Anti-Cancer Activities Of TNFα-Tumstatin-Modified Mesenchymal Stem Cells And Ribosomal Protein S25-Mediated Regulation Of P53

OBJECTIVETo investigate the anti-cancer activiti of lentivirus-TNFa-Tumstatin-modified mesenchymal stem cells on prostate cancer. To demonstrate the mechanisms by which ribosomal protein S25 regulates p53 protein and the biological consequences of S25-mediated regulation of p53.METHODSMSCs were isolated from various sources and characterized by flow cytometry. Recombinant lentiviral particles were produced in 293T cells by transient cotransfection involving a three-plasmid expression system. The transfection efficiency was analyzed by flow cytometry and the expression level of recombinant protein was verified by ELISA and Western blotting.The effect of recombinant TNFa-Tumstatin on cell viability was determined by MTT assay. The effect of the infusion protein on cell proliferation was determined by BrdUrd incorporation assay. Cells in early and late stages of apoptosis were detected using an Annexin V-FITC double staining assay. The endothelial tube formation assay was performed to analyze the effect of TNFa-Tumstatin on in vitro angiogenesis. Mouse xenograft tumor model was used to evaluate the anti-cancer effects of TNFa-Tumstatin in vivo. The proliferative, apoptotic tumor cells and tumor vessel density in vivo were visualized using immunohistochemical analysis and TUNEL assay. The mechanisms responsible for the anti-tumor activities of TNFα-Tumstatin were further investigated.The MDM2-interacting proteins were identified using yeast 2-hyrid assay. The coding sequence for ribosomal protein S25 was cloned and constructed into corresponding expression vectors, respectively. The interaction between S25 and MDM2 proteins was detected by immunoprecipitation, GST-pull down assay and Western blotting. The subcellular location of S25 protein was detected by immunofluorescent staining.The level of p53 protein after S25 overexpression or knockdown was determined by Western blotting. The effects of S25 on p53 function were evaluated by luciferase reporter gene assay, cell cycle distribution, cell apoptosis and proliferation assays. Immunoprecipitation and immunoblotting assay were used to detect the effects of S25 on p53 degradation, half-life and ubiquitination.RESULTSMSCs were successfully isolated from multiple tissue sources and the isolated MSCs were positive for CD13, CD29, CD44 and CD105, but negative for CD31, CD34, CD45 and HLA-DR. High titer of lentivirus was produced and efficiently transduced MSCs were prepared. The expression of recombinant gene and protein was detectable after lentiviral transduction. MTT assays indicated that the supernatant from transduced MSCs could inhibit the growth of prostate cancer cells (PC3 and LNCaP) in vitro. The growth inhibitory effect was due to both suppression of proliferation and induction of apoptosis. The transduced MSCs also inhibited the growth of xenograft tumor in vivo. TNFα-Tumstatin decreased the phosphorylation of ERK-1/2 and Akt, increased the ratio of pro-apoptotic/anti-apoptotic protein (Bax/Bcl2) and activated caspase-3 and 8 in prostate cancer cells both in vitro and in vivo.S25 bound to MDM2 and inhibited the E3 ligase activity of MDM2, resulting in the stabilization and activation of p53 protein. The MDM2-binding and nucleolar location domains of S25 were both required for p53 regulation. Knockdown of S25 attenuated the activation of p53 after ribosomal stress. MDMX was involved in S25-mediated regulation of MDM2 E3 ligase activity.CONCLUSIONOur results indicate that lentivirus-TNFα-Tumstatin transduction of MSCs inhibits the growth of prostate cancer cells and tumors, which provides a basis for future pre-clinical and clinical studies of MSC-based gene therapy for prostate cancer.Our data suggests that S25 can be added to the growing list of MDM2 regulatory binding partners. S25 is a novel regulator of p53. Our findings provide further insight into how ribosomal proteins regulate the p53 signaling pathway and potential candidate drug for therapeutic intervention of cancers that harber wild-type p53.