Histone acetylation and histone deacetylase activity of magnesium valproate in tumor and peripheral blood of patients with cervical cancer. A phase I study

Abstract

Background: The development of cancer has been associated with epigenetic alterations such as aberrant histone deacetylase (HDAC) activity. It was recently reported that valproic acid is an effective inhibitor of histone deacetylases and as such induces tumor cell differentiation, apoptosis, or growth arrest.

Methods: Twelve newly diagnosed patients with cervical cancer were treated with magnesium valproate after a baseline tumor biopsy and blood sampling at the following dose levels (four patients each): 20 mg/kg; 30 mg/kg, or 40 mg/kg for 5 days via oral route. At day 6, tumor and blood sampling were repeated and the study protocol ended. Tumor acetylation of H3 and H4 histones and HDAC activity were evaluated by Western blot and colorimetric HDAC assay respectively. Blood levels of valproic acid were determined at day 6 once the steady-state was reached. Toxicity of treatment was evaluated at the end of study period.

Results: All patients completed the study medication. Mean daily dose for all patients was 1,890 mg. Corresponding means for the doses 20-, 30-, and 40-mg/kg were 1245, 2000, and 2425 mg, respectively. Depressed level of consciousness grade 2 was registered in nine patients. Ten patients were evaluated for H3 and H4 acetylation and HDAC activity. After treatment, we observed hyperacetylation of H3 and H4 in the tumors of nine and seven patients, respectively, whereas six patients demonstrated hyperacetylation of both histones. Serum levels of valproic acid ranged from 73.6-170.49 microg/mL. Tumor deacetylase activity decreased in eight patients (80%), whereas two had either no change or a mild increase. There was a statistically significant difference between pre and post-treatment values of HDAC activity (mean, 0.36 vs. 0.21, two-tailed t test p < 0.0264). There was no correlation between H3 and H4 tumor hyperacetylation with serum levels of valproic acid.

Conclusion: Magnesium valproate at a dose between 20 and 40 mg/kg inhibits deacetylase activity and hyperacetylates histones in tumor tissues.

Figure 1

Western blots for anti-acetylated H3 and H4 histones pre- and post-treatment in patients receiving a dose of 20 mg/kg. Patients 2 and 4 are missed due to insufficient sample. Positive and negative controls are HeLa cells treated or not with trichostatinA. Loading control are gels stained with coomassie blue. Graphs in the inferior panel are HDAC activity expressed as optical densities (ODs), in the same scale as positive and negative controls that are also HeLa cells extracts with and without trichostatinA treatment. At the bottom are values of valproic acid in serum.

Figure 2

Western blots for anti-acetylated H3 and H4 histones pre- and post-treatment in patients receiving a dose of 30 mg/kg. Positive and negative controls are HeLa cells treated or not with trichostatin A. Loading control are gels stained with coomassie blue. Graphs in the inferior panel are HDAC activity expressed as ODs, in the same scale as positive and negative controls that are also HeLa cells extracts with and without trichostatinA treatment. At the bottom are values of valproic acid in serum.

Figure 3

Western blots for anti-acetylated H3 and H4 histones pre- and post-treatment in patients receiving a dose of 40 mg/kg. Positive and negative controls are HeLa cells treated or not with trichostatin A. Loading control are gels stained with coomassie blue. Graphs in the inferior panel are HDAC activity expressed as ODs, in the same scale as the positive and negative controls that are also HeLa cells extracts with and without trichostatin A treatment. At the bottom are values of valproic acid in serum.

Figure 4

Western blots for anti-acetylated H3 and H4 histones pre- and post-treatment in PBMN cells of patients 1, 2, 5, and 8. Patient 1 and 2 received a dose of 20 mg/kg, whereas patients 5 and 8 each received a dose of 30 mg/kg. Positive and negative controls are HeLa cells treated or not with thrichostatin A. Loading control are gels stained with Coomassie blue.