toxicity, thereby offering a way to individualize therapy while minimizing toxicity. In our research we studied the effect of 16 SNPs in 6 genes on cognition in a sample of healthy older adults. We found that SNPs that affect serotonin, dopamine and glutamate levels in the brain influence cognition in a healthy sample of older adults, possibly in a domain specific manner. This allowed us to identify a group of healthy adults who inherently have lower cognitive functioning in some domains but that is still within the normal range. In addition individuals with SNPs that previously were associated with lower levels of myeloperoxidase performed better on the executive functions, verbal memory, verbal IQ and IQ. SNPs associated with lower levels were also associated with improvement in self reported verbal and visual memory post chemotherapy. APOE E2 allele was associated with higher cognitive performance compared to other alleles. However we didn't see an effect of APOE post chemotherapy. In chapter five, the effects of 31 SNPs in 15 genes on cognition post chemotherapy were evaluated in community dwelling lymphoma patients. Changes in the domains of verbal memory, visual perceptual memory, and attention of the Multiple Ability Self Report Questionnaire were observed following chemotherapy, but only when groups were stratified by genotype. Contrary to what we might expect, patients showed improvements in function after chemotherapy. However, using patient stratification based on genotype, specific groups of patients had a measurable decline in cognitive function post chemotherapy. Interestingly a SNP in the DNA replication enzyme and the target of doxorubicin topoisomerase II was associated with varying degree of self reported attention; specifically the AA genotype of rs471692 was associated with statistically significant decline in attention post chemotherapy. This indicates that cognitive changes following chemotherapy can be subtle, and stratification by genotype helps us in identifying susceptible individuals and provides some insights on the inconsistencies that are frequently reported in the literature. These results allow for identifying genetic risk factors associated with chemotherapy-induced cognitive changes, which will ultimately help in developing therapeutic approaches for the management of those deficits. Strategies to avoid chemotherapy-induced cognitive changes will be prospectively evaluated in future studies and include alternative chemotherapy and less toxic regimens, intervention strategies to improve cognitive abilities, and drug therapy to improve cognition in patients who develop chemotherapy-induced cognitive changes. The overarching goals of our studies are to help improve cancer patients' quality of life while maintaining or improving cancer cure rates.

The sarcoplasmic reticulum is the intramuscular organelle responsible for the regulation of cytoplasmic calcium levels in muscle. This thesis investigates the effects of the cardioprotective drug, dexrazoxane, and its metabolite ADR-925 on doxorubicin induced calcium release from skeletal sarcoplasmic reticulum. Doxorubicin is a widely used antineoplastic agent. One of the major side effects of doxorubicin usage is chronic cardiotoxicity. Doxorubicin is a potent activator of the calcium release mechanism from the SR. The interaction between doxorubicin and the calcium release channel has been proposed as the possible underlying mechanism behind cardiotoxicity. A short overview of different hypotheses describing doxorubicin induced cardiotoxicity and proposed mechanisms of cardioprotection by dexrazoxane are presented. While dexrazoxane did not appear to affect the calcium permeability of the SR, its metabolite, ADR-925, modulates the ryanodine receptor complex. ADR-925 inhibits high affinity ryanodine binding to the ryanodine receptor/calcium release channel complex by decreasing the sensitivity of the receptor for stimulation by calcium. ADR-925's ability to inhibit doxorubicin stimulated ryanodine binding is independent of the doxorubicin concentration. These results demonstrate that ADR-925 directly affects the ryanodine receptor complex of the SR by desensitizing the receptor to activation by calcium. Furthermore, ADR-925 reduces the inhibitory effect of hydrogen peroxide on the ryanodine receptor/ calcium release channel. This suggests that ADR-925 may protect the SR from oxidative effects of free radicals. It has been somewhat controversial whether doxorubicin induced cardiotoxicity is due to a specific interaction with the calcium release mechanism of SR. The findings presented in this thesis which demonstrate that the cardioprotectant ADR-925 interacts directly with the ryanodine receptor from SR, further support the hypothesis that the ryanodine receptor is a primary target of doxorubicin's action.

Cardiotoxicity associated with many cancer drugs is a critical issue facing physicians these days and a huge hurdle that must be overcome for a side effects-free cancer therapy. Survival of cardiac myocytes is compromised upon the exposure to certain chemotherapeutic drugs. Unfortunately, the mechanisms implicated in cardiac toxicity and the pathways governing myocyte survival are poorly understood. The following thesis addresses the mechanisms underlying the cardiotoxicity of two anticancer drugs, doxorubicin (DOX) and Imatinib mesylate (Gleevec). Transcription factor GATA-4, has recently emerged as an indispensable factor in the adult heart adaptive response and cardiomyocyte survival. Therefore, the specific aim of this project was to determine the role of GATA-4, its upstream regulators, as well as partners in survival. A combination of cell and molecular techniques done on in vivo, and ex vivo models were utilized to tackle these issues. In this study, we confirmed the cardiotoxicity of the anticancer drug, Imatinib mesylate and found to be age dependent. GATA-4, already known to be implicated in DOX-induced toxicity, was confirmed as an Imatinib target. At the molecular level, we identified IGF-1 and AKT as upstream regulators of GATA-4. Moreover, we confirmed ZFP260 (PEX-1), a key regulator of the cardiac hypertrophic response, as a GATA-4 collaborator in common prosurvival pathways. Collectively, these results provide new insights on the mechanisms underlying drug-induced cardiotoxicity and raise the exciting possibility that cancer drugs are negatively affecting the same prosurvival pathway(s), in which GATA-4 is a critical component. Therapeutic interventions aimed at enhancing GATA-4 activity may be interesting to consider in the context of treatments with anticancer drugs.

<p>Traditionally, the goal of nanoparticle-based chemotherapy has been to decrease normal tissue toxicity by improving drug specificity to tumor. Relying on the EPR effect (Enhanced Permeability and Retention), a host of nanoparticles (from micelles and dendrimers to liposomes and lipidic nanoparticles) have been developed and tested for passive accumulation into tumor interstitium. Unfortunately, most nanoparticles achieve only suboptimal drug delivery to tumors, due to heterogeneity of tumor vessel permeability, limited nanoparticle penetration, and relatively slow drug release. However, recent developments in nanoparticle technology have occurred with the design and testing of a fast drug-releasing liposome triggered by local heat. This temperature-sensitive liposome formulation loaded with doxorubicin (Dox-TSL) has already shown substantial anti-tumor efficacy and is currently in clinical trials.</p><p> Previous pre-clinical work to understand the mechanism of efficacy has illustrated increases in overall drug concentration in the tumor, and an anti-vascular effect not observed with heat alone. These initial studies have also suggested that these liposomes may be the most efficacious when they are injected into a pre-heated tumor, with the hypothesis that in this treatment scheme the liposomes may be releasing inside the tumor vasculature. However, whether intravascular release is indeed occurring, and the subsequent implications this paradigm change in drug delivery could have are still unanswered questions. </p><p>The experiments presented herein aimed to investigate two effects: the existence and influence of intravascular drug release on drug delivery and distribution within the tumor, and the effect of drug delivery on subsequent anti-vascular effects. To investigate drug delivery, two mouse models were used. Dorsal window chambers implanted with FaDu human squamous carcinomas were used with real-time intravital confocal microscopy to evaluate time-resolved delivery of doxorubicin and liposome extravasation over the first 20 minutes of treatment. As a complimentary mouse model, flank FaDu tumors were also treated with Dox-TSL or treatment controls (doxorubicin with and without heat and Doxil with heat), and subsequently sectioned and histologicaly imaged to evaluate drug delivery and penetration depth, as well as impact on hypoxia and perfusion parameters. To investigate vascular effects, a GFP-eNos transgenic mouse model was used, also with window chamber confocal microscopy, to evaluate morphological changes occurring in the tumor vasculature following treatment.</p><p> The results presented herein demonstrate that contrary to the traditional liposome paradigm of extravasation and subsequent drug release, thermally sensitive liposomes release drug inside the tumor vasculature, and that the released free drug diffuses into the tumor interstitium. Real-time confocal imaging of doxorubicin delivery to murine tumor window chambers illustrates that intravascular drug release provides a mechanism to increase both the time that tumor cells are exposed to maximum drug levels and the penetration distance achievable by free drug diffusion. Histological analysis further confirms this finding, illustrating that drug delivered with Dox-TSL intravascular release can result in drug penetration levels up to 80 µm from vessels, in comparison with 40 µm achievable with free drug with heat. Further, Dox-TSL delivers drug to a higher percentage of a tumor's hypoxic area than possible with free drug with or without heat. Endothelial cells display marked morphological changes apparent immediately following treatment, with significant vascular destruction at 6 hours. However, heat had a similar influence on vascular morphology, underscoring the complexity of the anti-vascular effect, particularly in the more sensitive vasculature of a mouse model compared with reported human vascular heat tolerances. This work establishes intravascular release as a new paradigm in drug delivery to solid tumors, resulting in improved drug bioavailability, penetration depth, and enhanced delivery of drug to hypoxic regions of tumors.</p> / Dissertation

Multidrug resistance phenotype of tumor cells describes resistance to wide range of structurally unrelated anticancer agents and is a serious limitation to effective chemotherapy. It is a multifactor yet not fully elucidated phenomenon by the involvement of diverse cellular pathways. Aim of this study was to investigate the resistance mechanisms developed against docetaxel and doxorubicin that are widely used in the treatment of breast cancer in model cell line MCF-7. Resistant sublines were developed by application of drugs in dose increments and effect of docetaxel and doxorubicin on drug applied cells were investigated by cell viability assays. Expression analysis of P-gp, MRP1, BCRP, Bcl-2, Bax and &amp / #946 / -tubulin isotypes were performed by RT-PCR, qPCR, Western blot and immunocytochemistry. Genome-wide expression analysis was also performed by cDNA microarray. According to cell viability assays, drug applied cells developed varying degree of resistance to docetaxel and doxorubicin. Gene expression analysis demonstrated that de novo expression of P-gp contributed significantly to drug resistance. Expression levels of class II, III and V &amp / #946 / -tubulin isotypes increased in docetaxel resistant sublines. According to microarray analysis, a variety of genes showed significantly altered expression levels particularly drug metabolizing and detoxification enzymes (i.e. increased GPX1 and GSTP1 with decreased POR), survival proteins (e.g. decreased TRAIL together with increased decoy receptors and CD40), extracellular matrix components (e.g. increased integrin signaling), growth factors and cytokines (e.g. EGFR1, FGFR1, CTGF, IL6, IL8 and IL18 overexpression), epithelial-mesenchymal transition proteins (i.e. increased vimentin and N-cadherin with decreased E-cadherin and occludin) and microtubule dynamics related proteins (e.g. increased MAP1B and decreased MAP7). Development of cross-resistance and combined drug effects on resistant sublines were also studied. Results demonstrated that docetaxel and doxorubicin resistant cells developed cross-resistance to paclitaxel, vincristine, ATRA, tamoxifen and irradiation. Finally, modulatory effects of verapamil and promethazine in combined drug applications were investigated and verapamil and promethazine were shown to decrease MDR1 expression level thus reverse the MDR. They also showed synergic and additive effects in combined docetaxel and doxorubicin applications. Identification of resistance mechanisms may personalize chemotherapy potentially increasing efficacy of chemotherapy and life quality of patients.

Failure of chemotherapy in cancer patients because of development of drug resistance is a major problem. Alterations of DNA repair mechanisms and drug targets are among the important resistance mechanisms which are developed against topoisomerase II inhibitors etoposide and doxorubicin. Modifications in the expression levels of mismatch repair (MMR) genes due to resistance to topoisomerase II inhibitors are involved in breast cancer. In this study, etoposide resistant sublines were developed from MCF7 breast cancer cell line (MCF7/S) and the expression levels of TOP2A and two important MMR genes MSH2 and MLH1 were examined by real time qPCR. Previously developed doxorubicin resistant cells were also studied for comparison. Etoposide resistant sublines MCF7/1000E, MCF7/1250E and MCF7/2000E were approximately 2, 3 and 4 fold resistant relative to parental MCF7/S cells, respectively. MLH1, MSH2 and TOP2A expressions decreased in both etoposide and doxorubicin resistant sublines relative to MCF7/S cells. Expression levels of TOP2A in resistant sublines differ between 10-95 percent of the expression levels in the parental cells. In the sublines MCF7/200E, MCF7/500E, MCF7/750E and MCF7/1000E a decrease in TOP2A gene expression was determined. In sublines MCF7/1250E and MCF7/2000E fluctuations in the expression levels were observed. Among the doxorubicin resistant sublines (MCF7/600D and MCF7/1000D), in MCF7/1000D which is more resistant to doxorubicin, TOP2A expression level was higher. Expression levels of MSH2 decreased regularly as the resistance increased. However, in MCF7/1250E significant increase relative to MCF7/1000E was observed. In MCF7/2000E, expression levels of MSH2 again significantly decreased to 41 percent of the levels in parental cell line. Expression levels of MLH1 decreased significantly (18-58 percent) in etoposide resistant sublines relative to MCF7/S cells. In doxorubicin resistant sublines, a decrease in MLH1 gene expression was observed in MCF7/1000D. It can be concluded from the results that decrease in the expression levels of TOP2A, MSH2 and MLH1 genes may contribute to resistance together. Above a certain resistance level, sublines may develop new strategies for acquiring higher resistance. Whenever a strategy becomes limited, new strategies emerge. New approaches developed to overcome resistance in cancer chemotherapy should consider the molecular basis of resistance in different stages of the disease.