Augmentation of phenylbutyrate-induced differentiation of myeloid leukemia cells using all-trans retinoic acid

Abstract

Despite preliminary evidence of clinical activity of the putative differentiating agent sodium phenylbutyrate (PB) in the treatment of myeloid neoplasms, it has proven difficult to maintain therapeutic levels of PB above 0.5 mM, well below the ED 50 of 1-2 mM. We have studied the impact of combining PB with alltrans retinoic acid (ATRA) on the ML-1 myeloid leukemia cell line. ATRA augmented PB-induced differentiation, cell-cycle arrest, and apoptosis. ATRA augmented PB induction of the myelomonocytic marker CD11b at all doses of ATRA tested (0.0025-1 M). Although ATRA did not significantly affect the ED 50 of PB, the combination of ATRA (1 M) and PB (0.5 mM) augmented PB-induced CD11b expression eight-fold. Compared to PB alone, this combination of ATRA and PB induced greater cell cycle arrest (S-phase 14% vs 38%; G 0 /G 1 -phase cells 72% vs 52%) and greater apoptosis (24% vs 16% by TUNEL assay). Treatment with ATRA (0.5 M) in combination with PB (0.5 mM) led to significantly greater inhibition of colony formation (4.8% vs 48% inhibition). ATRA combined synergistically with PB to augment CD11b expression and inhibit colony formation. This combination also showed significant interaction in terms of S-phase inhibition. However, this interaction varied as a function of ATRA concentration: antagonistic at low concentrations of ATRA, synergistic at higher concentrations of ATRA. These data suggest that retinoids may significantly augment the cytostatic and differentiating activity of PB, leading to increased potency of the latter drug at clinically achievable doses.

Phenylbutyrate and Phenylacetate Induce Differentiation and Inhibit Proliferation of Human Medulloblastoma Cells

“…As the effects of most differentiation-inducing agents, including PB and PA, on malignant tumor cells are timeand dose-dependent, it has been suggested that higher doses and/or prolonged exposure may lead to irreversible arrest of cell proliferation and/or terminal differentiation (37,38). For PB, however, most of the published works were based on short-term exposure, ranging from 72 h (39,40) up to 12 days (11,16,18,19,31). To address the efficacy of the long-term exposure of medulloblastoma cells to PB, we performed the extensive washout experiments in vitro with cell proliferation assay.…”

“…Unlike the use of cytotoxic agents to kill tumor cells, differentiation therapy aims to restore the regulating machinery by overriding the defective signals or activating alternative pathways that will cause malignant tumor cells to mature and lose their malignant phenotype (5,6). Such induced differentiation could also cause tumor cells to become more responsive to normal growth regulatory signals or more sensitive to chemotherapy (7)(8)(9)(10) and other differentiation agents (11). The remarkable success of all-trans-retinoic acid in producing complete remission in patients with acute promyelocytic leukemia has sparked interest in differentiation induction as an alternative form of cancer chemotherapy (12).…”

Histone deacetylase inhibitor NVP-LAQ824 has significant activity against myeloid leukemia cells in vitro and in vivo

“…The increase in S phase was o10%, but was not necessarily associated with increased cell number, which was not examined. Yu et al 72 showed that, in response to very low concentrations of phenylbutyrate, there was a 5% increase in S phase in one figure, but no increase in cell numbers as determined by colony formation. In contrast, we have only observed a decrease in S phase in K562 cells using low concentrations of NVP-LAQ824.…”

Clinical and Experimental Applications of Sodium Phenylbutyrate

“…[58] In vitro studies have shown that phenylbutyrate can (i) induce differentiation and inhibit proliferation of AML cell lines and primary leukemic cells; [59,60] (ii) inhibit CFU-L production from bone marrow specimens from patients with MDS; [60] and (iii) induce differentiation associated with induction of p21 WAF1/CIP1 expression, hypophosphorylation of retinoblastoma protein, and G1 arrest in the ML-1 myeloid leukemia cell line. [59] At least some of the pharmacodynamic effects of phenylbutyrate seem to result because of its ability to inhibit HDACs, [61] considering that histone acetylation contributes to the regulation of the gene transcription. [62] The in vitro effects of decitabine, phenylbutyrate and TSA on clonogenic AML human cells have been investigated.…”