Histone deacetylase inhibitors induce apoptosis in both Type I and Type II endometrial cancer cells

Abstract

Objective: To characterize the molecular pathways involved in apoptosis following administration of histone deacetylase inhibitors to Type I and II endometrial cancer cells.

Methods: Ark2, Ishikawa, and AN3 cell lines representing both Type I and II endometrial cancers were treated with various concentrations of oxamflatin and HDAC inhibitor-1. Cell apoptosis was determined by flow cytometry, nuclear staining, Western blotting, and mitochondrial membrane potential assays.

Results: Compared to controls, there was a 95% reduction in the growth of Ark2 cells following administration of histone deacetylase inhibitors and this response was dose-dependent. These agents also caused profound morphologic changes and loss of mitochondrial membrane potentials consistent with the induction of apoptosis. Cleavage of PARP, caspase-9, and caspase-8 was detected, confirming the activation of apoptotic cascades in endometrial carcinoma cells. This effect was present in both serous and endometrioid cell types.

Conclusion: Our results suggest that oxamflatin and HDAC inhibitor-1 have potent cytotoxicity in endometrial cancer cells by inducing cell apoptosis. Histone deacetylase inhibitors are promising agents for the treatment of both Type I and II endometrial carcinoma.

Figure 1

Effects of HDAC inhibitors on endometrial cancer cell growth. Ark2, Ishikawa, and AN3 cells were treated with oxamflatin (Oxam, 0.25 μM), HDAC inhibitor-1 (HDAC-I1, 0.5 μM), or DMSO solvent as control (Con). Cell number was counted once a day following treatment. While both drugs significantly inhibited cell proliferation, the three cell lines exhibited different sensitivity profiles. Oxamflatin appears to be especially effective in Ark2 cells; Ishikawa cells are equally sensitivity to the two regents; AN3 cells showed stronger response to HDAC-I1 than to oxamflatin.

Figure 2

Dose–response of endometrial cancer cells to HDAC inhibitors. Ark2, Ishikawa, and AN3 cells were treated with increasing concentrations of oxamflatin (upper panel, 0, 0.25, and 0.75 μM) and HDAC-I1 (lower panel, 0, 0.5, and 1.5 μM) for 3 days and survival cells were harvested and counted. The cell number was compared between experiment and control groups and statistical significance (p<0.05) was marked by asterisks on the top of standard error bars.

Figure 3

(A) Nuclei staining assay. The three endometrial cancer cell lines were treated with oxamflatin (0.5 μM) and HDAC-I1 (1.0 μM) for 3 days. Nuclei condensation and fragmentation were detected by Hoechst staining of genomic DNA. Depending on cell type and drug used, a three- to nine-fold increase in apoptotic nuclei was observed. (B) Flow cytometry analysis of cell apoptosis. Following three days of treatment with oxamflatin (0.25 μM) and HDAC-I1 (0.5 μM), the cells were stained with biotin-labeled Annexin V and apoptotic cells counted by flow cytometry. The results confirm significant cell death induced by oxamflatin and HDAC-I1 treatment. Similar drug sensitivity profiles to those observed in cell growth experiments (Fig. 1) were found in the nuclei staining and flow cytometry experiments. Statistical significance between experimental and control groups (p<0.05) is marked by asterisks on the top of standard error bars.

Figure 4

Morphological changes induced by HDAC inhibitors. The three cell cultures were treated with oxamflatin (0.25 μM) and HDAC-I1 (0.5 μM) and pictures were taken after 3 days' treatment. Generally, upon treatment cells tend to become round and enlarged (indicated by solid arrows). Many dead cells can be seen floating in the medium (open arrows). Some treated cells form digitiform processes (indicated by arrowheads). The two reagents appear to induce similar morphological changes in given cell lines.

Figure 5

Loss of mitochondria membrane potentials. Cells were treated with oxamflatin (0.25 μM), HDAC-I1 (0.5 μM), or Thapsigargin (0.75 μM) as a positive control for 2 days. The loss of mitochondrial potential was detected with MitoTrack red (CMXRos). A significant increase (p<0.05, marked by asterisks) in the percentage of cells with dissipated mitochondria membrane potentials were observed following treatment with both HDAC inhibitors.

Figure 6

Western Blot analysis of apoptotic pathways. The cleavage of PARP, caspase-8, and caspase-9, was determined using specific antibodies as described under Materials and methods. The pro-apoptotic proteins and their cleaved products were indicated by arrows on the top and bottom, respectively. For each blot, β-actin levels were measured as a protein loading control. Oxamflatin (0.25 μM) and HDAC-I1 (0.5 μM) treatments resulted in significant activation of PARP in Ark2, Ishikawa and AN3 cells. Oxamflatin displayed preferential effects on caspase-9 cleavage in Ark2 in comparison to Ishikawa and AN3 cells. While oxamflatin induced caspase-8 cleavage in Ark2 cells, HDAC-I1 has little effect in this cell line.