Mode of interaction between butyroyloxymethyl-diethyl phosphate (AN-7) and doxorubicin in MCF-7 and resistant MCF-7/Dx cell lines.
OBJECTIVE
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 (14)C-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 (14)C-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.


CONCLUSIONS
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.
Engel
Dikla
D
Chemistry Department, Bar-Ilan University, Ramat Gan, 52900, Israel.
Nudelman
Abraham
A
Levovich
Inesa
I
Gruss-Fischer
Tal
T
Entin-Meer
Michal
M
Phillips
Don R
DR
Cutts
Suzanne M
SM
Rephaeli
Ada
A
D000107:Acetylation; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology* / Q000627:therapeutic use; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D002087:Butyrates / Q000494:pharmacology* / Q000627:therapeutic use; D045744:Cell Line, Tumor; D002470:Cell Survival; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D004317:Doxorubicin / Q000494:pharmacology* / Q000627:therapeutic use; D019008:Drug Resistance, Neoplasm; D004357:Drug Synergism; D005260:Female; D006801:Humans; D009943:Organophosphorus Compounds / Q000494:pharmacology* / Q000627:therapeutic use; D011355:Prodrugs / Q000494:pharmacology* / Q000627:therapeutic use; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D002087:Butyrates; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D009943:Organophosphorus Compounds; D011355:Prodrugs; D016159:Tumor Suppressor Protein p53; C502471:diethoxyphosphoryloxymethyl butanoate; D004317:Doxorubicin