Induction Of P21 Expression Research Articles

Abstract 2409: FLIP(L) determines colon cancer cell fate following p53 activation

Abstract Background: How p53 differentially activates cell cycle arrest versus cell death remains poorly understood. Understanding the mechanisms governing the switch from p53-induced cell-fates is important for optimising efficacy of p53-activating therapies, such as DNA damaging chemotherapy and radiotherapy. HDAC inhibitors (HDACi) have potential to enhance p53 induced cell death through enhancing p53 activation and altering regulation of other cell death regulatory proteins. Methods: We utilised a panel of matched p53 wild-type and deficient colorectal cancer cell line models to the potential for HDACi to augment cell death induced by direct and indirect p53 activating agents. A number of molecular (Western blot, RT-PCR), phenotypic (cell death) and functional genomic (RNA-seq, CRISPR, ChIP-seq) analyses were used to investigate the importance of p53 and its downstream transcriptional programs. Results: Here we report that upregulation of canonical pro-apoptotic p53 target genes in colon cancer cells imposes critical dependence on the long splice form of the caspase-8 regulator FLIP (FLIP(L)), which we identify as a direct p53 transcriptional target. Inhibiting FLIP(L) expression with siRNA or Class-I HDAC inhibitors promotes apoptosis in response to p53 activation by the MDM2 inhibitor Nutlin-3A, which otherwise predominantly induces cell-cycle arrest. When FLIP(L) upregulation is inhibited, apoptosis is induced in response to p53 activation via a ligand-independent TRAIL-R2/caspase-8 complex, which is distinct from the ligand-dependent DISC. Notably, FLIP(L) depletion inhibits p53-induced expression of the cell cycle regulator p21 and enhances p53-mediated upregulation of PUMA, with the latter activating mitochondrial-mediated apoptosis in FLIP(L)-depleted, Nutlin-3A-treated cells lacking TRAIL-R2/caspase-8. Conclusion: Acute p53-mediated transcriptional upregulation of FLIP(L) plays an unexpected nodal role in determining cell fate following p53 activation. This is mediated through two previously undescribed mechanisms, preventing apoptosis by a ligand-independent TRAIL-R2 complex and by suppressing expression of pro-apoptotic PUMA. Which, importantly imposes a critical dependence on FLIP(L) which can be overcome through combinations with class-I HDAC inhibitors such as Entinostat. Citation Format: Andrea Lees, Alexander J. McIntyre J. McIntyre, Nyree T. Crawford, Fiammetta Falcone, Chris McCann, Gerard P. Quinn, Jamie Z. Roberts, Thomas Sessler, Peter F. Gallagher, Gemma M. Gregg, Katherine McAllister, Kirsty M. McLaughlin, Wendy L. Allen, Caitriona Holohan, Laurence J. Egan, Aideen E. Ryan, Melissa Labonte-Wilson, Phillip D. Dunne, Mark Wappett, Vicky M. Coye, Patrick G. Johnston, Emma M. Kerr, Daniel B. Longley, Simon S. McDade. FLIP(L) determines colon cancer cell fate following p53 activation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2409.

2-O-Methylmagnolol, a Magnolol Derivative, Suppresses Hepatocellular Carcinoma Progression via Inhibiting Class I Histone Deacetylase Expression

Magnolia officinalis is widely used in Southeast Asian countries for the treatment of fever, headache, diarrhea, and stroke. Magnolol is a phenolic compound extracted from M. officinalis, with proven antibacterial, antioxidant, anti-inflammatory, and anticancer activities. In this study, we modified magnolol to synthesize a methoxylated derivative, 2-O-methylmagnolol (MM1), and investigated the use of MM1, and magnolol in the treatment of liver cancer. We found that both magnolol and MM1 exhibited inhibitory effects on the growth, migration, and invasion of hepatocellular carcinoma (HCC) cell lines and halted the cell cycle at the G1 phase. MM1 also demonstrated a substantially better tumor-suppressive effect than magnolol. Further analysis suggested that by inhibiting class I histone deacetylase expression in HCC cell lines, magnolol and MM1 induced p21 expression and p53 activation, thereby causing cell cycle arrest and inhibiting HCC cell growth, migration, and invasion. Subsequently, we verified the significant tumor-suppressive effects of magnolol and MM1 in an animal model. Collectively, these findings demonstrate the anti-HCC activities of magnolol and MM1 and their potential for clinical use.

FK506 induces lung lymphatic endothelial cell senescence and downregulates LYVE-1 expression, with associated decreased hyaluronan uptake

BackgroundTherapeutic lymphangiogenesis in an orthotopic lung transplant model has been shown to improve acute allograft rejection that is mediated at least in part through hyaluronan drainage. Lymphatic vessel endothelial hyaluronan receptor (LYVE-1) expressed on the surface of lymphatic endothelial cells plays important roles in hyaluronan uptake. The impact of current immunosuppressive therapies on lung lymphatic endothelial cells is largely unknown. We tested the hypothesis that FK506, the most commonly used immunosuppressant after lung transplantation, induces lung lymphatic endothelial cell dysfunction.MethodsLung lymphatic endothelial cells were cultured in vitro and treated with FK506. Telomerase activity was measured using the TRAP assay. Protein expression of LYVE-1 and senescence markers p21 and β-galactosidase was assessed with western blotting. Matrigel tubulation assay were used to investigate the effects of FK506 on TNF-α-induced lymphangiogenesis. Dual luciferase reporter assay was used to confirm NFAT-dependent transcriptional regulation of LYVE-1. Flow cytometry was used to examine the effects of FK506 on LYVE-1 in precision-cut-lung-slices ex vivo and on hyaluronan uptake in vitro.ResultsIn vitro, FK506 downregulated telomerase reverse transcriptase expression, resulting in decreased telomerase activity and subsequent induction of p21 expression and cell senescence. Treatment with FK506 decreased LYVE-1 mRNA and protein levels and resulted in decreased LEC HA uptake. Similar result showing reduction of LYVE-1 expression when treated with FK506 was observed ex vivo. We identified a putative NFAT binding site on the LYVE-1 promoter and cloned this region of the promoter in a luciferase-based reporter construct. We showed that this NFAT binding site regulates LYVE-1 transcription, and mutation of this binding site blunted FK506-dependent downregulation of LYVE-1 promoter-dependent transcription. Finally, FK506-treated lymphatic endothelial cells show a blunted response to TNF-α-mediated lymphangiogenesis.ConclusionFK506 alters lymphatic endothelial cell molecular characteristics and causes lymphatic endothelial cell dysfunction in vitro and ex vivo. These effects of FK506 on lymphatic endothelial cell may impair the ability of the transplanted lung to drain hyaluronan macromolecules in vivo. The implications of our findings on the long-term health of lung allografts merit more investigation.

Carbonic anhydrase IV inhibits cell proliferation in gastric cancer by regulating the cell cycle.

Carbonic anhydrase IV (CA4) is silenced in colorectal cancer. However, the effect of CA4 on the development of gastric cancer (GC) is poorly understood. The present study aimed to determine the role of CA4 in GC tumorigenesis and its underlying molecular mechanism. The levels of CA4 in GC cells and tissues were evaluated by reverse transcription-quantitative PCR and immunohistochemistry. CA4 expression was suppressed in GC cells and tissues compared with adjacent healthy tissues and normal human gastric epithelial cells, respectively. This reduced expression was significantly associated with tumor size, invasion and differentiation. Analyses with a real-time cell analyzer and clonogenic assays were conducted to validate the impact of CA4 on GC cell lines (AGS and HGC-27) and normal human gastric epithelial cell line (GES-1) proliferation. The effects of CA4 on the cell cycle in GC cells were determined by flow cytometry. The levels of CA4 and cell cycle-associated proteins were confirmed by western blotting. CA4 overexpression inhibited GC cell proliferation and reduced colony-forming ability, arrested the cell cycle in the G2/M phase, inhibited cyclin B1 and cyclin-dependent kinase 2 expression and induced p21 expression. These results indicate that CA4 may serve an important role in GC tumorigenesis by inhibiting cellular proliferation via regulating the expression of cell cycle-associated proteins. CA4 may serve as a diagnostic biomarker and a potential therapeutic target in GC.

The pseudo-caspase FLIP(L) regulates cell fate following p53 activation

p53 is the most frequently mutated, well-studied tumor-suppressor gene, yet the molecular basis of the switch from p53-induced cell-cycle arrest to apoptosis remains poorly understood. Using a combination of transcriptomics and functional genomics, we unexpectedly identified a nodal role for the caspase-8 paralog and only human pseudo-caspase, FLIP(L), in regulating this switch. Moreover, we identify FLIP(L) as a direct p53 transcriptional target gene that is rapidly up-regulated in response to Nutlin-3A, an MDM2 inhibitor that potently activates p53. Genetically or pharmacologically inhibiting expression of FLIP(L) using siRNA or entinostat (a clinically relevant class-I HDAC inhibitor) efficiently promoted apoptosis in colorectal cancer cells in response to Nutlin-3A, which otherwise predominantly induced cell-cycle arrest. Enhanced apoptosis was also observed when entinostat was combined with clinically relevant, p53-activating chemotherapy in vitro, and this translated into enhanced in vivo efficacy. Mechanistically, FLIP(L) inhibited p53-induced apoptosis by blocking activation of caspase-8 by the TRAIL-R2/DR5 death receptor; notably, this activation was not dependent on receptor engagement by its ligand, TRAIL. In the absence of caspase-8, another of its paralogs, caspase-10 (also transcriptionally up-regulated by p53), induced apoptosis in Nutlin-3A-treated, FLIP(L)-depleted cells, albeit to a lesser extent than in caspase-8-proficient cells. FLIP(L) depletion also modulated transcription of canonical p53 target genes, suppressing p53-induced expression of the cell-cycle regulator p21 and enhancing p53-induced up-regulation of proapoptotic PUMA. Thus, even in the absence of caspase-8/10, FLIP(L) silencing promoted p53-induced apoptosis by enhancing PUMA expression. Thus, we report unexpected, therapeutically relevant roles for FLIP(L) in determining cell fate following p53 activation.

Mithramycin selectively attenuates DNA-damage-induced neuronal cell death

DNA damage triggers cell death mechanisms contributing to neuronal loss and cognitive decline in neurological disorders, including traumatic brain injury (TBI), and as a side effect of chemotherapy. Mithramycin, which competitively targets chromatin-binding sites of specificity protein 1 (Sp1), was used to examine previously unexplored neuronal cell death regulatory mechanisms via rat primary neurons in vitro and after TBI in mice (males). In primary neurons exposed to DNA-damage-inducing chemotherapy drugs in vitro we showed that DNA breaks sequentially initiate DNA-damage responses, including phosphorylation of ATM, H2AX and tumor protein 53 (p53), transcriptional activation of pro-apoptotic BH3-only proteins, and mitochondrial outer membrane permeabilization (MOMP), activating caspase-dependent and caspase-independent intrinsic apoptosis. Mithramycin was highly neuroprotective in DNA-damage-dependent neuronal cell death, inhibiting chemotherapeutic-induced cell death cascades downstream of ATM and p53 phosphorylation/activation but upstream of p53-induced expression of pro-apoptotic molecules. Mithramycin reduced neuronal upregulation of BH3-only proteins and mitochondrial dysfunction, attenuated caspase-3/7 activation and caspase substrates’ cleavage, and limited c-Jun activation. Chromatin immunoprecipitation indicated that mithramycin attenuates Sp1 binding to pro-apoptotic gene promoters without altering p53 binding suggesting it acts by removing cofactors required for p53 transactivation. In contrast, the DNA-damage-independent neuronal death models displayed caspase initiation in the absence of p53/BH3 activation and were not protected even when mithramycin reduced caspase activation. Interestingly, experimental TBI triggers a multiplicity of neuronal death mechanisms. Although markers of DNA-damage/p53-dependent intrinsic apoptosis are detected acutely in the injured cortex and are attenuated by mithramycin, these processes may play a reduced role in early neuronal death after TBI, as caspase-dependent mechanisms are repressed in mature neurons while other, mithramycin-resistant mechanisms are active. Our data suggest that Sp1 is required for p53-mediated transactivation of neuronal pro-apoptotic molecules and that mithramycin may attenuate neuronal cell death in conditions predominantly involving DNA-damage-induced p53-dependent intrinsic apoptosis.

Overexpression of p21 Has Inhibitory Effect on Human Hematopoiesis by Blocking Generation of CD43＋ Cells via Cell-Cycle Regulation.

Background and Objectivesp21, an important member of the Cip/Kip family, is involved in inhibitory effects of RUNX1b overexpression during the early stage of human hematopoiesis.Methods and ResultsWe established a human embryonic stem cell (hESC) line with inducible expression of p21 (p21/hESCs). Overexpression of p21 did not influence either mesoderm induction or emergence of CD34+ cells, but it significantly decreased the production of CD43+ cells and changed the expression profile of hematopoiesis-related factors, leading to the negative effects of p21 on hematopoiesis.ConclusionsIn RUNX1b/hESC co-cultures when RUNX1b was induced from D0, perturbation of the cell cycle caused by upregulation of p21 probably prevented the appearance of CD43+ cells, but not CD34+ cells. The mechanisms via which CD34+ cells are blocked by RUNX1b overexpression remain to be elucidated.

‘MUSCADINE GRAPE SKIN EXTRACT INHIBITS ANDROGEN‐INDEPENDENT PROSTATE CANCER CELL GROWTH, INDUCING CELL‐CYCLE ARREST, AND DECREASING MIGRATION BY TARGETING HEAT SHOCK PROTEIN 40

Previously we demonstrated that muscadine grape skin extract (MSKE), a natural product, significantly inhibited androgen‐responsive prostate cancer cell growth by inducing apoptosis through the targeting of survival pathways. However, the therapeutic effect of MSKE on more aggressive androgen‐independent prostate cancer remains unknown. This study examined the effects of MSKE treatment in metastatic prostate cancer using complementary PC‐3 cells and xenograft model. MSKE significantly inhibited PC‐3 human prostate cancer cell tumor growth in vitro and in vivo. The growth‐inhibitory effect of MSKE appeared to be through the induction of cell‐cycle arrest. This induction was accompanied by a reduction in the protein expression of Hsp40 and cell‐cycle regulation proteins, cyclin D1 and NF‐kBp65. In addition, MSKE induced p21 expression independent of wild‐type p53 induced protein expression. Moreover, we demonstrate that MSKE significantly inhibited cell migration in PC‐3 prostate cancer cells. Overall, these results demonstrate that MSKE inhibits prostate tumor growth and migration, and induces cell‐cycle arrest by targeting Hsp40 and proteins involved in cell‐cycle regulation and proliferation. This suggests that MSKE may also be explored either as a neo‐adjuvant or therapeutic for castration resistant prostate cancer.

PRMT5-TRIM21 interaction regulates the senescence of osteosarcoma cells by targeting the TXNIP/p21 axis.

Osteosarcoma (OS) is the most common bone malignancy in adolescents and has poor clinical outcomes. Protein arginine methyltransferase 5 (PRMT5) has recently been shown to be aberrantly expressed in various cancers, yet its role in OS remains elusive. Here, we found that PRMT5 was overexpressed in OS and its overexpression predicted poor clinical outcomes. PRMT5 knockdown significantly triggered pronounced senescence in OS cells, as evidenced by the increase in senescence-associated β-galactosidase (SA-β-gal)-stained cells, induction of p21 expression, and upregulation of senescence-associated secretory phenotype (SASP) gene expression. In addition, we found that PRMT5 plays a key role in regulating DNA damaging agents-induced OS cell senescence, possibly, via affecting the repair of DNA damage. Furthermore, we found that TXNIP acts as a key factor mediating PRMT5 depletion-induced DNA damage and cellular senescence. Mechanistically, TRIM21, which interacts with PRMT5, was essential for the regulation of TXNIP/p21 expression. In summary, we propose a model in which PRMT5, by interaction with TRIM21, plays a key role in regulating the TXNIP/p21 axis during senescence in OS cells. The present findings suggest that PRMT5 overexpression in OS cells might confer resistance to chemotherapy and that targeting the PRMT5/TRIM21/TXNIP signaling may enhance the therapeutic efficacy in OS.

Selective PRMT5 Inhibitors Suppress Human CD8+ T Cells by Upregulation of p53 and Impairment of the AKT Pathway Similar to the Tumor Metabolite MTA.

Genetic alterations in tumor cells provide promising targets for antitumor therapy. Recently, loss of methylthioadenosine phosphorylase (MTAP), a deletion frequently occurring in cancer, has been shown to create vulnerability to the inhibition of the protein arginine methyltransferase 5 (PRMT5). MTAP deficiency leads to accumulation of methylthioadenosine (MTA), which reduces PRMT5 activity, and thus, sensitizes the tumor cells to selective PRMT5 inhibitors (PRMT5i). PRMT5i are investigated as a new strategy to selectively kill MTAP-deficient tumor cells by blocking residual PRMT5 activity, but also to treat PRMT5-overexpressing tumors. Although many studies investigated the role of PRMT5 in cancer, only little data exist about the effect of PRMT5 inhibition on immune cells. As we could show that the tumor metabolite MTA suppresses T cells, we asked whether selective PRMT5 inhibition is detrimental for T-cell immune responses. Therefore, we examined the effect of the synthetic PRMT5 inhibitor EPZ015666 on human CD8+ T cells in direct comparison with the naturally occurring PRMT5-inhibiting molecule MTA. Both compounds reduced T-cell proliferation, viability, and functionality. In addition, T-cell metabolism was impaired upon PRMT5 inhibition. These effects coincided with the induction of p53 expression and reduced AKT/mTOR signaling. Our data clearly demonstrate that PRMT5 activity is involved in various cellular processes of human CD8+ T cells associated with essential T-cell functions. Therefore, not only tumor cells, but also antitumor immune responses, are compromised by PRMT5 inhibitors. This emphasizes the importance of considering side effects on the immune system when developing new strategies to specifically target not only MTAP-deficient tumors.

Dihydroartemisinin suppresses bladder cancer cell invasion and migration by regulating KDM3A and p21.

Emerging evidences have shown that Dihydroartemisinin (DHA), used in malaria treatment, possess anti-cancer activity. However, the study of its potential functional roles and the anti-cancer mechanisms in bladder cancer is limited. We performed this study to elucidate the influence of DHA in the biological behavior of bladder cancer cells and tried to explore the molecular mechanism. The results of CCK-8 assay showed that DHA significantly inhibited bladder cancer cell 5637, UMUC3 and T24 proliferation and the inhibitory effect is dose- and time- dependent. Further mechanism study showed that DHA performed its function via down-regulating the expression of histone demethylase KDM3A and inducing p21 expression. Moreover, wound healing and transwell migration/invasion assays revealed that DHA inhibited the ability of migration and metastasis in bladder cancer cell line T24. Finally, flow cytometry and colony formation assays demonstrated that DHA significantly promoted apoptosis of T24 cells and suppressed tumorigenesis as expected. Taken together, our study identifies the anti-cancer capacity of DHA in bladder cancer and explores the underlying mechanism.

Genipin increases oxaliplatin-induced cell death through autophagy in gastric cancer.

Oxaliplatin is used for treatment in combination with many drugs. However, the survival rate is still low due to side effects and drug resistance. Therefore, the combination with natural products was required for increasing efficacy and reducing side effects.Genipin, a natural product derived from the Gardenia jasminoides, associated with anti-angiogenic, anti-proliferative, hypertension, inflammatory, and the Hedgehog pathway. It is not known that genipin increases the therapeutic effect of oxaliplatin in gastric cancer. In this study, we found that genipin sensitizes oxaliplatin-induced apoptosis for the first time using colony forming assay, FACS analysis, and western blotting in gastric cancer. Additionally, genipin induced p53 expression in AGS, MKN45, and MKN28 cells. Also, genipin induced autophagy and LC3 expression. Knockdown of LC3 decreased cell death enhanced by the combination of oxaliplatin and genipin. In summary, we showed that genipin increases the oxaliplatin-induced cell death via p53-DRAM autophagy. Based on this, we suggest that genipin is a sensitizer of oxaliplatin.

This Is a Title in Title Case: A Combination of Low-Dose Decitabine and Chidamide Resulted in Synergistic Effects on the Proliferation and Apoptosis of Human Myeloid Leukemia Cell Lines

Introduction: Myeloid leukemia is a malignant disease caused by both genetic and epigenetic changes. Aberrant DNA methylation and histone modifications are prevalent in cancers including leukemia and the process of epigenetic modifications is reversible, which makes them potential treatment targets using specific inhibitors. In this study, we sought to determine the antileukemic effects of low-dose decitabine (one of the most widely used DNA methyltransferase inhibitor) combined with chidamide (a novel orally active histone deacetylase inhibitor) on myeloid leukemia cells by detecting cell proliferation, cell cycle distribution and cell apoptosis to provide a promising regimen for clinical application. Methods: We conducted CCK-8 assay to determine the effect of low-dose decitabine combinated with chidamide on cell viability, cell cycle distribution analysis by use of flow cytometry accompanied with RT-qPCR and western blotting analysis to reveal its inhibitory mechanism of cell growth, and cell apoptotic analysis also by use of flow cytometry accompanied with RT-qPCR, western blotting analysis, intracellular ROS production and mitochondrial transmembrane potential assay to determine the apoptotic mechanism of combination of low-dose decitabine and chidamide in K562 and THP-1 cells. Result: Low-dose decitabine and chidamide alone played an inhibitory role in the proliferation of K562 and THP-1 cells in a dose- and time-dependent manner, and the combination of the two enhanced this effect, accompanied by induction of p21 expression and cell cycle arrest at G0/G1 phase. Chidamide instead of decitabine had pronounced effects on the histone H3 acetylation levels of leukemia cells. Meanwhile, combination of the two significantly induced cell apoptosis via mitochondrial transmembrane potential loss, resulting in upregulation of the cell apoptosis-related gene Bax and the protein cleaved PARP-1 and downregulation of antiapoptosis gene Bcl-2, and proteins, including pro-caspase 9, pro-caspase 3 and PARP-1. Conclusion: Our results provided evidence for the antileukemia effects of low-dose DNA methyltransferase inhibitor decitabine combinated with histone deacetylase inhibitor chidamide in preclinical myeloid leukemia models and suggested that combination of the two showed therapeutic potential for myeloid leukemia treatment. Disclosures No relevant conflicts of interest to declare.

Celecoxib suppresses lipopolysaccharide-stimulated oral squamous cell carcinoma proliferation in vitro and in vivo.

Periodontitis is one of the most common chronic oral inflammatory conditions worldwide and is associated with a risk of developing oral squamous cell carcinoma (OSCC). Porphyromonas gingivalis is a major pathogen in periodontitis, and its lipopolysaccharide (LPS) promotes the expression of cyclooxygenase-2 (COX-2) in OSCC both in vivo and in vitro. Celecoxib is a selective COX-2 inhibitor; however, its antitumor effects on P. gingivalis LPS-stimulated OSCC and the underlying molecular mechanism remain unclear. To elucidate the association between periodontitis and OSCC, the effect of P. gingivalis-derived LPS on OSCC cell proliferation was examined both in vitro and in vivo in the present study. The expression levels of COX-2 and p53 in OSCC cells with/without celecoxib treatment were determined via western blotting. The therapeutic potential of celecoxib in LPS-stimulated OSCC was evaluated by staining for Ki-67 and p21, as well as with terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling staining. LPS treatment significantly increased OSCC cell proliferation in vitro, and celecoxib significantly inhibited cell proliferation with/without LPS treatment. Celecoxib treatment of OSCC cells downregulated the protein expression levels of COX-2 compared with untreated cells, but there was little change in p53 expression. In the mouse xenograft model, oral administration of celecoxib significantly suppressed tumor growth, reduced the expression of Ki-67, increased the apoptosis index and induced p21 expression with/without LPS treatment. The results from the present study demonstrate that P. gingivalis' LPS can stimulate tumor growth by interacting with OSCC cells. In conclusion, these results suggest that celecoxib could be used for the effective prevention and treatment of LPS-stimulated OSCC.

Abstract 4739: Induction of cell death by HDAC, Aromatase and MDM2 inhibitors in MCF-7 cells

Abstract The histone deacetylase (HDAC) enzymes are evolving as key enzymes in the regulation of many physiological processes, including chromatin remodeling, genome stability, DNA repair, regulation of transcription, protein secretion, cellular metabolism, cell cycle progression, differentiation, migration, and cell death. Among different regulators of HDACs, suberoylanilide hydroxamic acid (SAHA) is a potent inhibitor and it is known to cause strong growth arrest, differentiation and apoptosis of many tumor types in both in vitro and in vivo experimental models. Interestingly, treatment of MCF-7 breast cancer cells with SAHA was found to trigger necroptosis, which is a caspase independent form of cell death. Typically, stimulation of death domain receptors (DDR) by suitable ligands lead to the activation of RIP1, RIP3 and MLKL proteins which are the critical regulators of necroptosis. It appears that, during SAHA induced necroptosis elevation of p21 may be the main signal that triggers recruitment of RIP1, Phospho-RIP3 and MLKL intracellularly in MCF-7 cells. Also, our results confirmed that the apoptosis marker such as BAX and caspases are not elevated by SAHA in MCF-7 cells after 24 hrs of treatment. In addition, the fluorescence assay conducted with SYTOX Green after 10 and 50 µM necrostatin pre-treatments, confirmed that SAHA treatment activates RIP1 mediated necroptosis pathway in MCF-7 cells. The induction of p21 expression was also down-regulated significantly by 10 and 50 µM necrostatin pre-treatments, which further suggests that p21 is up-stream of the necroptosis executioners in MCF-7 cells. On the other hand, when the MCF-7 cells were treated with Letrazole and RG-7388, BAX appears to be involved in mediating cell death. Interestingly, both Letrazole and RG-7388 were not able to elevate p21 in MCF-7 cells. So far, our results suggest that SAHA can induce BAX independent necroptosis while Letrazole and RG-7388 induce cell death through elevation of BAX and BAK in MCF-7 cells. (The generous support from the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida is gratefully acknowledged). Citation Format: Thiagarajan Venkatesan, Umamaheswari Natarajan, Vijayaraghavan Radhakrishnan, Shila Samuel, Appu Rathinavelu. Induction of cell death by HDAC, Aromatase and MDM2 inhibitors in MCF-7 cells [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 4739.

Abstract 75: Anti-tumor activity of a novel selenonucleoside via targeting Skp2 degradation in paclitaxel-resistant prostate cancer

Abstract Prostate cancer (PC) is the most common disease in men over age 50, and its prevalence rate has been gradually increasing since 1980. Taxane-derived anticancer agents are the primary agents used to treat metastatic prostate cancer patients; however, the side effects and acquired drug resistance limit the success of these therapies. Because there is no specific treatment for paclitaxel-resistant prostate cancer, it is necessary to develop new targets and therapeutic strategies to overcome the acquired resistance. In this study, the antitumor activity of a novel selenonucleoside (4′-selenofuranosyl-2,6-dichloropurine, LJ-2618), a third-generation nucleoside, and its plausible mechanisms of action in paclitaxel-resistant prostate cancer (PC-3-Pa) cells were investigated. The established PC-3-Pa cells exhibited over 100-fold resistance against paclitaxel compared to the paclitaxel-sensitive PC-3 cells. LJ-2618, however, effectively inhibited the proliferation of both cell lines with similar IC50values in vitro. In PC-3-Pa cells, the activated PI3K/Akt signaling pathway was suppressed by LJ-2618 treatment. In addition, Skp2 was found to be over-expressed in paclitaxel-resistant cells, and the transfection of Skp2 siRNA recovered the sensitivity of paclitaxel in PC-3-Pa cells. Furthermore, LJ-2618 significantly down-regulated Skp2 expression in PC-3-Pa cells by promoting degradation and inducing destabilization of Skp2, which triggers G2/M cell cycle arrest. In a xenograft mouse model implanted with PC-3-Pa cells, LJ-2618 (3 or 10 mg/kg) effectively inhibited tumor growth with the enhancement of Skp2 degradation and induction of p27 expression in tumor tissues. These findings suggest that LJ-2618 may have a potential for overcoming paclitaxel resistance via promoting Skp2 degradation and stabilizing p27 expression in PC-3-Pa cells. Therefore, the novel selenonucleoside LJ-2618 may lead to the development of a new treatment strategy for patients with paclitaxel-resistant, castration-resistant prostate cancer. This study was supported by a National Research Foundation of Korea (NRF) Grant funded by the Korean Government (NRF-2016M3A9B6903499). Citation Format: Woong Sub Byun, Minkyung Jin, Jinha Yu, Won Kyung Kim, Lak Shin Jeong, Sang Kook Lee. Anti-tumor activity of a novel selenonucleoside via targeting Skp2 degradation in paclitaxel-resistant prostate cancer [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 75.

P53 mediates PEDF‑induced autophagy in human umbilical vein endothelial cells through sestrin2 signaling

Autophagy is a conserved catabolic process by which cytoplasmic components are delivered into lysosomes for degradation. Pigment epithelium-derived factor (PEDF) has been reported to be associated with autophagy and can induce p53 expression; however, the mechanism relating PEDF with autophagy in endothelial cells remains poorly understood. The present study aimed to investigate the association between the PEDF-p53-sestrin pathway and autophagy in human umbilical vein endothelial cells (HUVECs). PEDF-induced autophagy was examined by fluorescence microscopy and western blot analysis. p53 small interfering (si)RNA and sestrin2 siRNA were constructed and transfected into HUVECs prior to PEDF treatment. The protein expression levels of microtubule-associated protein light chain 3 (LC3) I, LC3 II and p62 were evaluated by western blot analysis, and the mRNA expression levels of p53 and sestrin2 were determined using reverse transcription-quantitative polymerase chain reaction analysis. The regulation of mechanistic target of rapamycin (mTOR) was reflected by p70S6 kinase (p70S6K) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) protein expression levels, as determined by western blot analysis. PEDF could induce HUVEC autophagy by sequentially inducing p53 and sestrin2 expression, as observed by fluorescence microscopy and western blot analysis. Conversely, the induction of sestrin2 by PEDF was eliminated by p53 siRNA. In addition, p53 siRNA and sestrin2 siRNA could attenuate PEDF-induced HUVEC autophagy. Inhibition of mTOR may be the mechanism responsible for PEDF-induced autophagy; as p70S6K and 4E-BP1 phosphorylation levels were significantly upregulated in p53 siRNA-treated and sestrin2 siRNA-treated groups. The findings of the present study indicated that PEDF may trigger autophagy in HUVECs by inducing p53 and sestrin2 expression, and inhibiting mTOR expression; these findings may contribute to the improved understanding of diseases, including cancer and atherosclerosis.

Effects of SAHA and RG7388 on p21WAF1/CIP1 and p27KIP1 mediated Pathways in Cancer Cells

Epigenetic modifications can lead to significant influence on cancer development, growth and progression. The main epigenetic modifications observed in human are methylation and acetylation. Histone acetylations are largely regulated by histone deacetylases (HDACs) and, several HDAC inhibitors (HDACi) such as SAHA (Vorinostat), which are already approved for human use or currently in different stages of clinical trials, belong to the new class of drugs with promising effects on the cancer growth and metastatic process. The small molecule RG7388 is a newly developed inhibitor that is specific for an oncogene‐derived protein called MDM2, which is also in clinical trials for the treatment of various types of cancers. One of the unique characteristics of these two drugs is their ability to induce p21 expression through distinct mechanisms. A clear understanding related to the molecular mechanism whereby SAHA can induce cell cycle arrest and trigger necrosis, apoptosis or necroptosis is still evolving. Similarly, the ability of RG7388 for producing anticancer effect is undergoing thorough investigation, since it can produce p53 dependent and p53 independent effects. So far, our experiments with MCF‐7 breast cancer cells and LNCaP prostate cancer cells have confirmed the abilities of these two drugs to induce p21 expression through distinct mechanisms. In addition, these two drugs were expected trigger cell cycle arrest and cell death through mechanisms that are similar. Therefore, we performed experiments to measure the cell cycle arrest effects of SAHA and RG7388 by using the MCF‐7 and LNCaP cells. The cytotoxicity, cell cycle arrest and apoptosis/necroptosis effects of the treatments were assessed by using Trypan Blue Dye Exclusion (TBDE) method, and fluorescence assay with caspase 3/7 specific DEVD‐amc fluorogenic substrate. In addition, cell cycle and apoptosis related proteins were analyzed by immunoblotting methods. To this point, our results from MCF‐7 and LNCaP cells suggest that the cell death caused by SAHA treatment in both cell lines are through induction of p21WAF1/CIP1 and p27Kip1 levels, that could be independent of p53. On the other hand, treatment of LNCaP cells with RG7388 was able to induce p21WAF1/CIP1 expression through inhibition of MDM2 that was also causing significant cell death. However, the RG7388 treatment was not able to elevate p21WAF1/CIP1 in MCF‐7 cells, even though there was clear evidence of p53 elevation in these cells. Hence, we are suspecting that, there is some level of uncoupling of p53 mediated transcriptional induction of p21WAF1/CIP1 in MCF‐7 cells. As of now, our studies confirm that two different drugs, which have the ability to induce p21 expression, exhibit distinct mechanisms of cell death through p53 dependent and independent pathways. Since it points to an interesting interplay between multiple pathways, confirmation of the actual cell death mechanism induced by RG7388 in MCF‐7 and LNCaP cancer cells requires additional exploration.Support or Funding InformationThis research was supported by the Royal Dames of Cancer Research Inc., Fort Lauderdale, FloridaThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

8-Acetonyldihydronitidine inhibits the proliferation of human colorectal cancer cells via activation of p53

8-Acetonyldihydronitidine (8-AHN) is a potent antitumor compound extracted from Toddalia asiatica. However, the precise molecular antitumor mechanisms of 8-AHN have not been well elucidated. Here, we showed that 8-AHN significantly inhibited the proliferation of human colorectal cell lines via induction of G2/M cell cycle arrest and apoptosis. We found that the p53 played a central role in 8-AHN-induced cell proliferation inhibition. Mechanistically, 8-AHN induced p53 expression and enhanced transcriptional activity, subsequently elevating the expression of p53 target genes, including p21, FAS, and BAX, and then increased the level of activated caspase-3 and decreased the level of cyclin B and cyclin A. Moreover, pifithrin-α, the p53 inhibitor, markedly reversed the above responses induced by 8-AHN, and small interfering RNA-mediated knockdown of TP53 also significantly decreased 8-AHN-induced cell apoptosis. The experiments in vivo showed that 8-AHN significantly suppressed the growth of HCT116 xenograft tumors, associated with proliferation suppression and apoptosis induction in tumor tissues, without inducing any notable major organ-related toxicity. In summary, 8-AHN displays an antitumor effect through cell cycle arrest and apoptosis in colorectal cells via activating p53, which suggests that 8-AHN, exerted a therapeutic potential against colorectal cancer cells, and may be regarded as an effective lead compound.

Thyroid hormone receptor function in maturing ovine cardiomyocytes.

Plasma thyroid hormone (tri-iodo-l-thyronine; T3 ) concentrations rise near the end of gestation and is known to inhibit proliferation and stimulate maturation of cardiomyocytes before birth. Thyroid hormone receptors are required for the action of thyroid hormone in fetal cardiomyocytes. Loss of thyroid hormone receptor (TR)α1 abolishes T3 signalling via extracellular signal-related kinase and Akt in fetal cardiomyocytes. The expression of TRα1 and TRβ1 in ovine fetal myocardium increases with age, although TRα1 levels always remain higher than those of TRβ1. Near term fetal cardiac myocytes are more sensitive than younger myocytes to thyroid receptor blockade by antagonist, NH3, and to the effects of TRα1/α2 short interfering RNA. Although T3 is known to abrogate ovine cardiomyocyte proliferation stimulated by insulin-like growth factor 1, this effect is mediated via the genomic action of thyroid hormone receptors, with little evidence for non-genomic mechanisms. We have previously shown that the late-term rise in tri-iodo-l-thyronine (T3 ) in fetal sheep leads to the inhibition of proliferation and promotion of maturation in cardiomyocytes. The present study was designed to determine whether these T3 -induced changes are mediated via thyroid hormone receptors (TRs) or by non-genomic mechanisms. Fetal cardiomyocytes were isolated from 102±3 and 135±1days of gestational age (dGA) sheep (n=7 per age; term ∼145dGA). Cells were treated with T3 (1.5nm), insulin-like growth factor (IGF)-1 (1μgmL-1 ) or a combination in the presence of TR antagonist NH3 (100nm) or following short interfering RNA (siRNA) knockdown of TRα1/α2. Proliferation was quantified by 5-bromo-2'-deoxyuridine (BrdU) uptake (10μm). Western blots measured protein levels of extracellular signal-related kinase (ERK), Akt, TRα1/β1 and p21. Age specific levels of TRα1/β1 were measured in normal hearts from fetuses [95dGA (n=8), 135dGA (n=7)], neonates (n=8) and adult ewes (n=7). TRα1 protein levels were consistently >50% more than TRβ1 at each gestational age (P<0.05). T3 reduced IGF-1 stimulated proliferation by ∼50% in 100dGA and by ∼75% in 135dGA cardiomyocytes (P<0.05). NH3 blocked the T3 +IGF-1 reduction of BrdU uptake without altering the phosphorylation of ERK or Akt at both ages. NH3 did not suppress T3 -induced p21 expression in 100dGA cardiomyocytes in 135dGA cardiomyocytes, NH3 alone reduced BrdU uptake (-28%, P<0.05), as well as T3 -induced p21 (-75%, P<0.05). In both ages, siRNA knockdown of TRα1/α2 blocked the T3 +IGF-1 reduction of BrdU uptake and dramatically reduced ERK and Akt signalling in 135dGA cardiomyocytes. In conclusion, TRs are required for normal proliferation and T3 signalling in fetal ovine cardiomyocytes, with the sensitivity to TR blockade being age-dependent.