Antineoplastic effects of the DNA methylation inhibitor hydralazine and the histone deacetylase inhibitor valproic acid in cancer cell lines

Abstract

Background: Among the epigenetic alterations occurring in cancer, DNA hypermethylation and histone hypoacetylation are the focus of intense research because their pharmacological inhibition has shown to produce antineoplastic activity in a variety of experimental models. The objective of this study was to evaluate the combined antineoplastic effect of the DNA methylation inhibitor hydralazine and the histone deacetylase inhibitor valproic acid in a panel of cancer cell lines.

Results: Hydralazine showed no growth inhibitory effect on cervical, colon, breast, sarcoma, glioma, and head & neck cancer cell lines when used alone. On the contrary, valproic acid showed a strong growth inhibitory effect that is potentiated by hydralazine in some cell lines. Individually, hydralazine and valproic acid displayed distinctive effects upon global gene over-expression but the number of genes over-expressed increased when cells were treated with the combination. Treatment of HeLa cells with hydralazine and valproic acid lead to an increase in the cytotoxicity of gemcitabine, cisplatin and adriamycin. A higher antitumor effect of adriamycin was observed in mice xenografted with human fibrosarcoma cells when the animals were co-treated with hydralazine and valproic acid.

Conclusion: Hydralazine and valproic acid, two widely used drugs for cardiovascular and neurological conditions respectively have promising antineoplastic effects when used concurrently and may increase the antitumor efficacy of current cytotoxic agents.

Figure 1

In vitro growth inhibition by hydralazine, valproic acid and the combined treatment. Hydralazine had not growth inhibitory effects at the dose and conditions tested in none of the cell lines. Valproic acid induced a variable degree of inhibition in the cell lines. D54 glioma cell line had 80% growth inhibition whereas KB laryngeal carcinoma was the least but still inhibition was close to 50%. MCF-7 and SW480 were more sensitive to the combination of compounds although the difference was statistically significant only in SW480 cells (p < 0.001)

Figure 2

Hierarchical clustering revealed specific patterns induced by each treatment. Hydralazine, valproic acid and the combination down-regulated a similar set of genes. On the contrary, up-regulated genes were distinctive for each treatment. From 1,281 differentially expressed genes, 651 were up-regulated and 630 down-regulated. The number of genes over-expressed specific of hydralazine, valproic acid and the combination were 153, 178 and 352. Each row represents a gene, whereas each column corresponds to the treatment drug. The relative abundance of the gene in the tissue correlates with color intensity; red induced; green repressed; black no change. H: hydralazine, V: valproic acid, and HV: hydralazine and valproic acid.

Figure 3

Effect of cytotoxic drugs in combination with hydralazine and valproic acid in HeLa cells. HeLa cells were treated for 24 hours with cisplatin, adriamycin or gemcitabine at or close to IC⁵⁰ plus hydralazine and valproic acid. Afterwards medium was removed and fresh medium containing only hydralazine and valproic acid was added for additional 48 hours after which viability was measured. There was a statistically significant higher cytotoxicity of the chemotherapeutic agent when used in combination with hydralazine plus valproic acid. Viability with cisplatin was 37% and zero after co-treatment with hydralazine-valproic acid (p < 0.002). Corresponding values for adriamycin and gemcitabine alone or with co-treatment were 42% versus 27% (p < 0.020) and 45% versus 37% (p < 0.012) respectively.

Figure 4

Antitumor effect of the combination of hydralazine and valproic acid plus adriamycin in a nude mice xenograft model of fibrosarcoma. Control: solid line with open squares. Tumors in animal treated with adriamycin (dotted line with closed circles) and hydralazine-valproic acid and adriamycin (broken line with open triangles) had their tumors shrinkaged at day 21, however, in animals receiving hydralazine and valproic acid tumors failed to regrowth while in those with only adriamycin tumors increased in size shortly after day 21. Saline, or adriamycin injections were performed at days 7, 14 and 21. In the group with the three-drug combination, hydralazine and valproic acid were administered daily from day 1 (seven days before the first injection of adriamycin (day 7) until sacrifice (day 35).