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The prevention of tumor recurrence by the successful targeting of glioma stem cells endowed with a tumor-initiating capacity is deemed the key to the long-term survival of glioblastoma patients. Glioma stem cells are characterized by their marked therapeutic resistance; however, recent evidence suggests that they have unique vulnerabilities that may be therapeutically targeted. We investigated MDM2 expression levels in glioma stem cells and their non-stem cell counterparts and the effects of the genetic and pharmacological inhibition of MDM2 on the viability of these cells as well as downstream molecular pathways. The results obtained showed that MDM2 expression was substantially higher in glioma stem cells than in their non-stem cell counterparts and also that the inhibition of MDM2, either genetically or pharmacologically, induced a more pronounced activation of the p53 pathway and apoptotic cell death in the former than in the latter. Specifically, the inhibition of MDM2 caused a p53-dependent increase in the expression of BAX and PUMA and a decrease in the expression of survivin, both of which significantly contributed to the apoptotic death of glioma stem cells. The present study identified the MDM2–p53 axis as a novel therapeutic vulnerability, or an Achilles' heel, which is unique to glioma stem cells. Our results, which suggest that non-stem, bulk tumor cells are less sensitive to MDM2 inhibitors, may help guide the selection of glioblastoma patients suitable for MDM2 inhibitor therapy. [ABSTRACT FROM AUTHOR]

The p53 protein has appropriately been named the "guardian of the genome". In almost all human cancers, the powerful tumor suppressor function of p53 is compromised by a variety of mechanisms, including mutations with either loss of function or gain of function and inhibition by its negative regulators MDM2 and/or MDMX. We review herein the progress made on different therapeutic strategies for targeting p53. [ABSTRACT FROM AUTHOR]

Introduction: Glioma is the most common malignant brain tumor. TP53 is the most common mutant gene in human cancer. Wild-type p53 (wtp53) is a tumor suppressor protein whereas mutant p53 (mutp53) is an oncoprotein that promotes tumor cell proliferation. Our aim was to examine the inhibitory effects of berberine on the proliferation of human glioma cells via regulation of wtp53, mutp53, and their downstream molecules. Methods: We selected wtp53 cells (U87 cells) and mutp53 cells (U251 cells termed p53 R273H) to examine the inhibitory effects of berberine on human glioma cells. We used the CCK-8 kit to detect the toxic effect of berberine. Flow cytometry was used to detect the effect of berberine. Clone formation test was used to test the inhibitory effect of berberine on the proliferation of glioma cells. Western blot was used to detect the changes of related proteins such as p53, p-p53, p21 and cyclin D1. Lentivirus transduction was used to transduce wild-type p53 into U251 cells to further examine the effect of berberine. The nude mouse subcutaneous tumor model was used to detect the effect of berberine on inhibiting the proliferation of glioma cells in vivo. Results: Berberine promoted the phosphorylation of wtp53, increased the expression of p21 protein, reduced cyclin D1 content, and caused G1 phase arrest in U87 cells. Berberine also reduced mutp53 content and caused G2 phase arrest in U251 cells with a concurrent decrease in p21, cyclin D1, and cyclin B1 content. Transduction with wtp53 enhanced the effects on cell cycle arrest. Further, berberine significantly inhibited glioma growth in vivo mouse tumor model. Discussion: Glioma is a group of heterogeneous brain tumors with unique biological and clinical characteristics. Berberine can inhibit glioma cells through a variety of ways. Our research indicated that berberine inhibited the proliferation of glioma cells by interfering with wtp53 and mutp53. This indicates that berberine could be used as a potential drug to treat wild-type and mutant p53 glioma. [ABSTRACT FROM AUTHOR]

Background: Defects in modulating wild-type (wt) p53 and survivin are associated with a resistant disease in acute lymphoblastic leukemia (ALL). Yet, no wt-p53 and survivin modulating drugs have been approved for clinical application in ALL. Here, we investigated if in vitro eicosapentaenoic acid (EPA) concentrations equal to human plasma levels are able to target wt-p53 and survivin. Methods: Wt-p53 Molt-4 cells (ALL cell line) were treated with 50, 100, 150, and 200 µM of EPA after which cell number, viability, proliferation rate, survivin expression, wt-p53 accumulation, caspase-3 activation, and apoptosis were evaluated. Results: After 48- and 72-h treatments with EPA at concentrations ranging from 50 to 200 µM, cell proliferation rates were measured to be 71.5–32.6% and 68.2–13.7% and metabolic activities were measured to be 77–44% and 71–26%, respectively. Treatment with 50–200 µM of EPA for 48 h resulted in 14.1–74.6% and 69.5–45.5% decreases in survivin mRNA and protein levels, respectively. EPA induced 1.3–6 and 1.9–20-fold increases in caspase-3 activation and wt-p53 accumulation, respectively. Increase in wt-p53/survivin and caspase-3/survivin ratios from 1 in untreated cells to 20.3 and 5.8 was measured for 150 µM of EPA. Low necrotic rates ranging from 0.3% to 2.8% and an increase in the number of total apoptotic cells (early + late) ranging from 9.8% to 81% were also observed with increasing EPA concentrations. Conclusion: EPA induces strongly wt-p53 with a remarkable decrease in survivin expression, representing an attractive compound to modulate wt-p53 and survivin in ALL cells. [ABSTRACT FROM AUTHOR]

Prostate cancer is a common type of cancer in elderly men. The aim of the present study was to evaluate the effects of ultrasound exposure in combination with SonoVue microbubbles on liposome-mediated transfection of wild-type P53 genes into human prostate cancer cells. PC-3 human prostate cancer cells were exposed to ultrasound; duty cycle was controlled at 20% (2 sec on, 8 sec off) for 5 min with and without SonoVue microbubble echo-contrast agent using a digital sonifier (frequency, 21 kHz; intensity, 46 mW/cm2). The cells were divided into eight groups, as follows: Group A (SonoVue + wild-type P53), group B (ultrasound + wild-type P53), group C (SonoVue + ultrasound + wild-type P53), group D (liposome + wild-type P53), group E (liposome + SonoVue + wild-type P53), group F (liposome + wild-type P53 + ultrasound), group G (liposome + wild-type P53 + ultrasound + SonoVue) and the control group (wild-type P53). Following treatment, a hemocytometer was used to measure cell lysis, reverse transcription-quantitative polymerase chain reaction and western blotting were performed to detect P53 gene transfection efficiency, Cell Counting Kit-8 was employed to reveal cell proliferation and Annexin V/propidium iodide staining was used to determine cell apoptosis. Cell lysis was minimal in each group. Wild-type P53 gene and protein expression were significantly increased in the PC-3 cells in group G compared with the control and all other groups (P<0.01). Cell proliferation was significantly suppressed in group G compared with the control group and all other groups (P<0.01). Cell apoptosis levels in group G were significantly improved compared with the control group and all other groups (P<0.01). Thus, the results of the present study indicate that the use of low-frequency and low-energy ultrasound in combination with SonoVue microbubbles may be a potent physical method for increasing liposome gene delivery efficiency. [ABSTRACT FROM AUTHOR]

Genistein (GEN) has been previously shown to have a proapoptotic effect on cancer cells through a p53-dependent pathway, the mechanism of which remains unclear. One of its intracellular targets, APE1, protects against apoptosis under genotoxic stress and interacts with p53. In this current study, we explored the mechanism of the proapoptotic effect of GEN by examining the APE1–p53 protein–protein interaction. We initially showed that the p53 protein level was elevated in GEN-treated human non-small lung cancer A549 cells and cervical cancer HeLa cells. By examining both protein synthesis and degradation, we found that GEN enhances p53 intracellular stability by interfering with the interaction of APE1 and p53, which provided a plausible explanation for how GEN initiates apoptosis. Furthermore, we found that the interaction between APE1 and p53 is important for the degradation of p53 and is dependent on the redox domain of APE1 by utilizing the redox domain mutant APE1 C65A. Our data suggest that the degradation of wild-type p53 is blocked when the redox domain of APE1 is masked or interrupted. Based on this evidence, we hereby report a novel mechanism of p53 degradation through an APE1-mediated, redox-dependent pathway. [ABSTRACT FROM AUTHOR]

Abstract: Objective: The objective of this study is to investigate whether wild-type TP53 status in high-grade serous ovarian carcinoma is associated with poorer survival. Methods: Clinical and genomic data of 316 sequenced samples from The Cancer Genome Atlas (TCGA) ovarian high-grade serous carcinoma study were downloaded from TCGA data portal. Association between wild-type TP53 and survival was analyzed with Kaplan Meier method and Cox regression. The diagnosis of high-grade serous carcinomas was evaluated by reviewing pathological reports and high-resolution hematoxylin and eosin (H&E) images from frozen sections. The authenticity of wild-type TP53 in these tumor samples was assessed by analyzing SNP array data with ASCAT algorithm, reverse phase protein array (RPPA) data and RNAseq data. Results: Fifteen patients with high grade serous ovarian carcinomas were identified to have wild-type TP53, which had significantly shorter survival and higher chemoresistance than those with mutated TP53. The authenticity of wild-type TP53 status in these fifteen patients was supported by SNP array, RPPA, and RNAseq data. Except four cases with mixed histology, the classification as high grade serous carcinomas was supported by pathological reports and H&E images. Using RNAseq data, it was found that EDA2R gene, a direct target of wild-type TP53, was highly up-regulated in samples with wild-type TP53 in comparison to samples with either nonsense or missense TP53 mutations. Conclusion: Although patients with wild-type TP53 ovarian cancer were rare in the TCGA high grade ovarian serous carcinomas cohort, these patients appeared to have a poorer survival and were more chemoresistant than those with mutated TP53. Differentially expressed genes in these TP53 wild-type tumors may provide insight in the molecular mechanism in chemotherapy resistance. [Copyright &y& Elsevier]

Abstract: One of the best characterized resistance mechanisms of human cancer is multidrug resistance (MDR) mediated by P-glycoprotein (Pgp/MDR1) and multidrug-resistant related protein (MRP1). In addition to Pgp/MDR1 and MRP1, p53 inactivation or mutation might play a relevant role in therapeutic failure. This study involved 25 children (17 girls and 8 boys) aged 7 months to 10 years treated for unilateral Wilms’ tumor. 25 tissue samples of Wilms’ tumor and 5 samples of normal human kidneys were obtained from the Department of Pathological Anatomy, Jessenius Faculty of Medicine in Martin, Slovak Republic. We used an indirect immunohistochemical method to determine expression of Pgp/MDR1, MRP1 and wild-type p53 in 25 tissue samples of nephroblastoma. The minority of nephroblastoma specimens showed positivity for both MDR proteins, as well as for wild-type p53. 24% of tissue samples revealed positive results for Pgp/MDR1, 48% for MRP1 and 8% for wild-type p53. Furthermore, our study showed a statistically significant difference between p53 and MRP1 protein expression (p <0.01), but not between p53 and Pgp/MDR1 (p >0.05). No correlation was found between the expression of both multidrug resistance proteins (Pgp/MDR1 and MRP1) and the expression of wild-type p53. Immunohistochemical detection of the expression of MDR proteins and wild-type p53 at the time of diagnosis might assist in choosing specific chemotherapeutics to improve prognosis and therapy. [Copyright &y& Elsevier]

Abstract: The combination of gene therapy and chemotherapy may increase the therapeutic efficacy in the treatment of patients. In this work, the cationic polymer prodrug/plasmid nanocomplexes were designed to in vivo synergistically treat drug-resistant breast tumors. Cationic β-cyclodextrin-polyethylenimine-Dox (PC-Dox) conjugates were prepared for carrying wt p53 plasmid in the form of PC-Dox/p53 nanocomplexes to achieve synergistic cancer therapeutic effects of drug and gene therapies. Such PC-Dox/p53 nanocomplexes ensure that both drug and gene can be delivered to the same cancer cells. The physicochemical properties and Dox release profiles of the PC-Dox conjugates, as well as their antitumor activities in vitro and in vivo, were determined. mRNA expression and western blot experiments also proved that co-delivery of Dox with wt p53 plasmid from PC-Dox/wt p53 complexes could promote wt p53 gene expression largely. By investigating anticancer efficacy via multi-drug resistant MCF-7/Adr breast cancer cells, it was found that PC-Dox/wt p53 complexes promoted the inhibition of tumor growth in vivo and prolonged the survival time of tumor-bearing mice. With the efficient ability to co-deliver drug and gene, such multifunctional PC-Dox/pDNA complexes should have great potential applications in cancer therapy. [Copyright &y& Elsevier]

Abstract: Breast cancer resistance protein (BCRP) has been shown to confer multidrug resistance, but the mechanisms of its regulation are poorly understood. Here, we investigate the effects of wild-type and mutant p53, and nuclear factor kappa-B (NF-κB) (p50) on BCRP promoter activity in MCF-7 cells. Our results demonstrated that wild-type p53 markedly suppressed BCRP activity and enhanced the chemosensitivity of cells to mitoxantrone, whereas mutant p53 had little inhibitory effect. After inhibition of NF-κB, similar results were obtained. Following knockdown of endogenous p53, BCRP and p50 expressions were increased, and the chemosensitivity of the cells to mitoxantrone was decreased. We conclude that wild-type p53 acts as a negative regulator of BCRP gene transcription. [ABSTRACT FROM AUTHOR]

The study of the cancer secretome is gaining even more importance in cancers such as pancreatic ductal adenocarcinoma (PDAC), whose lack of recognizable symptoms and early detection assays make this type of cancer highly lethal. The wild-type p53 protein, frequently mutated in PDAC, prevents tumorigenesis by regulating a plethora of signaling pathways. The importance of the p53 tumor suppressive activity is not only primarily involved within cells to limit tumor cell proliferation but also in the extracellular space. Thus, loss of p53 has a profound impact on the secretome composition of cancer cells and marks the transition to invasiveness. Here, we demonstrate the tumor suppressive role of wild-type p53 on cancer cell secretome, showing the anti-proliferative, apoptotic and chemosensitivity effects of wild-type p53 driven conditioned medium. By using high-resolution SWATH-MS technology, we characterized the secretomes of p53-deficient and p53-expressing PDAC cells. We found a great number of secreted proteins that have known roles in cancer-related processes, 30 of which showed enhanced and 17 reduced secretion in response to p53 silencing. These results are important to advance our understanding on the link between wt-p53 and cancer microenvironment. In conclusion, this approach may detect a secreted signature specifically driven by wild-type p53 in PDAC. [ABSTRACT FROM AUTHOR]

Abstract: The tumor suppressor p53 controls a broad range of cellular responses. Induction of a transient (cell cycle arrest) or a permanent (senescence) block of cell proliferation, or the activation of cell death pathways in response to genotoxic stress comprise the major arms of the survival-death axis governed by p53. Due to these biological properties, inactivation of p53 is a crucial step in tumor development and progression, reflected by the high incidence of TP53 mutations in different types of human cancers. The remarkable potency of p53 in suppressing tumorigenic outgrowth has promoted the expectation that tumor cells expressing wild-type p53 (wtp53) should be more prone to elimination by cytotoxic treatments than tumor cells expressing mutant p53 (mutp53) with defunct wtp53 activities. However, recent findings yielded somewhat unexpected insights concerning the preponderance of the survival-promoting effects of wtp53 in cancer cells, a rather undesired property from the therapeutic point of view. In this commentary we will discuss the possibility that the developmentally established distinct patterns of wtp53 mediated responses in different tissues are an important factor in determining the ultimate outcome of cellular responses mediated by wtp53 in different types of tumor cells, with a particular focus on the divergent impact of wtp53 in malignant tumors of the central nervous system. We infer that a selective gain of pro-survival functions of wtp53 in cancer cells will confer a survival advantage that counteracts tumor therapy. [Copyright &y& Elsevier]

When normal cells come under stress, the wild-type (WT) p53 level increases resulting in the regulation of gene expression responsible for growth arrest or apoptosis. Here we show that elevated levels of WT p53 or its homologue, p73, inhibit expression of a number of cell cycle regulatory and growth promoting genes. Our analysis also identified a group of genes whose expression is differentially regulated by WT p53 and p73. We have infected p53-null H1299 human lung carcinoma cells with recombinant adenoviruses expressing WT p53, p73 or β-galactosidase, and have undertaken microarray hybridization analyses to identify genes whose expression profile is altered by p53 or p73. Quantitative real-time PCR verified the repression of E2F-5, centromere protein A and E, minichromosome maintenance proteins (MCM)-2, -3, -5, -6 and -7 and human CDC25B after p53 expression. 5-Fluorouracil treatment of colon carcinoma HCT116 cells expressing WT p53 results in a reduction of the cyclin B2 protein level suggesting that DNA damage may indeed cause repression of these genes. Transient transcriptional assays verified that WT p53 repressed promoters of a number of these genes. Interestingly, a gain-of-function p53 mutant instead upregulated a number of these promoters in transient transfection. Using promoter deletion mutants of MCM-7 we have found that WT p53-mediated repression needs a minimal promoter that contains a single E2F site and surrounding sequences. However, a single E2F site cannot be significantly repressed by WT p53. Many of the genes identified are also repressed by p21. Thus, our work shows that WT p53 and p73 repress a number of growth-related genes and that in many instances this repression may be through the induction of p21.Oncogene (2008) 27, 2583–2593; doi:10.1038/sj.onc.1210898; published online 5 November 2007 [ABSTRACT FROM AUTHOR]

Bombesin (BN) or gastrin-releasing peptide (GRP) can stimulate the growth of neoplasms such as breast cancer and small-cell lung carcinoma (SCLC). Antagonists of BN/GRP have been shown to inhibit these cancers. We evaluated whether antagonists of BN/GRP can suppress the growth of human non-SCLC (NSCLC) xenografted into nude mice. The effect of the administration of BN/GRP antagonist RC-3940-II on the growth of H460 and A549 NSCLC cell lines orthotopically xenografted into the intrapulmonary interstitium was examined. Protein levels of K-Ras, COX-2, Akt/pAkt, WT p53, Erk1/2, and lung resistance-related protein (LRP) in tumors were analyzed by Western blot analaysis, and receptors for BN/GRP were investigated by radioligand-binding studies. The effect of RC-3940-ll on the proliferation of H460 and A549 cells in vitro was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays. High-affinity receptors for BN/GRP were found on tumors. Treatment with RC-3940-Il significantly (P < 0.001) inhibited growth of H460 and A549 NSCLC xenografts by 30-50% and led to an improved performance status, compared with controls. In H460 NSCLC, the antitumor effect was associated with a significant (P < 0.001) reduction in protein levels of K-Ras, COX-2, pAkt, and pERK1/2 and with a major augmentation in the expression of WT p53, compared with controls. In A549 NSCLC, pAkt and LRP were significantly down-regulated. Our findings demonstrate the efficacy of BN/GRP antagonist RC-3940-lI for the treatment of NSCLC. The suppression of K-Ras, COX-2, pAkt, and LRP, as well as the up-regulation of WT p53 might contribute to the antitumor action of BN/GRP antagonists. [ABSTRACT FROM AUTHOR]

To clarify the role of p53 in 22 hepatitis C virus (HCV)-infected hepatocellular carcinomas (HCCs), we compared the gene expression profiles of HCCs with wild-type p53 (wt-p53) (n=17) and those with mutant-type p53 (mt-p53) (n=5) by oligonucleotide microarray analysis. Among 83 p53-related genes identified by a supervised learning method, 25 were underexpressed, and 58 were overexpressed in mt-p53 HCCs compared with wt-p53 HCCs. With a computer search, we identified consensus p53-binding sequences in the 3-kb region upstream of the translation initiation site in 59 of the 83 genes, suggesting that the in vivo p53-associated transcription system is very complicated. These data will provide additional insights into p53-related pathogenesis in HCV-infected HCC. [Copyright &y& Elsevier]

Abstract: Despite the lack of protective melanin and increased oxidative stress due to mM concentrations of epidermal H2 O2 in vitiligo, there is no significantly increased risk for chronic actinic damage and non-melanoma skin cancer. Therefore the question arises, which protective mechanisms could be involved in the skin of these patients preventing the initiation of these cancers. Recently an overexpression of p53 has been shown in vitiligo. Unfortunately there was no further characterization of this elevated p53. Employing a functional colour yeast assay, the study presented herein demonstrates for the first time the overexpression of a functioning wild-type p53 protein in both depigmented and ‘normal’ pigmented epidermis of patients with vitiligo compared with healthy controls. Surprisingly long-term narrowband UVB (311 nm) treatment does not alter this expression. Moreover, MDM-2, PCNA and p21 protein expression remain unchanged compared with healthy controls. This increased epidermal p53 in vitiligo coincides with decreased thioredoxin reductase (TR) protein levels in both depigmented and pigmented skin whereas mRNA expression is unaffected. Because TR is one transcriptional target of p53, these results support a wild-type functionality, which was further supported by the specific p53 FASAY yeast test. To our knowledge this is the first example of persistent elevated functioning wild-type p53 in humans. Based on our results we hypothesize that the low incidence for actinic damage, basal cell and squamous cell carcinoma as documented in vitiligo could well reside in a protective function of up-regulated wild-type p53. [ABSTRACT FROM AUTHOR]

Heat shock proteins (HSPs) are molecular chaperones that assist diverse cellular activities including protein folding, intracellular transportation, assembly or disassembly of protein complexes, and stabilization or degradation of misfolded or aggregated proteins. HSP40, also known as J-domain proteins (JDPs), is the largest family with over fifty members and contains highly conserved J domains responsible for binding to HSP70 and stimulation of the ATPase activity as a co-chaperone. Tumor suppressor p53 (p53), the most frequently mutated gene in human cancers, is one of the proteins that functionally interact with HSP40/JDPs. The majority of p53 mutations are missense mutations, resulting in acquirement of unexpected oncogenic activities, referred to as gain of function (GOF), in addition to loss of the tumor suppressive function. Moreover, stability and levels of wild-type p53 (wtp53) and mutant p53 (mutp53) are crucial for their tumor suppressive and oncogenic activities, respectively. However, the regulatory mechanisms of wtp53 and mutp53 are not fully understood. Accumulating reports demonstrate regulation of wtp53 and mutp53 levels and/or activities by HSP40/JDPs. Here, we summarize updated knowledge related to the link of HSP40/JDPs with p53 and cancer signaling to improve our understanding of the regulation of tumor suppressive wtp53 and oncogenic mutp53 GOF activities. [ABSTRACT FROM AUTHOR]

The tumor suppressor protein p53 is often called "the genome guardian" and controls the cell cycle and the integrity of DNA, as well as other important cellular functions. Its main function is to trigger the process of apoptosis in tumor cells, and approximately 50% of all cancers are related to the inactivation of the p53 protein through mutations in the TP53 gene. Due to the association of mutant p53 with cancer therapy resistance, different forms of restoration of p53 have been subject of intense research in recent years. In this sense, this review focus on the main currently adopted approaches for activation and reactivation of p53 tumor suppressor function, focusing on the synthetic approaches that are involved in the development and preparation of such small molecules. [ABSTRACT FROM AUTHOR]

Despite the high frequency of EGFR and TP53 genetic alterations in gliomas, little is known about their crosstalk during tumor progression. Here, we described a mutually exclusive distribution between mutations in these two genes. We found that wild-type p53 gliomas are more aggressive than their mutant counterparts, probably because the former accumulate amplifications and/or mutations in EGFR and show a stronger activation of this receptor. In addition, we identified a series of genes associated with vesicular trafficking of EGFR in p53 wild-type gliomas. Among these genes, TMEM167A showed the strongest implication in overall survival in this group of tumors. In agreement with this observation, inhibition of TMEM167A expression impaired the subcutaneous and the intracranial growth of wild-type p53 gliomas, regardless of the presence of EGFR mutations. In the absence of p53 mutations, TMEM167A knockdown reduced the acidification of intracellular vesicles, affecting the autophagy process and impairing EGFR trafficking and signaling. This effect was mimicked by an inhibitor of the vacuolar ATPase. We propose that the increased aggressiveness of wild-type p53 gliomas might be due to the increase in growth factor signaling activity, which depends on the regulation of vesicular trafficking by TMEM167A. [ABSTRACT FROM AUTHOR]