Antisense ferritin oligonucleotides inhibit growth and induce apoptosis in human breast carcinoma cells.
BACKGROUND
Ferritin is the major iron-storage protein which sequesters and detoxifies excess iron that is taken up by cells but is not utilized in normal metabolic processes. Human ferritin consists of various combinations of heavy (FerH, Mr 21,000) and light (FerL, Mr 19,000) chains and excess iron leads to an increase in the synthesis of both heavy and light chains.


METHODS
In this study four pairs of antisense oligodeoxynucleotides (ODNs) were synthesized: FerH-A1 and FerL-A1 were complementary to the 24-base pair sequence overlapping the starting codons of the FerH and FerL genes, respectively, but the sequences of FerH-A2 and FerL-A2 only covered the coding sequences of the ferritin genes. The corresponding sense chain sequences (FerH-S1, FerH-S2, FerL-S1 and FerL-S2) were used as controls.


RESULTS
Treatment with FerH-S1, FerH-A1, FerH-S2, FerH-A2, FerL-S1, FerL-A1, FerL-S2 and FerL-A2 at 40 microM, 25 microM, 30 microM, 17 microM, 45 microM, 18 microM, 40 microM and 26 microM, respectively, for 72 hours resulted in 50% inhibition of DNA synthesis (IC50) in MCF-7 breast carcinoma cells, as measured by [3H]-thymidine incorporation. FerH chain mRNA, FerL chain mRNA and total ferritin protein levels were significantly decreased by the IC50 concentrations of each of the antisense ODNs but were not inhibited by IC50 concentrations of sense ODNs, as measured by quantitative RT-PCR and microparticle enzyme immunoassay. However, antisense ferritin ODNs had no effect on the total iron concentration in MCF-7 cells. Incubation with IC50 concentrations of antisense ferritin ODNs caused reduction in cell volume, condensation of nuclear structures and lower levels of Bcl-2 mRNA and protein compared to control cells, but Bax mRNA and protein levels remained unchanged.


CONCLUSIONS
This study demonstrates that antisense ODNs to ferritin genes are about two-fold more cytotoxic than sense ODNs, and that antisense ODNs are specific inhibitors of ferritin gene expression at both the transcriptional and the translational levels. Further, the antisense ferritin ODNs promote programmed cell death with low ratios of Bcl-2 to Bax mRNA and protein expression providing evidence that antisense ferritin ODNs specifically inhibit MCF-7 breast carcinoma cell growth through increased apoptosis. Finally, since the IC50 concentrations of FerH-A1 and FerH-A2, and FerL-A1 and FerL-A2 are very similar for inhibition of DNA synthesis and gene expression in human breast carcinoma MCF-7 cells, it does not seem necessary for the antisense ODNs to overlap the starting codons of ferritin gene to maximize inhibition.
Yang
Ding Cheng
DC
Mastology Research Institute of The E.H.H. Breast Cancer Research and Treatment Center, Baton Rouge, LA 70816, USA.
Jiang
Xianpeng
X
Elliott
Robert L
RL
Head
Jonathan F
JF
D017209:Apoptosis / Q000187:drug effects; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002455:Cell Division / Q000187:drug effects; D004273:DNA, Neoplasm / Q000096:biosynthesis; D005293:Ferritins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*; D006131:Growth Inhibitors / Q000138:chemical synthesis / Q000235:genetics / Q000494:pharmacology; D006801:Humans; D007501:Iron / Q000378:metabolism; D020319:Oligodeoxyribonucleotides, Antisense / Q000138:chemical synthesis / Q000235:genetics / Q000494:pharmacology*; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; D006131:Growth Inhibitors; D020319:Oligodeoxyribonucleotides, Antisense; D012333:RNA, Messenger; D005293:Ferritins; D007501:Iron