Fluorescence imaging of biochemical relationship between ubiquitinated histone 2A and Polycomb complex protein BMI1.

Several in vitro experiments have highlighted that the Polycomb group protein BMI1 plays a pivotal role in determining the biological functions of the Polycomb Repressor Complex 1 (PRC1), including its E3-ligase activity towards the Lys119 of histone H2A to yield ubiquitinated uH2A. The role of BMI1 in the epigenetic activity of PRC1 is particularly relevant in several cancers, particularly Non-Small Cell Lung Cancer (NSCLC). In this study, using indirect immunofluorescence protocols implemented on a confocal microscopy apparatus, we investigated the relationship between BMI1 and uH2A at different resolutions, in cultured (A549) and clinical NSCLC tissues, at the single cell level. In both cases, we observed a linear dependence of uH2A concentration upon BMI1 expression at the single nucleus level, indicating that the association of BMI1 to PRC1, which is needed for E3-ligase activity, occurs linearly in the physiological BMI1 concentration range. Additionally, in the NSCLC cell line model, a minor pool of uH2A may exist in absence of concurrent BMI1 expression, indicating non-exclusive, although predominant, role of BMI1 in the amplification of the E3-ligase activity of PRC1. A pharmacological downregulator of BMI1, PTC-209, was also tested in this context. Finally, the absence of significant colocalization (as measured by the Pearson's coefficient) between BMI1 and uH2A submicron clusters hints to a dynamic model where PRC1 resides transiently at ubiquitination sites. Beside unveiling subtle functional relationships between BMI1 and uH2A, these results also validate the use of uH2A as downstream "reporter" for BMI1 activity at the nuclear level in NSCLC contexts.

Endogenously Triggerable Ultrasmall-in-Nano Architectures: Targeting Assessment on 3D Pancreatic Carcinoma Spheroids.

Several nanomaterials rely on the passive accumulation in the neoplasm target because of enhanced permeability and retention effect. On the other hand, directing nanomaterials to the target by employing the targeting agents may lead to a pivotal improvement in the efficacy of the treatment for a number of cancers. However, targeting moieties often lose their functionality upon injection in the bloodstream, leaving questions on their efficiency. Here, we assessed using a significant in vitro 3D model of pancreatic carcinoma the targeting efficiency of passion fruit-like nanoarchitectures (NAs) incorporated with a peptide that can recognize transferrin directly in the medium, thereby modulating protein solvation. NAs are biodegradable ultrasmall-in-nano platforms that combine the most appealing behaviors of noble metal nanomaterials with organism excretion of the building blocks by the renal pathway. Although the confocal images did not illustrate the significant differences in the targeting efficiency of the peptide-modified NAs, an improved internalization was quantitatively observed by inductively coupled plasma-mass spectrometry analysis. Our findings demonstrate that the peptide conjugation of NAs might be considered to enhance their theranostic potentials for this type of neoplasm.

Magnetic catechin-dextran conjugate as targeted therapeutic for pancreatic tumour cells.

BACKGROUND: Catechin-dextran conjugates have recently attracted a lot of attention due to their anticancer activity against a range of cancer cells. Magnetic nanoparticles have the ability to concentrate therapeutically important drugs due to their magnetic-spatial control and provide opportunities for targeted drug delivery. PURPOSE: Enhancement of the anticancer efficiency of catechin-dextran conjugate by functionalisation with magnetic iron oxide nanoparticles. METHODS: Modification of the coating shell of commercial magnetic nanoparticles (Endorem) composed of dextran with the catechin-dextran conjugate. RESULTS: Catechin-dextran conjugated with Endorem (Endo-Cat) increased the intracellular concentration of the drug and it induced apoptosis in 98% of pancreatic tumour cells placed under magnetic field. DISCUSSION: The conjugation of catechin-dextran with Endorem enhances the anticancer activity of this drug and provides a new strategy for targeted drug delivery on tumour cells driven by magnetic field. CONCLUSION: The ability to spatially control the delivery of the catechin-dextran by magnetic field makes it a promising agent for further application in cancer therapy.

Neuronal differentiation on anisotropic substrates and the influence of nanotopographical noise on neurite contact guidance.

Cells are exposed to specific directional physical signals determined by the micro/nano-environment that in vivo coexist with some degree of topographical noise. Particularly in the nervous system, cell contact sensing of the extracellular environment plays a primary role in defining neurite initiation and final brain wiring. Here we study neuronal cell response to directional stimuli by exploiting nanogratings with controlled amount of random nanotopographical noise. The impact of noise on neurite guidance and focal adhesions (FAs) development is investigated in NGF-differentiating PC12 cells by confocal and TIRF microscopy. We show that the loss of neurite guidance is not linear with noise, but is a threshold effect, correlating with changes in FA maturation and spatial organization. Finally nocodazole, a drug that increases cell contractility, can improve neurite alignment by promoting aligned FA maturation. We argue that these results show new possibilities for successful implant strategies particularly in the context of nerve-regeneration devices.

Cancer phototherapy in living cells by multiphoton release of doxorubicin from gold nanospheres.

Doxorubicin is a widely used but toxic cancer chemotherapeutic agent. In order to localize its therapeutic action and minimize side effects, it was covalently conjugated to peptide-encapsulated gold nanospheres by click-chemistry and then photo-released in a controlled fashion by a multiphoton process. Selective treatment of a chosen region in a 2D layer of U2Os cancer cells is shown by driving photorelease with 561 nm irradiation at µW power. These results show promising directions for the development of practical applications based on nanocarriers that can ensure drug delivery with high spatial and temporal control.

Neuronal polarity selection by topography-induced focal adhesion control.

Interaction between differentiating neurons and the extracellular environment guides the establishment of cell polarity during nervous system development. Developing neurons read the physical properties of the local substrate in a contact-dependent manner and retrieve essential guidance cues. In previous works we demonstrated that PC12 cell interaction with nanogratings (alternating lines of ridges and grooves of submicron size) promotes bipolarity and alignment to the substrate topography. Here, we investigate the role of focal adhesions, cell contractility, and actin dynamics in this process. Exploiting nanoimprint lithography techniques and a cyclic olefin copolymer, we engineered biocompatible nanostructured substrates designed for high-resolution live-cell microscopy. Our results reveal that neuronal polarization and contact guidance are based on a geometrical constraint of focal adhesions resulting in an angular modulation of their maturation and persistence. We report on ROCK1/2-myosin-II pathway activity and demonstrate that ROCK-mediated contractility contributes to polarity selection during neuronal differentiation. Importantly, the selection process confined the generation of actin-supported membrane protrusions and the initiation of new neurites at the poles. Maintenance of the established polarity was independent from NGF stimulation. Altogether our results imply that focal adhesions and cell contractility stably link the topographical configuration of the extracellular environment to a corresponding neuronal polarity state.

The effect of alternative neuronal differentiation pathways on PC12 cell adhesion and neurite alignment to nanogratings.

During development and regeneration of the mammalian nervous system, directional signals guide differentiating neurons toward their targets. Soluble neurotrophic molecules encode for preferential direction over long distances while the local topography is read by cells in a process requiring the establishment of focal adhesions. The mutual interaction between overlapping molecular and topographical signals introduces an additional level of control to this picture. The role of the substrate topography was demonstrated exploiting nanotechnologies to generate biomimetic scaffolds that control both the polarity of differentiating neurons and the alignment of their neurites. Here PC12 cells contacting nanogratings made of copolymer 2-norbornene ethylene (COC), were alternatively stimulated with Nerve Growth Factor, Forskolin, and 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cyclic (8CPT-2Me-cAMP) or with a combination of them. Topographical guidance was differently modulated by the alternative stimulation protocols tested. Forskolin stimulation reduced the efficiency of neurite alignment to the nanogratings. This effect was linked to the inhibition of focal adhesion maturation. Modulation of neurite alignment and focal adhesion maturation upon Forskolin stimulation depended on the activation of the MEK/ERK signaling but were PkA independent. Altogether, our results demonstrate that topographical guidance in PC12 cells is modulated by the activation of alternative neuronal differentiation pathways.

Development of a novel GFP-based ratiometric excitation and emission pH indicator for intracellular studies.

We report on the development of the F64L/S65T/T203Y/L231H GFP mutant (E2GFP) as an effective ratiometric pH indicator for intracellular studies. E2GFP shows two distinct spectral forms that are convertible upon pH changes both in excitation and in emission with pK close to 7.0. The excitation of the protein at 488 and 458 nm represents the best choice in terms of signal dynamic range and ratiometric deviation from the thermodynamic pK. This makes E2GFP ideally suited for imaging setups equipped with the most widespread light sources and filter settings. We used E2GFP to determine the average intracellular pH (pH(i)) and spatial pH(i) maps in two different cell lines, CHO and U-2 OS, under physiological conditions. In CHO, we monitored the evolution of the pH(i) during mitosis. We also showed the possibility to target specific subcellular compartments such as nucleoli (by fusing E2GFP with the transactivator protein of HIV, (Tat) and nuclear promyelocytic leukemia bodies (by coexpression of promyelocytic leukemia protein).