Molecular profiling of signalling proteins for effects induced by the anti-cancer compound GSAO with 400 antibodies.
BACKGROUND
GSAO (4-[N-[S-glutathionylacetyl]amino] phenylarsenoxide) is a hydrophilic derivative of the protein tyrosine phosphatase inhibitor phenylarsine oxide (PAO). It inhibits angiogenesis and tumour growth in mouse models and may be evaluated in a phase I clinical trial in the near future. Initial experiments have implicated GSAO in perturbing mitochondrial function. Other molecular effects of GSAO in human cells, for example on the phosphorylation of proteins, are still largely unknown.


METHODS
Peripheral white blood cells (PWBC) from healthy volunteers were isolated and used to profile effects of GSAO vs. a control compound, GSCA. Changes in site-specific phosphorylations, other protein modifications and expression levels of many signalling proteins were analysed using more than 400 different antibodies in Western blots.


RESULTS
PWBC were initially cultured in low serum conditions, with the aim to reduce basal protein phosphorylation and to increase detection sensitivity. Under these conditions pleiotropic intracellular signalling protein changes were induced by GSAO. Subsequently, PWBC were cultured in 100% donor serum to reflect more closely in vivo conditions. This eliminated detectable GSAO effects on most, but not all signalling proteins analysed. Activation of the MAP kinase Erk2 was still observed and the paxillin homologue Hic-5 still displayed a major shift in protein mobility upon GSAO-treatment. A GSAO induced change in Hic-5 mobility was also found in endothelial cells, which are thought to be the primary target of GSAO in vivo.


CONCLUSIONS
Serum conditions greatly influence the molecular activity profile of GSAO in vitro. Low serum culture, which is typically used in experiments analysing protein phosphorylation, is not suitable to study GSAO activity in cells. The signalling proteins affected by GSAO under high serum conditions are candidate surrogate markers for GSAO bioactivity in vivo and can be analysed in future clinical trials. GSAO effects on Hic-5 in endothelial cells may point to a new intracellular GSAO target.
Cadd
Verity A
VA
Cancer Research UK Cell Signalling Group, Weatherall Institute of Molecular Medicine, University of Oxford, UK. verity.cadd@cancer.org.uk
Hogg
Philip J
PJ
Harris
Adrian L
AL
Feller
Stephan M
SM
D000328:Adult; D000906:Antibodies / Q000378:metabolism*; D001152:Arsenicals / Q000494:pharmacology*; D002478:Cells, Cultured; D003470:Culture Media; D016895:Culture Media, Serum-Free; D004353:Drug Evaluation, Preclinical; D042783:Endothelial Cells / Q000187:drug effects; D005260:Female; D005978:Glutathione / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007962:Leukocytes / Q000187:drug effects* / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D008954:Models, Biological; D010770:Phosphotransferases / Q000378:metabolism; D040081:Protein Array Analysis; D015398:Signal Transduction; D013379:Substrate Specificity
C426645:4-(N-(S-glutathionylacetyl)amino)phenylarsenoxide; D000906:Antibodies; D001152:Arsenicals; D003470:Culture Media; D016895:Culture Media, Serum-Free; D010770:Phosphotransferases; D005978:Glutathione