泛素连接酶Cb1家族蛋白在三氧化二砷诱导肿瘤细胞凋亡和G2/M期阻滞中的作用机制研究
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摘要
泛素连接酶Cb1在三氧化二砷诱导肿瘤细胞凋亡或G2/M期阻滞中的作用机制研究
目的
三氧化二砷(Arsenic trioxide,ATO)是治疗以t(15;17)易位引起的PML/RARα融合蛋白为特征的急性早幼粒细胞白血病(APL)最有效的药物之一。ATO以相对低浓度(≤2μM)即可降解PML/RARα融合蛋白并诱导细胞凋亡。ATO对缺乏PML/RARα融合蛋白的许多肿瘤细胞均有抗癌活性,包括急性髓性白血病,淋巴细胞白血病,多发性骨髓瘤以及实体肿瘤等。然而,由于敏感性或耐药性的不同严重阻碍了临床应用。另外,ATO对大部分的实体肿瘤细胞虽然具有杀伤毒性但是敏感性差,而且大多伴随细胞周期的改变,G1期阻滞或G2/M期阻滞。目前对ATO在APL和实体肿瘤中产生不同作用的机制尚不清楚。
Phosphatidylinositol 3-kinase(PI3K)/Akt通路被认为是重要的存活信号通路。在多种肿瘤中研究表明PI3K/Akt的失调控可能造成肿瘤形成,转移和对化疗耐药。组成性的或诱导出的p-Akt介导重要底物分子的磷酸化,改变他们的活性从而启动或抑制凋亡。同时,PI3K/Akt信号通路也受到PTEN和Ras等信号分子的调节。
泛素连接酶Casitas B-lineage Lymphoma(Cb1)家族蛋白作为一种PI3K/Akt信号的负调节因子,在一些细胞如T细胞、破骨细胞的功能中起着非常重要的作用。Cb1蛋白能够与PI3K的p85亚单位结合,导致PI3K的泛素化和降解[34,35]而改变信号的传导。然而,Cb1蛋白是否通过调控PI3K/Akt信号参与ATO抗癌作用机制还不清楚。
胃癌是当今世界范围内第二位常见的恶性肿瘤。在我国,胃癌是肿瘤死亡的主要原因之一。晚期患者的预后较差,总体疗效不理想。寻找新的合适的药物或合理应用已知药物提高胃癌疗效甚为重要。临床实践证明毒副作用轻、疗效肯定的ATO成为最好的候选药物之一。因此有必要深入研究ATO对APL和胃癌的抗癌作用机制,尤其有必要确定在APL和胃癌中产生差异的确切机制。
为深入研究ATO抗癌机制,本实验首先以急性白血病细胞系NB4和HL60为ATO诱导凋亡模型,研究PI3K/Akt信号通路及泛素连接酶Cb1家族蛋白在此过程中的作用。其次以人胃癌细胞系MGC803,BGC823及SGC7901为模型,研究ATO对胃癌细胞的毒性作用,并研究了PI3K/Akt信号通路以及Cb1蛋白在这一过程中的调节机制。最后,进一步探讨了ATO在NB4和MGC803细胞中产生不同作用结果的分子学机制。
材料与方法
1、采用台盼蓝拒染法和MTT法测定细胞活力。
2、采用流式细胞仪通过PI染色进行细胞周期解析及凋亡判定。
3、采用Western blot检测Akt、p-Akt、bcl-2、p53、和Cb1-b、c-Cb1蛋白的表达。
4、统计学处理:每次实验重复3次,数据以(?)±s表示,采用SPSS13.0软件进行t检验和单因素方差分析。
实验结果
1、ATO抑制细胞增殖
台盼蓝拒染法检测ATO对NB4,HL60细胞活力的影响。ATO以剂量依赖性方式抑制NB4和HL60细胞增殖。抑制细胞增殖50%的药物浓度(IC_(50))24小时分别1.91μM和4.36μM。MTT法检测ATO对MGC803,BGC823,SGC7901细胞活力的影响。ATO以剂量依赖性方式抑制细胞增殖。抑制细胞增殖50%的药物浓度(IC_(50))24小时分别23.4μM,25.43μM,27.13μM。提示胃癌细胞对ATO敏感性差。
2、ATO诱导NB4,HL60细胞凋亡,诱导胃癌细胞G2/M期阻滞
流式细胞仪分析显示ATO分别作用NB4,HL60细胞24小时后,以剂量依赖方式增加细胞凋亡,即亚二倍体细胞百分数(细胞凋亡率)增加。ATO分别处理胃癌MGC803,BGC823,SGC7901细胞24小时后,倒置显微镜下观察,发现细胞仍然保持活力,圆而非帖壁的细胞较对照组多见。进一步以流式细胞仪分析,ATO处理胃癌细胞显示显著的G2/M期阻滞,而亚二倍体细胞无显著增加。提示ATO对胃癌细胞MGC803,BGC823,SGC7901的作用不同于ATO对急性早幼粒细胞白血病NB4细胞。
3、ATO对Akt活性的影响
分别以ATO诱导细胞凋亡和G2/M期阻滞的代表性浓度作用细胞不同的时间后检测磷酸化Akt(p-Akt)和Akt。Western blot解析结果显示,对照组NB4和HL60细胞均表达一定水平的p-Akt。ATO处理4小时后,二种细胞中均p-Akt显著增加,而后逐渐降至本底水平以下。而Akt水平在ATO作用前后没有变化。对照组胃癌细胞中均表达不同程度的p-Akt。在处理4小时后,也出现p-Akt短暂活化,随后降至本底水平以下。用25μM LN294002预处理细胞抑制PI3K/Akt信号传导。结果显示LY294002显著提高了ATO诱导的细胞凋亡百分比(NB4,HL60)。LY294002显著降低了ATO诱导胃癌细胞的G2/M期阻滞,同时显著提高了的细胞凋亡率(MGC803,BGC823,SGC7901)。进一步分析LY294002对p-Akt的影响,一致地显示LY294002预处理的细胞中ATO诱导的p-Akt的短暂活化消失。提示ATO诱导细胞凋亡和G2/M期阻滞过程中,p-Akt短暂活化为ATO本身的药物特性,并且在ATO抗癌作用中起着重要的调节作用。而p-Akt的最终失活是细胞走向死亡的重要原因。
4、ATO对Cb1蛋白表达的影响
据报道,Cb1为PI3K/Akt信号通路的负性调节因子。本研究检测了Cb1蛋白的表达及其功能。Western blot解析结果显示,ATO显著增加了NB4,HL60细胞中Cb1-b和c-Cb1蛋白表达,从4小时开始上升,至24小时达到平台。ATO也显著增加了胃癌细胞MGC803,BGC823,SGC7901细胞中Cb1-b和c-Cb1蛋白表达。Ps341能够抑制Cb1蛋白功能。10nMPs341预处理4小时后洗去药物,加入ATO分别处理白血病细胞和胃癌细胞。流式细胞分析结果显示Ps341预处理可部分减少ATO诱导细胞凋亡(NB4,HL60),而增强ATO诱导胃癌细胞G2/M期阻滞(MGC803,BGC823,SGC7901)。提示Cb1具有重要的调节作用。进一步地,Western blot解析结果显示Ps341预处理本身对细胞的p-Akt基础表达水平没有影响。但是,Ps341预处理显著延长了ATO诱导的PI3K/Akt的活化持续时间。提示Cb1抑制了ATO诱导的p-Akt过度活化。上调Cb1蛋白表达可能为ATO最终抑制PI3K/Akt信号通路,继而使细胞走向死亡的作用机制之一。
5、比较ATO诱导NB4细胞凋亡和MGC803细胞G2/M期阻滞过程中,bcl-2蛋白的变化
为了研究ATO诱导细胞凋亡和G2/M期阻滞产生不同结果的分子学机制,本实验分析了凋亡早期标志物bcl-2蛋白。分别以1μM和10μM ATO处理NB4细胞和MGC803细胞。在NB4细胞中,bcl-2蛋白从4小时开始下降,至24小时显著低于本底水平。然而,在MGC803细胞中,却没有bcl-2蛋白的下降,至16小时开始出现磷酸化bcl-2。这些结果提示MGC803细胞至少在10μM作用24小时以内没有诱导凋亡与未能下调bcl-2蛋白有关。在MGC803细胞中,bcl-2的磷酸化滞后于G2/M期阻滞的出现,提示bcl-2的磷酸化并不是导致G2/M期阻滞的关键性因子。
6、比较ATO诱导凋亡和G2/M期阻滞过程中,p53蛋白的变化
NB4细胞和MGC803细胞表达比较高水平的p53蛋白。10μM ATO处理MGC803细胞4小时开始出现p53蛋白表达显著下降。然而,1μM ATO处理NB4细胞后,p53蛋白却没有显著的改变。PI3K/Akt特异性抑制剂LY294002显著增加ATO处理的MGC803细胞中p53蛋白的表达,轻度增加NB4细胞p53表达。提示虽然ATO诱导NB4细胞凋亡没有p53的降解,但是短暂活化的PI3K/Akt信号在两种细胞中均抑制了p53蛋白表达。Ps341预处理进一步减少1ATO处理的胃癌细胞中p53蛋白表达,但是NB4细胞中p53却没有明显变化。进一步,在BGC823,SGC7901细胞中也见到与MGC803细胞中相似现象。提示p53功能的最终状态是使细胞发生凋亡还是G2/M期阻滞的关键因子。
结论
1、ATO抑制急性白血病细胞和胃癌细胞增殖,但是胃癌细胞敏感性最差。
2、ATO诱导NB4,HL60细胞凋亡,胃癌细胞MGC803,BGC823,SGC7901则G2/M期阻滞
3、ATO诱导细胞凋亡和G2/M期阻滞过程中均伴随短暂活化PI3K/Akt信号。
4、ATO诱导细胞凋亡和G2/M期阻滞过程中均上调Cb1-b和c-Cb1蛋白,Cb1蛋白抑制PI3'K/Akt信号的过度活化
5、ATO诱导NB4细胞凋亡时p53蛋白不变,而ATO诱导胃癌细胞G2/M期阻滞时p53蛋白显著下降,p53蛋白最终状态的不同是ATO产生不同结果的决定性因子。
Objective
Arsenic 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 Methods
1.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 analysis
Phase distributions of the cell cycle and hypodiploid DNA were determined by flow cytometry.
3.Western Blot Analysis
Western blotting was used to analyze Akt、p-Akt、bcl-2、p53、and Cb1-b、c-Cb1.
4.Statistical analysis
Data 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.
Results
1.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 ATO
Flow 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 proteins
After 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 ATO
It 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 protein
To 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 p53
To 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.
Conclution
1、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 cells
3、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.
目的
三氧化二砷(Arsenic trioxide,ATO)是治疗以t(15;17)易位引起的PML/RARα融合蛋白为特征的急性早幼粒细胞白血病(APL)最有效的药物之一。ATO以相对低浓度(≤2μM)即可降解PML/RARα融合蛋白并诱导细胞凋亡。ATO对缺乏PML/RARα融合蛋白的许多肿瘤细胞均有抗癌活性,包括急性髓性白血病,淋巴细胞白血病,多发性骨髓瘤以及实体肿瘤等。然而,由于敏感性或耐药性的不同严重阻碍了临床应用。另外,ATO对大部分的实体肿瘤细胞虽然具有杀伤毒性但是敏感性差,而且大多伴随细胞周期的改变,G1期阻滞或G2/M期阻滞。目前对ATO在APL和实体肿瘤中产生不同作用的机制尚不清楚。
Phosphatidylinositol 3-kinase(PI3K)/Akt通路被认为是重要的存活信号通路。在多种肿瘤中研究表明PI3K/Akt的失调控可能造成肿瘤形成,转移和对化疗耐药。组成性的或诱导出的p-Akt介导重要底物分子的磷酸化,改变他们的活性从而启动或抑制凋亡。同时,PI3K/Akt信号通路也受到PTEN和Ras等信号分子的调节。
泛素连接酶Casitas B-lineage Lymphoma(Cb1)家族蛋白作为一种PI3K/Akt信号的负调节因子,在一些细胞如T细胞、破骨细胞的功能中起着非常重要的作用。Cb1蛋白能够与PI3K的p85亚单位结合,导致PI3K的泛素化和降解[34,35]而改变信号的传导。然而,Cb1蛋白是否通过调控PI3K/Akt信号参与ATO抗癌作用机制还不清楚。
胃癌是当今世界范围内第二位常见的恶性肿瘤。在我国,胃癌是肿瘤死亡的主要原因之一。晚期患者的预后较差,总体疗效不理想。寻找新的合适的药物或合理应用已知药物提高胃癌疗效甚为重要。临床实践证明毒副作用轻、疗效肯定的ATO成为最好的候选药物之一。因此有必要深入研究ATO对APL和胃癌的抗癌作用机制,尤其有必要确定在APL和胃癌中产生差异的确切机制。
为深入研究ATO抗癌机制,本实验首先以急性白血病细胞系NB4和HL60为ATO诱导凋亡模型,研究PI3K/Akt信号通路及泛素连接酶Cb1家族蛋白在此过程中的作用。其次以人胃癌细胞系MGC803,BGC823及SGC7901为模型,研究ATO对胃癌细胞的毒性作用,并研究了PI3K/Akt信号通路以及Cb1蛋白在这一过程中的调节机制。最后,进一步探讨了ATO在NB4和MGC803细胞中产生不同作用结果的分子学机制。
材料与方法
1、采用台盼蓝拒染法和MTT法测定细胞活力。
2、采用流式细胞仪通过PI染色进行细胞周期解析及凋亡判定。
3、采用Western blot检测Akt、p-Akt、bcl-2、p53、和Cb1-b、c-Cb1蛋白的表达。
4、统计学处理:每次实验重复3次,数据以(?)±s表示,采用SPSS13.0软件进行t检验和单因素方差分析。
实验结果
1、ATO抑制细胞增殖
台盼蓝拒染法检测ATO对NB4,HL60细胞活力的影响。ATO以剂量依赖性方式抑制NB4和HL60细胞增殖。抑制细胞增殖50%的药物浓度(IC_(50))24小时分别1.91μM和4.36μM。MTT法检测ATO对MGC803,BGC823,SGC7901细胞活力的影响。ATO以剂量依赖性方式抑制细胞增殖。抑制细胞增殖50%的药物浓度(IC_(50))24小时分别23.4μM,25.43μM,27.13μM。提示胃癌细胞对ATO敏感性差。
2、ATO诱导NB4,HL60细胞凋亡,诱导胃癌细胞G2/M期阻滞
流式细胞仪分析显示ATO分别作用NB4,HL60细胞24小时后,以剂量依赖方式增加细胞凋亡,即亚二倍体细胞百分数(细胞凋亡率)增加。ATO分别处理胃癌MGC803,BGC823,SGC7901细胞24小时后,倒置显微镜下观察,发现细胞仍然保持活力,圆而非帖壁的细胞较对照组多见。进一步以流式细胞仪分析,ATO处理胃癌细胞显示显著的G2/M期阻滞,而亚二倍体细胞无显著增加。提示ATO对胃癌细胞MGC803,BGC823,SGC7901的作用不同于ATO对急性早幼粒细胞白血病NB4细胞。
3、ATO对Akt活性的影响
分别以ATO诱导细胞凋亡和G2/M期阻滞的代表性浓度作用细胞不同的时间后检测磷酸化Akt(p-Akt)和Akt。Western blot解析结果显示,对照组NB4和HL60细胞均表达一定水平的p-Akt。ATO处理4小时后,二种细胞中均p-Akt显著增加,而后逐渐降至本底水平以下。而Akt水平在ATO作用前后没有变化。对照组胃癌细胞中均表达不同程度的p-Akt。在处理4小时后,也出现p-Akt短暂活化,随后降至本底水平以下。用25μM LN294002预处理细胞抑制PI3K/Akt信号传导。结果显示LY294002显著提高了ATO诱导的细胞凋亡百分比(NB4,HL60)。LY294002显著降低了ATO诱导胃癌细胞的G2/M期阻滞,同时显著提高了的细胞凋亡率(MGC803,BGC823,SGC7901)。进一步分析LY294002对p-Akt的影响,一致地显示LY294002预处理的细胞中ATO诱导的p-Akt的短暂活化消失。提示ATO诱导细胞凋亡和G2/M期阻滞过程中,p-Akt短暂活化为ATO本身的药物特性,并且在ATO抗癌作用中起着重要的调节作用。而p-Akt的最终失活是细胞走向死亡的重要原因。
4、ATO对Cb1蛋白表达的影响
据报道,Cb1为PI3K/Akt信号通路的负性调节因子。本研究检测了Cb1蛋白的表达及其功能。Western blot解析结果显示,ATO显著增加了NB4,HL60细胞中Cb1-b和c-Cb1蛋白表达,从4小时开始上升,至24小时达到平台。ATO也显著增加了胃癌细胞MGC803,BGC823,SGC7901细胞中Cb1-b和c-Cb1蛋白表达。Ps341能够抑制Cb1蛋白功能。10nMPs341预处理4小时后洗去药物,加入ATO分别处理白血病细胞和胃癌细胞。流式细胞分析结果显示Ps341预处理可部分减少ATO诱导细胞凋亡(NB4,HL60),而增强ATO诱导胃癌细胞G2/M期阻滞(MGC803,BGC823,SGC7901)。提示Cb1具有重要的调节作用。进一步地,Western blot解析结果显示Ps341预处理本身对细胞的p-Akt基础表达水平没有影响。但是,Ps341预处理显著延长了ATO诱导的PI3K/Akt的活化持续时间。提示Cb1抑制了ATO诱导的p-Akt过度活化。上调Cb1蛋白表达可能为ATO最终抑制PI3K/Akt信号通路,继而使细胞走向死亡的作用机制之一。
5、比较ATO诱导NB4细胞凋亡和MGC803细胞G2/M期阻滞过程中,bcl-2蛋白的变化
为了研究ATO诱导细胞凋亡和G2/M期阻滞产生不同结果的分子学机制,本实验分析了凋亡早期标志物bcl-2蛋白。分别以1μM和10μM ATO处理NB4细胞和MGC803细胞。在NB4细胞中,bcl-2蛋白从4小时开始下降,至24小时显著低于本底水平。然而,在MGC803细胞中,却没有bcl-2蛋白的下降,至16小时开始出现磷酸化bcl-2。这些结果提示MGC803细胞至少在10μM作用24小时以内没有诱导凋亡与未能下调bcl-2蛋白有关。在MGC803细胞中,bcl-2的磷酸化滞后于G2/M期阻滞的出现,提示bcl-2的磷酸化并不是导致G2/M期阻滞的关键性因子。
6、比较ATO诱导凋亡和G2/M期阻滞过程中,p53蛋白的变化
NB4细胞和MGC803细胞表达比较高水平的p53蛋白。10μM ATO处理MGC803细胞4小时开始出现p53蛋白表达显著下降。然而,1μM ATO处理NB4细胞后,p53蛋白却没有显著的改变。PI3K/Akt特异性抑制剂LY294002显著增加ATO处理的MGC803细胞中p53蛋白的表达,轻度增加NB4细胞p53表达。提示虽然ATO诱导NB4细胞凋亡没有p53的降解,但是短暂活化的PI3K/Akt信号在两种细胞中均抑制了p53蛋白表达。Ps341预处理进一步减少1ATO处理的胃癌细胞中p53蛋白表达,但是NB4细胞中p53却没有明显变化。进一步,在BGC823,SGC7901细胞中也见到与MGC803细胞中相似现象。提示p53功能的最终状态是使细胞发生凋亡还是G2/M期阻滞的关键因子。
结论
1、ATO抑制急性白血病细胞和胃癌细胞增殖,但是胃癌细胞敏感性最差。
2、ATO诱导NB4,HL60细胞凋亡,胃癌细胞MGC803,BGC823,SGC7901则G2/M期阻滞
3、ATO诱导细胞凋亡和G2/M期阻滞过程中均伴随短暂活化PI3K/Akt信号。
4、ATO诱导细胞凋亡和G2/M期阻滞过程中均上调Cb1-b和c-Cb1蛋白,Cb1蛋白抑制PI3'K/Akt信号的过度活化
5、ATO诱导NB4细胞凋亡时p53蛋白不变,而ATO诱导胃癌细胞G2/M期阻滞时p53蛋白显著下降,p53蛋白最终状态的不同是ATO产生不同结果的决定性因子。
Objective
Arsenic 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 Methods
1.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 analysis
Phase distributions of the cell cycle and hypodiploid DNA were determined by flow cytometry.
3.Western Blot Analysis
Western blotting was used to analyze Akt、p-Akt、bcl-2、p53、and Cb1-b、c-Cb1.
4.Statistical analysis
Data 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.
Results
1.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 ATO
Flow 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 proteins
After 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 ATO
It 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 protein
To 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 p53
To 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.
Conclution
1、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 cells
3、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.
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43 Han Z,Wu K,Shen H,et al.Aktl/protein kinase B alpha is involved in gastric cancer progression and cell proliferation.Dig Dis Sci.2008 Jul;53(7):1801-10.
44 H.G Yu,Y.W.Ai,L.L.Yu,et al.Phosphoinositide 3-kinase/Akt pathway plays an important role in chemoresistance of gastric cancer cells against etoposide and doxorubicin induced cell death.Int.J.Cancer.2008;122(2):433-43
45 J.H.Kim,H.Y.Go,D.H.Jim,et al.Inhibition of the PI3K-Akt/PKB survival pathway enhanced an ethanol extract of Rhus verniciflua Stokes-induced apoptosis via a mitochondrial pathway in AGS gastric cancer cell lines.Cancer Lett.2008;265(2):197-205
46 Kandel ES,Skeen J,Majewski N,et al.Activation of Akt/protein kinase B overcomes a G_2/M cell cycle checkpoint induced by DNA damage.Mol Cell Biol.2002 Nov;22(22):7831-41
47 Q.Y.Zhang,Y.Y.Lu,Li,Zhang,et al.p53 gene mutation in Chinese gastric carcinoma cell lines,Chin.Biochem.J.1995;11 (3):311-315.
48 B.Vogelstein,D.Lane,A.J.Levine.Surfing the p53 network.Nature 2000;408:307-310
49 K.H.Vousden,D.P.Lane.p53 in health and disease.Nat.Rev.Mol.Cell Biol.2007;8:275-283
50 Y Joe,J.H.Jeong,S.Yang,et al.ATR,PML,and CHK2 play a role in arsenic trioxide-induced apoptosis,J.Biol.Chem.2006;281(39):28764-71
51 I.Louria-Hayon,T.Grossman,R.V.Sionov,et al.The promyelocyte leukemia protein protects p53 from Mdm2-mediated inhibition and degradation,J.Biol.Chem.2003;278(35):33134-41
52 B.F.Taylor,S.C.McNeely,H.L.Miller,et al.States,p53 suppression of arsenite-induced mitotic catastrophe is mediated by p21CIPl/WAFl,J.Pharmacol.Exp.Ther.2006;318(1):142-51
53 L.H.Yih,T.C.Lee.Arsenite induces p53 accumulation through an ATM-dependent pathway in human fibroblasts,Cancer Res.2000;60:6346-6352
54 O.Alsheich-Bartok,S.Haupt,I.Alkalay-Snir,et al.PML enhances the regulation of p53 by CK1 in response to DNA damage,Oncogene 2008;27(26):3653-61
55 Y Ogawara,S.Kishishita,T.Obata,et al.Akt enhances Mdm2-mediated ubiquitination and degradation of p53,J.Biol.Chem.2002;277(24):21843-50
56 J.Feng,R.Tamaskovic,Z.Yang,et al.Stabilization of Mdm2 via decreased ubiquitination is mediated by protein kinase B/Akt-dependent phosphorylation,J.Biol.Chem.2004;279(34):35510-7
57 Furukawa Y,Iwase S,Kikuchi J,et al.Phosphorylation of Bcl-2 protein by CDC2 kinase during G2/M phases and its role in cell cycle regulation.J Biol Chem.2000;275(28):21661-7
58 L.Zhang,G jia,W.M.Li,et al.Alteration of the ATM gene occurs in gastric cancer cell lines and primary tumors associated with cellular response to DNA damage,Mutat.Res.2004;557(1):41-51
1 1Jia P,Chen G,Huang X,et al.Arsentic trioxide induces multiple myeloma cell apoptosis via disruption of mitochondrialtransmembrane potentials and activation of caspase-3.Chin Med J(Engl),2001,114(1):19-24.
2 Pu Y S,Hour T C,Chen J,et al.Arsenic trioxide as a novel anticancer agent against human transitional carcinoma--characterizing its apoptotic pathway.AnticancerDrugs,2002,13(3):293-300.
3 Nakagawa Y,Akao Y,Morikawa H,et al.Arsenic trioxide-induced apoptosis through oxidative stress in cells of colon cancer cell lines.Life Sci,2002,70(19):2253-2269
4 Jie Wang 1,Lingna Li 1,Hui Cang,Guiying Shi,Jing Yi.NADPH oxidase-derived reactive oxygen species are responsible for the high susceptibility to arsenic cytotoxicity in acute promyelocytic leukemia cells Leukemia Research(2007)xxx-xxx
5 Baysan A,Yel L,Gollapudi S,Su H,Gupta S.Arsenic trioxide induces apoptosis via the mitochondrial pathway by upregulating the expression of Bax and Bim in human B cells.Int J Oncol.2007 Feb;30(2):313-8.
6 Katy P Y Siu,Judy YWChan,Fung K P.Effect of arsenic trioxide on human hepatocellular carcinoma HepG2 cells:Inhibition of proliferation and induction of apoptosis.Life Sci,2002,(71):275-285.
7 Zhu J,Okumura H,Ohtake S,et al.Arsenic trioxide induces apoptosis in leukemia/lympho cell lines via the CD95/CD95L system.Oncol Rep,2003,10(3):705-709.
8 李江涛,区庆嘉,刘颖斌,等.bcl-2mRNA,baxmRNA 表达在三氧化二砷诱导肝癌细胞凋亡中的意义.中华肝胆外科杂志,2003,9(4):214-217.
9 Liang X Q,Cao E H,Zhang Y,et al.A p53 target gene,PIG11,contributes to hemosensitivity of cells to arsenic trioxide.FEBS Lett,2004,569(1-3):94-98.
10 Zhang Jiuli,Li Jinlong,Xu Shiwen.Research Advances of Mechanism on Apoptosis of Tumor Cells Induced by Arsenic Trioxide.Chinese Agricultural Science Bulletin Vol.23 No.2 2007February
11 E Szegezdi1,2,S Cahill1,2,M Meyer1,2,M O'Dwyer3 and A Samali~*,1,2 TRAIL sensitisation by arsenic trioxide is caspase-8 dependent and involves modulation of death receptor components and Akt British Journal of Cancer(2006)94,398-406
12 Hong Zhang,George Duncan,Lixin Wang,Ping Liu,Hao.Arsenic trioxide initiates ER stress responses,perturbs calcium signalling and promotes apoptosis in human lens epithelial cells.Experimental Eye Research(2007)
13 张莉,王玲,宋维华,温薇,孙宏丽,吴兆菊,杨宝峰。三氧化二砷通过升高细胞内钙离子浓度介导肺癌细胞凋亡。中国药理学通报 2004 Aug;20(8):867-70
14 张兴荣,蔡洪培,邓志华,等.细胞内 Ca2+ 变化在砷剂诱导胰腺癌细胞株凋亡中的作用.第二军医大学学报,2001,22(5):422-424.
15 ADRIAN M.RAMOS,CARLOS FERNANDEZ,DONNA AMRA N,DIEGO ESTEBAN,ELENA DE BLAS,MARI'A A.PALACIOS AND PATRICIO ALLER1Pharmacologic Inhibitors of Extracellular Signal-Regulated Kinase(ERKs)and c-Jun NH2-Terminal Kinase (JNK)Decrease Glutathione Content and Sensitize Human Promonocytic Leukemia Cells to Arsenic Trioxide-Induced Apoptosis.JOURNAL OF CELLULAR PHYSIOLOGY 209:1006-1015(2006)
16 Ramos AM,Fernandez C,Amran D,Sancho P,de Blas E,Aller P Pharmacologic inhibitors of PI3K/Akt potentiate the apoptotic action of the antileukemic drug arsenic trioxide via glutathione depletion and increased peroxide accumulation in myeloid leukemia cells.Blood.2005 May 15;105(10):4013-20.Epub 2005 Jan 21.
17 Wen J,Cheng HY,Feng Y,Rice L,Liu S,Mo A,Huang J,Zu Y,Ballon DJ,Chang CC P38MAPK inhibition enhancing ATO-induced cytotoxicity against multiple myeloma cells..Br J Haematol.2008;140(2):169-80.
18 Ramos AM,Fernandez C,Amran D,Esteban D,de Blas E,Palacios MA,Aller P.Centro de Investigaciones Biol Pharmacologic inhibitors of extracellular signal-regulated kinase(ERKs)and c-Jun NH(2)-tenninal kinase(JNK)decrease glutathione content and sensitize human promonocytic leukemia cells to arsenic trioxide-induced apoptosis.J Cell Physiol.2006Dec;209(3):1006-15.
19 Nick Giafis,Efstratios Katsoulidis,Antonella Sassano,Martin S.Tallman,Linda S.Higgins, Angel R.Nebreda,Roger J.Davis,and Leonidas C.PlataniaslRole of the p38 Mitogen-Activated Protein Kinase Pathway in the Generation of Arsenic Trioxide—Dependent Cellular Responses Cancer Res 2006;66:(13).July 1,2006
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21 Markus Duechler,Maigorzata Stajczyk.Potentiation of arsenic trioxide cytotoxicity by Parthenolide and buthionine sulfoximine in murine and human leukemic cells.Cancer Chemother Pharmacol 2007 xxx-xxx
22 23Y.J.Choi,J.W.Park,S.I.Suh,K.C.Mun,J.H.Bae,D.K.Song,S.P.Kim,T.K.Kwon,Arsenic trioxide-induced apoptosis in U937 cells involve generation of reactive oxygen species and inhibition of Akt,Int J Oncol.21 (2002)603-10.
23 G Tabellini,A.Cappellini,P.L.Tazzari,F.Fala,A.M.Billi,L.Manzoli,L.Cocoo,A.M.Martelli,Phosphoinositide 3-kinase/Akt involvement in arsenic trioxide resistance of human leukemia cells.Cell Physiol.202(2)(2005)623-34
24 A.M.Ramos,C.Fernandez,D.Amran,P.Sancho,E.de Bias,P.Aller,Pharmacologic inhibitors of PBK/Akt potentiate the apoptotic action of the antileukemic drug arsenic trioxide via glutathione depletion and increased peroxide accumulation in myeloid leukemia cells,Blood 105(10)(2005)4013-20
25 G Tabellini,P.L.Tazzari,R.Bortul,C.Evangelisti,A.M.Billi,T.Grafone,G Martinelli,M.Baccarani,A.M.Martelli,Phosphoinositide 3-kinase/Akt inhibition increases arsenic trioxide-induced apoptosis of acute promyelocytic and T-cell leukaemias,Br.J.Haematol.130(5)(2005):716-25
26 D.P.Brazil,J.Park,B.A.Hemmings,PKB binding proteins:Getting in on the Akt,Cell.111 (3)(2002)293-303
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10 A.M.Ramos,C.Fernandez,D.Amran,et al.Pharmacologic inhibitors of PI3K/Akt potentiate the apoptotic action of the antileukemic drug arsenic trioxide via glutathione depletion and increased peroxide accumulation in myeloid leukemia cells.Blood 2005;105(10):4013-20
11 G Tabellini,P.L.Tazzari,R.Bortul,et al.Phosphoinositide 3-kinase/Akt inhibition increases arsenic trioxide-induced apoptosis of acute promyelocyte and T-cell leukaemias,Br.J.Haematol.2005;130(5):716-25
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25 D.P.Brazil,J.Park,B.A.Hemmings.PKB binding proteins:Getting in on the Akt,Cell.2002;111(3):293-303
26 C.P.Hornik,L.Segev,G.A.Shostak,et al.PBK/Akt and apoptosis:size matters.Oncogene 2003;22:8983-8998.
27 K.A.West,S.S.Castillo,P.A.Dennis.Activation of the PBK/Akt pathway and chemotherapeutic resistance.Drug Resist Updat.2002;5:234-248.
28 A.Cappellini,F.Ricci,C.Evangelisti,et al.Multidrug resistance-associated protein 1 expression is under the control of the phosphoinositide 3 kinase/Akt signal transduction network in human acute myelogenous leukemia blasts,Leukemia 2007;21:427-38.
29 A.M.Martelli,M.Nyakern,G.Tabellini,et al.Phosphoinositide 3-kinase/Akt signaling pathway and its therapeutical implications for human acute myeloid leukemia,Leukemia 2006;20:911-28.
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41 41 1 Ferlay J,Bray F,Pisani P,et al.GLOBOCAN 2000:Cancer Incidence,Mortality and Prevalence Worldwide.IARC Press,Lyon.
42 Cinti C,Vindigni C,Zamparelli A,et al.Activated Akt as an indicator of prognosis in gastric cancer.Virchows Arch.2008;453(5):449-55.
43 Han Z,Wu K,Shen H,et al.Aktl/protein kinase B alpha is involved in gastric cancer progression and cell proliferation.Dig Dis Sci.2008 Jul;53(7):1801-10.
44 H.G Yu,Y.W.Ai,L.L.Yu,et al.Phosphoinositide 3-kinase/Akt pathway plays an important role in chemoresistance of gastric cancer cells against etoposide and doxorubicin induced cell death.Int.J.Cancer.2008;122(2):433-43
45 J.H.Kim,H.Y.Go,D.H.Jim,et al.Inhibition of the PI3K-Akt/PKB survival pathway enhanced an ethanol extract of Rhus verniciflua Stokes-induced apoptosis via a mitochondrial pathway in AGS gastric cancer cell lines.Cancer Lett.2008;265(2):197-205
46 Kandel ES,Skeen J,Majewski N,et al.Activation of Akt/protein kinase B overcomes a G_2/M cell cycle checkpoint induced by DNA damage.Mol Cell Biol.2002 Nov;22(22):7831-41
47 Q.Y.Zhang,Y.Y.Lu,Li,Zhang,et al.p53 gene mutation in Chinese gastric carcinoma cell lines,Chin.Biochem.J.1995;11 (3):311-315.
48 B.Vogelstein,D.Lane,A.J.Levine.Surfing the p53 network.Nature 2000;408:307-310
49 K.H.Vousden,D.P.Lane.p53 in health and disease.Nat.Rev.Mol.Cell Biol.2007;8:275-283
50 Y Joe,J.H.Jeong,S.Yang,et al.ATR,PML,and CHK2 play a role in arsenic trioxide-induced apoptosis,J.Biol.Chem.2006;281(39):28764-71
51 I.Louria-Hayon,T.Grossman,R.V.Sionov,et al.The promyelocyte leukemia protein protects p53 from Mdm2-mediated inhibition and degradation,J.Biol.Chem.2003;278(35):33134-41
52 B.F.Taylor,S.C.McNeely,H.L.Miller,et al.States,p53 suppression of arsenite-induced mitotic catastrophe is mediated by p21CIPl/WAFl,J.Pharmacol.Exp.Ther.2006;318(1):142-51
53 L.H.Yih,T.C.Lee.Arsenite induces p53 accumulation through an ATM-dependent pathway in human fibroblasts,Cancer Res.2000;60:6346-6352
54 O.Alsheich-Bartok,S.Haupt,I.Alkalay-Snir,et al.PML enhances the regulation of p53 by CK1 in response to DNA damage,Oncogene 2008;27(26):3653-61
55 Y Ogawara,S.Kishishita,T.Obata,et al.Akt enhances Mdm2-mediated ubiquitination and degradation of p53,J.Biol.Chem.2002;277(24):21843-50
56 J.Feng,R.Tamaskovic,Z.Yang,et al.Stabilization of Mdm2 via decreased ubiquitination is mediated by protein kinase B/Akt-dependent phosphorylation,J.Biol.Chem.2004;279(34):35510-7
57 Furukawa Y,Iwase S,Kikuchi J,et al.Phosphorylation of Bcl-2 protein by CDC2 kinase during G2/M phases and its role in cell cycle regulation.J Biol Chem.2000;275(28):21661-7
58 L.Zhang,G jia,W.M.Li,et al.Alteration of the ATM gene occurs in gastric cancer cell lines and primary tumors associated with cellular response to DNA damage,Mutat.Res.2004;557(1):41-51
1 1Jia P,Chen G,Huang X,et al.Arsentic trioxide induces multiple myeloma cell apoptosis via disruption of mitochondrialtransmembrane potentials and activation of caspase-3.Chin Med J(Engl),2001,114(1):19-24.
2 Pu Y S,Hour T C,Chen J,et al.Arsenic trioxide as a novel anticancer agent against human transitional carcinoma--characterizing its apoptotic pathway.AnticancerDrugs,2002,13(3):293-300.
3 Nakagawa Y,Akao Y,Morikawa H,et al.Arsenic trioxide-induced apoptosis through oxidative stress in cells of colon cancer cell lines.Life Sci,2002,70(19):2253-2269
4 Jie Wang 1,Lingna Li 1,Hui Cang,Guiying Shi,Jing Yi.NADPH oxidase-derived reactive oxygen species are responsible for the high susceptibility to arsenic cytotoxicity in acute promyelocytic leukemia cells Leukemia Research(2007)xxx-xxx
5 Baysan A,Yel L,Gollapudi S,Su H,Gupta S.Arsenic trioxide induces apoptosis via the mitochondrial pathway by upregulating the expression of Bax and Bim in human B cells.Int J Oncol.2007 Feb;30(2):313-8.
6 Katy P Y Siu,Judy YWChan,Fung K P.Effect of arsenic trioxide on human hepatocellular carcinoma HepG2 cells:Inhibition of proliferation and induction of apoptosis.Life Sci,2002,(71):275-285.
7 Zhu J,Okumura H,Ohtake S,et al.Arsenic trioxide induces apoptosis in leukemia/lympho cell lines via the CD95/CD95L system.Oncol Rep,2003,10(3):705-709.
8 李江涛,区庆嘉,刘颖斌,等.bcl-2mRNA,baxmRNA 表达在三氧化二砷诱导肝癌细胞凋亡中的意义.中华肝胆外科杂志,2003,9(4):214-217.
9 Liang X Q,Cao E H,Zhang Y,et al.A p53 target gene,PIG11,contributes to hemosensitivity of cells to arsenic trioxide.FEBS Lett,2004,569(1-3):94-98.
10 Zhang Jiuli,Li Jinlong,Xu Shiwen.Research Advances of Mechanism on Apoptosis of Tumor Cells Induced by Arsenic Trioxide.Chinese Agricultural Science Bulletin Vol.23 No.2 2007February
11 E Szegezdi1,2,S Cahill1,2,M Meyer1,2,M O'Dwyer3 and A Samali~*,1,2 TRAIL sensitisation by arsenic trioxide is caspase-8 dependent and involves modulation of death receptor components and Akt British Journal of Cancer(2006)94,398-406
12 Hong Zhang,George Duncan,Lixin Wang,Ping Liu,Hao.Arsenic trioxide initiates ER stress responses,perturbs calcium signalling and promotes apoptosis in human lens epithelial cells.Experimental Eye Research(2007)
13 张莉,王玲,宋维华,温薇,孙宏丽,吴兆菊,杨宝峰。三氧化二砷通过升高细胞内钙离子浓度介导肺癌细胞凋亡。中国药理学通报 2004 Aug;20(8):867-70
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