Manner And Mechanism Of Death Tocotrienol Induced On Colon Cancer Cells

PurposeThe purpose of this study is to explore the way of tocotrienol-induced colon cancer cell death and death mechanism, throughing cultured the colon cancer cells SW620in vitro, and then using the experimental techniques of molecular biology.Method1. The manner of the death that tocotrienols induced human colon cancer cells SW620.Cultured in vitro SW620cells by adding different doses of delta-tocotrienol (1.2.5.5.10.15.20.25.30and40micromol/L) and ethanol (solvent control) for24h, MTT assay observed delta-tocotrienol SW620cell viability; according5,10,15,20μmol L δ-tocotrienol role in cell staining (AO/EB staining, DAPI staining) observed cell death; Add to delta-tocotrienol and then follow the dose of20micromol/L, using immunocytochemistry, DNA Ladder to detect cell death. Paclitaxel, as typical apoptosis-inducing agent on tumor cells observed in SW620cells, as a con trol experiment. Observed the method cell death of gamma-tocotrienol on HCT-8cells. as a parallel experiment.2. The mechanism of death that tocotrienol-induced human colon cancer cells SW620.Cultured in vitro SW620cells added20μmol/Lδ-tocotrienol for24h, immunocytochemistry and western blot method to detect calcium protein of the endoplasmic reticulum protein markers and Bip protein expression wnt pathway inhibitors FH535wnt pathway inhibition of cell, using Western methods to detect the Wnt pathway critical factor beta-catenin expression.ResultCompared with the ethanol control group, when colon cancer cells SW620was treated by delta-tocotrienol for24h after, each dose group can make SW620cells decreased survival (p<0.05), and cell viability with the delta-increasing doses of tocotrienol showed a reducing trend, the IC50value15.18μmol/1. when colon cancer cells HCT-8was treated by gamma-tocotrienol for24h after, each dose group can make the HCT-8cell viability decreased (p<0.05), and cell viability with gamma-tocotrienol dose increase showing a decreasing trend, the IC50value of32.69mol/L.2. When treated with tocotrienols colon cancer cells SW620and HCT-8cells, there were not only unregular nucleus,nuclear chromosomal DNA concentrated, with side crescent-shaped, but also DNA Ladder formation of apoptotic bodies generate typical withered the phenomenons of death, replaced by a large number of intracellular vacuoles. Autophagy marker LC3negative expression in the immunocytochemistry test. SW620cell death caused by the delta-tocotrienol paraptosis inhibitors may be inhibited. The typical apoptotic positive drug paclitaxel SW620nuclear DNA can generated gradient rupture.The preliminary results have confirmed tocotrienol-induced colon cancer cell death and inhibit SW620cells wnt pathway. As a control, the results show that the wnt pathway inhibitor FH535, FH535can lead SW620cells to death, morphology display a large number of vacuoles in the cytoplasm, and protein results show that the Wnt pathway critical factor beta-catenin expression used in this experiment decline.Immunocytochemistry, delta-tocotrienol role in colon cancer cells SW62024h after endoplasmic reticulum calcium, protein expression was positive, as the tocotrienols dose increases, the endoplasmic reticulum gradually the formation of vacuoles. Endoplasmic reticulum stress markers Bip protein expression increased.Conclusion1. Confirmed that tocotrienols induced a paraptosis death on colon cancer cells.2. This kind of death might be induced by inhibiting the activity of Wnt signaling pathway in colon cancer cells and causing endoplasmic reticulum stress after the SW620cells be treated with the tocotrienols.