BackgroundDoxorubicin (DOX) is the most widely used chemotherapeutic agent that has multimodal cytotoxicity. The main cytotoxic actions of DOX occur in the nucleus. The emergence of drug-resistant cancer cells that have the ability to actively efflux DOX out of the nucleus, and the cytoplasm has led to the search for a more effective derivative of this drug.Materials and methodsWe created a new derivative of DOX that was derived via simple conjugation of the 3′ amino group of DOX to the dexamethasone molecule.ResultsDespite having a lower cytotoxic activity in MCF-7 cells, the conjugated product, DexDOX, exerted its actions in a manner that was different to that of DOX. DexDOX rapidly induced MCF-7 cell apoptosis without entering the nucleus. Further analysis showed that Dex-DOX increased cytosolic oxidative stress and did not interfere with the cell cycle. In addition, the conjugated product retained its cytotoxicity in multidrug resistance-1-overexpressing MCF-7 cells that had an approximately 16-fold higher resistance to DOX.ConclusionWe have synthesized a new derivative of DOX, which has the ability to overcome multidrug resistance-1-induced resistance. This molecule may have potential as a future chemotherapeutic agent.

BACKGROUND AND PURPOSERecombinant human erythropoietin (rHuEPO) is currently the mainstay of renal anaemia treatment. Recently, rHuEPO has been shown to provide pleiotrophic tissue protection in various pathological conditions. However, the benefits of rHuEPO beyond anaemia treatment are limited because it increases red blood cell mass. Carbamylated erythropoietin (CEPO) is the first rHuEPO derivative that lacks erythropoietic activity but retains tissue protection properties. Since carbamylation targets lysine residues on rHuEPo, we hypothesized that targeted lysine modifications of rHuEPO may result in a novel non-erythropoietic erythropoietin. EXPERIMENTAL APPROACHrHuEPO was subjected to various targeted lysine modifications. In vitro cytoprotection and apoptosis were evaluated using P19 and HEK293 cells. In vivo erythropoiesis was performed by administering the derivatives to animals for 2 weeks. Renoprotection was tested on an ischaemia/reperfusion (I/R) model. KEY RESULTSWe synthesized a novel derivative, a glutaraldehyde erythropoietin (GEPO). This construct abolished in vivo erythropoiesis. Biochemical characterization showed that GEPO was more electrostatically negative than rHuEPO. Immunoprecipitation experiments revealed that GEPO bound to the IL3RB/EPOR heterotrimeric receptor and ameliorated cellular apoptosis via the activation of Bcl-2. Notably, Bcl-2 activation was suppressed by the JAK2 inhibitor, tyrphostin AG490. In vivo experiments showed that GEPO also ameliorated kidney damage due to I/R injury both functionally and histologically.

BackgroundsHomozygous 32-bp deletion of the chemokine receptor 5 gene (CCR5) is associated with resistance to human immunodeficiency virus (HIV) infection, while heterozygosity delays HIV progression. Bone marrow transplantation (BMT) from a 32/32 donor has been shown to cure an HIV-infected patient. However, the rarity of this mutation and the safety risks associated with current BMT protocols are the major obstacles to this treatment. Zinc finger nuclease (ZFN) targeting is a powerful method for achieving genomic disruption at specific DNA sites of interest.ResultsTaking advantage of the self-renewal and plasticity properties of stem cells, in this study, we successfully generated isogenic and six-cell clones of bone marrow-derived mesenchymal stem cells that carry the stop codon of the CCR5 gene by using a ZFN-mediated homology-directed repair technique. These cells were expandable for more than 5 passages, and thus show potential to serve as an individual’s cell factory. When Oct4 was overexpressed, the mutated cells robustly converted to CD34+ progenitor cells.ConclusionWe here reported the novel approach on generation of patients own CD34 cells from high fidelity ZFN-mediated HDR MSC clones. We believe that our approach will be beneficial in future HIV treatment.

We reported a simple genome editing approach that can generate human immunodeficiency virus-1 (HIV) coreceptor defective cells, which may be useful for latent viral eradication treatment. Samples of bone marrow leftover after diagnostic procedures and crude bone marrow from aviremic HIV patients were subjected to zinc finger nuclease-mediated stop codon insertion into chemokine receptor 5 (CCR5) loci. Locked nucleic acid-based polymerase chain reaction was used to estimate the amount of insertion in the expandable CD34 cells. The results showed that about 0.5% of CD34 cells carried stop codon insertions in CCR5 loci. Cells edited using this simple protocol have the potential to be infused back into the bone marrow.

In humans, mutations in the β-globin gene (HBB) have two important clinical manifestations: β-thalassemia and sickle cell disease. The progress in genome editing and stem cell research may be relevant to the treatment of β-globin-related diseases. In this work, we employed zinc-finger nuclease (ZFN)-mediated gene integration of synthetic β-globin cDNA into HBB loci, thus correcting almost all β-globin mutations. The integration was achieved in both HEK 293 cells and isolated dental pulp stem cell (DPSCs). We also showed that DPSCs with an artificial gene knock-in were capable of generating stable six-cell clones and were expandable at least 10-fold; therefore, they may serve as a personalized stem cell factory. In addition, transfection with non-integrated pCAG-hOct4 and culturing in a conditioned medium converted the genome-edited DPSCs to CD34 HSC-like cells. We believe that this approach may be useful for the treatment of β-globin-related diseases, especially the severe form of β-thalassemia.