P53 Post-translational Modifications Research Articles

A Sub-Group of Kidney-Transplant Recipients with Highly Aggressive Squamous Cell Carcinoma Expressing Phosphorylated Serine392p53.

Cutaneous squamous cell carcinomas in kidney-transplant recipients are frequent, with an increasing incidence linked to long immunosuppression durations and exposure to ultraviolet radiation. p53 is at the cornerstone of ultraviolet-induced DNA damage, but the role of p53 post-translational modifications in this context is not yet deciphered. Here, we investigated the phosphorylation status of p53 at Serine 392 in 25 cutaneous squamous cell carcinomas in kidney-transplant recipients, compared with 22 non-transplanted patients. Cutaneous squamous cell carcinomas in transplanted patients occurred after a median period of 19 years of immunosuppression, with a median number of 15 cutaneous squamous cell carcinomas and more aggressive histological and clinical characteristics. There was no significant difference between Ki67, p53, and pSer392p53 expression in the two groups. Using principal component analysis, we identified a cluster of exclusively transplanted patients with a median of 23 years of immunosuppression duration, significantly more aggressive biological characteristics, and higher pSer392p53 expression. pSer392p53 was expressed in the whole tumor, suggesting an early carcinogenic event in the course of prolonged immunosuppression. This high, diffuse pSer392p53 expression, corresponding to a high level of DNA damage, might be useful to identify aggressive cutaneous squamous cell carcinomas in kidney-transplant recipients to treat them more aggressively.

Regulating the p53 Tumor Suppressor Network at PML Biomolecular Condensates.

Simple SummaryThe cell nucleus is organized into different sub-nuclear compartments to control specific cellular processes, including PML nuclear bodies (NBs), also termed PML biomolecular condensates. PML-NBs form multi-protein complexes that are highly responsive to cellular stress, and regulate cell fate decisions in response to genome damage. By concentrating proteins that directly control the activity of the tumor suppressor p53, PML biocondensates guide the cellular response either towards induction of cell death or senescence through altering p53 modifications. In this review, we discuss the molecular mechanisms controlling PML condensate formation, and how they impact the regulation of p53 activity, and propose a list of promising candidate proteins that may contribute to the regulation of p53 at PML biomolecular condensates.By forming specific functional entities, nuclear biomolecular condensates play an important function in guiding biological processes. PML biomolecular condensates, also known as PML nuclear bodies (NBs), are macro-molecular sub-nuclear organelles involved in central biological processes, including anti-viral response and cell fate control upon genotoxic stress. PML condensate formation is stimulated upon cellular stress, and relies on protein–protein interactions establishing a PML protein meshwork capable of recruiting the tumor suppressor p53, along with numerous modifiers of p53, thus balancing p53 posttranslational modifications and activity. This stress-regulated process appears to be controlled by liquid–liquid phase separation (LLPS), which may facilitate regulated protein-unmixing of p53 and its regulators into PML nuclear condensates. In this review, we summarize and discuss the molecular mechanisms underlying PML nuclear condensate formation, and how these impact the biological function of p53 in driving the cell death and senescence responses. In addition, by using an in silico approach, we identify 299 proteins which share PML and p53 as binding partners, thus representing novel candidate proteins controlling p53 function and cell fate decision-making at the level of PML nuclear biocondensates.

P53 and cancer metabolism

<p indent="0mm">p53 has actions in cell cycle inhibition, induction of cell senescence and apoptosis and plays a role in tumor suppression. p53 has been reported to influence redox homeostasis and metabolic stability and also to mediate intracellular ferroptosis, thereby affecting the growth of cancer cells. Metabolic reprogramming of tumor cells is also modulated by p53 with resultant tumor inhibition. However, mutant p53 has altered metabolic regulatory functions from the wild-type protein and tumor inhibitory activity may be impaired. The current study reviews the metabolic regulatory activity of p53 and its involvement in the oxidative stress response and autophagy process in tumor cells. Post-translational modification of p53 and the functional changes of mutant p53 in tumor cells are discussed. Key roles of p53 in regulation of tumor metabolism are analyzed. The aim was to provide reference materials to aid the understanding of p53 regulatory functions and anticancer roles for research purposes.

Post-Translational Modifications of p53 in Ferroptosis: Novel Pharmacological Targets for Cancer Therapy.

The tumor suppressor p53 is a well-known cellular guardian of genomic integrity that blocks cell cycle progression or induces apoptosis upon exposure to cellular stresses. However, it is unclear how the remaining activities of p53 are regulated after the abrogation of these routine activities. Ferroptosis is a form of iron- and lipid-peroxide-mediated cell death; it is particularly important in p53-mediated carcinogenesis and corresponding cancer prevention. Post-translational modifications have clear impacts on the tumor suppressor function of p53. Here, we review the roles of post-translational modifications in p53-mediated ferroptosis, which promotes the elimination of tumor cells. A thorough understanding of the p53 functional network will be extremely useful in future strategies to identify pharmacological targets for cancer therapy.

P53 is functionally inhibited in clear cell renal cell carcinoma (ccRCC): a mechanistic and correlative investigation into genetic and molecular characteristics

PurposeAlthough p53 is rarely mutated in ccRCC, its overexpression has been linked to poor prognosis. The current study sought to elucidate the unique role of p53 in ccRCC with genomic, proteomic, and functional analyses.Materials and methodsData from the Cancer Genome Atlas (TCGA) were evaluated for genomic and proteomic characteristics of p53; a tissue micro array (TMA) study was carried out to evaluate the association of p53 and phosphorylated p53 (pp53) with clinical outcome. Mechanistic in vitro experiments were performed to confirm a pro-apoptotic loss of p53 in ccRCC and p53 isoforms as well as posttranslational modifications of p53 where assessed to provide possible reasons for a functional inhibition of p53 in ccRCC.ResultsA low somatic mutation rate of p53 could be confirmed. Although mRNA levels were correlated with poor prognosis and clinicopathological features, there was no monotonous association of mRNA levels with survival outcome. Higher p53 protein levels could be confirmed as poor prognostic features. In vitro, irradiation of ccRCC cell lines markedly induced levels of p53 and of activated (phosphorylated) p53. However, irradiated ccRCC cells demonstrated similar proliferation, migration, and p53 transcriptional activity like non-irradiated controls indicating a functional inhibition of p53. p53 isoforms and could not be correlated with clinical outcome of ccRCC patients.Conclusionsp53 is rarely mutated but the wildtype p53 is functionally inhibited in ccRCC. To investigate mechanisms that underlie functional inhibition of p53 may provide attractive therapeutic targets in ccRCC.

Deciphering the PTM codes of the tumor suppressor p53

The genome guardian p53 functions as a transcription factor that senses numerous cellular stresses and orchestrates the corresponding transcriptional events involved in determining various cellular outcomes, including cell cycle arrest, apoptosis, senescence, DNA repair, and metabolic regulation. In response to diverse stresses, p53 undergoes multiple posttranslational modifications (PTMs) that coordinate with intimate interdependencies to precisely modulate its diverse properties in given biological contexts. Notably, PTMs can recruit ‘reader’ proteins that exclusively recognize specific modifications and facilitate the functional readout of p53. Targeting PTM–reader interplay has been developing into a promising cancer therapeutic strategy. In this review, we summarize the advances in deciphering the ‘PTM codes’ of p53, focusing particularly on the mechanisms by which the specific reader proteins functionally decipher the information harbored within these PTMs of p53. We also highlight the potential applications of intervention with p53 PTM–reader interactions in cancer therapy and discuss perspectives on the ‘PTMomic’ study of p53 and other proteins.

A Comparison of p53 Isoform Profiles and Apoptosis Induced by Camptothecin or a Herbal Khat Extract (Catha Edulis (Vahl) Forssk. ex Endl.) in Leukemic Cell Lines: Exploring Cellular Responses in Therapy Development.

Simple SummaryThis study aimed at exploring the modulations of p53 in cell toxicity induced by an extract of the herb khat, a natural stimulant used by millions of people. We previously reported that khat-extract induced cell death by affecting mitochondrial function and the receptor- and mitochondria-mediated cell death pathways, in leukemic cell lines and cells of the oral cavity, in vitro. We included the cancer therapeutic camptothecin, which induces apoptosis in various cancer cell lines. By studying modulations of p53 full-length protein and p53 β/γ isoforms following exposure to khat-extract and camptothecin, we wished to elucidate differences and similarities resulting from the treatments using MOLM-13 and MV-4-11 leukemic cell lines. Our results demonstrate that molecular effects of the cytotoxic treatments resulted in different p53 isoforms patterns and post-translational modifications. We suggest that analysis of p53 modulations could be useful in the search for new chemical probes and experimental cancer therapeutics.Khat (Catha edulis (Vahl) Forssk. ex Endl.) is habitually used as a natural stimulant by millions of people, but is associated with adverse effects on gastrointestinal, cardiovascular and central neural systems. At the cellular level khat toxicity involves p53 induction and cell cycle arrest, decreased mitochondrial function and activation of receptor- and mitochondria-mediated cell death pathways. In this study we have examined an extract of khat for induction of p53 post-translational modifications (PTMs) and the functional role of p53 in khat-mediated cell death. Khat was shown to induce phosphorylation and acetylation of p53 in both the khat-sensitive MOLM-13 and the khat-resistant MV-4-11 cell line, but accumulation of the full-length p53 isoform was only observed in the khat sensitive cell line. Small molecule inhibitors of p38 MAP kinase sensitized MV-4-11 cells for khat-treatment without concomitant stabilization of p53. Experiments using a p53 knock-down cell line and murine p53 knock-out bone marrow cells indicated that p53 was redundant in khat-mediated cell death in vitro. We suggest that analysis of isoform patterns and p53 PTMs are useful for elucidation of biological effects of complex plant extracts, and that p53 protein analysis is particularly useful in the search for new chemical probes and experimental cancer therapeutics.

Immunohistochemical detection of p53 and pp53 Ser392 in canine hemangiomas and hemangiosarcomas located in the skin

Backgroundp53 protein is essential for the regulation of cell proliferation. Aberrant accumulation of it usually occurs in cutaneous malignancies. Mutant p53 is detected by immunohistochemistry because it is more stable than the wild-type p53. However, post-translational modifications of p53 in response to ultraviolet radiation are important mechanisms of wild-type p53 stabilization, leading to positive staining in the absence of mutation. The aims were: 1) to analyze the immunohistochemical expression of p53 and phospho-p53 Serine392 in canine skin endothelial tumours; and 2) to determine if any relationship exists between p53 and phospho-p53 Serine392 overexpression and cell proliferation.Resultsp53 and phospho-p53 Serine392 immunolabeling was examined in 40 canine cutaneous endothelial tumours (13 hemangiomas and 27 hemangiosarcomas). Their expression was associated with tumour size, hemangiosarcoma stage (dermal versus hypodermal), histological diagnosis and proliferative activity (mitotic count and Ki-67 index). Statistical analysis revealed a significant increase of p53 immunoreactivity in hemangiosarcomas (median, 74.61%; interquartile range [IQR], 66.97–82.98%) versus hemangiomas (median, 0%; IQR, 0–20.91%) (p < .001) and in well-differentiated hemangiosarcomas (median, 82.40%; IQR, 66.49–83.17%) versus hemangiomas (p = .002). Phospho-p53 Serine392 immunoreactivity was significantly higher in hemangiosarcomas (median, 53.80%; IQR, 0–69.50%) than in hemangiomas (median, 0%; IQR, 0.0%) (p < .001). Positive correlation of the overexpression of p53 and phospho-p53 Serine392 with mitotic count and Ki-67 index was found in the cutaneous vascular tumours (p < .001). The Ki-67 index of the hemangiomas (median, 0.50%; IQR, 0–2.80%) was significantly lower than that of the hemangiosarcomas (median, 34.85%; IQR, 23.88–42.33%) (p < .001), and that specifically of well-differentiated hemangiosarcomas (median, 24.60%; IQR, 15.45–39.35%) (p = .001). Immunolabeling of 18 visceral hemangiosarcomas showed that the p53 (median, 41.59%; IQR, 26.89–64.87%) and phospho-p53 Serine392 (median, 0%; IQR, 0–22.53%) indexes were significantly lower than those of skin (p = .001; p = .006, respectively).ConclusionsThe p53 and phospho-p53 Serine392overexpression together with high proliferative activity in hemangiosarcomas versus hemangiomas indicated that p53 might play a role in the acquisition of malignant phenotypes in cutaneous endothelial neoplasms in dogs. The Ki-67 index may be useful in distinguishing canine well-differentiated hemangiosarcomas from hemangiomas.

Pifithrin-\u03b1 alters p53 post-translational modifications pattern and differentially inhibits p53 target genes

Pifithrin-α (PFT-α) is a small molecule which has been widely used as a specific inhibitor of p53 transcription activity. However, its molecular mechanism of action remains unclear. PFT-α has also been described to display potent p53-independent activity in cells. In this study, we addressed the mechanism of action of PFT-α. We found that PFT-α failed to prevent the effects of Mdm2 inhibitor Nutlin-3 on cell cycle and apoptosis in several cancer cell lines. However, PFT-α rescued normal primary fibroblasts from growth inhibition by Nutlin-3. PFT-α displayed a very limited effect on p53-dependent transcription upon its activation by Nutlin-3. Moreover, PFT-α inhibitory effect on transcription was highly dependent on the nature of the p53 target gene. PFT-α attenuated post-translational modifications of p53 without affecting total p53 protein level. Finally, we found that PFT-α can decrease the level of intracellular reactive oxygen species through activation of an aryl hydrocarbon receptor (AHR)-Nrf2 axis in a p53-independent manner. In conclusion, PFT-α inhibits only some aspects of p53 function, therefore it should be used with extreme caution to study p53-dependent processes.

SNAP reverses temozolomide resistance in human glioblastoma multiforme cells through down‐regulation of MGMT

Glioblastoma multiforme (GBM) is the most frequently occurring and gravest primary tumor of the CNS in adults. The development of chemoresistance to temozolomide (TMZ), the first-line chemotherapy for GBM, is an important factor contributing to poor treatment outcomes. Down-regulation of O-6-methylguanine-DNA methyltransferase (MGMT) expression in GBM cells is an attractive strategy for overcoming TMZ resistance and improving outcomes. This study revealed that the nitric oxide (NO) donor S-nitroso-N-acetylpenicillamine (SNAP) exerts antitumorigenic effects on TMZ-sensitive and TMZ-resistant (TMZ-R) glioma cells. Pretreatment with SNAP not only induced apoptosis, mitochondrial dysfunction, and hypoxia-inducing factor 1, but also resensitized TMZ-R GBM cells to TMZ through down-regulation of MGMT expression. SNAP acted principally through post-translational modification of p53, phosphorylated N-myc downstream regulated gene 1, and MGMT protein stability in TMZ-R GBM cells. Additionally, when applied together, SNAP and TMZ enhanced the inhibition of tumor growth in vitro and in vivo. This study sheds new light on a potential strategy to overcome TMZ resistance in GBM and thus possesses the potential for prolonging survival of patients with GBM.-Tsai, C.-K., Huang, L.-C., Wu, Y.-P., Kan, I.-Y., Hueng, D.-Y. SNAP reverses temozolomide resistance in human glioblastoma multiforme cells through down-regulation of MGMT.

Cell Fate Regulation upon DNA Damage: p53 Serine 46 Kinases Pave the Cell Death Road.

Mild and massive DNA damage are differentially integrated into the cellular signaling networks and, in consequence, provoke different cell fate decisions. After mild damage, the tumor suppressor p53 directs the cellular response to cell cycle arrest, DNA repair, and cell survival, whereas upon severe damage, p53 drives the cell death response. One posttranslational modification of p53, phosphorylation at Serine 46, selectively occurs after severe DNA damage and is envisioned as a marker of the cell death response. However, the molecular mechanism of action of the p53 Ser46 phospho-isomer, the molecular timing of this phosphorylation event, and its activating effects on apoptosis and ferroptosis still await exploration. In this essay, the current body of evidence on the molecular function of this deadly p53 mark, its evolutionary conservation, and the regulation of the key players of this response, the p53 Serine 46 kinases, are reviewed and dissected.

Abstract LB-101: Combined p53 dynamics and post-translational modifications determine subsequent protein expression and cell fate

Abstract The tumor suppressor protein p53 is activated by a diverse repertoire of cellular stresses (e.g. oncogenic signalling, DNA damage) and as a transcription factor induces the expression of response pathways genes (including cell cycle arrest, senescence, apoptosis). While p53 is frequently lost during tumorigenesis, approximately 50% of human tumors retain intact p53, potentially rendering them sensitive to p53-induced cell death during radiotherapy. Recent single cell analysis from our lab has shown that irradiation of cells does not cause a simple sustained accumulation of p53, but rather, induces pulsatile p53 dynamic that oscillates at 5-hour periods resulting from a p53-Mdm2 negative feedback loop, leading to reversible cell cycle arrest. Disrupting this feedback loop by small molecules results in sustained p53 dynamics, leading to irreversible apoptosis or senescence. Moreover, extensive work by others has shown that post-translational modifications (PTM) on p53 also govern the cell fate decision process. Here we show that aside from differences in p53 dynamics following irradiation, multiple PTMs on p53 also differ, with the latter causing quantitative changes of p53 binding to interactions proteins and subsequent alterations in promoter binding. In contrast, p53 dynamics govern broad transcription dynamics of target genes. For example, pulsatile p53 dynamics result in several distinct patterns of mRNA transcription including a cluster of oscillating p53 target genes. However, these oscillatory mRNA dynamics are lost at the protein level. The only p53 target protein that oscillates is the Mdm2 negative regulator of p53, while all other targets gradually accumulate over time. Furthermore, sustained p53 dynamics over 24h results in far greater accumulation of nearly all target proteins compared to pulsatile p53 dynamics, which likely explains the differences in the reversibility of cell fate decisions. The results described here uncover a link between p53 dynamics and p53 PTMs providing an overall mechanistic cell fate selection process by p53, which have important implications both for understanding of cell signalling processing as well as improving p53-mediated therapies. Citation Format: Dan Lu, Kristina Holton, Antonina Hafner, Caroline DeHart, Jacek Sikora, Houjiang Zhou, Ashwini Jambhekar, Neil Kelleher, Jeremy Gunawardena, Galit Lahav. Combined p53 dynamics and post-translational modifications determine subsequent protein expression and cell fate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-101.

Interaction between p53 N terminus and core domain regulates specific and nonspecific DNA binding

The p53 tumor suppressor is a sequence-specific DNA binding protein that activates gene transcription to regulate cell survival and proliferation. Dynamic control of p53 degradation and DNA binding in response to stress signals are critical for tumor suppression. The p53 N terminus (NT) contains two transactivation domains (TAD1 and TAD2), a proline-rich region (PRR), and multiple phosphorylation sites. Previous work revealed the p53 NT reduced DNA binding in vitro. Here, we show that TAD2 and the PRR inhibit DNA binding by directly interacting with the sequence-specific DNA binding domain (DBD). NMR spectroscopy revealed that TAD2 and the PRR interact with the DBD at or near the DNA binding surface, possibly acting as a nucleic acid mimetic to competitively block DNA binding. In vitro and in vivo DNA binding analyses showed that the NT reduced p53 DNA binding affinity but improved the ability of p53 to distinguish between specific and nonspecific sequences. MDMX inhibits p53 binding to specific target promoters but stimulates binding to nonspecific chromatin sites. The results suggest that the p53 NT regulates the affinity and specificity of DNA binding by the DBD. The p53 NT-interacting proteins and posttranslational modifications may regulate DNA binding, partly by modulating the NT-DBD interaction.

The multiple mechanisms that regulate p53 activity and cell fate.

The tumour suppressor p53 has a central role in the response to cellular stress. Activated p53 transcriptionally regulates hundreds of genes that are involved in multiple biological processes, including in DNA damage repair, cell cycle arrest, apoptosis and senescence. In the context of DNA damage, p53 is thought to be a decision-making transcription factor that selectively activates genes as part of specific gene expression programmes to determine cellular outcomes. In this Review, we discuss the multiple molecular mechanisms of p53 regulation and how they modulate the induction of apoptosis or cell cycle arrest following DNA damage. Specifically, we discuss how the interaction of p53 with DNA and chromatin affects gene expression, and how p53 post-translational modifications, its temporal expression dynamics and its interactions with chromatin regulators and transcription factors influence cell fate. These multiple layers of regulation enable p53 to execute cellular responses that are appropriate for specific cellular states and environmental conditions.

Probing p53 Activation by Live-Cell Single-Molecule Chromatin Binding Measurements

Live-cell microscopy revealed the transient nature of transcription factors (TFs) binding to chromatin. However, it is still not clear whether the duration of these binding interactions can be tuned in response to an activating stimulus, and whether such modulation can control the transactivation of target genes. To cover this issue we focused on the tumor-suppressor p53 by combining live-cell single molecule tracking and single cell in-situ measurements of transcription. We show that the fraction of chromatin bound p53 and its residence time are increased following genotoxic stress. Moreover, we found that the modulation of p53 residence time on chromatin requires post translational modifications (PTMs) and correlates with the induction of transcription of target genes. These results allowed us to depict a model in which the modification state of the TF, that accompanies its increase, determines the TF transcriptional activity by tuning its residence time on target sites. Our findings could have an important relevance for those cancers that express an inactive wild type form of p53, like neuroblastoma (NS), in which has been recently reported a defect in PTMs of p53. Thus, we are currently performing SMT measurements in NS cell line to verify whether p53 binding kinetics are affected and we aim to restore the proper PTMs in order to rescue p53 binding kinetics and its transactivation potential in these cancers.

Modulatory Potential of Curcumin and Resveratrol on p53 Post-Translational Modifications during Gastric Cancer.

The combination approach is now a well-established treatment for cancer. The present study evaluated the potential of curcumin and resveratrol on p53 post-translational modifications during gastric cancer. We segregated rats into five groups that included normal controls, dimethylhydrazine (DMH) treated, DMH + curcumin treated, DMH + resveratrol treated, and DMH + curcumin + resveratrol treated. Morphological analyses of tumor nodules confirmed carcinogenesis in rats after 25 weeks of DMH administration. The DMH treatment significantly induced carcinogenesis, as evidenced by high tumor burden in DMH-treated rats compared with controls. Moreover, DMH treatment caused a significant increase in the protein expressions of p53 as well as p53 phosphorylation in the DMH-treated rats. In addition, a significant rise was observed in 14C glucose uptake and 3H-thymidin uptakes in DMH-treated rats. Furthermore, enzyme activities of lactate dehydrogenase and alkaline phosphatase also showed a significant rise. On the contrary, significant decline was noticed in the p53 acetylation at residue 382 of DMH-treated rats. Conversely, combined treatment with curcumin and resveratrol to DMH-treated rats resulted in significant moderation in the tumor burden. In addition, a significant rise in p53 acetylation was at residue 382 of DMH-treated rats after treatment with phytochemicals. Supplementation with phytochemicals significantly modulated other biophysical and biochemical indices to near normal levels. Therefore, we conclude that curcumin and resveratrol significantly modulated p53 post-translational modifications during gastric cancer.

Thiamine antagonists trigger p53-dependent apoptosis in differentiated SH-SY5Y cells

Accumulating evidences suggest that p53 is a key coordinator of cellular events triggered by oxidative stress often associated with the impairment in thiamine metabolism and its functions. However, there are limited data regarding the pursuant feedback between p53 transactivation and thiamine homeostasis. Impairment in thiamine metabolism can be induced experimentally via interference with the thiamine uptake and/or inhibition of the thiamin pyrophosphate–dependent enzymes using thiamine antagonists - amprolium (AM), oxythiamine (OT) or pyrithiamine (PT). We found that exposure of neuronally differentiated SH-SY5Y cells to AM, OT and PT triggered upregulation of p53 gene expression, post-translational modification of p53 via phosphorylation and activation of p53 DNA-binding activity. Phosphorylation of p53 at Ser20 was equally efficient in upregulation of thiamine transporter 1 (THTR1) by all antagonists. However, induction of the expressions of the pyruvate dehydrogenase E1 component subunit beta (PDHB) and oxoglutarate dehydrogenase (OGDH) required dual phosphorylation of p53 at Ser9 and Ser20, seen in cells treated with PT and OT. Moreover, pretreatment of the cells with a decoy oligonucleotide carrying wild-type p53-response element markedly attenuated OT-induced THTR1, PDHB and OGDH gene expression suggesting an important role of p53 in transactivation of these genes. Finally, analysis of gene and metabolic networks showed that OT triggers cell apoptosis through the p53-dependent intrinsic pathway.

Stimulatory effects of curcumin and quercetin on posttranslational modifications of p53 during lung carcinogenesis.

Lung cancer is responsible for increase in mortality due to cancer-related deaths, and new approaches are being explored for the betterment of the situation. In the present study, chemopreventive efficacy of curcumin and quercetin was investigated against benzo(a)pyrene (BP)-induced lung carcinogenesis. The mice were segregated into five groups, which included normal control, BP-treated, BP + curcumin-treated, BP + quercetin-treated, and BP + curcumin + quercetin-treated groups. The morphological and histological analyses of tumor nodules confirmed lung carcinogenesis22 weeks after weeks single intraperitoneal injection of BP at a dose of 100 mg/kg body weight to mice. Curcumin and quercetin when administered individually as well as in combination significantly elevated the expression of acetylated-p53, which was otherwise depressed due to BP treatment. Also, both the phytochemicals significantly reduced the BP-inflicted increased levels of phosphorylated-p53. Furthermore, observed increase in the number of apoptotic cells by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), assay and increased activities of caspase 3 and 9 confirmed the induction of apoptosis by curcumin and quercetin. Moreover, the histological slides also showed noticeable improvement in the histoarchitecture of lungs by phytochemicals. The present study concludes that prophylactic treatment with curcumin and quercetin induces apoptosis in the lungs by modulation of p53 posttranslational modifications.

Fusaric Acid Induces DNA Damage and Post-Translational Modifications of p53 in Human Hepatocellular Carcinoma (HepG2 ) Cells.

Fusaric acid (FA), a common fungal contaminant of maize, is known to mediate toxicity in plants and animals; however, its mechanism of action is unclear. p53 is a tumor suppressor protein that is activated in response to cellular stress. The function of p53 is regulated by post-translational modifications-ubiquitination, phosphorylation, and acetylation. This study investigated a possible mechanism of FA induced toxicity in the human hepatocellular carcinoma (HepG2 ) cell line. The effect of FA on DNA integrity and post-translational modifications of p53 were investigated. Methods included: (a) culture and treatment of HepG2 cells with FA (IC50 : 580.32 μM, 24 h); (b) comet assay (DNA damage); (c) Western blots (protein expression of p53, MDM2, p-Ser-15-p53, a-K382-p53, a-CBP (K1535)/p300 (K1499), HDAC1 and p-Ser-47-Sirt1); and (d) Hoechst 33342 assay (apoptosis analysis). FA caused DNA damage in HepG2 cells relative to the control (P < 0.0001). FA decreased the protein expression of p53 (0.24-fold, P = 0.0004) and increased the expression of p-Ser-15-p53 (12.74-fold, P = 0.0126) and a-K382-p53 (2.24-fold, P = 0.0096). This occurred despite the significant decrease in the histone acetyltransferase, a-CBP (K1535)/p300 (K1499) (0.42-fold, P = 0.0023) and increase in the histone deacetylase, p-Ser-47-Sirt1 (1.22-fold, P = 0.0020). The expression of MDM2, a negative regulator of p53, was elevated in the FA treatment compared to the control (1.83-fold, P < 0.0001). FA also inhibited cell proliferation and induced apoptosis in HepG2 cells as evidenced by the Hoechst assay. Together, these results indicate that FA is genotoxic and post-translationally modified p53 leading to HepG2 cell death. J. Cell. Biochem. 118: 3866-3874, 2017. © 2017 Wiley Periodicals, Inc.

Coumarin-chalcone hybrid instigates DNA damage by minor groove binding and stabilizes p53 through post translational modifications

S009-131, a coumarin-chalcone hybrid, had been shown to possess anti-proliferative and anti-tumour effect by triggering apoptosis. In this report, we investigated role of DNA damage signalling pathway in S009-131 induced cancer cell death. Here we show that S009-131 causes DNA damage by potential binding to the minor groove which led to the phosphorylation and activation of ATM and DNA-PK, but not ATR, at earlier time points in order to initiate repair process. S009-131 induced DNA damage response triggered activation of p53 through phosphorylation at its key residues. Pharmacological inhibition of PIKKs abrogated S009-131 induced phosphorylation of p53 at Ser 15. DNA damage induced phosphorylation resulted in reduced proteasomal degradation of p53 by disrupting p53-MDM2 interaction. Additionally, our docking studies revealed that S009-131 might also contribute to increased cellular p53 level by occupying p53 binding pocket of MDM2. Posttranslational modifications of p53 upon S009-131 treatment led to enhanced affinity of p53 towards responsive elements (p53-RE) in the promoter regions of target genes and increased transcriptional efficiency. Together, the results suggest that S009-131 cleaves DNA through minor groove binding and eventually activates PIKKs associated DNA damage response signalling to promote stabilization and enhanced transcriptional activity of p53 through posttranslational modifications at key residues.