Effect Of Triciribine On The DHL-60 Cell Polarization

BackgroundThe directional migration of neutrophils is associated with many pathological and physiological processes, such as acute infection, poisoning, malignant tumor, and tissue injury. And the directional migration of neutrophils is closely related to its polarity and chemotaxis.Neutrophils can sense no more than 2% of the discrepant concentration of chemoattractant between the head and trail, and then a large number of signaling molecules inside the cells are activated through the signal amplification effect. As a result, proteins and organelles were redistributed, especially the reconsitution of F-actin and myosin. Finally leading to the cell shape changes, then a wide lamellipodium and a narrow long uropod were set up. In general, this change is called the polarization of neutrophil. On this basis, the chemotactic effect of neutrophils is initiated with the lamellipodium forwarding and then the constantly adhering and dissociating of the uropod and matrix, followed by the neutrophils passing through the vescular wall as to migrate and recruit to the infection site. The general chemotaxins are N-formyl-methionyl-leucyl-phenylalanine (fMLP) which extracted from bacteria, interleukin 8 (IL-8) and c5a etc, among them, fMLP functioned to the strongest effect.Akt, which consists of about 480 amino acid residues, is a serine/threonine protein kinase, with a molecular weight of approximately 60 kDa. The structure of Akt is divided into N terminal (the PH domain), the center domain for catalytic structure and C terminal (similar to the regulatory domain of PK). There are three isomers of Akt:Akt1/PKBα, Akt2/PKBβ and Akt3/PKBγ. Phosphorylation, which is the main mechanism of the regulation of Akt, mainly depends on the phosphatidyl inositol 3 kinase (PI3K)-Akt-phosphoinositide dependent kinase (PDK) signaling pathway. Akt includes two phosphorylation sites, with located in the catalytic domain and regulating domain respectively, namely Thr308 and Ser473. Both the two sites phosphorylated at the same time can lead Akt to a completely active state. Small G-protein, named due to its molecular weight is as small as only 20-30 kDa, which roles as switches in varous cell responses because of its GTP enzyme activity. One of the common characteristics of small G-protein is that it will switch into an active form when combing with GTP, but turning back into a non-active form when GTP becomes GDP after hydrolyzing. During this time, the active form can activate downstream molecules. The small G-protein involved in our experiment is mainly the Rho family, such as Racl, Rac2, Cdc42 and rhoA. All they play roles in the regulation of cytoskeletal network constitution, and adjusting the polymerization or depolymerization of actin filaments in the cytoplasm, thereby affecting cell morphology. It has been identified that PI3K/Akt is the basis of the positive feedback loop in the process of F-actin polymerization and depolymerization to promote pseudopod and uropod formation. Through the strengthened interaction of PI3K/Akt and Rho family, cell axis was formed, which is towarding to the high concerntration of chemoattractant.PI3K/Akt can inhibit the occurrence of apoptosis genes, such as Bad genes and play an important biological role in the process of the occurrence and regulation in apoptosis. Most tumors are accompanied with Akt increase, sustained increase of Akt phosphorylated activity is closely related to the tumorigenesis, growth, migration and invasion of human tumor, which is also the main reason for the poor therapeutic effects of chemotherapy drugs.Triciribine (TCN) is a DNA synthesis inhibitor which can inhibit the activity of Akt. It has a broad-spectrum resistance of tumor cell proliferation. Todate, widely studies confirmed that TCN has been put into phase Ⅰ/Ⅱ clinical studies in the treatment of acute myeloid leukemia. But the effect of TCN on the neutrophil-like cells (dHL-60, induced from early immature myeloid leukemia cells) is remain unidentified.PurposeThis study mainly focuses on the inhibition of TCN to the polarity of dHL-60 through functioning on the Akt/Rho GTPases pathway after pretreatment of dHL-60 with TCN then incubation with fMLP. Besides, the effects of TCN on apoptosis, activity of Akt and small G-protein of dHL-60 were detected by using flow cytometry, Western blot and GST pull-down.Methods1. After thawing, HL-60 cells were cultured into exponential phase. Cells were collected and resuspended at the density of 1.5×105 into 15mL of complete medium containing 1.25% of DMSO, then differentiated for 4 days without replacing the medium. On the fourth day, cells were collected to start our experiments. During culture and differentiating, cells were incubated at 37℃ and 5% of CO2.2. The concentration gradient 0-100nM of fMLP was established by a Zigmond chamber, in which the change of the directional polarization of dHL-60 after pretreatment with TCN of different concentrations was observed.3. In a uniform concentration of fMLP, by using immunofluorescence technique under a confocal microscope, the impact of 5μM TCN on the polarity of F-actin was observed.4. By using GST-pull down, the impact of TCN on the activity of Cdc42 and Rac2 which had been treated with a uniform concentration of fMLP was detected.5. By using by a flow cytometry, the apoptosis rate of dHL-60 in different concentrations(OμM、5μM、60μM、80μM) of TCN was detected.6. By Western blotting, the impact of different concentrations of TCN(0μM, 0.1LM,1μM,5μM,10μM,20μM,M,40μM,60MM,80μM,100μM) on the phosphorylase activity of Akt in dHL-60 which had been pretreated with a uniform concentration of fMLP was detected. At different time points (0,15,30,60min), the impact of both 5μM and 60μM TCN on the phosphorylase activity of Akt in dHL-60 with and without a uniform concentration of fMLP pretreated was detected.7. The effect of 5μM TCN on Akt recruiting to the membrane was observed by using an immunofluorescence technique.8. Data was presented as the mean±standard. The one-way analysis of variance (one-way ANOVA) was used for the comparison of more than two means using SPSS 13.0 software. Homogeneity-of-variance test was used in pairwise comparisons among multiple groups. Using LSD method if variance was neat(P>0.05). If not (P <0.05), using Dynnett T3 method. Only if P<0.05, the difference was statistically significant.Results1. Compared with the control group, dHL-60 stimulated with fMLP had a higher polarization rate. Compared with the non-pretreated group, the polarization rate of dHL-60 decreased with increasing concentrations of TCN. When the pretreating with 5μM TCN, polarity of dHL-60 weakened a lot compared with 0μM TCN pretreatment, the tail became shorter and the direction was uncertain. What’s more, when the concentration rise to 10μM, the tail disappeared and the polarity became unapparent.2. When dHL-60 is on the resting state, F-actin evenly distributed on the cytoplasmic membrane. The polarity shape of dHL-60 obviously changed after treated with 100nM fMLP for 5 min. At the same time, F-actin showed a polarity distribution on the plasma membrane. It recruits at the lamellipodium or pseudopod, and the fluorescence enhanced.3. Compared with control group, activity of both Cdc42 and Rac2 in dHL-60 stimulated by fMLP increased obviously. But after pretreated with 5μM TCN for 1 hour, the activity decreased significantly.4. An hour later, detecting by flow cytometry, dHL-60 pretreated with different concentrations of TCN (0μM,5μM,60μM,80μM) confirmed that apoptosis rate also increased with increasing concentrations of TCN.5. dHL-60 treated with fMLP alone would lead to the phosphorylation of Akt at Thr308 and Ser437 obviously, but this effect was inhibited substantially after dHL-60 being pretreated with 5^M TCN for 1 hour, notably, the inhibitory effect rising as the concentration of TCN increasing. To avoid the drug toxicity of TCN to the cell, we determined an eclectic concentration of 5nM to apply in the subsequent experiments. Another point of view, phosphotylation of Akt at Thr308 and Ser473 in dHL-60 cells decreased obviously after stimulated by fMLP as the pretreatment time of TCN went by (0,15,30,60min).6. The immunofluorescence shows that Akt recruited to the cell membrane of dHL-60 cells after fMLP stimulation, but this recruitment was prevented after pretreamt of dHL-60 cells with 5μM TCN.ConclusionLow concentration of TCN inhibits polarity of dHL-60 through the Akt/Rho GTPase pathways, whereas high concentration of TCN induces apoptosis of dHL-60.