Dihydroxyquingdainone Induces Apoptosis in Leukaemia and Lymphoma Cells via the Mitochondrial Pathway in a Bcl-2- and Caspase-3-Dependent Manner and Overcomes Resistance to Cytostatic Drugs In Vitro.

Isatis tinctoria and its indigo dyes have already provided highly active anti-leukaemic lead compounds, with the focus mainly being on indirubin, whereas indigo itself is inactive. There are many more indigoids to find in this plant extract, for example, quingdainone, an indigoid derived from tryptanthrin. We present here a new synthesis of hitherto neglected substituted quingdainones, which is very necessary due to their poor solubility behaviour, and a structure-dependent anti-leukaemic activity study of a number of compounds. Substituted α-phenylaminoacrylic acid was synthesised by hydrogen sulfide extrusion from an analogue mercaptoacetic acid, available from the condensation of rhodanin and a substituted tryptanthrin. It is shown that just improving water solubility does not increase anti-leukaemic activity, since a quingdainone carboxylic acid is inactive compared to dihydroxyquingdainone. The most effective compound, dihydroxyquingdainone with an AC50 of 7.5 µmole, is further characterised, revealing its ability to overcome multidrug resistance in leukaemia cells (Nalm-6/BeKa) with p-glycoprotein expression.

GliPR1 knockdown by RNA interference exerts anti-glioma effects in vitro and in vivo.

INTRODUCTION: In human glioblastomas, glioma pathogenesis-related protein1 (GliPR1) is overexpressed and appears to be an oncoprotein. We investigated whether GliPR1 knockdown in glioma cells by RNA interference exerts anti-glioma effects. METHODS: Experiments used human glioblastoma cell lines transduced with GliPR1 shRNA (sh#301, sh#258). Transduction produced stringent doxycycline-dependent GliPR1 knockdown in clones (via lentiviral "all-in-one" TetOn-shRNA vector) or stable GliPR1 knockdown in polyclonal cells (via constitutive retroviral-shRNA vector). In vitro assessments included cellular proliferation and clonogenic survival. In vivo assessments in tumor-bearing nude mice included tumor growth and survival. RESULTS: Using doxycycline-dependent GliPR1 knockdown, shGliPR1-transduced U87-MG clones demonstrated reductions in cellular proliferation in the presence versus absence of doxycycline. Using stable GliPR1 knockdown, polyclonal shGliPR1-transduced U87-MG, A172, and U343-MG cells consistently showed decreased clonogenic survival and induced apoptosis (higher proportion of early apoptotic cells) compared to control shLuc-transduced cells. In tumor-bearing nude mice, using doxycycline-dependent GliPR1 knockdown, subcutaneous and cranial transplantation of the U87-MG clone 980-5 (transduced with GliPR1 sh#301) resulted in reduced subcutaneous tumor volume and cerebral tumor area in doxycycline-treated mice versus those left untreated. Using stable GliPR1 knockdown, nude mice cranially transplanted with polyclonal U87-MG cells transduced with GliPR1 sh#258 had significantly prolonged survival compared to mice cranially transplanted with control shLuc-transduced cells (41 versus 26 days; P < 0.001). CONCLUSION: GliPR1 knockdown in glioma cells decreased cellular proliferation, decreased clonogenic survival, and induced apoptosis in vitro, and reduced glioblastoma tumor growth and prolonged survival in vivo. These findings support that GliPR1 may have potential value as a therapeutic target.

Phenotypic, Morphological and Adhesive Differences of Human Hematopoietic Progenitor Cells Cultured on Murine versus Human Mesenchymal Stromal Cells.

Xenogenic transplantation models have been developed to study human hematopoiesis in immunocompromised murine recipients. They still have limitations and therefore it is important to delineate all players within the bone marrow that could account for species-specific differences. Here, we evaluated the proliferative capacity, morphological and physical characteristics of human CD34(+) hematopoietic stem and progenitor cells (HSPCs) after co-culture on murine or human bone marrow-derived mesenchymal stromal cells (MSCs). After seven days, human CD34(+)CD133(-) HSPCs expanded to similar extents on both feeder layers while cellular subsets comprising primitive CD34(+)CD133(+) and CD133(+)CD34(-) phenotypes are reduced fivefold on murine MSCs. The number of migrating HSPCs was also reduced on murine cells suggesting that MSC adhesion influences cellular polarization of HSPC. We used atomic force microscopy-based single-cell force spectroscopy to quantify their adhesive interactions. We found threefold higher detachment forces of human HSPCs from murine MSCs compared to human ones. This difference is related to the N-cadherin expression level on murine MSCs since its knockdown abolished their differential adhesion properties with human HSPCs. Our observations highlight phenotypic, morphological and adhesive differences of human HSPCs when cultured on murine or human MSCs, which raise some caution in data interpretation when xenogenic transplantation models are used.