Measuring dynamics of caspase-3 activity in living cells using FRET technique during apoptosis induced by high fluence low-power laser irradiation

Abstract

Using FRET technique, we have reported that high fluence LPLI can induce human lung adenocarcinoma cells (ASTC-a-1) apoptosis. The activation of caspase-3 plays an important role in the apoptotic process.

Measurement of two caspase activities simultaneously in living cells by a novel dual FRET fluorescent indicator probe

“…Although CFP-DEVD-YFP has frequently been used and reported as a caspase-3 fluorescent indicator and in monitoring apoptosis-related caspase-3 activity in living cells (24,33,35,40), the specificity of the DEVD cleavage site has not been carefully delineated in living cells. Indeed, we (data not shown) and others (9) have noted that z-DEVDfmk does not inhibit caspase-3 activity specifically.…”

“…Chemical substrates, including fluorogenic substrates and fluorochrome-labeled inhibitors of caspases (FLICAs), have been developed for this purpose (8,(21)(22)(23). More recently, genetically engineered probes have been described, consisting of a CFP-YFP fusion protein in which the fluorophores are separated by a very short FRET-permissive linker containing at least one caspase cleavage site, such as DEVD, LEVD, or another caspase-sensitive cleavage sequence (24)(25)(26)(27)(28)(29)(30)(31)(32)(33). The linker is cleaved by activated caspases, triggering decreased FRET because of the resultant physical separation of CFP and YFP molecules.…”

Análise da expressão e mecanismos de ação das proteínas Akt, Hsp90, mTOR e ciclina D1 em cultura de células de carcinoma epidermoide humano e células displásicas após irradiação com laser em baixa intensidade

“…Outros estudos já demonstraram que a LLLT, quando aplicada sob altas fluências, pode sim causar diminuição das células viáveis (209,221,251) . Mas apesar destes estudos terem encontrado efeitos inibitórios, (216,217,(252)(253)(254) .…”

“…Além de doses acima de 60 J/cm 2 terem induzido apoptose em células de adenocarcinoma de pulmão, por ativação de caspase-3 (221) , dois outros estudos mostraram que esta ativação de caspase-3 pode ser induzida pela geração de espécies reativas de oxigênio (ROS) por LLLT em altas fluências (299,300) . A geração de ROS leva ao estresse oxidativo e à perda das funções celulares, consequentemente levando à apoptose ou necrose das células (301,302) .…”

“…Por outro lado, altas doses de LLLT (HeNe; 632.8 nm; 40 60J/cm 2 ) tiveram o efeito inverso, inativando as PKCs (216) . De maneira condizente, estas altas doses de LLLT foram capazes de induzir apoptose em células de adenocarcinoma de pulmão (221) .…”