Phenylhexyl isothiocyanate has dual function as histone deacetylase inhibitor and hypomethylating agent and can inhibit myeloma cell growth by targeting critical pathways

Abstract

Histone deacetylase (HDAC) inhibitors are a new class of chemotherapeutic agents. Our laboratory has recently reported that phenylhexyl isothiocyanate (PHI), a synthetic isothiocyanate, is an inhibitor of HDAC. In this study we examined whether PHI is a hypomethylating agent and its effects on myeloma cells. RPMI8226, a myeloma cell line, was treated with PHI. PHI inhibited the proliferation of the myeloma cells and induced apoptosis in a concentration as low as 0.5 muM. Cell proliferation was reduced to 50% of control with PHI concentration of 0.5 muM. Cell cycle analysis revealed that PHI caused G1-phase arrest of RPMI8226 cells. PHI induced p16 hypomethylation in a concentration- dependent manner. PHI was further shown to induce histone H3 hyperacetylation in a concentration-dependent manner. It was also demonstrated that PHI inhibited IL-6 receptor expression and VEGF production in the RPMI8226 cells, and reactivated p21 expression. It was found that PHI induced apoptosis through disruption of mitochondrial membrane potential. For the first time we show that PHI can induce both p16 hypomethylation and histone H3 hyperacetylation. We conclude that PHI has dual epigenetic effects on p16 hypomethylation and histone hyperacetylation in myeloma cells and targets several critical processes of myeloma proliferation.

Figure 1

PHI suppresses growth and causes cell cycle arrest of RPMI8226 myeloma cells. (A). PHI suppresses growth of RPMI8226 myeloma cells. The myeloma cells were cultured with or without phenylhexyl isothiocyanate (PHI) at varying concentrations for 24, 48 or 72 hours. The cell number was recorded at each time point. Diamond (♦), control; Square (■), 0.1 μM; Triangle (), 0.5 μM; Dot (●); 5.0 μM. (B). PHI induces G1 growth arrest. The myeloma cells were cultured with 0.5 μM of PHI for 48 or 96 hours. The cellular DNA content was determined by flow cytometry. Values are means +/- SD from 3 independent experiments. Open bar, G1 phase; Black bar, G2M phase; Grid bar, S phase.

Figure 2

PHI induces p16 hypomethylation in RPMI8226 myeloma cells. The myeloma cells were cultured with or without phenylhexyl isothiocyanate (PHI) at varying concentrations for 10 days. Cellular DNA was extracted and bisulfite-converted as described in Material and Methods. Methylation-specific PCR was performed using primers specific for methylated and unmethylated DNA forms of p16, respectively. The PCR product was visualized after agarose gel electrophoresis. Decitabine (5-aza) was used as a positive control for DNA hypomethylation. M, methylated p16 fragment; U, unmethylated p16 fragment. The 150 bp marker position was indicated.

Figure 3

PHI inhibits histone deacetylation in RPMI8226 myeloma cells. The RPMI8226 myeloma cells were cultured with two different concentrations of PHI for 72 hours. The proteins were extracted from the cell lysates. The status of histone H3 acetylation was determined by Western blot as described in Material and Methods. β-actin level in the same blot was used as an internal loading control for protein amount.

Figure 4

PHI inhibits IL-6 receptor expression and reactivates p21 expression in RPMI8226 myeloma cells. The RPMI8226 myeloma cells were cultured with 5 μM of PHI for 24 and 48 hours, respectively. The proteins were extracted from the cell lysates. The expression level of IL-6 receptor subunits, gp80 and gp130, as well as p21 protein were determined by Western blot analysis as described in Material and Methods. β-actin level in the same blot was used as an internal loading control for protein amount.

Figure 5

PHI inhibits production of VEGF in RPMI8226 myeloma cells. (A). The cells were incubated with varying concentration of PHI for 24 and 48 hours, respectively. VEGF production was determined as described in the Material and Methods. Diamond (♦), control; Square (■), 0.1 μM; Triangle (), 5 μM; Cross (x), 10 μM. (B). Percent inhibition of VEGF production as compared with the control culture was calculated from the above experiments. Values are means +/- SD from 3 independent experiments. Open bar, 24 hours; Black bar, 48 hours.

Figure 6

PHI causes disruption of mitochondrial membrane potential in RPMI8226 myeloma cells. The myeloma cells were treated with 5 μM and 10 μM of PHI, respectively for 48 hours. The cells were then stained with JC-1 dye. The mitochondrial membrane potential was measured by flowcytometry as described in the Material and Methods. The shift-down of fluorescence from Red to Green indicates the collapse of mitochondrial membrane potential. CCCP was used as a positive control for the disruption of mitochondrial membrane potential. The percent of cells with the disruption of mitochondrial membrane potential was indicated.