Figure 4.

AM but not ATO demonstrated sustained induction reactive oxygen species (ROS) and apoptosis on Huh7 HCC cells. Huh7 cells were treated with 0.25–0.5 μM of AM, ATO, vacant PLGA microsphere (PLGA), or PBS as control (CT) for 3 or 6 days for ROS assay and six days for apoptosis assay. (A). Representative images of ROS levels by using converted dichlorofluorescein (DCF) (green fluorescence) as a surrogated marker. Bar = 50 μm. (B) ROS levels were measured by quantifying DCF fluorescent signals (adjusted by cell numbers and corrected with signals in CT; data: mean ± SD). No induction of ROS in cells treated with PLGA. After three days of incubation, both AM and ATO induced ROS in a dose-dependent manner (0.25–0.5 μM) (**p<.01). At the same concentration of drug, no difference was observed between AM and ATO in term of ROS induction at day 3. After six days of incubation, the induction of ROS in AM group was remained; but the induction of ROS in ATO group was abolished and reduced to the baseline (##p<.01). (C). Apoptosis was evaluated by using caspase-3 activity as a biomarker (data: mean ± SD). At day 6 of incubation, no induction of apoptosis was observed in cells treated with PLGA or ATO (0.25–0.5 μM). Induced apoptotic activity was observed in cells treated with AM (0.25–0.5 μM) (##p<.01). 0.5 μM of AM had higher proapoptotic activity than 0.25 μM of AM (**p<.01).