Differentiation-inducing factor-1 enhances 5-fluorouracil action on oral cancer cells inhibiting E2F1 and thymidylate synthase mRNAs accumulation

Purpose: Differentiation-inducing factor-1 (DIF-1) is a morphogen originally identified in the amoebozoan Dictyostelium discoideum. In mammalian cells, it has been shown to activate GSK3β, which in turn is expected to reduce levels of β-catenin and cyclin D1, thus mediating DIF-1 antiproliferative properties. Since this could alter the expression and activity of E2F1 transcription factor and consequently those of the prognostic marker/chemotherapy target thymidylate synthase (TS), we evaluated (1) whether DIF-1 could effectively regulate these genes, (2) whether it could interfere with cell viability, and (3) whether DIF-1 activity could enhance the efficacy of the TS inhibitor 5-fluorouracil (5-FU). Methods: We investigated the effects of DIF-1 in continuous human cell lines derived from two oral tumor histotypes (corresponding to an adenosquamous and a squamous carcinoma) and a gingival epithelium. We evaluated mRNA accumulation by means of quantitative real-time PCR and efficacy of drugs on cell viability by means of MTT assay. Results: DIF-1 inhibited the accumulation of E2F1 mRNA and reduces TS mRNA levels in tumor cell lines, but did not alter mRNA levels in the gingival counterpart. As a result, it inhibited proliferation preferentially of tumor cell in time- and concentration-dependent manner. Moreover, it enhanced cytotoxic effects of 5-FU only in tumor cell, whereas reduced them in the gingival counterpart. Conclusions: These findings suggest a tumor-specific action of DIF-1 on oral carcinoma cells. Thus, interfering with E2F1 and TS transcription, DIF-1 potentiates TS enzymatic inhibitors.