Combination Of Gene Silencing Research Articles

Metabolically distinct roles of NAD synthetase and NAD kinase define the essentiality of NAD and NADP in Mycobacterium tuberculosis.

Nicotinamide adenine dinucleotide (NAD) and its phosphorylated derivative (NADP) are essential cofactors that participate in hundreds of biochemical reactions and have emerged as therapeutic targets in cancer, metabolic disorders, neurodegenerative diseases, and infections, including tuberculosis. The biological basis for the essentiality of NAD(P) in most settings, however, remains experimentally unexplained. Here, we report that inactivation of the terminal enzyme of NAD synthesis, NAD synthetase (NadE), elicits markedly different metabolic and microbiologic effects than those of the terminal enzyme of NADP biosynthesis, NAD kinase (PpnK), in Mycobacterium tuberculosis (Mtb). Inactivation of NadE led to parallel reductions of both NAD and NADP pools and Mtb viability, while inactivation of PpnK selectively depleted NADP pools but only arrested growth. Inactivation of each enzyme was accompanied by metabolic changes that were specific for the affected enzyme and associated microbiological phenotype. Bacteriostatic levels of NAD depletion caused a compensatory remodeling of NAD-dependent metabolic pathways in the absence of an impact on NADH/NAD ratios, while bactericidal levels of NAD depletion resulted in a disruption of NADH/NAD ratios and inhibition of oxygen respiration. These findings reveal a previously unrecognized physiologic specificity associated with the essentiality of two evolutionarily ubiquitous cofactors. IMPORTANCE The current course for cure of Mycobacterium tuberculosis (Mtb)-the etiologic agent of tuberculosis (TB)-infections is lengthy and requires multiple antibiotics. The development of shorter, simpler treatment regimens is, therefore, critical to the goal of eradicating TB. NadE, an enzyme required for the synthesis of the ubiquitous cofactor NAD, is essential for survival of Mtb and regarded as a promising drug target. However, the basis of this essentiality was not clear due to its role in the synthesis of both NAD and NADP. Here, we resolve this ambiguity through a combination of gene silencing and metabolomics. We specifically show that NADP deficiency is bacteriostatic, while NAD deficiency is bactericidal due to its role in Mtb's respiratory capacity. These results argue for a prioritization of NAD biosynthesis inhibitors in anti-TB drug development.

Delivery of siRNA using hyaluronic acid-guided nanoparticles for downregulation of CXCR4.

In this study, effective transport of small interfering RNAs (siRNAs) via hyaluronic acid (HA) receptor was carried out with biodegradable HA and low-molecular weight polyethyleneimine (PEI)-based transport systems. Gold nanoparticles (AuNPs) capable of giving photothermal response, and their conjugates with PEI and HA, were also added to the structure. Thus, a combination of gene silencing, photothermal therapy and chemotherapy, has been accomplished. The synthesized transport systems ranged in size, between 25 and 690 nm. When the particles were applied at a concentration of 100 μg mL-1 (except AuPEI NPs) in vitro, cell viability was above 50%. Applying radiation after the conjugate/siRNA complex (especially those containing AuNP) treatment, increased the cytotoxic effect (decrease in cell viability of 37%, 54%, 13%, and 15% for AuNP, AuPEI NP, AuPEI-HA, and AuPEI-HA-DOX, respectively) on the MDA-MB-231 cell line. CXCR4 gene silencing via the synthesized complexes, especially AuPEI-HA-DOX/siRNA was more efficient in MDA-MB-231 cells (25-fold decrease in gene expression) than in CAPAN-1 cells. All these results demonstrated that the synthesized PEI-HA and AuPEI-HA-DOX conjugates can be used as siRNA carriers that are particularly effective, especially in the treatment of breast cancer.

Cell differentiation modifies the p53 transcriptional program through a combination of gene silencing and constitutive transactivation

The p53 transcription factor is a master regulator of cellular responses to stress that is commonly inactivated in diverse cancer types. Despite decades of research, the mechanisms by which p53 impedes tumorigenesis across vastly different cellular contexts requires further investigation. The bulk of research has been completed using in vitro studies of cancer cell lines or in vivo studies in mouse models, but much less is known about p53 action in diverse non-transformed human tissues. Here, we investigated how different cellular states modify the p53 transcriptional program in human cells through a combination of computational analyses of publicly available large-scale datasets and in vitro studies using an isogenic system consisting of induced pluripotent stem cells (iPSCs) and two derived lineages. Analysis of publicly available mRNA expression and genetic dependency data demonstrated wide variation in terms of expression and function of a core p53 transcriptional program across various tissues and lineages. To monitor the impact of cell differentiation on the p53 transcriptome within an isogenic cell culture system, we activated p53 by pharmacological inhibition of its negative regulator MDM2. Using cell phenotyping assays and genome wide transcriptome analyses, we demonstrated that cell differentiation confines and modifies the p53 transcriptional network in a lineage-specific fashion. Although hundreds of p53 target genes are transactivated in iPSCs, only a small fraction is transactivated in each of the differentiated lineages. Mechanistic studies using small molecule inhibitors and genetic knockdowns revealed the presence of two major regulatory mechanisms contributing to this massive heterogeneity across cellular states: gene silencing by epigenetic regulatory complexes and constitutive transactivation by lineage-specific transcription factors. Altogether, these results illuminate the impact of cell differentiation on the p53 program, thus advancing our understanding of how this tumor suppressor functions in different contexts.

Complete destruction of deep-tissue buried tumors via combination of gene silencing and gold nanoechinus-mediated photodynamic therapy

Cancer is one of the major diseases leading to human deaths. Complete destruction of deep tissue-buried tumors using non-invasive therapies is a grand challenge in clinical cancer treatments. Many therapeutic modalities were developed to tackle this problem, but only partial tumor suppression or delay growths were usually achieved. In this study, we report for the first time that complete destruction of deep tissue-buried tumors can be achieved by combination of gold nanoechinus (Au NEs)-mediated photodynamic therapy (PDT) and gene silencing under ultra-low doses of near infra-red (NIR) light irradiation (915 nm, 340 mW/cm2; 1064 nm, 420 mW/cm2) in the first and second biological windows. The average lifespan of the mice treated by the above combined therapy is beyond 40 days, which are ∼2.6 times longer than that (15 days) observed from the anticancer drug doxorubicin-treated group. The current study points out a new direction for the therapeutic design to treat deeply seated tumors in future cancer treatments.

Multifunctional Gold Nanorods for siRNA Gene Silencing and Photothermal Therapy

Cancer is a complex disease that usually requires several treatment modalities. A multifunctional nanotherapeutic system is designed, incorporating small interfering RNA (siRNA) and gold nanorods (Au NRs) for photothermal therapy. Surface-engineered Au NRs with polyethylenimine are synthesized using a layer-by-layer assembly and siRNA is absorbed on the surface. The siRNA is efficiently delivered into breast cancer cells, resulting in subsequent gene silencing. Cells are then irradiated with near-infrared (NIR) light, causing heat-induced anticancer activity. The combination of gene silencing and photothermal therapy results in effective inhibition of cell proliferation.

Markers of effective anti-tumor CTL populations for adoptive cell therapy (165.5)

Abstract Adoptive transfer of in vitro activated, tumor-specific CTL is a useful strategy for cancer immunotherapy. We compared in vitro culture conditions for ability to generate tumor-specific CTL that reject tumours in vivo, and for expression of molecular markers associated with effective anti-tumor activity. Specific CTL were obtained in short-term in vitro cultures of naïve CD8+ T cells and bone marrow DC loaded with peptide antigen, and were expanded in IL-2 for 2 days before transfer in vivo. The conditions of DC activation did not appear to affect the recovery, phenotype or effector function of cultured CTL, but had a substantial effect on the CTL’s ability to prevent tumour growth in vivo. Proteomic analysis of the total and surface proteome of different CTL populations was carried out using liquid chromatography and tandem mass spectrometry. Among more than 1,000 identified proteins, several were expressed with different abundance in CTL populations with varying anti-tumour activity. The functional significance of proteins showing the greatest differential expression is currently being examined using a combination of gene silencing and in vivo transfer studies. The identification of markers associated with effective anti-tumor function will provide useful means to understand and predict the functional activity of CTL populations for in vivo transfer.

Suppression of eNOS-derived superoxide by caveolin-1: a biopterin-dependent mechanism

In the vasculature, nitric oxide (NO) is generated by endothelial NO synthase (eNOS) in a calcium/calmodulin-dependent reaction. In the absence of the requisite eNOS cofactor tetrahydrobiopterin (BH(4)), NADPH oxidation is uncoupled from NO generation, leading to the production of superoxide. Although this phenomenon is apparent with purified enzyme, cellular studies suggest that formation of the BH(4) oxidation product, dihydrobiopterin, is the molecular trigger for eNOS uncoupling rather than BH(4) depletion alone. In the current study, we investigated the effects of both BH(4) depletion and oxidation on eNOS-derived superoxide production in endothelial cells in an attempt to elucidate the molecular mechanisms regulating eNOS oxidase activity. Results demonstrated that pharmacological depletion of endothelial BH(4) does not result in eNOS oxidase activity, whereas BH(4) oxidation gave rise to significant eNOS-oxidase activity. These findings suggest that the endothelium possesses regulatory mechanisms, which prevent eNOS oxidase activity from pterin-free eNOS. Using a combination of gene silencing and pharmacological approaches, we demonstrate that eNOS-caveolin-1 association is increased under conditions of reduced pterin bioavailability and that this sequestration serves to suppress eNOS uncoupling. Using small interfering RNA approaches, we demonstrate that caveolin-1 gene silencing increases eNOS oxidase activity to 85% of that observed under conditions of BH(4) oxidation. Moreover, when caveolin-1 silencing was combined with a pharmacological inhibitor of AKT, BH(4) depletion increased eNOS-derived superoxide to 165% of that observed with BH(4) oxidation. This study identifies a critical role of caveolin-1 in the regulation of eNOS uncoupling and provides new insight into the mechanisms through which disease-associated changes in caveolin-1 expression may contribute to endothelial dysfunction.

PH-Responsive Polymeric siRNA Carriers Sensitize Multidrug Resistant Ovarian Cancer Cells to Doxorubicin via Knockdown of Polo-like Kinase 1

Small interfering RNA (siRNA)-based therapies have great potential for the treatment of debilitating diseases such as cancer, but an effective delivery strategy for siRNA is elusive. Here, pH-responsive complexes were developed for the delivery of siRNA in order to sensitize drug-resistant ovarian cancer cells (NCI/ADR-RES) to doxorubicin. The electrostatic complexes consisted of a cationic micelle used as a nucleating core, siRNA, and a pH-responsive endosomolytic polymer. Cationic micelles were formed from diblock copolymers of dimethylaminoethyl methacrylate (pDMAEMA) and butyl methacrylate (pDbB). The hydrophobic butyl core mediated micelle formation while the positively charged pDMAEMA corona enabled siRNA condensation. To enhance cytosolic delivery through endosomal release, a pH-responsive copolymer of poly(styrene-alt-maleic anhydride) (pSMA) was electrostatically complexed with the positively charged siRNA/micelle to form a ternary complex. Complexes exhibited size (30-105 nm) and charge (slightly positive) properties important for endocytosis and were found to be noncytotoxic and mediate uptake in >70% of ovarian cancer cells after 1 h of incubation. The pH-responsive ternary complexes were used to deliver siRNA against polo-like kinase 1 (plk1), a gene upregulated in many cancers and responsible for cell cycle progression, to ovarian cancer cell lines. Treatment resulted in approximately 50% reduction of plk1 gene expression in the drug-resistant NCI/ADR-RES ovarian cancer cell model and in the drug-sensitive parental cell line, OVCAR8. This knockdown functionally sensitized NCI/ADR-RES cells to doxorubicin at levels similar to OVCAR8. Sensitization occurred through a p53 signaling pathway, as indicated by caspase 3/7 upregulation following plk1 knockdown and doxorubicin treatment, and this effect could be abrogated using a p53 inhibitor. To demonstrate the potential for dual delivery from this polymer system, micelle cores were subsequently loaded with doxorubicin and utilized in ternary complexes to achieve cell sensitization through simultaneous siRNA and drug delivery from a single carrier. These results show knockdown of plk1 results in sensitization of multidrug resistant cells to doxorubicin, and this combination of gene silencing and small molecule drug delivery may prove useful to achieve potent therapeutic effects.

Genetic polymorphisms in the 5' flanking region of glutathione S-transferase P1 affect promoter methylation

Glutathione-S-transferase P1 (GSTP1) is involved in thiol-mediated detoxification and breakdown of reactive oxygen species created by anticancer drug exposure. GSTP1 is also an inhibitor of c-Jun N-terminal kinase 1, a kinase involved in stress response, apoptosis and cellular proliferation. Hypermethylation of the GSTP1 promoter has been associated with gene silencing in prostate cancer, kidney cancer, and breast cancer, among others. Although frequently described, the mechanism underlying promoter hypermethylation of the GSTP1 gene is poorly understood. It has been reported that an ATAAA repeat of the GSTP1 promoter separates methylated from unmethylated CpGs in normal prostate tissue [1]. These separate methylation domains are lost in prostate cancer, and methylation extends throughout the whole promoter region. It has been proposed that hypermethylation of GSTP1 requires a combination of gene silencing and random seeds of methylation in prostate cancer cells, and that these combinatorial effects lead to histone deacetylation and subsequent chromatin remodeling [2]. To further elucidate the mechanisms underlying the hypermethylation of the GSTP1 promoter, we genotyped the (ATAAA) repeat and the linked SNPs in positions -354, -288, -287 and -282 in the GSTP1 promoter and we performed methylation analysis using mass spectrometry in tumor DNA from 82 breast cancer patients. The role of the different allelic variants on methylation status of the GSTP1 promoter and expression levels was assessed. We quantitatively determined the methylation status of six CpGs spanning the transcription start site of the GSTP1 promoter: -22, +8, +14, +38, +47 and +55. The average percentage methylation for each individual CpG for the 82 tumor samples analyzed was 16.9%, 30.3%, 18.2%, 21.2%, 18.6% and 8.1%, respectively. The average percentage methylation for all CpGs in all tumor samples was 19%. There was a correlation between the degree of methylation of the individual CpGs and their neighboring CpGs (P < 0.001). When correlating the extent of methylation to the mRNA levels previously assessed by whole genome gene-expression profiling of the same tumors, a significant inverse correlation was observed (P < 0.01). The methylation status of the three CpGs closest to the transcriptional start site was more highly associated with the level of GSTP1 mRNA expression than the CpGs further downstream of the +1 site. Furthermore, we observed differences in the degree of GSTP1 promoter methylation between the different tumor subclasses defined by whole-genome microarray analysis [3]. The methylation of the GSTP1 promoter was significantly lower in the basal subtype compared with the luminal subtype, which corresponded to elevated GSTP1 mRNA levels in the basal subtypes [4]. We further analyzed the impact of the most frequent haplotype structure of the GSTP1 promoter in relation to the extent of methylation, and a correlation was observed (P = 0.003) suggesting that haplotype structures can affect de novo methylation of adjacent sequences.