Syndecan-1 is a heparan sulfate proteoglycan (HSPG) commonly upregulated in AIDS-related B lymphoid malignancies. Tat is the main HIV-1 transactivating factor that has a major role in the ...pathogenesis of AIDS-related lymphomas (ARL) by engaging heparan sulfate proteoglycans (HSPGs), chemokine receptors and integrins at the lymphoid cell (LC) surface. Here B-lymphoid Namalwa cell clones that do not express or overexpress syndecan-1 (EV-Ncs and SYN-Ncs, respectively) were compared for their responsiveness with Tat: in the absence of syndecan-1, Tat induces a limited EV-Nc migration via C-X-C motif chemokine receptor 4 (CXCR4), G-proteins and Rac. Syndecan-1 overexpression increases SYN-Nc responsiveness to Tat and makes this response independent from CXCR4 and G-protein and dependent instead on pp60src phosphorylation. Tat-induced SYN-Nc migration and pp60src phosphorylation require the engagement of alpha sub(v) beta sub(3) integrin and consequent pp125FAK phosphorylation. This complex set of Tat-driven activations is orchestrated by the direct interaction of syndecan-1 with pp60src and its simultaneous coupling with alpha sub(v) beta sub(3). The Tat/syndecan-1/ alpha sub(v) beta sub(3 ) interplay is retained in vivo and is shared also by other syndecan-1 super(+) B-LCs, including BJAB cells, whose responsiveness to Tat is inhibited by syndecan-1 knockdown. In conclusion, overexpression of syndecan-1 confers to B-LCs an increased capacity to migrate in response to Tat, owing to a switch from a CXCR4/G-protein/Rac to a syndecan-1/ alpha sub(v) beta sub(3)/pp60src/pp125FAK signal transduction pathway that depends on the formation of a complex in which syndecan-1 interacts with Tat via its HS-chains, with alpha sub(v) beta sub(3) via its core protein ectodomain and with pp60src via its intracellular tail. These findings have implications in ARL progression and may help in identifying new therapeutical targets for the treatment of AIDS-associated neoplasia.
Abstract
Transfection efficiency and toxicity concerns remain a challenge for gene therapy. Nanoparticle-based gene delivery technique potentially overcomes these concerns and may be applicable to ...cancer gene therapy. Cell penetrating peptides (CPPs) have been broadly investigated to improve the transfection of genetic material (e.g., pDNA and siRNA). Our previous study demonstrated that an apoptosis inducer, angiotensin II type 2 receptor plasmid DNA (pAT2R) encapsulated in a modified HIV-1 TAT peptide (dTAT-pAT2R), significantly attenuated the growth of Lewis lung carcinoma (LLC) allograft in mouse lungs (Kawabata et al., Cancer Res, 2012). Here, we report a newly synthesized polylysine CPP (K9 peptide)-based gene therapy for lung cancer treatment. The pAT2R and K9 peptide (K9-pAT2R) complexes were condensed using calcium chloride (K9-pAT2R-Ca2+). The resulting complexes were small (∼150 nm) and showed high levels of gene expression in vitro. This simple non-viral formulation approach showed negligible cytotoxicity in several different human and mouse cell lines (human cervix, breast, kidney, and human and mouse lung cell lines). Additionally, this K9-pDNA-Ca2+ complex demonstrated cancer targeted gene delivery when administered via intravenous (IV) injection or intratracheal (IT) spray into LLC orthotopic allograft-bearing mice. Average lung weights (mg) of the K9-pAT2R-Ca2+ IT (190.6±48.3) and the K9-pAT2R-Ca2+ IV (201.6±67.0) treated groups were significantly smaller than that of the control PBS group (325.7±69.4, P<0.05). In histological examination of tumors in H&E stained lung, average numbers of tumor nodules in the lungs in the K9-pAT2R-Ca2+ IT (6.0±4.2) and the K9-pAT2R-Ca2+ IV (4.6±3.4) groups were also significantly smaller than that of the control PBS group (17.8±6.0, P<0.01). This tumor attenuating effect was not observed in K9-pLUC-Ca2+ IT and K9-pLUC-Ca2+ IV treatment, thus it is suggested that LLC tumor growth was attenuated by apoptosis inducer gene, pAT2R, delivery. Immunohistochemical analysis confirmed that the complex effectively delivered pAT2R to the cancer cells, where it was expressed mainly in cancer cells along with bronchial epithelial cells. A single administration of these complexes markedly attenuated lung cancer growth offering preclinical proof of concept for a novel non-viral gene delivery method exhibiting effective lung tumor gene therapy via either IV or IT administration. This work is supported in part by Savara Pharmaceuticals (CB and MT), Faculty of Pharmacy of King Abdulaziz University, Jeddah, Saudi Arabia (NAA), University of Kansas Macromolecule and Vaccine Stabilization Center (CB), Kansas State University Johnson Cancer Research Center (MT), NIH grants U43 CA165462 (MT), P20 GM103418 (MT), and Kansas Bioscience Authority Collaborative Cancer Research grant (MT).
Citation Format: Susumu Ishiguro, Nabil A. Alhakamy, Deepthi Uppalapati, Cory J. Berkland, Masaaki Tamura. Apoptosis inducer gene delivery by polylysine-calcium complexes attenuates mouse lung carcinoma allograft growth after intravenous injection or intratracheal spray. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3751.
Abstract
Extracellular vesicles (EVs) hold tremendous potential as drug delivery carriers for therapeutic nucleic acids such as microRNA (miRNA) due to their natural composition and ability to be ...engineered to contain targeting peptides on their surface. We have engineered HEK293T cells to secrete EVs by overexpressing Lamp-2A protein containing liver cancer targeting peptide PC94 and therapeutic pre-miR-199. The novel feature of this system is that both the cargo and the microvesicles are synthesized by the same cells, thus abrogating the need for loading synthetic oligonucleotides into EVs. The pre-miR-199a loop region was modified such that it resembled the HIV-1 transactivation response (TAR) RNA which was engineered into the Lamp2A intron. Correct splicing of the intron portion and processing of the mature miRNA was evaluated after its transfection into HEK293T cells. Computational modeling was used to study the interaction between the modified pre-miR-199a loop (TAR RNA) and the TAT peptide. This study exhibited a stable interaction between the two and also showed that the peptide is not bound to the stem portion of the RNA loop, permitting the insertion of any pre-miRNA sequence. EMSA gel shift, rDICER processing and luciferase assays were performed to study the correct binding, processing and functionality of the modified sequence. These results show that the modified loop is actively involved in binding with the TAT peptide and it enables the modified miRNA to be loaded in the microvesicles. In an effort to assess the loading of the therapeutic miR-199a-3p relative to other endogenous miRNAs contained within the EVs, we performed small RNA sequencing on RNA isolated from both the producing cells and purified EVs. Triplicate samples of RNA isolated from 3 different HEK293T producing cells were sequenced: wild type HEK293T and those stably transfected with the empty vector (empty) or the TAT/TAR pre-miR-199a (full). The expression of the miRNAs were ranked from the RNA sequenced in both the cells and EVs. Producing cells engineered to express miR-199a-3p containing the TAT/TAR loading motif ranked third when comparing the ratio of miRNAs loaded in the EVs from the full to empty cells. Interestingly, certain mature miRNAs were preferentially loaded into the EVs, including miR-451a, miR-122 and miR-1246. As reported by Villarroya-Beltri, et al., (Nature Comm., 2013) in human peripheral blood mononuclear cells, all three mature miRNAs contained 4 nucleotide “exo motifs” in their 3’ end that likely caused preferential loading into HEK293T EVs. Our data demonstrate that stably transfecting HEK293T cells with vectors expressing therapeutic miRNAs dramatically increases their loading into EVs. These mature miRNAs may be further engineered to contain exo motifs that could conceivably enhance their loading into EV drug carrier systems.
Citation Format: Dhruvitkumar S. Sutaria, Jinmai Jiang, Ola A. Elgamal, Ana-Clara P. Azevedo-Pouly, Ryan E. Pavlovicz, Chenglong Li, Mitch A. Phelps, Thomas D. Schmittgen. Engineering of hairpin loop enhances the loading of endogenously expressed pre-miRNA into extracellular vesicles. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2068.
Decitabine, which reverses hypermethylation of the p15 super(INK4B) gene in vitro, has been used to relieve cytopenias and blast excess in over 50% of patients with high-risk myelodysplastic syndrome ...(MDS). In this study, heme oxygenase-1 (HO-1) was overexpressed in MDS cell line SKM-1, which was closely related to resistance to decitabine-induced apoptosis. We aimed to further investigate the role of HO-1 in apoptosis induced by low-dose decitabine in SKM-1 cells. Upregulation of HO-1 by transfecting it into SKM-1 cells with lentivirus vector promoted cell proliferation and protected them against apoptosis. In contrast, downregulation of HO-1 enhanced decitabine-induced apoptosis but reduced accumulation of the S phase in cell cycle. To explore the mechanism, the expressions of cell cycle-related proteins were detected after the cells were treated by decitabine in each group. p15 super(INK4B) and CDK4 were overexpressed in SKM-1 cells in which HO-1 was inhibited, and the expression-depending apoptosis was related to the caspase-3 pathway. Even though HO-1 was silenced, the apoptotic rate never increased as the caspase-3 pathway was blocked. It is well known that p15 super(INK4B) dominantly regulates the S phase of the cell cycle. p15 super(INK4B) was herein demethylated more evidently in the group of SKM-1 cells in which HO-1 was downregulated, as well as in the mononuclear cells of patients suffering from MDS. In the case of poor prognosis, the mRNA level of HO-1 was raised. In conclusion, overexpression of HO-1 indicated resistance to demethylation of p15 super(INK4B) induced by decitabine.
Background: Cell-based insulin therapies can potentially improve glycemic regulation in insulin-dependent diabetic patients. Enteroendocrine cells engineered to secrete recombinant insulin have ...exhibited glycemic efficacy, but have been primarily studied as uncontrollable growth systems in immune incompetent mice. Furthermore, reports suggest that suboptimal insulin secretion remains a barrier to expanded application. Methods: Genetic and tissue engineering strategies were applied to improve recombinant insulin secretion from intestinal L-cells on both a per-cell and per-graft basis. Transduction of insulin-expressing GLUTag L-cells with lentivirus carrying an additional human insulin gene-enhanced secretion twofold. We infected cells with lentivirus expressing a luciferase reporter gene to track cell survival in vivo. To provide a growth-controlled and immune protective environment without affecting secretory capacity, cells were microencapsulated in barium alginate. Approximately 9x10 super(7) microencapsulated cells were injected intraperitoneally in immune competent streptozotocin-induced diabetic mice for therapeutic efficacy evaluation. Results: Graft insulin secretion was increased to 16 to 24 mU insulin per day. Transient normoglycemia was achieved in treated mice two days after transplantation, and endogenous insulin was sufficient to sustain body weights of treated mice receiving minimal supplementation. Conclusion: Glycemic efficacy of a bioartificial pancreas based on insulin-secreting enteroendocrine cells is insufficient as a standalone therapy, despite enhancement of graft insulin secretion capacity. Supplemental strategies to alleviate secretion limitations should be pursued.
Background The SCN5A-encoded voltage-gated sodium channel Na sub(V)1.5 is expressed in human jejunum and colon. Mutations in Na sub(V)1.5 are associated with gastrointestinal motility disorders. The ...rat gastrointestinal tract expresses voltage-gated sodium channels, but their molecular identity and role in rat gastrointestinal electrophysiology are unknown. Methods The presence and distribution of Scn5a-encoded Na sub(V)1.5 was examined by PCR, Western blotting and immunohistochemistry in rat jejunum. Freshly dissociated smooth muscle cells were examined by whole cell electrophysiology. Zinc finger nuclease was used to target Scn5a in rats. Lentiviral-mediated transduction with shRNA was used to target Scn5a in rat jejunum smooth muscle organotypic cultures. Organotypic cultures were examined by sharp electrode electrophysiology and RT-PCR. Key Results We found Na sub(V)1.5 in rat jejunum and colon smooth muscle by Western blot. Immunohistochemistry using two other antibodies of different portions of Na sub(V)1.5 revealed the presence of the ion channel in rat jejunum. Whole cell voltage-clamp in dissociated smooth muscle cells from rat jejunum showed fast activating and inactivating voltage-dependent inward current that was eliminated by Na super(+) replacement by NMDG super(+). Constitutive rat Scn5a knockout resulted in death in utero. Na sub(V)1.5 shRNA delivered by lentivirus into rat jejunum smooth muscle organotypic culture resulted in 57% loss of Scn5a mRNA and several significant changes in slow waves, namely 40% decrease in peak amplitude, 30% decrease in half-width, and 7 mV hyperpolarization of the membrane potential at peak amplitude. Conclusions & Inferences Scn5a-encoded Na sub(V)1.5 is expressed in rat gastrointestinal smooth muscle and it contributes to smooth muscle electrophysiology. The voltage-gated Na super(+) channel Na sub(V)1.5, encoded by the gene SCN5A, is expressed in human GI tract and is associated with functional GI disorders. Here we found that Scn5a is also expressed and functional in rat jejunal smooth muscle; knockdown of Scn5a expression with shRNA reduced the amplitude and half-widths of jejunal smooth muscle electrical slow waves. This study demonstrates a role for the voltage-gated mechanosensitive ion channel Na sub(V)1.5 in normal gastrointestinal motility in the rat and establishes the rat as an important model for study of the Scn5a-encoded Na sub(V)1.5 in gastrointestinal physiology and pathophysiology.
Small ruminant lentiviruses include viruses with diverse genotypes that frequently cross the species barrier between sheep and goats and that display a great genetic variability. These ...characteristics stress the need to consider the whole host range and to perform local surveillance of the viruses to opt for optimum diagnostic tests, in order to establish control programes. In the absence of effective vaccines, a comprehensive knowledge of the epidemiology of these infections is of major importance to limit their spread. This article intends to cover these aspects and to summarise information related to characteristics of the viruses, pathogenesis of the infection and description of the various syndromes produced, as well as the diagnostic tools available, the mechanisms involved in transmission of the pathogens and, finally, the control strategies that have been designed until now, with remarks on the drawbacks and the advantages of each one. We conclude that there are many variables influencing the expected cost and benefits of control programs that must be evaluated, in order to put into practice measures that might lead to control of these infections.
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