Background Prostate cancer is the major cause of cancer death in men and the androgen receptor (AR) has been shown to play a critical role in the progression of the disease. Our previous reports showed that knocking down the expression of the AR gene using a siRNA-based approach in prostate cancer cells led to apoptotic cell death and xenograft tumor eradication. In this study, we utilized a biodegradable nanoparticle to deliver the therapeutic AR shRNA construct specifically to prostate cancer cells. Materials & methods The biodegradable nanoparticles were fabricated using a poly(dl-lactic-co-glycolic acid) polymer and the AR shRNA constructs were loaded inside the particles. The surface of the nanoparticles were then conjugated with prostate-specific membrane antigen aptamer A10 for prostate cancer cell-specific targeting. Results A10-conjugation largely enhanced cellular uptake of nanoparticles in both cell culture- and xenograft-based models. The efficacy of AR shRNA encapsulated in nanoparticles on AR gene silencing was confirmed in PC-3/AR-derived xenografts in nude mice. The therapeutic property of A10-conjugated AR shRNA-loaded nanoparticles was evaluated in xenograft models with different prostate cancer cell lines: 22RV1, LAPC-4 and LNCaP. Upon two injections of the AR shRNA-loaded nanoparticles, rapid tumor regression was observed over 2 weeks. Consistent with previous reports, A10 aptamer conjugation significantly enhanced xenograft tumor regression compared with nonconjugated nanoparticles. Discussion These data demonstrated that tissue-specific delivery of AR shRNA using a biodegradable nanoparticle approach represents a novel therapy for life-threatening prostate cancers.

The localization of the platelet glycoprotein GP Ib-IX complex (GP Ib␣, GP Ib␤, and GP IX) to membrane lipid domain, also known as glycosphingolipid-enriched membranes (GEMs or raft) lipid domain, is essential for the GP Ib-IX complex mediated platelet adhesion to von Willebrand factor (vWf) and subsequent platelet activation. To date, the mechanism for the complex association with the GEMs remains unclear. Although the palmitate modifications of GP Ib␤ and GP IX were thought to be critical for the complex presence in the GEMs, we found that the removal of the putative palmitoylation sites of GP Ib␤ and GP IX had no effects on the localization of the GP Ib-IX complex to the GEMs. Instead, the disruption of GP Ib␣ disulfide linkage with GP Ib␤ markedly decreased the amount of the GEM-associated GP Ib␣ without altering the GEM association of GP Ib␤ and GP IX. Furthermore, partial dissociation with the GEMs greatly inhibited GP Ib␣ interaction with vWf at high shear instead of in static condition or under low shear stress. Thus, for the first time, we demonstrated that GP Ib␤/GP IX mediates the disulfide-linked GP Ib␣ localization to the GEMs, which is critical for vWf interaction at high shear.

MicroRNAs are a class of small non-coding RNAs that bind to the three prime untranslated region (3′-UTR) of target mRNAs. They cause a cleavage or an inhibition of the translation of target mRNAs, thus regulating gene expression. Here, we employed three prediction tools to search for potential miRNA target sites in the 3′-UTR of the human platelet glycoprotein (GP) 1BA gene. A luciferase reporter assay shows that miR-10a and -10b sites are functional. When miR-10a or -10b mimics were transfected into the GP Ibβ/GP IX-expressing cells, along with a DNA construct harboring both the coding and 3′-UTR sequences of the human GP1BA gene, we found that they inhibit the transient expression of GP Ibα on the cell surface. When the miR-10a or -10b mimics were introduced into murine progenitor cells, upon megakaryocyte differentiation, we found that GP Ibα mRNA expression was markedly reduced, suggesting that a miRNA-induced mRNA degradation is at work. Thus, our study identifies GP Ibα as a novel target of miR-10a and -10b, suggesting that a drastic reduction in the levels of miR-10a and -10b in the late stage of megakaryopoiesis is required to allow the expression of human GP Ibα and the formation of the GP Ib-IX-V complex.

Localization of the platelet glycoprotein Ib-IX complex to the membrane lipid domain is essential for platelet adhesion to von Willebrand factor (vWf) and subsequent platelet activation in vitro. Yet, the in vivo importance of this localization has never been addressed. We recently found that the disulfide linkage between Ibα and Ibβ is critical for the association of Ibα with the glycosphingolipid-enriched membrane (GEM) domain, in this study, we established a transgenic mouse model expressing this mutant human Ibα that is also devoid of endogenous Ibα (HαSSMα−/−). Characterization of this model demonstrated a similar dissociation of Ibα from murine platelet GEMs to that expressed in CHO cells, which correlates well with the impaired adhesion of the transgenic platelets to vWf ex vivo and in vivo. Furthermore, we bred our transgenic mice into an atherosclerosis-prone background (HαSSMα−/−ApoE−/− and HαWTMα−/−ApoE−/−). We observed that atheroma formation was significantly inhibited in mutant mice where fewer platelet-bound CD11c+ leukocytes were circulating (CD45+/CD11c+/CD41+) and residing in atherosclerotic lesions (CD45+/CD11c+), suggesting that platelet-mediated adhesion and infiltration of CD11c+ leukocytes may be one of the mechanisms. These observations provide the first in vivo evidence showing that the membrane GEMs is physiologically and pathophysiologically critical in the function of the GP Ib-IX complex.

Background: Localization of the GP Ib-IX complex to the lipid domain is mediated by the ␤ and IX subunits. Results: Mutations in ␤/IX TMDs inhibit GP Ib-IX localization to the lipid domain. Conclusion: Localization of the GP Ib-IX complex to the lipid domain is mediated by ␤/IX TMDs. Significance: The ␤/IX TMDs may be a novel therapeutic target.