The human ovarian cancer cell lines A2780 and SKOV3 were purchased from the American Type Culture Collection and cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FCS. All cells were cultured at 37°C in a humidified 5% CO₂ atmosphere. HDACis, TSA and SAHA, were purchased from Sigma and dissolved in DMSO.

Ovarian cancer tissues were obtained from 6 patients hospitalized in the Beijing Obstetrics and Gynecology Hospital affiliated to Capital Medical University before any clinical therapy. All research protocols in the present study were approved by our Ethics Committee, and all patients gave written informed consent to enroll in the study. These patients were 17–75 (mean, 47.1±13.5) years of age and were hospitalized between June 2012 and December 2013; ovarian cancer was confirmed by pathological diagnosis. Four patients were pathologically classified as serous adenocarcinoma and 2 as mucoid adenocarcinoma. According to the Federation International of Gynecology and Obstetrics (FIGO) staging system, 1 case was classified as FIGO stage I, 2 as FIGO stage II, 2 as FIGO stage III, and 1 as FIGO stage IV. Cells were isolated and cultured as previously described (15).

Cell viability was determined using an MTT assay. In brief, 5×10³ cells were plated into each well of 96-well plates at 72 h after the indicated treatments, after which 5 mg/ml MTT was added and incubated at 37°C for 4 h. Media were then removed, and 1 ml of DMSO was added to solubilize the MTT-formazan product. The MTT absorbance was then measured at 570 nm on a Multiscan JX ver 1.1 (Thermo Labsystems). Results are expressed as a percentage of the viable cells in the DMSO-treated group. Each data point is the mean ± SEM of six replicates.

Cells were stained with Annexin V and propidium iodide (PI) and the percentage of apoptotic cells were determined by flow cytometry as previously described (15). CellQuest software was used for data acquisition and analysis.

Quantitative PCR was conducted in ABI Prism 7000 using the SYBR-Green PCR Master Mix (Sigma) with the following set of primers: LIV1, 5′-GGT GAT GGC CTG CAC AAT TTC-3′ and 5-TTA ACG GTC ATG CCA GCC TTT AGT A-3; 18s RNA, 5′-AGT CCC TGC CCT TTG ACA CA-3′ and 5′-GAT CCG AGG GCC TCA CTA AAC-3′. 18s RNA was used as internal control. All primers were designed with the Primer3 software. A melting curve assay was performed to determine the purity of the amplified product. Contamination with genomic DNA was not detected in any of the analyzed samples. Each sample was assayed in triplicate, analysis of the relative gene expression data used the 2^−ΔCT method (15), and the results are expressed as fold induction compared with the untreated group.

Preparation of protein samples and western blotting were carried out as previously described (15). Antibodies against LIV1 were purchased from Novus Biologicals. Antibodies against Bcl-2, Bax, and caspase-3 were purchased from Cell Signaling Technology. Antibodies against β-actin were purchased from Santa Cruz Biotechnology.

A2780 and SKOV3 cells were stably transfected with PCEP4-CAT and AS-LIV1/FL-LIV1 and cultured for 24 h. Cells were then treated with 500 nmol/l TSA or 500 nmol/l apicidine for 24 h and plated in triplicate in 24-well plates at 50 cells/well. Plates were subsequently incubated for 14 days in a humidified incubator at 37°C, and colonies were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet and counted using a dissecting microscope (magnification, ×50). Three random fields were counted for each triplicate of samples, and average values were presented as the means ± SD.

To test whether expression of LIV1 is induced by TSA, we investigated the effects of TSA on the mRNA and protein expression of LIV1 in the ovarian cancer cells. A2780 and SKOV3 cells were chosen because they had moderate levels of LIV1. First, cells were treated with 500 nmol/l TSA for various lengths of time. As shown in Fig. 1A and B, transcription of LIV1 was highly induced. At 12 h post-treatment with TSA, transcription induction reached a maximal level (5.63±0.80-fold for A2780; 4.67±0.51-fold for SKOV3; P<0.05, compared with the basal transcriptional level). To address whether the induction of LIV1 transcription gave rise to the upregulated level of LIV1 protein, cultured A2780 and SKOV3 cells were treated with TSA or DMSO and examined for the LIV1 protein using western blotting. As expected, treatment of TSA significantly enhanced the protein levels of LIV1 in the ovarian cancer cells (Fig. 1C and D). Next, to determine whether the TSA-induced expression of LIV1 occurs in primary ovarian cancer cells, 6 primary tumor samples from patients with ovarian cancer were treated with 500 nmol/l TSA for different time periods. Again, TSA significantly induced the expression of LIV1 at a maximal level (patient 1 for example: 3 h, 15.3±3.24-fold; 6 h, 10.16±2.54-fold; and 12 h, 8.75±2.93-fold; P<0.05) as early as 3 h after treatment and the increase lasted up to 12 h in all of the samples examined (Table I).

To confirm whether expression of LIV1 significantly affects TSA-induced cell death in ovarian cancer cells, A2780 and SKOV3 cells were stably transfected with the LIV1 antisense plasmid AS-LIV1 or PCEP4-CAT followed by a 5-day treatment with TSA, and they were then examined for growth inhibition. AS-LIV1 was confirmed to significantly knockdown the basal and TSA-induced levels of LIV1 expression (Fig. 2A), and transfection of AS-LIV1 resulted in the resistance of the cells to TSA treatment (Fig. 2B and C). Furthermore, we treated the A2780 and SKOV3 cells stably transfected with AS-LIV1 or PCEP4-CAT with a TSA dose range between 300 and 700 nmol/l for 72 h and measured the cell viability using the MTT assay. As shown in Fig. 2D and E, knockdown of LIV1 decreased the TSA-induced killing efficiency in both the A2780 and SKOV3 cells at every dose, with a maximum effect observed at a 500 nmol/l concentration, where the rate of viable cells was increased over 29% in the A2780 and 24% in the SKOV3 cells. In addition, knockdown of LIV1 suppressed TSA- or SAHA (a structurally diverse HDACi)-induced killing and gave rise to more surviving colonies (Fig. 3A and B). Accordingly, knockdown of LIV1 made A2780 and SKOV3 cells resistant to TSA-induced apoptosis (Fig. 3C and D). A2780 and SKOV3 cells stably transfected with AS-LIV1 were much less sensitive to TSA-induced apoptosis than cells stably transfected with PCEP4-CAT (A2780, 18.7±3.6 vs. 49.06±4.3%, P<0.05; SKOV3, 16.28±2.9 vs. 38.13±3.5%, P<0.05).

To further verify whether LIV1 is capable of conferring a significant resistance to TSA-induced cell death in ovarian cancer cells from a contrasting perspective, we conducted LIV1 full-length plasmid FL-LIV1, which was confirmed to obviously increase the level of LIV1 expression (Fig. 4A). A2780 cells stably transfected with FL-LIV1 were much more sensitive to TSA-induced apoptosis than cells stably transfected with PCEP4-CAT (69.02±4.5 vs. 48.01±3.7%; P<0.05) (Fig. 4B). Moreover, overexpression of LIV1 promoted TSA- and SAHA-induced cell death and decreased the number of surviving colonies (Fig. 4C). Therefore, it appears that LIV1 modulates the killing efficacy of TSA and may be a critical regulator of cell growth and death in ovarian cancer cells.

Our previous data suggest that the inhibition of TSA-induced apoptosis by knockdown of LIV1 might be associated with its ability to disrupt intracellular zinc homeostasis (12). Zinc was shown to be a critical regulator of cell growth and death. Previous findings indicate that the mechanistic actions of zinc are shown through change in caspase enzyme activities, as well as the direct alteration of apoptotic regulator expression (16–19). As shown in Fig. 5, TSA induced apoptosis by decreasing endogenous levels of Bcl-2, enhancing levels of Bax and cleavage of procaspase-3. In contrast, the TSA-induced alteration mentioned above could be significantly reversed by LIV-1 knockdown, indicating that LIV1 plays a critical role rather than a by-phenomenon in TSA-mediated apoptosis.