Decitabine and suberoylanilide hydroxamic acid (SAHA) inhibit growth of ovarian cancer cell lines and xenografts while inducing expression of imprinted tumor suppressor genes, apoptosis, G2/M arrest, and autophagy

Abstract

Background: Epigenetic therapy has had a significant impact on the management of hematologic malignancies, but its role in the treatment of ovarian cancer remains to be defined. The authors previously demonstrated that treatment of ovarian and breast cancer cells with DNA methyltransferase and histone deacetylase (HDAC) inhibitors can up-regulate the expression of imprinted tumor suppressors. In this study, demethylating agents and HDAC inhibitors were tested for their ability to induce re-expression of tumor suppressor genes, inhibiting growth of ovarian cancer cells in culture and in xenografts.

Methods: Ovarian cancer cells (Hey and SKOv3) were treated with demethylating agents (5-aza-20-deoxycytidine [DAC] or 5-azacitidine [AZA]) or with HDAC inhibitors (suberoylanilide hydroxamicacid [SAHA] or trichostatin A [TSA]) to determine their impact on cellular proliferation, cell cycle regulation, apoptosis, autophagy, and re-expression of 2 growth inhibitory imprinted tumor suppressor genes: guanosine triphosphate-binding Di-RAS-like 3 (ARHI) and paternally expressed 3 (PEG3). The in vivo activities of DAC and SAHA were assessed in a Hey xenograft model.

Results: The combination of DAC and SAHA produced synergistic inhibition of Hey and SKOv3 cell growth by apoptosis and cell cycle arrest. DAC induced autophagy in Hey cells that was enhanced by SAHA. Treatment with both agents induced re-expression of ARHI and PEG3 in cultured cells and in xenografts, correlating with growth inhibition. Knockdown of ARHI decreased DAC-induced autophagy. DAC and SAHA inhibited the growth of Hey xenografts and induced autophagy in vivo.

Conclusions: A combination of DAC and SAHA inhibited ovarian cancer growth while inducing apoptosis, G2/M arrest, autophagy, and re-expression of imprinted tumor suppressor genes.

Figure 1. Antiproliferative activities of demethylating agents and histone deacetylase inhibitors on ovarian cancer cells

(A) Hey or (B) SKOv3 cells were treated with indicated concentrations of demethylating agents (DAC, AZA) and HDAC inhibitors (SAHA, TSA), individually or in combinations. Cell proliferation was determined on day 5 with SRB assays. Data plotted represent optical density of SRB stained cells treated with the different agents normalized to values for untreated control cells.

Figure 2. DAC and SAHA induce apoptosis in ovarian cancer cells

Ovarian cancer cells were treated with DAC and SAHA for 5 days at their respective IC₅₀ concentrations (see Table 1). Apoptosis in control and treated cells were determined by analysis of caspase 3/7 activities in (A) Hey or (B) SKOv3 cells or by flow cytometry in (C) Hey or (D) SKOv3 cells.

Figure 3. DAC and SAHA induce cell cycle arrest

(A) Hey or (B) SKOv3 cells were treated with DAC, SAHA or a combination at their respective IC₅₀. On days 1, 3, and 5, cells were harvested and the fraction of cells in G1, S and G2/M phases was determined by flow cytometry.

Figure 4. DAC induces autophagy in Hey cells but not in SKOv3 cells

(A) Hey and SKOv3 ovarian cancer cells were transfected with GFP-LC3 plasmids and treated with DAC, SAHA, or a combination at their respective IC₅₀ concentrations. Formation of punctate GFP-LC3 spots is indicative of autophagy. (B) Hey and SKOv3 cells were treated with DAC or SAHA for 1, 3, and 5 days. After staining with acridine orange, the fraction of autophagic cells was determined by flow cytometry. (C) Hey cells were treated with DAC or SAHA, or a combination for 5 days. Cells were then harvested and processed for TEM. The arrows indicate autophagosomes. N, nucleus; M, mitochondria.

Figure 5. DAC reactivates ARHI and PEG3 expression in ovarian cancer cells

(A) Hey and (B) SKOv3 cells were treated for 5 days with indicated concentrations of DAC, SAHA, or a combination. Cells were analyzed for cell proliferation by SRB staining (left column), and for ARHI (middle column) and PEG3 (right column) expression by real-time RT-PCR. The quantitative analyses were repeated at least two times and one representative experiment is shown.

Figure 6. ARHI expression is required for DAC and SAHA-induced autophagy in Hey ovarian cancer cells

Hey cells were transfected with control siRNA (siControl) or ARHI siRNA (siARHI) and treated with DAC, SAHA, or a combination at their respective IC₅₀ concentrations. Autophagic cells were determined by acridine orange staining. The figure shows results from one representative experiment.

Figure 7. DAC and SAHA inhibit growth of xenograft tumor and induce autophagy

(A) Mice were injected i.p. with Hey (1×10⁶) cells. Two days later, mice were injected i.p. with (1) saline (control) (2) DAC, (3) SAHA, and (4) a combination of DAC and SAHA as described in Materials and Methods. Five mice from each group were evaluated daily for survival. (B) Hey xenograft tumors were treated as indicated for 21 days before they were harvested and processed for TEM. The arrows indicate the autophagosomes. N, nucleus; M, mitochondria.

Figure 8. Treatment with a combination of DAC and SAHA reactivates the expression of ARHI and PEG3 in xenografts

Hey xenograft tumors were treated with a combination of DAC and SAHA for 21 days. Tumors from two mice were harvested and processed for (A) RNA extraction to quantify ARHI and PEG3 RNA transcripts by real-time RT-PCR or (B) immunohistochemical staining with anti-ARHI antibody.