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Effects and Mechanism of MDM2 Inhibitor on Proliferation and Apoptosis in Three Hmman Glioma Cell Lines with Different TP53 Status

Author: ZhaoXiaoMei
Tutor: ZhongPing；SunGuoTao
School: Henan University
Course: Pharmaceutical Analysis
Keywords: Glioma TP53 MDM2 inhibitors Nutlin - 3 Individualized treatment
CLC: R739.41
Type: Master's thesis
Year: 2011
Downloads: 57
Quote: 1
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Abstract

Background purposes: glioma is the most common malignant tumor of the nervous system, with rapid growth, invasive growth, a high degree of malignancy, short course, easy to relapse after surgery, the prognosis is poor and so on, a serious threat to people's health. Gliomas occur because as well as the development of mechanisms to be studied, there are many of. Tumor development is closely related to genetically modified, and there is a gene change and lesion effects continue to accumulate, the process of evolution. The genetic changes in oncogenes and tumor suppressor genes play a role in the most critical. Oncogene in tumor cells through a variety of ways abnormal activation Similarly, the tumor suppressor gene can also be a variety of ways abnormal inactivation. Meanwhile, the type of self-tumor gene changes, having a great difference in drug sensitivity. TP53 p53 protein expression product of the tumor suppressor gene in cell cycle regulation, the maintenance of the integrity of the cell genome to induce cell differentiation and apoptosis plays an important role. p53 signaling pathway plays an important role in the development of tumors. P53 signaling pathway in glioma development also plays a crucial role. MDM2 important role in regulating the level of p53 protein, and this role is closely related with the TP53 gene status Therefore, this study focuses on the MDM2-specific small molecule inhibitor Nutlin-3 with different TP53 gene status glioma the role and mechanism to provide experimental evidence for Nutlin-3 clinical applications as well as individualized treatment of glioma. Methods: glioma cell lines U87, A172 and U251 TP53 gene transcription of the cDNA cloned by RT-PCR, DNA sequencing method to determine whether the gene changes. On this basis, the application of Nutlin-3 to deal with three types of cells, cell viability was measured using the MTT method, Nutlin-3 of the three cells and clear differences; MTT assay was used Nutlin-3 Joint PI3K inhibitor LY294002 application of three plastic the role of stromal tumors. BAX and the difference in the amount of P21 mRNA expression by RT-PCR quantitative method to determine the three types of cells before and after treatment of p53 and p53 target genes MDM2, Western-blot was used to detect p53 protein, poly ADP-ribose polymerase (PARP) and changes in the amount of proliferating cell nuclear antigen (PCNA) before and after treatment, application Oligonucleotide pull-down detection of three types of cells in the DNA-binding activity of the p53 protein. Results: 1 TP53 gene was cloned into the cDNA fragments were sequenced to determine glioma cell lines U87 TP53 gene mutations are wild type; N239S of change and A172 cell lines, U251 cell line R273H change. The MTT cell activity assay: MDM2 inhibitor Nutlin-3 can significantly inhibit glioma cells U87 (25 mM, the inhibition rate of 36.97%), the growth of A172 (25 mM, 61.41%), while the U251 (25 mM, -18.27%) cells had no significant effect; PI3K inhibitor LY294002 at higher concentrations, the role of the three glioma cells; the Nutlin-joint LY294002 treatment, inhibition of glioma cell growth was stronger than Nutlin alone -3 or LY294002 3, RT-PCR semi-quantitative experiments showed that: Nutlin-3 treatment, glioma U251 cells p53 mRNA expression levels did not change significantly before and after processing U87, A172 cells, p53 mRNA expression levels rose significantly before and after drug treatment, p53 target genes P21 and Bax mRNA expression levels in U251 cells did not change much, MDM2 gene mRNA levels increased slightly, but not statistically significant (p lt; 0.01); U87 cells after treatment of p53 target genes MDM2, P21 and Bax mRNA expression level was significantly higher (p lt; 0.05); A172 cells of p53 target genes MDM2 and P21 mRNA expression levels were significantly higher (p lt; 0.01), BAX mRNA expression levels were significantly increased (p lt; 0.05). , Western-blot experiments show that: the absence of drug treatment, p53 protein in glioma U251 cells was significantly higher than U87 and A172 cells (p lt; 0.01); Nutlin-3 treatment, p53 protein levels in U251 cells slightly has been improved but not statistically significant. The drug treatment U87, p53 protein levels in A172 cells was significantly higher (p lt; 0.01), A172 cells increased particularly notable; drug treatment, apoptosis marker proteins PARP in U87 and U251 cells did not change significantly, and a small amount of caspase cutting fragments detected in A172 cells; cell proliferation marker protein PCNA levels did not change significantly in U251 and A172 cells in a significant reduction (p lt; 0.01), while in U87 cells. 5, Oligonucleotide pull-down experiments showed that: the wild-type p53 in U87 cells capable of binding to specific DNA sequences, and the N239S change type p53 protein in A172 cells also have binding activity, only U251 cells R273H mutant p53 protein with DNA binding activity. Conclusions: 1 glioma cells TP53 gene status, MDM2-specific small molecule inhibitor Nutlin-3 significantly different, suggesting that the clinical treatment decision based on the TP53 gene status need to MDM2 inhibitors. PI3K inhibitors in larger concentrations of the three nerve glioma have a role in MDM2 inhibitor United PI3K inhibitors significantly increased. A172 cells in the TP53 gene change does not affect the role of MDM2 inhibitors, indicating that the change does not change the function of the p53 protein, the Oligonucleotide pull-down experiments further support the conclusion. MDM2 inhibitors inhibit the proliferation of glioma cells (PCNA to change), promote apoptosis of glioma cells (BAX change, and PARP change) play a role. MDM2 inhibitor, processed to reduce the degradation of p53, the p53 protein levels increase to lead to an increase in the level of target gene expression, increased MDM2 removed part of the role of the MDM2 inhibitor, while the increase in the P21 will be further suppressed the MDM2 effect, suggesting that the p53-MDM2 feedback regulation ring importance in the treatment of tumors.

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CLC: > Medicine, health > Oncology > Nervous system tumors > Intracranial tumors and brain tumors
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