Small Interfering RNA Strategy Research Articles

LNCAROD promotes the proliferation and migration of gastric cancer: a bioinformatics analysis and experimental validation.

Gastric cancer (GC) is a heterogeneous disease at the molecular and clinical levels. The diffuse subtype is associated with more aggressive behavior and poor prognosis than the intestinal subtype. Epithelial-to-mesenchymal transition (EMT) may be involved in the diffuse mesenchymal phenotype. Long non-coding RNA (lncRNA) deregulation plays a vital role in GC development and progression. Here, we aimed to comprehensively disclose lncRNAs associated with GC diffuse/mesenchymal type. RNA-sequencing expression profiles of patients with stomach adenocarcinoma and the corresponding clinical data were downloaded from The Cancer Genome Atlas database. Differentially expressed lncRNAs related to tumor samples and diffuse subtype were identified. The lncRNA activating regulator of DKK1 (LNCAROD) was experimentally studied. Furthermore, a lncRNA-miRNA-mRNA network was constructed to identify potential biological functions of LNCAROD. LNCAROD expression was detected by reverse transcription-quantitative polymerase chain reaction in GC cell lines. LNCAROD expression was silenced using the small interference RNA strategy. Cell proliferation and migration were evaluated using colony formation assay, scratch wound healing, and Transwell migration assays. LNCAROD was significantly upregulated in some GC cells. The knocking down of LNCAROD significantly reduced cell proliferation and migration. LNCAROD-miR-181-PROX1 axis was introduced as a potential regulatory mechanism by which LNCAROD may exert its functions in cells. Our findings highlight that LNCAROD is involved in cell proliferation and migration in GC and supports its implicit role in regulating EMT. It may serve as a potential diagnostic and therapeutic target in GC. In addition, LNCAROD may function through the possible regulatory axis in GC development.

Chrysin, which targets PLAU, protects PC12cells from OGD/R-stimulated damage through repressing the NF-κB signaling pathway.

Cerebral ischemia reperfusion injury (CIRI) is a great challenge for the patients with brain ischemia, but its pathophysiological mechanism has not been clearly explored. This study aims to decipher the effect of chrysin and plasminogen activator urokinase (PLAU) in CIRI. The immune-related genes were collected from the ImmPort website, and the differentially expressed genes were determined based on the Gene Expression Omnibus (GEO) database. PC12 cells were used to establish an ischemic stroke model under the condition of oxygen-glucose deprivation and reoxygenation (OGD/R). Small interfering RNA strategy was employed to knock down the PLAU expression of PC12 cells. The proliferation and apoptosis rates of PC12 cells treated by OGD/R or/and chrysin were detected with Cell Counting Kit 8 (CCK-8) and flow cytometry. The protein and mRNA expressions were measured using western blotting and quantitative reverse transcription polymerase chain reaction (qRT-PCR). PLAU was identified as a candidate for CIRI treatment and expressed at higher levels in CIRI tissues compared with that in normal controls. Chrysin was determined as a crucial agent that could decrease the expression of PLAU. Chrysin significantly promoted the cell proliferation, inhibited the protein levels of PLAU, p–NF–κB, and p-IKκB in PC12 cells after OGD/R. Silencing of PLAU strengthened the protective effect of chrysin on PC12 cells treated by OGD/R, including the improvement of cell viability and suppression of apoptosis. Chrysin inactivated the NF-κB pathway via targeting PLAU in OGD/R-stimulated PC12 cells. Chrysin prevented PC12 cells from OGD/R-stimulated damage via decreasing PLAU expression and inactivating the NF-κB signaling pathway.

Inclisiran: a small interfering RNA strategy targeting PCSK9 to treat hypercholesterolemia

ABSTRACT Introduction Inclisiran is a novel posttranscriptional gene silencing therapy that inhibits proprotein convertase subtilisin/kexin type 9 (PCSK9) synthesis by RNA interference and has a potent, dose-dependent, durable effect in lowering LDL-C, and therefore is an effective drug to treat dyslipidemia, reducing the risk for acute cardiovascular (CV) events. It is safe and well-tolerated. Areas covered This paper aims to review the mechanism of action of inclisiran while evaluating its efficacy and safety in the treatment of dyslipidemia from data of the clinical trials in the ORION program. Expert opinion Data from the clinical trials in the ORION program demonstrated efficacy and safety of inclisiran in patients with dyslipidemia. Adverse events were similar in the inclisiran and placebo groups in the clinical trials, although injection-site reactions were more frequent with inclisiran than with placebo. Although the combination of efficacy and safety makes inclisiran a good option for the treatment of dyslipidemia compared to other PCSK9 targeting therapeutic strategies, however, further studies should exclude the possibility that inclisiran, through lower-affinity interactions, may influence other mRNAs in the physiological milieu.

Homeobox Genes in Cancers: From Carcinogenesis to Recent Therapeutic Intervention.

The homeobox (HOX) genes encoding an evolutionarily highly conserved family of homeodomain-containing transcriptional factors are essential for embryogenesis and tumorigenesis. HOX genes are involved in cell identity determination during early embryonic development and postnatal processes. The deregulation of HOX genes is closely associated with numerous human malignancies, highlighting the indispensable involvement in mortal cancer development. Since most HOX genes behave as oncogenes or tumor suppressors in human cancer, a better comprehension of their upstream regulators and downstream targets contributes to elucidating the function of HOX genes in cancer development. In addition, targeting HOX genes may imply therapeutic potential. Recently, novel therapies such as monoclonal antibodies targeting tyrosine receptor kinases, small molecular chemical inhibitors, and small interfering RNA strategies, are difficult to implement for targeting transcriptional factors on account of the dual function and pleiotropic nature of HOX genes-related molecular networks. This paper summarizes the current state of knowledge on the roles of HOX genes in human cancer and emphasizes the emerging importance of HOX genes as potential therapeutic targets to overcome the limitations of present cancer therapy.

Chemical Synthesis and Biological Application of Modified Oligonucleotides.

RNA plays a myriad of roles in the body including the coding, decoding, regulation, and expression of genes. RNA oligonucleotides have garnered significant interest as therapeutics via antisense oligonucleotides or small interfering RNA strategies for the treatment of diseases ranging from hyperlipidemia, HCV, and others. Additionally, the recently developed CRISPR-Cas9 mediated gene editing strategy also relies on Cas9-associated RNA strands. However, RNA presents numerous challenges as both a synthetic target and a potential therapeutic. RNA is inherently unstable, difficult to deliver into cells, and potentially immunogenic by itself or upon modification. Despite these challenges, with the help of chemically modified oligonucleotides, multiple RNA-based drugs have been approved by the FDA. The progress is made possible due to the nature of chemically modified oligonucleotides bearing advantages of nuclease stability, stronger binding affinity, and some other unique properties. This review will focus on the chemical synthesis of RNA and its modified versions. How chemical modifications of the ribose units and of the phosphatediester backbone address the inherent issues with using native RNA for biological applications will be discussed along the way.

Interleukin-1β/nuclear factor-κB signaling promotes osteosarcoma cell growth through the microRNA-181b/phosphatase and tensin homolog axis.

So far, microRNA has attracted plenty of interest due to its role in tumorigenesis. Reportedly, miR-181b may be involved in the tumorigenesis of osteosarcoma (OS). In the current study, we attempted to investigate the detailed function and mechanism of miR-181b in OS carcinogenesis. Herein, miR-181a, miR-181b, miR-181c, and miR-181d expressions in OS tissues were higher than that in nontumor tissue samples as examined real-time polymerase chain reaction. Via direct targeting, miR-181b negatively regulated the expression of phosphatase and tensin homolog (PTEN), a well-known tumor suppressor. Furthermore, a small interfering RNA strategy was used to find that interleukin (IL)-1B and nuclear factor-κB (NF-κB) regulate miR-181b and PTEN expression. Consequently, the repression of PTEN by miR-181b promotes OS cell proliferation. In summary, our data support a critical role for NF-κB-dependent upregulation of miR-181b, which further inhibited PTEN expression and promoted the cell proliferation of OS cell lines. The above findings represent a new pathway for the repression of PTEN and the promotion of cell proliferation upon IL-1β induction.

Small interference ITGA6 gene targeting in the human thymic epithelium differentially regulates the expression of immunological synapse-related genes

ABSTRACTThe thymus supports differentiation of T cell precursors. This process requires relocation of developing thymocytes throughout multiple microenvironments of the organ, mainly with thymic epithelial cells (TEC), which control intrathymic T cell differentiation influencing the formation and maintenance of the immunological synapse. In addition to the proteins of the major histocompatibility complex (MHC), this structure is supported by several adhesion molecules. During the process of thymopoiesis, we previously showed that laminin-mediated interactions are involved in the entrance of T-cell precursors into the thymus, as well as migration of differentiating thymocytes within the organ. Using small interference RNA strategy, we knocked-down the ITGA6 gene (which encodes the CD49f integrin α-chain) in cultured human TEC, generating a decrease in the expression of the corresponding CD49f subunit, in addition to modulation in several other genes related to cell adhesion and migration. Thymocyte adhesion to TEC was significantly impaired, comprising both immature and mature thymocyte subsets. Moreover, we found a modulation of the MHC, with a decrease in membrane expression of HLA-ABC, in contrast with increase in the expression of HLA-DR. Interestingly, the knockdown of the B2M gene (encoding the β-2 microglobulin of the HLA-ABC complex) increased CD49f expression levels, thus unraveling the existence of a cross-talk event in the reciprocal control of CD49f and HLA-ABC. Our data suggest that the expression levels of CD49f may be relevant in the general control of MHC expression by TEC and consequently the corresponding synapse with developing thymocytes mediated by the T-cell receptor.

Unexpected function of the phagocyte NADPH oxidase in supporting hyperglycolysis in stimulated neutrophils: key role of 6-phosphofructo-2-kinase.

The phagocyte NADPH oxidase 2 (Nox2) is an enzymatic complex that is involved in innate immunity, notably via its capacity to produce toxic reactive oxygen species. Recently, a proteomic analysis of the constitutively active Nox2 complex, isolated from neutrophil fractions, highlighted the presence of 6-phosphofructo-2-kinase (PFK-2). The purpose of this work was to study the relationship between PFK-2 and NADPH oxidase in neutrophils. Data have underlined a specific association of the active phosphorylated form of PFK-2 with Nox2 complex in stimulated neutrophils. In its active form, PFK-2 catalyzes the production of fructose-2,6-bisphosphate, which is the main allosteric activator of phosphofructo-1-kinase, the limiting enzyme in glycolysis. Pharmacologic inhibition of PFK-2 phosphorylation and cell depletion in PFK-2 by a small interfering RNA strategy led to a decrease in the glycolysis rate and a reduction in NADPH oxidase activity in stimulated cells. Surprisingly, alteration of Nox2 activity impacted the glycolysis rate, which indicated that Nox2 in neutrophils was not only required for reactive oxygen species production but was also involved in supporting the energetic metabolism increase that was induced by inflammatory conditions. PFK-2 seems to be a strategic element that links NADPH oxidase activation and glycolysis modulation, and, as such, is proposed as a potential therapeutic target in inflammatory diseases.-Baillet, A., Hograindleur, M.-A., El Benna, J., Grichine, A., Berthier, S., Morel, F., Paclet, M.-H. Unexpected function of the phagocyte NADPH oxidase in supporting hyperglycolysis in stimulated neutrophils: key role of 6-phosphofructo-2-kinase.

Deletion of Kvβ1.1 subunit leads to electrical and haemodynamic changes causing cardiac hypertrophy in female murine hearts.

What is the central question of this study? The goal of this study was to evaluate sex differences and the role of the potassium channel β1 (Kvβ1) subunit in the heart. What is the main finding and its importance? Genetic ablation of Kvβ1.1 in females led to cardiac hypertrophy characterized by increased heart size, prolonged monophasic action potentials, elevated blood pressure and increased myosin heavy chain α (MHCα) expression. In contrast, male mice showed only electrical changes. Kvβ1.1 binds the MHCα isoform at the protein level, and small interfering RNA targeted knockdown of Kvβ1.1 upregulated MHCα. Cardiovascular disease is the leading cause of death and debility in women in the USA, and cardiac arrhythmias are a major concern. Voltage-gated potassium (Kv) channels along with the binding partners; Kvβ subunits are major regulators of the action potential (AP) shape and duration (APD). The regulation of Kv channels by the Kvβ1 subunit is unknown in female hearts. In the present study, we hypothesized that the Kvβ1 subunit is an important regulator of female cardiac physiology. To test this hypothesis, we ablated (knocked out; KO) the KCNAB1 isoform 1 (Kvβ1.1) subunit in mice and evaluated cardiac function and electrical activity by using ECG, monophasic action potential recordings and echocardiography. Our results showed that the female Kvβ1.1 KO mice developed cardiac hypertrophy, and the hearts were structurally different, with enlargement and increased area. The electrical derangements caused by Kvβ1.1 KO in female mice included long QTc and QRS intervals along with increased APD (APD20-90% repolarization). The male Kvβ1.1 KO mice did not develop cardiac hypertrophy, but they showed long QTc and prolonged APD. Molecular analysis showed that several genes that support cardiac hypertrophy were significantly altered in Kvβ1.1 KO female hearts. In particular, myosin heavy chainα expression was significantly elevated in Kvβ1.1 KO mouse heart. Using a small interfering RNA strategy, we identified that knockdown of Kvβ1 increases myosin heavy chainα expression in H9C2 cells. Collectively, changes in molecular and cell signalling pathways clearly point towards a distinct electrical and structural remodelling consistent with cardiac hypertrophy in the Kvβ1.1 KO female mice.

Endophilin A2 protects H2O2-induced apoptosis by blockade of Bax translocation in rat basilar artery smooth muscle cells

BackgroundApoptosis plays a central role in maintaining the normal cell number and tissue homeostasis. Endophilins are a family of evolutionarily conserved proteins that have the critical role in endocytosis. Here, we determined whether endophilin A2 (EndoII) contributes to hydrogen peroxide (H2O2)-induced apoptosis in rat basilar artery smooth muscle cells (BASMCs) and the underlying mechanisms. Methods and resultsBy using small interference RNA (siRNA) and EndoII overexpression strategy, we found that EndoII siRNA knockdown reduced cell viability and promoted H2O2-induced cell apoptosis, evidenced by loss of mitochondrial membrane potential, release of cytochrome c, and activation of caspase-9, 3 and poly (ADP-ribose) polymerase (PARP). In contrast, EndoII overexpression showed opposite effects and inhibited H2O2-induced BASMCs apoptosis. Further studies revealed that there was a direct interaction between EndoII and Bax. Upon H2O2-induced apoptosis, the association of EndoII with Bax were significantly decreased, while the interaction of Bax/tBid were increased, accompanied by a translocation of Bax from cytosol to mitochondria. Knockdown of EndoII did not affect the expression of Bax, but further promoted the binding of Bax with tBid and favored the accumulation of Bax to mitochondria as well as Bax activation; whereas EndoII overexpression produced the opposite effects. In addition, EndoII siRNA aggravated, but EndoII overexpression alleviated, the reduction of Bcl-2 expression in H2O2-treated cells. ConclusionsThese data suggested a role of EndoII in protecting BASMCs apoptosis induced by H2O2, possibly by inhibiting the addressing of Bax to mitochondria. Targeting on EndoII may be a new strategy to treat apoptosis-associated diseases.

Gonadotropin-releasing hormone regulates human trophoblastic cell invasion via TWIST-induced N-cadherin expression.

GnRH and its receptor, GnRHR, were shown to promote trophoblastic cell invasion. Detection of elevated early development-related transcription factor TWIST and adhesion molecule N-cadherin in the invasive trophoblastic cells could suggest that GnRH promotes trophoblastic cell invasion through TWIST-regulated N-cadherin pathway. This study sought to investigate the regulatory effect of GnRH on TWIST and N-cadherin expression as well as invasiveness in human trophoblastic cells. The expression of GnRHR, TWIST, and N-cadherin was first examined in human first-trimester chorionic villi by immunohistochemistry. Expression levels of GnRHR, TWIST, and N-cadherin were tested in primary extravillous trophoblastic (EVT) cells and an immortalized EVT cell line HTR-8/SVneo cells with incubation of GnRH and its antagonist, Antide. Small interfering RNA strategy was used to study the roles of TWIST and N-cadherin in basal- and GnRH-regulated trophoblast invasion. Matrigel-mediated transwell invasion assays were employed to assess cell invasion capacity. GnRHR, TWIST, and N-cadherin were detected at the invasive site of first-trimester human placenta. GnRH treatment significantly increased TWIST and N-cadherin expression in primary EVT as well as HTR-8/SVneo cells. Pretreatment with the GnRH receptor antagonist Antide attenuated the effects of GnRH on TWIST and N-cadherin expression. Invasive capacity of primary EVT and HTR-8/SVneo cell was reduced following siRNA-mediated knockdown of either TWIST or N-cadherin. Furthermore, by knocking down endogenous TWIST, the expression level of N-cadherin was reduced as well as GnRH-induced HTR-8/SVneo cell invasion. Treatment with GnRH induces AKT phosphorylation and Phosphoinositide3-kinase inhibitor LY294002 attenuates the effects of GnRH on TWIST and N-cadherin expression and trophoblastic cell invasion. Our results suggest that GnRH acts via its receptor to induce AKT phosphorylation, which contributes to elevated TWIST expression. Increased levels of TWIST subsequently induce N-cadherin expression, which promotes human trophoblastic cell invasion in vitro.

Improving fruit quality in tomato (Lycopersicum esculentum Mill) under heat stress by silencing the vis 1 gene using small interfering RNA technology

Constantly elevated temperatures cause an array of physio-biochemical changes in tomato (Lycopersicum esculentum Mill.) which make the fruit ripen quickly and up to 50 % yield loss. The development of tomato cultivars, using genetic engineering approaches which delay ripening, offers a new way to keep tomatoes healthy under heat stress. Over-expression of small heat shock protein gene, viscosity 1 (vis 1) plays a role in increasing juice viscosity, early ripening and tissue soften which emphasizes the importance of this gene in premature ripening. The aim of this work was therefore to develop a useful system for silencing the vis1 gene using small interfering RNA strategy. Agrobacterium strain GV3101 harbouring the binary vector pICBV19 containing the gus and bar genes was used to adapt the transformation process in this study. The primers were designed to amplified the first exon of the vis 1 gene and the amplified fragment was used for cloning into the pFGC5941 at XhoI/NcoI site at sense orientation then additional fragment was subsequently cloned at BamHI/XbaI to form sense/antisense cloned fragment interrupted by the CHSA-intron Agrobacterium strain LBA4404 with the binary vector pFGC5941 which harbors vis1 gene under the control of the 35S promoter containing bar gene under the control of a mannopine synthase 2′(Mas2′) as selectable marker, was used to reduce the expression of vis1 gene in fruit. Polymerase chain reaction (PCR), RT–PCR and northern blotting analysis were applied to detect putative transgenic plants. Significantly, silencing of vis 1 gene was potently occur and new transgenic tomato cultivars were produced with enhanced ripening qualities for recommendation for growing under heat stress.

Autocrine positive regulatory feedback of glucocorticoid secretion: Glucocorticoid receptor directly impacts H295R human adrenocortical cell function

Glucocorticoid receptor (GR), a ubiquitous transcriptional factor, regulates target gene expression upon activation by glucocorticoids, notably cortisol, a corticosteroid hormone synthesized in the adrenal cortex. We thus hypothesized that both GR and cortisol might be involved in the regulation of adrenal physiology and steroidogenesis in an autocrine manner.In a cortisol-secreting human adrenocortical cell line (H295R), the GR-dependent signaling pathway was pharmacologically modulated either by dexamethasone (DEX), a GR agonist or by RU486, a GR antagonist, or was knocked-down by small interfering RNA strategy (SiRNA). We showed that GR activation, elicited by 48 h exposure to DEX, exerts a global positive regulatory effect on adrenal steroidogenesis as revealed by a 1.5- to 2-fold increase in cortisol, 11-deoxycortisol and 17-hydroxyprogesterone secretion associated with a significant enhanced expression of steroidogenesis factors such as StAR, CYP11A1, CYP21A2 and CYP11B1. In sharp contrast, RU486 treatment exerted opposite effects by decreasing both steroid production and expression of these steroidogenic factors. Likewise, GR repression by SiRNA also significantly reduced StAR, CYP11A1, and CYP11B1 mRNA levels. Interestingly, RU486 resulted in a significant CYP21A2 enzymatic blockade as demonstrated by a massive increase in 17-hydroxyprogesterone concentrations in RU486-treated H295R cell supernatants, while cortisol and 11-deoxycortisol secretions were reduced by more than 60%. Consistently, we also demonstrated that metabolic conversion of 17-hydroxyprogesterone into 11-deoxycortisol onto H295R cells was drastically blunted in the presence of RU 486. Finally, steady state levels of MC2R transcripts encoding for the ACTH receptor were significantly induced by DEX, unlikely through a direct GR-mediated transcriptional activation as opposed to CYP11A1 and FKBP5 target genes. These results could account for a higher glucocorticoid-elicited ACTH sensitivity of adrenocortical cells.Our study identifies a positive ultra-short regulatory loop exerted by GR on steroidogenesis in H295R cells, thus supporting a complex intra-adrenal GR-mediated feedback, likely relevant for human adrenocortical pathologies.

Loss of vitamin D receptor in chronic kidney disease: a potential mechanism linking inflammation to epithelial-to-mesenchymal transition

Both peritubular inflammation and tubular epithelial-to-mesenchymal transition (EMT) are critical events during the pathogenesis of renal fibrosis. However, the relationship between these two processes is unclear. Here, we investigated the potential role of the vitamin D receptor (VDR) in coupling peritubular inflammation and EMT. In a mouse model of unilateral ureteral obstruction (UUO), loss of VDR was observed as early as 1 day after surgery. In cultured proximal tubular epithelial HK-2 cells, proinflammatory TNF-α inhibited the expression of VDR in a dose- and time-dependant manner. Treatment with TNF-α sensitized HK-2 cells to EMT stimulated by transforming growth factor (TGF)-β1. Ectopic expression of VDR counteracted the synergistic effect of TNF-α and TGF-β1 on EMT. Furthermore, knockdown of VDR using a small interfering RNA strategy mimicked the effect of TNF-α on facilitating EMT. Either TNF-α treatment or a loss of VDR induced β-catenin activation and its nuclear translocation. The VDR ligand calcitriol reversed the VDR loss and inhibited EMT in the mouse UUO model, and late administration of active vitamin D was effective in restoring VDR expression as well, and reduced collagen accumulation and deposition compared with the vehicle control. β-Catenin expression induced by UUO was also significantly inhibited after the late administration of vitamin D. These results indicate that the early loss of VDR in chronic kidney diseases was likely mediated by proinflammatory TNF-α, which renders tubular cells susceptible to EMT. Our data suggest that loss of VDR couples peritubular inflammation and EMT, two key events in renal fibrogenesis.

Twist Modulates Human Trophoblastic Cell Invasion via Regulation of N-Cadherin

The invasion of extravillous cytotrophoblasts (EVT) into the underlying maternal tissues and vasculature is a key step in human placentation. The molecular mechanisms involved in the development of the invasive phenotype of EVT include many that were first discovered for their role in cancer cell metastasis. Previous studies have demonstrated that N-cadherin and its regulatory transcription factor Twist play important roles in the onset and progression of cancers, but their roles in human trophoblastic cell invasion is not clear. The goal of the study was to examine the role of Twist and N-cadherin in human trophoblastic cell invasion. Twist and N-cadherin mRNA and protein levels were determined by RT-PCR and Western blotting in human placental tissues, highly invasive EVT, and poorly invasive JEG-3 and BeWo cells. Whether IL-1β and TGF-β1 regulate Twist mRNA and protein levels in the EVT was also examined. A small interfering RNA strategy was employed to determine the role of Twist and N-cadherin in HTR-8/SVneo cell invasion. Matrigel assays were used to assess cell invasion. Twist and N-cadherin were highly expressed in EVT but were poorly expressed in JEG-3 and BeWo cells. IL-1β and TGF-β1 differentially regulated Twist expression in EVT in a time- and concentration-dependent manner. Small interfering RNA specific for Twist decreased N-cadherin and reduced invasion of HTR-8/SVneo cells. Similarly, a reduction in N-cadherin decreased the invasive capacity of HTR-8/SVneo cells. Twist is an upstream regulator of N-cadherin-mediated invasion of human trophoblastic cells.

Hook2 is involved in the morphogenesis of the primary cilium

Primary cilia originate from the centrosome and play essential roles in several cellular, developmental, and pathological processes, but the underlying mechanisms of ciliogenesis are not fully understood. Given the involvement of the adaptor protein Hook2 in centrosomal homeostasis and protein transport to pericentrosomal aggresomes, we explored its role in ciliogenesis. We found that in human retinal epithelial cells, Hook2 localizes at the Golgi apparatus and centrosome/basal body, a strategic partitioning for ciliogenesis. Of importance, Hook2 depletion disrupts ciliogenesis at a stage before the formation of the ciliary vesicle at the distal tip of the mother centriole. Using two hybrid and immunoprecipitation assays and a small interfering RNA strategy, we found that Hook2 interacts with and stabilizes pericentriolar material protein 1 (PCM1), which was reported to be essential for the recruitment of Rab8a, a GTPase that is believed to be crucial for membrane transport to the primary cilium. Of interest, GFP::Rab8a coimmunoprecipitates with endogenous Hook2 and PCM1. Finally, GFP::Rab8a can overcome Hook2 depletion, demonstrating a functional interaction between Hook2 and these two important regulators of ciliogenesis. The data indicate that Hook2 interacts with PCM1 in a complex that also contains Rab8a and regulates a limiting step required for further initiation of ciliogenesis after centriole maturation.

Knockdown of ornithine decarboxylase antizyme 1 causes loss of uptake regulation leading to increased N 1, N 11-bis(ethyl)norspermine (BENSpm) accumulation and toxicity in NCI H157 lung cancer cells

Ornithine decarboxylase antizyme 1 (AZ1) is a major regulatory protein responsible for the regulation and degradation of ornithine decarboxylase (ODC). To better understand the role of AZ1 in polyamine metabolism and in modulating the response to anticancer polyamine analogues, a small interfering RNA strategy was used to create a series of stable clones in human H157 non-small cell lung cancer cells that expressed less than 5-10% of basal AZ1 levels. Antizyme 1 knockdown clones accumulated greater amounts of the polyamine analogue N (1),N (11)-bis(ethyl)norspermine (BENSpm) and were more sensitive to analogue treatment. The possibility of a loss of polyamine uptake regulation in the knockdown clones was confirmed by polyamine uptake analysis. These results are consistent with the hypothesis that AZ1 knockdown leads to dysregulation of polyamine uptake, resulting in increased analogue accumulation and toxicity. Importantly, there appears to be little difference between AZ1 knockdown cells and cells with normal levels of AZ1 with respect to ODC regulation, suggesting that another regulatory protein, potentially AZ2, compensates for the loss of AZ1. The results of these studies are important for the understanding of both the regulation of polyamine homeostasis and in understanding the factors that regulate tumor cell sensitivity to the anti-tumor polyamine analogues.

A novel vascular disrupting agent plinabulin triggers JNK-mediated apoptosis and inhibits angiogenesis in multiple myeloma cells

AbstractPrevious studies have established a role of vascular-disrupting agents as anti- cancer agents. Plinabulin is a novel vascular-disrupting agent that exhibits potent interruption of tumor blood flow because of the disruption of tumor vascular endothelial cells, resulting in tumor necrosis. In addition, plinabulin exerts a direct action on tumor cells, resulting in apoptosis. In the present study, we examined the anti–multiple myeloma (MM) activity of plinabulin. We show that low concentrations of plinabulin exhibit a potent antiangiogenic action on vascular endothelial cells. Importantly, plinabulin also induces apoptotic cell death in MM cell lines and tumor cells from patients with MM, associated with mitotic growth arrest. Plinabulin-induced apoptosis is mediated through activation of caspase-3, caspase-8, caspase-9, and poly(ADP-ribose) polymerase cleavage. Moreover, plinabulin triggered phosphorylation of stress response protein JNK, as a primary target, whereas blockade of JNK with a biochemical inhibitor or small interfering RNA strategy abrogated plinabulin-induced mitotic block or MM cell death. Finally, in vivo studies show that plinabulin was well tolerated and significantly inhibited tumor growth and prolonged survival in a human MM.1S plasmacytoma murine xenograft model. Our study therefore provides the rationale for clinical evaluation of plinabulin to improve patient outcome in MM.

An Essential Role of STIM1, Orai1, and S100A8–A9 Proteins for Ca2+ Signaling and FcγR-Mediated Phagosomal Oxidative Activity

Phagocytosis is a process of innate immunity that allows for the enclosure of pathogens within the phagosome and their subsequent destruction through the production of reactive oxygen species (ROS). Although these processes have been associated with increases of intracellular Ca(2+) concentrations, the mechanisms by which Ca(2+) could regulate the different phases of phagocytosis remain unknown. The aim of this study was to investigate the Ca(2+) signaling pathways involved in the regulation of FcγRs-induced phagocytosis. Our work focuses on IgG-opsonized zymosan internalization and phagosomal ROS production in DMSO-differentiated HL-60 cells and neutrophils. We found that chelation of intracellular Ca(2+) by BAPTA or emptying of the intracellular Ca(2+) store by thapsigargin reduced the efficiency of zymosan internalization. Using an small interfering RNA strategy, our data establish that the observed Ca(2+) release occurs through two isoforms of inositol 1,4,5-triphosphate receptors, ITPR1 and ITPR3. In addition, we provide evidence that phagosomal ROS production is dependent on extracellular Ca(2+) entry. We demonstrate that the observed Ca(2+) influx is supported by ORAI calcium release-activated calcium modulator 1 (Orai1) and stromal interaction molecule 1 (STIM1). This result suggests that extracellular Ca(2+) entry, which is required for ROS production, is mediated by a store-operated Ca(2+) mechanism. Finally, our data identify the complex formed by S100A8 and S100A9 (S100 calcium-binding protein A8 and A9 complex), two Ca(2+)-binding proteins, as the site of interplay between extracellular Ca(2+) entry and intraphagosomal ROS production. Thus, we demonstrate that FcγR-mediated phagocytosis requires intracellular Ca(2+) store depletion for the internalization phase. Then phagosomal ROS production requires extracellular Ca(2+) entry mediated by Orai1/STIM1 and relayed by S100A8-A9 as Ca(2+) sensor.

Host DNA Repair Proteins in Response to Pseudomonas aeruginosa in Lung Epithelial Cells and in Mice

Although DNA repair proteins in bacteria are critical for pathogens' genome stability and for subverting the host defense, the role of host DNA repair proteins in response to bacterial infection is poorly defined. Here, we demonstrate, for the first time, that infection with the Gram-negative bacterium Pseudomonas aeruginosa significantly altered the expression and enzymatic activity of 8-oxoguanine DNA glycosylase (OGG1) in lung epithelial cells. Downregulation of OGG1 by a small interfering RNA strategy resulted in severe DNA damage and cell death. In addition, acetylation of OGG1 is required for host responses to bacterial genotoxicity, as mutations of OGG1 acetylation sites increased Cockayne syndrome group B (CSB) protein expression. These results also indicate that CSB may be involved in DNA repair activity during infection. Furthermore, OGG1 knockout mice exhibited increased lung injury after infection with P. aeruginosa, as demonstrated by higher myeloperoxidase activity and lipid peroxidation. Together, our studies indicate that P. aeruginosa infection induces significant DNA damage in host cells and that DNA repair proteins play a critical role in the host response to P. aeruginosa infection, serving as promising targets for the treatment of this condition and perhaps more broadly Gram-negative bacterial infections.