Selective and Functional-Group-Tolerant Photoalkylation of Imines by Energy-Transfer Photocatalysis.

Basic amines show broad bioactivity and remain a promising source of new medicines. The direct photoalkylation of imines offers a promising strategy for complex amines. However, the lack of efficient imine photoreactivity hinders this reaction and remains a fundamental limitation in organic photochemistry. We report an efficient photoalkylation of imines that provides primary amines directly without protecting or leaving groups. The transformation effects C-H addition across N-H imines under energy-transfer photocatalysis by a ketone. Our method is distinguished from organometallic, metal-catalyzed, and photoredox approaches to imine alkylation by its lack of protecting groups and its broad scope, which includes unactivated alkanes, protic substrates, basic amines, heterocycles, and ketone imines. We highlight this scope through the condensation and alkylation of two pharmaceutical ketones, providing complex amines succinctly. Our mechanistic analysis supports a three-step process, involving hydrogen-atom transfer to an imine triplet excited state, intersystem crossing, and radical recombination, with photocatalytic enhancement through energy transfer. We further show that N-H imines are more photoreactive than N-substituted imines, a distinction partially explained by sterics and side reactions. To fully explain this distinction, we introduce the thermodynamic parameter excited-state hydrogen-atom affinity, which is highly effective at predicting the photoreactivity of imines.

Shortwave Infrared Fluorofluorophores for Multicolor In Vivo Imaging.

Developing chemical tools to detect and influence biological processes is a cornerstone of chemical biology. Here we combine two tools which rely on orthogonality- perfluorocarbons and multiplexed shortwave infrared (SWIR) fluorescence imaging- to visualize nanoemulsions in real time in living mice. Drawing inspiration from fluorous and SWIR fluorophore development, we prepared two SWIR-emissive, fluorous-soluble chromenylium polymethine dyes. These are the most red-shifted fluorous fluorophores- "fluorofluorophores"-to date. After characterizing the dyes, their utility was demonstrated by tracking perfluorocarbon nanoemulsion biodistribution in vivo. Using an excitation-multiplexed approach to image two variables simultaneously, we gained insight into the importance of size and surfactant identity on biodistribution.

One-Pot Synthesis of Primary and Secondary Aliphatic Amines via Mild and Selective sp3 C-H Imination.

The direct replacement of sp3 C-H bonds with simple amine units (-NH2 ) remains synthetically challenging, although primary aliphatic amines are ubiquitous in medicinal chemistry and natural product synthesis. We report a mild and selective protocol for preparing primary and secondary aliphatic amines in a single pot, based on intermolecular sp3 C-H imination. The first C-H imination of diverse alkanes, this method shows useful site-selectivity within substrates bearing multiple sp3 C-H bonds. Furthermore, this reaction tolerates polar functional groups relevant for complex molecule synthesis, highlighted in the synthesis of amine pharmaceuticals and amination of natural products. We characterize a unique C-H imination mechanism based on radical rebound to an iminyl radical, supported by kinetic isotope effects, stereoablation, resubmission, and computational modeling. This work constitutes a selective method for complex amine synthesis and a new mechanistic platform for C-H amination.

A Circle RNA Regulatory Axis Promotes Lung Squamous Metastasis via CDR1-Mediated Regulation of Golgi Trafficking.

Lung squamous carcinoma (LUSC) is a highly metastatic disease with a poor prognosis. Using an integrated screening approach, we found that miR-671-5p reduces LUSC metastasis by inhibiting a circular RNA (circRNA), CDR1as. Although the putative function of circRNA is through miRNA sponging, we found that miR-671-5p more potently silenced an axis of CDR1as and its antisense transcript, cerebellar degeneration related protein 1 (CDR1). Silencing of CDR1as or CDR1 significantly inhibited LUSC metastases and CDR1 was sufficient to promote migration and metastases. CDR1, which directly interacted with adaptor protein 1 (AP1) complex subunits and coatomer protein I (COPI) proteins, no longer promoted migration upon blockade of Golgi trafficking. Therapeutic inhibition of the CDR1as/CDR1 axis with miR-671-5p mimics reduced metastasis in vivo. This report demonstrates a novel role for CDR1 in promoting metastasis and Golgi trafficking. These findings reveal an miRNA/circRNA axis that regulates LUSC metastases through a previously unstudied protein, CDR1. SIGNIFICANCE: This study shows that circRNA, CDR1as, promotes lung squamous migration, metastasis, and Golgi trafficking through its complimentary transcript, CDR1.

Histone deacetylase 11 inhibition promotes breast cancer metastasis from lymph nodes.

Lymph node (LN) metastases correspond with a worse prognosis in nearly all cancers, yet the occurrence of cancer spreading from LNs remains controversial. Additionally, the mechanisms explaining how cancers survive and exit LNs are largely unknown. Here, we show that breast cancer patients frequently have LN metastases that closely resemble distant metastases. In addition, using a microsurgical model, we show how LN metastasis development and dissemination is regulated by the expression of a chromatin modifier, histone deacetylase 11 (HDAC11). Genetic and pharmacologic blockade of HDAC11 decreases LN tumor growth, yet substantially increases migration and distant metastasis formation. Collectively, we reveal a mechanism explaining how HDAC11 plasticity promotes breast cancer growth as well as dissemination from LNs and suggest caution with the use of HDAC inhibitors.

Effect of various levels of dissolved oxygen on reactive oxygen species and cryocapacitation-like changes in bull sperm.

The present investigation was carried out to study the effect of various levels of dissolved oxygen (DO) on reactive oxygen species (ROS) and cryocapacitation-like changes in bull sperm. Egg yolk-Tris-glycerol (EYTG) extender was split into four subextenders; viz., Extender I (control; no flushing with liquid nitrogen (LN2 )), Extender II, Extender III and Extender IV were flushed with LN2 for 40, 16 and 8 min, respectively. The DO levels were standardized to 11.7, 2, 4 and 8 ppm, respectively, in control (Extender I), Extender II, Extender III and Extender IV. Ejaculates with mass motility of ≥ 3+ were divided into group I (diluted with Extender I), group II (diluted with Extender II), group III (diluted with Extender III) and group IV (diluted with Extender IV) up to 80 × 106  sperm/ml. Extended semen samples were packed in French mini straws (0.25 ml), equilibrated and cryopreserved. Semen samples were evaluated at prefreeze and post-thaw stage for various parameters (DO, progressive motility (PM), viability (VIB), acrosomal integrity (AI), hypo-osmotic swelling (HOS) test, ROS, cholesterol (C) and phospholipid (P). The percentage of PM, VIB, AI, HOS test, cholesterol (C) and phospholipid (P) levels, and capacitated sperm were significantly (p < 0.05) higher in groups III and IV as compared to groups I and II. However, the acrosome-reacted sperm (%; pattern AR) were significantly (p < 0.05) decreased in group III as compared to all other groups. Besides the proportion of sperm displaying tyrosine-phosphorylated pattern, EA (fluorescence at both equatorial and anterior acrosomal regions, i.e. high capacitation level) was significantly (p < 0.05) reduced in group III compared to all other groups. In conclusion, varying DO levels in the extender significantly affect sperm quality, ROS production and capacitation-like changes in bulls.

Factor XIIIA-expressing inflammatory monocytes promote lung squamous cancer through fibrin cross-linking.

Lung cancer is the leading cause of cancer-related deaths worldwide, and lung squamous carcinomas (LUSC) represent about 30% of cases. Molecular aberrations in lung adenocarcinomas have allowed for effective targeted treatments, but corresponding therapeutic advances in LUSC have not materialized. However, immune checkpoint inhibitors in sub-populations of LUSC patients have led to exciting responses. Using computational analyses of The Cancer Genome Atlas, we identified a subset of LUSC tumors characterized by dense infiltration of inflammatory monocytes (IMs) and poor survival. With novel, immunocompetent metastasis models, we demonstrated that tumor cell derived CCL2-mediated recruitment of IMs is necessary and sufficient for LUSC metastasis. Pharmacologic inhibition of IM recruitment had substantial anti-metastatic effects. Notably, we show that IMs highly express Factor XIIIA, which promotes fibrin cross-linking to create a scaffold for LUSC cell invasion and metastases. Consistently, human LUSC samples containing extensive cross-linked fibrin in the microenvironment correlated with poor survival.

Syntheses, structures, and stabilities of aliphatic and aromatic fluorous iodine(I) and iodine(III) compounds: the role of iodine Lewis basicity.

The title molecules are sought in connection with various synthetic applications. The aliphatic fluorous alcohols Rfn CH2OH (Rfn = CF3(CF2) n-1; n = 11, 13, 15) are converted to the triflates Rfn CH2OTf (Tf2O, pyridine; 22-61%) and then to Rfn CH2I (NaI, acetone; 58-69%). Subsequent reactions with NaOCl/HCl give iodine(III) dichlorides Rfn CH2ICl2 (n = 11, 13; 33-81%), which slowly evolve Cl2. The ethereal fluorous alcohols CF3CF2CF2O(CF(CF3)CF2O) x CF(CF3)CH2OH (x = 2-5) are similarly converted to triflates and then to iodides, but efforts to generate the corresponding dichlorides fail. Substrates lacking a methylene group, Rfn I, are also inert, but additions of TMSCl to bis(trifluoroacetates) Rfn I(OCOCF3)2 appear to generate Rfn ICl2, which rapidly evolve Cl2. The aromatic fluorous iodides 1,3-Rf6C6H4I, 1,4-Rf6C6H4I, and 1,3-Rf10C6H4I are prepared from the corresponding diiodides, copper, and Rfn I (110-130 °C, 50-60%), and afford quite stable Rfn C6H4ICl2 species upon reaction with NaOCl/HCl (80-89%). Iodinations of 1,3-(Rf6)2C6H4 and 1,3-(Rf8CH2CH2)2C6H4 (NIS or I2/H5IO6) give 1,3,5-(Rf6)2C6H3I and 1,2,4-(Rf8CH2CH2)2C6H3I (77-93%). The former, the crystal structure of which is determined, reacts with Cl2 to give a 75:25 ArICl2/ArI mixture, but partial Cl2 evolution occurs upon work-up. The latter gives the easily isolated dichloride 1,2,4-(Rf8CH2CH2)2C6H3ICl2 (89%). The relative thermodynamic ease of dichlorination of these and other iodine(I) compounds is probed by DFT calculations.

Effect of supplementation of recombinant Regucalcin in extender on cryopreservation of spermatozoa of water buffalo (Bubalus bubalis).

Elevated intracellular calcium concentration and oxidative damage are two major factors contributing to the poor fertility of cryopreserved spermatozoa. Regucalcin (RGN), also known as Senescence marker protein-30 (SMP-30), is a calcium-binding protein with multiple roles that include calcium homeostasis, anti-oxidative, anti-apoptosis, and anti-proliferation. In Drosophila, RGN is reportedly a putative cold-tolerance gene and a cytoprotective role for RGN against intracellular calcium elevation and oxidative stress was reported in P19 cell lines. Given that RGN has anticapacitatory effect and abundant in the male reproductive tract, we hypothesized that it may play a cryoprotective role for spermatozoa. We investigated this by including RGN, at three different concentrations (20, 40, and 60 µg/ml), as a supplement for Tris-egg yolk-based semen extender. Post-thaw metrics of progressive motility, acrosome integrity, and zona pellucida binding of spermatozoa were evaluated for three ejaculates of three clinically normal, breeding Murrah buffaloes. A concentration of 40 µg/ml of recombinant RGN supplemented during sperm freezing resulted in significant increases in the post-thaw progressive motility of spermatozoa (50.6 ± 3.5% vs 40.6 ± 2.6%; p < 0.01), acrosome integrity (53.3 ± 7.4 vs 75.6 ± 6.8; p < 0.05), and zona pellucida binding (31.6 ± 14.0 vs 191.9 ± 12.3 bound spermatozoa; p < 0.01) compared to control conditions without RGN. Thus, ∼1 µM recombinant RGN, which retains the ability to bind calcium, has a cryoprotective effect for buffalo spermatozoa in extender.

Syntheses of Families of Enantiopure and Diastereopure Cobalt Catalysts Derived from Trications of the Formula [Co(NH2CHArCHArNH2)3]3.

Aerobic reactions of CoX2 (X = OAc, Cl) or Co(ClO4)2 with (S,S)-1,2-diphenylethylenediamine [(S,S)-dpen] in CH3OH, followed by HCl or HClO4 additions, give the diastereomeric lipophobic salts Λ-[Co((S,S)-dpen)3]3+3Cl- [Λ-(S,S)-13+3Cl-] or Δ-(S,S)-13+3ClO4- (60-65%) with high degrees of selectivity. Anion metatheses (room temperature) and equilibrations (charcoal, CH3OH, 70 °C) show that the former is more stable than Δ-(S,S)-13+3Cl-, and the latter is more stable than Λ-(S,S)-13+3ClO4-. Additional anion metatheses lead to large families of lipophilic salts Λ- and Δ-(S,S)-13+2X-X'- [X/X' = Cl/BArf [BArf = B(3,5-C6H3(CF3)2)4], PF6/BArf, BF4/BArf, PhBF3/BArf, Cl/BArf20 [BArf20 = B(C6F5)4], BArf/BArf, BArf20/BArf20, BF4/BF4, PF6/PF6]. Mixed salts of the formula Λ- and Δ-[Co((S,S)-NH2CHArCHArNH2)3]3+2Cl-BArf- are similarly prepared (Ar = 4-C6H4n-Bu, 4-C6H4Cl, 4-C6H4CF3, 4-C6H4OCH3, α-naphthyl, ß-naphthyl, 2-C6H4OBn). The diastereotopic NHH' protons exhibit different 1H NMR signals; one shifts far downfield when X/X' = Cl/BArf (δ ca. 8.0 vs 4.0 ppm). This is believed to arise from hydrogen bonding between the two Cl- anions and the two C3 faces of the D3-symmetric trication, each of which feature three synperiplanar NH groups. When all of the anions are poor hydrogen-bond acceptors (e.g., BArf-, BF4-, ClO4-), equilibria favor Δ diastereomers.

Profiling of sperm gene transcripts in crossbred (Bos taurus x Bos indicus) bulls.

Crossbred cattle in some sectors of the world have a significant role in enhancing milk production thereby enhancing the per capita milk availability as a human food source. However, there are certain constraints associated with crossbred animals, such as disease susceptibility, increased reproductive problems, repeat breeding and poor seminal quality. The semen of crossbred bulls has a poor freezing capacity, increased cryo-damage, poor mass cell motility, greater percentages of dead/abnormal sperm and poor initial and post-freeze cell motility. The rejection rate of crossbred bulls for cryostorage of semen has been reported to be as great as 50% as a result of unacceptable semen quality. The identification of superior bulls using molecular technologies is needed which necessitates identification of the genes having a role in sperm function. The present study was, therefore, conducted to gain information on identification and expression of genes having a role in sperm motility in crossbred bulls. The gene transcripts in bulls with sperm of superior and inferior quality were profiled in Vrindavani crossbred cattle by microarray analyses and the results were verified by real time-quantitative PCR. Microarray analyses revealed 19,454 genes which were differentially expressed. At a two-fold cut off, 305 genes were differentially (P<0.01) expressed with 160 genes upregulated and 145 genes down regulated. Some of the upregulated candidate genes were further validated by RT-qPCR. These genes had a four to 16 fold upregulation in sperm with inferior motility as compared to sperm of crossbred bulls with superior motility.

Octahedral Werner complexes with substituted ethylenediamine ligands: a stereochemical primer for a historic series of compounds now emerging as a modern family of catalysts.

As reported by Alfred Werner in 1911-1912, salts of the formally D3 symmetric [Co(en)3]3+ (en = ethylenediamine) trication were among the first chiral inorganic compounds to be resolved into enantiomers, the absolute configurations of which are denoted Λ (left handed helix) or Δ (right handed helix). After a >100 year dormant period during which few useful reactions of these substitution inert complexes were described, carbon substituted derivatives have recently been found to be potent catalysts for enantioselective organic synthesis. This review systematically outlines the fascinating range of stereoisomers that can arise, such as conformers associated with the five membered chelate rings (λ/δ), alignment modes of the C-C bonds with the C3 symmetry axis (lel/ob), geometric isomers (fac/mer), and configurational diastereomers (R/S) arising from carbon stereocenters. These analyses demonstrate a profound stereochemical diversity that can be applied in catalyst optimization. Efforts are made to bridge the often orthogonal nomenclature systems inorganic and organic chemists employ to describe these phenomena.

Werner Complexes with ω-Dimethylaminoalkyl Substituted Ethylenediamine Ligands: Bifunctional Hydrogen-Bond-Donor Catalysts for Highly Enantioselective Michael Additions.

The racemic carbonate complex [Co(en)2 O2 CO](+) Cl(-) (en=1,2-ethylenediamine) and (S)-[H3 NCH((CH2 )n NHMe2 )CH2 NH3 ](3+) 3 Cl(-) (n=1-4) react (water, charcoal, 100 °C) to give [Co(en)2 ((S)-H2 NCH((CH2 )n NHMe2 )CH2 NH2 )](4+) 4 Cl(-) (3 a-d H(4+) 4 Cl(-) ) as a mixture of Λ/Δ diastereomers that separate on chiral-phase Sephadex columns. These are treated with NaOH/Na(+) BArf (-) (BArf =B(3,5-C6 H3 (CF3 )2 )4 ) to give lipophilic Λ- and Δ-3 a-d(3+) 3 BArf (-) , which are screened as catalysts (10 mol %) for additions of dialkyl malonates to nitroalkenes. Optimal results are obtained with Λ-3 c(3+) 3 BArf (-) (CH2 Cl2 , -35 °C; 98-82 % yields and 99-93 % ee for six ß-arylnitroethenes). The monofunctional catalysts Λ- and Δ-[Co(en)3 ](3+) 3 BArf (-) give enantioselectivities of <10 % ee with equal loadings of Et3 N. The crystal structure of Δ-3 a H(4+) 4 Cl(-) provides a starting point for speculation regarding transition-state assemblies.

Tris(1,2-diphenylethylenediamine)cobalt(III) Complexes: Chiral Hydrogen Bond Donor Catalysts for Enantioselective α-Aminations of 1,3-Dicarbonyl Compounds.

The enantiopure salt Δ-[Co((S,S)-dpen)3](3+)2Cl(-)B(C6F5)4(-) is an effective hydrogen bond donor catalyst for additions of 1,3-dicarbonyl compounds to di-tert-butyl azodicarboxylate in the presence of N-methylmorpholine (1.0:1.0:0.10) in CH3CN at 0 °C, as illustrated with educts derived from five- or six-membered ring ketones (99-88% yields, >99-91% ee) and cycloheptanone (94%, 72% ee) as well as 2-cyanocyclopentanone (92%, 45% ee) and an acyclic system (98%, >99% ee).

Induction of Vasculogenic Mimicry Overrides VEGF-A Silencing and Enriches Stem-like Cancer Cells in Melanoma.

The basis for resistance to VEGF inhibition is not fully understood despite its clinical importance. In this study, we examined the adaptive response to VEGF-A inhibition by a loss-of-function analysis using plasmid-based shRNA. Tumor xenografts that initially responded to VEGF-A inhibition underwent an adaptation in vivo, leading to acquired resistance. VEGF-A blockade in tumors was associated with HIF1α expression and an increase in CD144(+) vasculogenic mimicry (VM), leading to formation of channels displaying Tie-1 and MMP-2 upregulation. CD133(+) and CD271(+) melanoma stem-like cells (MSLC) accumulated in the perivascular niche. Tumor xenografts of melanoma cell populations that were intrinsically resistant to VEGF-A blockade did not exhibit any of these features, compared with nontarget control counterparts. Thus, melanomas that are initially sensitive to VEGF-A blockade acquire adaptive resistance by adopting VM as an alternate angiogenic strategy, thereby enriching for deposition of MSLC in the perivascular niche through an HIF1α-dependent process. Conversely, melanomas that are intrinsically resistant to VEGF-A blockade do not show any evidence of compensatory survival mechanisms that promote MSLC accumulation. Our work highlights the potential risk of anti-VEGF treatments owing to a selective pressure for an adaptive resistance mechanism that empowers the development of stem-like cancer cells, with implications for how to design combination therapies that can improve outcomes in patients.

Cobalt(III) Werner Complexes with 1,2-Diphenylethylenediamine Ligands: Readily Available, Inexpensive, and Modular Chiral Hydrogen Bond Donor Catalysts for Enantioselective Organic Synthesis.

In the quest for new catalysts that can deliver single enantiomer pharmaceuticals and agricultural chemicals, chemists have extensively mined the "chiral pool", with little in the way of inexpensive, readily available building blocks now remaining. It is found that Werner complexes based upon the D3 symmetric chiral trication [Co(en)3](3+) (en = 1,2-ethylenediamine), which features an earth abundant metal and cheap ligand type, and was among the first inorganic compounds resolved into enantiomers 103 years ago, catalyze a valuable carbon-carbon bond forming reaction, the Michael addition of malonate esters to nitroalkenes, in high enantioselectivities and without requiring inert atmosphere conditions. The title catalysts, [Co((S,S)-dpen)3](3+) ((S,S)-3 (3+)) 3X(-), employ a commercially available chiral ligand, (S,S)-1,2-diphenylethylenediamine. The rates and ee values are functions of the configuration of the cobalt center (Λ/Δ) and the counteranions, which must be lipophilic to solubilize the trication in nonaqueous media. The highest enantioselectivities are obtained with Λ and 2Cl(-)BArf (-), 2BF4 (-)BArf (-), or 3BF4 (-) salts (BArf (-) = B(3,5-C6H3(CF3)2)4 (-)). The substrates are not activated by metal coordination, but rather by second coordination sphere hydrogen bonding involving the ligating NH2 groups. Crystal structures and NMR data indicate enthalpically stronger interactions with the NH moieties related by the C3 symmetry axis, as opposed to those related by the C2 symmetry axes; rate trends and other observations suggest this to be the catalytically active site. Both Λ- and Δ-(S,S)-3 (3+) 2Cl(-)BArf (-) are effective catalysts for additions of ß-ketoesters to RO2CN=NCO2R species (99-86% yields, 81-76% ee), which provide carbon-nitrogen bonds and valuable precursors to α-amino acids.

Risk stratification of prostate cancer patients based on EPS-urine zinc content.

The early detection of prostate cancer is a life-saving event in patients harboring potentially aggressive disease. With the development of malignancy there is a dramatic reduction in the zinc content of prostate tissue associated with the inability of cancer cells to accumulate the ion. In the current study, we utilized endogenous zinc as a diagnostic biomarker for prostate cancer. We employed a novel fluorescent sensor for mobile zinc (ZPP1) to measure the concentration of zinc in thirty-nine patient samples of expressed prostatic secretion (EPS) in urine. We estimated the probability of classifying a subject as benign, low-risk, or high-risk as functions of the diagnostic test results using a non-informative prior Bayesian approach. Permutation tests and other non-parametric tests were also used. We demonstrated a significant trend in zinc score with disease and with disease risk (P = 0.03), and lack of a significant correlation between zinc score and PSA. We also showed that the proposed diagnostic is potentially superior to PSA for detecting high-risk disease. Considering that risk stratification represents an important unmet need, our method should advance the field of prostate cancer diagnostics and treatment planning.

Design of nanodrugs for miRNA targeting in tumor cells.

The delivery of oligonucleotide antagonists to cytosolic RNA targets such as microRNA represents an avenue for the post-transcriptional control of cellular phenotype. In tumor cells, oncogenic miRNAs, termed oncomirs, are tightly linked to processes that ultimately determine cancer initiation, progression, and response to therapy. Therefore, the capacity to redirect tumor cell fate towards therapeutically beneficial phenotypes holds promise in a future clinical scenario. Previously, we have designed "nanodrugs" for the specific inhibition of oncogenic microRNAs in tumor cells. The basic design of these nanodrugs includes dextran coated iron oxide nanoparticles, conjugated to a tumor-targeting peptide, and a locked nucleic acid (LNA)-modified antisense oligonucleotide that stably binds and inhibits the complementary mature miRNA. Here, we focus on elucidating an optimal nanodrug design for effective miRNA inhibition in tumor cells. Specifically, we investigate the choice of chemical linker for the conjugation of the oligonucleotide to the nanoparticles and evaluate the contribution of tumor-cell targeting to nanodrug uptake and functionality. We find that short labile linkers (SPDP; N-Succinimidyl 3-(2-pyridyldithio)-propionate) are superior to non-labile short linkers (GMBS; N-(gamma-Maleimidobutyryloxy)succinimide ester) or non-labile long linkers (PEG24; Succinimidyl-([N-maleimidopropionamido]-24ethyleneglycol)ester) in terms of their capacity to gain access to the cytosolic cellular compartment and to engage their cognate miRNA. Furthermore, using the nanodrug design that incorporates SPDP as a linker, we establish that the addition of tumor-cell targeting through functionalization of the nanodrug with the alphavbeta3-specific cyclic RGDfK-PEG peptide does not confer an advantage in vitro at long incubation times required for inhibition.

Detection of miRNA expression in intact cells using activatable sensor oligonucleotides.

We describe a technology for the profiling of miRNA expression in intact cells. The technology is based on sensor oligonucleotides that are cleavable, completely complementary to a target miRNA, and dual-labeled with a fluorescent dye and a quencher. Upon entering the cell, the sensor oligonucleotide binds its specific miRNA target through complementary base-pairing. This triggers assembly of the endogenous RNA Induced Silencing Complex (RISC) around the miRNA-sensor duplex and cleavage of the sensor oligonucleotide, resulting in separation between the dye and quencher, and a fluorescence turn-on. In the presented feasibility studies, we focus on a specific miRNA (miR-10b) implicated in breast cancer metastasis. Using a human breast adenocarcinoma cell line, we illustrate the application of this technology for miRNA detection with nanomolar sensitivity in both a cell-free system and intact cells.

Sequence-dependent combination therapy with doxorubicin and a survivin-specific small interfering RNA nanodrug demonstrates efficacy in models of adenocarcinoma.

The clinical management of cancer reflects a balance between treatment efficacy and toxicity. While typically, combination therapy improves response rate and time to progression compared with sequential monotherapy, it causes increased toxicity. Consequently, in cases of advanced cancer, emerging guidelines recommend sequential monotherapy, as a means to enhance quality of life. An alternative approach that could overcome nonspecific toxicity while retaining therapeutic efficacy, involves the combination of chemotherapy with targeted therapy. In the current study, we tested the hypothesis that combination therapy targeting survivin (BIRC5) and low-dose doxorubicin (Dox) will show enhanced therapeutic potential in the treatment of cancer, as compared to monotherapy with Dox. We demonstrate in both in vitro and in vivo models of breast cancer that combination therapy with a low dose of Dox and an anti-survivin siRNA nanodrug (MN-siBIRC5) is superior to mono-therapy with either low- or high-dose Dox alone. Importantly, therapeutic efficacy showed prominent sequence dependence. Induction of apoptosis was observed only when the cells were treated with Dox followed by MN-siBIRC5, whereas the reverse sequence abrogated the benefit of the drug combination. In vivo, confirmation of successful sequence dependent combination therapy was demonstrated in a murine xenograft model of breast cancer. Finally, to determine if the observed effect is not limited to breast cancer, we extended our studies to a murine xenograft model of pancreatic adenocarcinoma and found similar outcomes as shown for breast cancer.