O tipo biológico influenciado pela genética Bos taurus indicus garante maior adaptação aos animais em regiões com altas temperaturas, no entanto exibe alguns atributos de carcaça e associados à palatabilidade da carne inferiores quando comparados ao tipo biológico predominantemente Bos taurus taurus. Existe a hipótese de que tais diferenças estejam associadas ao conteúdo mitocondrial na musculatura destes animais. Objetivou-se avaliar as diferenças no metabolismo oxidativo, pela medida de respiração mitocondrial em fibras permeabilizadas submetidas a duas temperaturas (38,5 e 40 °C) utilizando respirômetro de alta resolução (Oroboros Instruments), do Longissimus lumborum imediatamente após abate, entre os tipos biológicos Brahman e Angus com taxa e extensão da proteólise distintas ao longo de 14 dias de maturação. Foram investigadas as atividades enzimáticas de lactato desidrogenase e citrato sintase, pH, bem como autólise de &mu;-calpaína e degradação de proteínas miofibrilares de 18 animais das raças Brahman (6), Brangus (6) e Angus (6). A taxa de acidificação das carcaças foi diferente entre os tipos biológicos (P = 0,049) e entre os tempos postmortem (P < 0,001), sendo que as carcaças dos animais Brahman às 6h postmortem apresentaram pH tão alto quanto as carcaças de Brangus e Angus às 3h posmortem. A atividade de citrato sintase 1h postmortem também foi diferente (P = 0,086) entre os tipos biológicos, sendo que maior atividade (P = 0,075) foi identificada em Brahman comparado a Brangus. O fluxo de consumo de oxigênio mitocondrial no estado 4 (ST4_O), apresentou interação (P = 0,050) entre os tipos biológicos e temperatura, assim como a respiração mitocondrial do sistema de transporte de elétrons desacoplado da produção de ATP (P = 0,047), ambos superiores para Brahman a 38,5 °C na comparação entre tipos biológicos. Todavia, ocorreu uma queda da ST4_O, apenas para os animais Brahman, quando a temperatura foi aumentada. A maior resistência a acidificação e maior respiração mitocondrial imediatamente após o abate para o tipo biológico Brahman, foram acompanhadas pela redução (P = 0,058) da autólise completa de &mu;-calpaína às 24h postmortem. A degradação da titina após 7d de maturação foi reduzida (P = 0,056) e se manteve menor (P = 0,003) aos 14d para Brahman comparado aos outros tipos biológicos. O aparecimento da banda 150 kDa da alfa-II-espectrina às 24h postmortem também é menor (P = 0,011) para Brahman, assim como a degradação da desmina (P = 0,019) e troponina-T (P = 0,001). As duas últimas proteínas tiveram sua degradação reduzida ao longo dos 14d postmortem para este tipo biológico. Portanto, o tipo biológico Brahman tem um mecanismo muscular adaptativo que envolve maior conteúdo mitocondrial, com maior vazamento de prótons pelo sistema de transporte de elétrons e que pode estar relacionado a redução da taxa de ativação da &mu;-calpaína, redução da degradação proteica e, consequentemente, a redução da taxa e da extensão do amaciamento da carne destes animais. / Biological type influenced by Bos taurus indicus ensures higher animals adaptation to warm environment, however exhibit less desirable carcass and palatability traits when compared to predominant Bos taurus taurus biological types. There is a hypothesis that muscle mitochondrial content is associated with those differences. It was aimed to evaluate the oxidative metabolism, measuring mitochondrial respiration in permeabilized fibers sumitted to two temperatures (38.5 and 40 °C) using high resolution respirometry (Oroboros Instruments), in Longissimus lumborum imediately after slaughter between biological types Brahman and Angus with different proteolysis rate and extension during 14d aging. Were investigated lactate dehydrogenase and citrate sinthase activities, pH, as well as &mu;-calpain autolysis and protein degradation between 18 animals from breeds Brahman (6), Brangus (6) and Angus (6). The carcass pH decline is different between biological types (P = 0.049) and between time postmortem (P < 0.001), with Brahman carcasses showing pH at 6h postmortem as high as Brangus and Angus pH at 3h postmortem. Citrate sinthase activity 1h postmortem is different (P = 0,086) between biological types, with Brahman showing higher (P = 0.075) values than Brangus animals. The mitochondrial oxygen flux consumption at state 4 (ST4_O) showed interaction (P = 0.050) between biological types and temperature, as well as respiration for electron transport system uncopled from ATP production (P = 0.047), both higher for Brahman at 38.5 °C in biological types comparison. However, decreased ST4_O was observed only for Brahman animals when temperature was increased. Higher resistance to pH decline and higher mitochondrial respiration after slaughter to Brahman cattle were followed by reduced (P = 0.058) complete &mu;-calpain autolysis at 24h postmortem. Titin degradation after 7d aging was reduced (P = 0.056) and kept lower (P = 0.031) after 14d to Brahman when compared to other biological types. The 150 kDa band from alpha-IIspectrin degradation at 24h postmortem is lower (P = 0.011) to Brahman, as well as desmin degradation (P = 0.019) and troponin-T (P = 0.001). Desmin and troponin-T had its degradation reduced extended until 14d aging for this biological type. Therefore, Brahman biological type has an adaptive muscular mechanism that involves higher mitochondrial content, with higher proton leak in electron transport system and that can be related with reduced rate for &mu;-calpain activation, reduced protein degradation and, consequentely, reduced rate and extension to beef tenderization for these animals.

Introdução: A hipertrofia cardíaca induzida por sobrecarga hemodinâmica crônica (HC) é caracterizada por espessamento das paredes do ventrículo esquerdo e do tecido intersticial. As atividades aumentadas de calpaína-1 e metaloproteinase de matriz(MMP)-2 são observadas em diferentes modelos de hipertensão arterial e estão relacionadas com as mudanças fisiopatológicas na HC. Por outro lado, a atividade de MMP-2 parece ser modulada positivamente por ativação de calpaína-1 em diferentes modelos. O objetivo deste trabalho é analizar se a calpaína-1 contribui para o aumento da atividade de MMP-2 no coração e se esse mecanismo resulta nas mudanças crônicas cardíacas na hipertensão renovascular. Métodos: Ratos Wistar submetidos ao modelo de 2-rins-1 clipe (2R-1C)(180-200g) e seus respectivos controles (Sham) foram tratados com verapamil (VRP), um bloqueador de canais para cálcio tipo L (BCCL, 8mg/kg/bid) ou veículo durante 8 semanas. O BCCL reduz as concentrações intracelulares de cálcio, o que leva à diminuição da ativação de calpaína-1, e então à possível modulação da atividade e expressão proteica de MMP-2. Pressão arterial sistólica (PAS) dos ratos foi monitorada durante 10 semanas de hipertensão por pletismografia de cauda. O ventrículo esquerdo (VE) foi analisado por histologia e ecocardiografia para avaliação das dimensões ventriculares. A atividade de calpaína-1 e MMP-2 foi avaliada por zimografia em gel. A expessão proteica de calpaína-1 e MMP-2 foi avaliada por western blot e imunofluorescência. Os corações foram submetidos à avaliação funcional por Langendorff. Todos os protocolos foram aprovados pelo Comitê de Ética em Pesquisa Animal da Faculdade de Medicina de Ribeirão Preto (43/2017). Resultados: Após 10 semanas, a PAS teve um aumento sustentado nos animais 2R-1C e o tratamento com VRP não foi capaz de reduzí-la em nenhum tempo de hipertensão. O peso corporal não apresentou diferença significativa entre os grupos. O grupo hipertenso teve um aumento da massa cardíaca quando comparado ao sham e o tratamento com verapamil reduziu esse parâmetro. A análise da espessura do ventrículo esquerdo demonstra que o VRP é capaz de reverter a HC induzida por sobrecarga pressórica nos animais hipertensos. Os animais 2R-1C apresentaram um aumento singificativo na expressão proteica e atividade de calpaína-1 e o VRP foi capaz de diminuir esses níveis. Foi observado aumento da atividade das isoformas de MMP-2 nos ratos 2R-1C quando comparados aos controles e o VRP foi capaz de reduzir a atividade da isoforma de 64kDa. A contratilidade cardíaca intrínseca dos animais 2R-1C sugere uma disfunção cardíaca quando comparados aos controles sham, embora a fração de ejeção desses animais esteja preservada. O VRP não foi capaz de alterar esses parâmetros. Conclusão: O VRP pode contribuir para a redução da hipertrofia cardíaca por diminuir a expressão proteica de calpaína-1 e MMP-2 na hipertensão renovascular. Apoio financeiro: Capes, CNPq, FAPESP / Introduction: The chronic hemodynamic overload-induced cardiac hypertrophy (CH) is characterized by thickening of the left ventricle walls and hypertrophy of the cardiomyocytes and interstitial tissue. Increased activity of calpain-1 and matrix metalloproteinase(MMP)-2 was observed in different models of arterial hypertension models and contributes to the pathophysiologic changes shown in CH. On the other hand, MMP-2 activity is also positively modulated by activation of calpain-1 in different animal models of cardiovascular diseases. The objectives here are to analyze whether calpain-1 contributes to increase the activity of MMP-2 in the heart and whether this mechanism results in chronic cardiac changes in the renovascular hypertension. Methods: Two kidney-one clip (2K1C) hypertensive male Wistar rats (180-200g) and their respective controls (Sham) were orally treated with verapamil (VRP), a L-type calcium channels blocker (LCCB, 8mg/kg/bid), or vehicle during 8 weeks. The LCCB reduces the intracellular concentration of calcium, thus decreasing the activation of calpain-1, and then may modulate the activity of MMP-2. Systolic blood pressure (SBP) was monitored in the rats during 10 weeks of hypertension. Left ventricle (LV) was analyzed by histology and echocardiography to evaluate ventricle thickening. Calpain- 1 and MMP-2 activities were analyzed by zymography and their expression by immunofluorescence and western blot. Hearts were submitted to functional evaluation by Langendorff. All the protocols were approved by the Ethical Committee in Animal Research of Ribeirao Preto Medical School (43/2017). Results: After 10 weeks, the systolic blood pressure had sustained increase and treatment with VRP was not able to decrease it in any time of hypertension. The body weight did not present significant changes between the groups. Hypertensive group had significant increase in the ventricle/body weight ratio (VW/BW) when compared to sham and treatment with VRP decreased it. Analysis of ventricle thickening showed that VRP is able to revert CHinduced pressure overload. The 2K-1C rats showed a significant increase in the activity and expression of calpain-1 in the heart and VRP reverted it. It was also observed increased activity of MMP-2 forms in the hypertensive rats and VRP decreased the 64kDa MMP-2 activity. The 2K-1C group had cardiac dysfunction when compared to controls groups, and VRP did not alter it. The ejection fraction was not changed in 2K- 1C rats. Conclusion: VRP decreased expression and activity of calpain-1 and MMP-2 in the hearts of 2K-1C rats and then contributed to ameliorate hypertension-induced cardiac hypertrophy

Introdução: A hipertrofia cardíaca induzida por sobrecarga hemodinâmica crônica (HC) é caracterizada por espessamento das paredes do ventrículo esquerdo e do tecido intersticial. As atividades aumentadas de calpaína-1 e metaloproteinase de matriz(MMP)-2 são observadas em diferentes modelos de hipertensão arterial e estão relacionadas com as mudanças fisiopatológicas na HC. Por outro lado, a atividade de MMP-2 parece ser modulada positivamente por ativação de calpaína-1 em diferentes modelos. O objetivo deste trabalho é analizar se a calpaína-1 contribui para o aumento da atividade de MMP-2 no coração e se esse mecanismo resulta nas mudanças crônicas cardíacas na hipertensão renovascular. Métodos: Ratos Wistar submetidos ao modelo de 2-rins-1 clipe (2R-1C)(180-200g) e seus respectivos controles (Sham) foram tratados com verapamil (VRP), um bloqueador de canais para cálcio tipo L (BCCL, 8mg/kg/bid) ou veículo durante 8 semanas. O BCCL reduz as concentrações intracelulares de cálcio, o que leva à diminuição da ativação de calpaína-1, e então à possível modulação da atividade e expressão proteica de MMP-2. Pressão arterial sistólica (PAS) dos ratos foi monitorada durante 10 semanas de hipertensão por pletismografia de cauda. O ventrículo esquerdo (VE) foi analisado por histologia e ecocardiografia para avaliação das dimensões ventriculares. A atividade de calpaína-1 e MMP-2 foi avaliada por zimografia em gel. A expessão proteica de calpaína-1 e MMP-2 foi avaliada por western blot e imunofluorescência. Os corações foram submetidos à avaliação funcional por Langendorff. Todos os protocolos foram aprovados pelo Comitê de Ética em Pesquisa Animal da Faculdade de Medicina de Ribeirão Preto (43/2017). Resultados: Após 10 semanas, a PAS teve um aumento sustentado nos animais 2R-1C e o tratamento com VRP não foi capaz de reduzí-la em nenhum tempo de hipertensão. O peso corporal não apresentou diferença significativa entre os grupos. O grupo hipertenso teve um aumento da massa cardíaca quando comparado ao sham e o tratamento com verapamil reduziu esse parâmetro. A análise da espessura do ventrículo esquerdo demonstra que o VRP é capaz de reverter a HC induzida por sobrecarga pressórica nos animais hipertensos. Os animais 2R-1C apresentaram um aumento singificativo na expressão proteica e atividade de calpaína-1 e o VRP foi capaz de diminuir esses níveis. Foi observado aumento da atividade das isoformas de MMP-2 nos ratos 2R-1C quando comparados aos controles e o VRP foi capaz de reduzir a atividade da isoforma de 64kDa. A contratilidade cardíaca intrínseca dos animais 2R-1C sugere uma disfunção cardíaca quando comparados aos controles sham, embora a fração de ejeção desses animais esteja preservada. O VRP não foi capaz de alterar esses parâmetros. Conclusão: O VRP pode contribuir para a redução da hipertrofia cardíaca por diminuir a expressão proteica de calpaína-1 e MMP-2 na hipertensão renovascular. Apoio financeiro: Capes, CNPq, FAPESP / Introduction: The chronic hemodynamic overload-induced cardiac hypertrophy (CH) is characterized by thickening of the left ventricle walls and hypertrophy of the cardiomyocytes and interstitial tissue. Increased activity of calpain-1 and matrix metalloproteinase(MMP)-2 was observed in different models of arterial hypertension models and contributes to the pathophysiologic changes shown in CH. On the other hand, MMP-2 activity is also positively modulated by activation of calpain-1 in different animal models of cardiovascular diseases. The objectives here are to analyze whether calpain-1 contributes to increase the activity of MMP-2 in the heart and whether this mechanism results in chronic cardiac changes in the renovascular hypertension. Methods: Two kidney-one clip (2K1C) hypertensive male Wistar rats (180-200g) and their respective controls (Sham) were orally treated with verapamil (VRP), a L-type calcium channels blocker (LCCB, 8mg/kg/bid), or vehicle during 8 weeks. The LCCB reduces the intracellular concentration of calcium, thus decreasing the activation of calpain-1, and then may modulate the activity of MMP-2. Systolic blood pressure (SBP) was monitored in the rats during 10 weeks of hypertension. Left ventricle (LV) was analyzed by histology and echocardiography to evaluate ventricle thickening. Calpain- 1 and MMP-2 activities were analyzed by zymography and their expression by immunofluorescence and western blot. Hearts were submitted to functional evaluation by Langendorff. All the protocols were approved by the Ethical Committee in Animal Research of Ribeirao Preto Medical School (43/2017). Results: After 10 weeks, the systolic blood pressure had sustained increase and treatment with VRP was not able to decrease it in any time of hypertension. The body weight did not present significant changes between the groups. Hypertensive group had significant increase in the ventricle/body weight ratio (VW/BW) when compared to sham and treatment with VRP decreased it. Analysis of ventricle thickening showed that VRP is able to revert CHinduced pressure overload. The 2K-1C rats showed a significant increase in the activity and expression of calpain-1 in the heart and VRP reverted it. It was also observed increased activity of MMP-2 forms in the hypertensive rats and VRP decreased the 64kDa MMP-2 activity. The 2K-1C group had cardiac dysfunction when compared to controls groups, and VRP did not alter it. The ejection fraction was not changed in 2K- 1C rats. Conclusion: VRP decreased expression and activity of calpain-1 and MMP-2 in the hearts of 2K-1C rats and then contributed to ameliorate hypertension-induced cardiac hypertrophy

O tipo biológico influenciado pela genética Bos taurus indicus garante maior adaptação aos animais em regiões com altas temperaturas, no entanto exibe alguns atributos de carcaça e associados à palatabilidade da carne inferiores quando comparados ao tipo biológico predominantemente Bos taurus taurus. Existe a hipótese de que tais diferenças estejam associadas ao conteúdo mitocondrial na musculatura destes animais. Objetivou-se avaliar as diferenças no metabolismo oxidativo, pela medida de respiração mitocondrial em fibras permeabilizadas submetidas a duas temperaturas (38,5 e 40 °C) utilizando respirômetro de alta resolução (Oroboros Instruments), do Longissimus lumborum imediatamente após abate, entre os tipos biológicos Brahman e Angus com taxa e extensão da proteólise distintas ao longo de 14 dias de maturação. Foram investigadas as atividades enzimáticas de lactato desidrogenase e citrato sintase, pH, bem como autólise de &mu;-calpaína e degradação de proteínas miofibrilares de 18 animais das raças Brahman (6), Brangus (6) e Angus (6). A taxa de acidificação das carcaças foi diferente entre os tipos biológicos (P = 0,049) e entre os tempos postmortem (P < 0,001), sendo que as carcaças dos animais Brahman às 6h postmortem apresentaram pH tão alto quanto as carcaças de Brangus e Angus às 3h posmortem. A atividade de citrato sintase 1h postmortem também foi diferente (P = 0,086) entre os tipos biológicos, sendo que maior atividade (P = 0,075) foi identificada em Brahman comparado a Brangus. O fluxo de consumo de oxigênio mitocondrial no estado 4 (ST4_O), apresentou interação (P = 0,050) entre os tipos biológicos e temperatura, assim como a respiração mitocondrial do sistema de transporte de elétrons desacoplado da produção de ATP (P = 0,047), ambos superiores para Brahman a 38,5 °C na comparação entre tipos biológicos. Todavia, ocorreu uma queda da ST4_O, apenas para os animais Brahman, quando a temperatura foi aumentada. A maior resistência a acidificação e maior respiração mitocondrial imediatamente após o abate para o tipo biológico Brahman, foram acompanhadas pela redução (P = 0,058) da autólise completa de &mu;-calpaína às 24h postmortem. A degradação da titina após 7d de maturação foi reduzida (P = 0,056) e se manteve menor (P = 0,003) aos 14d para Brahman comparado aos outros tipos biológicos. O aparecimento da banda 150 kDa da alfa-II-espectrina às 24h postmortem também é menor (P = 0,011) para Brahman, assim como a degradação da desmina (P = 0,019) e troponina-T (P = 0,001). As duas últimas proteínas tiveram sua degradação reduzida ao longo dos 14d postmortem para este tipo biológico. Portanto, o tipo biológico Brahman tem um mecanismo muscular adaptativo que envolve maior conteúdo mitocondrial, com maior vazamento de prótons pelo sistema de transporte de elétrons e que pode estar relacionado a redução da taxa de ativação da &mu;-calpaína, redução da degradação proteica e, consequentemente, a redução da taxa e da extensão do amaciamento da carne destes animais. / Biological type influenced by Bos taurus indicus ensures higher animals adaptation to warm environment, however exhibit less desirable carcass and palatability traits when compared to predominant Bos taurus taurus biological types. There is a hypothesis that muscle mitochondrial content is associated with those differences. It was aimed to evaluate the oxidative metabolism, measuring mitochondrial respiration in permeabilized fibers sumitted to two temperatures (38.5 and 40 °C) using high resolution respirometry (Oroboros Instruments), in Longissimus lumborum imediately after slaughter between biological types Brahman and Angus with different proteolysis rate and extension during 14d aging. Were investigated lactate dehydrogenase and citrate sinthase activities, pH, as well as &mu;-calpain autolysis and protein degradation between 18 animals from breeds Brahman (6), Brangus (6) and Angus (6). The carcass pH decline is different between biological types (P = 0.049) and between time postmortem (P < 0.001), with Brahman carcasses showing pH at 6h postmortem as high as Brangus and Angus pH at 3h postmortem. Citrate sinthase activity 1h postmortem is different (P = 0,086) between biological types, with Brahman showing higher (P = 0.075) values than Brangus animals. The mitochondrial oxygen flux consumption at state 4 (ST4_O) showed interaction (P = 0.050) between biological types and temperature, as well as respiration for electron transport system uncopled from ATP production (P = 0.047), both higher for Brahman at 38.5 °C in biological types comparison. However, decreased ST4_O was observed only for Brahman animals when temperature was increased. Higher resistance to pH decline and higher mitochondrial respiration after slaughter to Brahman cattle were followed by reduced (P = 0.058) complete &mu;-calpain autolysis at 24h postmortem. Titin degradation after 7d aging was reduced (P = 0.056) and kept lower (P = 0.031) after 14d to Brahman when compared to other biological types. The 150 kDa band from alpha-IIspectrin degradation at 24h postmortem is lower (P = 0.011) to Brahman, as well as desmin degradation (P = 0.019) and troponin-T (P = 0.001). Desmin and troponin-T had its degradation reduced extended until 14d aging for this biological type. Therefore, Brahman biological type has an adaptive muscular mechanism that involves higher mitochondrial content, with higher proton leak in electron transport system and that can be related with reduced rate for &mu;-calpain activation, reduced protein degradation and, consequentely, reduced rate and extension to beef tenderization for these animals.

Resistance to cisplatin (CDDP)-based therapy is a major hurdle to the successful treatment of human ovarian cancer (OVCA) and the chemoresistant phenotype in OVCA cells is associated with Akt-attenuated, p53-mediated apoptosis. Pro-apoptotic functions of p53 involve both transcription-dependent and -independent signaling pathways and dysfunctional localization and/or inactivation of p53 contribute to the development of chemoresistance. PARC is a cytoplasmic protein regulating p53 subcellular localization and subsequent function. Little is known about the molecular mechanisms regulating PARC. Although PARC contains putative caspase-3 cleavage sites, and CDDP is known to induce the activation of caspases and calpains and induce proteasomal degradation of anti-apoptotic proteins, if and how PARC is regulated by CDDP in OVCA is unknown. Here we present evidence that CDDP promotes calpain-mediated PARC down-regulation, mitochondrial and nuclear p53 accumulation and apoptosis in chemosensitive but not resistant OVCA cells. Inhibition of Akt is required to sensitize chemoresistant cells to CDDP in a p53-dependent manner, an effect enhanced by PARC down-regulation. CDDP-induced PARC down-regulation is reversible by inhibitor of calpain but not of caspase-3 or the 26S proteasome. Furthermore, in vitro experiments confirm the ability of calpain in mediating Ca2+-dependent PARC down-regulation. The role of Ca2+ in PARC down-regulation was further confirmed as ionomycin induced PARC down-regulation in both chemosensitive and chemoresistant ovarian cancer cells. The data presented here implicates the regulation of p53 subcellular localization and apoptosis by PARC as a contributing factor in CDDP resistance in OVCA cells and Ca2+/calpain in PARC post-translational processing and chemosensitivity.

Resistance to cisplatin (CDDP)-based therapy is a major hurdle to the successful treatment of human ovarian cancer (OVCA) and the chemoresistant phenotype in OVCA cells is associated with Akt-attenuated, p53-mediated apoptosis. Pro-apoptotic functions of p53 involve both transcription-dependent and -independent signaling pathways and dysfunctional localization and/or inactivation of p53 contribute to the development of chemoresistance. PARC is a cytoplasmic protein regulating p53 subcellular localization and subsequent function. Little is known about the molecular mechanisms regulating PARC. Although PARC contains putative caspase-3 cleavage sites, and CDDP is known to induce the activation of caspases and calpains and induce proteasomal degradation of anti-apoptotic proteins, if and how PARC is regulated by CDDP in OVCA is unknown. Here we present evidence that CDDP promotes calpain-mediated PARC down-regulation, mitochondrial and nuclear p53 accumulation and apoptosis in chemosensitive but not resistant OVCA cells. Inhibition of Akt is required to sensitize chemoresistant cells to CDDP in a p53-dependent manner, an effect enhanced by PARC down-regulation. CDDP-induced PARC down-regulation is reversible by inhibitor of calpain but not of caspase-3 or the 26S proteasome. Furthermore, in vitro experiments confirm the ability of calpain in mediating Ca2+-dependent PARC down-regulation. The role of Ca2+ in PARC down-regulation was further confirmed as ionomycin induced PARC down-regulation in both chemosensitive and chemoresistant ovarian cancer cells. The data presented here implicates the regulation of p53 subcellular localization and apoptosis by PARC as a contributing factor in CDDP resistance in OVCA cells and Ca2+/calpain in PARC post-translational processing and chemosensitivity.

Resistance to cisplatin (CDDP)-based therapy is a major hurdle to the successful treatment of human ovarian cancer (OVCA) and the chemoresistant phenotype in OVCA cells is associated with Akt-attenuated, p53-mediated apoptosis. Pro-apoptotic functions of p53 involve both transcription-dependent and -independent signaling pathways and dysfunctional localization and/or inactivation of p53 contribute to the development of chemoresistance. PARC is a cytoplasmic protein regulating p53 subcellular localization and subsequent function. Little is known about the molecular mechanisms regulating PARC. Although PARC contains putative caspase-3 cleavage sites, and CDDP is known to induce the activation of caspases and calpains and induce proteasomal degradation of anti-apoptotic proteins, if and how PARC is regulated by CDDP in OVCA is unknown. Here we present evidence that CDDP promotes calpain-mediated PARC down-regulation, mitochondrial and nuclear p53 accumulation and apoptosis in chemosensitive but not resistant OVCA cells. Inhibition of Akt is required to sensitize chemoresistant cells to CDDP in a p53-dependent manner, an effect enhanced by PARC down-regulation. CDDP-induced PARC down-regulation is reversible by inhibitor of calpain but not of caspase-3 or the 26S proteasome. Furthermore, in vitro experiments confirm the ability of calpain in mediating Ca2+-dependent PARC down-regulation. The role of Ca2+ in PARC down-regulation was further confirmed as ionomycin induced PARC down-regulation in both chemosensitive and chemoresistant ovarian cancer cells. The data presented here implicates the regulation of p53 subcellular localization and apoptosis by PARC as a contributing factor in CDDP resistance in OVCA cells and Ca2+/calpain in PARC post-translational processing and chemosensitivity.

Hypochlorous acid (HOCl) is a powerful oxidant produced by activated phagocytes at sites of inflammation to kill a wide range of pathogens. Yet, it may also damage and kill the neighbouring host cells. The abundance of dead macrophages in atherosclerotic plaques and their colocalization with HOCl-modified proteins implicate HOCl may play a role in killing macrophages, contributing to disease progression. The first part of this research was to investigate the cytotoxic effect and cell death mechanism(s) of HOCl on macrophages. Macrophages require efficient defense mechanism(s) against HOCl to function properly at inflammatory sites. The second part of the thesis was to examine the antioxidative effects of glutathione (GSH) and 7,8-dihydroneopterin (7,8-NP) on HOCl-induced cellular damage in macrophages. GSH is an efficient scavenger of HOCl and a major intracellular antioxidant against oxidative stress, whereas 7,8-NP is secreted by human macrophages upon interferon-γ (IFN-γ) induction during inflammation and can also scavenge HOCl. HOCl caused concentration-dependent cell viability loss in human monocyte derived macrophage (HMDM) cells above a specific concentration threshold. HOCl reacted with HMDMs to cause viability loss within the first 10 minutes of treatment, and it posed no latent effect on the cells afterwards regardless of the HOCl concentrations. The lack of caspase-3 activation, rapid influx of propidium iodide (PI) dye, rapid loss of intracellular ATP and cell morphological changes (cell swelling, cell membrane integrity loss and rupture) were observed in HMDM cells treated with HOCl. These results indicate that HOCl caused HMDM cells to undergo necrotic cell death. In addition to the loss of intracellular ATP, HOCl also caused rapid loss of GAPDH enzymatic activity and mitochondrial membrane potential, indicating impairment of the metabolic energy production. Loss of the mitochondrial membrane potential was mediated by mitochondrial permeability transition (MPT), as blocking MPT pore formation using cyclosporin A (CSA) prevented mitochondrial membrane potential loss. HOCl caused an increase in cytosolic calcium ion (Ca2+) level, which was due to both intra- and extra-cellular sources. However, extracellular sources only contributed significantly above a certain HOCl concentration. Preventing cytosolic Ca2+ increase significantly inhibited HOCl-induced cell viability loss. This suggests that cytosolic Ca2+ increase was associated with HOCl-induced necrotic cell death in HMDM cells, possibly via the activation of Ca2+-dependent calpain cysteine proteases. Calpain inhibitors prevented HOCl-induced lysosomal destabilisation and cell viability loss in HMDM cells. Calpains induced HOCl-induced necrotic cell death possibly by degrading cytoskeletal and other cellular proteins, or causing the release of cathepsin proteases from ruptured lysosomes that also degraded cellular components. The HOCl-induced cytosolic Ca2+ increase also caused mitochondrial Ca2+ accumulation and MPT activation-mediated mitochondrial membrane potential loss. MPT activation, like calpain activation, was also associated with the HOCl-induced necrotic cell death, as preventing MPT activation completely inhibited HOCl-induced cell viability loss. The involvement of both calpain activation and MPT activation in HOCl-induced necrotic cell death in HMDM cells implies a cause and effect relationship between these two events. HMDM cells depleted of intracellular GSH using diethyl maleate showed increased susceptibility towards HOCl insult compared to HMDM cells with intact intracellular GSH levels, indicating that intracellular GSH played an important role in protecting HMDM cells against HOCl exposure. Intracellular GSH level in each HMDM cell preparation directly correlated with HOCl concentration required to kill 50% of population for each cell preparation, indicating intracellular GSH concentrations determine the efficiency of GSH in preventing HOCl-induced damage to HMDM cells. Intracellular GSH and cell viability loss induced by 400 μM HOCl were significantly prevented by 300 μM extracellular 7,8-NP, indicating that added 7,8-NP is an efficient scavenger of HOCl and out-competed intracellular GSH for HOCl. The amount of 7,8-NP synthesized by HMDM cells upon IFN-γ induction was too low to efficiently prevent HOCl-mediated intracellular GSH and cell viability loss. HOCl clearly causes HMDM cells to undergo necrosis when the concentration exceeds the intracellular GSH concentrations. Above this concentration HOCl causes oxidative damage to the Ca2+ ion channels on cell and ER membranes, resulting in an influx of Ca2+ ions into the cytosol and possibly the mitochondria. The rise in Ca2+ ions triggers calpain activation, resulting in the MPT-mediated loss of mitochondrial membrane potential, lysosomal instability and cellular necrosis.

Resistance to cisplatin (CDDP)-based therapy is a major hurdle to the successful treatment of human ovarian cancer (OVCA) and the chemoresistant phenotype in OVCA cells is associated with Akt-attenuated, p53-mediated apoptosis. Pro-apoptotic functions of p53 involve both transcription-dependent and -independent signaling pathways and dysfunctional localization and/or inactivation of p53 contribute to the development of chemoresistance. PARC is a cytoplasmic protein regulating p53 subcellular localization and subsequent function. Little is known about the molecular mechanisms regulating PARC. Although PARC contains putative caspase-3 cleavage sites, and CDDP is known to induce the activation of caspases and calpains and induce proteasomal degradation of anti-apoptotic proteins, if and how PARC is regulated by CDDP in OVCA is unknown. Here we present evidence that CDDP promotes calpain-mediated PARC down-regulation, mitochondrial and nuclear p53 accumulation and apoptosis in chemosensitive but not resistant OVCA cells. Inhibition of Akt is required to sensitize chemoresistant cells to CDDP in a p53-dependent manner, an effect enhanced by PARC down-regulation. CDDP-induced PARC down-regulation is reversible by inhibitor of calpain but not of caspase-3 or the 26S proteasome. Furthermore, in vitro experiments confirm the ability of calpain in mediating Ca2+-dependent PARC down-regulation. The role of Ca2+ in PARC down-regulation was further confirmed as ionomycin induced PARC down-regulation in both chemosensitive and chemoresistant ovarian cancer cells. The data presented here implicates the regulation of p53 subcellular localization and apoptosis by PARC as a contributing factor in CDDP resistance in OVCA cells and Ca2+/calpain in PARC post-translational processing and chemosensitivity.

Wolfram syndrome is a genetic disorder characterized by diabetes and neurodegeneration. Two causative genes have been identified so far, WFS1 and WFS2, both encoding endoplasmic reticulum (ER) localized transmembrane proteins. Since WFS1 is involved in the ER stress pathway, Wolfram syndrome is considered an ER disease. Despite the underlying importance of ER dysfunction in Wolfram syndrome, the molecular mechanism linking ER to the death of β cells and neurons has not been elucidated. The endoplasmic reticulum (ER) is an organelle that forms a network of enclosed sacs and tubes that connect the nuclear membrane and other organelles including Golgi and mitochondria. ER plays critical functions in protein folding, protein transport, lipid metabolism, and calcium regulation. Dysregulation of ER function disrupts normal cell metabolism and activates an array of anti-survival pathways, eventually leading to disease state. Here we show that calpain is involved in both prototypes of Wolfram syndrome. Calpain 2 activity is negatively regulated by WFS2 protein, and hyper-activation of calpain 2 by WFS2-knockdown leads to cell death. Calpain hyper-activation is also present in WFS1 loss of function cells due to the high cytosolic calcium. Extensive calpain activation exists in the Wolfram syndrome mouse model as well as in patient cells. A compound screen targeting ER homeostasis reveals that dantrolene, a ryanodine receptor inhibitor, can prevent cell death in cell models of Wolfram syndrome. Our results demonstrate that the pathway leading to calpain activation provides potential therapeutic targets for Wolfram syndrome and other ER diseases.