Modulation Of P53 Research Articles

P-118 - Protective effect of quercetin-quinone derivative in senescent human fibroblasts

Gradual accumulation of oxidatively damaged cell structures along with impairment of redox homeostasis has been suggested as a key factor in ageing and cellular senescence. Anti-ageing effects were reported for a number of phenol- and quinone-type compounds. By using a model of senescent human fibroblasts (strain VH-10), potential beneficial effects of semisynthetic compound, 4-O-(2-chloro-1,4-naphtoquinone-3-yloxy) quercetin (CHNQ) were investigated. CHNQ significantly suppressed senescence markers, SAβ-galactosidase activity and lipofuscin-related autofluorescence. Moreover, CHNQ, more efficiently than its precursors, restored mitochondrial membrane potential linked probably to observed decrease in ROS levels. In addition, CHNQ-treated cells showed increased LC3 turnover pointing to improvement of autophagy flux. By contrast, the compounds tested restored neither the gradual decline in proliferation, nor the cell cycle arrest at G2/M phase. Yet, modulation of p21 and p53 proteins dependently on the persistence time of senescent cells in culture was found. In conclusion, the result of our study point to protective effect of CHNQ on functionalities of senescent fibroblasts, without influencing their proliferative lifespan [VEGA 2/0041/17,2/0029/16,APVV-15–0308, ITMS 26240220040].

P-146 - Effects of paraquat in the reproductive function and on redox state of adult male rats

Paraquat (PQ) is an extremely toxic herbicide. The effects of the in vivo exposure to PQ on male fertility and on redox state were investigated in testis and sperm from adult male rats. Adult Wistar male rats received ip injection of 10 mg PQ/Kg body weight or saline during 5 days. After euthanasia, testes and epididymis were dissected and sperm cell viability, motility and teratospermia were evaluated. Moreover, the enzymatic activity of superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST) and mieloperoxidase (MPO), as well as the GSH levels and p53 expression were assessed in sperm cells and testis. Our data showed that the number, viability and total motility of sperm cells were significantly decreased by PQ exposure. Indeed, we observed an abnormal morphology; identifying teratospermia in PQ exposed animals. We also observed GSH depletion, which was associated with decreased activity of SOD and MPO, while CAT and GST were unaltered by PQ treatment. Moreover, PQ exposure increased p53 expression. The PQ-induced male reproductive toxicity is associated with oxidative stress and p53 modulation.

Inhibition of Proliferation and Induction of Apoptosis by Thymoquinone via Modulation of TGF Family, p53, p21 and Bcl-2α in Leukemic Cells.

Adult T-cell leukemia (ATL) is an aggressive form of malignancy caused by human T- cell lymphotropic virus 1 (HTLV-1). Currently, there is no effective treatment for ATL. Thymoquinone has been reported to have anti-cancer properties. The aim of this study is to investigatthe effects of TQ on proliferation, apoptosis induction and the underlying mechanism of action in both HTLV-1 positive (C91-PL and HuT-102) and HTLV-1 negative (CEM and Jurkat) malignant T-lymphocytes. Cells were incubated with different thymoquinone concentrations for 24h. Cell cytotoxicity was assayed using the CytoTox 96® Non-Radioactive Cytotoxicity Assay Kit. Cell proliferation was determined using CellTiter 96® Non-Radioactive Cell Proliferation. Cell cycle analysis was performed by staining with propidium iodide. Apoptosis was assessed using cell death ELISA kit. The effect of TQ on p53, p21, Bcl-2 protein expression was determined using Western blot analysis while TGF mRNA expression was determined by RT-PCR. At non-cytotoxic concentrations of TQ, it resulted in the inhibition of proliferation in a dose dependent manner. Flow cytometric analysis revealed a shift in the cell cycle distribution to the PreG1 phase which is a marker of apoptosis. Also TQ increase DNA fragmentation. TQ mediated its anti-proliferative effect and apoptosis induction by an up-regulation of TGFβ1, p53 and p21 and a down-regulation of TGF-α and Bcl-2α. Thymoquinone presents antiproliferative and proapoptotic effects in ATL cells. For this reason, further research is required to investigate its possible application in the treatment of ATL.

Evaluation of Anticancer Activity of Water‐Soluble Curcumin through the Induction of Apoptosis by p53 and p21 Modulation

AbstractAmide containing water soluble curcumin derivatives were synthesized and studied for their anti‐cancer activity. The synthesized water soluble compounds were cytotoxic when tested in vitro on HeLa cell and induced a p53 mediated apoptosis as evidenced by immunoblot analysis. Further DNA binding studies were also carried out to investigate the interaction between these synthesized curcumin compounds and HS‐DNA. The binding constant values (0.8167X104 M−1of 5a, 0.5443 X103 M−1 of 5b) suggested a good binding propensity to DNA. Physical properties such as stability, solubility, and log p values were investigated for the synthesized compounds.

Dram1 regulates DNA damage-induced alternative autophagy.

Autophagy is an evolutionarily conserved process that degrades subcellular constituents. Mammalian cells undergo two types of autophagy; Atg5-dependent conventional autophagy and Atg5-independent alternative autophagy, and the molecules required for the latter type of autophagy are largely unknown. In this study, we analyzed the molecular mechanisms of genotoxic stress-induced alternative autophagy, and identified the essential role of p53 and damage-regulated autophagy modulator (Dram1). Dram1 was sufficient to induce alternative autophagy. In the mechanism of alternative autophagy, Dram1 functions in the closure of isolation membranes downstream of p53. These findings indicate that Dram1 plays a pivotal role in genotoxic stress-induced alternative autophagy.

A Novel Hydroxamate-Based Compound WMJ-J-09 Causes Head and Neck Squamous Cell Carcinoma Cell Death via LKB1-AMPK-p38MAPK-p63-Survivin Cascade.

Growing evidence shows that hydroxamate-based compounds exhibit broad-spectrum pharmacological properties including anti-tumor activity. However, the precise mechanisms underlying hydroxamate derivative-induced cancer cell death remain incomplete understood. In this study, we explored the anti-tumor mechanisms of a novel aliphatic hydroxamate-based compound, WMJ-J-09, in FaDu head and neck squamous cell carcinoma (HNSCC) cells. WMJ-J-09 induced G2/M cell cycle arrest and apoptosis in FaDu cells. These actions were associated with liver kinase B1 (LKB1), AMP-activated protein kinase (AMPK) and p38 mitogen-activated protein kinase (p38MAPK) activation, transcription factor p63 phosphorylation, as well as modulation of p21 and survivin. LKB1-AMPK-p38MAPK signaling blockade reduced WMJ-J-09’s enhancing effects in p63 phosphorylation, p21 elevation and survivin reduction. Moreover, WMJ-J-09 caused an increase in α-tubulin acetylation and interfered with microtubule assembly. Furthermore, WMJ-J-09 suppressed the growth of subcutaneous FaDu xenografts in vivo. Taken together, WMJ-J-09-induced FaDu cell death may involve LKB1-AMPK-p38MAPK-p63-survivin signaling cascade. HDACs inhibition and disruption of microtubule assembly may also contribute to WMJ-J-09’s actions in FaDu cells. This study suggests that WMJ-J-09 may be a potential lead compound and warrant the clinical development in the treatment of HNSCC.

Chemical modulation of transcription factors.

Transcription factors (TFs) constitute a diverse class of sequence-specific DNA-binding proteins, which are key to the modulation of gene expression. TFs have been associated with human diseases, including cancer, Alzheimer's and other neurodegenerative diseases, which makes this class of proteins attractive targets for chemical biology and medicinal chemistry research. Since TFs lack a common binding site or structural similarity, the development of small molecules to efficiently modulate TF biology in cells and in vivo is a challenging task. This review highlights various strategies that are currently being explored for the identification and development of modulators of Myc, p53, Stat, Nrf2, CREB, ER, AR, HIF, NF-κB, and BET proteins.

Novel Allosteric Mechanism of P53 Activation by Small Molecules for Targeted Anticancer Therapy

Given the immense significance of p53 restoration for anti-cancer therapy, elucidation of the mechanisms of action of p53-activating molecules is of the utmost importance. Here we report a discovery of a novel allosteric modulation of p53 by small molecules, which is an unexpected turn in the p53 story. We identified a structural element involved in p53 regulation, whose targeting by RITA, PpIX and licofelone blocks the binding of p53 inhibitors, MDM2 and MDMX. Deletion and mutation analysis followed by molecular modelling, identified the key p53 residues S33 and S37 targeted by RITA and PpIX. We propose that the binding of small molecules to the identified site induces a conformational trap preventing p53 from the interaction with MDM2 and MDMX. These results point to a high potential of allosteric activators. Our study provides basis for the development of therapeutics with a novel mechanism of action, thus extending the p53 pharmacological potential.

LQFM030 reduced Ehrlich ascites tumor cell proliferation and VEGF levels

AimsThis study reports the biological properties of LQFM030 in vivo, a molecular simplification of the compound nutlin-1. Main methodsEhrlich ascites tumor (EAT)-bearing mice were treated intraperitoneally with LQFM030 (50, 75 or 150mg/kg) for 10days to determine changes in ascites tumor volume, body weight, cytotoxicity and angiogenesis. Moreover, flow cytometric expression of p53 and p21 proteins and caspase-3/7, −8 and −9 activation were investigated in EAT cells from mice treated. Acute oral systemic toxicity potential of LQFM030 in mice was also investigated using an alternative method. Key findingsTreatment of EAT-bearing mice with LQFM030 resulted in a marked decline in tumor cell proliferation and the vascular endothelial growth factor (VEGF) levels along with enhanced survival of the mice. Apoptotic tumor cell death was detected through p53 and p21 modulation and increase of caspase-3/7, −8 and −9 activity. LQFM030 also showed orally well tolerated, being classified in the UN GHS category 5 (LD50>2000–5000mg/Kg). SignificanceLQFM030 seems to be a promising antitumor candidate for combinatory therapy with typical cytotoxic compounds, reducing the toxicity burden while allowing a superior anticancer activity. Moreover, these data also open new perspectives for LQFM030 as an antiangiogenic agent for treatment of diseases involving VEGF overexpression.

The inhibitory effects of capillarisin on cell proliferation and invasion of prostate carcinoma cells.

Capillarisin (Cap), an active component of Artemisia capillaris root extracts, is characterized by its anti-inflammatory, anti-oxidant and anti-cancer properties. Nevertheless, the functions of Cap in prostate cancer have not been fully explored. We evaluated the potential actions of Cap on the cell proliferation, migration and invasion of prostate carcinoma cells. Cell proliferation and cell cycle distribution were measured by water-soluble tetrazolium-1 and flow cytometry assays. The expression of cyclins, p21, p27, survivin, matrix metallopeptidase (MMP2 and MMP9) were assessed by immunoblotting assays. Effects of Cap on invasion and migration were determined by wound closure and matrigel transmigration assays. The constitutive and interlukin-6 (IL-6)-inducible STAT3 activation of prostate carcinoma cells were determined by immunoblotting and reporter assays. Capillarisin inhibited androgen-independent DU145 and androgen-dependent LNCaP cell growth through the induction of cell cycle arrest at the G0/G1 phase by upregulating p21 and p27 while downregulating expression of cyclin D1, cyclin A and cyclin B. Cap decreased protein expression of survivin, MMP-2, and MMP-9 and therefore blocked the migration and invasion of DU145 cells. Cap suppressed constitutive and IL-6-inducible STAT3 activation in DU145 and LNCaP cells. Our data indicate that Cap blocked cell growth by modulation of p21, p27 and cyclins. The inhibitory effects of Cap on survivin, MMP-2, MMP-9 and STAT3 activation may account for the suppression of invasion in prostate carcinoma cells. Our data suggest that Cap might be a therapeutic agent in treating advanced prostate cancer with constitutive STAT3 or IL-6-inducible STAT3 activation.

The Oncogenic Role of Tribbles 1 in Hepatocellular Carcinoma Is Mediated by a Feedback Loop Involving microRNA-23a and p53.

Hepatocellular carcinoma (HCC) is a common malignancy associated with a high risk of recurrence and metastasis and a poor prognosis. Here, we examined the involvement of the pseudokinase Tribbles 1 (TRIB1), a scaffold protein associated with several malignancies, in HCC and investigated the underlying mechanisms. TRIB1 was upregulated in HCC tissues and cell lines in correlation with low levels of p53. TRIB1 gain and loss of function experiments indicated that TRIB1 promoted HCC cell viability concomitant with the downregulation of p53, and induced HCC cell migration, invasion, and epithelial-mesenchymal transition. TRIB1 was identified as a target of microRNA-23a (miR-23a), and miR-23a overexpression downregulated TRIB1 and upregulated p53 in HCC cells. Ectopic expression of TRIB1 upregulated β-catenin and its effectors c-myc and MMP-7 in a p53-dependent manner. TRIB1 silencing inhibited tumor growth and promoted apoptosis in vivo via a mechanism that would involve the modulation of p53 and β-catenin signaling. The present results indicate that TRIB1 promotes HCC tumorigenesis and invasiveness via a feedback loop that involves the modulation of its expression by miR-23a with the likely downregulation of p53, and suggest the involvement of the β-catenin signaling pathway. These findings suggest potential targets for the treatment of HCC and therefore merit further investigation.

Punica granatum L. Fruit Aqueous Extract Suppresses Reactive Oxygen Species-Mediated p53/p65/miR-145 Expressions followed by Elevated Levels of irs-1 in Alloxan-Diabetic Rats.

Objective Reactive oxygen species (ROS) is an apoptosis inducer in pancreatic β-cells that stimulates p53/p65 mediated microRNA (miR)-145 expression. Punica granatum L. (pomegranate) is an antioxidant fruit that attenuates ROS generation. This study examines the effects of pomegranate fruit aqueous extract (PGE) on the levels of ROS, p53, p65, miR-145, and its target insulin receptor substrate 1 (irs-1) mRNA in Alloxan-diabetic male Wistar rats. Materials and Methods In this experimental study, diabetic rats received different doses of PGE. The effects of the PGE polyphenols were examined through a long-term PGE treatment period model, followed by an evaluation of the plasma and tissue contents of free fatty acids (FFAs), triglycerides (TG), and glycogen compared with diabetic controls (DC)and normal controls (NC). We used real-time polymerase chain reaction (PCR) to investigate the modulation of p53, p65, miR-145, and irs-1 expression levels. Results There was a noticeable reduction in fasting blood glucose (FBG) and ROS generation compared to DC. We observed marked decreases in p53, p65, miR-145 expression levels followed by an elevated level of irs-1, which contributed to improvement in insulin sensitivity. ConclusionPGE administration downregulated miR-145 levels in Alloxan-diabetic Wistar rats by suppression of ROS-mediated p53 and p65 overexpression.

153 - Modulation of p53 by DUSP4 Serves a Novel Therapeutic Strategy in the Treatment of Ischemic Heart Disease via Promoting Angiogenesis

After an ischemia and reperfusion (I/R) injury, reestablishing blood flow to the infarct region can salvage myocardium by clearing apoptotic cells, removing scar tissue, stimulating angiogenesis, and recruiting progenitor cells for tissue regeneration. Stimulating the growth of new microcirculation in the infarct area can help revive cardiac function. Angiogenesis is a process of developing new blood vessels from the existing vessels. Our previous work on Dual specificity phosphatase 4 (DUSP4) demonstrated that DUSP4-/- hearts sustain a larger infarct and have poor functional recovery, when isolated hearts were subjected to I/R. Uncontrolled p38 activation is the main effectors for this functional alteration. In this study, DUSP4 overexpression in endothelial cells was used to investigate the role of DUSP4 in the development of vascular function, and its role against oxidant stress. DUSP4 overexpression promotes angiogenesis and tube formation (increased by 57.3% ± 6.2% versus control; p

Combined Virtual and Experimental Screening for CK1 Inhibitors Identifies a Modulator of p53 and Reveals Important Aspects of in Silico Screening Performance

A compound collection of pronounced structural diversity was comprehensively screened for inhibitors of the DNA damage-related kinase CK1. The collection was evaluated in vitro. A potent and selective CK1 inhibitor was discovered and its capacity to modulate the endogenous levels of the CK1-regulated tumor suppressor p53 was demonstrated in cancer cell lines. Administration of 10 μM of the compound resulted in significant increase of p53 levels, reaching almost 2-fold in hepatocellular carcinoma cells. In parallel to experimental screening, two representative and orthogonal in silico screening methodologies were implemented for enabling the retrospective assessment of virtual screening performance on a case-specific basis. Results showed that both techniques performed at an acceptable and fairly comparable level, with a slight advantage of the structure-based over the ligand-based approach. However, both approaches demonstrated notable sensitivity upon parameters such as screening template choice and treatment of redundancy in the enumerated compound collection. An effort to combine insight derived by sequential implementation of the two methods afforded poor further improvement of screening performance. Overall, the presented assessment highlights the relation between improper use of enrichment metrics and misleading results, and demonstrates the inherent delicacy of in silico methods, emphasizing the challenging character of virtual screening protocol optimization.

PARP-1 and p53 Regulate the Increased Susceptibility to Oxidative Death of Lymphocytes from MCI and AD Patients.

Mild cognitive impairment (MCI) is a clinically detectable initial stage of cognitive deterioration with a high conversion rate to dementia. There is increasing evidence that some of the cerebral alterations present in Alzheimer type dementia can be found in peripheral tissues. We have previously shown that lymphocytes from Alzheimer’s disease (AD) patients have increased susceptibility to hydrogen peroxide (H2O2)-induced death that depends on dementia severity. We here investigated whether lymphocytes from MCI patients show increased vulnerability to death, and explored the involvement of Poly [ADP-ribose] polymerase (PARP-1) and p53 in the regulation of this process. Lymphocytes from 16 MCI and 10 AD patients, and 15 healthy controls (HCs) were submitted to increasing concentrations of H2O2 for 20 h. Cell death was determined by flow cytometry, in the presence or absence of PARP-1 inhibitors (3-aminobenzamide (3-ABA) or Nicotinamide (NAM)), or the p53 inhibitor (nutlin-3) or stabilizer (pifithrin-α). PARP-1 and p53 mRNA levels were determined by quantitative PCR (qPCR). Lymphocytes from MCI patients showed increased susceptibility to death, attaining intermediate values between AD and controls. PARP inhibitors -3-ABA and NAM- markedly protected from H2O2-induced death, making the difference between MCI and controls disappear, but not the difference between AD and controls. PARP-1 mRNA expression was increased in MCI lymphocytes. Modulation of p53 with Nutlin-3 or pifithrin-α did not modify the H2O2-induced death of lymphocytes from MCI or AD patients, but augmented the death in control lymphocytes attaining levels similar to MCI and AD. Accordingly, p53 mRNA expression was increased in AD and MCI lymphocytes compared to controls. In all, these results show that increased oxidative death is present in lymphocytes at the MCI stage. PARP-1 has a preponderant role, with complete death protection achieved with PARP inhibition in MCI lymphocytes, but not in AD, suggesting that PARP-1 might have a protective role. In addition, deregulations of the p53 pathway seem to contribute to the H2O2-induced death in MCI and AD lymphocytes, which show increased p53 expression. The results showing a prominent protective role of PARP inhibitors opens the door to study the use of these agents to prevent oxidative death in MCI patients.

TRAIL enhances quinacrine-mediated apoptosis in breast cancer cells through induction of autophagy via modulation of p21 and DR5 interactions.

Previously, we reported that quinacrine (QC) may cause apoptosis in breast and colon cancer cells by activating the death receptor 5 (DR5), resulting in autophagic cell death through p21 modulation. Here, we systematically evaluated the combined role of p21 and DR5 and their crosstalk in QC-mediated autophagy and apoptosis in breast cancer cells using in vitro and in vivo models. Multiple breast cancer-derived cell lines (MCF-7, ZR-75-1, T47D, MDA-MB-231 and MCF-10A-Tr) and a mouse xenograft model were used. Also, multiple assays, including Western blotting, immunoprecipitation, staining for autophagy and apoptosis, gene silencing, hematoxylin and eosin staining, immunohistochemistry, cell viability assessment, fluorescence imaging and cell sorting were used. We found that QC activates p21 and DR5 in combination with the apoptosis inducer TRAIL in the breast cancer-derived cells tested. Combined TRAIL and QC treatment increased autophagy and apoptosis by increasing the interaction between, and co-localization of, p21 and DR5 in the death-inducing signaling complex (DISC). We found that this combination also inhibited the mTOR/PI3K/AKT signaling cascade and modulated reactive oxygen species (ROS) and nitric oxide (NO) production. Reductions in autophagy and apoptosis in DR5-knockout cells and a lack of change in p21-DR5-silenced cells were noted after TRAIL + QC treatment. This result explains dependence of the death (autophagy and apoptosis) cascade on these two key regulatory proteins. In addition, we found in an in vivo mouse xenograft model that increased expression and enhanced co-localization of p21 and DR5 after TRAIL + QC treatment supported a joint regulatory role of these proteins in the co-prevalence of autophagy and apoptosis. Our data suggest that a combined treatment of TRAIL and QC causes cell death in breast cancer-derived cells via autophagy and apoptosis by increasing the interaction of p21 and DR5, as indicated by both in vitro and in vivo studies.

HZ-6d targeted HERC5 to regulate p53 ISGylation in human hepatocellular carcinoma

Manipulating the posttranslational modulator of p53 is central in the regulation of its activity and function. ISGylated p53 can be degraded by the 20S proteasome. During this process, HERC5/Ceb1, an IFN-induced HECT-type E3 ligase, mediated p53 ISGylation. In this study, we indicated that HERC5 was over-expressed in both HCC tissue samples and cell lines. Knockdown of HERC5 significantly induced the expression of p53, p21 and Bax/Bcl-2 in HCC cells, resulting in apoptosis augment. Whereas, opposite results were obtained by using HERC5 over-expression. On this basis, we screened a 7, 11-disubstituted quinazoline derivative HZ-6d that could bind to the HERC5 G-rich sequence in vitro. Interestingly, HZ-6d injection effectively delayed the growth of xenografts in nude mice. In vitro, HZ-6d significantly inhibited cell growth, suppressed cell migration, induced apoptosis in HCC cells. Further studies demonstrated the anti-cancer effect of HZ-6d was associated with down-regulation of HERC5 and accumulation of p53. Collectively, we demonstrated that HZ6d is a HERC5 G-quadruplex ligand with anti-tumor properties, an action that may offer an attractive idea for restoration of p53 function in cancers.

Abstract 1372: Towards novel strategies of targeting specific vulnerabilities of T-PLL cells

Abstract T-cell prolymphocytic leukemia (T-PLL) is a mature T-cell neoplasm usually presenting at an aggressive phase and a marked resistance to conventional cytostatics, i.e. alkylators. The monoclonal antibody alemtuzumab induces high response rates, but virtually all patients relapse and the mostly elderly individuals are often ineligible for allogeneic stem cell transplantation. Overall, the treatment options for T-PLL are scarce and its general prognosis with average survival times of <3 yrs. remains poor. In agreement with the pronounced amenability of T-cell tumors to intervention in the altered epigenetic code, recent reports also implicate a high potential of epigenetic targeting in T-PLL. In fact, profiling of anti-nucleoside sensitivities, including clinically used agents like fludarabine or nelarabine, revealed that the purine analog cladribine showed the highest T-PLL specific activity (LD50s: 0.16µM in T-PLL; 2.95µM in healthy-donor T-cells, 2.81µM in PBMC), potentially through its epigenetically active pyrimidine function. Fittingly, high-throughput genomic analysis of 95 T-PLL revealed alterations enriched in DNA repair genes and histone / DNA epigenetic modifiers. Extensive ex vivo drug screens (306 substances, 39 T-PLL) corroborated the genomic data and identified major clusters of T-PLL-active substances. The CDK inhibitor SNS-032 induced the highest T-PLL specific responses, followed by p53 activators, BCL2-family antagonists, and modulators of protein acetylation and chromatin accessibility, all surpassing the effects of JAK/STAT-inhibitors. The BCL2 inhibitor ABT199 (Venetoclax) profoundly affected T-PLL cell viability (LD50=6.9nM), although apoptosis was induced more efficiently in CLL cells (LD50=2.4nM). This slightly lower cytotoxicity in T-PLL might be explained by the more heterogeneous BCL2 expression in this disease than in CLL (flow-cytometry and gene expression arrays; 70 T-PLL). The best-performing substance of the set, SNS-032, selectively and profoundly induced apoptosis in T-PLL (LD50=0.19µM vs 0.47µM in normal T-cells vs 0.88µM in PBMC). Recognized as a CDK inhibitor, it antagonizes transcription via targeting CDK7 and -9. We identified associations of protein levels of the transcription factor MYC with the in vitro responses to SNS-032. Amplified cMYC is one of the genomic hallmarks of T-PLL and implicated in its aggressive phenotype. Hence it may be a target of SNS-032 or serve as a biomarker for SNS-032 sensitivity.In summary, we identified several substances with marked anti-leukemic activity in T-PLL. In vitro studies confirmed their potency and selectivity that could be aligned with identified protein and genomic-lesional target patterns. Follow-up studies of members of the major clusters of most T-PLL-active compounds including their best-informed combinations and pre-clinical in-vivo testing will guide in the design of promising clinical trials in T-PLL. Note: This abstract was not presented at the meeting. Citation Format: Sabine Puetzer, Emma Andersson, Alexandra Schrader, Lesley Varghese, M Hülsemann, Petra Mayer, Marc-Henri Stern, Sebastian Newrzela, L Frenzel, Satu Mustjoki, Marco Herling. Towards novel strategies of targeting specific vulnerabilities of T-PLL cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1372. doi:10.1158/1538-7445.AM2017-1372

Astaxanthin from shrimp efficiently modulates oxidative stress and allied cell death progression in MCF-7 cells treated synergistically with β-carotene and lutein from greens

This study investigated the synergistic efficacy of keto-carotenoid astaxanthin (AST, from shrimp) plus hydrocarbon (β-carotene, BC) and hydroxyl (lutein, L) carotenoids (from greens) on molecular events in MCF-7 cells. MCF-7 cells were treated with either of carotenoid (20 μM, AST or BC or L) separately or the mixture of them (an equimolar concentration of carotenoids mixture, CM) or saponified carotenoid extract from shrimp (SSCE) for 48 h and analyzed cellular uptake, cytotoxicity, and apoptosis. The IC50 and combination-index values of AST co-treatment with a lower concentration of BC and L (5 μM) exhibited enhanced cytotoxicity and oxidative stress as compared with individual carotenoids or SSCE. Further, higher cellular uptake/accumulation of AST along with BC and L found to synergistically induce apoptosis through modulation of cyclin D1, p53, Bax and Bcl-2 expressions by arresting cell cycle at G0/G1 phase. Further, CM or SSCE treatments are unlikely to affect proliferation of normal breast epithelial cells (MCF-10A). The results of selective killing of MCF-7 cells demonstrated a greater insight on the synergistic effect of shrimp AST plus BC and L. It is concluded that consumption of shrimp along with green leafy vegetables helps in combating cancer chemoprevention.

TRIM8 restores p53 tumour suppressor function by blunting N-MYC activity in chemo-resistant tumours

BackgroundTRIM8 plays a key role in controlling the p53 molecular switch that sustains the transcriptional activation of cell cycle arrest genes and response to chemotherapeutic drugs. The mechanisms that regulate TRIM8, especially in cancers like clear cell Renal Cell Carcinoma (ccRCC) and colorectal cancer (CRC) where it is low expressed, are still unknown. However, recent studies suggest the potential involvement of some microRNAs belonging to miR-17-92 and its paralogous clusters, which could include TRIM8 in a more complex pathway.MethodsWe used RCC and CRC cell models for in-vitro experiments, and ccRCC patients and xenograft transplanted mice for in vivo assessments. To measure microRNAs levels we performed RT-qPCR, while steady-states of TRIM8, p53, p21 and N-MYC were quantified at protein level by Western Blotting as well as at transcript level by RT-qPCR. Luciferase reporter assays were performed to assess the interaction between TRIM8 and specific miRNAs, and the potential effects of this interaction on TRIM8 expression. Moreover, we treated our cell models with conventional chemotherapeutic drugs or tyrosine kinase inhibitors, and measured their response in terms of cell proliferation by MTT and colony suppression assays.ResultsWe showed that TRIM8 is a target of miR-17-5p and miR-106b-5p, whose expression is promoted by N-MYC, and that alterations of their levels affect cell proliferation, acting on the TRIM8 transcripts stability, as confirmed in ccRCC patients and cell lines. In addition, reducing the levels of miR-17-5p/miR-106b-5p, we increased the chemo-sensitivity of RCC/CRC-derived cells to anti-tumour drugs used in the clinic. Intriguingly, this occurs, on one hand, by recovering the p53 tumour suppressor activity in a TRIM8-dependent fashion and, on the other hand, by promoting the transcription of miR-34a that turns off the oncogenic action of N-MYC. This ultimately leads to cell proliferation reduction or block, observed also in colon cancer xenografts overexpressing TRIM8.ConclusionsIn this paper we provided evidence that TRIM8 and its regulators miR-17-5p and miR-106b-5 participate to a feedback loop controlling cell proliferation through the reciprocal modulation of p53, miR-34a and N-MYC. Our experiments pointed out that this axis is pivotal in defining drug responsiveness of cancers such ccRCC and CRC.