The Role of Intracellularly Released Formaldehyde and Butyric Acid in the Anticancer Activity of Acyloxyalkyl Esters

Previous studies described a family of anticancer histone deacetylase inhibitor prodrugs of formula Me(CH(2))(2)COOCH(R)OR(1), which upon intracellular hydrolysis release acids and aldehydes. This study examines the mechanisms by which the prodrugs affect tumor cells and the contribution of the released aldehyde (formaldehyde or acetaldehyde) and acids to their anticancer activity. Type I prodrugs release 2 equiv of a carboxylic acid and 1 equiv of an aldehyde, and of Type II release 2 equiv of acids and 2 equiv of an aldehyde. SAR studied inhibition of proliferation, induction of differentiation and apoptosis, histone acetylation, and gene expression. Formaldehyde, measured intracellularly, was the dominant factor affecting proliferation and cell death. Among the released acids, butyric acid elicited the greatest antiproliferative activity, but the nature of the acid had minor impact on proliferation. In HL-60 cells, formaldehyde-releasing prodrugs significantly increased apoptosis. The prodrugs affected to a similar extent the wild-type HL-60 and MES-SA cell lines and their multidrug-resistant HL-60/MX2 and MES-Dx5 subclones. In a cell-free histone deacetylase (HDAC) inhibition-assay only butyric acid inhibited HDAC activity. The butyric acid and formaldehyde induced cell differentiation and increased p53 and p21 levels, suggesting that both affect cancer cells, the acid by inhibiting HDAC and the aldehyde by an as yet unknown mechanism.