Previously we found that 3,4-(methylenedioxy)-1-(2′,3′- epoxypropyl)-benzene (safrole oxide) induced a typical apoptosis in A549 human lung cancer cells. In this study, we further investigated which caspases were activated by safrole oxide during the apoptosis. The data showed that the activity of caspase-3, -8, and -9 was significantly enhanced by the compound, which suggested that safrole oxide might be used as a caspase promoter to initiate lung cancer cell apoptosis.

No description available.

A novel small molecule, 1-ethoxy-3-(3,4-methylenedioxyphenyl)-2-propanol (EOD), was synthesized in our laboratory. Previously, we reported pharmacological properties of EOD, triggering apoptosis in Human umbilical vein endothelial cells (HUVECs). Here, we further investigated the effects of EOD on the growth of A549 human lung cancer cells. EOD treatment induced apoptosis in A549 cells via up-regulating the expression of P53 protein, blocking cell cycle partly at G1 phase, and ultimately activating caspase-3. In contrast, caspase-8 might be irrelevant to EOD-triggered apoptosis. This study indicated that EOD might be a potential chemopreventive agent for lung cancer. The work would encourage us to add more novel compounds to our 'library' of small molecules derived through modern synthetic organic chemistry, and would drive us to determine the proteins that the compounds target.

<p>Cancer patients undergoing chemotherapy have an elevated risk for thrombosis. Although thrombosis is a common complication in cancer patients, the mechanisms of chemotherapy-induced thrombosis remain unclear. We investigated the procoagulant effects of lung cancer chemotherapy agents (carboplatin, paclitaxel, cisplatin, and gemcitabine) on endothelial cells, A549 cells, and monocytes. We also investigated the <em>in </em>vivo procoagulant effects of the aforementioned chemotherapeutic agents as well as the anti-angiogenic agent bevacizumab. Tissue factor (TF) activity, TF antigen and phosphatidylserine (PS) levels were measured on chemotherapy-treated human umbilical vein endothelial cells (HUVEC), A549 cells, and monocytes. Treatment of HUVECs, A549 cells, and monocytes with lung cancer single agent and combination chemotherapy resulted in significant increases in TF activity. However, only cisplatin- and gemcitabine- treated monocytes were found to have increased TF antigen levels. PS exposure was increased only on HUVEC and monocytes treated with cisplatin/gemcitabine combination therapy. Interestingly, addition of paclitaxel to carboplatin resulted in reduced levels of PS exposure on monocytes. This study is the first to explore the procoagulant effects of lung cancer chemotherapy agents on monocyte and A549 cell TF activity levels, as well as to investigate the mechanisms by which lung cancer agents may promote TF decryption on these cell lines<strong>.</strong> Our <em>in vivo</em> results demonstrated that treatment of healthy mice with bevacizumab, paclitaxel and carboplatin moderately increased plasma TAT levels in healthy mice. These studies reveal potential mechanisms by which lung cancer chemotherapy may increase the risk of thrombosis. These studies reveal potential mechanisms by which lung cancer chemotherapy agents induce a hypercoagulable state.</p> / Master of Science (MSc)

In recent years, respiratory diseases have emerged as a leading cause of mortality across the globe. In the United States alone respiratory diseases are the fourth leading cause of deaths annually. Moreover, with the rapid increase of industrialization and urbanization, the occurrences of respiratory diseases are expected to remain high with strong chances of increasing in the future. To ameliorate the epidemic of respiratory disease, it is first important to understand its underlying mechanisms. Respiratory research studies in animals have elucidated the chronological order of the pathological events and systemic responses inside the lung, but understanding the response of individual cell types inside the lung is necessary to outline the initiators and mediators of the pathological events. Many research studies have aimed to understand the behavior of individual cell types, from the lung, under different pathological conditions specific to the respiratory system. However, the cell culture systems used in most of these studies were limited by the absence of the dynamic cell growth environment present in actual lung tissues. The lung exists in a mechanically active environment, where different amounts of circumferential and longitudinal expansion and contraction occur during breathing movements. Thus, simulating the biomechanical environment in in vitro cell culture models may improve the cellular functionality and the outcome of the research studies. Moreover, the stimulation of biomechanical forces in in vitro cell cultures provides the advantage of mimicking the mechanical environment, related to different pathological conditions. In our study we used a dynamic in vitro cell culture system capable of implementing cyclic equibiaxial deformation in cell monolayers to stimulate different biomechanical environments similar to conditions inside the lung. The dynamic cell growth condition was used to determine the effects of ventilator-induced lung injury and nano-material/pollutant exposure in A549 cell cultures. Examples of such pollutants are diesel particulate matter, multi-walled carbon nanotubes, and single-walled carbon nanotubes. Our results indicated that the dynamic cell growth condition specific to ventilator induced lung injury facilitated an increase in inflammatory and tissue remodeling activities in A549 cells. Under the nano-material/pollutant exposure assessment studies, the dynamic cell growth condition induced changes in inflammation and oxidative stress level which closely resembled those in in vivo studies.

No description available.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Streptococcus agalactiae, ou Streptococcus do grupo B (GBS), é um importante patógeno oportunista que causa pneumonia, sepse e meningite em recém-nascidos e infecções em adultos imunocomprometidos. O pulmão aparentemente é o portal de entrada para o EGB na corrente sanguínea o que pode evoluir para uma septicemia. Os mecanismos de virulência relevantes envolve a habilidade do EGB em penetrar e sobreviver intracelularmente em células hospedeiras. Neste trabalho, foram analisados os mecanismos moleculares da apoptose epitelial induzida pelo EGB, e a produção de óxido nítrico (NO) e espécies reativas de oxigênio (ROS) em células epiteliais respiratórias A549 durante a infecção por EGB. Todas as amostras de EGB exibiram a capacidade de aderir e invadir células A549. A sobrevivência intracelular do EGB em células A549 ocorreu durante 24 h de incubação sem replicação do patógeno. No entanto, a amsotra 88641-V isolada de vagina não sobreviveu após 0,5 h de interação. O EGB promoveu a perda de viabilidade do epitélio durante a infecção. As alterações morfológicas em células A549 infectadas com o EGB incluem arredondamento celular, condensação nuclear, encolhimento celular e perda de contato célula-célula e célula-substrato. A dupla marcação AV/IP revelou que amostras de EGB sorotipo III induziram apoptose enquanto amostras do sorotipo V induziram morte celular semelhante a necrose em células A549. Caspase-3 foi ativada durante a apoptose induzida por EGB em células epiteliais. No entanto, a ativação de caspases-8 e -9 foi detectada apenas para a amostra 88641-V e as amostras EGB do sorotipo III, respectivamente. Experimentos comparativos de Immunoblotting revelaram que o EGB induziu um aumento da expressão Bim, uma proteína pró-apoptótica e diminuiu a expressão de Bcl-2 e Bcl-xL, proteínas anti-apoptóticas. As células A549 apresentaram perda de potencial de membrana mitocondrial &#916;&#968;m e co-localização com o Bax. Ensaio de espectrometria de massa identificou a proteína PI-2a, uma proteína estrutural de pili, que exibe atividade carboxipepdidase. Descobrimos que os dois sorotipos (III e V) induziram a produção ROS e NO em células A549. Em conclusão, a apoptose induzida pelo EGB em células A549 é um mecanismo importante de virulência, resultando na destruição de tecidos, escape do sistema imune do hospedeiro com espalhamento bacteriano e, em consequência, a doença invasiva ou uma infecção sistémica. / Streptococcus agalactiae, or group B Streptococcus (GBS), is an important opportunistic pathogen that causes pneumonia, sepsis, and meningitis in neonates and severe diseases in immunocompromised adults. The lung is the apparent portal of entry for GBS into the bloodstream, after which septicemia may ensue. A relevant virulence mechanism involves the ability of GBS to penetrate and to survive intracellularly within these host cells. In this work, we analyzed the molecular mechanisms of GBS-induced epithelial apoptosis, and nitric oxide (NO) and reactive oxygen species (ROS) production by lung epithelial cell line A549 cells during infection with GBS. All GBS exhibited the ability to adhere and to invade A549 cells. The survival of GBS within A549 cells without replication was shown during 24 h incubation. However, the 88641-V strain isolated from vagina did not survive after 0.5 h of interaction. GBS promoted the loss of viability of the epithelium during infection. The morphological changes in A549 cells infected with GBS included cell rounding, nuclear condensation, cellular shrinkage and loss of cell-cell contact and cell-substrate. The double staining AV / IP revealed that GBS serotype III induced apoptosis while GBS serotype V induced like necrosis cell death in A549 cells. Caspase-3 was activated during GBS-induced endothelial apoptosis. However, activation of caspases-8 and -9 was detected only by GBS 88641-V and GBS-III, respectively. Comparative immunoblotting experiments revealed that GBS induced an increasing pro-apoptotic Bim expression and decreasing anti-apoptotic Bcl-2 and Bcl-XL expression. A549 cells exhibited loss of mitochondrial membrane potential &#916;&#968;m with Bax colocalization. Mass spectrometry assay identified protein PI-2a, a structural protein pili, which exhibit carboxipepdidase activity. We found that both serotypes (III and V) induced ROS and NO production in A549 cells. In conclusion, apoptosis of A549 cell induced by GBS is an important virulence mechanism resulting in tissue destruction, escape from the host immune system with bacterial spreading and, in consequence, invasive disease or systemic infection.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Streptococcus agalactiae, ou Streptococcus do grupo B (GBS), é um importante patógeno oportunista que causa pneumonia, sepse e meningite em recém-nascidos e infecções em adultos imunocomprometidos. O pulmão aparentemente é o portal de entrada para o EGB na corrente sanguínea o que pode evoluir para uma septicemia. Os mecanismos de virulência relevantes envolve a habilidade do EGB em penetrar e sobreviver intracelularmente em células hospedeiras. Neste trabalho, foram analisados os mecanismos moleculares da apoptose epitelial induzida pelo EGB, e a produção de óxido nítrico (NO) e espécies reativas de oxigênio (ROS) em células epiteliais respiratórias A549 durante a infecção por EGB. Todas as amostras de EGB exibiram a capacidade de aderir e invadir células A549. A sobrevivência intracelular do EGB em células A549 ocorreu durante 24 h de incubação sem replicação do patógeno. No entanto, a amsotra 88641-V isolada de vagina não sobreviveu após 0,5 h de interação. O EGB promoveu a perda de viabilidade do epitélio durante a infecção. As alterações morfológicas em células A549 infectadas com o EGB incluem arredondamento celular, condensação nuclear, encolhimento celular e perda de contato célula-célula e célula-substrato. A dupla marcação AV/IP revelou que amostras de EGB sorotipo III induziram apoptose enquanto amostras do sorotipo V induziram morte celular semelhante a necrose em células A549. Caspase-3 foi ativada durante a apoptose induzida por EGB em células epiteliais. No entanto, a ativação de caspases-8 e -9 foi detectada apenas para a amostra 88641-V e as amostras EGB do sorotipo III, respectivamente. Experimentos comparativos de Immunoblotting revelaram que o EGB induziu um aumento da expressão Bim, uma proteína pró-apoptótica e diminuiu a expressão de Bcl-2 e Bcl-xL, proteínas anti-apoptóticas. As células A549 apresentaram perda de potencial de membrana mitocondrial &#916;&#968;m e co-localização com o Bax. Ensaio de espectrometria de massa identificou a proteína PI-2a, uma proteína estrutural de pili, que exibe atividade carboxipepdidase. Descobrimos que os dois sorotipos (III e V) induziram a produção ROS e NO em células A549. Em conclusão, a apoptose induzida pelo EGB em células A549 é um mecanismo importante de virulência, resultando na destruição de tecidos, escape do sistema imune do hospedeiro com espalhamento bacteriano e, em consequência, a doença invasiva ou uma infecção sistémica. / Streptococcus agalactiae, or group B Streptococcus (GBS), is an important opportunistic pathogen that causes pneumonia, sepsis, and meningitis in neonates and severe diseases in immunocompromised adults. The lung is the apparent portal of entry for GBS into the bloodstream, after which septicemia may ensue. A relevant virulence mechanism involves the ability of GBS to penetrate and to survive intracellularly within these host cells. In this work, we analyzed the molecular mechanisms of GBS-induced epithelial apoptosis, and nitric oxide (NO) and reactive oxygen species (ROS) production by lung epithelial cell line A549 cells during infection with GBS. All GBS exhibited the ability to adhere and to invade A549 cells. The survival of GBS within A549 cells without replication was shown during 24 h incubation. However, the 88641-V strain isolated from vagina did not survive after 0.5 h of interaction. GBS promoted the loss of viability of the epithelium during infection. The morphological changes in A549 cells infected with GBS included cell rounding, nuclear condensation, cellular shrinkage and loss of cell-cell contact and cell-substrate. The double staining AV / IP revealed that GBS serotype III induced apoptosis while GBS serotype V induced like necrosis cell death in A549 cells. Caspase-3 was activated during GBS-induced endothelial apoptosis. However, activation of caspases-8 and -9 was detected only by GBS 88641-V and GBS-III, respectively. Comparative immunoblotting experiments revealed that GBS induced an increasing pro-apoptotic Bim expression and decreasing anti-apoptotic Bcl-2 and Bcl-XL expression. A549 cells exhibited loss of mitochondrial membrane potential &#916;&#968;m with Bax colocalization. Mass spectrometry assay identified protein PI-2a, a structural protein pili, which exhibit carboxipepdidase activity. We found that both serotypes (III and V) induced ROS and NO production in A549 cells. In conclusion, apoptosis of A549 cell induced by GBS is an important virulence mechanism resulting in tissue destruction, escape from the host immune system with bacterial spreading and, in consequence, invasive disease or systemic infection.

No description available.

Le stress oxydatif joue un rôle prépondérant en tant que messager secondaire dans la régulation de nombreux processus cellulaires tels que l’apoptose, la survie et la prolifération et serait impliqué dans l’ensemble des étapes de la carcinogenèse pulmonaire. L’activité physique et la nutrition sont deux facteurs pouvant moduler le stress oxydatif et les mécanismes associés. La bétaïne et la C-Phycocyanine sont deux micronutriments reconnus pour avoir des effets antioxydants, anti-inflammatoires et antiprolifératifs. Récemment notre équipe a montré in vitro qu’un traitement en bétaïne et/ou la C-phycocyanine diminuait la viabilité des cellules A549 (carcinome pulmonaire). L’objectif général de ce travail de thèse était d’évaluer l’effet de facteurs nutritionnels (bétaïne, C-PC ou activité physique) sur la croissance tumorale de cellules A549 implantées chez le rat Nude et de déterminer les mécanismes sous-jacents. Dans un premier temps, nous avons étudié l’effet d’une supplémentation nutritionnelle (bétaïne ou C-phycocyanine) associée ou non à la pratique d’une activité physique volontaire (roue d’activité) sur l’équilibre redox et l’inflammation, chez des rats sains. Nous avons montré que la bétaïne et la C-phycocyanine augmentaient les défenses antioxydantes tandis que l’activité physique volontaire n’avait pas d’effet si elle n’était pas couplée à une supplémentation. Nous avons également mis en évidence que C-phycocyanine inhibait l’augmentation de Cox-2 musculaire induite par l’activité physique. Dans un second temps, nous avons étudié l’effet de la bétaïne et/ou la C-Phycocyanine sur la croissance des cellules A549 implantées chez des rats Nude. Nous avons montré que ces deux micronutriments associés ou non ralentissaient la croissance des tumeurs pulmonaires, au travers de mécanismes communs (activation de NF-kappaB, augmentation de la peroxydation lipidique et de l’expression de cytokine pro-inflammatoire (IL-1-beta, Cox-2 et TNF-alpha) au sein de la tumeur) et de mécanismes propres à chaque micronutriment. La C-phycocyanine a induit une diminution du ratio AKT phosphorylé / AKT total et une augmentation du ratio p38 phosphorylé / p38 total, mécanisme en faveur de l’apoptose et de l’autophagie. La bétaïne associée à la C-phycocyanine a augmenté le ratio caspase-3 / pro-caspase-3. Dans un dernier temps, nous avons évalué l’effet de l’activité physique volontaire sur la croissance tumorale des cellules A549 implantées chez des rats Nude. Nous avons mis en évidence que l’activité physique volontaire ralentissait la croissance des tumeurs pulmonaires induites, sans différence significative avec la bétaïne et/ou la C-phycocyanine. Il apparait que l’augmentation de la peroxydation lipidique, l’activation de la MAPK p38 et de NF-kappaB, et l’inhibition d’AKT, favorisant la mort cellulaire soient impliquées dans cette diminution tumorale. Un régime enrichi en bétaïne et/ou C-phycocyanine ralentit la croissance cellulaire d’adénocarcinome pulmonaire implanté chez le rat, suggérant leur intérêt dans l’action anti-carcinogène pulmonaire. L’activité physique semble jouer sur les mêmes mécanismes. Nos résultats méritent d’être confirmés par des protocoles à plus large échelle et suggèrent de possibles applications chez des patients porteurs de tumeurs pulmonaires. / Oxidative stress seems to play a crucial role as a secondary messenger in the regulation of several cellular processes such as apoptosis, survival and proliferation, and could be involved in all steps of the lung carcinogenesis (i.e. initiation, promotion and progression). Physical activity and nutrition are two factors able to modulate oxidative stress and associated mechanisms. Betaine and C-phycocyanin are two known micronutrients having antioxidant, anti-inflammatory and anti-proliferative effects. Previously, our team showed that betaine and/or C-phycocyanin treatment decreased the viability of A549 cells in vitro (pulmonary adenocarcinoma cell line). The main objective of this work was to evaluate the effect of nutritional factors (betaine, C-phycocyanin) or physical activity on growth of implanted A549 cells in Nude rats and to determine underlying mechanisms.Firstly, we studied the effect of nutritional supplementation (betaine or C-phycocyanin) combined or not with voluntary physical activity (wheel running) on redox balance and inflammation in healthy rats. We showed that betaine and C-phycocyanin increased antioxidant defenses, whereas voluntary physical activity did not have an effect when it was not associated with micronutrient supplementation. We also observed that C-phycocyanin inhibited physical activity-induced muscle Cox-2 activity increase.Secondly, we studied the effect of betaine and/or C-phycocyanin on growth of implanted A549 cells in Nude rats. We showed that these two micronutrients, whether associated or supplied separately, slowed down the lung tumour growth through similar mechanisms (NF-kappaB activation and increase of lipid peroxidation and expression of pro-inflammatory cytokines (IL-1beta, Cox-2 et TNF-alpha) in tumour). Also, some mechanisms were specific for each micronutrient or their combination. C-phycocyanin induced a decrease of phosphorylated AKT / total AKT ratio, and an increase of phosphorylated p38 / total p38 ratio, both mechanisms promoting apoptosis and autophagy. On the other hand, betaine associated with C-phycocyanin increased caspase-3 / pro-caspase-3 ratio.Finally, we studied the effect of voluntary physical activity on growth of implanted A549 cells in Nude rats. We showed that voluntary physical activity slowed down the lung tumour growth, without significant difference if animals were supplied with betaine or/and C-phycocyanin. It seems that the increase of lipid peroxidation, NF-kappaB and p38 activation, and AKT inhibition, all having a role in promotion of a cell death, are responsible for the tumour growth slowdown following the physical activity. In conclusion, diet enriched with betaine or/and C-phycocyanin slows down the growth of pulmonary adenocarcinoma cells implanted in rats, suggesting their interest in anti-cancer activity. Physical activity seems to act on similar mechanisms as these micronutrients. Our results have to be confirmed with further studies, but are already suggesting a potential application in lung cancer patients.