Inhibition Of MDM2-p53 Interaction By Adenovirus Delivery Of Ribosomal Protein L23 Stabilizes P53 And Induces Cell Cycle Arrest And Apoptosis In Gastric Cancer

Functional integrity of the p53 gene is an essential cellular defense againstneoplastic transformation. In fact, nearly all of the different kinds of humanmalignancies analyzed thus far were shown to contain mutations of the p53 geneor alternations in the p53 regulating pathway. Mutations in the p53 gene arefound in about 50% of all human tumors. Even so, half of human tumors retainwild-type p53, which are thought to have inadequate p53 function due toabnormalities in p53 regulation or defective signaling in the p53 pathway.Murine double minute 2 (MDM2), is an oncogene which encodes a proteinbest characterized for its role in the inactivation and degradation of wt p53. Innormal cells, steady-state levels of wt p53 are maintained at very low levels bycontinuous degradation by MDM2, which is essential for cells to hold p53function in check during normal development. The MDM2 gene itself is a downstream target of p53, thus forming a tight autoregulatory feedback loop.Consistent with this notion, gene amplification and over-expression of MDM2have been shown in a variety of tumors, which results in the inactivation of wtp53 protein, with an effect similar to that of mutations in the p53 gene.Interfering with the MDM2-p53 feedback loop leads to p53 (wt) activation,ultimately preventing neoplasia. Ribosomal proteins L23 (RPL23) was reportedto be able to inhibit MDM2-mediated p53 ubiquitination through direct bindingto MDM2, and subsequently induce p53 levels as well as its activity. In addition,RPL23 could interact with MDM2 in both the nucleus and the cytoplasm,which meant MDM2 was retained in the cytoplasm and the nucleus as acomplex, and this complex formation represented one more mechanism bywhich RPL23 indirectly inhibited the MDM2-p53 binding. Based on the knownmolecular properties of the RPL23 protein, we proposed a rationale for usingRPL23 as a target for cancer gene therapy.Objective:To investigate whether by disrupting the interaction between MDM2 andp53, adenovirus-mediated delivery of human ribosomal protein L23 (Ad-RPL23)stabilizes p53 protein, specifically activates the p53 pathway, and has in vitroand in vivo antitumor activity against gastric cancer harboring wt-p53.Methods:1. The expression of p53 and MDM2 in gastric cancer was determined byimmunohistochemical staining; 2. Adenovirus expressing a his-tagged humanribosomal protein L23 (Ad-RPL23) was constructed, and immunoblotting,immunoprecipitation, and immunofluorescence staining were applied to verifythe inhibition of MDM2-p53 interaction by Ad-RPL23; 3. Using an in vitrosystem with cultured gastric cancer MKN45 and AGS cells carrying wt-p53, the effects of Ad-RPL23 transfection on cell cycle and apoptosis were investigated,and studies using nude mice were applied further to ascertain the antitumoreffect of Ad-RPL23 in vivo.Results:1. Expression of p53 and MDM2 in gastric cancer94 primary gastric adenocarcinomas were assessed for MDM2 and p53expression using immunohistochemistry. It was found that p53 protein (mt-p53)was detected in 36% (34/94) of primary gastric cancer, and the percentage ofp53-positive cells was highly variable ranging from 10% to 99% (median, 30%),indicating that more cancer cells in gastric cancer express wt-p53; MDM2protein was located in the cytoplasm and nucleus of gastric cancer cells, and thepositive rate of MDM2 expression in primary gastric cancer was 62.8% (59/94).In addition, immunohistochemical staining for MDM2 revealed that expressionof MDM2 in lymph node metastasis was significantly higher than that inprimary gastric focus, indicating that overexpression of MDM2 were related tometastasis of gastric cancer.2. Construction and verification of Ad-RPL23Adenoviruses expressing a his-tagged human ribosomal protein L23 (Ad-RPL23)was constructed using the adenovirus expression system of WuhanGenesil Biotechnology Co., Ltd. Consequently, by immunoblotting,immunoprecipitation, and immunofluorescence staining, we showed that p53was accumulated in gastric cancer MKN45 cells through disruption of theinteraction between MDM2 and p53 by adenovirus delivered L23. Further, weprovided evidence that the accumulation of p53 induced by Ad-RPL23 waslargely attributable to decreased degradation of the protein.3. The growth inhibitory effect of Ad-RPL23 on in vitro and in vivo human gastric cancer cell linesIn MKN45 and AGS cells harboring wt-p53, inhibition of MDM2-p53interaction by Ad-RPL23 stabilized p53 and thus resulted in substantial cellcycle arrest at G₁-S checkpoint, which was mediated largely by up-regulation ofp21, a transcriptional target of p53. We also showed that in MKN45 cells withwt-p53, inhibition of MDM2-p53 interaction by Ad-RPL23 resulted insignificant apoptotic cell death, which was associated with up-regulation of twowell-established transcriptional targets of p53, the proapoptotic proteins Bax andPUMA. Of note, no effects of Ad-RPL23 on cell cycle progression or apoptosiswere found in MGC803 cells with mt-p53, indicating the specificity ofexogenous RPL23 in activation of the p53 pathway. Encouraging results werealso obtained in the Ad-RPL23 treatment for subcutaneous tumors of MKN45gastric cancer. In conclusion, the data presented in this study verified thatinhibition of MDM2-p53 interaction by Ad-RPL23 could stabilize p53 andinduce cell cycle arrest and apoptosis in gastric cancer cells carrying wt-p53.Conclusion:Exogenous RPL23 induced wt-p53 stabilization and activation may be anovel therapeutic approach for patients with gastric cancer.