oa Histone Deacetylase Inhibitor Trichostatin A (TSA) and DNA Methyltransferase Inhibitor 5-aza-2′-deoxycitidine (5-AZA) Enhanced the Cisplatin (CDDP) Induced Cytotoxicity of Neuroblastoma Cells, in Vitro

Neuroblastoma is a childhood cancer that is frequently treated with CDDP. Upon chemotherapeutic treatment, neuroblastoma patients might develop drug resistance. A large body of evidence associates the cytotoxic effect of CDDP in cancer cells with the formation of DNA adducts and thus interference with the DNA replication of cancer cells but also with the increase of the intracellular calcium concentration ([Ca²⁺]i) that leads to apoptosis. Nevertheless, it is not understood how epigenetic mechanisms, such as DNA methylation and chromatin modification might influence the effect of CDDP on cytotoxicity and [Ca²⁺]i of neuroblastoma cells.

Therefore, here we investigate in human neuroblastoma cell lines SH-SY5Y and IMR-32 the changes in cytotoxicity and calcium homeostasis induced by the treatment with epigenetic modulators: TSA and 5-AZA but also in combination with CDDP.

Treatment schemes for calcium homeostasis experiments were performed in four different groups: A) treatment with CDDP only (control); B) acute treatment with 5-AZA or TSA (without CDDP); C) pre-treatment with both 5-AZA and TSA (before CDDP application), D) pre-treatment with 5-AZA (before CDDP). For cytotoxicity test: the Trypan Blue Cytotoxicity test was performed using the automated cell counter ViCellXR (Beckman Coulter, Germany), for exposure times ranging 24–72 h. The concentrations used were for 5-AZA ranging 1–200 μM while for TSA 1–10 μM were used.

The results showed significant increase of neurblastoma cells cytotoxicity upon treatment, especially for the combinatory treatment 5-AZA and TSA. We observed a time and concentration dependent increased cytotoxicity. The most efficient treatment was the combination 5-AZA and TSA, showing additive effects. TSA showed higher efficiencity triggering cytotoxic effects in neuroblastoma cells than 5AZA. Furthermore, the combinatory treatment of neuroblastoma cells with epigenetic modulators and CDDP increased the efficiency of CDDP treatment in a synergistic manner, effect that could be confirmed in IMR32 cells. Thus, the combination 5-AZA/CDDP or TSA/CDDP or 5-AZA /TSA/CDDP was more effective in triggering cell death of neuroblastoma cells than each of the compound alone.

For the investigation of changes in [Ca²⁺]i we performed calcium imaging studies using the calcium sensitive dye Fluo-4-AM in Tyrode's buffer with calcium (1.5 mM). Fluorescent images were taken with Olympus Microscope BX51 Wi with Xenon Arc Burner and “Xcellence rt” software. CDDP (1 μM) treatment (control group) increased the [Ca²⁺]i (30.3% ± 11.7) group “A” in SH-SY5Y cells. This effect of CDDP was reversed by the pretreatment with 5-AZA and TSA group “C” (5.1% ± 5.1) (p