<p>Antitumour properties of the cyanoguanidine CHS 828 and analogues were discovered in 1997. CHS 828 is presently in clinical phase I/II trials. This thesis encompasses in vitro studies of the kinetics and mode of cell death induced in the human cell line U-937 GTB, by CHS 828 and the standard antitumour drug etoposide.</p><p>Etoposide induces apoptosis in U-937 GTB within 4 h. The cells exhibited apoptotic morphology, including condensed and fragmented nuclei and formation of apoptotic bodies, activation of caspase 3 and 8, and DNA fragmentation, visualised by TdT-mediated dUTP nick end-labelling (TUNEL).</p><p>CHS 828 induced few and weak signs of apoptosis. Metabolic activity was the only parameter affected during the first 24 h of exposure. After ~30 h, proliferation (DNA synthesis) and protein synthesis ceased, and viability started to decrease towards 10% at 72 h. Morphology and ultrastructure of dying/dead cells showed predominant necrosis. The decrease in viability was postponed by protein synthesis inhibition or maintenance of ATP levels by 3-aminobenzamide. In addition, 3-aminobenzamide switched morphology towards apoptosis. </p><p>Continuous co-exposure to CHS 828 and etoposide resulted in impressive cell kill synergy in U-937 GTB cells at effect levels of 30-70%. Pre-exposure to CHS 828 for 18 h or more, on the other hand, resulted in diminished cell kill and inability to activate the apoptotic machinery upon etoposide stimulation, evaluated by morphology and caspase activity.</p><p>In summary, CHS 828 induced cell death is predominantly non-apoptotic, does not involve caspases and can be postponed by maintained protein synthesis and ATP levels.</p>

Antitumour properties of the cyanoguanidine CHS 828 and analogues were discovered in 1997. CHS 828 is presently in clinical phase I/II trials. This thesis encompasses in vitro studies of the kinetics and mode of cell death induced in the human cell line U-937 GTB, by CHS 828 and the standard antitumour drug etoposide. Etoposide induces apoptosis in U-937 GTB within 4 h. The cells exhibited apoptotic morphology, including condensed and fragmented nuclei and formation of apoptotic bodies, activation of caspase 3 and 8, and DNA fragmentation, visualised by TdT-mediated dUTP nick end-labelling (TUNEL). CHS 828 induced few and weak signs of apoptosis. Metabolic activity was the only parameter affected during the first 24 h of exposure. After ~30 h, proliferation (DNA synthesis) and protein synthesis ceased, and viability started to decrease towards 10% at 72 h. Morphology and ultrastructure of dying/dead cells showed predominant necrosis. The decrease in viability was postponed by protein synthesis inhibition or maintenance of ATP levels by 3-aminobenzamide. In addition, 3-aminobenzamide switched morphology towards apoptosis. Continuous co-exposure to CHS 828 and etoposide resulted in impressive cell kill synergy in U-937 GTB cells at effect levels of 30-70%. Pre-exposure to CHS 828 for 18 h or more, on the other hand, resulted in diminished cell kill and inability to activate the apoptotic machinery upon etoposide stimulation, evaluated by morphology and caspase activity. In summary, CHS 828 induced cell death is predominantly non-apoptotic, does not involve caspases and can be postponed by maintained protein synthesis and ATP levels.

The focus of this investigation is about DNA topoisomerases, the molecular targets of clinically important chemotherapy, and mechanisms of drug resistance in human myeloma and leukemia cell lines. The ultimate goal of this investigation was to identify mechanism(s) of drug resistance to anticancer agents so that a strategy to overcome drug resistance could be conceived. We established an in vitro cell model by using human leukemia and myeloma cell lines to investigate possible mechanisms of drug resistance that are observed in confluent cells. Plateau cell densities demonstrated de novo drug resistance to commonly used chemotherapeutic agents that was independent of altered drug transport. We established that cellular drug resistance in these cells is a function of topo IIalpha subcellular localization and further demonstrate that topo IIalpha translocates to the cytoplasm in a cell-density dependent manner. We provide experimental data that supports the nuclear export of topo IIalpha as the most likely event contributing to drug resistance to topoisomerase II inhibitors, which occurs when transformed cells transition from log to plateau cell density. We provided a plausible nuclear export pathway for topo IIalpha, by identifying two Leptomycin B sensitive nuclear export signals, which are homologous to the binding sites recognized by the nuclear export receptor, exportin-1. Thus, topo IIalpha is likely to be exported from the nucleus at plateau cell densities when exportin-1 binds topo IIalpha. We confirmed that the nuclear export signals identified in topo IIalpha are functional when expressed in human myeloma cells transfected with an epitope-tagged topo IIalpha gene. Furthermore we demonstrate that the nuclear export signals can be abolished by site-directed mutagenesis of specific amino acids residues found in the nuclear export signal. Our data may have clinical relevance because plasma cells obtained from bone marrow aspirates of patients with multiple myeloma contain a cytoplasmic distribution of topo IIalpha. The potential implications of a functioning nuclear enzyme located in the cytoplasm of cells and theoretical mechanisms for overcoming the observed drug resistance are considered.

Objetivo: O objetivo do presente estudo foi descrever a relação entre as concentrações plasmáticas totais e livres tumorais do etoposídeo (ETO) e a inibição do crescimento do tumor observada em ratos Wistar portadores de tumor Walker- 256 (W256) utilizando a modelagem farmacocinética/farmacodinâmica (PK/PD). Métodos: Os procedimentos com animais foram aprovados no CEUA/UFRGS sob o número 22302. Os experimentos de farmacocinética foram realizados para determinar concentrações plasmáticas e livres em duas regiões do tumor sólido W256 através de microdiálise. Após a administração do ETO nas doses de 10 ou 20 mg/kg i.v. bolus em ratos Wistar portadores de tumor W256, amostras de sangue e microdialisado de tecido do centro e periferia do tumor foram coletadas simultaneamente, até 7 h pós-dose, para determinar o fator de penetração no tumor. Um método analítico por CLAE-UV foi desenvolvido e validado para quantificação do etoposídeo nas amostras de plasma e dialisado. Os experimentos de farmacodinâmica foram conduzidos em ratos portadores de tumor W256 que receberam ETO 5 e 10 mg/kg i.v. bolus uma vez ao dia por 8 e 4 dias, respectivamente. O volume dos tumores foram monitorados diariamente durante 30 dias. Análise não-compartimental dos dados de PK foi realizada no WinNonlin®. A modelagem dos dados PK e PK/PD foi realizada no Monolix®, utilizando abordagem populacional. Os dados PK/PD foram analisados usando o modelo Simeoni TGI modificado através da introdução de uma função Emax para descrever a relação nãolinear entre a concentração plasmática e tumoral e o efeito. Resultados e Discussão: O método por CLAE-UV foi desenvolvido e validado para quantificar as amostras de ETO em plasma e tecido. A penetração do ETO no tumor foi maior na periferia (61 ± 15 % e 61 ± 29 %) do que no centro do tumor (34 ± 6 % e 28 ± 11 %) após administração das doses 10 e 20 mg/kg, respectivamente (ANOVA, α = 0.05). Um modelo de 4 compartimentos compreendendo uma distribuição saturável (cinética de Michaelis-Menten) nos compartimentos tumorais a partir do compartimento central modelou simultaneamente os perfis de concentração-tempo do ETO em plasma e em ambas regiões do tumor. O modelo populacional PK/PD Simeoni TGI–Emax foi capaz de descrever o efeito antitumoral dependente do regime de administração do ETO utilizando concentrações totais plasmáticas ou livres no tumor, resultando em um maior k2max (potência máxima) para as concentrações livres (25,8 mL.μg-1.dia-1 - intratumoral vs. 12,6 mL.μg-1.dia-1 - plasma total). Conclusões: Os resultados mostram que a utilização das concentrações livres do fármaco no tumor para a modelagem PK/PD pode fornecer um melhor entendimento da relação farmacocinética e farmacodinâmica e melhoram a capacidade de previsão do modelo, considerando que a eficácia dos fármacos antineoplásicos no tratamento de tumores sólidos é dependente da capacidade do fármaco em se distribuir no tecido tumoral. / Objective: The aim of this study was to describe the relationship between total plasma and free interstitial tumor etoposide (ETO) concentrations and the drug tumor growth inhibition observed in a Walker-256 (W256) tumor-bearing Wistar rat model using the pharmacokinetic/pharmacodynamic (PK/PD) modeling. Methods: The experiments with animals were approved by CEUA/UFRGS (protocol number 22302). Pharmacokinetic experiments were conducted to determine total plasma and free intratumoral concentrations in two regions of W256 solid tumor by microdialysis. After administration of ETO 10 or 20 mg/kg i.v. bolus to W256 tumorbearing Wistar rats, blood and tissue microdialysate samples from tumor center and periphery were simultaneously collected up to 7h to determine the tumor penetration factor. An analytical HPLC-UV method was developed and validated for quantification of ETO in plasma and microdialysate samples. The pharmacodynamic experiments were conducted in W256 tumor-bearing rats that received ETO 5 or 10 mg/kg i.v. bolus every day for 8 and 4 days, respectively. Tumor volumes were monitored daily for 30 days. Non-compartmental analysis of PK data was performed in WinNonlin®. The PK and PK/PD modeling by population approach were performed using Monolix®. PK/PD data were analyzed using a modification of Simeoni TGI model by introducing an Emax function to describe the nonlinear relationship between tumor and plasma concentrations and effect. Results and Discussion: The HLPCUV method was developed and validated to determine plasma and tissue samples of ETO. ETO tumor penetration was higher in the tumor periphery (61 ± 15 % and 61 ± 29 %) than center (34 ± 6 % and 28 ± 11 %) following 10 and 20 mg/kg doses, respectively (ANOVA, α = 0.05). A 4-compartment structural model comprising a saturable distribution (Michaelis-Menten kinetics) into the tumor compartments from the central compartment simultaneously described the ETO concentration–time profiles in plasma and both tumor regions. The PK/PD population Simeoni TGI–Emax model was capable of describing the schedule-dependent antitumor effects of ETO using total plasma or free tumor concentrations obtained in a W256-tumor bearing Wistar rat model, resulting in higher k2max (maximal potency) for free concentrations (25.8 mL.μg-1.day-1 - intratumoral vs. 12.6 mL.μg-1.day-1 total plasma). Conclusions: The results showed that the use of free intratumoral drug concentrations in the PK/PD modeling can provide a better understanding of the pharmacokinetics and pharmacodynamics relationship and improve the forecasting ability of the models considering that the efficacy of antineoplastic drugs in the treatment of solid tumors is dependent on the drug ability to distribute into the tumor.

Introdução: O câncer de ovário é o mais prevalente e letal câncer ginecológico. A quimioterapia é um componente importante do tratamento sistêmico clássico com uma combinação de um agente platinado e um taxano, usualmente. Invariavelmente, câncer de ovário avançado torna-se resistente à quimioterapia. Objetivos: Com base em dados recentes que demonstram um possível papel das neurotrofinas na regulação de quimiosensibilidade, decidimos estudar o impacto do fator neurotrófico derivado de cérebro (BDNF) sobre a atividade antitumoral de diferentes classes de agentes antineoplásicos. Métodos: Para avaliar um possível efeito sinérgico entre BDNF e diferentes combinações de tratamento para câncer de ovário, as células foram expostas a cisplatina, etoposideo, doxorrubicina e paclitaxel concomitantemente com BDNF durante 48 horas. Administração sequencial de BDNF e quimioterapia foi realizada para avaliar o potencial de BDNF em modificar a resposta ao tratamento quimioterápico dependendo de qual agente é aplicado em primeiro lugar. Resultados: Houve uma redução da viabilidade de células OVCAR-3 quando expostas a cisplatina, doxorubicina e etoposideo concomitantemente com BDNF em 61,18% (SE±1.12, p=0.002), 38,96% (SE±1.08, p=0.001) e 49,63% (SE±1.17, p<0.001), respectivamente. BDNF também reduziu significativamente o efeito do paclitaxel e doxorrubicina quando usado antes da quimioterapia com uma redução de efeito de 53,46% (SE±3.48, p=0.001) e 48,25% (SE±1.25, p=0.018), respectivamente. Além disso, o BDNF utilizado sequencialmente à doxorrubicina foi capaz de reverter a quimiotoxicidade deste agente em 37,77% (SE±1.25, p=0.018). Conclusão: Utilizando a linhagem celular de câncer de ovário (OVCAR-3), BDNF exibiu um efeito sinérgico quando administrado concomitantemente com os agentes citotóxicos doxorrubicina, etoposideo e cisplatina. Observamos também um efeito protetor de BDNF quando aplicado 24 horas antes de doxorrubicina e paclitaxel. Notavelmente, quando BDNF foi administrado após a exposição a agentes antineoplásicos, uma reversão da citotoxicidade foi observada apenas para a doxorrubicina e não para os outros agentes. / Background: Ovarian cancer is the most prevalent and lethal of gynecological malignancies. Chemotherapy is an important component of the systemic treatment with a combination of a platinum complex and a taxane one of the classic treatments. Invariably, advanced ovarian cancer becomes resistant to chemotherapy. Objective: Based on recent data demonstrating a possible role of neurotrophins regulating chemosensitivity, we decided to study the impact of brain-derived neurotrophic factor (BDNF) on the antitumor activity of different classes of antineoplastic agents. Methods: Primarily, to evaluate a possible synergistic effect of BDNF and different ovarian cancer treatments combination, cells were exposed to cisplatin, etoposide, doxorubicin and paclitaxel concomitantly with BDNF for 48 hours. Sequential administration of BDNF and any of the agents was carried out to evaluate if BDNF has the potential of enhancing or protecting cells from the effects of treatment depending of each agent is applied first. Results: There were a reduction in viability of OVCAR-3 cells exposed to cisplatin, doxorubicin and etoposide when used concomitantly with BDNF in 61.18% (SE 1.12, p=0.002), 38.96% (SE 1.08, p=0.001) and 49.63% (SE 1.17, p<0.001) respectively. We also found that BDNF reduced significantly the effect of paclitaxel and doxorubicin when used before chemotherapy with a reduction of effect of 53.46% (SE±3.48, p=0.001) and 48.25% (SE±1.25, p=0.018), respectively. Furthermore, BDNF used sequentially to doxorubicin was able to reverse the chemotoxicity of this agent in 37.77% (SE 1.25, p=0.018). Conclusion: In conclusion, using the human ovarian carcinoma cell line OVCAR-3, BDNF exhibited a synergistic effect when administered concomitantly to the cytotoxic agents doxorubicin, etoposide and cisplatin. We have also observed a protective effect of BDNF when applied 24 hours before doxorubicin and paclitaxel. Notably, when BDNF was administered after the exposure to the antineoplastic agents, a reversal of cytotoxicity was observed only for doxorubicin and not for the other agents.

Objetivo: O objetivo do presente estudo foi descrever a relação entre as concentrações plasmáticas totais e livres tumorais do etoposídeo (ETO) e a inibição do crescimento do tumor observada em ratos Wistar portadores de tumor Walker- 256 (W256) utilizando a modelagem farmacocinética/farmacodinâmica (PK/PD). Métodos: Os procedimentos com animais foram aprovados no CEUA/UFRGS sob o número 22302. Os experimentos de farmacocinética foram realizados para determinar concentrações plasmáticas e livres em duas regiões do tumor sólido W256 através de microdiálise. Após a administração do ETO nas doses de 10 ou 20 mg/kg i.v. bolus em ratos Wistar portadores de tumor W256, amostras de sangue e microdialisado de tecido do centro e periferia do tumor foram coletadas simultaneamente, até 7 h pós-dose, para determinar o fator de penetração no tumor. Um método analítico por CLAE-UV foi desenvolvido e validado para quantificação do etoposídeo nas amostras de plasma e dialisado. Os experimentos de farmacodinâmica foram conduzidos em ratos portadores de tumor W256 que receberam ETO 5 e 10 mg/kg i.v. bolus uma vez ao dia por 8 e 4 dias, respectivamente. O volume dos tumores foram monitorados diariamente durante 30 dias. Análise não-compartimental dos dados de PK foi realizada no WinNonlin®. A modelagem dos dados PK e PK/PD foi realizada no Monolix®, utilizando abordagem populacional. Os dados PK/PD foram analisados usando o modelo Simeoni TGI modificado através da introdução de uma função Emax para descrever a relação nãolinear entre a concentração plasmática e tumoral e o efeito. Resultados e Discussão: O método por CLAE-UV foi desenvolvido e validado para quantificar as amostras de ETO em plasma e tecido. A penetração do ETO no tumor foi maior na periferia (61 ± 15 % e 61 ± 29 %) do que no centro do tumor (34 ± 6 % e 28 ± 11 %) após administração das doses 10 e 20 mg/kg, respectivamente (ANOVA, α = 0.05). Um modelo de 4 compartimentos compreendendo uma distribuição saturável (cinética de Michaelis-Menten) nos compartimentos tumorais a partir do compartimento central modelou simultaneamente os perfis de concentração-tempo do ETO em plasma e em ambas regiões do tumor. O modelo populacional PK/PD Simeoni TGI–Emax foi capaz de descrever o efeito antitumoral dependente do regime de administração do ETO utilizando concentrações totais plasmáticas ou livres no tumor, resultando em um maior k2max (potência máxima) para as concentrações livres (25,8 mL.μg-1.dia-1 - intratumoral vs. 12,6 mL.μg-1.dia-1 - plasma total). Conclusões: Os resultados mostram que a utilização das concentrações livres do fármaco no tumor para a modelagem PK/PD pode fornecer um melhor entendimento da relação farmacocinética e farmacodinâmica e melhoram a capacidade de previsão do modelo, considerando que a eficácia dos fármacos antineoplásicos no tratamento de tumores sólidos é dependente da capacidade do fármaco em se distribuir no tecido tumoral. / Objective: The aim of this study was to describe the relationship between total plasma and free interstitial tumor etoposide (ETO) concentrations and the drug tumor growth inhibition observed in a Walker-256 (W256) tumor-bearing Wistar rat model using the pharmacokinetic/pharmacodynamic (PK/PD) modeling. Methods: The experiments with animals were approved by CEUA/UFRGS (protocol number 22302). Pharmacokinetic experiments were conducted to determine total plasma and free intratumoral concentrations in two regions of W256 solid tumor by microdialysis. After administration of ETO 10 or 20 mg/kg i.v. bolus to W256 tumorbearing Wistar rats, blood and tissue microdialysate samples from tumor center and periphery were simultaneously collected up to 7h to determine the tumor penetration factor. An analytical HPLC-UV method was developed and validated for quantification of ETO in plasma and microdialysate samples. The pharmacodynamic experiments were conducted in W256 tumor-bearing rats that received ETO 5 or 10 mg/kg i.v. bolus every day for 8 and 4 days, respectively. Tumor volumes were monitored daily for 30 days. Non-compartmental analysis of PK data was performed in WinNonlin®. The PK and PK/PD modeling by population approach were performed using Monolix®. PK/PD data were analyzed using a modification of Simeoni TGI model by introducing an Emax function to describe the nonlinear relationship between tumor and plasma concentrations and effect. Results and Discussion: The HLPCUV method was developed and validated to determine plasma and tissue samples of ETO. ETO tumor penetration was higher in the tumor periphery (61 ± 15 % and 61 ± 29 %) than center (34 ± 6 % and 28 ± 11 %) following 10 and 20 mg/kg doses, respectively (ANOVA, α = 0.05). A 4-compartment structural model comprising a saturable distribution (Michaelis-Menten kinetics) into the tumor compartments from the central compartment simultaneously described the ETO concentration–time profiles in plasma and both tumor regions. The PK/PD population Simeoni TGI–Emax model was capable of describing the schedule-dependent antitumor effects of ETO using total plasma or free tumor concentrations obtained in a W256-tumor bearing Wistar rat model, resulting in higher k2max (maximal potency) for free concentrations (25.8 mL.μg-1.day-1 - intratumoral vs. 12.6 mL.μg-1.day-1 total plasma). Conclusions: The results showed that the use of free intratumoral drug concentrations in the PK/PD modeling can provide a better understanding of the pharmacokinetics and pharmacodynamics relationship and improve the forecasting ability of the models considering that the efficacy of antineoplastic drugs in the treatment of solid tumors is dependent on the drug ability to distribute into the tumor.

Introdução: O câncer de ovário é o mais prevalente e letal câncer ginecológico. A quimioterapia é um componente importante do tratamento sistêmico clássico com uma combinação de um agente platinado e um taxano, usualmente. Invariavelmente, câncer de ovário avançado torna-se resistente à quimioterapia. Objetivos: Com base em dados recentes que demonstram um possível papel das neurotrofinas na regulação de quimiosensibilidade, decidimos estudar o impacto do fator neurotrófico derivado de cérebro (BDNF) sobre a atividade antitumoral de diferentes classes de agentes antineoplásicos. Métodos: Para avaliar um possível efeito sinérgico entre BDNF e diferentes combinações de tratamento para câncer de ovário, as células foram expostas a cisplatina, etoposideo, doxorrubicina e paclitaxel concomitantemente com BDNF durante 48 horas. Administração sequencial de BDNF e quimioterapia foi realizada para avaliar o potencial de BDNF em modificar a resposta ao tratamento quimioterápico dependendo de qual agente é aplicado em primeiro lugar. Resultados: Houve uma redução da viabilidade de células OVCAR-3 quando expostas a cisplatina, doxorubicina e etoposideo concomitantemente com BDNF em 61,18% (SE±1.12, p=0.002), 38,96% (SE±1.08, p=0.001) e 49,63% (SE±1.17, p<0.001), respectivamente. BDNF também reduziu significativamente o efeito do paclitaxel e doxorrubicina quando usado antes da quimioterapia com uma redução de efeito de 53,46% (SE±3.48, p=0.001) e 48,25% (SE±1.25, p=0.018), respectivamente. Além disso, o BDNF utilizado sequencialmente à doxorrubicina foi capaz de reverter a quimiotoxicidade deste agente em 37,77% (SE±1.25, p=0.018). Conclusão: Utilizando a linhagem celular de câncer de ovário (OVCAR-3), BDNF exibiu um efeito sinérgico quando administrado concomitantemente com os agentes citotóxicos doxorrubicina, etoposideo e cisplatina. Observamos também um efeito protetor de BDNF quando aplicado 24 horas antes de doxorrubicina e paclitaxel. Notavelmente, quando BDNF foi administrado após a exposição a agentes antineoplásicos, uma reversão da citotoxicidade foi observada apenas para a doxorrubicina e não para os outros agentes. / Background: Ovarian cancer is the most prevalent and lethal of gynecological malignancies. Chemotherapy is an important component of the systemic treatment with a combination of a platinum complex and a taxane one of the classic treatments. Invariably, advanced ovarian cancer becomes resistant to chemotherapy. Objective: Based on recent data demonstrating a possible role of neurotrophins regulating chemosensitivity, we decided to study the impact of brain-derived neurotrophic factor (BDNF) on the antitumor activity of different classes of antineoplastic agents. Methods: Primarily, to evaluate a possible synergistic effect of BDNF and different ovarian cancer treatments combination, cells were exposed to cisplatin, etoposide, doxorubicin and paclitaxel concomitantly with BDNF for 48 hours. Sequential administration of BDNF and any of the agents was carried out to evaluate if BDNF has the potential of enhancing or protecting cells from the effects of treatment depending of each agent is applied first. Results: There were a reduction in viability of OVCAR-3 cells exposed to cisplatin, doxorubicin and etoposide when used concomitantly with BDNF in 61.18% (SE 1.12, p=0.002), 38.96% (SE 1.08, p=0.001) and 49.63% (SE 1.17, p<0.001) respectively. We also found that BDNF reduced significantly the effect of paclitaxel and doxorubicin when used before chemotherapy with a reduction of effect of 53.46% (SE±3.48, p=0.001) and 48.25% (SE±1.25, p=0.018), respectively. Furthermore, BDNF used sequentially to doxorubicin was able to reverse the chemotoxicity of this agent in 37.77% (SE 1.25, p=0.018). Conclusion: In conclusion, using the human ovarian carcinoma cell line OVCAR-3, BDNF exhibited a synergistic effect when administered concomitantly to the cytotoxic agents doxorubicin, etoposide and cisplatin. We have also observed a protective effect of BDNF when applied 24 hours before doxorubicin and paclitaxel. Notably, when BDNF was administered after the exposure to the antineoplastic agents, a reversal of cytotoxicity was observed only for doxorubicin and not for the other agents.

Introdução: O câncer de ovário é o mais prevalente e letal câncer ginecológico. A quimioterapia é um componente importante do tratamento sistêmico clássico com uma combinação de um agente platinado e um taxano, usualmente. Invariavelmente, câncer de ovário avançado torna-se resistente à quimioterapia. Objetivos: Com base em dados recentes que demonstram um possível papel das neurotrofinas na regulação de quimiosensibilidade, decidimos estudar o impacto do fator neurotrófico derivado de cérebro (BDNF) sobre a atividade antitumoral de diferentes classes de agentes antineoplásicos. Métodos: Para avaliar um possível efeito sinérgico entre BDNF e diferentes combinações de tratamento para câncer de ovário, as células foram expostas a cisplatina, etoposideo, doxorrubicina e paclitaxel concomitantemente com BDNF durante 48 horas. Administração sequencial de BDNF e quimioterapia foi realizada para avaliar o potencial de BDNF em modificar a resposta ao tratamento quimioterápico dependendo de qual agente é aplicado em primeiro lugar. Resultados: Houve uma redução da viabilidade de células OVCAR-3 quando expostas a cisplatina, doxorubicina e etoposideo concomitantemente com BDNF em 61,18% (SE±1.12, p=0.002), 38,96% (SE±1.08, p=0.001) e 49,63% (SE±1.17, p<0.001), respectivamente. BDNF também reduziu significativamente o efeito do paclitaxel e doxorrubicina quando usado antes da quimioterapia com uma redução de efeito de 53,46% (SE±3.48, p=0.001) e 48,25% (SE±1.25, p=0.018), respectivamente. Além disso, o BDNF utilizado sequencialmente à doxorrubicina foi capaz de reverter a quimiotoxicidade deste agente em 37,77% (SE±1.25, p=0.018). Conclusão: Utilizando a linhagem celular de câncer de ovário (OVCAR-3), BDNF exibiu um efeito sinérgico quando administrado concomitantemente com os agentes citotóxicos doxorrubicina, etoposideo e cisplatina. Observamos também um efeito protetor de BDNF quando aplicado 24 horas antes de doxorrubicina e paclitaxel. Notavelmente, quando BDNF foi administrado após a exposição a agentes antineoplásicos, uma reversão da citotoxicidade foi observada apenas para a doxorrubicina e não para os outros agentes. / Background: Ovarian cancer is the most prevalent and lethal of gynecological malignancies. Chemotherapy is an important component of the systemic treatment with a combination of a platinum complex and a taxane one of the classic treatments. Invariably, advanced ovarian cancer becomes resistant to chemotherapy. Objective: Based on recent data demonstrating a possible role of neurotrophins regulating chemosensitivity, we decided to study the impact of brain-derived neurotrophic factor (BDNF) on the antitumor activity of different classes of antineoplastic agents. Methods: Primarily, to evaluate a possible synergistic effect of BDNF and different ovarian cancer treatments combination, cells were exposed to cisplatin, etoposide, doxorubicin and paclitaxel concomitantly with BDNF for 48 hours. Sequential administration of BDNF and any of the agents was carried out to evaluate if BDNF has the potential of enhancing or protecting cells from the effects of treatment depending of each agent is applied first. Results: There were a reduction in viability of OVCAR-3 cells exposed to cisplatin, doxorubicin and etoposide when used concomitantly with BDNF in 61.18% (SE 1.12, p=0.002), 38.96% (SE 1.08, p=0.001) and 49.63% (SE 1.17, p<0.001) respectively. We also found that BDNF reduced significantly the effect of paclitaxel and doxorubicin when used before chemotherapy with a reduction of effect of 53.46% (SE±3.48, p=0.001) and 48.25% (SE±1.25, p=0.018), respectively. Furthermore, BDNF used sequentially to doxorubicin was able to reverse the chemotoxicity of this agent in 37.77% (SE 1.25, p=0.018). Conclusion: In conclusion, using the human ovarian carcinoma cell line OVCAR-3, BDNF exhibited a synergistic effect when administered concomitantly to the cytotoxic agents doxorubicin, etoposide and cisplatin. We have also observed a protective effect of BDNF when applied 24 hours before doxorubicin and paclitaxel. Notably, when BDNF was administered after the exposure to the antineoplastic agents, a reversal of cytotoxicity was observed only for doxorubicin and not for the other agents.

Currently available anticancer therapies are inadequate and spur demand for improved technologies. Among others, the utilization of nanocarriers for anticancer drug delivery has shown great potential in cancer treatment. Nanocarriers can improve the therapeutic efficiency of the drugs with minimization of the undesirable side effects. To evaluate potential application of this technology, two forms of nanocarriers have been studied: multi-walled carbon nanotubes (MWCNTs) and apoferritin. The aim of this study was to determine, whether given cytostatics (ellipticine, etoposide and doxorubicin) are bound to these nanotransporters and how are they released from them, especially depending on pH. Since the pH of the tumor cells is lower than the pH of healthy cells it would be preferred that the drugs would release from nanocarriers at the lower pH while at the physiological pH the release of the drug would be eliminated. The results found show that ellipticine is actually released from its MWCNT- and apoferrtin-encapsulated form at acidic pH (5.0), while at pH 7.4 its interaction with nanocarriers is stable. Ellipticine released from MWCNT is activated by microsomal enzymes to reactive metabolites (13- hydroxyellipticine and 12-hydroxyellipticine) forming DNA adducts. The results indicate that both...

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