| 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 identityThe 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 growthIn 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 cellsThe 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 cellsCell 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 profilesGene 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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