Piceatannol-Loaded Emulsomes Exhibit Enhanced Cytostatic and Apoptotic Activities in Colon Cancer Cells

Abstract

Piceatannol (PIC), a naturally occurring polyphenolic stilbene, has pleiotropic pharmacological activities. It has reported cytotoxic activities against different cancer cells. In the present study, PIC emulsomes (PIC-E) were formulated and assessed for cytotoxic activity. A Box-Behnken design was employed to investigate the influence of formulation factors on particle size and drug entrapment. After optimization, the formulation had a spherical shape with a particle size of 125.45 ± 1.62 nm and entrapment efficiency of 93.14% ± 2.15%. Assessment of cytotoxic activities indicated that the optimized PIC-E formula exhibited significantly lower IC₅₀ against HCT 116 cells. Analysis of the cell cycle revealed the accumulation of cells in the G2-M phase as well as increased cell fraction in the sub-G1 phase, an indication of apoptotic-enhancing activity. Staining of cells with Annexin V indicated increased early and late apoptosis. Further, the cellular contents of caspase - 3 and Bax/Bcl-2 mRNA expression were significantly elevated by PIC-E. In addition, the mitochondrial membrane potential (MMP) was disturbed and reactive oxygen species (ROS) production was increased. In conclusion, PIC-E exhibited superior cell death-inducing activities against HCT 116 cells as compared to pure PIC. This is mediated, at least partly, by enhanced pro-apoptotic activity, disruption of MMP, and stimulation of ROS generation.

Keywords: HCT 116 cells; apoptosis; emulsomes; mitochondrial membrane potential; piceatannol; reactive oxygen species.

Figure 1

Externally studentized residual plot versus run for (A) particle size and (B) entrapment efficiency% of PIC-E.

Figure 2

3D response surface plots for the effect of PIC concentration, Lipoid^® S 100 concentration, and the pH of the hydration medium on the particle size of PIC-E.

Figure 3

3D response surface plots for the effect of PIC concentration, Lipoid^® S 100 concentration, and pH of hydration medium on entrapment efficiency % of PIC-E.

Figure 4

TEM image of PIC-E (X 60,000).

Figure 5

In vitro release profile of PIC-E in phosphate-buffered saline (pH 7.4, 0.1 M) containing Tween 80 (0.1%) at 37 ± 0.5 °C.

Figure 6

Impact of PIC-E on cell cycle phases of HCT 116 cells. (a) Control, (b) Plain-E, (c) PIC-raw, (d) PIC-E, and (e) bar diagram of the different cycle phases. Cells were incubated for 24 h with plain-E, PIC-raw (0.1 µM) or PIC-loaded E (equivalent to 0.1 µM PIC). * Significantly different from control at p < 0.05. ^# Significantly different from plain-E at p < 0.05. ^$ Significantly different from PIC-raw at p < 0.05.

Figure 7

Effect of PIC-E on apoptotic and necrotic death in HCT 116 cells and impact of PIC-E on cell cycle phases of HCT 116 cells: (a) Control, (b) Plain-E, (c) PIC-raw, (d) PIC-E, and (e) bar diagram of different types of cell death. Cells were incubated for 24 h with plain-E, PIC-raw (0.1 µM), or PIC-E (equivalent to 0.1 µM PIC) * Significantly different from control at p < 0.05. ^# Significantly different from Plain-E at p < 0.05.

Figure 8

Effect of PIC-E on MMP in HCT 116 cells. Cells were incubated for 24 h with plain-E, PIC-raw (0.1 µM), or PIC-E (equivalent to 0.1 µM PIC) * Significantly different from control at p < 0.05. ^# Significantly different from plain-E at p < 0.05. ^$ Significantly different from PIC-raw at p < 0.05.

Figure 9

Effect of PIC-E on cleaved caspase-3 content in HCT 116 cells. Cells were incubated for 24 h with plain-E, PIC-raw (0.1 µM), or PIC- E (equivalent to 0.1 µM PIC) * Significantly different from control at p < 0.05. # Significantly different from plain-E at p < 0.05. $ Significantly different from PIC-raw at p < 0.05.

Figure 10

Effect of PIC-E on Bax and Bcl-2 mRNA expression content in HCT 116 cells; cells were incubated for 24 h with plain-E, PIC-raw (0.1 µM), or PIC- E (equivalent to 0.1 µM PIC) * Significantly different from control at p < 0.05. ^# Significantly different from plain-E at p < 0.05. ^$ Significantly different from PIC-raw at p < 0.05.

Figure 11

Effect of PIC-E on nitric oxide production in HCT 116 cells. Cells were incubated for 24 h with plain-E, PIC-raw (0.1 µM), or PIC- E (equivalent to 0.1 µM PIC).

Figure 12

Effect of PIC-E on ROS generation in HCT 116 cells. Cells were incubated for 24 h with plain-E, PIC-raw (0.1 µM), or PIC-E (equivalent to 0.1 µM PIC) * Significantly different from control at p < 0.05. ^# Significantly different from Plain-E at p < 0.05. ^$ Significantly different from PIC-raw at p < 0.05.