Studies of targeting and intracellular trafficking of an anti-androgen doxorubicin-formaldehyde conjugate in PC-3 prostate cancer cells bearing androgen receptor-GFP chimera.
The synthesis of a doxorubicin-formaldehyde conjugate bound to the nonsteroidal anti-androgen cyanonilutamide, via a cleavable tether, and binding of the construct to cell free androgen receptor (AR) as a function of tether design were previously reported. Cyanonilutamide bearing a linear alkyne tether bound to the AR better than other designs. Fluorescence microscopy studies of binding of the lead targeted drug, as well as various tethered cyanonilutamides, to the AR and subsequent trafficking of the resulting AR complex in live PC3 prostate cancer cells transfected with AR-green fluorescent protein (GFP) chimera are now described. Cyanonilutamide and cyanonilutamide bonded to a linear alkyne tether caused translocation of AR-GFP to the nucleus. In general, the ability of tethered cyanonilutamides to cause translocation paralleled their binding affinity for the AR. However, a noncleavable form of the lead cyanonilutamide-doxorubicin-formaldehyde conjugate bound to AR-GFP but the resulting complex did not translocate to the nucleus. Binding was apparent from the drugs inhibition of Mibolerone-induced translocation. Direct observation of anthraquinone fluorescence of targeted drug in PC3 cells showed initial cytosolic localization, independent of AR expression, with predominant nuclear localization after sufficient time for release of drug from the targeting moiety. The results indicate that doxorubicin-formaldehyde conjugate bonded to cyanonilutamide via a cleavable linear tether enters PC3 cells, resides in cytosol, binds to the AR if present, and ultimately releases doxorubicin or a doxorubicin derivative to the nucleus.
Cogan
Peter S
PS
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, USA.
Koch
Tad H
TH
D000726:Androgen Antagonists / Q000737:chemistry / Q000493:pharmacokinetics*; D004317:Doxorubicin / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000493:pharmacokinetics*; D005557:Formaldehyde / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006801:Humans; D007093:Imidazoles / Q000737:chemistry / Q000493:pharmacokinetics*; D008024:Ligands; D008297:Male; D008352:Mannich Bases / Q000737:chemistry / Q000493:pharmacokinetics*; D008856:Microscopy, Fluorescence; D011471:Prostatic Neoplasms; D011944:Receptors, Androgen / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
D000726:Androgen Antagonists; D007093:Imidazoles; D008024:Ligands; D008352:Mannich Bases; C494347:N-(5-(4-(3-(4-cyano-3-trifluoromethylphenyl)-5,5-dimethyl-2,4-dioxoimidazolidin-1-yl)but-2-ynyloxymethyl)-2-methoxybenzamidomethyl)doxorubicin; D011944:Receptors, Androgen; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D005557:Formaldehyde; D004317:Doxorubicin