The Effects Of Ubiquitin Ligase Cb1 Family Proteins On Arsenic Trioxide-induced Apoptosis And G2/M Phase Arrest

ObjectiveArsenic trioxide(As2O3,ATO)is very effective in the treatment of patients with acute promyelocytic leukemia(APL).APL is characterized by the PML/RARαfusion protein,a product of the t(15:17)translocation.Relatively low concentrations(≤2μM) of ATO induce apoptosis and downregulate the PML-RARαfusion protein.ATO also has potential anticancer activity against other kinds of cells which lack the PML-RARαfusion protein,such as acute myeloid leukemia(AML),multiple myeloma, lymphocytic leukemia,solid tumors.However,the sensitivity of different types of cells against ATO was much different and the low sensitivity of cells confined clinical application.In addition,ATO induces arrest in the G1 or G2/M phases of the cell cycle rather than apoptosis in most solid tumor cells.This different mechanism of ATO action on solid tumor cancer cells and APL cells is not well understood.The phosphatidylinositol 3-kinase(PI3K)/Akt pathway is considered as a critical survival-signaling pathway.Akt mediated phosphorylation may alter the activity of proteins such as p53,some Bcl-2 family members,caspase-9,and nuclear factorκB (NF-κB)and other transcription factors,which trigger or restrain apoptosis.And PI3K/Akt deregulation may contribute to tumorigenesis,metastasis,and resistance to chemotherapy.Actually,PI3K/Akt pathway is also modulated by several important signaling moleculars,such as PTEN and Ras.The casitas B-lineage Lymphoma(Cb1)family of ubiquitin ligases is a negative regulator of PI3K/Akt signaling in several cell types,including osteoblast and T cells. Cb1 proteins can interact with the p85-regulatory subunit of PI3K,resulting in PI3K ubiquitination and degradation.However,it is not clear whether Cb1 family members are involved in ATO action by regulating PI3K/Akt signaling.p53 is a critical determinant in controlling both cell cycle arrest and apoptosis.In several different types of cancer cells,the functional status of p53 determines the cellular response to ATO.Cells with normal p53 were resistant to ATO-induced apoptosis and were arrested in G1 phase,while cells lacking functional p53 were sensitive and were arrested in G2/M phase.Gastric cancer is the second most common cancer in the world,and still remains one of the major causes of cancer death in China.The prognosis for advanced gastric cancer is poor and the efficacy of chemotherapy is limited.Nevertheless,to be effective, this possibility requires the elaboration of strategies to increase the apoptotic action of agents and to search new drugs.In this study,we investigated the effect of Cb1-b on the apoptotic action of ATO in NB4 and HL60 cells.Secondly,we investigated the effect of ATO on gastric cancer cells.In addition,we further investigated the different mechanism of ATO action on gastric cancer cells and NB4 cells.Materials and Methods1.Cell viability assay The effect of ATO on cell proliferation was measured using the trypan blue exclusion assay and the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT) assay.2.Cell cycle phase analysisPhase distributions of the cell cycle and hypodiploid DNA were determined by flow cytometry. 3.Western Blot AnalysisWestern blotting was used to analyze Akt,p-Akt,bcl-2,p53,and Cb1-b,c-Cb1.4.Statistical analysisData are expressed as mean±SD.Differences between two groups were evaluated by Student's t-test.P＜0.05 was considered to be statistically significant.Results1.Growth inhibition by ATO The effect of ATO on the viability of NB4 and HL60 cells was examined by the trypan blue exclusion assay.Dose-dependent inhibition of cell growth was observed in both cell lines.The IC50 at 24 h was 1.91μM and 4.36μM respectively in NB4 and HL60 ceils.The effect of ATO on the viability of MGC803,BGC823,SGC7901 cells was examined by MTT assay.Dose-dependent inhibition of cell growth was observed in the cell lines.The IC50 at 24 h was 23.4μM,25.43μM and 27.13μM respectively.2.Induction of apoptosis in NB4 cells and G2/M phase arrest in gastric cancer cells by ATOFlow cytometry analysis showed that ATO increased the apoptotic(sub-G1) population in a dose-dependent manner at 24h in NB4 and HL60 cells.Upon observation under the microscope,gastric cancer cells remained viable and rounded up and detached from the culture dishes.Consistently,treatment with ATO induced a significant G2/M phase arrest,but not a significant increase in the sub-G1 population, in MGC803,BGC823,SGC7901 cells.These results indicated that the action of ATO in gastic cancer cells is different from in NB4 cells.3.Effects of ATO on activation and expression of Akt proteinsAfter treatment of ATO with the representative concentration for times,we analyzed the expression of Akt and p-Akt.NB4 and HL60 cells had a relatively high basal level of p-Akt expression.Phospho-Akt was strongly increased after 4h ATO treatment in both cell lines,while it was decreased to less than basal level after 24h in NB4 and HL60 cells.The similar phenomenon was shown in gastric cancer cells.The levels of Akt were unchanged upon ATO treatment in the cell lines.We used LY294002 to inhibit PI3K/Akt signaling.25μM of LY294002 significantly elevated the percentages of the sub-G1 population in ATO-treated NB4 and HL60 cells.While 25μM of LY294002 elevated the percentages of the sub-G1 cells and markedly reduced G2/M phase cells in gastric cancer cells.But phospho-Akt did not show transient increase at 4h.4.Up-regulation of the Cb1 family of ubiquitin ligases by ATOIt was reported thar Cb1 was a negative modulator of PI3K/Akt signaling pathway. We examined the expression levels of c-Cb1 and Cb1-b and their functions in the cell lines.Treatment with ATO significantly increased the levels of Cb1-b and c-Cb1 proteins in NB4 and HL60 cells starting at 4h,with maximal expression observed at 24h. Treatment with ATO also significantly increased the levels of Cb1-b and c-Cb1 proteins in gastric cancer cells.Pretreatment with 10nM of Ps341 can inhibit the function of Cb1. Flow cytometry analysis revealed that pretreatment with 10nM Ps341 significantly reduced ATO-induced apoptosis of NB4 and HL60 cells.While Ps341 significantly elevated ATO-induced G2/M phase arrest of gstric cancer cells.These results indicate that Cb1 might impact the action of ATO.To further confirm this presumption,we assessed the level of phospho-Akt.In both NB4 cells and MGC803 cells,Ps341 alone did not affect the basal level of phospho-Akt.But pretreatment with Ps341 persistently increased the level of phospho-Akt at 24h in NB4 cells and at 16h in MGC803 cells.So Ps341 markedly prolonged the activation of PI3K/Akt by ATO,which suggests that Cb1 inhibited the activation of PI3K/Akt by ATO.5.Effects of ATO on apoptosis-associated proteinTo research the reason why ATO failed to induce apoptosis in MGC803,we analyzed bcl-2 protein,an early marker of apoptosis.MGC803 were treated with 10μM ATO for 4-24h,while NB4 with 1μM.The level of bcl-2 protein was gradually decreased from 4h to 24h in NB4,while not in MGC803.ATO induced phosphorylation of bcl-2 which appeared from 16h.These results indicated the reason why ATO failed to induce apoptosis in MGC803 within 24h might be the failure of decreasing bcl-2 protein. ATO-mediated G2/M arrest was earlier than phosphorylation of bcl-2by ATO,which indicated that phosphorylation of bcl-2 was not the reason of G2/M arrest induced by ATO.6 Effects of ATO on cell cycle regulators p53To investigate the mechanism by which ATO induced G2/M arrest in MGC803 cells, we examined the levels of p53 protein by Western blot.MGC803 and NB4 expressed a relatively high basal level of p53.After ATO-treatment,the level of the p53 expression in MGC803 was significantly decreased in a time-dependent manner.Notably, down-regulation p53 appeared as early as 4h.However,there were no significant changes in NB4.These results indicated that the effects of ATO on apoptosis and cell cycle arrest depended on differential regulation of p53.We analyzed the effects of LY294002 on the expression of p53 protein.The combination of LY294002 and ATO significantly increased the expression of p53 protein in MGC803 cells.Slight up-regulation of p53 by LY294002 was seen in NB4 cells.These results indicated activation of PI3K/Akt led to degradation of p53.In addition,Ps341 combined with ATO further decreased the expression of p53 in MGC803 cells,but not in NB4 cells.In addition,the phenomena in MGC803 cells were also shown in BGC823 and SGC7901 cells.Altogether,the final function status of p53 was the determiner for the action of ATO,apoptosis or G2/M phase arrest.Conclution1,ATO inhibited the growth of AML and gastric cancer cells,but gastric cancer cells showed low sensitivity to ATO. 2,ATO induced apoptosis in NB4 and HL60 cells,while induced G2/M phase arrest in gastric cancer cells3,During ATO-induced apoptosis and G2/M phase arrest,ATO transiently activated PI3K/Akt signaling.4,ATO up-regulated Cb1-b and c-Cb1 proteins in the both processes of apoptosis and G2/M phase arrest and Cb1 inhibited the excessive activation of PI3K/Akt induced by ATO.5,ATO down-regulation p53 expression in gastric cancer cells during G2/M arrest while failed in NB4 cells.The final function status of p53 was the determiner for ATO-induced apoptosis and G2/M phase arrest.