Epidermal growth factor promotes protein degradation of epithelial protein lost in neoplasm (EPLIN), a putative metastasis suppressor, during epithelial-mesenchymal transition.

Aberrant expression of EGF receptors has been associated with hormone-refractory and metastatic prostate cancer (PCa). However, the molecular mechanism for EGF signaling in promoting PCa metastasis remains elusive. Using experimental models of PCa metastasis, we demonstrated that EGF could induce robust epithelial-mesenchymal transition (EMT) and increase invasiveness. Interestingly, EGF was found to be capable of promoting protein turnover of epithelial protein lost in neoplasm (EPLIN), a putative suppressor of EMT and tumor metastasis. Mechanistic study revealed that EGF could activate the phosphorylation, ubiquitination, and degradation of EPLIN through an extracellular signal-regulated kinase 1/2 (ERK1/2)-dependent signaling cascade. Pharmacological inhibition of the ERK1/2 pathway effectively antagonized EGF-induced EPLIN degradation. Two serine residues, i.e. serine 362 and serine 604, were identified as putative ERK1/2 phosphorylation sites in human EPLIN, whose point mutation rendered resistance to EGF-induced protein turnover. This study elucidated a novel molecular mechanism for EGF regulation of EMT and invasiveness in PCa cells, indicating that blockade of EGF signaling could be beneficial in preventing and retarding PCa metastasis at early stages.

Genistein enhances the efficacy of cabazitaxel chemotherapy in metastatic castration-resistant prostate cancer cells.

BACKGROUND: Cabazitaxel (Jevtana) has been approved for the treatment of metastatic castration-resistant prostate cancer (mCRPC). However, most patients progress and become chemoresistant, which remains a major challenge in the management of advanced PCa. In this study, we investigated whether genistein, an isoflavone abundant in soy products, could sensitize mCRPC cells to cabazitaxel treatment in experimental models. METHODS: The in vitro and in vivo effect of genistein in enhancing the response of mCRPC cells to cabazitaxel chemotherapy was evaluated in experimental models. RESULTS: Genistein increases the expression of pro-apoptotic protein Bax, activates apoptotic signals, and enhances the response to cabazitaxel treatment in mCRPC cells. In a PC3-luciferase xenograft model, the combined treatment with genistein and cabazitaxel significantly retarded the growth of mCRPC when compared to vehicle control, cabazitaxel, or genistein. Tissue staining confirmed the in vivo effect of genistein on the induction of Bax and activation of apoptosis. CONCLUSION: This study provided the first preclinical evidence supporting that genistein could be beneficial in improving cabazitaxel chemotherapy in mCRPC.

Pomegranate extract inhibits the bone metastatic growth of human prostate cancer cells and enhances the in vivo efficacy of docetaxel chemotherapy.

BACKGROUND: Docetaxel treatment is the only first-line chemotherapy with a survival benefit in metastatic castration-resistant prostate cancer (PCa). Nonetheless, most patients become docetaxel resistant and inevitably progress with no cure. In this study, we investigated the potential of pomegranate extract (PE) in targeting metastatic castration-resistant PCa and improving docetaxel chemotherapy. METHODS: The in vitro and in vivo effect of POMx, a PE formula currently approved for clinical trials, in metastatic castration-resistant PCa cells was evaluated in experimental models. RESULTS: We demonstrated that POMx exhibited potent in vitro cytotoxicity in metastatic castration-resistant PCa cells. Mechanistic studies identified survivin as a novel molecular target that may mediate the anti-cancer activity of POMx, presumably through the inhibition of signal transducer and activator of transcription 3. The in vivo administration of POMx treatment effectively inhibited survivin, induced apoptosis, retarded C4-2 tumor growth in skeleton and significantly enhanced the efficacy of docetaxel in athymic nude mice. CONCLUSION: These results provide the first preclinical evidence that POMx may be effective in treating metastatic castration-resistant PCa and enhancing the efficacy of docetaxel chemotherapy. Prostate © 2013 Wiley Periodicals, Inc.

Probing and Enhancing Ligand-Mediated Active Targeting of Tumors Using Sub-5 nm Ultrafine Iron Oxide Nanoparticles.

Rationale: "Active targeting" based on the ligand-target affinity is a common strategy to precisely deliver nanoparticle (NP) imaging probes or drug carriers to the diseased tissue. However, such ligand-mediated active targeting inevitably takes place with prerequisite "passive targeting", driven by the enhanced permeability and retention (EPR) effect. Thus, the efficiency of active targeting in relation to off-targeted unbound NPs is of great importance in quantitative imaging of tumor biomarkers and delivery. With the notion that easy clearance of off-targeted uIONPs may lead to enhanced active targeting and tumor accumulation, we examined the NP size effect on "active targeting" of the transferrin receptor (TfR) using transferrin (Tf)-conjugated sub-5 nm (3 nm core) ultrafine iron oxide NPs (uIONPs) and larger IONPs (30 nm core). Methods: Green fluorescent dye (FITC)-labeled active targeting uIONPs (FITC-Tf-uIONPs) and red fluorescent dye (TRITC)-labeled passive targeting uIONPs (TRITC-uIONPs) were prepared. FITC-Tf-IONPs and TRITC-IONPs were used as comparison for the NP size effect. Multiphoton imaging, confocal fluorescence imaging, histological staining and computational analysis were applied to track different types of NPs in tumors at 1, 3 and 24 hours after co-injection of equal amounts of paired NPs, e.g., active targeting FITC-Tf-uIONPs and non-targeting TRITC-uIONPs, or FITC-Tf-IONPs and TRITC-IONPs into the same mice bearing 4T1 mouse mammary tumors. Results: Active targeting uIONPs exhibited an almost 6-fold higher level of tumor retention with deeper penetration comparing to non-targeting uIONPs at 24 hours after co-injection. However, accumulation of active targeting IONPs with a 30-nm core is only about 1.15-fold higher than non-targeting IONPs. The enhanced active targeting by uIONPs can be attributed to the size dependent clearance of unbound off-targeted NPs, as majority off-targeted uIONPs were readily cleared from the tumor by intravasation back into tumor blood vessels likely due to high interstitial pressure, even though they are not favorable for macrophage uptake. Conclusion: Ligand-mediated active targeting improves the delivery and accumulation of the sub-5 nm NPs. The improvement on active targeting is size-dependent and facilitated by NPs with sub-5 nm core sizes. Thus, sub-5 nm NPs may serve as favorable platforms for development of NP-based molecular imaging probes and targeted drug carriers.

Exerting Enhanced Permeability and Retention Effect Driven Delivery by Ultrafine Iron Oxide Nanoparticles with T1-T2 Switchable Magnetic Resonance Imaging Contrast.

Poor delivery efficiency remains a major challenge in nanomaterial-based tumor-targeted imaging and drug delivery. This work demonstrates a strategy to improve nanoparticle delivery and intratumoral distribution using sub-5 nm (3.5 nm core size) ultrafine iron oxide nanoparticles (uIONP) that can easily extravasate from the tumor vasculature and readily diffuse into the tumor tissue compared to the iron oxide nanoparticle (IONP) with larger sizes, followed by self-assembling in the acidic tumor interstitial space to limit their re-entering into circulation. By combining enhanced extravasation and reduced intravasation, we achieved improved delivery and tumor retention of nanoparticles. Multiphoton imaging of mice bearing orthotopic tumors co-injected with fluorescent dye-labeled nanoparticles with different sizes showed that uIONPs exhibited more efficient extravasation out of tumor vessels and penetrated deeper into the tumor than larger sized IONP counterparts. Moreover, in vivo magnetic resonance imaging revealed that uIONPs exhibited "bright" T1 contrast when dispersed in the tumor vasculature and peripheral area at 1 h after intravenous administration, followed by emerging "dark" T2 contrast in the tumor after 24 h. Observed T1-T2 contrast switch indicated that uIONPs single-dispersed in blood with T1 contrast may self-assemble into larger clusters with T2 contrast after entering the tumor interstitial space. Improved passive targeting and intratumoral delivery along with increased tumor retention of uIONPs are due to both easy extravasation into the tumor when single-dispersed and restricting intravasation back into circulation after forming clusters, thus exerting the enhanced permeability and retention effect for nanoparticle delivery to tumors.

Reducing non-specific binding and uptake of nanoparticles and improving cell targeting with an antifouling PEO-b-PgammaMPS copolymer coating.

One of the major limitations impeding the sensitivity and specificity of biomarker targeted nanoparticles is non-specific binding by biomolecules and uptake by the reticuloendothelial system (RES). We report the development of an antibiofouling polysiloxane containing amphiphilic diblock copolymer, poly(ethylene oxide)-block-poly(gamma-methacryloxypropyl trimethoxysilane) (PEO-b-PgammaMPS), for coating and functionalizing high quality hydrophobic nanocrystals such as iron oxide nanoparticles and quantum dots. These PEO-b-PgammaMPS-coated nanocrystals were colloidally stable in biological medium and showed low non-specific binding by macromolecules after incubation with 100% fetal bovine serum. Both in vitro experiments with macrophages and in vivo biodistribution studies in mice revealed that PEO-b-PgammaMPS copolymer-coated nanocrystals have an antibiofouling effect that reduces non-specific cell and RES uptake. Surface functionalization with amine groups was accomplished through co-crosslinking the polysiloxane coating layer and (3-Aminopropyl)trimethoxysilane in aqueous solution. Tumor integrin alpha(v)beta(3) targeting peptide cyclo-RGD ligands were conjugated on the nanoparticles through a heterobifunctional linker. The resulting integrin alpha(v)beta(3) targeting nanoparticle conjugates showed improved cancer cell targeting with a stronger affinity to U87MG glioma cells, which have a high expression of alpha(v)beta(3) integrins, but minimal binding to MCF-7 breast cancer cells with low expression of alpha(v)beta(3) integrins.