The Effect Of Overexpression Of RhoGDI2 On Differentiation In Leukemia HL-60 Cell Induced By Diallyl Disulfide

Objective: Our previous studies have shown that dads(diallyl disulfide) can inhibit the proliferation of human leukemia HL-60 cells dose dependently in vitro and vivo, recent proteomics found that dads can reduce expression of RhoGDI2 protein. According to the preliminary study, through building a stable overexpression of the RhoGDI2 in human leukemic HL-60 cell line to explore the impact of DADS on the biological behavior of HL-60 cells.Methods: The pIRES2-EGFP-RhoGDI2 eukaryotic expression vector was transfected into HL-60 cells to construct stable RhoGDI2 overexpression of HL-60 cell line. By CCK-8, Transwell experiment, Flow cytometry, NBT and immunofluorescence to detect the biological behavior of RhoGDI2 overexpression of HL-60 cells, such as proliferation, migration and invasion and differentiation effects. Using Western blot method to prove that DADS could downregulated RhoGDI2 expression to effect the Rac1/Pak1/LIMK1 signaling pathway of HL-60 cells.Results1. Establish a stable HL-60 cells line of overexpressed RhoGDI2.We can find green fluorescence in two groups of transfection cells, RhoGDI2/HL-60 and p IRES2-EGFP/HL-60. Western Blot and QT-PCR prove that the expression level of RhoGDI2 protein and m RNA in RhoGDI2/HL-60 are higher than that of HL-60 and p IRES2-EGFP/HL-60. It shows that we have established a stable HL-60 cells line of overexpressed RhoGDI2.2.The effects of DADS on biological behavior of the human leukemia HL-60 cells with overexpression of RhoGDI2.CCK-8 experiment showed that the inhibition of cell proliferation was higher obviously in the group of non-transfection and negative transfection after treated with DADS, when compared to the group of overexpression of RhoGDI2(P<0.05). No significant difference was found between the group of DADS and ARTA(P>0.05). It indicates that overexpression of RhoGDI2 could reduce the effect of DADS on cell proliferation.NBT experiment display, that the reducing power was significantly enhanced in non-transfected group, negative transfected group and the high expressed group after treated with DADS, when compared to untreated cells(P<0.05). The group of high expression has a lower significantly reducing power than the group of non-transfection and negative transfection(P<0.05). No significant difference was found between the group of DADS and ARTA(P>0.05).Transwell migration and invasion experiment showed that the number of cells through the film and matrigel membrane were reduced significantly in non-transfected group, negative transfected group and the high expressed group after treated with DADS(P<0.05). The number of cells through the film and matrigel membrane in the high expression group were more than non-transfected group and negative transfected group(P<0.05). No significant difference was found between the group of DADS and ARTA(P>0.05). It suggests that the mechanism of DADS inhibiting the migrated and invasive ability of HL-60 cells was related with downregulation of RhoGDI2 expression.Immunofluorescence experiment showed that the expression of CD11 b were enhanced significantly in non-transfected group, negative transfected group and the overexpressed group after treated with DADS, the expression of CD33 were reduced significantly in non-transfected group, negative transfected group and the high expressed group after treated with DADS.Flow cytometry experiment showed that overexpression of RhoGDI2 cells in the S/G1 phase cell ratio was significantly higher than that of negative transfection group and non transfection group(P < 0.05), suggesting that overexpressed RhoGDI2 can enhance the proliferation of HL-60 cells., and DADS induced the HL-60 cells of G0/G1 arrest(P<0.05). No significant difference was found between the group of DADS and ARTA(P>0.05).3. The effects of DADS on the Rac1-LIMK1 signaling pathways in overexpressed RhoGDI2 of HL-60 cells.Western blot results revealed that the expression of Rac1, Pak1, LIMK1 and RhoGDI2 were inhibited in HL-60 cells treated with DADS for 24h(P<0.05). QT-PCR and Western blot results showed, that the expression of Rac1, Pak1, LIMK1 and RhoGDI2 were decreased significantly in non-transfected group, negative transfected group and the overexpressed group after treated with DADS(P<0.05). The expression levels of Rac1, Pak1, LIMK1 and RhoGDI2 did not change significantly in non-transfected group, negative transfected group and the overexpressed group after treated with DADS(P>0.05).Conclusion1. DADS can down-regulate the expression of RhoGDI2 through Rac1/Pak1/LIMK1 signaling pathway inducting the differentiation in HL-60 cells.2. RhoGDI2 overexpression can increase the proliferation, migration and invasion and inhibit differentiation in HL-60 cells.3. RhoGDI2 overexpression can impair the differentiation in HL-60 cells induced by DADS.