We in the beginning investigated whether romidepsin could inhibit cell proliferation

We in the beginning investigated whether romidepsin could inhibit cell proliferation. with wild-type or mutant p53 and those made up of Alk mutations. The decrease in cell proliferation is usually accompanied by caspase-dependent apoptosis as shown by PARP cleavage, an accumulation of cells in the sub-G1 phase of the cell cycle and the ability of a pan-caspase inhibitor to reduce cell death. Romidepsin inhibits the growth of subcutaneous NB xenografts in a dose dependent manner in immunocompromised mice. Furthermore, romidepsin induces expression of genes such as p21 and expression of p75 and NTRK (TrkA) which are more highly expressed in the tumors from NB patients that have a good prognosis. These studies support continued investigations into the therapeutic activity of romidepsin in NB. was the first histone deacetylase inhibitor to demonstrate clinical anti-tumor activity in patients.11 Although TSA and romidepsin target the same pathway, the anti-proliferative effect of romidepsin is 10-fold greater than that of TSA, and the IC50 of romidepsin on histone acetylation is much lower than that of TSA.12 Much like other HDAC inhibitors, romidepsin has been shown to induce cell cycle arrest, cellular differentiation, apoptosis and alter gene expression in a variety of adult malignancies.10, 12, 13 A pediatric phase I investigation of romidepsin has determined the maximally tolerated dose14 and a preliminary assessment indicated inhibition of tumor cell growth in 3 of 4 NB cell lines.15 We have shown that HDAC inhibitors such as MS-27C275 can mediate potent and antitumor activity against a broad panel of pediatric solid tumors including NB.16 Previous studies focused on regulation of NB tumor cell growth;15 in this study, we detail mechanisms of cell cycle regulation and induction of apoptosis and gene regulation induced by romidepsin in NB tumor cells. Results Romidepsin inhibits NB cell growth in a dose-dependent manner The characteristics of the NB cell lines used in this study are detailed in Table 1. We in the beginning investigated whether romidepsin could inhibit cell proliferation. Cells cultured with numerous concentrations of romidepsin for 72 h. Romidepsin (0.5C30 ng/mL) resulted in a dose-dependent decrease in cell viability of all NB cell lines as measured by the MTT or MTS assay (Fig 1A). Both MYCN amplified and non-amplified cell lines showed comparable dose-dependent inhibition of growth with the IC80 concentration of romidepsin for 4, 8 and 24 h. Protein lysates were evaluated for acetylation by monitoring the acetylation of lysines on histone H3 (Ac-H3) by a quantitative-immunoblot analysis (Fig 2) with selected examples of Western analysis detailed in Fig. 2 inset. Accumulation of acetylated histones was seen as early as 4 h after romidepsin treatment in all cell lines (except IMR32) and increased further at 24 h. Open in a separate window Physique 2- Acetylation of histones after romidepsin treatment. NB cell lines were treated with IC80 concentration of romidepsin for 4, 8 and 24 h, protein was extracted and analyzed for Ac-H3 analysis by immunoblot assay. Blots were (±)-Ibipinabant reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels as loading controls. Normalized values are plotted as ratio of (±)-Ibipinabant control. Inset- Representative western analyses of acetylated Histone H3. Romidepsin induces apoptosis Because inhibition of cell growth may be due to cell cycle arrest or induction of apoptosis, NB cell lines were exposed to romidepsin (IC80 concentration as determined for each cell collection) for 4, 8 and 24 h and DNA content was assessed by FACS analysis. For all the cell lines, there was a significant increase in cells with sub-G1 DNA content with a corresponding decrease in cells in the G1 phase, consistent with apoptosis (Fig 3A and ?andB).B). There was no evidence of cell cycle arrest in the G1 or G2/M phases of the cell cycle as has been seen in some human tumor cell lines.9, 12 In all the NB cell lines, there was a demonstrable increase in apoptotic cells by 8 h. To determine if the cell death induced by treatment of NB cells with romidepsin is usually caspase-dependent, cells were pretreated with 20 M Z-VAD-FMK, a broad-spectrum caspase-3 inhibitor, and were incubated with or without romidepsin for 48 hours. Cell viability was assessed using the MTS assay. Z-VAD-FMK significantly inhibited romidepsin-induced cell death in all NB cell lines tested (Fig 3C). Although Z-VAD-FMK alone did not have any.In some experiments, cell growth was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (Sigma Chemical Company, St. phase of the cell cycle and the ability of a pan-caspase inhibitor to reduce cell death. Romidepsin inhibits the growth of subcutaneous NB xenografts in a dose dependent manner in immunocompromised mice. Furthermore, Rabbit Polyclonal to MYO9B romidepsin induces expression of genes such as p21 and expression of p75 and NTRK (TrkA) which are more highly expressed in the tumors from NB patients that have a good prognosis. These studies support continued investigations into the therapeutic activity of romidepsin in NB. was the first histone deacetylase inhibitor to demonstrate clinical anti-tumor activity in patients.11 Although TSA and romidepsin target the same pathway, the anti-proliferative effect of romidepsin is 10-fold greater than that of TSA, and the IC50 of romidepsin on histone acetylation is much lower than that of TSA.12 Much like other HDAC inhibitors, romidepsin has been shown to induce cell cycle arrest, cellular differentiation, apoptosis and alter gene expression in a variety of adult malignancies.10, 12, 13 A pediatric phase I investigation of romidepsin has determined the maximally tolerated dose14 and a preliminary assessment indicated inhibition of tumor cell growth in 3 of 4 NB cell lines.15 We have shown that HDAC inhibitors such as MS-27C275 can mediate potent and antitumor activity against a broad panel of pediatric solid tumors including NB.16 Previous studies focused on regulation of NB tumor cell growth;15 in this study, we detail mechanisms of cell cycle regulation and induction of apoptosis and gene regulation induced by romidepsin in NB tumor cells. Results Romidepsin inhibits NB cell growth in a dose-dependent manner The characteristics of the NB cell lines used in this study are detailed in Table 1. We in the beginning investigated whether romidepsin could inhibit cell proliferation. Cells cultured with numerous concentrations of romidepsin for 72 h. Romidepsin (0.5C30 ng/mL) resulted in a dose-dependent decrease in cell viability of all NB cell lines as measured by the MTT or MTS assay (Fig 1A). Both MYCN amplified and non-amplified cell lines showed comparable dose-dependent inhibition of growth with the IC80 concentration of romidepsin for 4, 8 and 24 h. Protein lysates were evaluated for acetylation by monitoring the acetylation of lysines on histone H3 (Ac-H3) by a quantitative-immunoblot analysis (Fig 2) with selected examples of Western analysis detailed in Fig. 2 inset. Accumulation of acetylated histones was seen as early as 4 h after romidepsin treatment in all cell lines (except IMR32) and increased further at 24 h. Open in a (±)-Ibipinabant separate window Physique 2- Acetylation of histones after romidepsin treatment. NB cell lines were treated with IC80 concentration of romidepsin for 4, 8 and 24 h, protein was extracted and analyzed for Ac-H3 analysis by immunoblot assay. Blots were reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels as loading controls. Normalized values are plotted as ratio of control. Inset- Representative western analyses of acetylated Histone H3. Romidepsin induces apoptosis Because inhibition of cell growth may be due to cell cycle arrest or induction of apoptosis, NB cell lines were exposed to romidepsin (IC80 concentration as determined for each cell collection) for 4, 8 and 24 h and DNA content was assessed by FACS analysis. For all the cell lines, there was a significant increase in cells with sub-G1 DNA content with a corresponding decrease in cells in the G1 phase, consistent with apoptosis (Fig 3A and ?andB).B). There was no evidence of cell cycle arrest in the G1 or G2/M phases of the cell cycle as has been seen in some human tumor cell lines.9, 12 In all the NB cell lines, there was a demonstrable increase in apoptotic cells by 8 h. To determine if the cell death induced by treatment of NB cells with romidepsin is usually caspase-dependent, cells were pretreated with 20 M Z-VAD-FMK, a broad-spectrum caspase-3 inhibitor, and were incubated with or without romidepsin for 48 hours. Cell viability was assessed using the MTS assay. Z-VAD-FMK significantly inhibited romidepsin-induced cell death in all NB cell lines tested (Fig 3C). Although Z-VAD-FMK alone did.