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Papel do H2O2 na resistência adquirida ao cancro: alterações na organização das biomembranas
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A membrana plasmática é uma barreira que controla a entrada e a saída de compostos na célula, ao separar o interior desta do meio extracelular. Este trabalho teve como objectivo elucidar o papel do peróxido de hidrogénio (H2O2), uma molécula sinalizadora, na alteração das jangadas lipídicas e domínios caveolae da membrana plasmática e estabelecer a possível resistência de células tumorais (HeLa) adaptadas ao H2O2, aos fármacos anti-cancro cis-diamminedicloroplatina (II) (cisplatina) e doxorrubicina. As células HeLa quando expostas a um estado estacionário de 12,5 μM de H2O2 durante 6 h adaptam-se ao H2O2, apresentando um aumento de sobrevivência quando expostas a doses letais deste agente. Durante a exposição ao H2O2 ocorreu um aumento da peroxidação lipídica da membrana, mas este aumento não comprometeu a viabilidade celular. A marcação de células HeLa com a subunidade B da toxina da cólera, a qual detecta a presença de jangadas lipídicas, mostrou uma diminuição de intensidade aos 30 min e uma ligeira acumulação de proteína aos 360 min de exposição a 12,5 μM de H2O2. Estudos usando imunofluorescência sugerem um aumento dos níveis de caveolina-1 para tempos curtos de exposição ao H2O2 em estado estacionário, facto comprovado por Western blot. Resultados preliminares usando Western blot e a análise por imunofluorescência sugerem que a pré-exposição ao H2O2 aumenta os níveis de caveolina-1 fosforilada. Estudos de sobrevivência indicam que células HeLa adaptadas ao H2O2 se tornaram mais resistentes a uma exposição a doses letais de cisplatina mas menos resistentes aquando da exposição à doxorrubicina. Estes resultados mostram, numa linha celular humana, que concentrações baixas e adaptativas de H2O2 alteram rapidamente, em poucos minutos a organização da membrana plasmática. A adaptação ao H2O2 influencia a resistência das células à utilização dos fármacos cisplatina e doxorrubicina, o que poderá ter importância na terapia anti-cancro, no desenho de terapias adjuvantes.
The plasma membrane is a barrier that controls the entry and exit of compounds in the cell, separating the intracellular content from the extracellular medium. This study aimed to elucidate the role of hydrogen peroxide (H2O2), a signaling molecule, in the alteration of lipid rafts and caveolae domains in plasma membrane and establishment of possible resistance of tumor cells (HeLa) adapted to H2O2, to anti-cancer drugs cisplatin and doxorubicin. When HeLa cells were exposed to a steady state of 12,5 μM of H2O2 for 6 h, it was observed that cells adapt to H2O2, showing a survival advantage when exposed to lethal doses of this agent. During this exposure to H2O2, it was observed an increase in lipid peroxidation of the membrane, though this increase did not affect cell viability. The labeling of HeLa cells with the B subunit of cholera toxin, which detects the presence of lipid rafts, showed a decrease in intensity at 30 min and a slight accumulation at 360 min of exposure to 12,5 μM H2O2. Studies using immunofluorescence suggest an increasing in the levels of caveolin-1 for short periods of exposure to H2O2 steady-state, which was confirmed by Western blot. Preliminary results using Western blot and immunofluorescence suggested that pre-exposure to 12,5 μM H2O2 also increased the levels of phosphorylated caveolin-1. Concerning the resistance of tumor cells to lethal doses of the anti-cancer drugs cisplatin and doxorubicin, adaptation to H2O2, increased resistance to cisplatin, having the opposite effect when was used doxorubicin-induced cell death. These results show that, in a human cell line, low and adaptive concentrations of H2O2 change, in a few minutes, the organization of the plasma membrane. Adaptation to H2O2 confers resistance of HeLa cells to the drug cisplatin, while increasing doxorubicin anti-tumor action. Altogether, these observations may have implications to anti-cancer therapy, namely in the design of adjuvant therapy.
The plasma membrane is a barrier that controls the entry and exit of compounds in the cell, separating the intracellular content from the extracellular medium. This study aimed to elucidate the role of hydrogen peroxide (H2O2), a signaling molecule, in the alteration of lipid rafts and caveolae domains in plasma membrane and establishment of possible resistance of tumor cells (HeLa) adapted to H2O2, to anti-cancer drugs cisplatin and doxorubicin. When HeLa cells were exposed to a steady state of 12,5 μM of H2O2 for 6 h, it was observed that cells adapt to H2O2, showing a survival advantage when exposed to lethal doses of this agent. During this exposure to H2O2, it was observed an increase in lipid peroxidation of the membrane, though this increase did not affect cell viability. The labeling of HeLa cells with the B subunit of cholera toxin, which detects the presence of lipid rafts, showed a decrease in intensity at 30 min and a slight accumulation at 360 min of exposure to 12,5 μM H2O2. Studies using immunofluorescence suggest an increasing in the levels of caveolin-1 for short periods of exposure to H2O2 steady-state, which was confirmed by Western blot. Preliminary results using Western blot and immunofluorescence suggested that pre-exposure to 12,5 μM H2O2 also increased the levels of phosphorylated caveolin-1. Concerning the resistance of tumor cells to lethal doses of the anti-cancer drugs cisplatin and doxorubicin, adaptation to H2O2, increased resistance to cisplatin, having the opposite effect when was used doxorubicin-induced cell death. These results show that, in a human cell line, low and adaptive concentrations of H2O2 change, in a few minutes, the organization of the plasma membrane. Adaptation to H2O2 confers resistance of HeLa cells to the drug cisplatin, while increasing doxorubicin anti-tumor action. Altogether, these observations may have implications to anti-cancer therapy, namely in the design of adjuvant therapy.
Descrição
Tese de mestrado em Bioquímica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2011
Palavras-chave
Peróxido de hidrogénio Jangadas lipídicas Caveolae Adaptação Resistência Cisplatona Doxorubicina Teses de mestrado - 2011