Study On Preparation And Anti-tumor Activity Of Sphingoid Bases From Hippocampus Trimaculatus Leach

Hippocampus trimaculatus Leach belongs to the teleost family Syngnathidae,which is widely distributed in tropical or subtropical shallow waters.H.trimaculatus Leach is one of the most important species in traditional Chinese medicine and has been studied for its diverse biological activities,such as anti-aging,anti-fatigue,anti-inflammation and antioxidant properties.The aim of the present study was to evaluate the anti-tumor activity of sphingoid bases from H.trimaculatus Leach through apoptosis induction.Furthermore,the possible mechanisms of sphingoid base-induced apoptosis were also elucidated.The major research and results are as follow.1.Separation,purification and structure identification of sphingoid bases from H.trimaculatus LeachTotal lipids were extracted with ethanol through ultrasonication from H.trimaculatus Leach.Total lipids were applied to medium-pressure column chromatography(MPCC)and eluted by chloroform,acetone and methanol,respectively.The acetone elution was saponified with 0.4 N KOH in methanol and degraded with aqueous methanolic 1 N HCL.The raw sphingoid bases were purified by MPCC and HP-20 macroporous resin.The composition and structure of sphingoid bases were determined by thin-layer chromatography(TLC),infrared spectroscopy(IR)and gas chromatography-mass spectrometry(GC-MS).A single spot on TLC was visualized by ninhydrin solution.The purple and red spot indicated that the elution from MPCC were sphingoid bases.The spectrum of sphingoid bases was used to speculate its functionalities.The results from GC-MS analysis showed that the major sphingoid bases were d16:0,d18:0 and d18:2.2.The anti-tumor effect of sphingoid bases in K562 cellsWe first analyzed the effect of sphingoid bases on cell viability by MTT and trypan blue staining.The results showed that sphingoid bases effectively inhibited K562 cell viability in a dose-dependent manner.The IC50 of sphingoid bases in 24 and 48 h were approximately 37.17 and 31.01 ?g/mL respectively.Furthermore,the sphingoid bases-induced apoptosis was evaluated by Annexin V assay.The results showed that sphingoid bases caused cell death by inducing apoptosis in a dose-dependent manner after 24 h treatments.The results of cell cycle revealed that sphingoid bases increased the number of K562 cells with sub-G0/G1 region(hypodiploid DNA content indicative of apoptosis)left to the G0/G1 phase and arrested cell cycle at the S phase.Therefore,we can conclude that sphingoid bases potently inhibited the viability of K562 cells and the inhibitory effect was associated with the increase of sub-G0/G1 region and accumulation of cells in the S phase.Those results indicated that the anti-tumor of sphingoid bases was associated with apoptosis.3.The possible mechanism of sphingoid base-induced apoptosisWe first investigated the effect of sphingoid bases on Bcl-2 and Bax expression by RT-PCR.The results indicated that the treatment of sphingoid bases up-regulated the mRNA expression of Bax and down-regulated the expression of Bcl-2,further decreasing the ratio of Bcl-2 to Bax in K562 cells.The balance of Bcl-2/Bax was crucial to mitochondrial membrane potential(MMP).Therefore,we investigated the effect of sphingoid bases on mitochondrial membrane potential.The results showed that dose-dependent dropping of fluorescent signal was observed in K562 cells treated with sphingoid bases,indicating the loss of transmembrane potential induced by sphingoid bases.Loss of MMP results in the release of cytochrome c from the mitochondrial intermembrane space into the cytosol.The formation of a multimeric cytochrome c and Apaf-1 complex can trigger the activation of procaspase-9,which subsequently activating the members of caspases.We also investigated the effect of sphingoid bases on caspase-3,8 and 9 expression by RT-PCR and Western blot.The results showed that sphingoid base-induced apoptosis was accompanied by increased expression of caspase 3 and 9 in a dose-dependent manner.The results indicated that mitochondrial pathways can trigger the activation of initiator procaspase-9,the caspase-9 subsequently induces the cleavage of procaspase-3,further inducing cell apoptosis.In addition,sphingoid base-induced apoptosis was accompanied by the activation of caspase-3 and 8 in K562 cells.The data demonstrated that the death receptor pathway may be involved in inducing the death of cancer cells in the presence of sphingoid bases.In conclusion,these results demonstrated that sphingoid bases from H.trimaculatus Leach triggered mitochondria-and death receptor-mediated apoptosis in K562 cells.4.The effect of sphingoid bases on COX-2 in K562 cellsWe investigated the effect of sphingoid bases on the expression of cyclooxygenase(COX-1 and COX-2)by mouse cyclooxygenase ELISA kits.The result showed that the treatment of sphingoid bases significantly decreased the expression of COX-2 compared to the control group.Those results demonstrated that sphingoid bases may inhibit the expression of COX-2 and the formation of new blood vessels,and promote the cell apoptosis,which can inhibit the proliferation of K562 cells.