Effects Of MDM2-p53 Feedbackloop On The Chemotherapy Sensitivity Of Ovarian Cancer Cell Lines To Cisplatin

[Objective:] Ovarian carcinoma is one of the worst tumors among malignancies of the female genital tract with the highest morbidity and threat the health and safe of women's life. The main measures of therapy is operation and chemical drugs. The main reason that leads to the failure of chemotherapy of the ovarian cancer is the drug resistance which is evident especially at an advanced stage where the five-year living rate is only 30 persent. There is a great difference between the average living time and recurrent rate of patients who are drug resistant or drug sensitive. It's crucial to increase the drug sensitivity and decrease drug resistance especially to the common used cisplatin in order to improve the clinical prognosis. Thus finding a new and effective way to crack the ovarian cancer is going on keen research now.The mechanism of the drug resistance of ovarian cancer is very complicated with many factors involved in different levels. There are also many changes in high resistant ovarian cancer cells. MDM2(murine double minute 2)was originally identified as an amplified gene carried on double minutes in a mouse tumor fibrous cell line 3T3. Over-expression of MDM2 produced a transformed phenotype. Then it has been founded expressing in a series human tumor. Human MDM2 is located in 12ql3-14.The modulation of its expression is pretty complicated and there are several different mRNA types. So far it has been found that MDM2 have at least seven different mdm2mRNA and five different mdm2 protein(that is P90, P85, P76, P74, P57). 36 percent of sarcoma have the amplification of MDM2 and 5 to 10 percent of human carcinoma have the over expression of MDM2. MDM2 is one of the downstream regulated genes of p53 which can be transcriptionallyinduced by p53 and binds to p53 in return to block its ability to function as a transciption factor. Thus MDM2 and p53 formed a autoregulatory feedback loop which keep a low level of p53 in a normal cell. This mechanism is modulated by a lot of genes and other factors. There are more and more evidences that MDM2-p53 autoregulatory feedback loop has a key role not only in the occurrence and development but also in the therapy of tumors. There have been some researches focused on the relationship of p53 and chemosensitivity. The results have shown either no association or an association. Resistance to cisplatin has been demonstrated in human cell lines transfected either with wild type p53 or a dominant-negative mutant p53. As a contrast, little is known about the connection of MDM2 and chemosensitivity. But with the keen modulationary relationship of MDM2 and p53, the mechanism of MDM2 to the biological behavior of the cell has received a sever concentration.[Methods and Results]: In this research, We transfected the eukaryotic expression vector PCMV-MDM2 which contain full length human MDM2 and its control vector PCMV to the human ovarian cancer cell line A2780(WT-p53) and SKOV-3(dominant-null p53). Then the difference of drug sensitivity of these two cell lines with different status of p53 was investigated. Western blotting assay was used to examine the protein expression of p53. We have observed in our research that parental A2780 and A2780 with empty vector had an obvious p53 expression after the radiation treatment, whereas A2780 cells which had been transfected with MDM2 had no p53 expression due to the inhibition role of MDM2 to p53. Thus, we can say that MDM2 had been transfected in an efficient way. In SKOV-3, there wasn't MDM2 expression. But after thansfected, the cells had an high level of MDM2 expression. Then we tested the change of drug sensitivity through several ways include clonogenic survival assay, the trypan blue exclusion assay and IC50 value. The trypan blue exclusion assay demonstrates that A2780-MDM2 have a substantially higher percentage of trypan blue stainedcells than A2780-V in response to cisplatin in every time point; The rate of formation of clones in A2780-MDM2 was much lower compared with A2780 and A2780-V (P<0.01) . A2780-MDM2 showed a sign...