Histone Deacetylase Inhibitor M344 Inhibits Cell Proliferation and Induces Apoptosis in Human THP-1 Leukemia Cells

Abstract

Histone acetylation plays an important role in the silencing and activation of genes involved in tumoregenesis. Trichostatin A, originally identified as an anti-fungal drug, is a potent inhibitor of histone deacetylase (HDAC) with potential anti-tumor activity. In this study, we investigated the effect of M344, an amide analogues of trichostatin A, on the growth and differentiation of THP-1 human leukemia cells. We showed that at low doses, (< 0.2 muM), M344 could inhibit the growth of THP-1 cells at G1 phase in vitro with low cytotoxic effect. Low dose of M344 exerted some differentiating effect on THP-1 cells as judged by the expression of c-fms proto-oncogene (M-CSF receptor) and appearance of adherent cells. Growth arrest induced by M344 is associated with increased levels of cyclin-dependent protein kinase inhibitor p21 and cyclin E, in agreement with G1 phase arrest. At higher doses (2 muM), M344 could induce THP-1 cells to undergo apoptosis, which was associated with the cleavage of PARP, cytochrome c release and activation of both caspases-8, -9, followed by the activation of caspase-3. In addition, M344 could increase the levels of pro-apoptotic protein Bax but decreased the levels of anti-apoptotic protein XIAP. M344 is a potent activator of NF-kappaB transcription factor. RT-PCR assay showed that the M344 could transiently increase IL-1 expression yet markedly decreased TNF-alpha expression. Our results show that M344 is a potent growth inhibitor and inducer of apoptosis in human leukemia cells and suggest potential therapeutic strategies of HDAC inhibitors for patients with leukemias.

Fig 1

The effects of M344 on cell growth and c-fms expression. A) THP-1 cells were treated with increasing doses of M344. After 24 hrs, cells were stained by trypan blue solution and counted under light microscope. B) THP-1 cells were incubated with 0.2 μM and 2 μM M344, respectively. After indicated time, cells were stained by trypan blue solution and unstained cells were counted under light microscope. C) THP-1 cells were treated with different doses of M344 for 24 hr, or with 0.2 μM of M344 for indicated time periods. Cells were harvested, washed and lysed in SDS-loading buffer. The samples were subjected to western blot assay for c-fms protein level. β-actin level was used as an equal loading control. D) THP-1 cells (10⁵/ml) were treated with various doses of M344 for 24 hr. Adherent cells and nonadherent cells were measured to determine the percentage of adherent cells. E) THP-1 cells were treated with M344 for various time periods. Thereafter, cells were harvested and stained with acridine orange. Apoptotic cells were measured under a fluorescence microscope. F) THP-1 cells were incubated 2 μM M344 for various time periods as indicated. C-fms level was determined by western blot as described in Materials and Methods.

Fig 2

Apoptosis induction by HDAC inhibitor M344. A) THP-1 cells were treated with various doses of M344 for 24hr. Cells were harvested and lysed in SDS-loading buffer. Total protein concentration was determinate and sample was subjected to western blot assay with anti-XIAP, Bax, and Bcl-2 antibodies. B) THP-1 cells were treated with 2 μM of M344 for various time periods as indicated. Total cell lysates were subjected to western blot assay with anti-XIAP, Bax, Bcl-2 and anti-PARP antibodies. C) THP-1 cells were treated M344 for indicated time periods. The activity of caspase-3 was analyzed as described in Materials and Methods. D) THP-1 cells were treated with 2 μM of M344 for indicated time periods; cytosolic fractions were obtained and subjected to western blot assay for cytochrome c protein. β-actin was used as equal loading control.

Fig 3

Cell cycle regulation by HDAC inhibitor M344. A) THP-1 cells were treated with 0.2 μM of M344 for 48 hrs. Cells were harvested and stained with propidium Iodide staining solution followed by Flow Cytometry assay to determine the cell cycle stage. B) THP-1 cells were treated with various doses of M344 for 24 hr, or with 2 μM of M344 for indicated time periods. At the end of incubation, cells were harvested and lysed in SDS-loading buffer. Samples were subjected to western blot assay. β-actin level was used as an equal loading control.

Fig 4

Induction of NF-κB activation by HDAC inhibitor M344. A) THP-1 cells were treated with indicated doses of M344 for 3 hrs, or with 2 μM of M344 for indicated time periods. After incubation, cell nuclear extract was isolated and total protein concentration was determinate. Samples were subjected to EMSA by running through 4% non-denaturing acrylamide gel at 100 v for 30 min. The gel was dried and exposed to X-ray film. B) THP-1 cells were treated with 2 μM of M344 for 0, 6 and 12 hrs and nuclear extractions were obtained; western blot was employed to assay the nuclear p65 Rel A level in equal amount of nuclear extraction. GAPDH was used as equal loading control.

Fig 5

Effect of M344 on the expression of inflammatory cytokines in THP-1 cells. A) THP-1 cells were treated with 0.2 μM and 2 μM of M344, separately for up to 48 hrs. Cells were then harvested at indicated time and total RNA was extracted followed by RT-PCR analysis. One μg of total RNA for each sample was used to generate the cDNA through reverse transcription reaction. PCR assays for individual cytokine were conducted as described in Materials and Methods.