Figure 1.
The cell viability of SW620 cells and HCT-8 cells.
The panels showed γ-tocotrienol (I) and PTX (II) in SW620 cells and HCT-8 cells (III). The cells were treated with various concentrations of γ-tocotrienol or PTX for 24 h. Cell viability was determined by MTT assay. Data were expressed as means ± standard deviation (n = 6). *P<0.05, compared to the negative control group.
Figure 2.
The morphological changes of cell death in SW620 cells treated with γ-tocotrienol, FH535 or PTX.
SW620 cells were treated with γ-tocotrienol, FH535 or PTX for 6 and 24 h. The morphological changes of SW620 cells were detected under a light microcopy (I, Magnification: ×100), a fluorescent microscope stained with DAPI (II, magnification: ×200) and AO/EB (III, magnification: ×100). The cells treated with control (A), 15 (B), 30 (C), 45 (D) and 60 µmol/L (E) for 24 h as well as 60 µmol/L of γ-tocotrienol (F) for 6 h, or the cells treated with PTX at doses of 0(A), 0.2(B), 0.4(C), 0.8(D), 1.2(E) µmol/L, or FH535 at doses of 0(A), 0.625(B), 1.25(C), 2.5(D) µmol/L and DMSO (E) for 24 h. After treatment, the cells showed the slow growth, loosed tight contacts amongst cells and reduction of cell size, turn round of cell shape and cytoplasmic vacuolar when compare a spindly and large and grew close together, with prominent nucleoli and abundant cytoplasm in control group. Red arrows indicate the vacuoles.
Figure 3.
The morphological changes of cell death in HCT-8 cells.
HCT-8 cells were treated with 0, 20, and 40 µmol/L of γ-tocotrienol for 24 h. The morphological changes of HCT-8 cells were detected under a light microcopy (I, Magnification: ×200). The cells were stained with DAPI (II, Magnification: ×200). Both figure showed that the cells treated with control (A), 20 (B), 40 µmol/L(C) for 24 h. Arrows indicate the vacuoles.
Figure 4.
Morphological changes of paraptosis-like cell death were observed in SW620 cells by transmission electron microscopy (TEM).
After treatment with 15 (B), 30 (C), 45 (D) and 60 µmol/L (E) of γ-tocotrienol for 24 h, SW620 cells showed the swelling of mitochondria or ER (red arrowheads) and extensive cytosolic vacuolization (red arrows). The micrographs (A) provided normal cells control. The significant swellings of mitochondria or ER were observed after treated with 60 µmol/L of γ-tocotrienol for 6 h (F).
Figure 5.
Cell death was examined in SW620 cells by flow cytometry.
The cells treated with 0 (A), 15 (B), 30 (C), 45 (D) and 60 µmol/L (E) of γ-tocotrienol for 24 h. The percentage of early apoptosis was obtained from lower right quadrant (annexin V-EGFP+, PI–). *P<0.05, compared to the same dose of each group.
Figure 6.
The cellular caspase-3 activity and DNA ladder were examined in SW620 cells or HCT-8 cells.
The cellular caspase-3 activity was determined in SW620 cells (I) and HCT-8 cells (II) treated with different doses of γ-tocotrienol as well as DNA ladder in SW620 cells treated with PBS (A), 1.0 and 1.5 µmol/L of PTX or γ-tocotrienol (D) for 24 h. There are no difference between the treated and control groups.
Figure 7.
The cell viability of γ-tocotrienol with or without z.VAD.fmk.
The SW620 cells were treated with different doses of 15, 30, 45 and 60 µmol/L of γ-tocotrienol with or without z.VAD.fmk for 24 h. The cell viability was examined by MTT assay. *P<0.05, compared to the control group.
Figure 8.
The mRNA expression levels of Wnt-1, β-catenin, c-jun and cyclin D1 were detected in SW620 cells treated with different concentration of γ-tocotrienol by RT-PCR.
γ-Tocotrienol significantly decreased the mRNA expression of β-catenin, c-jun and cyclin D1 in SW620 cells (P<0.05).
Figure 9.
The protein expressions of Wnt-1, β-catenin, c-jun, cyclin D1 and β-actin in SW620 cells.
The cells treated with 15, 30, 45 and 60 µmol/L of γ-tocotrienol (A and B), or PTX at 0.2, 0.4, 0.8 and 1.2 µmol/L (C), or FH535 at 1.25, 2.5, 5 and 15 µmol/L for 24 h (D). *P<0.05, compared to the negative control group.
Table 1.
The sequences of primers.