Professional antigen-presenting cells, such as dendritic cells (DCs) and macrophages, are target cells for gene therapy of infectious disease and cancer. However, transduction of DCs and macrophages has proved difficult by most currently available gene transfer methods. Several recent studies have shown that lentiviral vector systems can efficiently transduce many nondividing and differentiated cell types. In this study, we examined the gene transfer to DCs and macrophages using a lentiviral vector system. Human DCs were propagated from the adherent fraction of peripheral blood mononuclear cells (PBMCs) by culture in medium containing GM-CSF, IL-4, and TNF-alpha. Human macrophages were propagated from adherent PBMCs in medium containing GM-CSF. High titers of a replication-defective vesicular stomatitis virus glycoprotein G pseudotyped HIV-1-based vector encoding the enhanced yellow fluorescent protein were produced. In immature DCs (culture days 3 and 5), transduction efficiencies of 25 to 35% were achieved at a multiplicity of infection of 100. However, the transduction efficiency was decreased in more mature DCs (culture day 8 or later). Furthermore, monocyte-derived macrophages were also transduced by the lentiviral vector system. In addition, Alu-LTR PCR demonstrated the integration of the HIV-1 provirus into the cellular genome of the transduced DCs and macrophages. Allogeneic mixed lymphocyte reactions revealed similar antigen-presenting functions of untransduced and lentivirally transduced DCs. Thus, the results of this study demonstrate that both PBMC-derived DCs and macrophages can be transduced by lentiviral vectors.
We have previously reported that leukemic dendritic cells (DC) can be generated ex vivo from myelomonocytic precursors in chronic myelogenous leukemia. In this study we report the generation of DC from acute myelogenous leukemia (AML) cells and their potent ability to stimulate leukemia-specific cytolytic activity in autologous lymphocytes. DC were generated in vitro using granulocyte-macrophage colony-stimulating factor +interleukin-4 in combination with either tumor necrosis factor- or CD40 ligand (CD40L). Cells from 19 AML patients with a variety of chromosomal abnormalities were studied for their ability to generate DC. In all but 1 case, cells with the morphology, phenotypic characteristics, and T-cell stimulatory properties of DC could be generated. These cells expressed high levels of major histocompatibility complex class I and class II antigens as well as the costimulatory molecules B7-2 and ICAM-1. In three cases these cells were determined to be of leukemic origin by fluorescence in situ hybridization for chromosomal abnormalities or Western blotting for the inv(16) fusion gene product. Autologous lymphocytes cocultured with AML-derived DC (DC-AL) were able to lyse autologous leukemia targets, whereas little cytotoxicity was noted against autologous, normal cells obtained from the patients during remission. We conclude that leukemia derived DC may be useful for immunotherapy of many AML patients.
Overall, these findings indicate that MR is associated with a reduction in prostate cancer development in the TRAMP model and supports the continued development of MR as a potential PCa prevention strategy.
BackgroundDietary methionine restriction (MR) improves healthspan in part by reducing adiposity and by increasing insulin sensitivity in rodent models. The purpose of this study was to determine whether MR inhibits tumor progression in breast cancer xenograft model and breast cancer cell lines.MethodsAthymic nude mice were injected with MCF10AT1 cells in Matrigel® and fed a diet containing either 0.86 % methionine (control fed, CF), or 0.12 % methionine (MR) for 12 weeks. Plasma amino acid concentrations were measured by UPLC, and proliferation and apoptosis were examined using RT-PCR, immunohistochemistry, and Cell Titer 96® Aqueous One Solution Cell Proliferation assay.ResultsMice on the MR diet had reduced body weight and decreased adiposity. They also had smaller tumors when compared to the mice bearing tumors on the CF diet. Plasma concentrations of the sulfur amino acids (methionine, cysteine, and taurine) were reduced, whereas ornithine, serine, and glutamate acid were increased in mice on the MR diet. MR mice exhibited decreased proliferation and increased apoptosis in cells that comprise the mammary glands and tumors of mice. Elevated expression of P21 occurred in both MCF10AT1-derived tumor tissue and endogenously in mammary gland tissue of MR mice. Breast cancer cell lines MCF10A and MDA-MB-231 grown in methionine-restricted cysteine-depleted media for 24 h also up-regulated P21 and P27 gene expression, and MDA-MB-231 cells had decreased proliferation.ConclusionMR hinders cancer progression by increasing cell cycle inhibitors that halt cell cycle progression. The application of MR in a clinical setting may provide a delay in the progression of cancer, which would provide more time for conventional cancer therapies to be effective.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2367-1) contains supplementary material, which is available to authorized users.
We have previously reported that leukemic dendritic cells (DC) can be generated ex vivo from myelomonocytic precursors in chronic myelogenous leukemia. In this study we report the generation of DC from acute myelogenous leukemia (AML) cells and their potent ability to stimulate leukemia-specific cytolytic activity in autologous lymphocytes. DC were generated in vitro using granulocyte-macrophage colony-stimulating factor +interleukin-4 in combination with either tumor necrosis factor- or CD40 ligand (CD40L). Cells from 19 AML patients with a variety of chromosomal abnormalities were studied for their ability to generate DC. In all but 1 case, cells with the morphology, phenotypic characteristics, and T-cell stimulatory properties of DC could be generated. These cells expressed high levels of major histocompatibility complex class I and class II antigens as well as the costimulatory molecules B7-2 and ICAM-1. In three cases these cells were determined to be of leukemic origin by fluorescence in situ hybridization for chromosomal abnormalities or Western blotting for the inv(16) fusion gene product. Autologous lymphocytes cocultured with AML-derived DC (DC-AL) were able to lyse autologous leukemia targets, whereas little cytotoxicity was noted against autologous, normal cells obtained from the patients during remission. We conclude that leukemia derived DC may be useful for immunotherapy of many AML patients.
Collectively, these preliminary findings support the effectiveness of daily liposomal GSH administration at elevating stores of GSH and impacting the immune function and levels of oxidative stress.
Hormone refractory prostate cancer poses a huge problem and standard of care chemotherapy has not been very successful. We used a novel strategy to combine properties of 2 well-studied class of compounds (selenium and COX-2 inhibitor) and examined the resulting effectiveness against prostate cancer. Bearing in mind that sulfonamide moiety and pyrazole ring is important for the proapoptotic activity of Celecoxib, we synthesized a selenium derivative, Selenocoxib-1, by modifying Celecoxib at position 3 of the pyrazole ring. The PAIII cells derived from a metastatic prostate tumor that arose spontaneously in a Lobund-Wistar (LW) rat were used to examine the efficacy of Selenocoxib-1 in vitro. In addition, human metastatic prostate cancer cells, PC-3M, were tested for antitumor effect of Selenocoxib-1 in vitro. The IC 50 in PAIII and PC-3M cells for Selenocoxib-1 was about 5 lM, while for Celecoxib it was more than 20 lM. Selenocoxib-1 induced apoptosis in a dosedependent manner in the PAIII cells. COX-2 expression in PAIII cells was downregulated by Celecoxib and Selenocoxib-1 at 20 and 5 lM, respectively; the COX-2 activity was, however, not affected by Selenocoxib-1. Following treatment with Selenocoxib-1, PAIII cells resulted in dose-dependent decrease in HIF-1a, p-AKT and Bcl-2 levels. A reduction in weights was observed in subcutaneous tumors produced by PAIII cells pretreated with Selenocoxib-1 as compared to Celecoxib in LW rats. Further, following 1 week Selenocoxib-1 treatment of PAIII tumors resulted in significant reduction of tumor weights. This study demonstrates that Selenocoxib-1 is more effective against prostate cancer than Celecoxib.
Alternative strategies are needed to control growth of advanced and hormone refractory prostate cancer. In this regard, we investigated the efficacy of methylseleninic acid (MSeA), a penultimate precursor to the highly reactive selenium metabolite, methylselenol, to inhibit growth of invasive and hormone refractory rat (PAIII) and human (PC-3 and PC-3M) prostate cancer cells. Our results demonstrate that MSeA inhibits PAIII cell growth in vitro as well as reduces weights of tumors generated by PAIII cells treated ex vivo. A significant reduction in the number of metastatic lung foci by MSeA treatment was also noted in Lobund-Wistar rats. The PAIII cells along with PC-3, DU145 and PC-3M cells undergo apoptosis after MSeA treatments in both normoxia and hypoxia. Treatment of metastatic rat and human prostate cancer cell lines with MSeA decreased hypoxiainducible factor-1a (HIF-1a) levels in a dose-dependent manner. Additionally, HIF-1a transcription activity both in normoxic and hypoxic conditions is reduced after MSeA treatment of prostate cancer cells. Furthermore, VEGF and GLUT1, downstream targets of HIF-1a, were also reduced in prostate cancer cells after MSeA treatment. Our study illustrates the efficacy of MSeA in controlling growth of hormone refractory prostate cancer by downregulating HIF-1a, which is possibly occurring through stabilization or increase in prolyl hydroxylase activity.In men with advanced prostate cancer, hormone therapy is accepted as the initial treatment of choice and produces good responses in most patients. However, many patients relapse and become resistant to further hormone manipulation. Radical prostatectomy is recommended for treatment of localized prostate cancer. Unfortunately, local recurrences occur in up to one-third of patients by 5 years after surgery.1 Furthermore, a combination of hormone therapy with radical prostatectomy in patients with localized disease resulted in 5-year disease-free survival of 64%. 2Effective radiation therapy requires the presence of oxygen.3 As a result of rapid mitotic growth and clonal expansion, tumor cells are usually forced away from vessels beyond effective diffusion distance of oxygen and thrive remarkably well within a hypoxic environment. This tumor hypoxia may lead to resistance to radiation and chemotherapy.4 Hypoxia within solid tumors induces hypoxia-inducible factor (HIF)-1a, 5 and its elevation in prostate cancer cells may attribute to enhanced growth rates, survival and increased metastatic potential. 6HIF-1 is a heterodimer composed of an inducible a-subunit that confers the sensitivity to oxygen and a constitutively expressed b-subunit, aryl hydrocarbon receptor nuclear translocator.7 Under normoxic conditions after a post-translational hydroxylation by prolyl hydroxylase (PHD), HIF-1a interacts with tumor suppressor von-Hippel-Lindau protein and is rapidly degraded via ubiquitin-dependent proteasome pathway. 8 Hypoxia induces a rapid increase in HIF-1a protein stability and transcriptional activity, 9 resulting in the activat...
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