Thailandepsins are new small molecule class I HDAC inhibitors with potent cytotoxic activity in ovarian cancer cells: a preclinical study of epigenetic ovarian cancer therapy

Romidepsin (FK228) was obtained from Gloucester Pharmaceuticals, Celgene Corporation, Cambridge, MA. The TDPs, TDP-A and TDP-B, were discovered, patented, and provided by the Cheng group [12]. Dimethyl sulfoxide (DMSO) (Sigma Chemical Co., St Louis, MO), at a concentration of 0.01%, was used as a vehicle.

The epithelial ovarian cancer cell lines SKOV-3, OVCAR-8 and NCI/ADR-RES were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum and penicillin/streptomycin, and passaged using standard methods [8, 13]. SKOV-3 (American Type Culture Collection, Manassas, VA), OVCAR-8, and NCI/ADR-RES cells (National Cancer Institute, Bethesda, MD) are well-characterized as part of the National Cancer Institute 60 Cancer Panel [14, 15]. UWB1.289 (Brca1 null) and UWB1.289 + BRCA1 (Brca1 wild type) cell lines (American Type Culture Collection) were maintained as previously described [16]. All cell lines were used within 6 months of receipt and tested negative for mycoplasma prior to the following experiments.

Cell viability assays using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma) were conducted using established methods [8]. SKOV3 and OVCAR8 cells were seeded at a density of 1250 cells/well in 384 well plates (Corning Life Sciences, Lowell, MA), while NCI/ADR-RES cells were seeded at 1750 cells/well. Absorbance was measured at 540 nm using a Spectramax M5 spectrophotometer (Molecular Devices, Sunnyvale, CA). Cell viability was determined by measuring the percentage of viable treated cells compared to controls.

SKOV-3 ovarian cancer cells were fixed, permeabilized and stained with anti-caspase 3 (Cell Signaling Technology, Inc, Danvers, MA) as per established protocols [8, 17]. Binding of the primary antibody was detected with Alexa Flour anti-mouse IgG 488 secondary antibodies (Invitrogen, Carlsbad, CA), as appropriate. Nuclei were counterstained with DAPI (Millipore Corp., Billerica, MA). Fluorescence microscopy images were captured in 24-bit TIFF format with a Zeiss Axiocam HRC camera (Carl Zeiss MicroImaging Inc.) using Zeiss Axiovision 3.1 software, and analyzed as previously described [8, 17].

Whole cell protein isolation, Western Blotting and signal detection were performed as described from NCI/ADR-RES, OVCAR-8 and SKOV-3 ovarian cancer cells [13, 18]. For histone extraction, cell pellets were resuspended in an extraction buffer: PBS containing 0.5% Triton X100 (v/v) (Sigma), 2 mM phenylmethylsulfonyl fluoride (Sigma) and 1:100 dilutions of protease and phosphatase inhibitor cocktails (Sigma). Cells were lysed on ice for 10 min and centrifuged for 10 min at 2000 rpm at 4°C. The pellet was resuspended in 0.2 N hydrochloric acid (Sigma) supplemented with protease and phosphatase inhibitors and acid extracted overnight at 4°C. The samples were centrifuged for 10 min at 2000 rpm at 4°C, and the supernatant was collected for further analysis.

The following antibodies were used: anti-phospho H2AX (Ser¹³⁹) and anti-histone H3, (Millipore); anti-P-glycoprotein (PgP), anti-acetylated α-tubulin, anti-α-tubulin and anti-β-Actin (Sigma); and anti-Poly (ADP-ribose) polymerase (PARP) (Cell Signaling, Beverly, MA). Where applicable, corresponding levels of β-Actin, α-tubulin or histone H3 were determined to ensure equal protein loading.

Our group's experience with FK228 has led us to speculate that there are unique features of FK228 that contribute to its potency in ovarian cancer cells. The strong inhibitory activity against class I HDACs compared to class II HDACs is well-known [11]. It is possible that this preferential class I HDAC binding contributes to the cytotoxic effects of FK228. The TDPs and FK228 share a conserved bicyclic depsipeptide structure that differs significantly from SAHA, a hydroxamic acid (Figure 1). In this study we asked if TDPs, HDAC inhibitors with similar chemical and class I HDAC binding properties, produce similar biological effects in ovarian cancer cells.

The antiproliferative and cytotoxic activities of TDPs have been compared to FK228 in sulphorhodamine B cell growth assays in the NCI60 panel of cancer cell lines [12]. Of the seven ovarian cancer cell lines present in the panel, SKOV-3 cells were the most sensitive, NCI/ADR-RES cells were profoundly resistant and OVCAR-8 cells exhibited an intermediate response. Therefore, we chose this subset of the NCI60 panel for further investigation of the cytotoxic effects of the TDPs. In addition, we included the well-characterized isogenic Brca1 null and wild type (WT) cell lines in our study, because of the high incidence of BRCA1 dysfunction in ovarian cancer [16].

First, we performed MTT assays to independently test the effects of FK228 compared to those of the TDPs on ovarian cancer cell viability in vitro. As shown in Figure 2A, TDP-B displayed similar inhibitory effects on cell viability as an equal concentration of FK228 (10 nM) in the three NCI60 cell lines and the isogenic Brca1 wild-type and null cell pair. Although TDP-A (10 nM) induced significant inhibitory effects on cell viability in all cell lines except NCI/ADR-RES, the magnitude of inhibition was consistently less than that observed for TDP-B (Figure 2A). The NCI/ADR-RES ovarian cancer cells were relatively resistant to FK228 and both TDP compounds. Interestingly, we have published that NCI/ADR-RES cells are exquisitely sensitive to SAHA, which is structurally different from the FK228-like compounds [8]. NCI/ADR-RES cell lines highly express the multidrug resistant gene mdr1/ABCB1 that encodes for PgP [15]. FK228 is known to induce PgP [19‐21], suggesting that further upregulation of PgP contributes to relative resistance to FK228. Similar to FK228, the TDPs upregulated PgP in NCI/ADR-RES cells (Figure 2B). This activation of PgP may contribute to the NCI/ADR-RES cellular resistance to the TDPs. In contrast, PgP was not detectable in the relatively sensitive cell lines, SKOV-3 and OVCAR-8, either in treated or untreated cells.

Brca1 null cells, which harbor a truncating Brca1 mutation, are known to be more sensitive than their wild-type counterparts to various agents that promote DNA damage [16]. In the MTT assays, the Brca1 status of the cells was similarly observed to determine sensitivity to the small molecule HDAC inhibitors, since FK228 and the TDPs exerted greater effects in the Brca1 null cells.

Although TDP-A has previously been shown to exhibit the greatest growth inhibitory effects across cell lines, the cytotoxic activity of TDP-A is lower than FK228 and TDP-B in ovarian cancer cells [12]. One possible mechanism for the observed differences in cytotoxicity is differential induction of apoptosis. Therefore, we examined the effects of TDP-A and TDP-B on apoptosis using two independent assays. Caspase 3 activation measured by immunofluorescence staining for cleaved caspase 3 was similarly induced by FK228 and TDP-B compared to controls in SKOV-3 cells (Figure 3), while fewer TDP-A-treated cells displayed activated caspase-3. Western blot analysis of PARP cleavage was performed to validate these findings. Similarly, cleaved PARP was activated by FK228 and TDP-B to a greater extent than by TDP-A (Figure 4A). As anticipated by the relative resistance to FK228 and the TDPs, there was no evidence of apoptosis induction by these HDAC inhibitors in NCI/ADR-RES cells.

We have demonstrated previously that pH2AX (a sensitive histone mark of DNA damage) is a useful indicator of HDAC cytotoxicity and a potential mechanism for inducing apoptosis in ovarian cancer [8, 22]. Prolonged induction of pH2AX foci after genotoxic injury is associated with irreparable DNA damage and apoptosis [22]. Therefore, we evaluated the activation of pH2AX 24 h after exposure to FK228, TDP-A and TDP-B. In relatively sensitive ovarian cancer cells OVCAR-8 and SKOV-3, FK228 and TDP-B induced pronounced upregulation of pH2AX expression compared to controls (Figure 4B). However, in TDP-A treated cells, pH2AX activation was not as robust. In the NCI/ADR-RES cells, pH2AX was minimally activated, reflecting the relative resistance to FK228-like drugs.

Inhibition of class I HDACs is known to contribute to DNA damage and cytotoxicity in ovarian cancer cells [7, 8]. The cellular responses we observed in the studies presented here support our hypothesis that FK228-like compounds that target class I HDACs exhibit potent antitumor activity. Enzymatic assays comparing TDP-A and TDP-B to FK228 have shown that both TDPs bind to class I HDACs with 30-100 times more potency than to class II HDACs [12] (Figure 5A). Therefore, we tested the effects of TDP-A and TDP-B on a target of class II HDAC6 inhibition, tubulin acetylation [23]. We evaluated tubulin acetylation by Western blot after treatment with TDPs and FK228 at drug concentrations that inhibited cell viability and induced markers of apoptosis and DNA damage response (Figure 5B). However, we found no significant change in acetylated tubulin compared to DMSO treated controls. These results suggest that HDAC6 inhibition is not critical for the antitumor effects of TDP-A and TDP-B, and are in line with previous data showing that low levels of tubulin acetylation do not correlate with the biological activity of HDAC inhibitors in ovarian cancer cells [8].