Small Interfering RNA Oligonucleotides Research Articles

Abstract 12705: Identification and Characterization of Solbinsiran, a GalNAc Conjugated siRNA Targeting Angiopoietin-Like 3

Introduction: While statins play an important role in reducing LDL-C levels and the risk of ASCVD events, residual risk from apolipoprotein B (apoB)-containing lipoprotein particles persists among patients with or at high risk of ASCVD. Hypothesis: Blocking ANGPTL3 activity or preventing its expression is a therapeutic approach to reduce apoB-containing lipoprotein particles. Solbinsiran (LY3561774), a novel Dicer-substrate small interfering RNA oligonucleotide, was designed to reduce the levels of ANGPTL3 protein expression. Methods: Nucleotide base sequences of a library of anti-ANGPTL3 siRNAs were selected using an in silico RNAi prediction algorithm on ANGPTL3 mRNA sequences conserved in humans and cynomolgus monkeys. Through a large-scale in vitro screen of the anti-ANGPTL3 library in human cells, the candidate solbinsiran sequence was identified as an efficacious siRNA. Solbinsiran in vivo efficacy was measured by RT-qPCR of liver from CD-1 female mice overexpressing human ANGPTL3 . The degree and durability of ANGPTL3 mRNA knockdown and circulating protein level reduction by solbinsiran was evaluated in cynomolgus monkeys administered single dose subcutaneous injections of either PBS or 3 mg/kg solbinsiran. Blood samples to measure ANGPTL3 protein were collected on Study Days -8, -5, and 0, and weekly after dosing until Day 113. Ultrasound-guided core needle liver biopsies were collected on Study Days 5, 28, 56, 84, and 113. Total RNA derived from the liver biopsy samples to measure ANGPTL3 mRNA was subjected to RT-qPCR analysis. Results: When given a single dose at 1 mg/kg subcutaneously, solbinsiran reduced human ANGPTL3 mRNA by 65% in hepatocytes as compared to vehicle-treated mice. A single dose of solbinsiran reduced liver ANGPTL3 mRNA expression by up to 72.9% (p<0.0001) and serum ANGPTL3 protein expression by 68.6% (p<0.001). Solbinsiran also displayed a long duration of activity, as indicated by a greater than 50% mRNA knockdown remaining 12 weeks after a single dose. Conclusions: These data supported the advancement of solbinsiran to Phase 1 clinical testing in participants with mixed dyslipidemia (ClinicalTrials.gov Identifier: NCT04644809).

Golden opportunity to treat glioblastoma

Cancer Glioblastoma (GBM) is a difficult cancer to treat, in part because of the blood–brain and blood–tumor barriers. Kumthekar et al. studied the drug NU-0129, which consists of a gold nanoparticle core conjugated to small interfering RNA oligonucleotides targeting the oncogene Bcl2L12 , for the treatment of GBM. After performing toxicology studies in cynomolgus monkeys, the authors conducted a first-in-human single-arm clinical trial of very-low-dose NU-0129 in eight patients with recurrent GBM. By analyzing gold accumulation, the authors found that NU-0129 passed through the blood–brain barrier and accumulated in the tumor, where it reduced Bcl2L12 protein abundance. These results suggest that spherical nucleic acid conjugates can potentially treat GBM. Sci. Transl. Med. 13 , eabb3945 (2021).

Impact of histone methyltransferase SUV420H2 in breast cancer.

Breast cancer is the most common type of cancer in women worldwide. Triple methylation of H4 lysine 20 (H4K20me3), a key component of epigenetic regulation of genomic integrity, is catalyzed by the methyltransferase, SUV420H2. Data on the expression status of SUV420H2 in breast cancer are limited. In the present study, the influence of SUV420H2 suppression on the proliferation of breast cancer cells was experimentally investigated. Subsequently, SUV420H2 expression was assessed in resectable breast cancer along with H4K20me3 status. SUV420H2 expression was knocked down in breast cells using small interfering RNA oligonucleotides. SUV420H2 expression was determined semi-quantitatively at the mRNA level. H4K20me3 was measured on extracted histone proteins using an approach similar to ELISA. Suppression of the SUV420H2 gene resulted in increased cell proliferation. Although the median SUV420H2 expression values were similar in tumor tissues and non-cancerous regions in the entire cohort (0.0022 and 0.0015, respectively; P=0.46), there was a notable difference in expression between tumor tissues and the adjacent non-cancerous region in the majority of patients. Increased SUV420H2 expression in tumors compared with healthy tissue was predominantly observed in patients with early-stage breast cancer, whereas reduced SUV420H2 expression was observed in tumors more frequently in patients with advanced stage diseases. There was no association between SUV420H2 expression and the tissue levels of H4K20me3. The results showed that SUV420H2 exhibited anti-proliferative activity in vitro, and exhibits a heterogeneous expression pattern in breast cancer tissues.

Kallistatin Inhibits Atherosclerotic Inflammation by Regulating Macrophage Polarization.

Kallistatin (KS) has been recognized as a plasma protein with anti-inflammatory functions. Macrophages are the primary inflammatory cells in atherosclerotic plaques. However, it is unknown whether KS plays a role in macrophage development and the pathogenesis of atherosclerosis. This study investigated the role of KS in macrophage development, a key pathological process in atherosclerosis. An atherosclerosis model was established in ApoE-/- mice via partial left carotid artery (PLCA) ligation. An adenovirus vector (Ad. HKS) containing the human KS gene was delivered via the tail vein before PLCA ligation. The mice were divided into two groups: the PLCA + Ad. HKS and PLCA + adenovirus vector (Ad. Null) groups and followed for 2 and 4 weeks. Human KS was expressed in the mice after KS gene delivery. In addition, KS significantly inhibited plaque formation and reduced inflammation in the plaques and liver 4 weeks after gene delivery. Moreover, KS gene delivery significantly increased the expression of interleukin-10 and Arginase 1, which are M2 macrophage markers, and reduced the expression of inducible nitric oxide synthase and monocyte chemotactic protein 1, which are M1 macrophage markers. Furthermore, in cultured RAW 264.7 macrophages, KS significantly stimulated M2 marker expression and differentiation and decreased M1 marker expression, as determined by flow cytometry and real-time polymerase chain reaction. These effects were blocked by Krüppel-like factor 4 small-interfering RNA oligonucleotides. These findings demonstrate that KS inhibits atherosclerotic plaque formation and regulates M1/M2 macrophage polarization via Krüppel-like factor 4 activation.

SOCS3 participates in cholinergic pathway regulation of synovitis in rheumatoid arthritis

ABSTRACTStimulation of the cholinergic inflammatory pathway can attenuate collagen-induced arthritis (CIA) and inhibit synovitis by Janus kinase (JAK) 2 and signal transducer and activator of transcription (STAT) 3 signaling. Suppressor of cytokine signaling (SOCS) protein can also regulate the inflammatory processes and activate JAK/STAT signal transduction, but its involvement in rheumatoid arthritis (RA) has not been demonstrated. This study investigated the effect of SOCS on cholinergic pathway regulation of synovitis in the fibroblast-like synoviocytes (FLSs) of RA and CIA mice. The effects of nicotine on SOCS1 and SOCS3 protein expression in FLSs were assayed by western blotting before and after transfection with a small interfering RNA oligonucleotide (SOCS3-siRNA or control-siRNA). Interleukin-6 was measured by enzyme-linked immunosorbent assay of SOCS3-siRNA and control-siRNA transfected FLS culture supernatants. Histopathological evaluation and immunohistochemical staining of SOCS3 were performed in joint tissue sections of control, CIA model, vagotomy, and nicotine-treated DBA/1 mice. Nicotine increased SOCS3 expression in the FLSs of RA. The inhibitory effect of nicotine on inflammatory factors was abolished by siRNA knockdown of SOCS3 protein expression. Nicotine increased the expression of SOCS3 protein in the synovium and reduced synovitis and bone erosion in CIA mice.

Downregulation of β-catenin blocks fibrosis via Wnt2 signaling in human keloid fibroblasts.

Keloid is a disorder of fibroproliferative diseases that occurs in wounds, characterized by an exaggerated response to injury. The key factor responsible for the disease process has not been identified. This study sought to elucidate the role of β-catenin in the regulation of keloid phenotypes and signaling. Expression of β-catenin in keloid and normal non-keloid samples was measured by real-time polymerase chain reaction. Knockdown of β-catenin was achieved by delivering small interfering RNA to target β-catenin. Cell proliferation, cell cycle progression, and apoptosis of keloid cells were measured by functional assays in vitro. The proteins related to keloid fibrosis were measured by Western blotting. β-catenin expression was significantly upregulated in keloid tissue samples compared with the normal non-keloid age-adjusted skin sample counterparts. Functionally, targeting β-catenin with lipofection-delivered small interfering RNA oligonucleotide inhibited the proliferation and cell cycle arrest in G0/G1 phase and increased apoptosis of fibroblast cells, accompanied by downregulation of Wnt2 and cyclin D1 as well as the phosphorylation level of glycogen synthase kinase 3 beta in the keloid fibrosis. Our study supports a crucial role of β-catenin in the regulation of fibroproliferation and extracellular matrix deposition. Targeting β-catenin using small interfering RNA oligonucleotide may be a promising approach for preventing excessive fibroproliferative development after wound healing and may lead to the development of novel strategies for restoring keloid diseases.

MicroRNA-132 promotes estradiol synthesis in ovarian granulosa cells via translational repression of Nurr1.

BackgroundEstrogen synthesis is an important function of the mammalian ovary. Estrogen plays important roles in many biological processes, including follicular development, oocyte maturation and endometrial proliferation, and dysfunctions in estrogen synthesis contribute to the development of polycystic ovary syndrome and premature ovarian failure. Classical signaling cascades triggered by follicle-stimulating hormone induce estrogen synthesis via the upregulation of Cyp19a1 in granulosa cells (GCs). This study aimed to determine the effect of microRNA-132 (miR-132) on estradiol synthesis in GCs.MethodsPrimary mouse GCs were collected from ovaries of 21-day-old immature ICR mice through follicle puncture. GCs were cultured and treated with the stable cyclic adenosine monophosphate analog 8-Br-cAMP or transfected with miR-132 mimics, Nurr1-specific small interfering RNA oligonucleotides and Flag-Nurr1 plasmids. Concentrations of estradiol and progesterone in culture medium were determined by an automated chemiluminescence-based assay. Quantitative real time PCR and western blot were performed to identify the effect of miR-132 on Cyp19a1, Cyp11a1 and an orphan nuclear receptor-Nurr1 expression in GCs. Direct suppression of Nurr1 via its 3'-untranslated region by miR-132 were further verified using luciferase reporter assays.ResultsThe expression level of miR-132 in cultured mouse GCs was significantly elevated during 48 h of treatment with 8-Br-cAMP. The synthesis of estradiol increased after the overexpression of miR-132 in mouse GCs. The real-time PCR results demonstrated that miR-132 induced the expression of Cyp19a1 significantly. Nurr1, an orphan nuclear receptor that suppresses Cyp19a1 expression, was found to be a direct target of miR-132. Nurr1 was suppressed by miR-132, as indicated by a luciferase assay and Western blotting. The knockdown of Nurr1 primarily elevated the synthesis of estradiol and partially attenuated the miR-132-induced estradiol elevation, and the ectopic expression of Flag-Nurr1 abrogated the stimulatory effect of miR-132 on estradiol synthesis in mouse GCs.ConclusionsOur findings suggest that miR-132 is involved in the cAMP signaling pathway and promotes estradiol synthesis via the translational repression of Nurr1 in ovarian GCs.

Small molecule inhibition of group I p21-activated kinases in breast cancer induces apoptosis and potentiates the activity of microtubule stabilizing agents.

IntroductionBreast cancer, the most common cause of cancer-related deaths worldwide among women, is a molecularly and clinically heterogeneous disease. Extensive genetic and epigenetic profiling of breast tumors has recently revealed novel putative driver genes, including p21-activated kinase (PAK)1. PAK1 is a serine/threonine kinase downstream of small GTP-binding proteins, Rac1 and Cdc42, and is an integral component of growth factor signaling networks and cellular functions fundamental to tumorigenesis.MethodsPAK1 dysregulation (copy number gain, mRNA and protein expression) was evaluated in two cohorts of breast cancer tissues (n = 980 and 1,108). A novel small molecule inhibitor, FRAX1036, and RNA interference were used to examine PAK1 loss of function and combination with docetaxel in vitro. Mechanism of action for the therapeutic combination, both cellular and molecular, was assessed via time-lapse microscopy and immunoblotting.ResultsWe demonstrate that focal genomic amplification and overexpression of PAK1 are associated with poor clinical outcome in the luminal subtype of breast cancer (P = 1.29 × 10−4 and P = 0.015, respectively). Given the role for PAK1 in regulating cytoskeletal organization, we hypothesized that combination of PAK1 inhibition with taxane treatment could be combined to further interfere with microtubule dynamics and cell survival. Consistent with this, administration of docetaxel with either a novel small molecule inhibitor of group I PAKs, FRAX1036, or PAK1 small interfering RNA oligonucleotides dramatically altered signaling to cytoskeletal-associated proteins, such as stathmin, and induced microtubule disorganization and cellular apoptosis. Live-cell imaging revealed that the duration of mitotic arrest mediated by docetaxel was significantly reduced in the presence of FRAX1036, and this was associated with increased kinetics of apoptosis.ConclusionsTaken together, these findings further support PAK1 as a potential target in breast cancer and suggest combination with taxanes as a viable strategy to increase anti-tumor efficacy.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0564-5) contains supplementary material, which is available to authorized users.

Activation of Nicotinic Receptors Inhibits TNF-α-Induced Production of Pro-inflammatory Mediators Through the JAK2/STAT3 Signaling Pathway in Fibroblast-Like Synoviocytes.

It was recently demonstrated that stimulation of the nicotine receptor attenuates collagen-induced arthritis and inhibits cytokine release in mice. We elucidated the possible intracellular signaling mechanism of the cholinergic anti-inflammatory pathway in fibroblast-like synoviocytes (FLSs). Levels of interleukin (IL)-6, IL-10, and monocyte chemoattractant protein (MCP)-1 in culture supernatants of tumor necrosis factor (TNF)-α-stimulated FLSs were measured using an enzyme-linked immunosorbent assay (ELISA). FLSs were transfected with a small interfering RNA oligonucleotide (STAT3 siRNA or control siRNA). AG490, a specific inhibitor of JAK2, was added 16h before nicotine, and blocker of nAChR was added 30min before nicotine. Activation of signal transducers and activators of transcription (STAT) such as STAT1 and STAT3 were detected using Western blotting. Nicotine downregulated production of IL-6 and MCP-1 in RA-FLSs induced by TNFα in a concentration-dependent manner, and IL-10 levels were not significantly different after nicotine pretreatment. Nicotine-induced activation of STAT3 (but not STAT1) and deactivation of STAT3 decreased the anti-inflammatory effect of nicotine. AG490 inhibited the phosphorylation of STAT1 and STAT3 and decreased the TNF-α-induced production of pro-inflammatory mediators in RA-FLSs. A α7nAChR antagonist abrogated the anti-inflammatory effects of nicotine and suppressed STAT3 activity. In conclusion, nicotine has an anti-inflammatory effect on RA by downregulating production of IL-6 and MCP-1 in FLSs, and this is mediated through activation of the JAK2-STAT3 signal pathway.

Downregulated expression of metastasis associated in colon cancer 1 (MACC1) reduces gallbladder cancer cell proliferation and invasion

Metastasis associated in colon cancer 1 (MACC1), a key regulator of the hepatocyte growth factor (HGF)/MET signaling pathway, has been implicated in multiple human cancers. However, little is known regarding its expression and biological function in human gallbladder cancer (GBC). In this study, we focused on the clinical significance and biological functions of MACC1 in GBC and found that MACC1 protein overexpression was frequently detected in GBC tissues. Patients with MACC1-positive tumors had worse overall survival than patients with MACC1-negative tumors. Furthermore, treatment of GBC lines with MACC1-targeting small interfering RNA oligonucleotides (MACC1-siRNA) significantly reduced the proliferation of GBC-SD and OCUG-1 cell lines and diminished both anchorage-independent growth on soft agar and cell migration. These data indicate that MACC1 acts as a putative oncogene in GBC and could be a novel diagnostic and therapeutic target for GBC.

Relocalization of Junctional Adhesion Molecule A during Inflammatory Stimulation of Brain Endothelial Cells

Junctional adhesion molecule A (JAM-A) is a unique tight junction (TJ) transmembrane protein that under basal conditions maintains endothelial cell-cell interactions but under inflammatory conditions acts as a leukocyte adhesion molecule. This study investigates the fate of JAM-A during inflammatory TJ complex remodeling and paracellular route formation in brain endothelial cells. The chemokine (C-C motif) ligand 2 (CCL2) induced JAM-A redistribution from the interendothelial cell area to the apical surface, where JAM-A played a role as a leukocyte adhesion molecule participating in transendothelial cell migration of neutrophils and monocytes. JAM-A redistribution was associated with internalization via macropinocytosis during paracellular route opening. A tracer study with dextran-Texas Red indicated that internalization occurred within a short time period (~10 min) by dextran-positive vesicles and then became sorted to dextran-positive/Rab34-positive/Rab5-positive vesicles and then Rab4-positive endosomes. By ~20 min, most internalized JAM-A moved to the brain endothelial cell apical membrane. Treatment with a macropinocytosis inhibitor, 5-(N-ethyl-N-isopropyl)amiloride, or Rab5/Rab4 depletion with small interfering RNA oligonucleotides prevented JAM-A relocalization, suggesting that macropinocytosis and recycling to the membrane surface occur during JAM-A redistribution. Analysis of the signaling pathways indicated involvement of RhoA and Rho kinase in JAM-A relocalization. These data provide new insights into the molecular and cellular mechanisms involved in blood-brain barrier remodeling during inflammation.

A novel role of Id-1 in regulation of epithelial-to-mesenchymal transition in bladder cancer

Background and objectiveInhibitor of differentiation or DNA binding -1 (Id-1) has been shown to be increased in several types of advanced cancer, and to be associated with aggressive and metastatic abilities of cancer cells. Recently, more and more evidence indicates that epithelial-to-mesenchymal transition (EMT) is an important mechanism taking place during tumor invasion and metastasis, but the molecular pathways underlying EMT have not been clearly established. This study was to investigate the expression of Id-1 in bladder cancer and its association with EMT. Materials and methodsA total of 169 tissues, consisting of 147 primary bladder cancers and 22 adjacent normal tissues were included in this study. Id-1, E-cadherin, and β-catenin were examined immunohistochemically in paraffin sections. The pBabe-Id-1 expression retroviral vector and retroviral vectors containing an Id-1-specific small interfering RNA oligonucleotides (si-Id-1) were transfected into 2 bladder cancer cell lines respectively. Then, we used Western blotting and immunofluorescent staining to detect the cellular expression of epithelial markers and mesenchymal markers. The invasion and migration ability of bladder cancer cells were identified by type I collagen invasion assay and wound closure assay. ResultsWe demonstrated that increased Id-1 expression was associated with advanced tumor stage and grade. In addition, the increased Id-1 expression in bladder tumors was also correlated with decreased membranous E-cadherin and β-catenin expression. In vitro, studies showed that inactivation of the Id-1 gene conferred morphologic transition of bladder cancer cells from a fibroblastic to epithelial appearance, and overexpression of Id-1 could lead to acquisition of a fibroblastic spindle cell phenotype accompanied by loss of cell-to-cell contacts. By Western blotting and immunofluorescent staining, we showed that the expression level of Id-1 was correlated with the expression of mesenchymal markers but was inversely correlated with the expression of epithelial markers. Moreover, results of collagen invasion and wound closure assays showed ectopic Id-1 expression led to increased ability of invasion and migration. ConclusionsOur results suggest that Id-1 may play roles in tumor progression and EMT activation in bladder cancer.

Binding tendency with oligonucleotides and cell toxicity of cetyltrimethyl ammonium bromide-coated single-walled carbon nanotubes

Functionalized carbon nanotubes (CNTs) were made for the delivery of genes and drugs and CNT-based biosensors. The basis of CNTs is for binding with biomolecules in biomedical applications. The binding tendency with small interfering RNA oligonucleotides and cytotoxicity of cetyltrimethyl ammonium bromide (CTAB)-coated single-walled carbon nanotubes (SWNTs) were studied. The field emission scanning electron microscopy and transmission electron microscopy results show that a SWNT suspension in CTAB solution was well-dispersed and stable. CTAB is the cross-linker between SWNTs and oligonucleotides. The CTAB-coated SWNTs have less cytotoxicity to human umbilical vein endothelial cells than single SWNTs and the cytotoxicity of CTAB-coated SWNTs depended on the concentration of CTAB-coated SWNTs.

A Chemosensitization Screen Identifies TP53RK, a Kinase that Restrains Apoptosis after Mitotic Stress

Taxanes are very effective at causing mitotic arrest; however, there is variability among cancer cells in the apoptotic response to mitotic arrest. The variability in clinical efficacy of taxane-based therapy is likely a reflection of this variability in apoptotic response, thus elucidation of the molecular mechanism of the apoptotic response to mitotic stress could lead to improved clinical strategies. To identify genes whose expression influences the rate and extent of apoptosis after mitotic arrest, we screened a kinase-enriched small interfering RNA library for effects on caspase activation in response to maximally effective doses of paclitaxel, a PLK1 inhibitor, or cisplatin. Small interfering RNA oligonucleotides directed against an atypical protein kinase, TP53RK, caused the greatest increase in caspase-3/7 activation in response to antimitotic agents. Time-lapse microscopy revealed that cells entered mitosis with normal kinetics, but died after entry into mitosis in the presence of paclitaxel more rapidly when TP53RK was depleted. Because expression levels of TP53RK vary in cancers, TP53RK levels could provide a molecular marker to predict response to antimitotic agents. TP53RK inhibition may also sensitize cancers to taxanes.

Role of External Loops of Human Ceruloplasmin in Copper Loading by ATP7B and Ccc2p

Ceruloplasmin is a multicopper oxidase required for correct iron homeostasis.Previously, we have identified a ceruloplasmin mutant associated with the iron overload disease aceruloplasminemia, which was unable to acquire copper from the mammalian pump ATP7B but could be produced in an enzymatically active form in yeast. Here, we report the expression of recombinant ceruloplasmin in the yeast Pichia pastoris and the study of the role of five surface-exposed loops in copper incorporation by comparing the efficiencies of mammalian ATP7B and yeast Ccc2p. The possibility to "mix and match" mammalian and yeast multicopper oxidases and copper ATPases can provide clues on the molecular features underlying the process of copper loading in multicopper oxidases.

Abstract 1612: Validation of CSE1L/hCAS as a potential therapeutic target in pancreatic cancer

Abstract Background: The nuclear export protein CSE1L/hCAS has recently gained attention as a putative oncogene, which is amplified in several human malignancies. The role of hCAS in pancreatic cancer is unknown. In this work, we sought to evaluate the expression of hCAS in pancreatic cancer cell lines and tumor tissue and to further explore the potential of hCAS as a therapeutic target. Methods: The nuclear and cytosolic hCAS protein expression was determined in human pancreatic cancer cell lines and tumor tissues and compared with those of immortalized normal pancreatic ductal cells and normal pancreatic tissues. The cellular effects of hCAS knockdown using small interfering RNA oligonucleotides were investigated in MiaPaCa-2 human pancreatic cancer cells with regard to proliferation (MTT assay), cell death (Trypan blue and Flow cytometry), and malignant transformation (colony formation assay). Results: hCAS protein expression increased in pancreatic cancer cell lines relative to the immortalized pancreatic ductal epithelial cell line. Immunohistochemical staining of a human pancreatic tumor tissue microarray showed that hCAS expression was higher in pancreatic tumors compared with their normal counterparts. There was increasing nuclear to cytoplasmic staining with progression from normal pancreatic duct to increasing grade of pancreatic intraductal neoplasia to invasive pancreatic cancer. Treatment with hCAS targeting small interfering RNAs effectively reduced pancreatic cell growth in tissue culture, induced apoptosis, and inhibited growth in soft agar. Conclusion: This is the first report of hCAS expression and cellular distribution in human pancreatic cancer. Our data suggest that hCAS might be an attractive target for human pancreatic cancer prevention and therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1612.

Abstract 1280: Inhibition of cellular FLICE-inhibitory protein and induction of Death Receptor 5 expression is a major component of δ- tocotrienol-induced apoptosis in pancreatic cancer cells

Abstract Background: Previous pre-clinical studies have demonstrated that δ-tocotrienol, is the most bioactive natural vitamin E compound against pancreatic cancer. δ-Tocotrienol induces growth arrest and apoptosis in several human pancreatic cancer cell lines; however, the underlying mechanism remains unknown. In this study, we evaluated the role of the extrinsic and intrinsic apoptotic signaling pathways in δ-tocotrienol-induced apoptosis. Also, we investigated the modulation of cellular FLICE-inhibitory protein (c-FLIP) and death receptor 5 (DR5) by δ-tocotrienol, and the involvement of c-FLIP down-regulation and DR5 up-regulation in δ-tocotrienol-induced apoptosis. Methods: We examined δ-tocotrienol induced apoptosis in human pancreatic cancer cell lines in vitro and in vivo. We used western blot and immunohistochemistry to determine the expression of DR4, DR5, Caspase 8, Caspase 9, Caspase 3, c-FLIP, and PARP. The effect of combination treatment with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and δ-tocotrienol was also determined. We examined the cellular effects of DR5 knockdown using small interfering RNA oligonucleotides, and c-FLIP overexpression using c-FLIP cDNA, on δ-tocotrienol-induced apoptosis using various assays. Results: Delta-tocotrienol activated caspase-8 and its downstream caspases but not caspase-9. Caspase-8 specific inhibitor abrogated δ-tocotrienol-induced apoptosis whereas caspase-9 specific inhibitor did not, indicating that δ-tocotrienol induces caspase-8 -dependent apoptosis. c-FLIP was rapidly down-regulated by δ-tocotrienol in all of the tested cell lines. Delta-tocotrienol up-regulated DR4 and DR5 significantly and enhanced TRAIL-induced apoptosis. Enforced expression of ectopic c-FLIP, and DR5 knockdown partially abolished δ-tocotrienol-induced apoptosis. δ-Tocotrienol inhibition of pancreatic tumor growth in SCID mice was associated with significant induction of apoptosis, activation of caspase 8, and down-regulation of c-FLIP expression. Conclusion: We conclude that DR5 up-regulation and c-FLIP down-regulation contributes to δ-tocotrienol-induced apoptosis in pancreatic cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1280.

Abstract 222: Inhibition of cellular FLICE-inhibitory protein and induction of death receptor 5 expression is a major component of δ-tocotrienol-induced apoptosis in pancreatic cancer cells

Abstract Background: Previous pre-clinical studies have demonstrated that δ-tocotrienol, is the most bioactive natural vitamin E compound against pancreatic cancer. δ-Tocotrienol induces growth arrest and apoptosis in several human pancreatic cancer cell lines; however, the underlying mechanism remains unknown. In this study, we evaluated the role of the extrinsic and intrinsic apoptotic signaling pathways in δ-tocotrienol induced apoptosis and investigated the modulation of cellular FLICE-inhibitory protein (c-FLIP) and death receptor 5 (DR5) by δ-tocotrienol and the involvement of c-FLIP down-regulation and DR5 up-regulation in δ-tocotrienol-induced apoptosis. Methods: We examined δ-tocotrienol induced apoptosis in human pancreatic cancer cell lines in vitro and in vivo. We used western blot and immunohistochemistry to determine the expression of DR4, DR5, Caspase 8, Caspase 9, Caspase 3, C-FLIPs and PARP. The effect of combination treatment with TRAIL and δ-tocotrienol was also determined. We examined the cellular effects of DR5 knockdown using small interfering RNA oligonucleotides, and c-FLIP overexpression using cDNA, on δ-tocotrienol-induced apoptosis using various assays. Results: Delta-tocotrienol activated caspase-8 and its downstream caspases but not caspase-9, whereas caspase-8 specific inhibitor abrogated δ-tocotrienol-induced apoptosis indicating that δ-tocotrienol induces caspase-8-dependent apoptosis. c-FLIP was rapidly down-regulated by δ-tocotrienol in all of the tested cell lines. δ-Tocotrienol up-regulated DR4 and DR5 significantly and enhanced tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis. Enforced expression of ectopic c-FLIP, and DR5 knockdown partially abolished δ-tocotrienol-induced apoptosis. δ-Tocotrienol inhibition of pancreatic tumor growth in SCID mice was associated with significant induction of apoptosis, activation of caspase 8, and down-regulation of c-FLIP expression. Conclusion: We conclude that DR5 up-regulation and c-FLIP down-regulation contributes to δ-tocotrienol-induced apoptosis in pancreatic cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 222.

Phosphorylation of VACM-1/Cul5 by Protein Kinase A Regulates Its Neddylation and Antiproliferative Effect

Expression of the VACM-1/cul5 gene in endothelial and in cancer cell lines in vitro inhibits cellular proliferation and decreases phosphorylation of MAPK. Structure-function analysis of the VACM-1 protein sequence identified consensus sites specific for phosphorylation by protein kinases A and C (PKA and PKC) and a Nedd8 protein modification site. Mutations at the PKA-specific site in VACM-1/Cul5 ((S730A)VACM-1) sequence resulted in increased cellular growth and the appearance of a Nedd8-modified VACM-1/Cul5. The aim of this study was to examine if PKA-dependent phosphorylation of VACM-1/Cul5 controls its neddylation status, phosphorylation by PKC, and ultimately growth. Our results indicate that in vitro transfection of rat adrenal medullary endothelial cells with anti-VACM-1-specific small interfering RNA oligonucleotides decreases endogenous VACM-1 protein concentration and increases cell growth. Western blot analysis of cell lysates immunoprecipitated with an antibody directed against a PKA-specific phosphorylation site and probed with anti-VACM-1-specific antibody showed that PKA-dependent phosphorylation of VACM-1 protein was decreased in cells transfected with (S730A)VACM-1 cDNA when compared with the cytomegalovirus-transfected cells. This change was associated with increased modification of VACM-1 protein by Nedd8. Induction of PKA activity with forskolin reduced modification of VACM-1 protein by Nedd8. Finally, rat adrenal medullary endothelial cells transfected with (S730A)VACM-1/cul5 cDNA and treated with phorbol 12-myristate 13-acetate (10 and 100 nm) to induce PKC activity grew significantly faster than the control cells. These results suggest that the antiproliferative effect of VACM-1/Cul5 is dependent on its posttranslational modifications and will help in the design of new anticancer therapeutics that target the Nedd8 pathway.

Orphan Nuclear Receptor DAX-1 Acts as a Novel Corepressor of Liver X Receptor α and Inhibits Hepatic Lipogenesis

DAX-1 (dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on X chromosome, gene 1) is a member of the nuclear receptor superfamily that can repress diverse nuclear receptors and has a key role in adreno-gonadal development. Our previous report has demonstrated that DAX-1 can inhibit hepatocyte nuclear factor 4alpha transactivity and negatively regulate gluconeogenic gene expression (Nedumaran, B., Hong, S., Xie, Y. B., Kim, Y. H., Seo, W. Y., Lee, M. W., Lee, C. H., Koo, S. H., and Choi, H. S. (2009) J. Biol. Chem. 284, 27511-27523). Here, we further expand the role of DAX-1 in hepatic energy metabolism. Transfection assays have demonstrated that DAX-1 can inhibit the transcriptional activity of nuclear receptor liver X receptor alpha (LXRalpha). Physical interaction between DAX-1 and LXRalpha was confirmed Immunofluorescent staining in mouse liver shows that LXRalpha and DAX-1 are colocalized in the nucleus. Domain mapping analysis shows that the entire region of DAX-1 is involved in the interaction with the ligand binding domain region of LXRalpha. Competition analyses demonstrate that DAX-1 competes with the coactivator SRC-1 for repressing LXRalpha transactivity. Chromatin immunoprecipitation assay showed that endogenous DAX-1 recruitment on the SREBP-1c gene promoter was decreased in the presence of LXRalpha agonist. Overexpression of DAX-1 inhibits T7-induced LXRalpha target gene expression, whereas knockdown of endogenous DAX-1 significantly increases T7-induced LXRalpha target gene expression in HepG2 cells. Finally, overexpression of DAX-1 in mouse liver decreases T7-induced LXRalpha target gene expression, liver triglyceride level, and lipid accumulation. Overall, this study suggests that DAX-1, a novel corepressor of LXRalpha, functions as a negative regulator of lipogenic enzyme gene expression in liver.