Mechanism Of Tocotrienol Induced Cell Death On Colon Carcinoma Cells Via Wnt Signaling Pathway

ObjectiveThe purpose of this study was to determine the mechanism of tocotrienols induced cell death on the Wnt signaling pathway in colon carcinoma cells (SW620) by the method of cell culture in vitro and molecular biological techniques.Methods1. The effects of tocotrienol on the proliferation of SW620 cellsAfter treated with different concentrations ofδ-tocotrienol or y-tocotrienol for 24h, cell viability was tested by MTT assay. The SW620 cells were treated with 0,5,10,15,20μmol/Lδ-tocotrienol and 15,30,45,60μmol/Lγ-tocotrienol respectively. Then, the cell death was determined via DAPI and AO/EB staining assay, transmission electron microscopy assay, flow cytometry assay, Caspase-3 activity assay, cell viability with/without z.VAD.fmk.2. The mechanism of tocotrienol induced cell death on SW620 cellsThe SW620 cells were treated with 0,5,10,15,20μmol/Lδ-tocotrienol and 15, 30,45,60μmol/Lγ-tocotrienol respectively. After treated for 24h, wnt-1,β-catenin,cyclin Dl,c-jun and MMP-7 mRNA and protein expression were observed by Real-time RT-PCR and Western blotting assay.Results1. Compared with control group, both y-tocotrienol andδ-tocotrienol dose-dependently decreased SW620 cell viability (P<0.05). The IC50 value of y-tocotrienol andδ-tocotrienol on SW620 cells was 31.35,15.2μmol/L respectively.2. The morphological assays showed that classic apoptotic changes were absent in tocotrienol-treated cells, such as cell shrinkage, membrane blebbing, chromatin condensation with margination at the nuclear periphery and formation of apoptotic body, but there were a plenty of large or small cytoplasmic vacuoles in treated cells. Swelling of mitochondria was observed from transmission electron microscopy and mitochondria had vacuolized. 3. The cell viability in both the 8-tocotrienol groups andδ-tocotrienol with z.VAD.fmk groups were decreased in a dose-dependent manner. There was no difference within the same concentration ofδ-tocotrienol with or without z.VAD.fmk (P>0.05). When compared to the negative control group, there was not a significant increase in cell apoptosis ratio an the levels of caspase-3 activity in the 8-tocotrienol-treated groups (P>0.05). The results of y-tocotrienol were the similar toδ-tocotrienor's.4. After treated withδ-tocotrienol for 24h, the expression ofδ-catenin and wnt-1 mRNA and proteins were reduced.δ-Tocotrienol had no effects on the mRNA expression of cyclin Dl and MMP-7, but decreased the cyclin D1 and MMP-7 protein expression.5. In the treated groups, y-tocotrienol reduced the expression ofβ-catenin, c-jun and cyclin Dl mRNA and proteins in varying degrees. y-Tocotrienol decreased the wnt-1 and MMP-7 protein expression, but had no effects on wnt-1 and MMP-7 mRNA expression.Conclusions1. It is a study to prove the inhibitory effect of tocotrienols on SW620 cells. The inhibitory effect ofδ-tocotrienols is stronger than y-tocotrienol's.2. Tocotrienols can induce paraptosis-like cell death on SW620 cells.3. Having an influence on the Wnt signaling pathways may be one of the mechanisms of paraptosis-like cell death induced by tocotrienols on SW620 cells.