ABSTRACT
Direct delivery of therapeutic genes is a promising approach for treating cancers and other diseases. The current human viral vectors, however, suffer from several drawbacks, including poor cell-type specificity and difficult large-scale production. The M13 phage provides an alternative vehicle for gene therapy with engineerable specificity, but the low transduction efficiency seriously limits its translational application. In this work, we discovered important factors of cells and phages that greatly influence the phage transduction. The up-regulation of PrimPol or the down-regulation of DMBT1 in cells significantly enhanced the phage transduction efficiency. Furthermore, we found that the phage transduction efficiency was inversely correlated with the phage size. By carefully reconstructing the phage origin with the gene of interest, we designed "TransPhage" with a minimal length and maximal transduction efficiency. We showed that TransPhage successfully transduced the human cells with an excellent efficiency (up to 95%) comparable to or superior to that of the adeno-associated virus vectors. Moreover, we showed that TransPhage's tropism was specific to the cells that overexpress the target antigen, whereas adeno-associated viruses (AAVs) promiscuously infected many cell types. Using TransPhage as a gene therapy vehicle, we invented an NK-cell-mediated immunotherapy in which a membrane-bound fragment crystallizable region was introduced to cancer cells. We showed in vitro that the cancer cells expressing the membrane-bound fragment crystallizable (Fc) were effectively killed by CD16+ NK cells through an antibody-dependent cell-mediated cytotoxicity (ADCC)-like mechanism. In the xenograft mouse model, the administration of TransPhage carrying the membrane-bound Fc gene greatly suppressed tumor growth.
Subject(s)
Gene Transfer Techniques , Neoplasms , Humans , Mice , Animals , Genetic Vectors , Bacteriophage M13 , Genetic Therapy , Killer Cells, Natural , Neoplasms/genetics , Neoplasms/therapy , Calcium-Binding Proteins , DNA-Binding Proteins , Tumor Suppressor Proteins/genetics , DNA-Directed DNA Polymerase , DNA Primase/genetics , Multifunctional EnzymesABSTRACT
Melanotan II (MTII), a synthetic analogue of the alpha-melanocyte stimulating hormone (α-MSH), has been applied for skin tanning in humans. However, the carcinogenic consequence of topical MTII has been equivocal. This study aims to delineate the anti-neoplastic efficacy and mechanism of MTII using the B16-F10 melanoma model in vitro and in vivo. It was found that, despite a lack of influence on proliferation, MTII potently inhibited the migration, invasion, and colony-forming capability of melanoma cells. Moreover, topical MTII application significantly attenuated the tumor progression in mice bearing established melanoma. Histological analysis revealed that MTII therapy induced apoptosis while inhibiting the proliferation and neovaluarization in melanoma tissues. By immunoblot and immunohistochemical analysis, it was found that MTII dose-dependently increased the phosphatase and tensin homolog (PTEN) protein level while reducing PTEN phosphorylation, which resulted in the inhibition of AKT/nuclear factor kappa B (NFκB) signaling. Consistently, MTII treatment inhibited cyclooxygenase II (COX-2) expression and prostaglandin E2 (PGE2) production in melanoma cells. Finally, studies of antibody neutralization suggest that the melanocortin 1 receptor (MC1R) plays a critical role in MTII-induced PTEN upregulation and melanoma suppression. Together, these results indicate that MTII elicits PTEN upregulation via MC1R, thereby suppressing melanoma progression through downregulating COX-2/PGE2 signaling. Hence, topical MTII therapy may facilitate a novel therapeutic strategy against melanoma.
Subject(s)
Antineoplastic Agents/administration & dosage , Cyclooxygenase 2 Inhibitors/administration & dosage , Cyclooxygenase 2/metabolism , Melanoma, Experimental/drug therapy , PTEN Phosphohydrolase/metabolism , Peptides, Cyclic/administration & dosage , Signal Transduction/drug effects , alpha-MSH/analogs & derivatives , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclooxygenase 2/genetics , Dinoprostone/genetics , Dinoprostone/metabolism , Humans , Immunohistochemistry , Melanoma, Experimental/metabolism , Mice , Models, Biological , PTEN Phosphohydrolase/genetics , Phosphorylation , Receptor, Melanocortin, Type 1/genetics , Receptor, Melanocortin, Type 1/metabolism , Up-Regulation/drug effects , alpha-MSH/administration & dosageABSTRACT
The 2 1Π state of NaH has been observed up to the last bound vibrational level using pulsed optical-optical double resonance fluorescence depletion spectroscopy. A total of 20 rovibrational energy levels ( v = 2-4 and J = 1-9) were assigned to this electronic state by means of comparing the successive rovibrational spectra to the eigenvalues of the ab initio potential energy curve. The decrease of background fluorescence near the atomic asymptotic limit Na(3d) + H(1s) is an indication of reaching the dissociation limit of the NaH 2 1Π state. Unobserved rovibrational levels ( v = 0 and 1) are due to poor Franck-Condon overlap of 2 1Π â A 1Σ+ transition within the accessible rovibrational levels of intermediate A 1Σ+ state of this work.
