Grb2抑制剂peptidimer-c抗K_（562）细胞的实验研究
摘要
Grb2分子是细胞内一个重要的连接蛋白,在细胞内信号传递中起重要作用,尤其介导由生长因子诱发的Ras活化,并启动了MAPK通路上的系列磷酸化反应,参与细胞生长和分化的调控。一些肿瘤的恶性生长总伴随着Grb2蛋白表达的增加,Grb2分子SH3的突变可抑制Grb2的信号传递功能,从而影响肿瘤细胞的生长、增殖,及其恶性化。Cussac等设计并合成了可与Grb2-SH3特异性结合的二聚肽peptidimer-c,能阻断Grb2与下游分子的连接。本研究以Bcr-abl阳性表达的K562细胞为对象,观察peptidimer-c对K562细胞生长和增殖的影响,探讨其可能的作用机制。
一、peptidimer-c的合成、鉴定
应用N端基团保护的Fmoc化学,固相合成针对Grb2-SH3的二聚肽peptidimer,载体肽penetratin以及含有载体肽的二聚肽peptidimer-c,用C18层析柱纯化合成的肽,高压液相色谱技术(HPLC)分析肽的纯度,质谱法分析肽的结构,应用pull-down实验,观察peptidimer与K562细胞裂解物中Grb2分子的结合。
结果显示,所合成的肽纯度高,HPLC图谱上只见一样品峰而无其它杂峰,质谱分析显示,所合成的化合物与设计的肽是一致的,peptidimer-c的分子量为4794.0,penetratin分子量为2246.7。为了观察peptidimer与K562细胞中Grb2的结合,将合成的peptidimer和penetratin分别结合于琼脂糖凝胶微球(CNBr-Sepharose)上,并与K562细胞裂解液共孵育,Western blot结果显示,peptidimer可与K562细胞中的Grb2分子特异性结合,而penetratin不与Grb2结合。
二、peptidimer-c对K562细胞生长增殖的影响
在慢性粒细胞白血病中,Bcr-abl可激活胞内多条信号传导通路,维持细胞存活和增生,以及相应的生物学功能。Grb2介导的Ras-MAPK是其主要的信号途径,并与细胞生长的调控息息相关。本部分应用苔盼兰拒染法,观察不同浓度peptidimer-c,作用不同时间对K562增殖的影响,应用克隆形成法观察peptidimer-c对K562细胞生长的影响,通过WST-1法了解peptidimer-c对K562的细胞毒作用,并与Gleevec作比较,分析二者对K562细胞作用的差异。进一步,通过克隆形成实验,探讨peptidimer-c与常用的CML治疗药物Gleevec,羟基脲,及阿糖胞苷联合应用,对K562克隆形成的影响,应用金式公式计算q值,并根据q值的大小,制定两种药物的合并效应。
苔盼兰计数法结果提示,peptidimer-c可明显抑制K562细胞的生长,且在加药后短时间内(3-6h)即有明显作用。peptidimer-c对K562的生长增殖的抑制呈浓度依赖型,而非时间依赖型。单用载体penetratin,未见明显的增殖抑制。而Gleevec则在加药24h后才出现明显的生长增殖抑制,其作用方式既表现为浓度依赖性,亦表现时间依赖性。peptidimer-c与Gleevec对K562均有细胞毒作用,且Gleeve的细胞毒作用远大于peptidimer-c。WST-1检测结果显示,peptidimer-c杀伤K562细胞的半致死剂量为(17.85±2.10)μM,而Gleevec的半致死剂量为(0.25±0.05)μM。
在甲基纤维素半固体培养体系中,K562细胞的克隆形成受peptidimer-c抑制,并随着peptidimer-c浓度的增加克隆数逐渐减低,在浓度达到2μM以上时,即有显著性抑制。几种化合物对K562克隆形成抑制的半致死剂量为:peptidimer-c(3.90±0.86)μM,Gleevec(0.03±0.02)μM,羟基脲(15.00±7.07)μM,阿糖胞苷(0.014±0.012)μM。peptidimer-c分别与Gleevec,阿糖胞苷,羟基脲联合应用时,对K562克隆形成抑制均表现为相加作用或协同作用,其中1.5μM peptidimer-c与0.05μM Gleevec联合应用,表现协同作用,1.5μM peptidimer-c与0.006μM阿糖胞苷或0.01μM阿糖胞苷联用,也显示协同抑制效应。提示peptidimer-c可与其它类型的药物合用,以提高抗肿瘤的综合效应。
三、peptidimer-c诱导K562细胞的凋亡
细胞生长的抑制总是伴随着细胞的死亡,包括坏死和凋亡。本部分应用免疫印迹技术,观察peptidimer-c对K562细胞主要信号分子的影响;通过光镜和透射电镜形态观察,TUNEL法,流式细胞术检测亚二倍体形成、annexin V/PI比例、Caspase-3和Fas的表达水平,探讨peptidimer-c诱导K562细胞死亡的方式和可能的机制。
结果表明,用peptidimer-c处理K562细胞后3-6h,细胞就发生明显形态学改变,细胞涨大,进而出现皱缩,并进入死亡阶段,透射电镜观察提示,经peptidimer-c处理,细胞形态改变,核内异染色质增多,边集现象明显,出现典型的凋亡特征。胞内内质网,线粒体结构改变。随着peptidimer-c浓度增加,坏死细胞比例加大。Annexin V/P测定结果也证实,中低浓度peptidimer-c主要诱导K562细胞凋亡,高浓度(27μM)的peptidimer-c在诱导凋亡的同时,直接导致细胞坏死。peptidimer-c的作用可使K562细胞Caspase-3的比例显著增加,提示其通过Caspase-3途径促进K562细胞凋亡,但peptidimer-c并不增加K562细胞死亡受体Fas以及Bcl-2的表达,可以确定,Fas-FasL系统不是peptidimer-c诱导K562细胞的凋亡途径,也与Bcl-2相关途径无关。peptidimer-c通过影响MAPK、PI3K、STAT5信号通路而抑制细胞生长、增殖。
四、peptidimer-c对K562细胞周期的改变
细胞生长的调节是通过细胞周期的调控得以实现的。本部分应用碘化丙啶一步插入DNA定量荧光染色法,检测peptidimer-c作用后,K562细胞周期各时项的比例,应用免疫印迹法(Western blot)和免疫细胞化学染色法观察K562细胞周期素Cyclin A, Cyclin B,以及周期素依赖性激酶Cdk2,p-Cdk2,,Cdk1, p-Cdk1的表达,探讨peptidimer-c对K562细胞周期的影响及其机制。
结果显示,随着peptidimer-c浓度的增加,K562细胞的S期比例明显上升,同时,G0/G1期比例以及G2/M期比例下降,将2细胞阻滞于S期,而Penetratin不改变K562细胞周期。Gleevec亦可使K562细胞周期比例发生改变,但却使细胞阻滞于G0/G1期。Western blot检测结果提示,随着peptidimer-c浓度加大,K562细胞的Cyclin A水平被显著抑制,同时P-Cdk2表达水平亦降低,而CyclinB,Cdk1以及p-Cdk1的表达均未改变。免疫细胞化学染色结果亦显示,peptidimer-c可明显降低K562细胞CyclinA
的表达;对Cdk2的抑制不明显,但却使Cdk2分子在细胞核内的分布发生变化,呈现不均一分布,并向核的一边浓聚。以上结果说明,peptidimer-c通过下调细胞内S期相关蛋白CyclinA和p-Cdk2的表达而使K562细胞阻滞于S期,抑制了细胞周期进程。
五、peptidimer-c对K562细胞基因表达谱的影响
基因芯片法技术通过同时对细胞或个体内的大量基因表达的平行分析,实现从整体上分析细胞表达状况的信息。本部分采用美国Affimetrix公司的人类U133 Plus3.0基因表达谱芯片,探讨经peptidimer-c作用后K562细胞基因表达谱的改变,并用RT-PCR方法对部分有显著差异的基因进行验证。
结果显示,应用人类U133 plus 3.0基因表达谱芯片检测经peptidimer-c处理后K562细胞,发现有455个上调表达的基因,以及74个下调表达基因,涉及细胞凋亡,血管生成,细胞周期,信号转导,细胞趋化,蛋白合成、蛋白折叠,转录因子,以及一些功能不明的基因等等。RT-PCR检测结果与基因表达谱芯片检测结果吻合。
凋亡相关基因分析表明,petidimer-c可能通过上调TNF及其受体家族成员,和JUN家族,启动K562细胞凋亡;通过抑制热休克蛋白导致细胞死亡。
细胞周期相关基因分析表明,petidimer-c可能通过上调细胞周期抑制因子P21、低氧诱导基因95(Hi95, sestrin2)等改变细胞周期进程。
Grb2 (growth factor receptor-bound protein 2) is an important adaptor protein in the cells, and plays a pivotal role in the cellular signal transduction. Grb2 activates MAPK cascade which contributes to the cellular growth and differentiation by Ras activation. In some cancer cells, Grb2 is over-expressed, and the mutation of Grb2 could inhibit its signal transferation and impair the cell proliferation and transformation. In 1999, a peptide dimmer (peptidimer-c) was designed to bind the two domains of Grb2 and blocked the link of Grb2 and the down-stream molecule Sos which is a guanine nucleotide exchange factor for Ras. K562 is a Bcr-abl positive cell line. Grb2 plays an important role in the Bcr-abl induced signal transductian. In this study the effect of peptidimer-c on K562 cells was investigated and the possible mechanisms would be disussed in the following five sections.
1. The synthesis of peptides and identity
The peptidimer ( VPPPVPPRRR-K-RRRPPVPPPV ), penetratin (RQIKIWFQNRRMKWKK) and peptidimer-c (peptidimer linked to penetratin) were synthesized by solid-phase synthesis using Fmoc chemistry, and purified by high performance liquid chromatography (HPLC) on a C18 column. Purity was evaluated by HPLC, and the identity of the peptides was checked by electrospray mass spectroscopy(MS). A pull-down assay was used to observe the specific binding of peptidimer to the Grb2 from K562 cells.
The result showed that a single protein peak was observed by HPLC, which meant that the peptide was in high purity. MS analysis showed the peptides were coinsident with the design. The molecular weight of peptidimer-c was 4794.0 and that of the penetratin was 2246.7. In order to determine the binding of peptidimer to Grb2, the peptidimer and penetratin were respectively coupled to CNBr sepharose beads and incubated with cell lysate of K562. Pull down assay demonstrated that the peptidimer, not the penetratin, could bind to Grb2 specifically.
2. The influence of peptidimer-c on K562 cell growth
In chronic myelogenous leukemia (CML) cells, Bcr-abl fusion protein activates many signal pathways to maintain the cell survival, proliferation and transformation. Among these signalings, the MAPK cascade activated by Ras is the key pathway, and the coupling of Bcr-abl and Ras is mediated by Grb2. In this section we investigated the inhibition of peptidimer-c on K562cell proliferation by trypan blue exclusion assay; the cytostatic effect by clonogenic assay, and the cytotoxicity by WST-1 method. A further experiment was performed with clonogenic assay to explore the effect of peptidimer alone and in combination with Gleevec, Hydroxyurea and Cytarabine respectively. Jing’s method was used for analysis.
The results showed that the peptidimer-c could inhibit the proliferation of K562 significantly in a dose-dependent manner, shortly (3-6h) after the cells were exposed to the drug, and the penetratin alone did not influence the cell proliferation. Gleevec inhibited the growth of K562 not only in a dose-dependent manner, but also in a time-dependent manner. WST-l test showed the cytotoxicity of peptidimer-c or gleevec on K562 cells, the IC50 of peptidimer-c was (17.85±2.10)μM and the IC50 of gleevec was (0.25±0.05)μM.
In the methylcellulose semi-solid medium system, the colony formation of K562 was greatly decreased by peptidimer-c as compared to the penetratin, and the colony number decreased as the dose of peptidimer-c increased. For IC50 value of colony formation on K562, peptidimer-c was (3.9±0.86)μM, Gleevec was (0.03±0.02)μM, Hydroxyurea was (15.00±7.07)μM, and cytarabine was (0.014±0.012)μM. There were synergistic effects of peptidimer-c with Gleevec, Hydroxyurea or Cytarabine on K562 by colonogenic assay. Combination of 1.5μM peptidimer-c and 0.05μM Gleevec showed synergistic effect on K562, as well as the combination of 1.5μM peptidimer-c and 0.006μM or 0.01μM Cytarabine. These results suggested that combination of peptidimer-c with other drugs would increase the anti-cancer effects.
3. peptidimer-c induced apoptosis in K562 cells
The inhibition of cell growth is always concomitant with the cell death, including necrosis and apoptosis. By morphological observation, TUNEL technique, flow cytometry for determining the hypodiploid formation; annexin V/PI percentage; and expression of caspase-3, Fas and Bcl-2, K562 cell death induced by peptidimer-c would be analyzed in this section.Some signal molecules were detected by Western blot to observe the influence of peptidimer-c on the signal pathways of K562.
The results showed that under the microscope, the K562 cells got some morphological changes: cells became swelling and crimpling after 6h exposure to the peptidimer-c. At the same time, it was found that the hetero-chromatin increased and accumulated in the margin of nucleus under the electronic microscope. The structure of endoplasmic reticulum and mitochondria changed. The percentage of necrosis increase as the dose of peptidimer-c increased. Annexin V/PI test demonstrated that medium or low dose of peptidimer-c mainly induced the apoptosis of K562 cells, and high dose of peptidimer-c (27μM) induced both the apoptosis and the necrosis of K562 cell. Peptidimer-c might induce the apoptosis of K562 by activating the caspase-3. Neither the Fas expression, nor the Bcl-2 of the K562 cells was changed.Peptidiemer-c may inhibit the growth of K562 cells by decreasing the transduction of MAPK, PI3K, and STAT5 pathways.
4. Peptidimer-c changed the cell cycle phase distribution of K562 cells
Cell growth is regulated by the control of cell cycle progression. In this section, the cell cycle modifications of K562 by peptidimer-c were detected with PI staining method in flow cytometer and analyzed by ModFit software. The role of the cyclins (Cyclin A/B) and cylin-dependent kinases (Cdk2, P-Cdk2, Cdk1 P-Cdk1) on cell cycle modification were investigated by western blot and immunocytochemistry techniques.
The results showed that the percentage of K562 cells in S phase inceased, and those of G0/G1 phase and G2/M phase decreased when K562 cells were treated with increasing concentrations of peptidimer-c for 6h. Peptidimer-c induced an S phase arrest in K562 cells, and penetratin had no effect on the cell cycle phase distribution. Gleeve also changed the cell cycle phase distribution, but it induced a G0/G1 phase arrest in K562 cells. Western blot demonstrated that the peptidimer-c treatment resulted in a significant reduction in the protein levels of cyclin A and P-cdk2, and a slight decrease in the protein level of Cdk2 by high dose of peptidimmer-c. Peptidimer-c did not influence the protein expression level of cyclin B, Cdk1, P-Cdk. Immunocytochemistical results also indicated that the cyclin A level decreased in K562 treated with increasing peptidimer-c, and the Cdk2 level decreased a little. Peptidimer-c resulted in the change of Cdk2 distribution in the nucleus. Cdk2 protein was enriched in the inner margin of the nuclear membrane. These results suggested that peptidimer led to an S phase arrest of K562 cells by down-regulating the cyclin A and P-Cdk2 and inhibited the cell cycle progression.
5. The influence of peptidimer-c on K562 gene expression profiles
Gene expression profiles analysis is a powerful technology to gain valuable information of cells in general by a synchronous detection of plentiful genes in the cells or the organisms. In this section, the human U133 plus 3.0 gene expression profiles from Affimetrix Company were used to detect the gene expression changes in K562 cells treated with poptidimer-c. RT-PCR assays were performed to confirm some significantly changed genes.
The results showed that the expression of 455 genes in K562 cells treated with peptidimer-c was up-regulated and that of 74 genes was down-regulated in the gene profiles. These genes are those for apoptosis, angiogenesis, cell cycle, signal transduction, chemotaxis, protein synthesis, protein folding, transcript factors, and some other unknown functions. The results of RT-PCR were coincident with those of gene expression profiles.
Apoptosis related genes analysis suggested that peptidimer-c may induce apoptosis of K562 cells by activating TNF/TNFR family and the JUN family, and lead to cell death by inhibiting some hot shock proteins (HSP).
Cell cycle related genes analysis indicated that peptidimer-c also modified the cell cycle progression of K562 through up-regulation of cyclin-dependent kinase inhibitor P21, and the hypoxia-induced gene 95(Hi95, sestrin2).
一、peptidimer-c的合成、鉴定
应用N端基团保护的Fmoc化学,固相合成针对Grb2-SH3的二聚肽peptidimer,载体肽penetratin以及含有载体肽的二聚肽peptidimer-c,用C18层析柱纯化合成的肽,高压液相色谱技术(HPLC)分析肽的纯度,质谱法分析肽的结构,应用pull-down实验,观察peptidimer与K562细胞裂解物中Grb2分子的结合。
结果显示,所合成的肽纯度高,HPLC图谱上只见一样品峰而无其它杂峰,质谱分析显示,所合成的化合物与设计的肽是一致的,peptidimer-c的分子量为4794.0,penetratin分子量为2246.7。为了观察peptidimer与K562细胞中Grb2的结合,将合成的peptidimer和penetratin分别结合于琼脂糖凝胶微球(CNBr-Sepharose)上,并与K562细胞裂解液共孵育,Western blot结果显示,peptidimer可与K562细胞中的Grb2分子特异性结合,而penetratin不与Grb2结合。
二、peptidimer-c对K562细胞生长增殖的影响
在慢性粒细胞白血病中,Bcr-abl可激活胞内多条信号传导通路,维持细胞存活和增生,以及相应的生物学功能。Grb2介导的Ras-MAPK是其主要的信号途径,并与细胞生长的调控息息相关。本部分应用苔盼兰拒染法,观察不同浓度peptidimer-c,作用不同时间对K562增殖的影响,应用克隆形成法观察peptidimer-c对K562细胞生长的影响,通过WST-1法了解peptidimer-c对K562的细胞毒作用,并与Gleevec作比较,分析二者对K562细胞作用的差异。进一步,通过克隆形成实验,探讨peptidimer-c与常用的CML治疗药物Gleevec,羟基脲,及阿糖胞苷联合应用,对K562克隆形成的影响,应用金式公式计算q值,并根据q值的大小,制定两种药物的合并效应。
苔盼兰计数法结果提示,peptidimer-c可明显抑制K562细胞的生长,且在加药后短时间内(3-6h)即有明显作用。peptidimer-c对K562的生长增殖的抑制呈浓度依赖型,而非时间依赖型。单用载体penetratin,未见明显的增殖抑制。而Gleevec则在加药24h后才出现明显的生长增殖抑制,其作用方式既表现为浓度依赖性,亦表现时间依赖性。peptidimer-c与Gleevec对K562均有细胞毒作用,且Gleeve的细胞毒作用远大于peptidimer-c。WST-1检测结果显示,peptidimer-c杀伤K562细胞的半致死剂量为(17.85±2.10)μM,而Gleevec的半致死剂量为(0.25±0.05)μM。
在甲基纤维素半固体培养体系中,K562细胞的克隆形成受peptidimer-c抑制,并随着peptidimer-c浓度的增加克隆数逐渐减低,在浓度达到2μM以上时,即有显著性抑制。几种化合物对K562克隆形成抑制的半致死剂量为:peptidimer-c(3.90±0.86)μM,Gleevec(0.03±0.02)μM,羟基脲(15.00±7.07)μM,阿糖胞苷(0.014±0.012)μM。peptidimer-c分别与Gleevec,阿糖胞苷,羟基脲联合应用时,对K562克隆形成抑制均表现为相加作用或协同作用,其中1.5μM peptidimer-c与0.05μM Gleevec联合应用,表现协同作用,1.5μM peptidimer-c与0.006μM阿糖胞苷或0.01μM阿糖胞苷联用,也显示协同抑制效应。提示peptidimer-c可与其它类型的药物合用,以提高抗肿瘤的综合效应。
三、peptidimer-c诱导K562细胞的凋亡
细胞生长的抑制总是伴随着细胞的死亡,包括坏死和凋亡。本部分应用免疫印迹技术,观察peptidimer-c对K562细胞主要信号分子的影响;通过光镜和透射电镜形态观察,TUNEL法,流式细胞术检测亚二倍体形成、annexin V/PI比例、Caspase-3和Fas的表达水平,探讨peptidimer-c诱导K562细胞死亡的方式和可能的机制。
结果表明,用peptidimer-c处理K562细胞后3-6h,细胞就发生明显形态学改变,细胞涨大,进而出现皱缩,并进入死亡阶段,透射电镜观察提示,经peptidimer-c处理,细胞形态改变,核内异染色质增多,边集现象明显,出现典型的凋亡特征。胞内内质网,线粒体结构改变。随着peptidimer-c浓度增加,坏死细胞比例加大。Annexin V/P测定结果也证实,中低浓度peptidimer-c主要诱导K562细胞凋亡,高浓度(27μM)的peptidimer-c在诱导凋亡的同时,直接导致细胞坏死。peptidimer-c的作用可使K562细胞Caspase-3的比例显著增加,提示其通过Caspase-3途径促进K562细胞凋亡,但peptidimer-c并不增加K562细胞死亡受体Fas以及Bcl-2的表达,可以确定,Fas-FasL系统不是peptidimer-c诱导K562细胞的凋亡途径,也与Bcl-2相关途径无关。peptidimer-c通过影响MAPK、PI3K、STAT5信号通路而抑制细胞生长、增殖。
四、peptidimer-c对K562细胞周期的改变
细胞生长的调节是通过细胞周期的调控得以实现的。本部分应用碘化丙啶一步插入DNA定量荧光染色法,检测peptidimer-c作用后,K562细胞周期各时项的比例,应用免疫印迹法(Western blot)和免疫细胞化学染色法观察K562细胞周期素Cyclin A, Cyclin B,以及周期素依赖性激酶Cdk2,p-Cdk2,,Cdk1, p-Cdk1的表达,探讨peptidimer-c对K562细胞周期的影响及其机制。
结果显示,随着peptidimer-c浓度的增加,K562细胞的S期比例明显上升,同时,G0/G1期比例以及G2/M期比例下降,将2细胞阻滞于S期,而Penetratin不改变K562细胞周期。Gleevec亦可使K562细胞周期比例发生改变,但却使细胞阻滞于G0/G1期。Western blot检测结果提示,随着peptidimer-c浓度加大,K562细胞的Cyclin A水平被显著抑制,同时P-Cdk2表达水平亦降低,而CyclinB,Cdk1以及p-Cdk1的表达均未改变。免疫细胞化学染色结果亦显示,peptidimer-c可明显降低K562细胞CyclinA
的表达;对Cdk2的抑制不明显,但却使Cdk2分子在细胞核内的分布发生变化,呈现不均一分布,并向核的一边浓聚。以上结果说明,peptidimer-c通过下调细胞内S期相关蛋白CyclinA和p-Cdk2的表达而使K562细胞阻滞于S期,抑制了细胞周期进程。
五、peptidimer-c对K562细胞基因表达谱的影响
基因芯片法技术通过同时对细胞或个体内的大量基因表达的平行分析,实现从整体上分析细胞表达状况的信息。本部分采用美国Affimetrix公司的人类U133 Plus3.0基因表达谱芯片,探讨经peptidimer-c作用后K562细胞基因表达谱的改变,并用RT-PCR方法对部分有显著差异的基因进行验证。
结果显示,应用人类U133 plus 3.0基因表达谱芯片检测经peptidimer-c处理后K562细胞,发现有455个上调表达的基因,以及74个下调表达基因,涉及细胞凋亡,血管生成,细胞周期,信号转导,细胞趋化,蛋白合成、蛋白折叠,转录因子,以及一些功能不明的基因等等。RT-PCR检测结果与基因表达谱芯片检测结果吻合。
凋亡相关基因分析表明,petidimer-c可能通过上调TNF及其受体家族成员,和JUN家族,启动K562细胞凋亡;通过抑制热休克蛋白导致细胞死亡。
细胞周期相关基因分析表明,petidimer-c可能通过上调细胞周期抑制因子P21、低氧诱导基因95(Hi95, sestrin2)等改变细胞周期进程。
Grb2 (growth factor receptor-bound protein 2) is an important adaptor protein in the cells, and plays a pivotal role in the cellular signal transduction. Grb2 activates MAPK cascade which contributes to the cellular growth and differentiation by Ras activation. In some cancer cells, Grb2 is over-expressed, and the mutation of Grb2 could inhibit its signal transferation and impair the cell proliferation and transformation. In 1999, a peptide dimmer (peptidimer-c) was designed to bind the two domains of Grb2 and blocked the link of Grb2 and the down-stream molecule Sos which is a guanine nucleotide exchange factor for Ras. K562 is a Bcr-abl positive cell line. Grb2 plays an important role in the Bcr-abl induced signal transductian. In this study the effect of peptidimer-c on K562 cells was investigated and the possible mechanisms would be disussed in the following five sections.
1. The synthesis of peptides and identity
The peptidimer ( VPPPVPPRRR-K-RRRPPVPPPV ), penetratin (RQIKIWFQNRRMKWKK) and peptidimer-c (peptidimer linked to penetratin) were synthesized by solid-phase synthesis using Fmoc chemistry, and purified by high performance liquid chromatography (HPLC) on a C18 column. Purity was evaluated by HPLC, and the identity of the peptides was checked by electrospray mass spectroscopy(MS). A pull-down assay was used to observe the specific binding of peptidimer to the Grb2 from K562 cells.
The result showed that a single protein peak was observed by HPLC, which meant that the peptide was in high purity. MS analysis showed the peptides were coinsident with the design. The molecular weight of peptidimer-c was 4794.0 and that of the penetratin was 2246.7. In order to determine the binding of peptidimer to Grb2, the peptidimer and penetratin were respectively coupled to CNBr sepharose beads and incubated with cell lysate of K562. Pull down assay demonstrated that the peptidimer, not the penetratin, could bind to Grb2 specifically.
2. The influence of peptidimer-c on K562 cell growth
In chronic myelogenous leukemia (CML) cells, Bcr-abl fusion protein activates many signal pathways to maintain the cell survival, proliferation and transformation. Among these signalings, the MAPK cascade activated by Ras is the key pathway, and the coupling of Bcr-abl and Ras is mediated by Grb2. In this section we investigated the inhibition of peptidimer-c on K562cell proliferation by trypan blue exclusion assay; the cytostatic effect by clonogenic assay, and the cytotoxicity by WST-1 method. A further experiment was performed with clonogenic assay to explore the effect of peptidimer alone and in combination with Gleevec, Hydroxyurea and Cytarabine respectively. Jing’s method was used for analysis.
The results showed that the peptidimer-c could inhibit the proliferation of K562 significantly in a dose-dependent manner, shortly (3-6h) after the cells were exposed to the drug, and the penetratin alone did not influence the cell proliferation. Gleevec inhibited the growth of K562 not only in a dose-dependent manner, but also in a time-dependent manner. WST-l test showed the cytotoxicity of peptidimer-c or gleevec on K562 cells, the IC50 of peptidimer-c was (17.85±2.10)μM and the IC50 of gleevec was (0.25±0.05)μM.
In the methylcellulose semi-solid medium system, the colony formation of K562 was greatly decreased by peptidimer-c as compared to the penetratin, and the colony number decreased as the dose of peptidimer-c increased. For IC50 value of colony formation on K562, peptidimer-c was (3.9±0.86)μM, Gleevec was (0.03±0.02)μM, Hydroxyurea was (15.00±7.07)μM, and cytarabine was (0.014±0.012)μM. There were synergistic effects of peptidimer-c with Gleevec, Hydroxyurea or Cytarabine on K562 by colonogenic assay. Combination of 1.5μM peptidimer-c and 0.05μM Gleevec showed synergistic effect on K562, as well as the combination of 1.5μM peptidimer-c and 0.006μM or 0.01μM Cytarabine. These results suggested that combination of peptidimer-c with other drugs would increase the anti-cancer effects.
3. peptidimer-c induced apoptosis in K562 cells
The inhibition of cell growth is always concomitant with the cell death, including necrosis and apoptosis. By morphological observation, TUNEL technique, flow cytometry for determining the hypodiploid formation; annexin V/PI percentage; and expression of caspase-3, Fas and Bcl-2, K562 cell death induced by peptidimer-c would be analyzed in this section.Some signal molecules were detected by Western blot to observe the influence of peptidimer-c on the signal pathways of K562.
The results showed that under the microscope, the K562 cells got some morphological changes: cells became swelling and crimpling after 6h exposure to the peptidimer-c. At the same time, it was found that the hetero-chromatin increased and accumulated in the margin of nucleus under the electronic microscope. The structure of endoplasmic reticulum and mitochondria changed. The percentage of necrosis increase as the dose of peptidimer-c increased. Annexin V/PI test demonstrated that medium or low dose of peptidimer-c mainly induced the apoptosis of K562 cells, and high dose of peptidimer-c (27μM) induced both the apoptosis and the necrosis of K562 cell. Peptidimer-c might induce the apoptosis of K562 by activating the caspase-3. Neither the Fas expression, nor the Bcl-2 of the K562 cells was changed.Peptidiemer-c may inhibit the growth of K562 cells by decreasing the transduction of MAPK, PI3K, and STAT5 pathways.
4. Peptidimer-c changed the cell cycle phase distribution of K562 cells
Cell growth is regulated by the control of cell cycle progression. In this section, the cell cycle modifications of K562 by peptidimer-c were detected with PI staining method in flow cytometer and analyzed by ModFit software. The role of the cyclins (Cyclin A/B) and cylin-dependent kinases (Cdk2, P-Cdk2, Cdk1 P-Cdk1) on cell cycle modification were investigated by western blot and immunocytochemistry techniques.
The results showed that the percentage of K562 cells in S phase inceased, and those of G0/G1 phase and G2/M phase decreased when K562 cells were treated with increasing concentrations of peptidimer-c for 6h. Peptidimer-c induced an S phase arrest in K562 cells, and penetratin had no effect on the cell cycle phase distribution. Gleeve also changed the cell cycle phase distribution, but it induced a G0/G1 phase arrest in K562 cells. Western blot demonstrated that the peptidimer-c treatment resulted in a significant reduction in the protein levels of cyclin A and P-cdk2, and a slight decrease in the protein level of Cdk2 by high dose of peptidimmer-c. Peptidimer-c did not influence the protein expression level of cyclin B, Cdk1, P-Cdk. Immunocytochemistical results also indicated that the cyclin A level decreased in K562 treated with increasing peptidimer-c, and the Cdk2 level decreased a little. Peptidimer-c resulted in the change of Cdk2 distribution in the nucleus. Cdk2 protein was enriched in the inner margin of the nuclear membrane. These results suggested that peptidimer led to an S phase arrest of K562 cells by down-regulating the cyclin A and P-Cdk2 and inhibited the cell cycle progression.
5. The influence of peptidimer-c on K562 gene expression profiles
Gene expression profiles analysis is a powerful technology to gain valuable information of cells in general by a synchronous detection of plentiful genes in the cells or the organisms. In this section, the human U133 plus 3.0 gene expression profiles from Affimetrix Company were used to detect the gene expression changes in K562 cells treated with poptidimer-c. RT-PCR assays were performed to confirm some significantly changed genes.
The results showed that the expression of 455 genes in K562 cells treated with peptidimer-c was up-regulated and that of 74 genes was down-regulated in the gene profiles. These genes are those for apoptosis, angiogenesis, cell cycle, signal transduction, chemotaxis, protein synthesis, protein folding, transcript factors, and some other unknown functions. The results of RT-PCR were coincident with those of gene expression profiles.
Apoptosis related genes analysis suggested that peptidimer-c may induce apoptosis of K562 cells by activating TNF/TNFR family and the JUN family, and lead to cell death by inhibiting some hot shock proteins (HSP).
Cell cycle related genes analysis indicated that peptidimer-c also modified the cell cycle progression of K562 through up-regulation of cyclin-dependent kinase inhibitor P21, and the hypoxia-induced gene 95(Hi95, sestrin2).
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