Design of ultrahigh-affinity and dual-specificity peptide antagonists of MDM2 and MDMX for P53 activation and tumor suppression

Peptide inhibition of the interactions of the tumor suppressor protein P53 with its negative regulators MDM2 and MDMX activates P53

Immunohistochemical expression of p53 and murine double minute 2 protein in odontogenic keratocyst versus variants of ameloblastoma

Introduction: Oncogenes and tumor suppressor genes play a major role in cancer formation, growth, and progression. One of the important findings in this area is that murine double minute 2 (MDM2) oncogene is a negative regulator of wild-type p53. In tumors, expressing wild-type p53, inhibition of MDM2 expression will stabilize p53 and allow it to perform its proapoptotic function, while simultaneously preventing MDM2 from exerting its p53-independent oncogenic effects. The intracellular levels of p53 are tightly regulated by MDM2, as it is a key player in autoregulatory feedback loop under nonstressed conditions. The p53-MDM2 relationship is vital not only for essential functions of the cell, but it also appears to be an integrated part of the complex cellular network which supports the importance of this affair and is a hallmark for its coexistence. Subjects and Methods: This study was designed to identify immunohistochemically the expression of p53 and MDM2 gene using monoclonal antibody in 60 cases of formalin-fixed paraffin-embedded tissue blocks, of which 20 cases were of solid multicystic ameloblastoma (SMA), 20 cases were of odontogenic keratocyst (OKC), and 20 cases were of unicystic ameloblastoma (UA). Results: Immunoexpression of p53 and MDM2 was highest in OKC followed by SMA and was minimum in UA. Further results showed positive correlation between both the molecules. Conclusion: The studied showed that the relationship has a significant role in cancer etiology and progression and therefore is an important topic for future research which should help in the development of new therapeutic agent against cancer

Effect of miR-513a-3p targeting MDM2 on proliferation, migration and invasion of gastric cancer cells / 中华肿瘤杂志

Objective@#To investigate the effects of miR-513a-3p on proliferation, migration and invasion of gastric cancer cells and its mechanism.@*Methods@#The miR-NC (miR-negative control mimics), miR-513a-3p (miR-513a-3p mimics), anti-miR-NC, anti-miR-513a-3p, si-NC, si-MDM2 (murine double minute 2), miR-513a-3p+ pcDNA3.1 (co-transfected with miR-513a-3p and pcDNA3.1), miR-513a-3p+ pcDNA3.1-MDM2 (co-transfected with miR-513a-3p and pcDNA3.1-MDM2) were transfected into BGC-823 cells, respectively. The expression of miR-513a-3p was detected by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), and the protein expressions of cyclin D1, MMP-2, p21, E-cadherin, MDM2 were detected by western blot. The viability of BGC-823 cells of each group was detected by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay. The migration and invasion of each group were detected by Transwell, the targeting relationship between miR-513a-3p and MDM2 was detected by double luciferase reporter gene assay.@*Results@#The expression of miR-513a-3p in gastric epithelial cells GES-1 was 0.76±0.08, significantly higher than 0.21±0.02 in gastric cancer cells BGC-823 and 0.34±0.03 in MGC-803, respectively (P<0.05). The cell viabilities of the miR-NC group at 24 h, 48 h and 72 h were 0.57±0.05, 1.03±0.10, 1.43±0.14, respectively, while those of the miR-513a-3p group were 0.36±0.03, 0.48±0.05, and 0.63±0.06, respectively. The migration and invasion numbers of miR-NC group were 130±11.80 and 117±10.60, respectively, those of miR-513a-3p group were 58±5.64 and 50±5.13, respectively, and the differences were statistically significant (P<0.05). The cell viabilities of the si-NC group at 24 h, 48 h and 72 h were 0.53±0.05, 0.95±0.10, 1.36±0.14, respectively. Those of the si-MDM2 group were 0.39±0.04, 0.57±0.06, and 0.80±0.08, respectively. The cell migration and invasion of the si-NC group were 141±12.02 and 109±10.60, respectively, while those of the MDM2 group were 66±6.67 and 61±6.18, respectively, and the differences were statistically significant (P<0.05). The cell viabilities of the miR-513a-3p+ pcDNA3.1 group at 24 h, 48 h and 72 h were 0.34±0.03, 0.46±0.05, and 0.61±0.06, respectively. Those of miR-513a-3p+ pcDNA3.1-MDM2 group were 0.48±0.05, 0.82±0.08, 1.17±0.12, respectively. The migration and invasion of miR-513a-3p+ pcDNA3.1 group were 56±5.71 and 51±5.16, respectively, while those of miR-513a-3p+ pcDNA3.1-MDM2 group were 113±10.28 and 104±10.02, respectively, and the differences were statistically significant (P<0.05).@*Conclusion@#miR-513a-3p may inhibit the proliferation, migration and invasion of gastric cancer cells through targeting regulation of MDM2, which will provide new targets for the prevention and treatment of gastric cancer.

Role of MDM2-p53 signaling pathway in the development of colorectal cancer / 国际生物医学工程杂志

Objective To investigate the mechanism of MDM2-p53 signaling pathway in the development of colorectal cancer and correlation between p53 with clinicopathological parameters, so as to further analyze the effect of p53 on prognosis. Methods The colorectal cancer tissues and the adjacent normal tissues from 86 cases of patients with colorectal cancer were collected . The expression of p53 and murine double minute 2 (MDM2) in colorectal cancer and adjacent normal tissues were detected by immunohistochemistry, Western Blot and real-time fluorescence quantitative polymerase chain reaction (RT-PCR). The prognosis of the patients was analyzed by the Kaplan-Meier survival curves. Results The protein expression and the mRNA expression of p53 and MDM2 in colorectal cancer tissues were significantly higher than that in the adjacent non-cancerous tissues (all P<0.01). A positive correlation was observed between the expression of p53 and MDM2 (r=0.785). The expression of p53 in colorectal cancer tissues were correlated well with the degree of tumor differentiation, TNM stage, lymph node metastasis and infiltration depth (all P<0.05). Survival analysis demonstrated that the mean overall survival time in p53 high expression group was (53.92±1.56) months which was significantly lower than that in p53 low expression group of (69.16±3.72) months, and the difference was statistically significant (χ2=14.78, P<0.01). Conclusions The risk and prognosis of colorectal cancer are closely related to the MDM2-p53 signaling pathway. p53 can be used as a potential target for the prognosis and treatment of colorectal cancer.

Murine double minute 2 309 T ＞G polymorphism is associated with gastrointestinal cancer risk especially in Asians: a meta-analysis / 肿瘤研究与临床

Objective To comprehensively evaluate the relationship between murine double minute 2 (MDM2) gene promoter SNP309 T＞G polymorphism and the susceptibility of gastrointestinal cancer.Methods The China National Knowledge Infrastructure (CNKI),WanFang database,SpringerLink database and PubMed were retrieved to get all case-control research literatures (2005-2012) on the relationship of MDM2 gene SNP309 T＞G and gastrointestinal cancer susceptibility.Meta-analysis with RevMan 4.2 was used to combine OR values of the relationship between SNP309 T＞G and gastrointestinal cancer susceptibility.A sensitivity analysis and tested publication bias were made with all selected literatures' data.Results A total of 17 domestic and foreign qualified papers were included in this study.Twenty case-control studies including 5 183 cases and 6 660 controls were identified for the present meta-analysis.A significant association was detected between the MDM2 SNP309 T＞G polymorphism and gastrointestinal cancer risk.The meta-aualysis showed that the combined odds ratio (OR) for GG genotype was 2.23 (95 ％ CI =1.73-2.89,P ＜ 0.01) compared with that for TG + TT genotypes.There was no statistical significance for the evaluation of publication bias.Conclusion The GG genotype of MDM2 SNP309 may increase gastrointestinal cancer risk in Asians.

Overexpression and amplification of Murine double minute 2 as a diagnostic tool in large lipomatous tumor and its correlation with Ki67 proliferation index: An institutional experience.

Context: Well-differentiated liposarcoma (WDLPS) is the most common type of liposarcoma and sometimes can be diffi cult to distinguish from large lipoma due to the similar morphology. Aims: This study proposed to evaluate the expression and amplifi cation of Murine double minute 2 (MDM2) gene and determine its correlation with Ki67 proliferation index. Settings and Design: This study used cross-sectional design. Materials and Methods: This study enrolled 37 cases of lipomatous tumors with >5 cm in size. Eighteen cases of WDLPS and 19 cases of lipoma were stained for MDM2 and Ki67 immunohistochemistry, followed by MDM2 in situ hybridization in 12 selected cases. Statistical Analysis Used: MDM2 overexpression and amplification status for both groups were compared using Chi-square test, with the alternative of Fisher’s exact test. Correlation test between MDM2 overexpression and clinical characteristics with the Ki67 proliferation index were performed using Pearson’s test with the alternative of Spearman’s rho test. Results: MDM2 overexpression was detected in all WDLPS cases and in 3 (16%) of lipoma cases with signifi cance difference (P = 0.000), whereas MDM2 amplifi cation was found in all WDLPS and in 1 of lipoma cases (P = 0.200). There was a strong correlation between MDM2 overexpression and higher Ki67 proliferation index (r = 0.645, P = 0.000). Conclusion: Evaluation of MDM2 overexpression can be used as a useful adjunct to differentiate WDLPS from large lipoma and seems to be related with Ki67 proliferation index.

Role of murine double minute 2 in apoptosis of acute lymphoblastic leukemia EU-4 cells induced by berberine / 中华实用儿科临床杂志

Objective To explore the role of murine double minute 2 (MDM2) in apoptosis of acute lymphoblastic leukemia EU-4 cells induced by berberine.Methods EU-4 cells at logarithm growth phase were chosen to carry out the experiments.EU-4 cells were incubated with different doses of berberine for 72 hours,and the final doses of berberine were 0,1,10 and 100 μmoL/L,respectively.Then the apoptosis rate of EU-4 cells were detected by flow cytometry,and the expression levels of MDM2 protein were tested by Western blot.The levels of MDM2 gene were examined by means of real-time PCR at 0,24,48 and 72 hours after incubation of EU-4 cells with 100 μmol/L berberine,respectively.EU-4 cells in MDM2 small interfering RNA(siRNA) group were transfected with 150 nmol/L MDM2 siRNA,and the control siRNA group were transfected with 150 nmol/L control siRNA.The apoptosis rates of EU-4 cells were detected 48 hours after transfection by flow cytometry.Results The apoptosis rates of EU-4 cells were increased in a dosedependent manner,and the highest apoptosis rate was up to(60.13 ± 4.21)％,which was incubated with 100 μmol/L berberine.Significant differences were found in all groups (F =280.56,P ＜ 0.05).Berberine treatment suppressed the protein expression of MDM2 in a dose-dependent manner(F =73.82,P ＜ 0.01).The mRNA expression of MDM2 was inhibited in a time-dependent manner by 100 μmol/L berberine(F =45.37,P ＜0.01).The apoptosis rates of control siRNA group and MDM2 siRNA group were(11.09 ± 1.63) ％ and (29.84 ± 1.75) ％,respectively.Significant difference was found between the 2 groups (t =-13.57,P ＜ 0.01).Conclusions Berberine can inhibit the expression of MDM2 in a dose-and time-dependent manner,which may be one of the mechanisms of apoptosis induced by berberine in leukemia cells.

Immunohistochemical expression of protein 53, murine double minute 2, B-cell lymphoma 2, and proliferating cell nuclear antigen in odontogenic cysts and keratocystic odontogenic tumor.

Introduction: Even though odontogenic cysts share a similar histogenesis, they show different growth and differentiation profile due to differences in the proliferative cellular activity. Aims: We perform an immunohistochemical assessment of protein 53 (p53), proliferating cell nuclear antigen (PCNA), B-cell lymphoma 2 (bcl-2), and murine double minute 2 (MDM2) expression in odontogenic cysts and keratocystic odontogenic tumor analyzing their correlation with the biological behavior of these lesions. Materials and Methods: By the streptavidin-biotin-peroxidase method with antibodies against p53, PCNA, bcl-2, and MDM2 proteins, 11 radicular cysts, 11 dentigerous cysts, and 11 keratocystic odontogenic tumor were analyzed. The non-parametric Mann-Whitney U-test and Kruskall-Wallis test (P ≤ 0.05) were used to analyze the data. Results: Immunopositivity for PCNA was observed in all cases appraised, predominantly in the suprabasal layer of keratocystic odontogenic tumor epithelial lining (SD ± 19.44), but no significant differences were found among the groups of lesions. Bcl-2 immunoexpression was observed especially in the basal layer of keratocystic odontogenic tumor. PCNA LI was significantly higher than bcl-2 LI in keratocystic odontogenic tumor. MDM2 and p53 immunoexpression were not detected in the lesions studied. Among the evaluated lesions, the keratocystic odontogenic tumor showed different immunoexpression of the proliferation and apoptosis markers. Conclusion: The results of this study suggest that the keratocystic odontogenic tumor presents distinct biological behavior of the odontogenic cysts, as for the processes of proliferation, apoptosis, and differentiation, reinforcing the information in favor of the neoplastic nature of this lesion.

Study of the Murine Double Minute 2 status in patients with gastric and colorectal carcinomas and its correlation with prognostic factors.

Background: Gastric and colorectal cancers are the second and the fourth most common cancers in Iran, respectively. The presence of Murine Double Minute 2 (MDM2) has been identified in many cancers and its relationship with prognosis is under investigation. This study aimed to assess the status of MDM2 and its relationship with prognostic factors in gastric and colorectal carcinoma. Materials and Methods: This study was performed on 99 paraffin blocks of gastric and colorectal cancers, during the years 2001 to 2007 from Mostafa Khomeini Hospital, Tehran, Iran. Tissue sections were prepared, stained with Hematoxylin and Eosin and immunohistochemistry to evaluate for MDM2 expression. The type of tumor, lymph node involvement and tumor grade was determined. Results: Of the 99 cases, 34.3% and 65.7% cases were diagnosed with gastric and colorectal adenocarcinoma, respectively. The average tumor size was 5.5 cm. MDM2 expression level was 82.4% and 90.8% in gastric and colorectal adenocarcinoma, respectively. No statistical difference was found between MDM2 expression and various prognostic factors; however, significant correlation was observed between gastric (P = 0.03) and colorectal (P = 0.03) tumor size and the percentage of MDM2 immunoreactivity. Conclusion: Considering the role of MDM2 in cell growth and its positive correlation with tumor size (an established prognostic factor), it can be indirectly concluded that MDM2 is also important in prognosis. However, additional investigation is needed.

Murine double minute 2: a novel mineralocorticoid-responsive gene involved in aldosterone-induced human mesangial cells line proliferation / 中华肾脏病杂志

Objective To investigate whether and how murine double minute 2(MDM2) was involved in aldosterone (ALD)-induced human mesangial cells line (HMCLs) proliferation. Methods RT-PCR and immunofluorescence were used to confirm the expression of MDM2 in HMCLs. Western blotting was used to estimate the relationship between ALD dose and MDM2 expression. Spironolactone, a mineralocorticoid receptor (MR) blocker, was used to estimate the role of MR on the up-regulation of MDM2 induced by ALD. Cycloheximide (CHX), a protein synthesis inhibitor, was used to estimate whether the rapid nongenomic mechanism was involved in the upregulation. To confirm the relationship among ALD, MDM2 expression and proliferation of HMCLs, small interference RNA of MDM2 was applied. Results Both MR and 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) mRNAs were detected in HMCLs. MDM2 protein expression was also detected in both the nucleus and the cytoplasm. ALD significantly stimulated MDM2 expression, which implied that MDM2 was a novel mineralocorticoid-responsive gene in HMCLs. MR was involved in this process as spironolactone did not promote the expression of MDM2 mRNA or protein. ALD with CHX did not increase the expression of MDM2 protein, which indicated it was not directly regulated by the rapid nongenomic mechanisms. MDM2 protein was decreased by using the transfection of MDM2 siRNA and ALD did not promote the cell proliferation of HMCLs under the same conditions. All of which implied that MDM2 participated in ALQ-induced HMCLs proliferation. Conclusions MDM2 is a novel mineralocorticoid-responsive gene in HMCLs. MR is involved in ALD-induced MDM2 expression which is inhibited by spironolactone. The increased expression of MDM2 protein induced by ALD is not directly regulated by the rapid nongenomic mechanisms. MDM2 participates in ALD induced HMCLs proliferation.

Polymorphisms in promoter sequences of MDM2, p53, and p16INK4a genes in normal Japanese individuals

Research has been conducted to identify sequence polymorphisms of gene promoter regions in patients and control subjects, including normal individuals, and to determine the influence of these polymorphisms on transcriptional regulation in cells that express wild-type or mutant p53. In this study we isolated genomic DNA from whole blood of healthy Japanese individuals and sequenced the promoter regions of the MDM2, p53, and p16INK4a genes. We identified polymorphisms comprising 3 nucleotide substitutions at exon 1 and intron 1 regions of the MDM2 gene and 1 nucleotide insertion at a poly(C) nucleotide position in the p53 gene. The Japanese individuals also exhibited p16INK4a polymorphisms at several positions, including position -191. Reporter gene analysis by using luciferase revealed that the polymorphisms of MDM2, p53, and p16INK4a differentially altered luciferase activities in several cell lines, including the Colo320DM, U251, and T98G cell lines expressing mutant p53. Our results indicate that the promoter sequences of these genes differ among normal Japanese individuals and that polymorphisms can alter gene transcription activity.

Immunohistochemical Expression of p53, p21, and mdm2 Proteins in Human Papillomavirus Positive and Negative Invasive Uterine Cervical Carcinomas

BACKGROUND: In the uterine cervical carcinoma, the inactivation of p53 protein by human papillomavirus(HPV) E6 protein has been reported to play a greater role in carcinogenesis than the mutation of the p53 gene. Therefore, the mutation of the p53 gene is rare. p21 and mdm2 proteins are induced by wild-type p53 protein and are involved in the cell cycle regulatory mechanism. METHODS: Immunohistochemical staining for p53, p21 and mdm2 proteins was performed in 26 HPV-positive and 13 HPV-negative invasive cervical carcinomas together with 5 non-neoplastic cervical tissues. RESULTS: The frequencies of the expression of p53, p21 and mdm2 proteins were 82.1%, 84.6% and 66.7%, respectively. The expression of p53 protein was less frequently demonstrated in HPV-positive cases than HPV-negative cases, which was statistically a negative correlation(p=0.018). The expression of p53 and p21 proteins was statistically significant(p=0.000). CONCLUSIONS: p53, p21 and mdm2 proteins were highly expressed in both HPV-positive and HPV-negative cervical carcinomas. Significantly higher expression of p53 protain in HPV-negative cases necessitate a further study for investigating the role of p53 protein accumulation in carcinogenesis of HPV-negative cervical carcinomas. The relationship between the expression of p53 protein and p21/mdm2 proteins may indicate that p21 and mdm2 proteins also have a role in carcinogenesis, where p53 protein plays a fundamental role.

Expressions of p63 mRNA and murine double minute 2 in esophageal cancer and molecular mechanisms involved / 中国医师杂志

Objective To explore the expressions and the roll of p63mRNA and murine double minute 2(MDM-2) protein in esophageal squamous cell cancer and the prognosis of the cancer.Methods The expressions of p63mRNA and MDM-2 protein in 38 cases of esophageal squamous cell carcinoma tissues and the corresponding esophageal normal tissues were detected using reverse transcriptase polymerase chain reaction(RT-PCR) and immunohistochemistry. Results The positive rates of deltaNp63 mRNA in all malignant and normal tissues were 63.5% and 52.63% respectively,without significant difference.The expression of the DeltaNp63 mRNA was significantly higher in low differentiated group than in well and moderate differentiated group,without significant difference in age,gender,lymph node metastasis and clinical stage.Only 1 of 38 cases of esophageal squamous cell carcinoma tissues presented TAp63 mRNA expression and all esophageal normal tissues had no TAp63 mRNA expression.The positive rates of MDM-2 protein in esophageal squamous cell carcinoma tissues and normal tissues were 65.79% and 5% respectively,with significant difference.The expression level of the MDM-2 protein was higher significantly in low differentiated group than in well and moderate differentiated group,with significant difference in clinical stage and without significant difference in age,gender and lymph node metastasis.The DeltaNp63 mRNA expression in the esophageal squamous cell carcinoma tissues was positively related to MDM-2 protein expression.Conclusion The major isotype expressed in the esophageal cancer and epithelia is DeltaNp63,which may play an important role in the carcinogenesis,development and prognosis of the esophageal squamous cell carcinoma.TAp63 is not the major isotype expressed in the esophageal cancer and epithelia,and not related to the carcinogenesis of esophageal squamous cell carcinoma.High expression of MDM-2 protein is associated with the carcinogenesis of esophageal squamous cell cancer and related to the prognosis of esophageal squamous cell carcinoma.DeltaNp63 regulates the cell cycle and apoptosis through the pathway of MDM2 and causes the carcinogenesis of esophageal squamous cell cancer.

Expression of MDM-2 and p53 Proteins in Gastric Adendegrees Carcinoma and Its Relationship with Clinicopathologic Factors / 대한암학회지

PURPOSE: MDM-2 is an oncoprotein that inhibits p53 tumor-suppressor protein. These abnor malities have a role in tumorigenesis through inactivation of p53 function. To determine the clini copathological and prognostic value of MDM2 abnormalities in gastric adendegrees Carcinoma, MDM-2& p53 protein expression were analysed in surgically resected materials of gastric adendegrees Carcinoma. MATERIALS AND METHODS: Fifty cases which had got follow-up after surgical resection were immunohistdegrees Chemically studied with p53 and MDM-2 antibodies. We defined variable clinico pathologic factors for expression of p53 and MDM-2 protein and analysed their relationships. RESULTS: Immunohistdegrees Chemical stain revealed expression of MDM-2 protein as a 52.0% (26/50) and p53 protein 20.0% (10/50), respectively. But their expressions were not assdegrees Ciated with clinicopathological factors such as T-factor, N-factor, stage, histology and differentiation. Overall, p53-negative patients seemed to have a better prognosis regardless of MDM-2 protein status (P= 0.057). MDM-2 protein status was considered to have no play as a prognostic factor. CONCLUSION: In the gastric adendegrees Carcinoma, p53 protein expression seemed to have a inverse relationship with clinical outcomes but MDM-2 protein expression, which was observed more frequently than those of p53, seemed not to be prognostic indicator.