Oroxylin A (OA), a natural compound extracted from Scutellaria baicalensis, demonstrates preventive potential against ultraviolet B (UVB)-induced non-melanoma skin cancer (NMSC), the most prevalent cancer worldwide with increasing incidence. Utilizing SKH-1 hairless mice exposed to UVB, this study showed that OA delayed NMSC onset and alleviated acute skin damage. Mechanistic investigations revealed its dual action: inhibiting inflammation and enhancing nucleotide excision repair (NER) by stabilizing XPA, a crucial deoxyribonucleic acid (DNA) repair protein. This stabilization occurred through OA's interaction with glucose-regulated protein 94 (GRP94), which disrupted murine double minute 2 (MDM2)-mediated XPA ubiquitination and proteasomal degradation. By maintaining XPA levels, OA expedited photoproduct clearance and diminished genomic instability, ultimately impeding NMSC development. These findings suggest OA as a promising chemopreventive agent targeting the GRP94/MDM2-XPA axis to counteract UVB-induced carcinogenesis.
Animals ; Ultraviolet Rays/adverse effects* ; Skin Neoplasms/prevention & control* ; Flavonoids/pharmacology* ; Mice ; Xeroderma Pigmentosum Group A Protein/genetics* ; Humans ; Proto-Oncogene Proteins c-mdm2/genetics* ; DNA Repair/drug effects* ; Scutellaria baicalensis/chemistry* ; Mice, Hairless ; Skin/radiation effects*

OBJECTIVE: To investigate the value of using MDM2 amplification probe and DDIT3 dual-color, break-apart rearrangement probe fluorescence in situ hybridization (FISH) technique in the diagnosis of liposarcoma. METHODS: In the study, 62 cases of liposarcoma diagnosed in Peking University First Hospital from January 2015 to December 2019 were analysed for clinicopathological information. Of these 62 cases of liposarcoma, all were analysed for MDM2 amplification and 48 cases were analysed for DDIT3 rearrangement using a FISH technique. Our study aimed to evaluate the status of MDM2 and DDIT3 by FISH in liposarcoma and correlate it with diagnosis of different subtypes of liposarcoma. The subtypes of liposarcoma were classified according to the FISH results, combined with the relevant clinicopathological features. RESULTS: The patients aged 31-89 years (mean: 59 years) with a 1.75:1 male to female ratio. Histologically, there were 20 cases of atypical lipomatous tumour/well-differentiated liposarcoma (ALT/WDLPS), 26 cases of dedifferentiated liposarcoma (DDLPS), 13 myxoid liposarcoma (MLPS) and 3 pleomorphic liposarcoma (PLPS). Tumors with DDLPS (23/26) and WDLPS (8/20) were localized retroperitoneally, while both tumours of MLPS and PLPS were localized extra-retroperitoneally, and the difference of sites among the four subtypes of liposarcoma was statistically significant (P < 0.05). Histologically, varied mucoid matrix could be observed in the four subtypes of liposarcoma, and the difference was statistically significant (P < 0.05). MDM2 gene amplification was demonstrated in all cases of ALT/WDLPS and DDLPS (100%, 20/20 and 26/26 respectively); DDIT3 gene rearrangement was noted only in MLPS (100%, 13/13); most cases of DDLPS (96.2%, 25/26) and ALT/WDLPS (83.3%, 5/6, 6 cases selected for detection) demonstrated the picture of amplification of the DDIT3 telomeric tag. According to the instructions of DDIT3 break-apart rearrangement probe, the 5' telomere probe and 3' centromere probe spanned but did not cover the DDIT3 gene itself, on the contrary, the 5' telomere probe covered the CDK4 gene, while the DDIT3 and CDK4 gene were located adjacent to each other on chromosome, therefore, when the amplification signal appeared on the telomeric tag of the DDIT3 rearrangement probe, it indeed indicated the CDK4 gene amplification rather than the DDIT3 gene rearrangement. Then the 10 cases with DDIT3 telomeric tag amplification were selected for CDK4 and DDIT3 gene amplification probe FISH tests, and all the cases showed CDK4 gene amplification (100%, 10/10) and two of the 10 cases demonstrated co-amplification of CDK4 and DDIT3 (20%, 2/10); DDIT3 polysomy detected by DDIT3 gene rearrangement probe was found in 1 case of DDLPS and 2 cases of PLPS (66.7%, 2/3) with morphology of high-grade malignant tumour and poor prognosis. CONCLUSION Our results indicate that a diagnosis of different subtype liposarcoma could be confirmed based on the application of MDM2 and DDIT3 FISH, combined with clinicopathological findings. It is also noteworthy that atypical signals should be correctly interpreted to guide correct treatment of liposarcomas.
Male ; Female ; Humans ; In Situ Hybridization, Fluorescence/methods* ; Cyclin-Dependent Kinase 4/metabolism* ; Liposarcoma/pathology* ; Lipoma/pathology* ; Gene Amplification ; Transcription Factor CHOP/genetics* ; Proto-Oncogene Proteins c-mdm2/metabolism*

Objective: To investigate the clinicopathological features and differential diagnoses of paratesticular liposarcoma. Methods: The cases were collected from 2012-2020, from the archives of the Department of Pathology, Peking University Third Hospital, with diagnosis confirmed by histology, immunostaining and FISH tests. Results: Totally 19 patients were enrolled (including 11 in-hospital patients and 8 consultant cases). The patients aged 37-84 years (mean 57 years). The preoperative clinical diagnoses were spermatic cord/inguinal masses (nine patients), scrotal masses (seven patients), and inguinal hernia (three patients). Six lesions recurred after local resection, including one case extending from pelvic liposarcoma. Histologically, there were 10 cases of well-differentiated liposarcoma (WDLPS) and nine cases of dedifferentiated liposarcoma (DDLPS). WDLPSs mostly showed the combined features of lipoma-like, inflammatory and sclerosing subtypes (six patients); the other four WDLPSs had pure lipoma-like subtype features. DDLPSs were low-grade (three patients) or high-grade (six patients), with the morphology resembling myxofibrosarcoma, inflammatory myofibroblastoma, spindle cell sarcoma, pleomorphic undifferentiated sarcoma and pleomorphic liposarcoma. Intense inflammatory cells infiltration was commonly observed in five WDLPSs and two DDLPSs. Ossification was observed in three tumors. Immunohistochemically, the tumors were positive for MDM2 (8/10) and CDK4 (10/10), which were expressed in lipo-differentiating cells, spindle cells in WDLPS, and in dediffferentiated components. S-100 was only expressed by lipocytes (10/10). CD34 expression was positive and diffuse in the stromal cells of WDLPSs and focal or diffuse in dedifferentiated areas (10/10). FISH tests with an MDM2 gene probe were positive (12/12). Conclusions: Paratesticular liposarcoma may be overlooked by both clinicians and pathologists. WDLPS and DDLPS predominate, showing various histologic divergences. The presence of amplification of the 12q14-q15 region (containing the MDM2 and CDK4 genes) is helpful for making the correct diagnosis.
Adult ; Genital Neoplasms, Male/surgery* ; Humans ; In Situ Hybridization, Fluorescence ; Liposarcoma/surgery* ; Male ; Proto-Oncogene Proteins c-mdm2/genetics* ; Soft Tissue Neoplasms

Objective: To investigate the value of MDM2 RNA in situ hybridization (RNA-ISH) in diagnosing atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WDL) and dedifferentiated liposarcoma (DDL). Methods: A total of 26 ALT/WDL/DDLs diagnosed from March 2017 to May 2019 in West China Hospital, Sichuan University, Chengdu, China and 18 control cases were included. MDM2 RNA-ISH was performed on all samples and compared with the fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) regarding their performance in detecting MDM2. Results: All samples were detected successfully using the three methods. Among 26 ALT/WDL/DDLs, all cases showed MDM2 amplification and positivity for MDM2 RNA-ISH (26/26, 100%). Twenty-four (24/26, 92.3%) of the 26 tested cases were positive for MDM2 IHC while two of them were negative. Eighteen control cases were all negative for MDM2 FISH and RNA-ISH, and 15 (15/18) cases were negative for MDM2 IHC. The sensitivity and specificity of RNA-ISH were both 100%, and those of MDM2 IHC were 92.3% and 83.3%, respectively. Diffuse staining was identified in all MDM2 RNA-ISH positive ALT/WDL/DDLs, but identified in only 8/24 (33.3%) of the MDM2 IHC positive cases. Among the 11 ALT/WDL/DDL samples evaluated on tissue microarray, the positive rate of MDM2 RNA-ISH was 100% with diffuse staining in all cases. The positive rate of MDM2 IHC was 9/11 while only 1 of the 9 cases showed diffuse staining. The result of MDM2 RNA-ISH was identical to that of MDM2 FISH and was overall consistent with that of MDM2 IHC (Kappa=0.763, P<0.001). Conclusions: In ALT/WDL/DDLs, results of MDM2 RNA-ISH are highly consistent with those of FISH. MDM2 RNA-ISH is more sensitive and more specific and has more diffuse positive signals than the IHC. The findings indicate that MDM2 RNA-ISH is highly valuable for the diagnosis and differential diagnosis of ALT/WDL/DDLs.
Biomarkers, Tumor/genetics* ; Gene Amplification ; Humans ; In Situ Hybridization, Fluorescence ; Liposarcoma/genetics* ; Proto-Oncogene Proteins c-mdm2/genetics* ; RNA

Objective: To investigate the clinicopathologic and molecular genetic characteristics of myxoid pleomorphic liposarcoma (MPLPS). Methods: Six cases of MPLPS diagnosed and consulted in Fujian Provincial Hospital from 2015 to 2021 were collected for histomorphological observation, immunohistochemistry, and fluorescence in situ hybridization (FISH) detection of DDIT3 (CHOP) gene translocation and MDM2/CDK4 gene amplification. Results: There were four males and two females, aged 26-74 years (mean 53.8 years). The tumor size was 3.8-16.0 cm (mean 11.8 cm). All six cases had similar histopathologic features, showing overlapping histologic morphology of myxoid liposarcoma and pleomorphic liposarcoma. Four cases (4/6) were positive for S-100 protein, and the Ki-67 index was 50%-95%. All cases (6/6) were negative for DDIT3 (CHOP) translocation and MDM2/CDK4 amplification by FISH. TP53 (p.R248w) germline mutation was found in one case. Conclusions: MPLPS is a rare subtype of liposarcoma, characterized by overlapping morphology of myxoid liposarcoma and pleomorphic liposarcoma. Genetically, a few of them have TP53 gene germline mutations, but they lack of DDIT3 (CHOP) translocation or MDM2/CDK4 amplification.
Female ; Humans ; Immunohistochemistry ; In Situ Hybridization, Fluorescence ; Liposarcoma/pathology* ; Liposarcoma, Myxoid/diagnosis* ; Male ; Molecular Biology ; Proto-Oncogene Proteins c-mdm2/genetics* ; Translocation, Genetic

OBJECTIVETo study the relationship and clinicopathological significance of Numb,MDM2 and p53 expression in human pancreatic cancer.

METHODSThe expression of Numb,MDM2 and p53 proteins in 65 cases of paired paraffin embedded pancreatic ductal adenocarcinoma (PDAC) specimens and adjacent non-cancerous pancreas was detected by immunohistochemistry (IHC). The relationship among their expression and clinicopathological characters was analyzed.Westem blot was used to examine their expression in 16 paired fresh PDAC specimens and adjacent non-cancerous pancreatic tissues. Meanwhile,Numb expression in Capan-2, PANC-1 and AsPC-1 pancreatic cancer cells with different differentiation were detected by immunofluorescence (IF) , Westem blot and quantitative real-time (qRT) -PCR, respectively. Paired sample t-test, χ(2) test, Kaplan-Meier and Cox regression were used to analyze the results of our experiments, respectively.

RESULTSIHC showed that there was no differential expression of Numb in PDAC and adjacent pancreas (t = 1.746, P = 0.086) , while the expression of MDM2 and p53 was significantly increased in PDAC, compared to that in paired normal pancreas (t = 3.294, P = 0.002; t = 3.152, P = 0.002, respectively) .Numb expression was negatively associated with tumor size (χ² = 5.206, P = 0.023), differentiation (χ² = 7.802, P = 0.005) and UICC stage (χ² = 4.770, P = 0.029), while expression of MDM2 and p53 was positively associated with tumor T and TNM stage, respectively (χ² = 5.182, P = 0.023; χ² = 6.448, P = 0.011) . Correlation analysis showed a negative association between Numb and MDM2 (r = -0.283, P = 0.023) , but there was no relationship of them with p53 (P > 0.05) .Univariate and multivariate analysis revealed that Numb was a protective prognostic indicator for patients with PDAC (χ² = 5.408, P = 0.020). Moreover, patients with Numb positive and MDM2 negative expression had a significantly better overall survival (χ² = 5.868, P = 0.015). Western blot showed that Numb expression was much higher in well differentiated PDAC than that in paired normal pancreas (t = 1.092, P = 0.020) , while the expression of MDM2 and p53 was significantly increased in 16 cases of PDAC (t = 3.263, P = 0.005; t = 3.607, P = 0.003, respectively). Numb expression was gradually increased in pancreatic cancer cells with the increasing degree of cell differentiation detected by IF, Westem blot and qRT-PCR.

CONCLUSIONSNumb acts as a tumor suppressor gene in the development of PDAC. Numb, MDM2 and p53 might coordinately participate in the development of PDAC.

Carcinoma, Pancreatic Ductal ; genetics ; Humans ; Immunohistochemistry ; Kaplan-Meier Estimate ; Membrane Proteins ; metabolism ; Neoplasm Staging ; Nerve Tissue Proteins ; metabolism ; Pancreas ; metabolism ; Pancreatic Neoplasms ; genetics ; Prognosis ; Proto-Oncogene Proteins c-mdm2 ; metabolism ; Tumor Suppressor Protein p53 ; metabolism

OBJECTIVETo study the role and possible mechanism of glioma-associated oncogene-1 (Gli1) in regulating the cell invasion and migration of pancreatic cancer cells.

METHODSQuantitative real-time (qRT) -PCR was used to detect the effect of siRNA interference on Gli1, murine double minute 2 (MDM2) and p53 genes. Cell invasion and migration assays were used to observe the effect of Gli1, MDM2 and p53 silence on cell invasion and migration in p53 wild-type Capan-2 pancreatic cancer cells, respectively. Meanwhile, immunoblotting (IB) was used to detect the protein level of matrix metalloproteinase (MMP) -9, phospho-excelluar signal-regulated kinase (pERK) and phosphorylation protein kinase B (pAKT) in Gli1-silencing Capan-2 cells. The data were analyzed by paired t-test.

RESULTSqRT-PCR showed that the expression of Gli1, MDM2 and p53 is down-regulated 70.5% and 74.5%, 61.8% and 65.3%, and 73.8% and 78.2% after siRNA interference, compared with the mock and siRNA control groups, respectively. Cell invasion (94 ± 8) and migration (143 ± 8) in p53 wild-type Capan-2 cells transfected with Gli1siRNA were significantly decreased, compared with the siRNA control group (150 ± 7, 190 ± 10) (t = 6.584, P = 0.022; t = 8.266, P = 0.014) , while MDM2 silence inhibited cell invasion (experiment group:85 ± 12, control group: 138 ± 6) and migration (experiment group: 127 ± 9, control group:180 ± 10) in the same cells, respectively (t = 5.097, P = 0.036;t = 4.860, P = 0.040). However, cell invasion (experiment group: 153 ± 11, control group: 106 ± 7) and migration (experiment group: 209 ± 13, control group: 164 ± 8) in p53-silencing Capan-2 cells were significantly enhanced (t = 4.669, P = 0.043; t = 4.990, P = 0.038). IB showed that Gli1 silence down-regulated MMP-9 but not pERK and pAKT protein expression.

CONCLUSIONGli1 might contribute to the cell invasion and migration in pancreatic cancer via the regulation of MDM2, p53 and MMP-9 expression.

Animals ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Matrix Metalloproteinase 9 ; metabolism ; Mice ; Neoplasm Invasiveness ; Oncogene Proteins ; genetics ; metabolism ; Pancreas ; metabolism ; Pancreatic Neoplasms ; metabolism ; pathology ; Proto-Oncogene Proteins c-akt ; metabolism ; Proto-Oncogene Proteins c-mdm2 ; metabolism ; RNA, Small Interfering ; genetics ; Trans-Activators ; genetics ; metabolism ; Transfection ; Tumor Suppressor Protein p53 ; metabolism ; Zinc Finger Protein GLI1

Mdm2 and Mdm4 are two key negative regulators of the tumor suppressor p53. Deletion of either Mdm2 or Mdm4 induces p53-dependent early embryonic lethality in knockout mouse models. The tissue-specific deletion of Mdm2 induces p53-dependent apoptosis, whereas the deletion of Mdm4 induces both p53-dependent apoptosis and cell cycle arrest. Compared to Mdm4 deletion, Mdm2 deletion causes more severe phenotypic defects. Disrupting the Mdm2 and Mdm4 interaction using knockin mice models causes embryonic lethality that can be completely rescued by the concomitant loss of p53, suggesting that Mdm2 and Mdm4 heterodimerization is critical to inhibit p53 activity during embryogenesis. Overexpression of Mdm2 and Mdm4 in mice induces spontaneous tumorigenesis, which clearly indicates that Mdm2 and Mdm4 are bona fide oncogenes. Studies from these mouse models strongly suggest that blocking Mdm2- and Mdm4-mediated p53 inhibition is an appealing therapeutic strategy for cancer patients with wild-type p53 alleles.
Animals ; Apoptosis ; Cell Cycle Checkpoints ; Mice ; Mice, Knockout ; Models, Animal ; Proto-Oncogene Proteins ; genetics ; metabolism ; Proto-Oncogene Proteins c-mdm2 ; genetics ; metabolism ; Tumor Suppressor Protein p53 ; antagonists & inhibitors ; genetics ; metabolism ; Ubiquitin-Protein Ligases ; genetics ; metabolism

OBJECTIVETo construct a short hairpin (sh)RNA targeting the gene encoding the MDM2 oncoprotein in order to investigate its role in human hepatocellular carcinoma (HCC) and its potential for use as a gene therapy strategy to inhibit HCC growth in vivo.

METHODSSmall interfering (si)RNAs were designed targeting the MDM2 gene (siMDM2-1 and siMDM2-2) and unrelated sequences (negative control) and cloned into the expression plasmid pGCSilencer-U6-neo-GFP. A HCC mouse model was established by subcutaneous inoculation of HepG2 cells (2 x 10(6) in 0.2 ml) into 20 nude mice. The inoculated mice were divided into four equal groups for tumor-localized injections of saline, negative control siRNA plasmid, siMDM2-1 plasmid, and siMDM2-2 plasmid. Tumor growth was observed daily (by caliper measurement) for one month, when mice were sacrificed by cervical dislocation. The tumor mass was resected for analysis of tumor inhibition rate (% = [(average tumor weight of control group - average tumor weight of treatment group) / average tumor weight of control group x 100]) and effects on MDM2 and p53 mRNA and protein expression (by reverse transcription- PCR and western blotting, both normalized to beta-actin). Significance of between-group differences was assessed by one-way ANOVA or LSD test; pairwise comparisons were made by the Chi-squared test.

RESULTSsiMDM2-1 and siMDM2-2 suppressed the xenografted tumor growth remarkably (60.6% and 54.6% inhibition rates, respectively), significantly reduced the expression ofMDM2 gene (62.8% and 61.6%) and protein (60.7% and 59.5%), and significantly increased p53 gene (47.1% and 45.6%) and protein (45.9% and 44.3%) (all, P < 0.05).

CONCLUSIONshRNA-mediated silencing of the MDM2 gene effectively inhibits HCC tumorigenesis of subcutaneously xenografted HepG2 cells in nude mice, and the mechanism may involve p53.

Animals ; Carcinoma, Hepatocellular ; genetics ; pathology ; Cell Proliferation ; Hep G2 Cells ; Humans ; Liver Neoplasms ; genetics ; pathology ; Male ; Mice ; Mice, Nude ; Plasmids ; Proto-Oncogene Proteins c-mdm2 ; genetics ; metabolism ; RNA Interference ; RNA, Messenger ; genetics ; RNA, Small Interfering ; Transfection ; Tumor Suppressor Protein p53 ; metabolism ; Xenograft Model Antitumor Assays

OBJECTIVETo investigate the combined effects between the two polymorphisms murine double minute 2 (MDM2) rs2279744 T→G and P53 rs1042522 G→C on the genetic susceptibility of breast cancer.

METHODSA total of 600 female patients with diagnosed breast cancer were consecutively recruited from the Yuhang district, Hangzhou city during March 2001 to May 2009. In the same period as the cases were collected, 600 healthy women living in Yuhang district, Hangzhou city were selected from a nutritional survey conducted. Peripheral blood lymphocytes were obtained from the study subjects and the demographic information were collected through questionnaires. PCR-restriction fragment length polymorphism (PCR-RFLP) was used for genotyping MDM2 rs2279744 T→G and P53 rs1042522 G→C. Logistic regression analysis was used to analyze the combined effects of the two polymorphisms on breast cancer risk.

RESULTSThe frequency of MDM2 rs2279744 GG, TG and TT genotypes were 31.5% (189/600), 45.5% (273/600), 23.0% (138/600) in case group and 19.0% (114/600), 49.2% (295/600), 31.8% (191/600) in control group. The frequency of P53 rs1042522 GG, GC and CC genotypes were 23.1% (139/600), 50.2% (301/600), 26.7% (160/600) in case group and 30.5% (183/600), 51.3% (308/600), 18.2% (109/600) in control group. Logistic regression analysis showed that carriers with rs2279744 TG, GG genotypes had a significant increased risk for developing breast cancer compared with rs2279744 TT carriers (OR = 1.31, 95%CI: 0.97 - 1.73 for TG; OR = 2.24, 95%CI: 1.61 - 3.09 for GG). When comparing with rs1042522 GG carriers, carriers with rs1042522 GC, CC genotypes had a significant increased risk for developing breast cancer (OR = 1.34, 95%CI: 0.94 - 1.68 for GC; OR = 1.89, 95%CI: 1.35 - 2.68 for CC). The united analysis of this two polymorphisms showed that compared with individuals carrying rs2279744 TT and rs1042522 GG (the frequency were 4.8% (29/600) in case group and 11.5% (69/600) in control group), carries with rs2279744 TG/GG and rs1042522 GC/GG genotypes (the frequency were 95.2% (571/600) in case group and 88.5% (531/600) in control group) showed significant higher risk in the susceptibility to breast cancer (OR = 2.30, 95%CI: 1.39 - 3.82 for TG/GC + GG; OR = 2.14, 95%CI: 1.29 - 3.55 for TT + GC/CC; OR = 2.86, 95%CI: 1.80 - 4.53 for TG/GG + GC/CC). The combination of MDM2 rs2279744 T→G and P53 rs1042522 G→C contributed to a significantly higher risk of breast cancer than did any one of the variant (P = 0.046). The risk of susceptibility to breast cancer was much higher when this two polymorphisms both variant.

CONCLUSIONSThe MDM2 rs2279744 T→G and P53 rs1042522 G→C may be risk factor for breast cancer. Significant combined effects between the two polymorphisms may contribute to the genetic susceptibility to breast cancer.

Adult ; Breast Neoplasms ; genetics ; Case-Control Studies ; Female ; Genetic Predisposition to Disease ; Genotype ; Humans ; Middle Aged ; Polymorphism, Single Nucleotide ; Proto-Oncogene Proteins c-mdm2 ; genetics ; Risk Factors ; Tumor Suppressor Protein p53 ; genetics