| Objective: Malignant mesothelioma, also known as the diffuse malignant mesothelioma, is originated from the pleura, peritoneum and covering skin cells in pericardium cavity, and it is an extremely rare and high maliganant carcinoma with highly local invasion ability. Malignant pleural mesothelioma is accounted for approximately 70% of pleura primary tumor, and malignant peritoneal mesothelioma also occupies a large proportion. Today the cause and mechanism of nausea mesothelioma is still unclear, but they are directly related to almost all types of overexposure asbestos substances. As since 1970, especially in industrialized and developing countries, the production and consumption of asbestos substances has been increased as well as the malignant mesothelioma. Now for this disease with a long incubation period, we do not have very effective preventive measures. And the number of patients with malignant mesothelioma will probably continue to grow in the next few decades.Although now there are a lot of treatments, malignant mesothelioma is still an intractable disease. Typical early symptoms of malignant mesothelioma are lacking of specificity, which are not easily detected. Malignant mesothelioma grows and invades fast in pleural cavity or peritoneal cavitysurgery, the operational treatment is only suitable for early stages, and the recurrence rate is very high even after radical pleural resection. Radiation therapy for malignant mesothelioma is not sensitive, so it is only used in part cases of palliative care. Therefore, for patients suffering from this difficult disease, chemotherapy has been widely recognized. Today’s chemotherapy treatment programs is mainly pemetrexed and cisplatin-based, the prognosis is poor, with a median survival of only 12.1 months. Therefore, a new therapeutic strategies based on the molecular mechanisms of malignant mesothelioma is very necessary for prolong survival and improving quality of life.Malignant mesothelioma generally contains p16INK4 A and p14 ARF gene defects. Although it also contains wild-type p53 gene, the p53-mediated apoptosis fails to be completed due to the limited expression of p53 protein. The selective proliferating adenovirus possesses superior selective proliferation capacity and is highly expressed in tumor cells while lowly proliferates or does not proliferate in normal cells. Furthermore, it can also activate the p53 pathway of tumor cells, express p53 protein and induce apoptosis. MDM2(murine double minute2) protein small molecular inhibitor can inhibit MDM2 protein function and downregulate proteasome function through binding with MDM2, thus suppressing the degradation pathway of p53 protein, upregulating p53 protein level and leading to cell apoptosis. In this study, the combination scheme of selective proliferating adenovirus and the small-molecule inhibitor of MDM2: p53 gene was expressed in the selective proliferating adenovirus of tumor cells. Combined with the inhibitor of MDM2, whether the anti-tumor effect of selective proliferating adenovirus will be increased and whether the growth of malignant mesothelioma cells will be inhibited to promote the apoptosis were tested. As shown from the result, the expression level of p53 was increased and the phosphorylation of p53 was promoted in the selective proliferating adenovirus of tumor cells. Meanwhile, the inhibitor of MDM2 could inhibit the expression level of MDM2 in the malignant mesothelioma cells effectively, suppress proteasome function, and decrease p53 degradation, leading to the accumulation of p53 protein. Hence, the expression level of p21 was increased, the expression level of Rb phosphorylation was reduced, and the caspase-3 and caspase-8 were cut, thereby resulting in the increased carcinoma apoptosis. Method: The experiment select and use malignant mesothelioma cell line culturing in vitro: MSTO-211 H, NCI-H28, EHMES-10, NCI-H2052, EHMES-1, NCI-2452 and JMN-1B. In MTT experiment, detect inhibited sensitivity of selective adenovirus(with ONYX-015) and MDM2 protein inhibitor(with Nutlin-3a) on above malignant mesothelioma cell line with cells in logarithmic phase. Use Trypan blue dye exclusion assay to detect the inhibitory effect of ONYX-015 on the growth of above malignant mesothelioma cell line. Detect the changes of Nutlin-3a on periodic variation by using flow cytometry period analysis method. Use Western blot to analyze the changes of apoptosis of periodic variation caused by the combination of ONYX-015 and Nutlin-3a. Finally, detect the coordinate repression of the combination of ONYX-015 and Nutlin-3a on malignant mesothelioma with MTT experiment.Result: 1 The analysis results of MTTHuman malignant mesothelioma cell line is the effect of MSTO-211 H, NCI-H28, EHMES-10, NCI-H2052, EHMES-1, NCI-2452 and JMN-1B treated with different concentration of ONYX-015 on cell viability. It is shown that MSTO-211 H, NCI-H28, NCI-H2052, EHMES-10 cell line is relatively sensitive. According to IC50, MSTO-211 H cell is most sensitive to Nutlin-3a, the next are NCI-H28 and EHMES-10 and last is NCI-H2052 cell line. However, EHMES-1, NCI-2452 and JMN-1B cell line are not sensitive to ONYX-015. Since EHMES-1, NCI-2452 and JMN-1B are mutation-type p53 cell lines and MSTO-211 H, NCI-H28, NCI-H2052, EHMES-10 are wild-type p53 cell lines, so it indicates that the wild-type p53 cell lines are sensitive to ONYX-015 while the mutation-type cell lines are not. After being treated with ONYX-015, wild-type p53 cell lines as: MSTO-211 H, NCI-H28, NCI-H2052 and EHMES-10 become relatively sensitive. According to IC50, NCI-H28 cell is the most sensitive to Nutlin-3a, the next are MSTO-211 H and EHMES-10 cell lines and last is NCI-H2052. 2 Trypan blue dye exclusion assayComparing each group, it is shown that ONYX-015 inhibit the growth of malignant mesothelioma cells in MSTO-211 H, NCI-H28 cell lines dependent on the dosage while control groups are treated with adenovirus Ad-Lac Z and the growth of malignant mesothelioma cells is not inhibited. 3 The analysis result flow cell cycleUse ONYX-015 and β-Ad-Lac Z to infect malignant mesothelioma cell line with NCI-H28, MSTO-211 H and Ad-Lac Z as negative control. ONYX-015 causes NCI-H28 cell to obviously increase in sub-G1 and decease in S and proportionally increase in G2/M. MSTO-211 H cells increase in sub-G1 and stops to grow in G0/G1. Compared with NCI-H28 cells and MSTO-211 H cells in sub-G1, NCI-H28 cells in sub-G1 increase more obviously, which indicates NCI-H28 cells are more sensitive to apoptosis mediated by ONYX-015 but there is no obvious difference between negative control groups and blank control groups. 4 The analysis result of Western blot 4. 1 Use Western blot to detect molecular pathway of ONYX-015 mediated by MSTO-211 H and NCI-H28 cells. ONYX-015 induces p53 phosphorylation to increase, rising the expression level of p21 in the downstream and lowering phosphorylation Rb, which indicates that ONYX-015 makes molecular pathway mediated by p53 active. 4. 2 In the two cell lines of MSTO-211 H and NIC-H28, ONYX-015 does not induce caspase-9 to cut after being treated but induce caspase-3 and caspase-8 to cut,and it increases the expression level of Akt and phosphorylation Akt. It indicates from above results that ONYX-015 makes extrinsic apoptotic pathways mediated by death receptors active but it is impossible to involve in endogenous apoptotic pathways.5 The analysis of MTT on the synergistic effect of ONYX-015 and Nutlin-3a NCI-H28 and MSTO-211 H of human malignant mesothelioma cells are treated with different concentration of ONYX-015 and Nutlin-3a, and made experiment with appropriate untied drug concentration. Then it is analyzed with Calcu Syn software developed by University of Cambridge and the CI of drug combination shows that the application of combination of ONYX-015 and Nutlin-3a has synergetic inhibitory effect on malignant mesothelioma cell.Conclusion:1 ONYX-015 inhibits the growth of malignant mesothelioma.2 ONYX-015 induces apoptosis on the malignant mesothelioma cell with wild p53.3 ONYX-015 makes molecular pathway and exogenous apoptosis pathway mediated by p53.4 Nutlin-3a inhibits the growth of malignant mesothelioma.5 The combination of ONYX-015 and Nutlin-3a has synergetic inhibited effect on malignant mesothelioma cells. |
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