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Low molecular weight proteins and protein assemblies can now be investigated using cryo-electron microscopy (EM) as a complement to traditional structural biology techniques. It is important, however, to not lose sight of the dynamic information inherent in macromolecules that give rise to their exquisite functionality. As computational methods continue to advance the field of biomedical imaging, so must strategies to resolve the minute details of disease-related entities. Here, we employed combinatorial modeling approaches to assess flexible properties among low molecular weight proteins (~100 kDa or less). Through a blend of rigid body refinement and simulated annealing, we determined new hidden conformations for wild type p53 monomer and dimer forms. Structures for both states converged to yield new conformers, each revealing good stereochemistry and dynamic information about the protein. Based on these insights, we identified fluid parts of p53 that complement the stable central core of the protein responsible for engaging DNA. Molecular dynamics simulations corroborated the modeling results and helped pinpoint the more flexible residues in wild type p53. Overall, the new computational methods may be used to shed light on other small protein features in a vast ensemble of structural data that cannot be easily delineated by other algorithms. [ABSTRACT FROM AUTHOR]

The novel series of furan‐bearing pyrazolo[3,4‐b]pyridines were designed as cyclin‐dependent kinase 2 (CDK2) inhibitors and as p53–murine double minute 2 (MDM2) inhibitors. The newly synthesized compounds were screened for their antiproliferative activity toward hepatocellular carcinoma (HepG2) and breast cancer (MCF7) cell lines. The most active compounds on both cell lines were additionally evaluated for their in vitro CDK2 inhibitory activity. Compounds 7b and 12f displayed enhanced activity (half‐maximal inhibitory concentration [IC50] = 0.46 and 0.27 µM, respectively) in comparison to the standard roscovitine (IC50 = 1.41 ± 0.03 µM), in addition to, cell cycle arrest at S phase and G1/S transition phase in MCF7 cells treated with both compounds, respectively. Moreover, the most active spiro‐oxindole derivative against MCF7 cell line, 16a, exhibited enhanced inhibitory activity against p53–MDM2 interaction in vitro (IC50 = 3.09 ± 0.12 µM) compared to nutlin, and increased the levels of both p53 and p21 by nearly fourfold in comparison to the negative control. Molecular docking studies demonstrated the plausible interaction patterns of the most potent derivatives 17b and 12f in the CDK2 binding pocket and the spiro‐oxindole 16a with p53–MDM2 complex, respectively. Consequently, the new chemotypes 7b, 12f, and 16a can be presented as promising antitumor hits for further studies and optimization. [ABSTRACT FROM AUTHOR]

Reduced Glutathione (GSH) was found in all mammalian tissues, as antioxidant is plays an important role in number of biochemical processes that includes repair of oxidativestress damage, defense against free radicals, apoptosis, signal transduction and gene expression. Cisplatin (CDDP) as common chemotherapy used against many forms of human cancers. Unfortunately, like many chemotherapy drugs the CDDP have adverse effects on cancers patients such as cytotoxicity on normal tissue and decreases levels of glutathione. The current studywas verified the effect of GSH by using different dosages (10, 20, 30 and 40 mg/kg) on Swiss albino mice, then chosen the best dose of GSH to use with one acute dose (2.5mg/kg) of CDDP, through measuring two parameters: Micronucleus rate MN as Cytotoxic test and Tumor Suppressor Protein p53 levels as a Genotoxic test. Results of combination group showed significant decrease in number of micronuclei (7.48%) compared to CDDP group (9.64%) and significant increase in levels of p53(164.63pg/ml) compared to CDDP group (47.26 pg/ml). Although, results when compared to control group (2.60%) it still significant increases in formation of MN rate, also significant increases in p53 values when compared to control group (72.27pg/ml). This means that GSH may be has ability to improve the cytotoxicity of CDDP and reduces its toxicity on normal cells of cancers patients. [ABSTRACT FROM AUTHOR]

Background: CDKN2A, encoding two important tumor suppressor proteins p16 and p14, is a tumor suppressor gene. Mutations in this gene and subsequently the defect in p16 and p14 proteins lead to the downregulation of RB1/p53 and cancer malignancy. To identify the structural and functional effects of mutations, various powerful bioinformatics tools are available. The aim of this study is the identification of high-risk non-synonymous single nucleotide variants in the CDKN2A gene via bioinformatics tools. Materials and Methods: Among the identified polymorphisms in this gene, 353 missense variants are retrieved from the national center for biotechnology information/single nucleotide polymorphism database (NCBI/dbSNP). Then, the pathogenicity of missense variants are considered using different bioinformatics tools. The stability of these mutant proteins, conservation of amino acids and the secondary and tertiary structural changes are analyzed by bioinformatics tools. After the identification of high-risk mutations, the changes in the hydrophobicity of high-risk amino acid substitutions are considered. Results: Deleterious single nucleotide polymorphisms (SNPs) were screened step by step using the bioinformatics tools. The results obtained from the set of bioinformatics tools identify high-risk mutations in CDKN2A gene. Conclusion: 18 high-risk mutations including L16R/Q, G23D/R/S, L32P, N42K, G55D, G67D/R, P81R, H83R, G89D/S, A102E, G101R, G122R, and V126D were identified. According to the previous experimental studies, the association of L16R, G23D/R/S, L32P, G67R, H83R, G89D, G101R, and V126D amino acid substitutions with various cancers has been confirmed. [ABSTRACT FROM AUTHOR]

Oligomerization plays a crucial role in the structure and function of peptides and proteins, wherein sequence variations can affect the oligomeric stability of the biomolecule. In this study, an artificial neural network classifier that can predict the oligomeric state of peptides is presented, using the p53 tetramerization domain and associated mutants as the model system. The FASGAI vectors were utilized as the peptide descriptors, and the resulting binary classifier exhibits satisfactory predictive ability as demonstrated by a test set accuracy of 86%. [ABSTRACT FROM AUTHOR]

Prostate cancer is the second leading cause of cancer death in men and about one in nine will be diagnosed in his lifetime. Loss of PTEN has been considered as one of the major factors leading to the origin of prostate cancer through modulating PI3K/AKT signaling pathways. In this study, we have prepared a multifunctional antioxidant nanoliposome containing PTEN plasmid and cerium oxide nanoparticles (CeNPs). The efficient delivery of PTEN plasmid to human prostate cancer cells (PC‐3) leads to restoration of the expression of lost PTEN protein in the cell cytoplasm. The delivered superoxide dismutase (SOD)‐mimetic CeNPs were also found to decrease the cytoplasmic free radical levels in prostate cancer cells. The above two activities induced DNA fragmentation and micronucleus formation in prostate cancer cells. Furthermore, it was also found that these multifunctional antioxidant nanoliposomes inhibit the PI3K/AKT signaling pathway to negatively regulate the cell viability of prostate cancer cells. The mRNA expression pattern of other relevant proteins predominantly involved in cancer cell proliferation and apoptosis suggested that the high PTEN expression could control the synthesis of oncogenic proteins. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 3152–3164, 2018. [ABSTRACT FROM AUTHOR]

Interferon regulatory factor-1 (IRF-1) is a tumor suppressor gene, which encodes a mammalian transcription factor that serves various vital functions in a cell, such as cell cycle regulation, immunomodulation, and antiviral response. We report full-length human IRF-1 cDNA cloning and expression in E. coli/BL21 cells with complete solubilisation of recombinant protein. We cloned the gene by the RT-PCR technique using ORF-specific primers followed by expression of recombinant IRF-1 in E. coli under GST fusion system. The profound expression of recombinant protein was observed after inducing with 0.5 mM IPTG for 3 h at 37 °C. We observed few degradation products of low molecular mass along with full-length fusion protein. We successfully minimized the formation of low molecular mass degradation products of GST-huIRF-1 protein at 16 °C. Simultaneously, we achieved the expression of recombinant protein in soluble fraction of E. coli/BL21 cells at 20 °C with higher yield, which is crucial to the study of the biological functions of any protein. We further confirmed it by the immunoblotting technique using anti-IRF-1 and anti-GST antibodies under the induction of E. coli cells harboring the IRF-1 recombinant plasmid after sonicated and fractioned fractions. This work will serve as a platform for characterizing the recombinant protein that may pave the way to understand molecular mechanism of tumour suppression caused by this molecule. [ABSTRACT FROM AUTHOR]

The retinoblastoma protein (Rb) and the homologous pocket proteins p107 and p130 negatively regulate cell proliferation by binding and inhibiting members of the E2F transcription factor family. The structural features that distinguish Rb from other pocket proteins have been unclear but are critical for understanding their functional diversity and determining why Rb has unique tumor suppressor activities. We describe here important differences in how the Rb and p107 C-terminal domains (CTDs) associate with the coiled-coil and marked-box domains (CMs) of E2Fs. We find that although CTD–CM binding is conserved across protein families, Rb and p107 CTDs show clear preferences for different E2Fs. A crystal structure of the p107 CTD bound to E2F5 and its dimer partner DP1 reveals the molecular basis for pocket protein–E2F binding specificity and how cyclin-dependent kinases differentially regulate pocket proteins through CTD phosphorylation. Our structural and biochemical data together with phylogenetic analyses of Rb and E2F proteins support the conclusion that Rb evolved specific structural motifs that confer its unique capacity to bind with high affinity those E2Fs that are the most potent activators of the cell cycle. [ABSTRACT FROM AUTHOR]

The tumor suppressor protein, promyelocytic leukemia protein (PML), was originally identified in acute promyelocytic leukemia due to a chromosomal translocation between chromosomes 15 and 17. PML is the core component of subnuclear structures called PML nuclear bodies (PML-NBs), which are disrupted in acute promyelocytic leukemia cells. PML plays important roles in cell cycle regulation, survival and apoptosis, and inactivation or down-regulation of PML is frequently found in cancer cells. More than 120 proteins have been experimentally identified to physically associate with PML, and most of them either transiently or constitutively co-localize with PML-NBs. These interactions are associated with many cellular processes, including cell cycle arrest, apoptosis, senescence, transcriptional regulation, DNA repair and intermediary metabolism. Importantly, PML inactivation in cancer cells can occur at the transcriptional-, translational- or post-translational-levels. However, only a few somatic mutations have been found in cancer cells. A better understanding of its regulation and its role in tumor suppression will provide potential therapeutic opportunities. In this review, we discuss the role of PML in multiple tumor suppression pathways and summarize the players and stimuli that control PML protein expression or subcellular distribution. [ABSTRACT FROM AUTHOR]

p63 is a transcription factor of p53 gene family, which are involved in development, differentiation and cell response to stress; however, its roles in ischemic preconditioning (IPC) in the brain are not clear. In the present study, we investigated the effect of IPC on p63 immunoreactivity caused by 5 min of transient cerebral ischemia in gerbils. IPC was induced by subjecting the gerbils to 2 min of transie ischemia 1 day prior to 5 min of transient ischemia. The animals were randomly assigned to four groups (sham-operated-group, ischemia-operated-group, IPC plus (+)-sham-operated-group and IPC + ischemia-operated-group). The number of viable neurons in the stratum pyramidale of the hippocampal CA1 region (CA1) was significantly increased by IPC + ischemia-operated-group compared with that in the ischemia-operated-group 5 days after ischemic insult. We found that strong p63 immunoreactivity was detected in the CA1 pyramidal neurons in the sham-operated-group, and the immunoreactivity was decreased with time after ischemia-reperfusion. In addition, strong p63 immunoreactivity was newly expressed in microglial cells of the CA1 region from 2 days after ischemia-reperfusion. In all the IPC + sham-operated-groups, p63 immunoreactivity in the CA1 pyramidal neurons was similar to that in the sham-operated-group, and the immunoreactivity was well maintained in the IPC + ischemia-operated-groups after cerebral ischemia. In brief, our present findings show that IPC dramatically protected the reduction of p63 immunoreactivity in the pyramidal neurons of the CA1 region after ischemia-reperfusion, and this result suggests that the expression of p63 may be necessary for neurons to survive after transient cerebral ischemia. [ABSTRACT FROM AUTHOR]

Background: Exportin 1 (XPO1), also called chromosome region maintenance 1 (CRM1), is the sole exportin mediating transport of many multiple tumor suppressor proteins out of the nucleus. Aim and methods: To verify the hypothesis that XPO1 inhibition affects prostate cancer (PCa) metastatic potential, orally available, potent and selective, SINE compounds, Selinexor (KPT- 330) and KPT-251, were tested in preclinical models known to generate bone lesions and systemic tumor spread. Results: In vitro, Selinexor reduced both secretion of proteases and ability to migrate and invade of PCa cells. SINEs impaired secretion of pro-angiogenic and pro-osteolytic cytokines and reduced osteoclastogenesis in RAW264.7 cells. In the intra-prostatic growth model, Selinexor reduced DU145 tumor growth by 41% and 61% at the doses of 4 mg/Kg qd/5 days and 10 mg/Kg q2dx3 weeks, respectively, as well as the incidence of macroscopic visceral metastases. In a systemic metastasis model, following intracardiac injection of PCb2 cells, 80% (8/10) of controls, 10% (1/10) Selinexor- and 20% (2/10) KPT-251-treated animals developed radiographic evidence of lytic bone lesions. Similarly, after intra-tibial injection, the lytic areas were higher in controls than in Selinexor and KPT-251 groups. Analogously, the serum levels of osteoclast markers (mTRAP and type I collagen fragment, CTX), were significantly higher in controls than in Selinexor- and KPT-251-treated animals. Importantly, overall survival and disease-free survival were significantly higher in Selinexor- and KPT-251-treated animals when compared to controls. Conclusions: Selective blockade of XPO1-dependent nuclear export represents a completely novel approach for the treatment of advanced and metastatic PCa. [ABSTRACT FROM AUTHOR]

Background: The tumor suppressor genes CADM1/TSLC1 and DAL- 1/4.1B are frequently inactivated by promoter methylation in non-small cell lung cancer. The proteins they encode, CADM1 and 4.1B, form a complex in human epithelial cells and are involved in cell-cell adhesion. Methods: Expression of CADM1 and 4.1B proteins was examined by immunohistochemistry in 67 primary breast cancer and adjacent noncancerous tissues. CADM1 and 4.1B messenger RNA (mRNA) was detected by reverse-transcription polymerase chain reaction (RT-PCR). The methylation status of the CADM1 and 4.1B promoters was determined quantitatively by bisulfite treatment followed by pyrosequencing. Results: CADM1 and 4.1B protein signals were detected along the cell membrane in normal mammary epithelia. By contrast, 47 (70%) and 49 (73%) of 67 primary breast cancers showed aberrant CADM1 and 4.1B staining, respectively. Aberrant CADM1 staining was more frequently observed in pT2 and pT3 tumors and for stages II and III ( P = 0.045 and P = 0.020, respectively), while aberrant 4.1B staining was more often observed in tumors with lymph node metastasis, for pT2 and pT3 tumors, and for stages II and III ( P = 0.0058, P = 0.0098, and P = 0.0007, respectively). Furthermore, aberrant CADM1 and 4.1B expression was preferentially observed in invasive relative to noninvasive lesions from the same specimen ( P = 0.036 and P = 0.0009, respectively). Finally, hypermethylation of CADM1 and 4.1B genes was detected in 46% and 42% of primary breast cancers, respectively. Conclusions: Our findings suggest that aberrant CADM1 and 4.1B expression is involved in progression of breast cancer, especially in invasion into the stroma and metastasis. [ABSTRACT FROM AUTHOR]

An endogenous 17β-estradiol (E2) metabolite, 2-methoxyestradiol (2-ME2), has been reported to exhibit estrogen receptor (ER)-independent anti-angiogenic and anti-tumor effects. Several mechanisms have been proposed for 2-ME2 actions, but there is a lack of evidence for a common pathway for all of the cell-types sensitive to this metabolite. We have examined potential alterations in p53 in response to 2-ME2, E2 and the microtubule disruptor taxol in T47D breast cancer cells. Cells were cultured for six days in medium depleted of endogenous steroids or effectors. Semi-confluent cells were treated with 2-ME2 (1 nM - 10 µM), 10 nM E2 and//or 1 µM taxol and subjected to SDS-PAGE and Western blot analysis, quantitative analysis, or laser-scanning confocal microscopy. Western blot analysis revealed a concentration-dependent biphasic trend in p53 levels. Addition of 10 nM - 1 µM 2-ME2 induced significant up-regulation in p53, and this response gradually diminished to levels comparable to the control upon treatment with higher concentrations (2.5 - 10 µM). The observed upregulation of p53 induced by 2-ME2 is inhibited by concurrent treatment with 1 µM taxol. Cell quantitation revealed a significant decrease (50 - 90%%) in cell number upon treatment with 1 - 10 µM 2-ME2 with minimal effect at lower concentrations. No additional effect on cell proliferation was observed when taxol was combined with 10 nM or 1 µM 2-ME2. In a concentration dependent manner, treatment with 2-ME2 for 24 h differentially influenced cellular localization of p53. These results may aid in further understanding the relationship between steroid receptors, tumor suppressor proteins, and effects of hormone metabolites on breast cancer cells. [ABSTRACT FROM AUTHOR]

The human Septin 4 gene ( Sept4) encodes two major protein isoforms; Sept4_i1 (H5/PNUTL2) and Sept4_i2/ARTS. Septins have been traditionally studied for their role in cytokinesis and their filament-forming abilities, but subsequently have been implicated in diverse functions, including membrane dynamics, cytoskeletal reorganization, vesicle trafficking, and tumorigenesis. ARTS is localized at mitochondria and promotes programmed cell death (apoptosis). These features distinguish ARTS from any other known human septin family member. This review compares the structural and functional properties of ARTS with other septins. In addition, it describes how a combination of two distinct promoters, differential splicing, and intron retention leads to the generation of two different Sept4 variants with diverse biological activity. [ABSTRACT FROM AUTHOR]

Inactivation of the von Hippel-Lindau tumor suppressor protein (pVHL) causes the most common form of kidney cancer. pVHL is part of a complex that polyubiquitinates the alpha subunit of the heterodimeric transcription factor HIF. In the presence of oxygen, HIF1α is prolyl hydroxylated by EglN1 (also called PHD2); this modification recruits pVHL, which then targets HIF1α for proteasomal degradation. In hypoxic or pVHL-defective cells, HIF1α accumulates, binds to HIF1β, and transcriptionally activates genes such as VEGF. VEGF inhibitors and mTOR inhibitors, which indirectly affect HIF, are now approved for the treatment of kidney cancer. EglN1 is a 2-oxoglutarate-dependent dioxygenase; such enzymes can be inhibited with drug-like small molecules and EglN1 inhibitors are currently being tested for the treatment of anemia. EglN2 (PHD1) and EglN3 (PHD3), which are EglN1 paralogs, appear to play HIF-independent roles in cell proliferation and apoptosis, respectively, and are garnering interest as potential cancer targets. A number of JmjC-containing proteins, including RBP2 and PLU-1, are 2-oxoglutarate-dependent dioxygenases that demethylate histones. Preclinical data suggest that inhibition of RBP2 or PLU-1 would suppress tumor growth. [ABSTRACT FROM AUTHOR]

Estrogen receptor α (ERα) plays an important role in the onset and progression of breast cancer, whereas p53 functions as a major tumor suppressor. We previously reported that ERα binds to p53, resulting in inhibition of transcriptional regulation by p53. Here, we report on the molecular mechanisms by which ERa suppresses p53's transactivation function. Sequential ChIP assays demonstrated that ERa represses p53-mediated transcriptional activation in human breast cancer cells by recruiting nuclear receptor corepressors (NCoR and SMRT) and histone deacetylase 1 (HDAC1). RNAI-mediated down-regulation of NCoR resulted in increased endogenous expression of the cyclin-dependent kinase (CDK)inhibitor α (CDKNIA) gene, a prototypic transcriptional target of p53. While 17β-estradiol (E2) enhanced ERa binding to p53 and inhibited p21 transcription, antiestrogens decreased ERα recruitment and induced transcription. The effects of estrogen and antiestrogens on p21 transcription were diametrically opposite to their known effects on the conventional ERE-containing ERα target gene, pS2/TFF1. These results suggest that ERα uses dual strategies to promote abnormal cellular proliferation: enhancing the transcription of ERE-containing proproliferative genes and repressing the transcription of p53-responsive antiproliferative genes. Importantly, ERα binds to p53 and inhibits transcriptional activation by p53 in stem/progenitor cell-containing murine mammospheres, suggesting a potential role for the ER-p53 interaction in mammary tissue homeostasis and cancer formation. Furthermore, retrospective studies analyzing response to tamoxifen therapy in a subset of patients with ER-positive breast cancer expressing either wild-type or mutant p53 suggest that the presence of wild-type p53 is an important determinant of positive therapeutic response. [ABSTRACT FROM AUTHOR]

Insulin-like growth factor binding-protein-7 (IGFBP7) was obtained from our previous colonic adenocarcinoma (CRC) and normal mucosa suppression subtraction hybridization (SSH) cDNA libraries. By RT-PCR and immunohistochemistry, we found that IGFBP7 was overexpressed in CRC tissue compared to normal tissue. However, our in vitro experiments performed in 10 CRC cell lines showed that IGFBP7 expressed only in SW480 and Caco2 cell lines, which implied an underlying reversible regulatory mechanism. Using methylation-specific PCR (MSP) and bisulfite sodium PCR (BSP), we found that its expression was associated with DNA hypomethylation of exon1. This was further supported by the in vitro study which showed restored IGFBP7 expression after demethylation agent 5-aza-2′-deoxycytidine treatment. Correlation analysis between IGFBP7 expression and prognosis indicated that overexpression of IGFBP7 in CRC tissue correlated with favourable survival. Investigation of the functional role of IGFBP7 through transfection studies showed that IGFBP7 protein could inhibit growth rate, decrease colony formation activity, and induce apoptosis in RKO and SW620 cells, suggesting it a potential tumor suppressor protein in colorectal carcinogenesis. In conclusion, our study clearly demonstrated that IGFBP7 plays a potential tumor suppressor role against colorectal carcinogenesis and its expression is associated with DNA hypomethylation of exon 1. [ABSTRACT FROM AUTHOR]

The expression pattern of VEGF, p53 and ICAM-1 was studied in conjunctiva of diabetic patients with and without retinopathy. All patients underwent a complete ophthalmic examination, including retinal fluorescein angiography. Indirect immunoperoxidase method was performed on 20 eyes of 20 patients with type II diabetes without DR and on 5 eyes of 5 patients with PDR. A control study was performed on 6 normal conjunctiva undertaken during cataract surgery. Immunoreactivity of VEGF, p53 and ICAM-1 was found in epithelial, fibroblast and vascular endothelial cells. For the same duration of diabetes, a strong to moderate or weak immunoreactivity was observed in the conjunctiva of patients without retinopathy. In patients with PDR, the expression was strong for all these proteins. The immunoreactivity was correlated between VEGF, p53 and ICAM-1. In the normal conjunctiva, a weak to negative immunostaining was observed. The presence of these proteins in the conjunctiva of diabetic patients without retinopathy may add new data in the pathogenesis of diabetic retinopathy. Further studies are needed to confirm this hypothesis. [ABSTRACT FROM AUTHOR]

The mammalian protein PSF contains a DNA-binding domain (DBD) that coordinately represses multiple oncogenic genes in human cell lines, indicating a role for PSF as a human tumor-suppressor protein. PSF also contains two RNA-binding domains (RBD) that form a complex with a noncoding VL30 retroelement RNA, releasing PSF from a gene and reversing repression. Thus, the DBD and RBD in PSF are linked by a mechanism of reversible gene regulation involving a noncoding RNA. This mechanism also could apply to other regulatory proteins that contain both DBD and RBD. The mouse genome has multiple copies of VL30 retroelements that are developmentally regulated. and mouse cells contain VL30 RNAs that have normal and pathological roles in gene regulation. Human chromosome 11 has a VL30 retroelement, and a VL30 EST was identified in human blastocyst cells, indicating that the PSF-VL30 RNA regulatory mechanism also could function in human cells. [ABSTRACT FROM AUTHOR]

While the tumor suppressor protein p53 regulates the cell cycle to prevent cell damage, it also triggers apoptosis and prevents cancer. These inhibitory functions may disappear once the p53 gene is mutated. Under these circumstances, the detection of p53 protein concentrations can have significant clinical applications. In this study, nickel phthalocyanine (NiPc) was coated on a gold electrode to produce a modified Au/NiPc electrode. p53 antibodies were bonded to the Au/NiPc electrode by the Ni+2 ion in NiPc, which can be self-assembled with the imidazole group of the p53 protein. The Au/NiPc/anti-p53 electrode was subsequently dripped with a buffer solution of bovine serum albumin (BSA) to form the Au/NiPc/anti-p53/BSA electrode, which was used for the detection of p53 antigen under 10 mM potassium ferricyanide/potassium ferrocyanide (K3Fe(CN)6/K4Fe(CN)6) solution by cyclic voltammetry and differential pulse voltammetry analyses. The linear detection range and the sensitivity for the p53 antigen were 0.1–500 pg/mL and 60.65 μA/Log (pg/mL)-cm2, respectively, with a detection time of 90–150 s. In addition, Au/NiPc/anti-p53 (100 ng/mL)/BSA electrodes were tested for specificity using glucose, bovine serum albumin, histidine, ascorbic acid, uric acid, prostate-specific antigen, human serum albumin, and human immunoglobulin G. All p-values were <0.0005, indicating an outstanding specificity. [ABSTRACT FROM AUTHOR]

Background: We reported previously that phenethyl isothiocyanate (PEITC), a dietary compound, can reactivate p53R175H mutant in vitro and in SK-BR-3 (p53R175H) breast xenograft model resulting in tumor inhibition. Because of the diversity of human cancers with p53 mutations, these findings raise important questions whether this mechanism operates in different cancer types with same or different p53 mutations. In this study, we investigated whether PEITC recuses mutant p53 in prostate cancer cells harboring different types of p53 mutants, structural and contact, in vitro and in vivo. Methods: Cell proliferation, cell apoptosis and cell cycle arrest assays were performed to examine the effects of PEITC on prostate cancer cell lines with p53 mutation(s), wild-type p53, p53 null or normal prostate cells in vitro. Western blot analysis was used to monitor the expression levels of p53 protein, activation of ATM and upregulation of canonical p53 targets. Immunoprecipitation, subcellular protein fraction and qRT-PCR was performed to determine change in conformation and restoration of transactivation functions/ inhibition of gain-of-function (GOF) activities to p53 mutant(s). Mice xenograft models were established to evaluate the antitumor efficacy of PEITC and PEITC-induced reactivation of p53 mutant(s) in vivo. Immunohistochemistry of xenograft tumor tissues was performed to determine effects of PEITC on expression of Ki67 and mutant p53 in vivo. Results: We demonstrated that PEITC inhibits the growth of prostate cancer cells with different "hotspot" p53 mutations (structural and contact), however, preferentially towards structural mutants. PEITC inhibits proliferation and induces apoptosis by rescuing mutant p53 in p53R248W contact (VCaP) and p53R175H structural (LAPC-4) mutant cells with differential potency. We further showed that PEITC inhibits the growth of DU145 cells that co-express p53P223L (structural) and p53V274F (contact) mutants by targeting p53P223L mutant selectively, but not p53V274F. The mutant p53 restored by PEITC induces apoptosis in DU145 cells by activating canonical p53 targets, delaying cells in G1 phase and phosphorylating ATM. Importantly, PEITC reactivated p53R175H and p53P223L/V274F mutants in LAPC-4 and DU145 prostate xenograft models, respectively, resulting in significant tumor inhibition. Conclusion: Our studies provide the first evidence that PEITC's anti-cancer activity is cancer cell type-independent, but p53 mutant-type dependent. [ABSTRACT FROM AUTHOR]

Cancer research has been heavily geared towards genomic events in the development and progression of cancer. In contrast, metabolic regulation, such as aberrant metabolism in cancer, is poorly understood. Alteration in cellular metabolism was once regarded simply as a consequence of cancer rather than as playing a primary role in cancer promotion and maintenance. Resurgence of cancer metabolism research has identified critical metabolic reprogramming events within biosynthetic and bioenergetic pathways needed to fulfill the requirements of cancer cell growth and maintenance. The tumor suppressor protein p53 is emerging as a key regulator of metabolic processes and metabolic reprogramming in cancer cells—balancing the pendulum between cell death and survival. This review provides an overview of the classical and emerging non-classical tumor suppressor roles of p53 in regulating mitochondrial dynamics: mitochondrial engagement in cell death processes in the prevention of cancer. On the other hand, we discuss p53 as a key metabolic switch in cellular function and survival. The focus is then on the conceivable roles of p53 in breast cancer metabolism. Understanding the metabolic functions of p53 within breast cancer metabolism will, in due course, reveal critical metabolic hotspots that cancers advantageously re-engineer for sustenance. Illustration of these events will pave the way for finding novel therapeutics that target cancer metabolism and serve to overcome the breast cancer burden. [ABSTRACT FROM AUTHOR]