The human first trimester extravillous trophoblast (EVT) cell line (HTR-8) cultured in DMEM/F12 and two human CC cell lines (JAR and JEG-3) cultured in DMEM were obtained from the American Type Culture Collection (Manassas, VA, USA). These cells were supplemented with 10% FBS and incubated in a 37°C humidified incubator containing 5% CO₂. Notch activity was blocked by a 10 μM concentration of DAPT (Sigma, St. Louis, MO, USA), added at each day of culturing.

Total RNA was extracted from above-mentioned cell lines according to RNeasy Mini kit (Qiagen, USA) manufacturer’s protocol and quantitated by spectrophotometry. According to the manufacturer’s protocol, mRNA was then reverse transcribed using Revert Aid™ First Strand cDNA Synthesis kit (MBI Fermentas, St. Leon-Rot, Germany). The SYBRR Premix Ex Taq™ II system (Takara, Dalian, China) and the Bio-Rad CFX96™ Real-time system (Bio-Rad, CA, USA) were used to perform real-time quantitative PCR. Following the reverse transcription, 1 μl primer (10 μM, summarized in Table I), 12.5 μl SYBRR Premix Ex Taq II, 2 μl cDNA and 8.5 μl dH₂O mixed together, pre-degeneration for 95°C, 30 sec, one repeat, and PCR reaction, 95°C 5 sec followed by 60°C, 30 sec, 35 repeats, and the dissociation stage, 95°C, 15 sec followed by 60°C, 30 sec, and 95°C, 15 sec, then the data were collected and analyzed. β-actin (25) was used as the internal control. The threshold cycle (Ct) value for triplicate reactions was averaged. Melting curve for the primers was analyzed to confirm the specificity of the PCR product. The relative expression of mRNA for each target gene was calculated as follows: ΔCt = Ct(target) - Ct(β-actin), ΔΔCt = ΔCt - ΔCt(calibrator), and the fold changes in mRNAs were calculated through relative quantification (2^−ΔΔCt).

Cells were harvested at 80–90% confluence, and washed with 4°C PBS three times. Total cellular protein lysates were reconstituted with RIPA buffer [50 mM Tris (pH 8.0), 0.1% SDS, 150 mM NaCl, 1% NP40 and 0.5% sodium deoxycholate] and proteinase inhibitors [1 mM PMSF (Sigma)]. The denatured protein samples was separated by SDS-PAGE and electrophoretically transferred to nitrocellulose (NC) membranes. The NC membranes were blocked with 5% skim milk at room temperature for 1 h, then incubated in different monoclonal or polyclonal antibodies with different dilutions by 5% BSA overnight at 4°C and washed with TBST three times (with Tween-20, pH 7.6). Subsequently, membranes were incubated in secondary antibodies (Licor, Rockford, IL, USA) in the dark for 1 h. The NC membranes were scanned with Odyssey detection system (Licor). MG-132 (Sigma) was applied to inhibit the proteasome-dependent degradation if necessary (10 μM, 4 h before the protein harvest) (26). β-actin (25) were used as the loading controls.

The NCBI accession number for the Homo sapiens HIF-1α gene sequence is NM_001530.3. The CDS of HIF-1α was used to construct lentiviral vectors. HIF-1α cDNA was amplified and cloned into the lentiviral (lv) vector GV358 (Genechem, Shanghai, China), a lentiviral vector encoding the enhanced green fluorescent protein (EGFP) gene cDNA downstream of the ubi promoter (Lenti-ubiluc). To prepare lentiviral particles, the lentiviral vector overexpressing HIF-1α gene (lv-HIF-1α), pHelper 1.0 plasmid and pHelper 2.0 helper plasmid were co-transfected into 293T cells according to the manufacturer’s protocol. The lentiviral vector were used to infect JAR and JEG-3 cells at a multiplicity of infection (MOI) of 40 and 100, respectively. Lentivirus vectors only expressing EGFP gene (lv-NC) was used as a transfection control. Seventy-two hours after infection, EGFP expression was observed to evaluate the infection efficiency. Cells were harvested 96 h after infection. The expression level of HIF-1α was examined by qPCR and western blotting. These transfectants were selected by 2 or 1 μg/ml puromycin respectively (Sigma) to establish the stable clone cell lines and analyze with fluorescent microscopy. After the DAPT treatment was performed, the cells were harvested for further analysis.

To assay cell migration, JAR and JEG-3 cells after lentiviral transfection (lv-HIF-1α and lv-NC) were scratched in 6-well plates with pipette tip when the cells were at 80% confluence. Cells were incubated in serum-free medium, and the width of the scratches was measured at 0 and 24 h after scratching. DMSO (0.1%) or 10 μM DAPT was added to medium.

Cell invasion were performed by Boyden chamber assay. The 8-mm pore size upper chamber with 50 μl Matrigel (Sigma) was incubated for 4 h. For migration, the serum-free medium suspension with 5×10⁴ cells was added in the upper chamber, and 800 μl medium with 20% serum was added in the lower chamber of 24-well plate. Cells (5×10⁴/well) were seeded in the same medium in a well without chamber simultaneously. 0.1% DMSO or 10 μM DAPT was added to medium in upper and lower chambers. After 24 h, the chambers were washed with PBS (pH 7.4) three times. The non-invaded cells of upper surface in chamber were removed with a cotton swab. The cells of lower surface was fixed with 4% formalin for 10 min, then stained with crystal violet (0.01% in ethanol, Sigma) for 20 min followed by PBS washing three times. Five random visions were taken by inverted microscope, and the cell number was counted.

Considering that the JAR cell line is more easily infected by lentiviruses than the JEG-3 cell line (unpublished data), the JAR cells transfected with lv-NC or lv-HIF-1α were used in vivo. Cells were cultured and collected in a single-cell suspension in PBS (pH 7.4). Suspension (200 μl) (5×10⁶ lv-NC or lv-HIF-1α cells) was implanted under the dorsal skin of 4- to 6-week-old female SCID mice to form a subcutaneous tumor. Based on the references (27–29) and previous studies, the mice were randomly divided into four groups (n=5): i) JAR (lv-NC) treated with 0.1% DMSO diluted with saline; ii) JAR (lv-NC) treated with DAPT (10 mg/kg); iii) JAR (lv-HIF-1α) treated with 0.1% DMSO diluted with saline; iv) JAR (lv-HIF-1α) treated with DAPT (10 mg/kg). The mice were treated with DAPT or DMSO intraperitoneally six times at 3-day intervals (on days 1, 4, 7, 10, 13 and 16). Tumors were observed for growth, swelling, and diabrosis, and the tumor volumes were measured daily. The following experiment was conducted when the tumors reached 15–75 mm³ in size.

All mice were induced with 4% isoflurane and maintained on 2–2.5% isoflurane in preparation for SA-PET/CT scans. Subsequently, ¹⁸F-FDG tracer were injected intravenously under general anesthesia. The PET/CT imaging was carried out for evaluation of uptake in the subcutaneous tumors (30). A CT scan was obtained for reconstruction and further analysis of the PET/CT data. Standardized uptake value (SUV) were used to compare variations among groups.

To establish a model for monitoring of Notch signaling in choriocarcinoma metastasis, the JAR cells transfected with lv-NC or lv-HIF-1α (200 μl, 5×10⁶ cells) were injected intravenously into femal SCID mice. The groups and treatment were the same as the growth assay. Their metastasis was monitored by using In Vivo Imaging System (IVIS; Xenogen, Alameda, CA, USA) at the end of treatment. Animal studies were approved by the Ethics Committee of our Hospital.

Each of the experiments were performed in triplicate and repeated three times. Statistical analysis was carried out using the SPSS 18.0 statistical software package (SPSS Inc., Chicago, IL, USA). Summary data are showed as means ± SEM. Group means were compared using the appropriate version of the Student’s unpaired t-test. P-values were 2-sided. Test results were considered significant at P<0.05.

We examine the mRNA and protein expression levels of HIF-1α in CC cell lines and HTR-8. The expression levels of HIF-1α were markedly upregulated in JAR and JEG-3 cells, as compared to the expression level of a control HTR-8 cell line (Fig. 1A, P<0.01).

To mimic the hypoxic microenvironment and further define our understanding of HIF-1α in choriocarcinoma, JAR and JEG-3 cells were transfected with lentivirus of HIF-1α (lv-HIF-1α) to establish stable cell lines that upregulated expression of HIF-1α in CC cells. Simultaneously, EGFP expression was observed in CC cells transfected with the corresponding lv-NC. Compared to the lv-NC cells, the mRNA and protein level of HIF-1α were significantly upregulated in lv-HIF-1α cells (P<0.01, respectively, Fig. 1B and C). Consistently, upregulation of HIF-1α promotes corresponding HIF-1β expression on mRNA and protein level (P<0.01).

In order to investigate whether HIF-1α induces EMT of CC cells, inverted microscopy was used revealing that Lv-HIF-1α cells formed a spindle-like monolayer with more elongated and larger gaps between cells than lv-NC cells which formed a cobblestone-like monolayer (Fig. 2A). Western blot analyses further confirmed that the EMT phenotype is induced by over-expression of HIF-1α in CC cells. HIF-1α markedly decreased E-cadherin, CK-18 and CK-19 expression in lv-HIF-1α cells, the reciprocal changes associated with the EMT phenotypic transition (P<0.01, respectively, Fig. 2B and C).

To study HIF-1α-induced EMT with malignant behavior, the scratch wound-healing assay and Matrigel membrane invasion assay were used. The extent of migration of CC cells with exogenous HIF-1α into the scratched area was elevated (Fig. 3A, P<0.05). Similarly, the invasive capability was significantly enhanced when HIF-1α was upregulated (Fig. 3B, P<0.05). Consistent with the data, MMP2 and MMP9 were upregulated by HIF-1α overexpression (Fig. 3C, P<0.01).

The elevated Notch response after mimicked hypoxia was observed at two levels in the Notch signaling pathway. On the receptor side, the level of N1ICD was elevated in JAR cells subjected to HIF-1α. On the downstream side, upregulation of Hes1 mRNA and protein was observed in CC cells, simultaneously (P<0.01, Fig. 4A). The DAPT data would suggest that the inhibitor blocked the conformation of N1ICD protein to abrogate Notch signaling, then Hes1 mRNA and protein were decreased in JAR cells (P<0.01, Fig. 4A). Similar data were obtained for the JEG-3 cell line (P<0.01, Fig. 4B).

To evaluate the involvement of Notch in HIF-1α-induced EMT in CC cells, 0.1% DMSO or 10 μM DAPT was added in culture medium. Inhibition of Notch signaling abolished the downregulation of E-cadherin, CK-18, CK-19 mRNA and protein levels observed in lv-HIF-1α cells (P<0.01, respectively, Fig. 5). Because Snail-1 is the crucial regulator of E-cadherin expression, the data would hint that E-cadherin expression is mediated via regulating Snail-1 at the transcriptional and protein level. Upregulation of Snail-1 by exogenous HIF-1α was abrogated by DAPT treatment in CC cells (P<0.01, Fig. 5).

Given the fundamental interaction between Notch signaling and HIF-1α-induced EMT with enhanced ability of migration/invasion, we considered whether HIF-1α-induced upregulation of MMPs require Notch signaling. The data revealed that MMP2 and MMP9 were upregulated by HIF-1α but substantially blocked in the presence of DAPT (P<0.01, respectively, Fig. 6).

To determine whether the observed antitumor activity in vitro by Notch inhibitors could be extended in vivo, DAPT was injected intraperitoneally into SCID mice with tumor xenografts of the JAR cells transfected with lv-NC or lv-HIF-1α six times at 3-day intervals. Here, SA-PET/CT scans of subcutaneous tumors were performed on mice and SUV of ¹⁸F-FDG binding in vivo scans are provided in Fig. 7A. ¹⁸F-FDG uptake in nodules of the lv-HIF-1α mice treated with DMSO was high, while the nodules of lv-NC mice treated with DMSO were at low levels. DAPT-treated groups showed only slight ¹⁸F-FDG activity in the tumors of the lv-NC group, unlike the high level of activity in the lv-HIF-1α group (Fig. 7A). After PET/CT scans, we removed the red-brown transplantation tumor (Fig. 7B). The transplantation tumors grew in elliptical shapes with the purplish-black covering skin. The DMSO groups showed that HIF-1α alone significantly promoted tumor growth. Compared with the DMSO group, DAPT treatment significantly inhibited tumor growth (P<0.05, Fig. 7C).

To assess the capacity of suppression of endogenous Notch signaling as a treatment for choriocarcinoma cell metastasis, JAR cells stably expressing EGFP were injected intravenously into SCID mice as an in vivo model of metastasis. In mice treated with DAPT or DMSO six times at 3-day intervals from the time of cell injection. Under IVIS system, macroscopic appearance of metastatic tumors were detected in lung of 85% (17/20) mice at 30 days after intravenous injection. Positive signals were detected in 100% (5/10, lv-HIF-1α) and 100% (5/5, lv-NC) of mice treated with DMSO, respectively. Because there were multiple metastatic lesions found in the lungs, positive signals from isolated lesions were integrated for the measurement of photon counts in each mouse. The DMSO groups showed that the levels of detectable EGFP signals for the lv-HIF-1α mice were stronger than lv-NC mice. In contrast, 80% (4/5, HIF-1α) and 60% (3/5, lv-NC) of mice treated with DAPT had positive signals. In contrast to increased areas of detectable EGFP in DMSO-treated lv-HIF-1α and lv-NC mice, the EGFP-positive areas were limited in mice with DAPT treatment (Fig. 8). No side effect was observed throughout the experiments in the tested mice.