Figure 3.

Cellular Entry of PC-SA Liposome and Its Role in Induction of Apoptosis

(A and B) Uptake of Rh123-PC-SA liposomes in B16F10 cells in the presence of inhibitors of endocytosis and macropinocytosis as determined by fluorescence-activated cell sorting (FACS). (A) PC-SA liposomes utilize clathrin and an actin-dependent endocytosis pathway mechanism for entry into target cells. (B) Shown are B16F10 cells without Rh123-PC-SA (grey peak), with Rh123-PC-SA (red peak), and with Rh123-PC-SA in the presence of cytochalasin D (blue peak), chlorpromazine (yellow), and amiloride (green). (C–F) PC-SA treatment for 2 hr after cellular entry induces multiple hallmarks of apoptosis in B16F10, K562, and U937 cells and has negligible effect on RAW264.7 cells. (C) Depolarization of the mitochondrial potential of cells treated with 140 μg/mL of PC-SA, stained with JC-1 and analyzed in a flow cytometer. The percentage of cells expressing green and red fluorescence is indicated. (D) To determine the effect of PC-SA liposomes on reactive oxygen species (ROS) generation, the B16F10, K562, U937, and RAW 264.7 cell lines were loaded with H2DCFDA after treatment with 40 and 140 μg/mL of PC-SA liposomes. The relative increase in fluorescence intensity of H2DCFDA as percent of untreated cells was measured. (E) Apoptosis (sub-G0/G1 accumulation and G2M) as assessed by FACS after treatment with 140 μg/mL of PC-SA liposomes. (F) Early and late apoptosis as determined by annexin V/PI staining by FACS after treatment with 140 μg/mL of PC-SA liposomes. All data represent the mean of triplicate experiments, with error bars indicating the SEM.