Inhibition of growth of human breast carcinoma cells by an antisense oligonucleotide targeted to the transferrin receptor gene.
Transferrin receptor expression is controlled by the amount of iron required by the cell to maintain its metabolism and therefore tumor cells in a highly proliferative state have a high density of transferrin receptors. In this study, phosphorothioated antisense TfR oligonucleotides (TfR-ODna) targeted to the sequences of TfR mRNA including the AUG initiation codon and the control sense chain (TfR-ODns) were synthesized. The rate of cellular DNA synthesis was determined by [3H]-thymidine incorporation. Administering TfR-ODna to three morphologically distinct breast cancer cell lines (MCF-7, T47D, and MDA-MB-231) and a normal breast cell line (MCF-12A) caused specific inhibition of tumor cell growth. The IC50 (50% inhibition of DNA synthesis) of the TfR-ODna for the MCF-7, T47D and MDA-MB-231 cells were 0.5, 0.5, and 1.0 microM, respectively, whereas the MCF-12A normal breast cells were about 30 times (IC50 of 30 microM) less sensitive to TfR-ODna than the breast cancer cells. The cytotoxicity of the antisense TfR-ODna was 10 to 60 times greater than that of the sense chain (TfR-ODns). TfR mRNA and protein synthesis were demonstrated by RT-PCR and immunohistochemical staining, respectively. Approximately 50% inhibition of the expression of TfR mRNA was observed when breast cancer cells were treated with 1 microM antisense TfR ODNa for 72 hrs but 1 microM antisense only caused 14% inhibition in normal breast cells. The decreased cytotoxicity and inhibition of TfR gene expression when the tumor cells were treated with the same concentration (1 microM) of TfR-ODns demonstrated the specificity of the TfR-ODna for blocking the target TfR gene. The combined cytotoxicities to human breast tumor MCF-7 cells of the antisense TfR-ODna and the iron chelator deferoxamine (DFO) or the ribonucleotide reductase inhibitor hydroxyurea were observed in this study. IC50s (50% inhibition of DNA synthesis) for DFO and hydroxyurea individually were 0.3 microM and 250 microM, respectively. The CalcuSyn program was used to determine the combined effects among the agents and synergism (Combined Indexes (CI) < 1) were found with the following two combinations: TfR-ODna (0.007 microM to 0.15 microM) with DFO (0.15 microM to 5 microM) and TfR-ODna (0.007 microM to 0.15 microM) with hydroxyurea (50 microM to 800 microM). However, inhibition by TfR-ODns was not synergistic with either DFO or hydroxyurea. The synergistic effects on inhibition of DNA synthesis between TfR-ODna and DFO or hydroxyurea suggest that inhibition of breast cancer cell growth by TfR-ODna is produced by depletion of iron pools that are required for DNA synthesis in tumor cells. The fact that TfR-ODna specifically decreases cell viability and proliferation, and reduces TfR mRNA and protein expression in human breast carcinoma cells without affecting normal breast cells, suggests that the antisense oligonucleotide to the transferrin receptor may be a novel therapeutic approach in breast cancer.
Yang
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DC
Mastology Research Institute of The Elliott Mastology Center, Baton Rouge, LA 70816, USA. emcmri@iamerica.net
Jiang
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Elliott
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Head
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JF
D001943:Breast Neoplasms / Q000188:drug therapy*; D002455:Cell Division; D002470:Cell Survival; D004247:DNA / Q000378:metabolism; D003676:Deferoxamine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D007150:Immunohistochemistry; D020128:Inhibitory Concentration 50; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D011990:Receptors, Transferrin / Q000096:biosynthesis / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D011990:Receptors, Transferrin; D004247:DNA; D003676:Deferoxamine; D006918:Hydroxyurea