RNAi mediated Tiam1 gene knockdown inhibits invasion of retinoblastoma

T lymphoma invasion and metastasis protein (Tiam1) is up-regulated in variety of cancers and its expression level is related to metastatic potential of the type of cancer. Earlier, Tiam1 was shown to be overexpressed in retinoblastoma (RB) and we hypothesized that it was involved in invasiveness of RB. This was tested by silencing Tiam1 in RB cell lines (Y79 and Weri-Rb1) using siRNA pool, targeting different regions of Tiam1 mRNA. The cDNA microarray of Tiam1 silenced cells showed gene regulations altered by Tiam1 were predominantly on the actin cytoskeleton interacting proteins, apoptotic initiators and tumorogenic potential targets. The silenced phenotype resulted in decreased growth and increased apoptosis with non-invasive characteristics. Transfection of full length and N-terminal truncated construct (C1199) clearly revealed membrane localization of Tiam1 and not in the case of C580 construct. F-actin staining showed the interaction of Tiam1 with actin in the membrane edges that leads to ruffling, and also imparts varying invasive potential to the cell. The results obtained from our study show for the first time that Tiam1 modulates the cell invasion, mediated by actin cytoskeleton remodeling in RB

EpCAM aptamer mediated cancer cell specific delivery of EpCAM siRNA using polymeric nanocomplex

BACKGROUND: Epithelial cell adhesion molecule (EpCAM) is overexpressed in solid tumors and regarded as a putative cancer stem cell marker. Here, we report that employing EpCAM aptamer (EpApt) and EpCAM siRNA (SiEp) dual approach, for the targeted delivery of siRNA to EpCAM positive cancer cells, efficiently inhibits cancer cell proliferation. RESULTS: Targeted delivery of siRNA using polyethyleneimine is one of the efficient methods for gene delivery, and thus, we developed a novel aptamer-PEI-siRNA nanocomplex for EpCAM targeting. PEI nanocomplex synthesized with EpCAM aptamer (EpApt) and EpCAM siRNA (SiEp) showed 198 nm diameter sized particles by dynamic light scattering, spherical shaped particles, of 151 ± 11 nm size by TEM. The surface charge of the nanoparticles was -30.0 mV using zeta potential measurements. Gel retardation assay confirmed the PEI-EpApt-SiEp nanoparticles formation. The difference in size observed by DLS and TEM could be due to coating of aptamer and siRNA on PEI nanocore. Flow cytometry analysis revealed that PEI-EpApt-SiEp has superior binding to cancer cells compared to EpApt or scramble aptamer (ScrApt) or PEI-ScrApt-SiEp. PEI-EpApt-SiEp downregulated EpCAM and inhibited selectively the cell proliferation of MCF-7 and WERI-Rb1 cells. CONCLUSIONS: The PEI nanocomplex fabricated with EpApt and siEp was able to target EpCAM tumor cells, deliver the siRNA and silence the target gene. This nanocomplex exhibited decreased cell proliferation than the scrambled aptamer loaded nanocomplex in the EpCAM expressing cancer cells and may have potential for EpCAM targeting in vivo

Attenuated total reflectance fourier transform infrared spectroscopy: an analytical technique to understand therapeutic responses at the molecular level

Rapid monitoring of the response to treatment in cancer patients is essential to predict the outcome of the therapeutic regimen early in the course of the treatment. The conventional methods are laborious, time-consuming, subjective and lack the ability to study different biomolecules and their interactions, simultaneously. Since; mechanisms of cancer and its response to therapy is dependent on molecular interactions and not on single biomolecules, an assay capable of studying molecular interactions as a whole, is preferred. Fourier Transform Infrared (FTIR) spectroscopy has become a popular technique in the field of cancer therapy with an ability to elucidate molecular interactions. The aim of this study, was to explore the utility of the FTIR technique along with multivariate analysis to understand whether the method has the resolution to identify the differences in the mechanism of therapeutic response. Towards achieving the aim, we utilized the mouse xenograft model of retinoblastoma and nanoparticle mediated targeted therapy. The results indicate that the mechanism underlying the response differed between the treated and untreated group which can be elucidated by unique spectral signatures generated by each group. The study establishes the efficiency of non-invasive, label-free and rapid FTIR method in assessing the interactions of nanoparticles with cellular macromolecules towards monitoring the response to cancer therapeutics

Competitive inhibition of survivin using a cell-permeable recombinant protein induces cancer-specific apoptosis in colon cancer model

Endogenous survivin expression has been related with cancer survival, drug resistance, and metastasis. Therapies targeting survivin have been shown to significantly inhibit tumor growth and recurrence. We found out that a cell-permeable dominant negative survivin (SurR9-C84A, referred to as SR9) competitively inhibited endogenous survivin and blocked the cell cycle at the G1/S phase. Nanoencapsulation in mucoadhesive chitosan nanoparticles (CHNP) substantially increased the bioavailability and serum stability of SR9. The mechanism of nanoparticle uptake was studied extensively in vitro and in ex vivo models. Our results confirmed that CHNP-SR9 protected primary cells from autophagy and successfully induced tumor-specific apoptosis via both extrinsic and intrinsic apoptotic pathways. CHNP-SR9 significantly reduced the tumor spheroid size (three-dimensional model) by nearly 7-fold. Effects of SR9 and CHNP-SR9 were studied on 35 key molecules involved in the apoptotic pathway. Highly significant (4.26-fold, P≤0.005) reduction in tumor volume was observed using an in vivo mouse xenograft colon cancer model. It was also observed that net apoptotic (6.25-fold, P≤0.005) and necrotic indexes (3.5-fold, P≤0.05) were comparatively higher in CHNP-SR9 when compared to void CHNP and CHNP-SR9 internalized more in cancer stem cells (4.5-fold, P≤0.005). We concluded that nanoformulation of SR9 did not reduce its therapeutic potential; however, nanoformulation provided SR9 with enhanced stability and better bioavailability. Our study presents a highly tumor-specific protein-based cancer therapy that has several advantages over the normally used chemotherapeutics

Synthesis and characterization of surface-enhanced Raman-scattered gold nanoparticles

In this paper, we report a simple, rapid, and robust method to synthesize surface-enhanced Raman-scattered gold nanoparticles (GNPs) based on green chemistry. Vitis vinifera L. extract was used to synthesize noncytotoxic Raman-active GNPs. These GNPs were characterized by ultraviolet-visible spectroscopy, dynamic light-scattering, Fourier-transform infrared (FTIR), transmission electron microscopy (TEM), X-ray diffraction (XRD), and Raman spectroscopy. The characteristic surface plasmon-resonance band at ~528 nm is indicative of spherical particles, and this was confirmed by TEM. The N–H and C–O stretches in FTIR spectroscopy indicated the presence of protein molecules. The predominant XRD plane at (111) and (200) indicated the crystalline nature and purity of GNPs. GNPs were stable in the buffers used for biological studies, and exhibited no cytotoxicity in noncancerous MIO-M1 (Müller glial) and MDA-MB-453 (breast cancer) cell lines. The GNPs exhibited Raman spectral peaks at 570, 788, and 1,102 cm-1. These new GNPs have potential applications in cancer diagnosis, therapy, and ultrasensitive biomarker detection

Erratum to: Toxicogenomics of nanoparticulate delivery of etoposide: potential impact on nanotechnology in retinoblastoma therapy

To develop a suitable formulation with high entrapment efficiency, etoposide-loaded poly(lactide-co-glycolide) nanoparticles (NPs) were formulated by single emulsion-solvent evaporation method by changing different formulation parameters such as drug loading, choice of organic solvent and percentage of emulsifier polyvinyl alcohol. The NPs showed higher entrapment efficiency, ~86% (with 15% (w/w) drug loading). The physicochemical parameters revealed smooth topology with size range (240–320 nm), a negative zeta potential (~19 mV) and in vitro sustained-release activity (~60% drug release in 40 days). Greater anti-proliferative activity ~100 times was observed with NPs (IC(50) = 0.002 μg/ml) than that of native etoposide (IC(50) = 0.2 μg/ml) in retinoblastoma cell line (Y-79). These NPs demonstrated greater (G1/S) blocking and decreased mitochondrial membrane potential as measured by flow cytometry. There was upregulation of apoptotic gene activity in NPs than native etoposide, as revealed through microarray analysis. However, this is the first ever report demonstrating the intricate modulation of genetic network affected by NPs. Collectively, these results suggest that etoposide-loaded NPs could be potentially useful as a novel drug delivery system for retinoblastoma in the future. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12645-010-0010-4) contains supplementary material, which is available to authorized users

Production forecasting of coconut: Variation in number of bearing palms and productivity in selected districts in India

Forecasting of coconut production in the country was attempted for three consecutive years from 2006-07. Stratified multistage sampling design was employed. At district level, forecasting of production was arrived by multiplying average predicted yield of palms with the 'harvested-area' of the crop in that district. Ratio estimator was constructed to obtain forecasting at different administrative levels. The all India forecasts of coconut production in the years 2006-07 to 2008-09 were obtained as 13448, 16331 and 14183 million nuts against the published values of 15840, 14743 and 15729 in order. On observing noticeable reduction in area under coconut in Kerala, the all India forecasting was revised as separately working out the forecasts for Kerala and rest of India and adding. By following this approach, the per cent difference of forecasts with published values were observed to be reduced from 15.1, -10.8, and 9.8 to 10.5, -5.2, and 6.0 in order in the years 2006-07 to 2008-09

Production forecasting of coconut: Variation in number of bearing palms and productivity in selected districts in India

Forecasting of coconut production in the country was attempted for three consecutive years from 2006-07. Stratified multistage sampling design was employed. At district level, forecasting of production was arrived by multiplying average predicted yield of palms with the 'harvested-area' of the crop in that district. Ratio estimator was constructed to obtain forecasting at different administrative levels. The all India forecasts of coconut production in the years 2006-07 to 2008-09 were obtained as 13448, 16331 and 14183 million nuts against the published values of 15840, 14743 and 15729 in order. On observing noticeable reduction in area under coconut in Kerala, the all India forecasting was revised as separately working out the forecasts for Kerala and rest of India and adding. By following this approach, the per cent difference of forecasts with published values were observed to be reduced from 15.1, -10.8, and 9.8 to 10.5, -5.2, and 6.0 in order in the years 2006-07 to 2008-09