A nanoformulation of siRNA and its role in cancer therapy: In vitro and in vivo evaluation

Overexpression of anti-apoptotic Bcl-2 is often observed in a wide variety of human cancers. It prevents the induction of apoptosis in neoplastic cells and contributes to resistance to chemotherapy. RNA interference has emerged as an efficient and selective technique for gene silencing. The potential to use small interfering RNA (siRNA) as a therapeutic agent for the treatment of cancer has elicited a great deal of interest. However, insufficient cellular uptake and poor stability have limited its therapeutic applications. The purpose of this study was to prepare chitosan nanoparticles via ionic gelation of chitosan by tripolyphosphate for effective delivery of siRNA to silence the anti-apoptotic Bcl-2 gene in neoplastic cells. Chitosan nanoparticles loaded with siRNA were in the size range 190 to 340 nm with a polydispersive index ranging from 0.04 to 0.2. They were able to completely bind with siRNA, provide protection against nuclease degradation, and enhance the transfection. Cell culture studies revealed that nanoparticles with entrapped siRNA could efficiently silence the antiapoptotic Bcl-2 gene. Studies on Swiss albino mice showed that siRNA could be effectively delivered through nanoparticles. There was significant decrease in the tumor volume. Blocking the expression of anti-apoptotic Bcl-2 can enhance the sensitivity of cancerous cells to anti-cancer drugs and the apoptosis rate. Therefore, nanoformulations with siRNA can be promoted as an adjuvant therapy in combination with anti-cancer drugs.     

The Effect of Molecular Weight,PK, and Valency on Tumor Biodistribution and Efficacy of Antibody-Based Drugs

Poor drug delivery and penetration of antibody-mediated therapies pose significant obstacles to effective treatment of solid tumors. This study explored the role of pharmacokinetics, valency, and molecular weight in maximizing drug delivery. Biodistribution of a fibroblast growth factor receptor 4 (FGFR4) targeting CovX-body (an FGFR4-binding peptide covalently linked to a nontargeting IgG scaffold; 150 kDa) and enzymatically generated FGFR4 targeting F(ab)₂ (100 kDa) and Fab (50 kDa) fragments was measured. Peak tumor levels were achieved in 1 to 2 hours for Fab and F(ab)₂versus 8 hours for IgG, and the percentage injected dose in tumors was 0.45%, 0.5%, and 2.5%, respectively, compared to 0.3%, 2%, and 6% of their nontargeting controls. To explore the contribution of multivalent binding, homodimeric peptides were conjugated to the different sized scaffolds, creating FGFR4 targeting IgG and F(ab)₂ with four peptides and Fab with two peptides. Increased valency resulted in an increase in cell surface binding of the bivalent constructs. There was an inverse relationship between valency and intratumoral drug concentration, consistent with targeted consumption. Immunohistochemical analysis demonstrated increased size and increased cell binding decreased tumor penetration. The binding site barrier hypothesis suggests that limited tumor penetration, as a result of high-affinity binding, could result in decreased efficacy. In our studies, increased target binding translated into superior efficacy of the IgG instead, because of superior inhibition of FGFR4 proliferation pathways and dosing through the binding site barrier. Increasing valency is therefore an effective way to increase the efficacy of antibody-based drugs.     

crw1 - A Novel Maize Mutant Highly Susceptible to Foliar Damage by the Western Corn Rootworm Beetle

Western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is the most destructive insect pest of corn (Zea mays L.) in the United States. The adult WCR beetles derive their nourishment from multiple sources including corn pollen and silks as well as the pollen of alternate hosts. Conversely, the corn foliage is largely neglected as a food source by WCR beetles, leading to a perception of a passive interaction between the two. We report here a novel recessive mutation of corn that was identified and named after its foliar susceptibility to corn rootworm beetles (crw1). The crw1 mutant under field conditions was exceptionally susceptible to foliar damage by WCR beetles in an age-specific manner. It exhibits pleiotropic defects on cell wall biochemistry, morphology of leaf epidermal cells and lower structural integrity via differential accumulation of cell wall bound phenolic acids. These findings indicate that crw1 is perturbed in a pathway that was not previously ascribed to WCR susceptibility, as well as implying the presence of an active mechanism(s) deterring WCR beetles from devouring corn foliage. The discovery and characterization of this mutant provides a unique opportunity for genetic analysis of interactions between maize and adult WCR beetles and identify new strategies to control the spread and invasion of this destructive pest.     

Extracellular solvent stable cold-active lipase from psychrotrophic Bacillus sphaericus MTCC 7526: partial purification and characterization

Psychrotropic Bacillus sphaericus producing solvent stable cold-active lipase upon growth at low temperature was isolated from Gangotri glacier. Optimal parameters for lipase production were investigated and the strain was able to produce lipase even at 15 °C. An incubation period of 48 h and pH 8 was found to be conducive for cold-active lipase production. The addition of trybutyrin as substrate and lactose as additional carbon source increased lipase production. The enzyme was purified up to 17.74-fold by ammonium sulphate precipitation followed by DEAE cellulose column chromatography. The optimum temperature and pH for lipase activity were found to be 15 °C and 8.0, respectively. The lipase was found to be stable in the temperature range 20–30 °C and the pH range 6.0–9.0. The protein retained more than 83 % of its initial activity after exposure to organic solvents. The lipase exhibited significant stability in presence of acetone and DMSO retaining >90 % activity. The enzyme activity was inhibited by 10 mM CuSO₄ and EDTA but showed no loss in activity after incubation with other metals or inhibitors examined in this study.     

Ligand-Induced Protein Mobility in Complexes of Carbonic Anhydrase II and Benzenesulfonamides with Oligoglycine Chains

This paper describes a biophysical investigation of residual mobility in complexes of bovine carbonic anhydrase II (BCA) and para-substituted benzenesulfonamide ligands with chains of 1–5 glycine subunits, and explains the previously observed increase in entropy of binding with chain length. The reported results represent the first experimental demonstration that BCA is not the rigid, static globulin that has been typically assumed, but experiences structural fluctuations upon binding ligands. NMR studies with ¹⁵N-labeled ligands demonstrated that the first glycine subunit of the chain binds without stabilization or destabilization by the more distal subunits, and suggested that the other glycine subunits of the chain behave similarly. These data suggest that a model based on ligand mobility in the complex cannot explain the thermodynamic data. Hydrogen/deuterium exchange studies provided a global estimate of protein mobility and revealed that the number of exchanged hydrogens of BCA was higher when the protein was bound to a ligand with five glycine subunits than when bound to a ligand with only one subunit, and suggested a trend of increasing number of exchanged hydrogens with increasing chain length of the BCA-bound ligand, across the series. These data support the idea that the glycine chain destabilizes the structure of BCA in a length-dependent manner, causing an increase in BCA mobility. This study highlights the need to consider ligand-induced mobility of even “static” proteins in studies of protein-ligand binding, including rational ligand design approaches.     

Inhibition of copper-induced aggregation of human γD-crystallin by a diimine molecule and investigations on their molecular interactions

Communicated by Ramaswamy H. Sarma     

IGFBP3 gene promoter methylation analysis and its association with clinicopathological characteristics of colorectal carcinoma

Molecular Biology Reports - Promoter methylation mediated silencing of tumor suppressor genes plays an important role in the tumorigenesis of colorectal carcinoma (CRC). Tumor suppressor gene,...     

A Critical Role for ISGylation,Ubiquitination and,SUMOylation in Brain Damage: Implications for Neuroprotection

Neurochemical Research - Post-translational modification (PTMs) of proteins by ubiquitin and ubiquitin-like modifiers such as interferon-stimulated gene 15 (ISG15) and small ubiquitin-related...     

Chloro and bromo-pyrazole curcumin Knoevenagel condensates augmented anticancer activity against human cervical cancer cells: design,synthesis, in silico docking and in vitro cytotoxicity analysis

Abstract

With an endeavor to develop novel curcumin analogs as potential anti-cancer agents, we designed and synthesized a series of Knoevenagel condensates by clubbing pyrazole carbaldehydes at the active methylene carbon atom of the curcumin backbone. Molecular docking studies were carried out to target the proposed derivatives on human kinase β (IKKβ), a potential anti-cancer target. The chloro derivative displayed five hydrogen bond interactions with a docking score of ?11.874?kcal/mol higher than curcumin (docking score =??7.434?kcal/mol). This was supported by the fact that the propellant shaped derivatives fitted aptly into the binding pocket. Molecular simulations studies were also conducted on the lead molecule and the results figured out that the stable complexes were developed as the minimal deviations per residue of protein within the range of 0.11–0.92 Å. The screened compounds were synthesized, characterized and evaluated in vitro for cytotoxicity against cervical cancer cell line, HeLa using standard cell proliferation assay. Chloro derivative and bromo analog demonstrated IC₅₀ (half maximal inhibitory concentration) value of 14.2 and 18.6 µg/ml, respectively, significantly lower than 42.4 µg/ml of curcumin and higher than 0.008 µg/ml of paclitaxel. Induction of apoptosis was evaluated in the terms of cleavage of caspase-3 enzyme and they also exhibited 69.6 and 65.4% of apoptosis significantly higher than 19.9% induced by curcumin. In conclusion, chloro and bromo derivatives must be evaluated under a set of stringent in vitro and in vivo parameters for translating in to a clinically viable product.

Communicated by Ramaswamy H. Sarma     

Epistatic role of base excision repair and mismatch repair pathways in mediating cisplatin cytotoxicity

Base excision repair (BER) and mismatch repair (MMR) pathways play an important role in modulating cis-Diamminedichloroplatinum (II) (cisplatin) cytotoxicity. In this article, we identified a novel mechanistic role of both BER and MMR pathways in mediating cellular responses to cisplatin treatment. Cells defective in BER or MMR display a cisplatin-resistant phenotype. Targeting both BER and MMR pathways resulted in no additional resistance to cisplatin, suggesting that BER and MMR play epistatic roles in mediating cisplatin cytotoxicity. Using a DNA Polymerase β (Polβ) variant deficient in polymerase activity (D256A), we demonstrate that MMR acts downstream of BER and is dependent on the polymerase activity of Polβ in mediating cisplatin cytotoxicity. MSH2 preferentially binds a cisplatin interstrand cross-link (ICL) DNA substrate containing a mismatch compared with a cisplatin ICL substrate without a mismatch, suggesting a novel mutagenic role of Polβ in activating MMR in response to cisplatin. Collectively, these results provide the first mechanistic model for BER and MMR functioning within the same pathway to mediate cisplatin sensitivity via non-productive ICL processing. In this model, MMR participation in non-productive cisplatin ICL processing is downstream of BER processing and dependent on Polβ misincorporation at cisplatin ICL sites, which results in persistent cisplatin ICLs and sensitivity to cisplatin.