Fluorescence Lifetime of 1-pyrenebutyric Acid Detects Ros Variation after Treatment with Anticancer Drug Adriamycin

Tumor hypoxia is causally related with resistance to adriamycin (ADR), and hyperbaric oxygen therapy has been shown to enhance the chemotherapeutic effect of the drug. It is believed that exposure to oxygen leads to an increase of intracellular reactive oxygen species (ROS) concentration. To monitor ROS involvement in this improved anticancerous effect, we used a new method of ROS detection based on measurement of 1-pyrenebutyric acid (PBA) fluorescence lifetime [1]. Here, we quantify ROS variations after cell treatment with ADR under 2%O2 or 21%O2. The cell behaviour was also followed by videomicrofluorimetry allowing to record and analyse numerical images of different fluorescent probes labelling the cells. This tool combined to multifactorial analysis give us informations about cell cycle state [2] and apoptosis induction. Methods: We recorded the fluorescence lifetime decays using time-resolved microfluorimetry on human lymphoblastic cells CCRF-CEM loaded with PBA. Emission is recorded through a 404nm band-pass filter after excitation of single living cells with a pulsed nitrogen laser (337nm, 3ns). ROS quantity variations were calculated using the Stern-Volmer equation [1]. Measurements were daily performed on adriamycin-treated cells (1.7μM) incubated under 2%O2 or 21%O2 during 144h. Concomitantly, we recorded numerical images of cell populations stained with Hoescht 33342, Rhodamine 123 and Nile Red, which respectively labelled DNA, mitochondria and plasma membrane. The collected parameters were computed in a multifactorial approach (typology and factorial analysis) to classify the cells in the different cell cycle phases. Results: We observed a faster and more significant ROS production by adriamycin when treated cells were incubated under 21%O2. ADR immediately blocks the cell proliferation at once under 2%O2 and 21%O2. No significant variation of the cell cycle distribution was observed after increasing the oxygen level during incubation. However, we noted a slight increase of the number of cells in early state of apoptosis under 21%O2. Moreover, trypan blue exclusion assay showed that cell viability was less considerable under 21%O2. Conclusions: In our in-vitro model, we showed a slight increase of the anticancerous effect of adriamycin after increasing the oxygen level up to 21%O2 during cell incubation. DNA alteration by adriamycin seems to be independent of the ROS production by the drug. However, ROS appears to be involved in the improvement of the drug anticancerous effect, certainly throught apoptosis pathway.