The role of topoisomerases and RNA transcription in the action of the antitumour benzonaphthyridine derivative SN 28049.
OBJECTIVE
SN 28049 (N-[2-(dimethylamino)ethyl]-2,6-dimethyl-1-oxo-1,2-dihydrobenzo[b]-1,6-naphthyridine-4-carboxamide) is a DNA intercalating drug that binds selectively to GC-rich DNA and shows curative activity against the Colon 38 adenocarcinoma in mice. We wished to investigate the roles of topoisomerase (topo) I, topo II and RNA transcription in the action of SN 28049.


METHODS
We used clonogenic assays to study the cytotoxicity of SN 28049; RNA interference and enzyme assays to examine the role of topo I in SN 28049 action; 3H uridine incorporation and reporter assays to study its effects on transcription; and RT-PCR to examine its ability to reduce endogenous h-TERT expression.


RESULTS
In clonogenic assays, SN 28049 showed a biphasic cytotoxic dose response curve in H460 cells typical of acridine derivatives such as N-[2-(dimethylamino)ethyl]acridine-4-carboxamide (DACA) although it was approximately 16-fold more potent. Down-regulation of topo IIalpha in HTETOP cells reduced the cytotoxicity of SN 28049, establishing its action as a topo IIalpha poison. Surprisingly, down-regulation of topo I in H460 cells by RNA interference sensitised them to the actions of SN 28049 and other topo II poisons. SN 28049 also inhibited topo I-mediated relaxation of supercoiled plasmid DNA. SN 28049 was also an inhibitor of transcription in HEK293 cells and was more potent at reducing luciferase expression from a GC-rich SP-1 binding promoter than from a non-GC-rich AP-1 binding promoter. The drug also reduced luciferase reporter gene expression driven by the SP-1-binding survivin promoter as well as reducing endogenous h-TERT expression in HEK293 cells whose promoter also contains SP-1 binding sites.


CONCLUSIONS
We conclude that SN 28049 has a complex action that may involve poisoning of topo IIalpha, suppression of topo I and inhibition of gene transcription from promoters with SP-1 sites. These actions may contribute to the promising experimental solid tumour anticancer activity of SN 28049.
Bridewell
David J A
DJ
Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019 Auckland, New Zealand. d.bridewell@auckland.ac.nz
Porter
Andrew C G
AC
Finlay
Graeme J
GJ
Baguley
Bruce C
BC
D000970:Antineoplastic Agents / Q000494:pharmacology*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004264:DNA Topoisomerases, Type I / Q000378:metabolism / Q000502:physiology*; D004250:DNA Topoisomerases, Type II / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D008156:Luciferases / Q000235:genetics; D009287:Naphthyridines / Q000494:pharmacology*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D034622:RNA Interference; D012334:RNA, Neoplasm / Q000096:biosynthesis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013752:Tetracycline / Q000494:pharmacology; D014158:Transcription, Genetic / Q000502:physiology; D014529:Uridine / Q000378:metabolism
D000970:Antineoplastic Agents; C533598:N-(2-(dimethylamino)ethyl)-2,6-dimethyl-1-oxo-1,2-dihydroxybenzo(b)-1,6-naphthyridine-4-carboxamide; D009287:Naphthyridines; D011500:Protein Synthesis Inhibitors; D012334:RNA, Neoplasm; D008156:Luciferases; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II; D013752:Tetracycline; D014529:Uridine