Effects And Mechanism Of MDM2 Inhibitor On Proliferation And Apoptosis In Three Hmman Glioma Cell Lines With Different TP53 Status

Background and objectiveGlioma is one of the most common malignant tμMors in central nervous system. Malignant gliomas are characterized by its invasiveness and dissemination, resulting in frequent tμMor recurrence after surgical resection and/or conventional chemotherapy and radiation therapy. Current combinations of surgical therapy, radiotherapy and chemotherapy regimens do not significantly improve long-term survival of the patients with malignant glioma. The abnormal behaviors demonstrated by cancer cells are the result of a series of mutations in key regulatory genes. The cells become progressively more abnormal as more genes become damaged. The transition from a normal, healthy cell to a cancer cell is step-wise progression that requires genetic changes in several different oncogenes and tμMor suppressors. The growing awareness of differences in inter-individual response to radiochemical exposure calls for a new approach in the therapy and follow-up of cancer patients. P53 acts as a tμMor suppressor in many tμMor types, induces growth arrest or apoptosis depending on the physiological circμMstances and cell type. TP53 is frequently mutated or inactivated in about 60% of cancers. P53 is ubiquitinated by MDM2, which leads to proteasomal degradation. The goal is to improve the therapy results of Nutlin-3 by evaluating inter-individual differences in TP53 status.MethodsGlioma cell lines U87, A172 and U251 were used to evaluate the effects of MDM2 inhibitor Nutlin-3. First, the whole coding sequence of the p53 gene was cloned from cDNA by RT-PCR and sequenced. Second, the MTT assay was used to measure the activity of cell proliferation. Third, the effects of combination of Nutlin-3 and PI3k inhibitor, LY294002, were detected. Fourth, after the drug treatment, the mRNA levels of p53 and the target genes of p53, MDM2, P21 and BAX were compared with the control by semi-quantitative RT-PCR. Fifth, Western-blots were used to detect the changes of p53, PARP and PCNA protein after the treatment. Sixth, the specific DNA binding capacity of p53 protein in three cell lines was evaluated by oligonucleotide pull-down.Results1. The cloned coding sequence of p53 was sequenced. The results indicated that TP53 gene in U87 is wild-type, but N239S in A172, R273H in U251.2. MTT assays showed that Nutlin-3 can significantly inhibit the growth of U87 (25μM, inhibition rate 36.97%) and A172 (61.41%), but no effects on U251. The PI3K inhibitor, LY294002, can inhibit all three cells under high concentration. The effects of combination of Nutlin-3 and LY294002 are stronger than alone.3. Semi-quantitative RT-PCR analysis indicated that the p53 mRNA level didn't change with Nutlin-3 treatment in U251 cells, and increased remarkably in U87 and A172 (p<0.01). After the treatment, the expression levels of p53 target genes, P21 and BAX, maintained, but that of MDM2 increased in U251. The Nutlin-3 treatment improved the expression of all the genes in U87 and A172.4. Without drug treatment, the protein level of p53 is higher in U251 than in U87 and A172. After the treatment, the p53 protein didn't change in U251 and increased significantly in U87 and A172. The cleavage of poly (ADP-ribose) polymerase (PARP) - a biochemical marker for apoptosis - was induced by Nutlin-3 in A172 .The PCNA (Proliferating Cell Nuclear Antigen) decreased in U87, but not changed in U251 and A172.5. Oligonucleotide pull-down analysis showed that wild-type p53 in U87 and N239S p53 in A172 can effectively binding DNA, but R273H p53 in U251.Conclusions1. The effects of MDM2 inhibitor Nutlin-3 are different on gliomas with different TP53 status, which indicated the importance for the use of Nutlin-3 in clinic.2. The PI3K inhibitor has effects on gliomas under high concentration. And combination of MDM2 inhibitor and PI3K inhibitor give more strong effects.3. The genetic change in A172 doesn't influence the effects of Nutlin-3, which indicates that N239S p53 have normal function. And oligonucleatide pull-down analysid supported this conclusion.4. The mechanisms of MDM2 inhibitor are inhibition of proliferation (PCNA change) and promotion of apoptosis (BAX change and PARP change).5. MDM2 inhibitor inhibits the degradation of p53, which promotes the expression of MDM2, and increased MDM2 will improve the degradation of p53, increased P21 will inhibit the effects of MDM2. Collectively, our results indicate that the p53-MDM2 feedback cycle play important role in glioma therapy.