Cell-cycle arrest and p53-independent induction of apoptosis in vitro by the new anticancer drugs 5-FdUrd-P-FdCydOct and dCydPam-P-FdUrd in DU-145 human prostate cancer cells.
OBJECTIVE
Current therapies have limited impact on the progression of metastatic hormone-refractory prostate cancer. Therefore, we investigated the utility of new heterodinucleoside phosphate dimers of 5-fluorodeoxyuridine (5-FdUrd) in p53-mutated and androgen-independent DU-145 human prostate tumour cells.


METHODS
The effects of the dimers were assessed in vitro by a cell proliferation assay for cytotoxicity, flow cytometry for cell cycle distribution, confocal laser scanning microscopy for the detection of apoptotic bodies, poly(ADP-ribose) polymerase cleavage for caspase 3 activity and by a thymidylate synthetase assay.


RESULTS
The new dimers N4-palmitoyl-2'-deoxycytidylyl-(3'-->5')-5-fluoro-2'-deoxyuridine (dCydPam-P-FdUrd) and 2'-deoxy-5-fluorouridylyl-(3'-->5')-2'-deoxy-5-fluoro-N4-octade cylcytidine (5-FdUrd-P-FdCydOct) caused marked cytotoxicity with IC50 values of 3-4 microM. 5-FdUrd-P-FdCydOct at 200 microM was capable of eradicating 100% of tumour cells whereas 10% of the cells were resistant to 5-FdUrd. Cytotoxicity was caused by a dramatic S-phase arrest, resulting in an increase of this cell population from 34% to 85% with 5-FdUrd-P-FdCydOct and to 81% with dCydPam-P-FdUrd. S-phase arrest was followed by apoptosis, as shown by 85% of the cells staining positive for Apo 2.7 antibody, a six- to eight-fold increased caspase 3 activity and DNA fragmentation. Thymidylate synthase activity was inhibited by 50% at 0.6-0.7 microM dimer concentration. The dimers were hydrolysed in vitro by phosphodiesterase I and human serum to the corresponding nucleosides and nucleoside monophosphates.


CONCLUSIONS
The new dimers dCydPam-P-FdUrd and 5-FdUrd-P-FdCydOct are effective prodrugs of 5-FdUrd and have potential value for the treatment of p53-mutated and hormone-independent human prostate carcinomas.
Cattaneo-Pangrazzi
R M
RM
Department of Pathology, University Hospital Zürich, Switzerland.
Schott
H
H
Wunderli-Allenspach
H
H
Rothen-Rutishauser
B
B
Guenthert
M
M
Schwendener
R A
RA
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects*; D003599:Cytoskeleton / Q000187:drug effects / Q000648:ultrastructure; D053938:DNA Fragmentation; D019281:Dimerization; D005434:Flow Cytometry; D005467:Floxuridine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology / Q000627:therapeutic use; D005455:Fluorescent Antibody Technique; D006801:Humans; D006868:Hydrolysis; D008297:Male; D018613:Microscopy, Confocal; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000494:pharmacology* / Q000627:therapeutic use; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011471:Prostatic Neoplasms / Q000188:drug therapy / Q000473:pathology*; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D014407:Tumor Cells, Cultured
C408702:2'-deoxy-5-fluorouridylyl-(3'-5')-2'-deoxy-5-fluoro-N(4)-octadecylcytidine; D000964:Antimetabolites, Antineoplastic; C408701:N(4)-palmitoyl-2'-deoxycytidylyl-(3'-5')-5-fluoro-2'-deoxyuridine; D009838:Oligodeoxyribonucleotides; D005467:Floxuridine; D013940:Thymidylate Synthase; D010727:Phosphoric Diester Hydrolases; D043264:Phosphodiesterase I; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases