In vitro anti-cancer efficacy evaluation of cholesteryl butyrate solid lipid nanoparticles in human cancer cell lines with different p53 status

F Foglietta1, R Canaparo1, E Imbalzano1, C Musicanti2, P Gasco2, GP Zara1, L Serpe1. 1Università degli Studi di Torino, Drug Science and Technology, 10125, Italy, 2Nanovector srl, 10144, Italy

The short-chain fatty acid butyrate, a metabolite naturally presents in human beings as a result of bacterial fermentation, is an inhibitor of histone deacetylase (HDAC), thus able to regulate gene expression by modifying the acetylation status of nuclear proteins.

In vitro studies have shown that butyrate, in its conventional formulation of sodium salt (Na-but) is able to play a wide range of effects, including anti-tumor effect, suggesting its use as an anti-cancer drug. Nevertheless, in in vivo studies butyrate showed unfavourable pharmacological properties. In an attempt to overcome these limitations to its clinical use, researchers are exploring various delivery systems including butyric acid as cholesteryl butyrate solid lipid nanoparticles (Chol-but).

The aim of this study is to evaluate the effectiveness of butyrate as Chol-but compared to the conventional formulation of Na-but in human tumor cell lines that differ in the p53 status, i.e., HL-60, derived from acute promyelocytic leukemia that carries mutant p53 and MCF7, derived from breast cancer that carries wild type p53. Cell growth was evaluated by WST-1 assay, cell death mechanism and cell cycle distribution by flow cytometric analysis, cellular uptake by fluorescence microscopy, gene expression by real time RT-PCR and inhibition of HDAC by ELISA assay. Data are expressed as mean ± sem of three separate experiments and statistical comparisons between treatment groups were performed with analysis of variance (two-way ANOVA) and the threshold of significance was calculated by the Bonferroni’s test.

The cell growth of HL-60 and MCF7 was evaluated at increasing concentrations (0.01, 0.10, 0.50, 1.00 mM) of Na-but and Chol-but at 24 and 48 h. In HL-60 at 24 h Chol-but treatment determined a reduction of 80% of cell growth compared to control cells (p<0.001) and cells treated with Na-but at the same concentration (p<0.001). In MCF7 at 48 h Chol-but and Na-but treatment at 1.00 mM resulted in both a reduction of approximately 45% of cell growth compared with control (p<0.01). Moreover, in HL-60 was possible to highlight a significant increase in percentages of apoptotic cells on the whole population at 12 h after treatment with Chol-but 0.50 mM (59.10% ± 3.94; p<0.01) compared to control (11.9% ± 0.84) and cells treated with Na-but (18.26% ± 5.62) and a block in the G0/G1 phase of the cell cycle compared to control and cells treated with Na-but. By analyzing a series of genes involved in the mechanism of action of butyrate we observed a significant increase in the expression of CDKN1A gene (p<0.001 vs control cells), responsive to butyrate, following treatment with 0.50 mM Chol-but at 12 h and, in general, a different kinetics in gene transcription regulation with Na-but and Chol-but. In MCF7, at 1.00 mM, it was not possible to highlight differences between Na-but and Chol-but level in induction of apoptosis and cell cycle, however, a slight increase in the expression of CDKN1A with both formulations was observed.

Moreover, an increase in HDAC inhibition with Chol-but 0.50 mM was observed compared to Na-but (p<0.05) in HL-60 at 12 h exposure while either Chol-but and Na-but were unable to induce a significant decrease in HDAC inhibition in MCF-7 at 48 h exposure.

Therefore, our in vitro results have shown that Chol-but lead to different anti-cancer efficacy according to the cell line p53 status.