Genetic manipulation of adhesion GPCR CD97/ADGRE5 modulates invasion in patient-derived glioma stem cells.

INTRODUCTION: Effective glioblastoma (GBM) treatment is limited by high invasiveness and heterogeneity. Current therapies target proliferating Glioma Stem Cell (GSC) subpopulations while sparing invading GSCs, which eventually engender tumor recurrence after treatment. Surface receptor CD97/ADRGE5 is associated with invasion and metastasis regulation in non-CNS cancers. Although CD97 expression level positively correlates with poor GBM patient prognosis, its role in this tumor is unclear. METHODS: Here, we examined CD97 function in primary patient-derived GSCs (pdGSCs) obtained from five GBM tumors, belonging to three major genetic subtypes. We compared endogenous CD97 levels in pdGSCs to the corresponding patient MRI's radiographic invasion pattern aggressiveness. We manipulated CD97 levels in these pdGSCs by knockdown and overexpression and analyzed: (i) stem and subtype marker expression, (ii) in vitro invasive properties, and (iii) cell proliferation. RESULTS: Endogenous CD97 levels in pdGSCs positively correlated with radiographic invasion pattern aggressiveness on patient MRIs, and in vitro invasion rate. CD97 knockdown decreased pdGSC invasion rates in vitro, most markedly in mesenchymal subtype pdGSCs, as well as classical subtype pdGSCs. Invasion rates in vitro increased after CD97 overexpression predominately in proneural subtype pdGSCs. In the pdGSC line with the lowest endogenous CD97 level, CD97 overexpression increased the proliferation rate almost threefold. CONCLUSIONS: For the first time in pdGSCs, we have shown that CD97 knockdown decreases and overexpression increases invasion rate in vitro. The effect of CD97 on invasion is pdGSC subtype-dependent. Future in vivo and mechanistic studies are needed for validation. Pharmacologic CD97 inhibitors should be identified, as they may potentially therapeutically diminish GBM invasion.

PBK/TOPK interacts with the DBD domain of tumor suppressor p53 and modulates expression of transcriptional targets including p21.

PBK/TOPK (PDZ-binding kinase, T-LAK-cell-originated protein kinase) is a serine-threonine kinase that is overexpressed in a variety of tumor cells but its role in oncogenesis remains unclear. Here we show, by co-immunoprecipitation experiments and yeast two-hybrid analysis, that PBK/TOPK physically interacts with the tumor suppressor p53 through its DNA-binding (DBD) domain in HCT116 colorectal carcinoma cells that express wild-type p53. PBK also binds to p53 mutants carrying five common point mutations in the DBD domain. The PBK-p53 interaction appears to downmodulate p53 transactivation function as indicated by PBK/TOPK knockdown experiments, which show upregulated expression of the key p53 target gene and cyclin-dependent kinase inhibitor p21 in HCT116 cells, particularly after genotoxic damage from doxorubicin. Furthermore, stable PBK/TOPK knockdown cell lines (derived from HCT116 and MCF-7 cells) showed increased apoptosis, G(2)/M arrest and slower growth as compared to stable empty vector-transfected control cell lines. Gene microarray studies identified additional p53 target genes involved in apoptosis or cell cycling, which were differentially regulated by PBK knockdown. Together, these data suggest that increased levels of PBK/TOPK may contribute to tumor cell development and progression through suppression of p53 function and consequent reductions in the cell-cycle regulatory proteins such as p21. PBK/TOPK may therefore be a valid target for antineoplastic kinase inhibitors to sensitize tumor cells to chemotherapy-induced apoptosis and growth suppression.

Antimicrobial activity of clavines.

The antimicrobial activity of two clavine-type ergot alkaloids (agroclavine, festuclavine), and 16 derivatives against four human pathogenic bacteria and Candida albicans was determined. It is shown that all ergolines tested with one exception exhibit antibacterial properties against one to four bacteria species. The most active compounds are 6-allyl-6-norfestuclavine (minimal inhibitory concentration (MIC) 30 micrograms/ml against Staphylococcus aureus), 1-propyl-6-norfestuclavine (MIC 60 micrograms/ml against Escherichia coli), 6-cyano-6-norfestuclavine (MIC 250 micrograms/ml against Pseudomonas aeruginosa), and 1-methyl-agroclavine (MIC 200 micrograms/ml against Proteus vulgaris). 1-Allyl-6-norfestuclavine and 1-propyl-6-norfestuclavine showed a broad action spectrum: the growth of all four bacteria species and of Candida albicans was inhibited. The most effective antifungal compounds are 1-propyl-6-norfestuclavine and 6-cyano-6-norfestuclavine (MIC 250 micrograms/ml). Three alkaloids of different structure (codeine, emetine, quinine) are inactive up to 500 micrograms/ml against the bacteria species and C. albicans. The acute toxicity (mouse) is remarkably diminished by the modifications of the natural clavines.

Clavines. New antibiotics with cytostatic activity.

The cytostatic potentials of ten ergolines were determined in the L5178y mouse lymphoma cell system; six of them belong to the clavines (agroclavine, 1-propyl-agroclavine, 1-propyl-festuclavine, 1-allyl-festuclavine, 6-cyano-6-nor-festuclavine and 1-hydroxymethyl-festuclavine) and four to the lysergic acid derivatives (methylergometrine, lysergic acid amide, isolysergic acid amide and lysergic acid diethylamide). It is shown that agroclavine (ED50: 3.9 microM), 1-propyl-agroclavine (3.5 microM), 1-propylfestuclavine (4.3 microM) and 1-allyl-festuclavine (4.3 microM) are potent cytostatic agents. Up to 2 X ED50 concentration the inhibitory effect was completely reversible. Incorporation studies suggested that the compounds inhibit DNA synthesis; this assumption was also supported by the findings which revealed that after incubation with these clavines, the cells showed slight 'unbalanced growth'. 6-Cyano-6-nor-festuclavine was less inhibitory (ED50 11.8 microM). 1-Hydroxymethyl-festuclavine and all lysergic acid derivatives tested were without any detectable activity.