Synergistic Cytotoxicity of a Toxin Targeting the Epidermal Growth Factor Receptor and the Glycosylated Triterpenoid SO1861 in Prostate Cancer.

Treatment of advanced prostate cancer lacks specificity and curative intent. Therefore, the need for new targeted therapeutic approaches is high. In the present study, we generated the new targeted toxin EGF-PE24mutΔREDLK binding to the epidermal growth factor receptor (EGFR) on the surface of prostate cancer cells. It consists of the human epidermal growth factor (EGF) as binding domain and a de-immunized variant of Pseudomonas Exotoxin A (PE), called PE24mutΔREDLK, as toxin domain. The toxin domain contains a deletion of the C-terminal KDEL-like motif REDLK to prevent its transport from sorting endosomes via the KDEL receptor mediated pathway into the cytosol, where it can inhibit cellular protein biosynthesis and induce apoptosis. Indeed, REDLK deletion resulted in a strong decrease in cytotoxicity of the targeted toxin in prostate cancer cells compared to the parental targeted toxin EGF-PE24mut. However, addition of the plant glycosylated triterpenoid SO1861, which is known to mediate the release of biomolecules from endolysosomal compartments into the cytosol, resulted in an up to almost 7,000-fold enhanced synergistic cytotoxicity. Moreover, combination of PE24mutΔREDLK with SO1861 led to a cytotoxicity that was even 16- to 300-fold enhanced compared to that of EGF-PE24mut. Endolysosomal entrapment of the non-toxic targeted toxin EGF-PE24mutΔREDLK followed by activation through enhanced endosomal escape therefore represents a new promising approach for the future treatment of advanced prostate cancer with high efficacy and diminished side effects.

ZBTB18 inhibits SREBP-dependent lipid synthesis by halting CTBPs and LSD1 activity in glioblastoma.

Enhanced fatty acid synthesis is a hallmark of tumors, including glioblastoma. SREBF1/2 regulate the expression of enzymes involved in fatty acid and cholesterol synthesis. Yet, little is known about the precise mechanism regulating SREBP gene expression in glioblastoma. Here, we show that a novel interaction between the co-activator/co-repressor CTBP and the tumor suppressor ZBTB18 regulates the expression of SREBP genes. In line with our findings, metabolic assays and glucose tracing analysis confirm the reduction in several phospholipid species upon ZBTB18 expression. Our study identifies CTBP1/2 and LSD1 as co-activators of SREBP genes and indicates that the functional activity of the CTBP-LSD1 complex is altered by ZBTB18. ZBTB18 binding to the SREBP gene promoters is associated with reduced LSD1 demethylase activity of H3K4me2 and H3K9me2 marks. Concomitantly, the interaction between LSD1, CTBP, and ZNF217 is increased, suggesting that ZBTB18 promotes LSD1 scaffolding function. Our results outline a new epigenetic mechanism enrolled by ZBTB18 and its co-factors to regulate fatty acid synthesis that could be targeted to treat glioblastoma patients.

Epidermal Growth Factor Based Targeted Toxin for the Treatment of Bladder Cancer.

BACKGROUND/AIM: Reports on over-expression of the epidermal growth factor receptor (EGFR) in bladder cancer and its function in tumorigenesis have suggested to target this antigen. MATERIALS AND METHODS: We generated the targeted toxin EGF-PE40 consisting of the human epidermal growth factor (EGF) as the binding domain and PE40, a truncated version of Pseudomonas Exotoxin A, as the toxin domain. EGF-PE40 was tested on EGFR-expressing bladder cancer cells in view of binding via flow cytometry, and cytotoxicity via WST viability assay. Induction of apoptosis was examined by western blot. RESULTS: The targeted toxin specifically triggered cytotoxicity in the bladder cancer cells with 50% inhibitory concentration (IC50) values in the low nanomolar or picomolar range, and was about 1,250- to 1,500-fold more cytotoxic than the EGFR inhibitor erlotinib. Cytotoxicity of EGF-PE40 was based on the induction of apoptosis. CONCLUSION: EGF-PE40 represents a promising candidate for the future treatment of bladder cancer.

Pseudomonas Exotoxin A Based Toxins Targeting Epidermal Growth Factor Receptor for the Treatment of Prostate Cancer.

The epidermal growth factor receptor (EGFR) was found to be a valuable target on prostate cancer (PCa) cells. However, EGFR inhibitors mostly failed in clinical studies with patients suffering from PCa. We therefore tested the targeted toxins EGF-PE40 and EGF-PE24mut consisting of the natural ligand EGF as binding domain and PE40, the natural toxin domain of Pseudomonas Exotoxin A, or PE24mut, the de-immunized variant thereof, as toxin domains. Both targeted toxins were expressed in the periplasm of E.coli and evoked an inhibition of protein biosynthesis in EGFR-expressing PCa cells. Concentration- and time-dependent killing of PCa cells was found with IC50 values after 48 and 72 h in the low nanomolar or picomolar range based on the induction of apoptosis. EGF-PE24mut was found to be about 11- to 120-fold less toxic than EGF-PE40. Both targeted toxins were more than 600 to 140,000-fold more cytotoxic than the EGFR inhibitor erlotinib. Due to their high and specific cytotoxicity, the EGF-based targeted toxins EGF-PE40 and EGF-PE24mut represent promising candidates for the future treatment of PCa.

Activity of plasma membrane beta-galactosidase and beta-glucosidase.

Human fibroblasts produce ceramide from sialyllactosylceramide on the plasma membranes. Sialidase Neu3 is known to be plasma membrane associated, while only indirect data suggest the plasma membrane association of beta-galactosidase and beta-glucosidase. To determine the presence of beta-galactosidase and beta-glucosidase on plasma membrane, cells were submitted to cell surface biotinylation. Biotinylated proteins were purified by affinity column and analyzed for enzymatic activities on artificial substrates. Both enzyme activities were found associated with the cell surface and were up-regulated in Neu3 overexpressing cells. These enzymes were capable to act on both artificial and natural substrates without any addition of activator proteins or detergents and displayed a trans activity in living cells.