Squalene in Nanoparticles Improves Antiproliferative Effect on Human Colon Carcinoma Cells Through Apoptosis by Disturbances in Redox Balance

Abstract

Squalene, a triterpene found in extra virgin olive oil, has therapeutic properties in diseases related to oxidative stress, such as cancer. However, its hydrophobic nature and susceptibility to oxidation limit its bioavailability outside of olive oil. To expand its applications, alternative delivery methods are necessary. The objective of the present study was to examine the impact of squalene encapsulated in PLGA (poly(lactic-co-glycolic) acid) nanoparticles (PLGA + Sq) on the proliferation of human colon carcinoma Caco-2 cells, as well as its underlying mechanism of action. The findings demonstrated that PLGA + Sq exert no influence on differentiated cells; however, it is capable of reducing the proliferation of undifferentiated Caco-2 cells through apoptosis and cell cycle arrest in the G1 phase. This effect was initiated by the release of cytochrome c into the cytoplasm and the subsequent activation of caspase-3. Furthermore, squalene exhibited pro-oxidant activity, as evidenced by an increase in intracellular ROS (reactive oxygen species) levels. The results of the squalene effect on genes associated with cell death, inflammation, and the cell cycle indicate that its antiproliferative effect may be post-transcriptional. In conclusion, PLGA + Sq demonstrate an antiproliferative effect on Caco-2 cells through apoptosis by altering redox balance, suggesting squalene's potential as a functional food ingredient for colorectal cancer prevention.

Keywords: Caco-2 cells; PLGA; ROS; apoptosis; nanoparticles; squalene.

Figure 1

Viability effect on undifferentiated Caco-2 cells incubated with different squalene concentrations using DMEM, DMSO, EtOH, and PLGA vehicles for 72 h.

Figure 2

Time and dose-response viability effect of a range of PLGA + Sq on undifferentiated Caco-2 cells.

Figure 3

Differentiated Caco-2 cells incubated for 72 h with 70 or 140 μg/mL PLGA + Sq. PLGA 70 or 140 μg/mL (without Sq) represent the PLGA-NPs required for the indicated Sq concentration. C, control, refers to untreated cells.

Figure 4

In vitro cellular uptake of squalene. Caco-2 cells were incubated with 140 μg/mL of PLGA + Sq NPs and PLGA NPs for a period of 24 h. * p < 0.05 vs. PLGA.

Figure 5

Incubation of undifferentiated Caco-2 cells for 72 h. (A) Negative control, referring to the untreated cells, (B) PLGA, (C) PLGA + Sq at IC₅₀ concentration (140 μg/mL). Percentages of alive (A3), necrotic (A1), early apoptotic (A4) and late apoptotic (A2) cells are indicated.

Figure 6

Undifferentiated Caco-2 cells with mitochondrial cytochrome c after 72 h incubation with/without PLGA or Sq (140 μg/mL). (A) Negative control, referring to the untreated cells, (B) PLGA, (C) PLGA + Sq. A1: cytochrome c released, A2: cytochrome c retained, A3 and A4: debris and dead cells.

Figure 7

Percentage of undifferentiated Caco-2 cells with active caspase-3 after 72 h incubation with/without PLGA + Sq (140 μg/mL). * p < 0.05 vs. control.

Figure 8

Measurement of the cell cycle after a 72 h incubation on undifferentiated Caco-2 cells (A) Negative control, referring to the untreated cells, (B) PLGA, (C) PLGA + Sq (IC₅₀). From left to right: red peak: G1, black hatched peak: S, red peak: G2.

Figure 9

Measurement of ROS levels on undifferentiated Caco-2 cells after 24 h incubation with PLGA + Sq and PLGA alone. PLGA 70 or 140 μg/mL (without Sq) represents the PLGA-NPs required for the indicated Sq concentration. * p < 0.05 vs. negative control (without PLGA and Sq). C, control, refers to untreated cells.

Figure 10

Cell viability measurement after pretreatment of cells with 5 mM NAC for 2 h followed by treatment of cells with 140 μg/mM PLGA + Sq for 72 h. * p < 0.05 vs. control. C, control, refers to untreated cells.

Figure 11

Differentiated Caco-2 cells incubated with 70 or 140 mg/mL PLGA in the presence or absence of squalene. PLGA 70 or 140 μg/mL (without Sq) represents the PLGA-NPs required for the indicated Sq concentration. C, control, refers to untreated cells.

Figure 12

Evaluation of the effect of PLGA + Sq nanoparticles on cytotoxicity and ROS modulation in undifferentiated and differentiated Caco-2 cells, and AML12 cells [16,49].