Search

OBJECTIVE: Repurposing represents a promising approach to safely accelerate the clinical application of therapeutics with anti-cancer activity. In this study, we examined whether inhibition of the anti-apoptotic Bcl-2 family proteins Bcl-2 and Bcl-xL enhances the biological effects of the repurposed CUSP9 regimen in an in vitro setting of glioblastoma. METHODS: We applied MTT assays to assess cellular proliferation. Annexin V/PI and TMRE staining were used to examine apoptosis. Western blotting, RT-PCR and specific knockdown experiments using siRNA were employed to examine molecular mechanisms of action. RESULTS: Bcl-2/Bcl-xL inhibition by the BH3 mimetic ABT263, yielded synergistic anti-proliferative effects across a wide panel of established and primary cultured glioblastoma cells when combined with CUSP9 which had been reduced to only one tenth of its original concentration (CUSP9 1/10). The combination treatment also led to enhanced apoptosis with loss of mitochondrial membrane potential and activation of caspases. On the molecular level, CUSP9 1/10 counteracted ABT263-mediated upregulation of Mcl-1. Silencing of Mcl-1 enhanced ABT263-mediated apoptosis, indicating that Mcl-1 is crucial for the induction of cell death conveyed by the combination treatment. Levels of Mcl-1 mRNA were not decreased following combination therapy, and co-treatment with cycloheximide showed reduced protein stability, pointing towards a post-translational mechanism of action. CONCLUSION: These data suggest that Bcl-2/Bcl-xL inhibition enhances the susceptibility of glioblastoma cells towards CUSP9, allowing dramatic dose reduction and potentially decreased toxicity when applied clinically. A clinical trial involving the original CUSP doses (CUSP9v3) is currently ongoing in our institution (NCT02770378). The Bcl-2/Bcl-xL inhibitor ABT263 is in clinical trials and might represent a valuable adjunct to the original CUSP. more...

Poly-[(trans-1,2-diaminocyclohexane) platinumj-carboxyamylose (“poly-plat”), 5-sulfosalicylato-trans -(1,2-diaminocyclohexane) platinum (SSP), and 4-hydroxy-∝-sulfonylphenylacetato (trans 1,2-diaminocyclohexane) platinum (II) (SAP) are second generation analogs of cisplatin (CDDP) with higher efficacy and potency than cisplatin. This is particularly true of “poly-plat” which contains 1/5 the platinum of CDDP. In order to understand the mechanism of action of these compounds, isolated murine peritoneal macrophages in culture medium were treated with “poly-plat”, SSP, or SAP (5 μg/ml) for 2 h. Drug containing medium was then replaced with fresh medium and the cells were allowed to incubate at 37° C (5% CO2) for 24 h. Supernatants were collected at 0.5, 1, 2, and 24 h post-treatment for immunocytochemical analysis. Confocal microscopy studies demonstrated an increase in the number of lysosomes in the treated macrophages, but only “poly-plat” and SSP treated macrophages were stimulated to form cytoplasmic extensions at 2 h and 24 h. more...

We describe a gas chromatographic method for measuring submicrogram quantities of dianhydrogalactitol, a promising antineoplastic agent (currently undergoing clinical trials in humans), in plasma. The drug is first extracted from blood plasma by saturating the aqueous phase with solid potassium carbonate and extracting with isopropanol/chloroform (9/1 by vol). It is then converted to the corresponding n-butaneboronic ester by reaction at room temperature with butaneboronic acid and chromatographed on an SE-30 (3%) column, with flame ionization detection. Practicality of the method for monitoring drug distribution was demonstrated by administering dianhydrogalactitol in therapeutic doses to a dog and monitoring its concentrations in blood for the next 2 h. more...