The Anticancer And Anti-inflammation Effects Of ?-Tocotrienol And Their Molecular Mechanisms

?-tocotrienol is an important ingredient of vitamin E.However,most of the vitamin E studies were focused on tocopherols,in particular on?-tocopherol.Roughly3%of the studies of vitamin E,were focused on tocotrienols.Surprisingly,in some cancer species,the anti-inflammatory effect and anti-cancer effects of tocotrienol are superior than that of tocopherol.Moreover,?-tocotrienol has a superior anticancer effect,comparing to?-tocotrienol and other components of vitamin E.But the molecular mechanism were not further studied.Nasopharyngeal carcinoma(NPC)is a cancer with a high incidence in south China,southeast Asia,north Africa,the Middle East and the arctic.Therefore,it is of great significance to find chemopreventive food in our daily diet for the sake of health of people in areas with high incidence of NPC.In this study saponification method and supercritical CO₂ extraction were used to extract?-tocotrienol,and?-tocotrienol was identified by HPLC,using RAW264.7 and CNE1 cells as in vitro model,the anti-inflammatory effect and anti-cancer effect of?-tocotrienol on macrophage RAW264.7 in vitro model and on nasopharyngeal carcinoma cells in vitro model were detected.Furthermore,the molecular mechanisms of anti-inflammatory and anti-cancer effect of anti-cancer were assessed,for further research of use of?-tocotrienol,and further use of?-tocotrienol as functional food,and developed as health care products to provide some scientific theory basis.1.Extraction and determination of?-tocotrienolThe rice bran is dried and evenly crushed and put into a high-pressure container with a water jacket.The CO₂ flow rate was kept constant in the high pressure vessel.Then the rice bran oil was extracted by CO₂ supercritical extraction.The separation and detection by high performance liquid chromatography(HPLC)showed that the purity of?-tocotrienol in rice bran oil was 132.7 g/kg.2.Inhibitory effect and molecular mechanism of?-tocotrienol on the anti-inflammatory in vitro model of macrophage RAW264.7In order to assess the anti-inflammatory effect of?-tocotrienol on RAW264.7cells induced by LPS in vitro,this study used RAW264.7 cells to detect the inhibitory effect of?-tocotrienol on RAW264.7 cells induced by LPS.Firstly,microscopic observation and MTS analysis showed that 0-80 M?-tocotrienol had no significant toxicity to RAW264.7 cells.Lipopolysaccharide is one of the most powerful exogenous stimulants to activate the body's immune system and induce septic shock.At a low concentration(less than 1nmol/L),it can activate the body's immune system and stimulate the release of inflammatory cytokines by immune cells.Lipopolysaccharide(LPS)induced results show that 20,40,80?M?-tocotrienol can significantly inhibit RAW264.7 lipopolysaccharide is activate the body's immune system and induce the body happens septic shock material of one of the most typical and strongest exogenous stimuli,at low concentrations(less than 1 nmol/L),you can activate the body's immune system,immune cells release inflammatory factor.Compared with the control group,the m RNA expressions and protein expressions of proinflammatory factors TNF-?,IL-6,IL-1?,i NOS were significantly increased in the LPS group,while the m RNA and protein expressions were decreased in the?-tocotrienol group,showing a dose-dependent relationship.3.Studies on the effect of?-tocotrienol on inducing apoptosis and inhibiting proliferation of CNE1 in nasopharyngeal carcinoma cells and its molecular mechanismNasopharyngeal cancer(NPC)is a cancer with a relatively high incidence in southern China,southeast Asia,northern Africa,the Middle East and the arctic.It kills many people every year at a rate of 25-50/100,000.So it is very important for the health of people with high incidence of nasopharyngeal cancer to find tumor chemoprophylaxis in our diet.This study based on the observation of delta-triene of phenol,the influence of the nasopharyngeal carcinoma CNE1 cells induced apoptosis and proliferation,found in the?-tocotrienol after processing,the optical microscope and cell morphology Hoechst 33258 fluorescent dye has obvious between 24-72 h has the effect of inducing apoptosis,and along with the MTS test results show that the?-tocotrienol of CNE1 cells proliferation inhibition rate at 0-80?M obviously dose dependent features.The results showed that the effects of?-tocotrienol on the key target genes of inducing cancer apoptosis and inhibiting cell proliferation were studied by RT-PCR and western blot.Western blotting analysis showed that Bcl-2were decreased,Bax,caspase-3 and caspase-9 were significantly increased after treatment with different concentrations of?-tocotrienol in nasopharyngeal carcinoma cells,while caspase-8 was not significantly changed.The results suggested that?-tocotrienol promotes apoptosis by activating caspase-3 and caspase-9.Through real-time fluorescent quantitative polymerase chain reaction and western blotting,the expressions of p16,p18,p21,p27 and p53 in CDKI were significantly up-regulated and dose-dependent after treatment with different concentrations of?-tocotrienol,but the expressions of p15,p18 and p19 were not significantly changed.?-tocotrienol may inhibit the proliferation of cancer cells by down-regulating the expression of cyclin,inhibiting the activity of CDK,and up-regulating some CDKI expressions to block cell cycle.The results suggested that?-tocotrienol induced cell cycle arrest in CNE1cells via p16/CDK4/cyclin D1 siganaling pathway.Further analysis was done by bioinformatics,it is identified by our results that 169 genes were up-regulated while167 gene were down-regulated.It was found that?-tocotrienol did not only intervened MAPK signal pathway,but also intervened in multiple signaling pathways involved in the resistance to the effect of nasopharyngeal carcinoma cell proliferation.Thus,?-tocotrienol triggers apoptosis in CNE1 cells through Caspase-3 signaling.Both of the signalings were controlled by NF-?B.To sum up,our work suggested the underlying pathway of anticancer effect of?-tocotrienol in CNE1 cells was though NF-?B signaling pathway.