The Effect Of Diallyl Disulfide On Differentiation In HL-60Cells Of Overexpression Cofilin1

Objective: To research the effect of diallyl disulfide (DADS) on cofilin1expression,proliferation, migration, invasion and differentiation of HL-60cells, and investigateits molecular mechanisms. Searching for more effective treatment of leukemia.Methods: The effects of DADS on the expression of cofilin1, p-cofilin1and thefactors of Rac1-ROCK1-LIMK1signal pathway in HL-60cells was detected byRT-PCR and Western blotting. The recombinant plasmidpcDNA3.1-CFL1-IRES2-EGFP that overexpression of cofilin1was stably transfectedinto HL-60cells, to construct HL-60/pcDNA3.1/cofilin1-EGFP cell lines. Theexpression of cofilin1mRNA and protein in cofilin1/HL-60cells was detected byRT-PCR and Western blot, as well as the effects of DADS on expression of cofilin1mRNA and protein in cofilin1/HL-60cells was detected by RT-PCR and Western blot.The effects of DADS on the differentiation of HL-60cells and cofilin1/HL-60cellswas detected by Immunofluorescence, NBT experiment, CCK-8experiment andTranswell experiment. The effects of DADS on the factors of Rac1-ROCK1-LIMK1signal pathway in cofilin1/HL-60cells was detected by RT-PCR and Western blotting.Results:1. The effects of DADS on the expression of cofilin1in HL-60cells.RT-PCR and Western blot assays showed, the expression of cofilin1and p-cofilin1inHL-60cells were inhibited in a time dependent model when treated with DADS for0h,6h,12h,24h,48h (P<0.05).2. The effects of DADS on the proliferation,migration, invasion and reducing ability of HL-60cells. NBT expriment revealed thatthe cells showed a enhanced reduction ability in time-dependent model after treatedwith DADS for24h,48h,72h (P<0.05). CCK-8expriment showed, DADS can inhibitthe proliferation ability of HL-60cells in a time dependent model(P<0.05). Transwellmigration and invasion expriment showed that the migration and invasion ability ofHL-60were inhibited significantly after treated with DADS (P<0.05). No significant difference was found between the DADS groups and ATRA groups (P>0.05).3. Theeffects of DADS on the expression of cofilin1in cofilin1/HL-60cells. Highexpressive vector of cofilin1, namely pcDNA3.1-CFL1-IRES2-EGFP, wasconstructed and stably transfected into HL-60cells. RT-PCR and Western blotanalysis showed that the expression of cofilin1mRNA and protein were increasedsignificantly in cofilin1/HL-60cells, when compared to the empty Vector group andthe non-transfection group, suggests the cofilin1/HL-60cell lines was successfullyconstructed. RT-PCR and Western blot analysis showed that the expression ofcofilin1and p-cofilin1were different significantly in the cells of the non-transfectiongroup, the empty Vector group and the overexpression of cofilin1group which treatedwith DADS, when compared to the untreated groups (P<0.05).4. The effect of DADSon the proliferation, migration, invasion and reducing ability in cofilin1/HL-60cells.NBT experiment displayed, the reducing ability of the cells was significantlyenhanced in the non-transfection group, the empty Vector group and theoverexpression of cofilin1group that treated with DADS, when compared to theuntreated cells (P<0.05). The reducing ability of cofilin1/HL-60cells was lowersignificantly when compared the group of non-transfection and the empty Vector(P<0.05). No significant difference was found between the cells of DADS and ATRAgroups (P>0.05). CCK-8experiment showed, the proliferation ability of cells wasinhibited obviously in the group of non-transfection, empty Vector andoverexpression of cofilin1that treated with DADS, when compared to the untreatedcells (P<0.05). The inhibitions of DADS on the groups of non-transfection and emptyVector was enhanced than the group of cofilin1/HL-60(P<0.05). No significantdifference was found between the group of DADS and ATRA (P>0.05). It indicatesthat overexpression of cofilin1could reduce the effect of DADS on the cellularproliferation ability. Transwell migration and invasion experiment showed that thenumber of cells through the film and matrigel membrane were reduced significantly inthe non-transfection group, the empty Vector group and the overexpression of cofilin1group which treated with DADS than the untreated group (P<0.05). The number ofcells through the film and matrigel membrane in the overexpression of cofilin1group were more than the non-transfection group and the empty Vector group (P<0.05). Nosignificant difference was found between the group of DADS and ATRA (P>0.05). Itsuggests that the mechanism of DADS on the migration and invasion ability of HL-60cells was related with the downregulation of cofilin1expression. Immunofluorescenceexperiment showed that the expression of CD11b was enhanced significantly in thenon-transfection group, the empty Vector group and the overexpression group whichtreatde with DADS than the untreated group; the expression of CD33was reducedsignificantly in the non-transfection group, the empty Vector group and theoverexpression group which treatde with DADS than the untreated group.5. Theeffects of DADS on the Rac1-ROCK1-LIMK1signaling pathway in HL-60cells.RT-PCR and Western blot results revealed that the expression of Rac1, ROCK1,LIMK1and p-LIMK1were inhibited in a time dependent model in HL-60cellstreated with DADS for0h,6h,12h,24h,48h (P<0.05).6. The effects of DADS on theRac1-ROCK1-LIMK1signaling pathway in cofilin1/HL-60cells. RT-PCR andWestern blot results showed that the expression of Rac1, ROCK1, LIMK1andp-LIMK1were decreased significantly in the non-transfection group, the emptyVector group and the overexpression group that treated with DADS than the untreatedgroup (P<0.05). The expression level of Rac1, ROCK1, LIMK1and p-LIMK1did notchange significantly between the non-transfection group, the empty Vector group andthe overexpression group that treated with DADS (P>0.05). Conclusion:1. DADScan inhibit the proliferation, migration, invasion ability of HL-60cells and can induceit to differentiate by down-regulating the expression of cofilin1.2. DADS can inhibitthe proliferation, migration, invsion ability of HL-60cells and induce it todifferentiate though antagonizing Rac1-ROCK1-LIMK1signaling pathway whichcould down-regulated the expression and phosphorylation of cofilin1.