Simultaneous Treatment with P53 Overexpression and Interferon γ Exerts a Dramatic Increase in Apoptosis Induction of U87 Cells

Background: Gliomas possess low immunogenicity, which is an inevitable hinder in front of cancer immunotherapy. Different interferons (IFNs) may proceed apoptosis instead in p53-dependent or independent pathways. P53 induces the anti-inflammatory programmed cell death in cancer cells; on the other hand, IFN gamma (IFNγ) is a modulatory/pro-inflammatory cytokine. There are contradictory reports of whether this cytokine can possess an anti- or pro-cancerous impact on tumors. Hence, we aimed to investigate the possible cooperative apoptotic effect of the P53 and IFNγ over expressions on the U87 glioblastoma cell line. Materials and Methods: The P53 expressing vector was amplified by Escherichia coli BL21. This vector was confirmed by the aid of sequencing. At the next step, U87 cells were transfected using lipofectamine. Cells were treated with P53 vector and/or IFNγ. The type of cellular death investigated by flow cytometry and the expression level of cleaved caspase-3 protein was also precisely demonstrated by western blotting. Results: Sequencing results revealed that inserted P53 was identical with human P53. Western blot results revealed that both IFNγ and P53 overexpression could up-regulate cleaved caspase-3 protein expression in this cell line. Interestingly, flow cytometry data determined that concurrent treatment with P53 exogenous overexpression and IFNγ induces about 70% apoptosis in U87; more than the sum of cell death occurs after IFNγ or P53 overexpression alone (~18%+21%=39%). Conclusion: The present study results showed that p53-overexpression and IFNγ could ultimately induce up-regulation of the caspase-3 and ultimately significant apoptosis increasing in the U87 cell line. Although IFNγ is believed to be a pro-inflammatory cytokine and P53 is an anti-inflammatory agent, our results demonstrated that they could act synergistically to induce apoptosis in U87 cells. [GMJ.2021;10:e2270]

p53 Inhibition Protects against Neuronal Ischemia/Reperfusion Injury by the p53/PRAS40/mTOR Pathway

The underlying mechanisms of cerebral ischemia/reperfusion (I/R) injury are unclear. Within this study, we aimed to explore whether p53 inhibition exerts protective effects via the p53/PRAS40/mTOR pathway after stroke and its potential mechanism. Both an in vitro oxygen-glucose deprivation (OGD) model with a primary neuronal culture and in vivo stroke models (dMCAO or MCAO) were used. We found that the infarction size, neuronal apoptosis, and autophagy were less severe in p53 KO mice and p53 KO neurons after cerebral I/R or OGD/R injury. By activating the mTOR pathway, p53 knockdown alleviated cerebral I/R injury both in vitro and in vivo. When PRAS40 was knocked out, the regulatory effects of p53 overexpression or knockdown against stroke disappeared. PRAS40 knockdown could inhibit the activities of the mTOR pathway; moreover, neuronal autophagy and apoptosis were exacerbated by PRAS40 knockdown. To sum up, in this study, we showed p53 inhibition protects against neuronal I/R injury after stroke via the p53/PRAS40/mTOR pathway, which is a novel and pivotal cerebral ischemic injury signaling pathway. The induction of neuronal autophagy and apoptosis by the p53/PRAS40/mTOR pathway may be the potential mechanism of this protective effect.

Insight into the Protective Effect of Salidroside against H2O2-Induced Injury in H9C2 Cells

Salidroside is the important active ingredient of Rhodiola species, which shows a wide range of pharmacological activities such as antioxidative stress, anti-inflammation, and antiliver fibrosis. In this paper, we aimed to study the protective effect and mechanism of salidroside against H2O2-induced oxidative damage in H9C2 cells by determining cell proliferation rate, intracellular reactive oxygen species (ROS) level, antioxidant enzyme activities, and the expression of apoptosis-related proteins. The results showed that salidroside significantly alleviated cell growth inhibition induced by H2O2 treatment in H9C2 cells, decreased the levels of intracellular ROS and malondialdehyde (MDA), and increased the activity of superoxide dismutase (SOD) and catalase (CAT); meanwhile, salidroside upregulated the expression of Bcl-2 while downregulated the expression of Bax, p53, and caspase-3 in H2O2-treated H9C2 cells. Furthermore, the antiapoptotic effect of salidroside was almost eliminated by the knockdown of Bcl-2. In the further exploration, the Bcl-2 expression was decreased by the p53 overexpression and increased by p53 knockdown in H2O2-treated H9C2 cells. Consequently, salidroside could protect H9C2 cells against H2O2-induced oxidative damage, and the underlying mechanism may be related to scavenging intracellular ROS, increasing the activities of intracellular antioxidant enzymes and inhibiting the expression of apoptosis-related proteins.

A rare case of genital porokeratosis clinically misdiagnosed as lichen planus

Introduction/Objective Porokeratosis is a clonal disorder of keratinization characterized by atrophic patches surrounded by a histologically distinctive hyperkeratotic ridge-like border called the cornoid lamella. Porokeratosis occurring on the genital area is considered a rare entity and much more unusual when in isolated form Methods/Case Report This is a case of a 54-year old man who presented with a penile lesion that has been present for 10 years, unchanged, non-bleeding and non-pruritic. There was no history of genital ulcer or inguinal swelling, neither any history of sexual promiscuity. The patient has a history of basal cell carcinoma on the right nasal area managed with Mohs surgery and scalp actinic keratoses treated with liquid nitrogen. On examination, there was a solitary 6-mm red linear macule with collarette of white scale on the corona of the penis. There was no similar lesion seen on other body parts. His blood investigations, including venereal disease research laboratory test (VDRL) and HIV screening, were negative. The initial clinical diagnosis was lichen planus, and triamcinolone was administered for three months with minimal benefit. This failed steroid management prompted a shave biopsy Results (if a Case Study enter NA) Histologic sections showed a column of hyperkeratosis arising from the epidermis (cornoid lamella) with absent granular layer and dyskeratotic cells beneath the cornoid lamella, confirming the diagnosis of porokeratosis limited to the genitalia Conclusion Porokeratosis is considered a pre-malignant condition based on clonal proliferation, dyskeratotic cells, abnormal keratinocyte maturation and p53 overexpression. Malignant progression to Bowen’s disease and squamous cell carcinoma has been documented in 6.9 to 30% of cases.

T2-FLAIR mismatch sign for diagnosis of 1p19q non-codeleted or ATRX mutant astrocytoma

Aims The World Health Organisation (WHO) classification of adult gliomas has undergone significant revision in recent years, with current emphasis on the role of the molecular biomarkers IDH, 1p19q, ATRX, and p53 for classification of glioblastoma, astrocytoma, and oligodendroglioma. When correctly applied the T2-FLAIR mismatch sign is reported to have 100% specificity for WHO grade II or III IDH mutant 1p19q non-codeleted astrocytoma. We sought to verify this classic imaging-molecular correlate in our cohort at a single tertiary level neurosurgical referral centre in the United Kingdom. Method Data were gathered by searching the histopathology database for cases between 2014 and 2019 containing the keywords ‘IDH Mutant’ AND ‘Astrocytoma’ or ‘Glioblastoma’ or ‘Oligodendroglioma’ in the report. Inclusion criteria: Biopsy/resection proven IDH mutant tumours in adults (age >17). A strict application of the T2-FLAIR mismatch sign was used when evaluating MRI. Native T2 signal was required to be homogenous or near homogenous, with hypointense signal on T2 weighted FLAIR except for a hyperintense peripheral rim. In addition, the T2-FLAIR mismatch sign was not applied to tumours showing any unequivocal contrast enhancement or macrocystic change. Results 66/185 cases were excluded for reasons of insufficient imaging, duplication, 1p19q partial deletion/unknown + ATRX wild type/unknown, IDH wild type/negative, Grade IV histology. 119 cases fit the inclusion criteria, all IDH positive. Group 1 comprised 49 (39%) 1p19q codeleted tumours, or oligodendrogliomas. ATRX was wild type (78%), unknown (18%), or mutated (<1%). Group 2 comprised 37 (29%) 1p19q non-codeleted tumours, or astrocytomas. ATRX was mutated (70%), unknown (22%), wild type (5%), or equivocal (3%). Group 3 comprised 41 (32%) 1p19q unknown tumours, all ATRX mutated, p53 expressed (83%). When p53 status was unaltered/equivocal, microscopy was convincingly astrocytic. Groups 2 and 3 comprised the astrocytomas (61%). T2-FLAIR mismatch was positive in 5 1p19q non-codeleted astrocytomas, 5 1p19q unknown ATRX mutant astrocytomas, and no 1p19q co-deleted oligodendrogliomas. Test sensitivity and specificity was 14% and 100% for 1p19q non-codeletion, 13% and 100% for ATRX mutation. Conclusion Although relatively uncommon, when present and correctly applied we confirm 100% specificity of the T2-FLAIR mismatch sign for IDH mutant 1p19q non-codeleted astrocytoma. However, if 1p19q status is unknown, clear astrocytic histology and ATRX mutation and/or p53 overexpression is also considered sufficient to diagnose astrocytoma. When 1p19q status is unavailable we also report 100% specificity of T2-FLAIR mismatch for ATRX mutated astrocytomas. T2-FLAIR mismatch was not observed in any 1p19q codeleted oligodendrogliomas or ATRX wild type tumours. More accurate methods of non-invasive glioma diagnosis will help improve neurohistopathological correlation, prognostication, and guide the tempo of the pre-operative planning phase.

Gain-of-Function Mutant TP53 R248Q Overexpressed in Epithelial Ovarian Carcinoma Alters AKT-Dependent Regulation of Intercellular Trafficking in Responses to EGFR/MDM2 Inhibitor

As the most common gene mutation found in cancers, p53 mutations are detected in up to 96% of high-grade serous ovarian carcinoma (HGSOC). Meanwhile, mutant p53 overexpression is known to drive oncogenic phenotypes in cancer patients and to sustain the activation of EGFR signaling. Previously, we have demonstrated that the combined inhibition of EGFR and MDM2-p53 pathways, by gefitinib and JNJ-26854165, exerts a strong synergistic lethal effect on HGSOC cells. In this study, we investigated whether the gain-of-function p53 mutation (p53R248Q) overexpression could affect EGFR-related signaling and the corresponding drug inhibition outcome in HGSOC. The targeted inhibition responses of gefitinib and JNJ-26854165, in p53R248Q-overexpressing cells, were extensively evaluated. We found that the phosphorylation of AKT increased when p53R248Q was transiently overexpressed. Immunocytochemistry analysis further showed that upon p53R248Q overexpression, several AKT-related regulatory proteins translocated in unique intracellular patterns. Subsequent analysis revealed that, under the combined inhibition of gefitinib and JNJ-26854165, the cytonuclear trafficking of EGFR and MDM2 was disrupted. Next, we analyzed the gefitinib and JNJ-26854165 responses and found differential sensitivity to the single- or combined-drug inhibitions in p53R248Q-overexpressing cells. Our findings suggested that the R248Q mutation of p53 in HGSOC caused significant changes in signaling protein function and trafficking, under EGFR/MDM2-targeted inhibition. Such knowledge could help to advance our understanding of the role of mutant p53 in ovarian carcinoma and to improve the prognosis of patients receiving EGFR/MDM2-targeted therapies.

TERTp mutations and p53 expression in head and neck cutaneous basal cell carcinomas with different aggressive features

Cutaneous basal cell carcinoma (cBCC) is an economic burden to health services, due to its great morbidity and increasing incidence in old people. Infiltrative cBCCs and cBCCs with micronodular pattern are considered as more aggressive. The role of p53 expression and TERTp mutation on cBCC behavior remains to be clarified. We aimed to assess TERTp mutations and p53 expression in relation to the cBCC histological subtype in a cohort of patients referred to an ENT Department of a tertiary Hospital of Northern Portugal. We performed a retrospective clinicopathological and histological review of the head and neck cBCCs followed-up at the otorhinolaryngology department of Trás-os-Montes e Alto Douro hospital (January 2007–June 2018). We assessed TERTp mutations in 142 cBCCs and p53 protein expression, through immunohistochemistry, in 157 cBCCs. We detected TERTp mutations in 43.7% of cBCCs and p53 overexpression in 60.5% of cBCCs. We spotted association of p53 overexpression and TERTp mutation with necrosis. In the infitrative-growth pattern cBCCs, there was no significant association with the clinical and histological features evaluated, except for necrosis. In the indolent-growth cBCCs, we identified a significant association of TERTp mutation status with female sex, necrosis, multiple cBCCs, and p53 positive expression. Our results suggest that TERTp mutation may be useful to identify more aggressive features in the indolent-growth pattern cBCCs (nodular and superficial subtypes). Further studies with larger cohorts are warranted to clarify the relevance of TERTp mutation in cBCCs.

Clinical Features and Molecular Markers on Diffuse Midline Gliomas With H3K27M Mutations: A 43 Cases Retrospective Cohort Study

PurposeDiffuse midline gliomas (DMG) with H3K27M mutations have been identified as a rare distinctive entity with unique genetic features, varied molecular alterations, and poor prognosis. The current study aimed to evaluate the clinical characteristics and profile of molecular markers on patients with a DMG harboring H3K27M mutations, and explore the impact of this genetic makeup on overall survival.MethodsWe retrospectively analyzed 43 consecutive patients diagnosed with a DMG harboring H3K27M mutations (age range 3 to 75 years) and treated in a tertiary institution within China between January 2017 to December 2019. Various clinical and molecular factors were evaluated to assess their prognostic value in this unique patient cohort.ResultsThe median overall survival (OS) was 12.83 months. Preoperative Karnofsky Performance Score (KPS) and adjuvant radiotherapy were found to be independent clinical parameters influencing the OS by multivariate analysis (p = 0.027 and p < 0.001 respectively). Whereas extent of tumor resection failed to demonstrate statistical significance. For molecular markers, P53 overexpression was identified as a negative prognostic factor for overall survival by multivariate analysis (p = 0.030).ConclusionLow preoperative KPS, absence of radiotherapy and P53 overexpression were identified as predictors of a dismal overall survival in patients with DMG and H3K27M mutations.

LncRNA STK4 antisense RNA 1(STK4-AS1) affects the osteosarcoma cell cycle by regulating p53 protein

Background: LncRNA STK4-AS1 has been identified as a potential biomarker associated with multiple cancers. We proposed that STK4-AS1 plays a role in the proliferation of osteosarcoma by regulating its cell cycle. Methods: We compared the expression of STK4-AS1 in osteosarcoma vs normal samples both in clinical tissues and cell lines. We overexpressed and knockdown STK4-AS1 in p53 expressing osteosarcoma cells U2OS and p53 muted expressing osteosarcoma cells MG63 and analyzed cell viability and cell cycle. We overexpressed p53 in STK4-AS1 knockdown cells to explore the association of STK4-AS1 and p53 in the cell cycle. Results: The STK4-AS1 expression was higher in osteosarcoma tissue than adjacent normal bone tissues and was higher in osteosarcoma cell lines (U2OS, MG63, and SAOS-2) than in osteoblast cell lines (hFOB and HOB). Knockdown of STK4-AS1 in U2OS decreased the cell viability, increased cells in the G0/G1 phase, decreased cells in the S and G2/M phase, decreased expression of cyclin A and B, increased p53 and p21, and had no effect on cyclin D and cyclin E, while overexpression did the opposes. MG63 cell viability was not affected by altered STK4-AS1 levels. P53 overexpression in STK4-AS1 knockdown cells recovered cell viability, p21, cyclin A, and cyclin B expression. Conclusion: LncRNA STK4-AS1 affected p53 expressing osteosarcoma cells U2OS cell viability through regulating cell cycle, which is mediated by p53/p21 pathway.

Impact of p53 and BCL2 Expression on Prognosis in Patients with Acute Myeloid Leukaemia

Introduction Acute myeloid leukaemia (AML) is a heterogenous disorder that arises from clonal expansion of malignant hematopoietic precursor cells. The tumor suppressor p53 gene plays an important role in regulation of the cell cycle and apoptosis. Somatic mutations of the p53 gene have been reported in 5-10% of all patients with AML, although the rate is higher in therapy-related disease and elderly patients. Alteration or loss of p53 is one of the most powerful independent indicators of poor outcome. BCL2 positivity in AML and its impact on prognosis is less well described. Methods This is a single-centre, retrospective analysis of AML patients treated over a ten year period (2006-2016). Patients were identified through our pathology database. P53 expression (as a surrogate marker for tp53 mutation) and BCL2 expression were analysed by immunohistochemical (IHC) analysis of marrow biopsies (greater than 30% was considered positive). We analyzed AML presentation, management and overall survival (OS), and correlated these with presence of p53 mutation and BCL2 expression. Results We identified 48 patients; the majority were elderly (median age 68.5 years). Thirty-four patients (71%) haddenovopresentation of AML, 6 had underlying Myelodysplastic Syndrome (13%), 4(8%) progressed to AML from underlying Myelofibrosis, and 8% had therapy-associated AML. Patient and disease characteristics are outlined inTable 1. Median blast count by IHC of bone marrow biopsies was 70%. The results of cytogenetic analysis were available for 38 patients (79%). Fourteen patients (37%) had normal cytogenetics, but a wide range of genetic abnormalities were identified(Table 1). NPM1 (for 22 patients) and FLT3 mutation analysis (for 33 patients) were available, and reported as positive in 4(18%) and 5(15%) respectively. p53 overexpression was identified in 6 patients (12.5%). 50% of these had secondary AML, and the remainder weredenovopresentations. Four of these patients (66%) had complex cytogenetics, one had deletion 5q and cytogenetic analysis was not available for the remaining patient. Median age of patients who were p53 positive was 60 years, compared to 70 years in those without p53 overexpression. BCL2 expression analysis showed a median value of 60% (range 5-100%). BCL2 positivity was seen in 39 patients (81.2%); half of this cohort (15 patients) had normal cytogenetics and only five patients (16%) had abnormalities that indicated poor risk cytogenetics (complex, deletion 7q, etc.). Thirty-nine patients (83%) received active treatment and nine received supportive care only due either to frailty or patient choice. Twenty-two patients (60%) had induction chemotherapy, 7(19%) received Azacitadine, and a further 7 patients had low-dose cytarabine. Nineteen patients (51%) had relapsed/refractory disease. Five patients (13.5%) with BCL2 positive disease received Venetoclax as a single agent for relapsed or refractory disease. Median OS was 11.5 months (range 0-72); 39 patients (81%) had died at the time of analysis. 26 died from disease progression (79%), and 7(19%) of sepsis. One patient was lost to follow up. OS in those with wild-type p53 was 11.5 months, which compared favorably to 7.5 months in those with p53 mutation. BCL2 overexpression also conferred a poorer prognosis; median OS was 10 months compared to 17 months in BCL2 negative disease. Conclusion This study reflects the potential use of p53 and BCL2 in real-life practice in assessing prognosis in AML patients outside of a clinical trial setting. We confirmed the previously reported poor prognosis of p53 alterations in AML (OS 7.5 months), and the study also indicates a poorer prognosis in patients with BCL2 positivity. The widespread availability of IHC techniques for trephine biopsies and the potential for rapid turn-around times in routine clinical laboratories suggests that further studies of the prognostic impact of such gene alterations are warranted. Disclosures No relevant conflicts of interest to declare.