siRNA Gene Knockdown Research Articles

Disruption of endocytic trafficking protein Rab7 impairs invasiveness of cholangiocarcinoma cells.

Alterations and mutations of endo-lysosomal trafficking proteins have been associated with cancer progression. Identification and characterization of endo-lysosomal trafficking proteins in invasive cholangiocarcinoma (CCA) cells may benefit prognosis and drug design for CCA. To identify and characterize endo-lysosomal trafficking proteins in invasive CCA. A lysosomal-enriched fraction was isolated from a TNF-α induced invasive CCA cell line (KKU-100) and uninduced control cells and protein identification was performed with nano-LC MS/MS. Novel lysosomal proteins that were upregulated in invasive CCA cells were validated by real-time RT-PCR. We selected Rab7 for further studies of protein level using western blotting and subcellular localization using immunofluorescence. The role of Rab7 in CCA invasion was determined by siRNA gene knockdown and matrigel transwell assay. Rab7 mRNA and protein were upregulated in invasive CCA cells compared with non-treated controls. Immunofluorescence studies demonstrated that Rab7 was expressed predominantly in invasive CCA cells and was localized in the cytoplasm and lysosomes. Suppression of Rab7 translation significantly inhibited TNF-α-induced cell invasion compared to non-treated control (p= 0.044). Overexpression of Rab7 in CCA cells was associated with cell invasion, supporting Rab7 as a novel candidate for the development of diagnostic and therapeutic strategies for CCA.

Efficient Receptor Mediated siRNA Delivery in Vitro by Folic Acid Targeted Pentablock Copolymer-Based Micelleplexes

Novel, biocompatible polyplexes, based on the combination of cationic pentablock copolymers with folic acid functionalized copolymers, were designed and developed for target-specific siRNA delivery. The resulting micelleplexes spontaneously formed polymeric micelles with a hydrophobic core surrounded directly by a cationic poly-2-(4-aminobutyl)-oxazole (PABOXA) and subsequently shielded by hydrophilic poly-2-methyl-oxazole (PMOXA) layer. The described micelleplexes form highly stable particles even in complete serum after 24 h compared with the highly cationic polymer PEI, which show aggregate formation in serum containing buffer solution. Targeted siRNA delivery and gene knockdown could be shown using green fluorescent protein (GFP) expressing HeLa cells, resulting in ∼31% and ∼8% suppression of the expression of GFP for targeted and nontargeted micelleplexes, respectively. Comparison studies of folic-receptor positive HeLa cells with normal folic-receptor-negative HEK293 cells revealed involvement of receptor mediated cellular uptake of fluorescently labeled siRNA. The new designed nanocarrier showed no cytotoxicity, having a potential application. The presented concept of shielding a nucleic-acid complexing cationic chains with a stealth layer and combining it with receptor ligand overcomes typical problems with undesired protein and cell interactions in delivery of nucleic acids using polymeric systems, opening new doors for application if RNA inhibition in the organism.

Validation of Synthetic CRISPR Reagents as a Tool for Arrayed Functional Genomic Screening.

To date, lentiviral-based CRISPR-Cas9 screens have largely been conducted in pooled format. However, numerous assays are not amenable to pooled approaches, and lentiviral screening in arrayed format presents many challenges. We sought to examine synthetic CRISPR reagents in the context of arrayed screening. Experiments were performed using aberrant DNA replication as an assay. Using synthetic CRISPR RNAs targeting the known control gene GMNN in HCT-116 cells stably expressing Cas9, we observed statistically significant phenotype among the majority of transfected cells within 72 hours. Additional studies revealed near complete loss of GMNN protein and editing of GMNN DNA. We next conducted a screen of synthetic CRISPR RNAs directed against 640 ubiquitin-related genes. Screening identified known and novel DNA replication regulators that were also supported by siRNA gene knockdown. Notably, CRISPR screening identified more statistically significant hits than corresponding siRNA screens run in parallel. These results highlight the possibility of using synthetic CRISPR reagents as an arrayed screening tool.

Overexpression and knock-down studies highlight that a disintegrin and metalloproteinase 28 controls proliferation and migration in human prostate cancer.

Prostate cancer is one of the most prevalent cancers in men. It is critical to identify and characterize oncogenes that drive the pathogenesis of human prostate cancer. The current study builds upon previous research showing that a disintegrin and metallproteinase (ADAM)28 is involved in the pathogenesis of numerous cancers. Our novel study used overexpression, pharmacological, and molecular approaches to investigate the biological function of ADAM28 in human prostate cancer cells, with a focus on cell proliferation and migration. The results of this study provide important insights into the role of metalloproteinases in human prostate cancer.The expression of ADAM28 protein levels was assessed within human prostate tumors and normal adjacent tissue by immunohistochemistry. Immunocytochemistry and western blotting were used to assess ADAM28 protein expression in human prostate cancer cell lines. Functional assays were conducted to assess proliferation and migration in human prostate cancer cells in which ADAM28 protein expression or activity had been altered by overexpression, pharmacological inhibition, or by siRNA gene knockdown.The membrane bound ADAM28 was increased in human tumor biopsies and prostate cancer cell lines. Pharmacological inhibition of ADAM28 activity and/or knockdown of ADAM28 significantly reduced proliferation and migration of human prostate cancer cells, while overexpression of ADAM28 significantly increased proliferation and migration.ADAM28 is overexpressed in primary human prostate tumor biopsies, and it promotes human prostate cancer cell proliferation and migration. This study supports the notion that inhibition of ADAM28 may be a potential novel therapeutic strategy for human prostate cancer.

Nanoparticle-mediated siRNA delivery assessed in a 3D co-culture model simulating prostate cancer bone metastasis

siRNA has emerged as a potential therapeutic for the treatment of prostate cancer but effective delivery remains a major barrier to its clinical application. This study aimed to develop and characterise a 3D in vitro co-culture model to simulate prostate cancer bone metastasis and to assess the ability of the model to investigate nanoparticle-mediated siRNA delivery and gene knockdown. PC3 or LNCaP prostate cancer cells were co-cultured with hFOB 1.19 osteoblast cells in 2D on plastic tissue culture plates and in 3D on collagen scaffolds mimicking the bone microenvironment. To characterise the co-culture model, cell proliferation, enzyme secretion and the utility of two different gene delivery vectors to mediate siRNA uptake and gene knockdown were assessed. Cell proliferation was reduced by∼50% by day 7 in the co-culture system relative to monoculture (PC3 and LNCaP co-cultures, in 2D and 3D) and an enhanced level of MMP9 (a marker of bone metastasis) was secreted into the media (1.2–4-fold increase depending on the co-culture system). A cationic cyclodextrin gene delivery vector proved significantly less toxic in the co-culture system relative to the commercially available vector Lipofectamine 2000®. In addition, knockdown of both the GAPDH gene (minimum 15%) and RelA subunit of the NF-κB transcription factor (minimum 20%) was achieved in 2D and 3D cell co-cultures. Results indicate that the prostate cancer-osteoblast in vitro co-culture model was more physiologically relevant vs the monoculture. This model has the potential to help improve the design and efficacy of gene delivery formulations, to more accurately predict in vivo performance and, therefore, to reduce the risk of product failure in late-stage clinical development.

Local anesthetic bupivacaine induced ovarian and prostate cancer apoptotic cell death and underlying mechanisms in vitro.

Retrospective studies indicate that the use of regional anesthesia can reduce cancer recurrence after surgery which could be due to ranging from immune function preservation to direct molecular mechanisms. This study was to investigate the effects of bupivacaine on ovarian and prostate cancer cell biology and the underlying molecular mechanisms. Cell viability, proliferation and migration of ovarian carcinoma (SKOV-3) and prostate carcinoma (PC-3) were examined following treatment with bupivacaine. Cleaved caspase 3, 8 and 9, and GSK-3β, pGSK-3βtyr216 and pGSK-3βser9 expression were assessed by immunofluorescence. FAS ligand neutralization, caspase and GSK-3 inhibitors and GSK-3β siRNA were applied to further explore underlying mechanisms. Clinically relevant concentrations of bupivacaine reduced cell viability and inhibited cellular proliferation and migration in both cell lines. Caspase 8 and 9 inhibition generated partial cell death reversal in SKOV-3, whilst only caspase 9 was effective in PC-3. Bupivacaine increased the phosphorylation of GSK-3βTyr216 in SKOV-3 but without measurable effect in PC3. GSK-3β inhibition and siRNA gene knockdown decreased bupivacaine induced cell death in SKOV-3 but not in PC3. Our data suggests that bupivacaine has direct ‘anti-cancer’ properties through the activation of intrinsic and extrinsic apoptotic pathways in ovarian cancer but only the intrinsic pathway in prostate cancer.

EPHB4 Protein Expression in Vascular Smooth Muscle Cells Regulates Their Contractility, and EPHB4 Deletion Leads to Hypotension in Mice

EPH kinases are the largest family of receptor tyrosine kinases, and their ligands, ephrins (EFNs), are also cell surface molecules. This work presents evidence that EPHB4 on vascular smooth muscle cells (VSMCs) is involved in blood pressure regulation. We generated gene KO mice with smooth muscle cell-specific deletion of EPHB4. Male KO mice, but not female KO mice, were hypotensive. VSMCs from male KO mice showed reduced contractility when compared with their WT counterparts. Signaling both from EFNBs to EPHB4 (forward signaling) and from EPHB4 to EFNB2 (reverse signaling) modulated VSMC contractility. At the molecular level, the absence of EPHB4 in VSMCs resulted in compromised signaling from Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) to myosin light chain kinase (MLCK) to myosin light chain, the last of which controls the contraction force of motor molecule myosin. Near the cell membrane, an adaptor protein GRIP1, which can associate with EFNB2, was found to be essential in mediating EPHB4-to-EFNB reverse signaling, which regulated VSMC contractility, based on siRNA gene knockdown studies. Our research indicates that EPHB4 plays an essential role in regulating small artery contractility and blood pressure.

Knock-Down of Id1 and Id3 Proteins Induces Apoptosis in Human Colon Carcinoma (HCT116) Cells

Id (Inhibitor of differentiation) proteins belong to the Helix-Loop-Helix (HLH) group of transcription factors and they are characterized by the absence of their DNA-binding domain. Id proteins are involved in cellular processes. These biological roles include cell growth, differentiation, senescence, apoptosis, angiogenesis and neoplastic transformation. This work aimed at investigating the consequences of down-regulating Id proteins on growth and pro-apoptotic functions in an in vitro model using HCT116 by siRNA gene knock-down. This objective was achieved by using antisense oligonucleotides complementary to Id1, Id2 and Id3 mRNA on human epithelial colon carcinoma cell line (HCT116). Silencing of Id1 and Id3 in HCT116 resulted in widespread cell death but not S-phase arrest. Interestingly, knock-down of Id2 neither resulted in induction of apoptosis nor cell growth arrest. However, since it was able to reduce viable cell populations of HCT116 at 48 h post-transfection, it may imply that cell death and growth arrest following down-regulation of Id2 may occur via a different mechanism. These observations suggest that Id1 and Id3 could provide some therapeutic opportunities to cure colonic cancers.

Abstract 4987: Stathmin regulates cell migration, invasion and transendothelial migration via RhoA activation in neuroblastoma

Abstract Neuroblastoma is an aggressive childhood cancer with dismal 5-year survival rates, primarily due to metastatic disease. The microtubule protein stathmin is highly expressed in neuroblastoma, and silencing stathmin decreases metastatic disease in a neuroblastoma orthotopic mouse model1. However, stathmin's role in the metastatic cascade and mechanism of action in this process is unknown. Our aim was to investigate the role of stathmin in neuroblastoma metastasis. Methods: To study the role of stathmin (STMN) in neuroblastoma, siRNA gene knockdown (using two individual siRNAs STMNsiRNA1 and STMNsiRNA2) or a non-targeting control siRNA (ContsiRNA) was performed in both SK-N-BE(2) and SH-SY5Y cell lines. 3D spheroid invasion assessed by counting and measuring cellular protrusion length into collagen I matrix. Stathmin expression was assessed by RT-qPCR and western blotting. Migration and invasion assays were performed using Boyden-chambers. Anoikis was assessed using Annexin V staining. Transendothelial migration was assessed by measurement of lysed migrated cells using a fluorescent plate reader. RhoA activation was assessed by RhoA activity pulldown and western blotting. Results: Stathmin suppression significantly decreased SK-N-BE(2) and SH-SY5Y cell migration (p<0.05) and invasion (p<0.0005). Spheroid invasion was significantly decreased in SK-N-BE(2) as measured by the number and length of cellular protrusions (p<0.05). Stathmin did not influence anoikis in either of the neuroblastoma cell lines, however stathmin knockdown significantly reduced transendothelial migration in both cell lines compared to ContsiRNA cells (p<0.0001). To determine whether stathmin's effect on transendothelial migration was mediated via ROCK signalling SK-N-BE(2) cells were treated with two separate ROCK inhibitors. Importantly, ROCK inhibition restored transendothelial migration levels in stathmin knockdown cells to those of ContsiRNA cells (STMNsiRNA1:86.0±4.9%; STMNsiRNA2:66.2±4.5%; control:80.95±10.85%; p>0.25), indicating stathmin mediates transendothelial migration via inhibition of ROCK signalling. Importantly, stathmin suppression dramatically increased RhoA activation (upstream regulator of ROCK signalling) indicating that stathmin's effect on ROCK signalling is mediated via its effects on its regulator RhoA. Conclusions: Stathmin suppression interferes with key metastatic events via RhoA/ROCK signalling in neuroblastoma. Targeting stathmin may provide a novel treatment approach for metastatic neuroblastoma. 1 Byrne, FL, Yang, L, Phillips, PA, Hansford, LM, Fletcher, JI, Ormandy, CJ, McCarroll, JA & Kavallaris, M. (2013) Oncogene [epub Feb 11] Citation Format: Christopher M. Fife, Frances L. Byrne, Sharon M. Sagnella, Thomas P. Davis, Joshua A. McCarroll, Maria Kavallaris. Stathmin regulates cell migration, invasion and transendothelial migration via RhoA activation in neuroblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4987. doi:10.1158/1538-7445.AM2014-4987

Seed sequence-matched controls reveal limitations of small interfering RNA knockdown in functional and structural studies of hepatitis C virus NS5A-MOBKL1B interaction.

Hepatitis C virus (HCV) is a widespread human pathogen causing liver cirrhosis and cancer. Similar to the case for other viruses, HCV depends on host and viral factors to complete its life cycle. We used proteomic and yeast two-hybrid approaches to elucidate host factors involved in HCV nonstructural protein NS5A function and found that MOBKL1B interacts with NS5A. Initial experiments with small interfering RNA (siRNA) knockdown suggesting a role in HCV replication led us to examine the interaction using biochemical and structural approaches. As revealed by a cocrystal structure of a core MOBKL1B-NS5A peptide complex at 1.95 Å, NS5A binds to a hydrophobic patch on the MOBKL1B surface. Biosensor binding assays identified a highly conserved, 18-amino-acid binding site in domain II of NS5A, which encompasses residues implicated in cyclophilin A (CypA)-dependent HCV RNA replication. However, a CypA-independent HCV variant had reduced replication in MOBKL1B knockdown cells, even though its NS5A does not interact with MOBKL1B. These discordant results prompted more extensive studies of MOBKL1B gene knockdowns, which included additional siRNAs and specifically matched seed sequence siRNA controls. We found that reduced virus replication after treating cells with MOBKL1B siRNA was actually due to off-target inhibition, which indicated that the initial finding of virus replication dependence on the MOBKL1B-NS5A interaction was incorrect. Ultimately, using several approaches, we found no relationship of the MOBKL1B-NS5A interaction to virus replication. These findings collectively serve as a reminder to investigators and scientific reviewers of the pervasive impact of siRNA off-target effects on interpretation of biological data. Our study illustrates an underappreciated shortcoming of siRNA gene knockdown technology. We initially identified a cellular protein, MOBKL1B, as a binding partner with the NS5A protein of hepatitis C virus (HCV). MOBKL1B siRNA, but not irrelevant RNA, treatment was associated with both reduced virus replication and the absence of MOBKL1B. Believing that HCV replication depended on the MOBKL1B-NS5A interaction, we carried out structural and biochemical analyses. Unexpectedly, an HCV variant lacking the MOBKL1B-NS5A interaction could not replicate after cells were treated with MOBKL1B siRNA. By repeating the MOBKL1B siRNA knockdowns and including seed sequence-matched siRNA instead of irrelevant siRNA as a control, we found that the MOBKL1B siRNAs utilized had off-target inhibitory effects on virus replication. Collectively, our results suggest that stricter controls must be utilized in all RNA interference (RNAi)-mediated gene knockdown experiments to ensure sound conclusions and a reliable scientific knowledge database.

The TRPC channel blocker SKF 96365 inhibits glioblastoma cell growth by enhancing reverse mode of the Na+/Ca2+ exchanger and increasing intracellular Ca2+

SKF 96365 is well known for its suppressing effect on human glioblastoma growth by inhibiting pre-activated transient receptor potential canonical (TRPC) channels and Ca(2+) influx. The effect of SKF 96363 on glioblastoma cells, however, may be multifaceted and this possibility has been largely ignored. The effects of SKF 96365 on cell cycle and cell viability of cultured human glioblastoma cells were characterized. Western blot, Ca(2+) imaging and patch clamp recordings were used to delineate cell death mechanisms. siRNA gene knockdown provided additional evidence. SKF 96365 repressed glioblastoma cell growth via increasing intracellular Ca(2+) ([Ca(2+) ]i ) irrespective of whether TRPC channels were blocked or not. The effect of SKF 96365 primarily resulted from enhanced reverse operation of the Na(+) /Ca(2+) exchanger (NCX) with an EC50 of 9.79 μM. SKF 96365 arrested the glioblastoma cells in the S and G2 phases and activated p38-MAPK and JNK, which were all prevented by the Ca(2+) chelator BAPTA-AM or EGTA. The expression of NCX in glioblastoma cells was significantly higher than in normal human astrocytes. Knockdown of the NCX1 isoforms diminished the effect of SKF 96365 on glioblastoma cells. At the same concentration, SKF 96365 blocks TRPC channels and enhances the reverse mode of the NCX causing [Ca(2+) ]i accumulation and cytotoxicity. This finding suggests an alternative pharmacological mechanism of SKF 96365. It also indicates that modulation of the NCX is an effective method to disrupt Ca(2+) homeostasis and suppress human glioblastoma cells.

Distinct Pathways Regulated by RET and Estrogen Receptor in Luminal Breast Cancer Demonstrate the Biological Basis for Combination Therapy

We investigated directed therapy based on TFAP2C-regulated pathways to inform new therapeutic approaches for treatment of luminal breast cancer. TFAP2C regulates the expression of genes characterizing the luminal phenotype including ESR1 and RET, but pathway cross talk and potential for distinct elements have not been characterized. Activation of extracellular signal-regulated kinases (ERK) and AKT was assessed using phosphorylation-specific Western blot. Cell proliferation was measured with MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] after siRNA (small interfering RNA) gene knockdown or drug treatment. Cell cycle, Ki-67, and cleaved caspase 3 were measured by fluorescence-activated cell sorting. Tumorigenesis was assessed in mice xenografts. Knockdown of TFAP2C or RET inhibited GDNF (glial cell line-derived neurotrophic factor)-mediated activation of ERK and AKT in MCF-7 cells. Similarly, sunitinib, a small-molecule inhibitor of RET, blocked GDNF-mediated activation of ERK and AKT. Inhibition of RET either by gene knockdown or by treatment with sunitinib or vandetanib reduced RET-dependent growth of luminal breast cancer cells. Interestingly, knockdown of TFAP2C, which controls both ER (estrogen receptor) and RET, demonstrated a greater effect on cell growth than either RET or ER alone. Parallel experiments using treatment with tamoxifen and sunitinib confirmed the increased effectiveness of dual inhibition of the ER and RET pathways in regulating cell growth. Whereas targeting the ER pathway altered cell proliferation, as measured by Ki-67 and S-phase, anti-RET primarily increased apoptosis, as demonstrated by cleaved caspase 3 and increased TUNEL (terminal deoxyneucleotidyl transferase dUTP nick end labeling) expression in xenografts. ER and RET primarily function through distinct pathways regulating proliferation and cell survival, respectively. The findings inform a therapeutic approach based on combination therapy with antiestrogen and anti-RET in luminal breast cancer.

Enhanced Intracellular siRNA Delivery using Bioreducible Lipid‐Like Nanoparticles

A new library of lipid-like nanoparticles (lipidoids) comprising disulfide bond is developed for siRNA delivery. Bioreducible lipidoids deliver siRNA with greater efficiency than nonbioreducible lipidoids with similar chemical structures. A siRNA release investigation, as well as an intracellular siRNA trafficking study, reveals that the degradation of bioreducible lipidoid in a strongly reductive intracellular environment boosts siRNA release and enhances siRNA gene knockdown efficiency.

Ubiquitin C-terminal hydrolase L1 interacts with choline transporter in cholinergic cells

Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is a deubiquitinating enzyme, which is highly expressed in neuronal cells. Previous studies have indicated that UCHL1 is involved in cognitive function, neurodegenerative diseases, and neuromuscular junction development. Acetylcholine (Ach) is a critical neurotransmitter in these functions. Yet, the effect of UCHL1 on the cholinergic system has not been reported. In this study, using a cholinergic neuronal cell line, SN56, as an invitro model, we detected the physical interaction of UCHL1 and high affinity choline transporter (CHT), which is a key protein regulating Ach re-synthesis. Reduction of UCHL1 by siRNA gene knockdown significantly increased poly-ubiquitinated CHT and decreased native CHT protein level, but did not affect CHT mRNA expression. Biotinylation assay showed that UCHL1 is localized only in the cytosol of the cells and that the gene knockdown of UCHL1 significantly reduced cytosolic CHT but had no significant effect on membrane CHT level. These data provide novel and potentially important evidence that UCHL1 may play a role in the regulation of cholinergic function by affecting CHT ubiquitination and degradation.

Abstract B01: Phosphorylation of pro-oncogenic c-Src at serine-17 by protein kinase G-Iα promotes chemoresistance/cell proliferation in human non-small cell lung cancer cells.

Abstract We previously showed that PKG-Iα promotes cell survival in neural cells (N1E-115 neuroblastoma and NG108-15 neuroblastoma-glioma hybrid cells) and significantly contributes to serine-155 phosphorylation of BAD, an apoptosis-regulating protein (Johlfs and Fiscus 2010). We also found, by using both gene knockdown and pharmacological inhibitors (ODQ, DT-2 and DT-3) to decrease PKG-Iα kinase activity, that PKG-Iα promotes cell proliferation and chemoresistance in human ovarian cancer cells, involving a novel interaction between PKG-Iα and the oncogenic protein c-Src (Fiscus et al 2012, Leung et al 2010). PKG-Iα activity was dependent on c-Src-catalyzed tyrosine phosphorylation of PKG-Iα; and, reciprocally, c-Src activation was dependent on expression and kinase activity of PKG-Iα;. We hypothesized that activation of c-Src involves PKG-Iα-catalyzed phosphorylation at serine-17 of c-Src, because amino acids around serine-17 provide consensus sequence for PKG-I, and tαhat this plays an important role in proliferation and chemoresistance. As c-Src has been proposed to promote cell proliferation and chemoresistance in NSCLC cells, the present study aimed to: 1) determine if c-Src increases PKG-Iα kinase activity in NSCLC cells using our newly-developed near-infrared-fluorescence (NIRF)-kinase assay, 2) determine if serine-17-phosphorylation of c-Src, in intact cancer cells, is dependent on PKG-Iα expression and kinase activity (using siRNA gene knockdown and pharmacological inhibitors), and 3) determine if inhibition/knockdown of c-Src and PKG-Iα alters chemoresistance and proliferation of NSCLC cells. Protein expression of c-Src and PKG-Iα was determined by conventional Western blot analysis and new ultrasensitive capillary-electrophoresis-based NanoPro100/1000 (ProteinSimple, Santa Clara, CA, USA). NanoPro100/1000 allows clear separation and identification of different c-Src and PKG-Iαphospho-forms (a novel way of identifying the activation of c-Src and PKG-Iα). We found PKG-Iα was able to directly catalyze phosphorylation of serine-17 of c-Src, increasing autophosphorylation of c-Src at tyrosine-419, the activation site of c-Src. Inhibition of PKG-Iα kinase activity using DT-2 or silencing of PKG-Iα expression using siRNA dramatically reduced the intracellular phosphorylation of c-Src at serine-17. Both kinase-activity inhibition and knockdown of PKG-Iα caused significant increases in apoptosis, synergistically enhancing apoptosis induced by the chemotherapeutic agent cisplatin, and dramatically decreased cell proliferation/colony formation in NSCLC cells. This novel reciprocal relationship between c-Src and PKG-Iα provides a new target for the development of new anti-cancer therapeutic agents. Fiscus RR, Leung EL, Wong JC, Johlfs MG (2012). Nitric oxide/protein kinase G-Ialpha promote c-Src activation, proliferation and chemoresistance in ovarian cancer. In: Farghaly S (ed). Ovarian Cancer - Basic Science Perspective. Intech Open Access Publisher. pp 315-334. Johlfs MG, Fiscus RR (2010). Protein kinase G type-Ialpha phosphorylates the apoptosis-regulating protein Bad at serine 155 and protects against apoptosis in N1E-115 cells. Neurochem Int 56: 546-553. Leung EL, Wong JC, Johlfs MG, Tsang BK, Fiscus RR (2010). Protein kinase G type Ialpha activity in human ovarian cancer cells significantly contributes to enhanced Src activation and DNA synthesis/cell proliferation. Mol Cancer Res 8: 578-591. Citation Format: Mary G. Johlfs, Ronald R. Fiscus, Priyatham Gorjala, Renee Coffman, Harry Rosenberg. Phosphorylation of pro-oncogenic c-Src at serine-17 by protein kinase G-Iα promotes chemoresistance/cell proliferation in human non-small cell lung cancer cells. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B01.

Abstract P4-19-05: Breast cancer campaign tissue bank human cell culture programme: Generating new biomaterials for breast research

Abstract Background: In-vitro model systems provide an important tool in breast cancer research and, over recent years, there have been considerable advances in their construction. However, despite these advances, the majority of models still employ a narrow range of established cell lines, which frequently are not appropriate for the in-vivo cell type they are representing1. Method: As part of the Breast Cancer Campaign Tissue Bank, we have developed a cell culture programme which feeds into the main tissue collection and involves the systematic generation of materials from normal, high-risk, cancer-containing and malignant breast tissue in order to make available a breadth of material to the research community. Following informed patient consent, fresh tissue is retrieved and, using tissue digestion and magnetic bead technology2, purified cell populations are isolated, characterized and frozen down, or further processed for DNA and RNA isolation. Results: We have isolated purified populations of luminal epithelial, myoepithelial and fibroblast cells from normal breast, risk-reduction (BRCA1 and BRCA2 mutated) breast and from morphologically normal breast tissue surrounding breast cancer. We have generated matched surround and tumour-associated fibroblasts, as well as matched tumour epithelial and tumour-associated fibroblast cell isolates. These cells have been incorporated into 2- and 3-D culture models and we have demonstrated successful genetic manipulation, with siRNA gene knockdown and gene over-expression through retroviral transduction. Immortalisation of selected cell populations is currently underway. Intact organoids and tissue explants have also been generated for in-vitro experimentation. Matched frozen and FFPE samples are available, together with full clinicopathological data. The research community can access these biomaterials via a web-based search portal, with full user support. Conclusion: Through this cell culture programme we aim to make available a wider and more appropriate range of materials for breast research, which should allow more clinically relevant model systems to be developed and add value to breast cancer biobanking.

Culture optimisation of CD34‐positive corneal stromal stem cells and transdifferentiation into corneal epithelial cells

AbstractPurpose The integrity of the corneal epithelium is essential for clarity of vision. Under normal circumstances, superficial epithelial cells are shed into the tear film and regenerated by epithelial stem/progenitor cells located at the limbus. Damage or depletion of the limbal cells leads to conditions such as limbal stem cell deficiency (LSCD). Current treatment involves transplantation of limbal epithelial cells expanded ex‐vivo; however cell numbers are limited. In this study, we identify a source of multipotent stem cells located in the corneal stroma that express CD34 upon isolation. These cells demonstrate an ability to transdifferentiate into corneal epithelial cells.Methods CD34 expression diminishes during traditional tissue‐culture plastic propagation; in this study we optimised culture conditions for maintained and efficient expansion of CD34+ cells. We investigated the role of CD34 in epithelial transdifferentiation of stromal cells, using siRNA gene knockdown and analysis by immunocytochemistry, flow cytometry and qPCR.Results Early results suggest a three‐dimensional environment and semi‐solid medium intended for hematopoietic culture demonstrated extended CD34 expression. When cells were transferred to epithelial differentiation medium they showed an epithelial morphology, significantly increased cytokeratin 3 and 19 expression, and considerable upregulation of genes related to corneal epithelial cells (ABCG2, DeltaN63, LEF1, HES1, FRZB1, KRT19, DTC and CDH1).Conclusion This work will help produce methodologies to create cell banks for generation of corneal epithelium, from a corneal stromal stem cell source, leading to improved surgical and visual outcomes in LSCD patients.

Korean red ginseng extract prevents APAP‐induced hepatotoxicity through metabolic enzyme regulation: The role of ginsenoside Rg3, a protopanaxadiol

Inappropriate use of acetaminophen (APAP) can lead to morbidity and mortality secondary to hepatic necrosis. We evaluated the beneficial effect and molecular mechanism of Korean red ginseng (KRG) on the APAP-mediated hepatotoxicity and identified a major component of KRG for hepatoprotection. Survival test, liver function test, histopathological study, APAP-metabolic profiling and gene expression were examined in mice. We determined the enzyme expression and upstream signalling in H4IIE cells analysed by RT-PCR, immunoblotting, siRNA gene knockdown and promoter-luciferase assay. High doses of KRG reduced mortality at the LD50 of APAP. APAP increased AST and ALT activities, which were abrogated by low doses of KRG. These protective effects were consistent with the results from histopathological examinations. KRG altered APAP metabolic profiles through inhibition of cytochrome P450 2E1 and induction of glutathione S-transferase A2 (GSTA2). Knockdown of GSTA2 catalyses the conjugation of glutathione reversed KRG-mediated protection against N-acetyl-p-benzoquinone imine in H4IIE cells. The nuclear Nrf2 and C/EBPβ, which are essential transcriptional factors for GSTA2 were increased by KRG. These effects were downstream of multiple signalling, including PI3K, JNK or PKA. Ginsenoside Rg3 but not Rb1, Rc and Rg1 significantly increased GSTA2 protein expression. Rg3 resulted in the transcriptional activation of GSTA2 downstream of the multiple cellular signalling. These results demonstrate that KRG is efficacious in protection against APAP-induced hepatotoxicity and mortality through metabolic regulation and that Rg3 is a major component of KRG for the GST induction, implying that Rg3 should be considered to be a potential hepatoprotective agent.

Zyxin Regulates Cell Migration and Differentiation in EMT during Chicken AV Valve Morphogenesis

During heart valve development, epithelial-mesenchymal transformation (EMT) is a key process for valve formation. EMT leads to the generation of mesenchymal cells that will eventually become the interstitial cells (fibroblasts) of the mature valve. During EMT, cell architecture and motility change markedly; significant changes are also observed in various signaling pathways. Here we systematically examined the expression, localization, and function of zyxin, a focal adhesion protein, in EMT during atrioventricular (AV) valve morphogenesis. Expression and localization studies showed that zyxin was expressed in the AV canal region during crucial stages of valve development. An in vitro 3D collagen gel culture system was used to determine zyxin function either after siRNA gene knockdown or after overexpression. Our studies revealed that zyxin overexpression inhibited endocardial cell migration and cell differentiation and also led to a decrease in the number of migrating mesenchymal cells. Moreover, correlative cytoskeletal changes were apparent in response to both overexpression and knockdown treatments. Thus, zyxin appears to play a role as a regulator of cell migration and differentiation during EMT in chicken AV valve formation.

Abstract 3431: βIII-tubulin in glioblastoma: An emerging multifactorial survival factor role in chemotherapy response and tumor formation.

Abstract Glioblastoma (GBM) is an aggressive primary brain tumour with a very poor prognosis. Current treatment is usually surgical resection, radiotherapy and chemotherapy, however, GBM's highly resistant nature renders treatment ineffective. ßIII-tubulin is a neuronal specific microtubule protein that is aberrantly expressed in GBM and it has been implicated in a range of epithelial cancers with poor prognosis and high aggressiveness1. In gliomas, its high expression correlates with higher tumour grade2, however the functional relevance of this expression is unknown. Our aim was to investigate the functional and mechanistic role of ßIII-tubulin in GBM drug sensitivity and tumorigenesis. Methods: To study the functional role of βIII-tubulin in GBM, siRNA gene knockdown was used in two GBM cell lines, U87 and U87vIII and in a primary GBM neurosphere culture. Non-targeting siRNA was used as a control. Knockdown levels were assessed by western blotting. Apoptosis was measured by AnnexinV staining. Drug sensitivity of cell lines was tested using drug-treated clonogenic assays. Senescence was assessed by using cytoplasmic staining for sa-β-galactosidase. Neurosphere formation was assessed by soft-agar growth assay. Results: Specific βIII-tubulin knockdown achieved a 94% and 52% reduction in ßIII-tubulin levels in GBM cell lines and neurospheres respectively, compared to control siRNA. βIII-tubulin knockdown in U87vIII cells resulted in a significant increase in sensitivity to both the DNA-damaging agent temozolomide (TMZ) and tubulin binding agents epothilone B and paclitaxel compared to control (p<0.01). Increased sensitivity to TMZ following βIII-tubulin knockdown in U87 and U87vIII cells was associated with a significant increase in drug-induced apoptosis (p<0.01). TMZ is known to induce senescence3,4 and βIII-tubulin knockdown significantly increased TMZ-induced senescence in U87 (βIII-tub siRNA: 84.1±2.6%, p<0.05, compared to ctrl siRNA: 58.1±7.1%) and U87vIII (βIII-tub siRNA: 83.9±4.5%, p<0.01, compared to ctrl siRNA: 56.3±4.3%) cells. Further validation using a 3D-growth model of primary human GBM neurospheres, showed that neurosphere formation was significantly reduced (30.7%, p<0.01) upon βIII-tubulin knockdown compared to ctrl siRNA. Conclusions: Our data strongly suggests that aberrant expression of βIII-tubulin in glioblastoma may play an important role in intrinsic drug sensitivity to broad classes of chemotherapy drugs. Targeting βIII-tubulin increases temozolomide sensitivity by enhancing drug-induced apoptosis and senescence. Furthermore, we have identified a potential role for βIII-tubulin in GBM tumorigenesis. 1 Kavallaris. Nature Rev Cancer, 10:194-204, 2010 2 Katsetos et al. Arch Pathol Lab Med, 125:613-624, 2001 3 Mhaidat. Br J Cancer, 97:1225-1233, 2007 4 Gunther. Br J Cancer, 88:463-469, 2003 Citation Format: Gorjana Mitic, Maria Tsoli, David S. Ziegler, Maria Kavallaris. βIII-tubulin in glioblastoma: An emerging multifactorial survival factor role in chemotherapy response and tumor formation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3431. doi:10.1158/1538-7445.AM2013-3431