Mode of interaction between butyroyloxymethyl-diethyl phosphate (AN-7) and doxorubicin in MCF-7 and resistant MCF-7/Dx cell lines

Dikla Engel, Abraham Nudelman, Inesa Levovich, Tal Gruss-Fischer, Michal Entin-Meer, Don R. Phillips, Suzanne M. Cutts, Ada Rephaeli*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


Purpose: To investigate the anticancer activity and mode of action of butyroyloxymethyl-diethyl phosphate (AN-7), a prodrug of butyric acid and formaldehyde, as a single agent and in combination with doxorubicin in human carcinoma MCF-7 and the multidrug resistant MCF-7 Dx cell lines. Methods: The anti-cancer activity of AN-7 as a single agent or in combination with doxorubicin was measured by the Hoechst cell viability and colony forming assays as well as by FACS analyses of cells stained with propidium iodide and annexin V-FITC. Modulations of protein expression and acetylation were measured by Western blot analyses. The number of doxorubicin-DNA adducts formed was evaluated using 14C-labeled doxorubicin. Results: The AN-7 and homologous prodrugs exhibited similar growth inhibition effects against drug resistant and sensitive cells, and elicited their anticancer effect partially by inhibition of HDAC. The AN-7 transiently augmented histone acetylation and increase of p21 expression. Synergy between AN-7 and doxorubicin was demonstrated in the sensitive and the resistant cell lines by viability and colony formation assays and was further confirmed by FACS analysis showing an increase in cell mortality. The number of doxorubicin-DNA adducts in total genomic DNA isolated from cells treated with 14C-labeled doxorubicin and AN-7 increased substantially compared to treatment with doxorubicin only. Treatment with AN-7 or doxorubicin increased p53 acetylation that was further potentiated by their combination. Conclusion: The AN-7 combined with doxorubicin overcame drug resistance; at least in part by the intracellularly releasable formaldehyde that augmented formation of doxorubicin-DNA adducts and butyric acid that induced histone and p53 acetylation. Since the use of doxorubicin is limited by toxicity, the combination could offer an effective treatment modality with lower toxicity for breast cancer.

Original languageEnglish
Pages (from-to)673-683
Number of pages11
JournalJournal of Cancer Research and Clinical Oncology
Issue number10
StatePublished - Oct 2006


FundersFunder number
Bar Ilan University
Israel Cancer Research Fund
Australian Research Council
Israel Cancer Association
Israel Science Foundation


    • AN-7
    • Breast cancer
    • CIP1/WAF1 (p21)
    • Doxorubicin
    • HDAC inhibition
    • p53 Tumor suppressor gene


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