Folate-targeted amphiphilic cyclodextrin.siRNA nanoparticles for prostate cancer therapy exhibit PSMA mediated uptake, therapeutic gene silencing in vitro and prolonged circulation in vivo.

In this study, a folate targeted cyclodextrin (CD) nanoparticle was prepared by co-formulating CD.siRNA complexes with DSPE-PEG5000-folate to target the prostate specific membrane antigen (PSMA). Targeted formulations showed increased uptake, relative to untargeted controls, in two prostate cancer cell lines expressing PSMA (VCaP and LNCaP). Competitive uptake studies, using excess folate, significantly reduced uptake of targeted nanoparticles in PSMA positive cell lines (P<0.001). Relative to untreated controls, folate-targeted nanoparticles significantly reduced the levels of RelA mRNA in VCaP and LNCaP cells by 44% and 22% respectively (P<0.001). In contrast there was no significant reduction in RelA mRNA in these cell lines by untargeted complexes. Pharmacokinetic (PK) data indicated that the incorporation of PEG into the formulation increased the circulation time of siRNA 8-fold. This study highlights the ability of incorporating a folate ligand into CD.siRNA nanoparticles to allow for targeted delivery of siRNA to prostate cancer cells via the PSMA.

ROS signalling, NADPH oxidases and cancer.

ROS (reactive oxygen species) have long been regarded as a series of destructive molecules that have a detrimental effect on cell homoeostasis. In support of this are the myriad antioxidant defence systems nearly all eukaryotic cells have that are designed to keep the levels of ROS in check. However, research data emerging over the last decade have demonstrated that ROS can influence a range of cellular events in a manner similar to that seen for traditional second messenger molecules such as cAMP. Hydrogen peroxide (H2O2) appears to be the main ROS with such signalling properties, and this molecule has been shown to affect a wide range of cellular functions. Its localized synthesis by the Nox (NADPH oxidase) family of enzymes and how these enzymes are regulated is of particular interest to those who work in the field of tumour biology.

Imatinib and Nilotinib inhibit Bcr-Abl-induced ROS through targeted degradation of the NADPH oxidase subunit p22phox.

Constitutive expression of the Bcr-Abl kinase in Chronic Myelogenous Leukaemia (CML) is known to produce elevated levels of Reactive Oxygen Species (ROS) which can enhance cell survival as well as generate genomic instability. Our laboratory has previously demonstrated that NADPH oxidase (Nox) activity contributes to intracellular-ROS levels in Bcr-Abl-positive cells, while inducing increased pro-survival signalling through the PI3K/Akt pathway. How Bcr-Abl signalling regulates Nox activity still remains to be elucidated. In this study, using the K562 CML cell line we showed that inhibition of Bcr-Abl signalling, by either Imatinib or Nilotinib, led to a significant reduction in ROS levels which was concurrent with the GSK-3ß dependent, post-translational down-regulation of the small membrane-bound protein p22phox, an essential component of the Nox complex. Furthermore, siRNA knockdown of p22phox in these cells established its importance in ROS production and proliferation. Taken together we believe our results provide a possible link between Bcr-Abl signalling and ROS production through Nox activity and demonstrate a novel mechanism of action associated with Imatinib and Nilotinib treatment in CML.

Redox-regulated growth factor survival signaling.

SIGNIFICANCE: Once the thought of as unwanted byproducts of cellular respiration in eukaryotes, reactive oxygen species (ROS) have been shown to facilitate essential physiological roles. It is now understood that ROS are critical mediators of intracellular signaling. Control of signal transduction downstream of growth factor receptors by ROS is a complex process whose details are only recently coming to light. RECENT ADVANCES: Indeed, recent evidence points to control of signal propagation by ROS at multiple levels in the typical cascade. Growth factor stimulation activates nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Noxs) at the membrane, producing superoxide in the extracellular matrix, which is catalyzed to the membrane-permeable hydrogen peroxide (H2O2) that mediates intracellular signaling events. CRITICAL ISSUES: The potential for H2O2, however, to disrupt cellular functions by damaging proteins and nucleic acids demands that its levels are kept in check by receptor-associated peroxiredoxins. This interplay of Nox and peroxiredoxin activity moderates levels of H2O2 sufficiently to modify signaling partners locally. Among the best studied of these partners are redox-controlled phosphatases that are inactivated by H2O2. Phosphatases regulate signal propagation downstream of receptors, and thus their inactivation allows a further level of control. Transmission of information further downstream to targets such as transcription factors, themselves regulated by ROS, completes this pathway. FUTURE DIRECTIONS: Thus, signal propagation or attenuation can be dictated by ROS at multiple points. Given the complex nature of these processes, we envisage the emerging trends in the field of redox signaling in the context of growth factor stimulation.

Inhibition of protein-tyrosine phosphatase 1B (PTP1B) mediates ubiquitination and degradation of Bcr-Abl protein.

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder characterized at the molecular level by the expression of Bcr-Abl, a chimeric protein with deregulated tyrosine kinase activity. The protein-tyrosine phosphatase 1B (PTP1B) is up-regulated in Bcr-Abl-expressing cells, suggesting a regulatory link between the two proteins. To investigate the interplay between these two proteins, we inhibited the activity of PTP1B in Bcr-Abl-expressing TonB.210 cells by either pharmacological or siRNA means and examined the effects of such inhibition on Bcr-Abl expression and function. Herein we describe a novel mechanism by which the phosphatase activity of PTP1B is required for Bcr-Abl protein stability. Inhibition of PTP1B elicits tyrosine phosphorylation of Bcr-Abl that triggers the degradation of Bcr-Abl through ubiquitination via the lysosomal pathway. The degradation of Bcr-Abl consequently inhibits tyrosine phosphorylation of Bcr-Abl substrates and the downstream production of intracellular reactive oxygen species. Furthermore, PTP1B inhibition reduces cell viability and the IC(50) of the Bcr-Abl inhibitor imatinib mesylate. Degradation of Bcr-Abl via PTP1B inhibition is also observed in human CML cell lines K562 and LAMA-84. These results suggest that inhibition of PTP1B may be a useful strategy to explore in the development of novel therapeutic agents for the treatment of CML, particularly because host drugs currently used in CML such as imatinib focus on inhibiting the kinase activity of Bcr-Abl.