The human HCC cell lines (HepG2 and SMMC-7721 were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences) were cultivated in DMEM medium supplemented with 10% FCS (fetal calf serum, Hyclone laboratories, Logan, UT, United States). All experiments were carried out using a confluent monolayer of HCC cell cultures. Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO₂. The primary antibodies for Notch1 (120 kDa), E-cadherin (120 kDa), SNAIL1 (29 kDa), SNAIL2 (29 kDa), MMP-2 (74 kDa), XIAP (55 kDa), VEGF (31 kDa) and GAPDH (37 kDa) were purchased from Santa Cruz Biotechnology (SantaCruz, CA, United States). All secondary antibodies were obtained from Pierce (Rockford, IL, United States). Small interfering RNA (siRNA) targeting Notch1 and control siRNA (siCON) were obtained from Santa Cruz Biotechnology. Lipofectin^TM2000 was purchased from Life Technologies (Carlsbad, CA, United States). All other chemicals and solutions were purchased from Sigma-Aldrich unless otherwise indicated.

HepG2 and SMMC7721 cells were seeded in 96-well plates and treated with GSI-I or/and IL-24 for 48 h, separately. Then, 10 μL of 3-(4,5-dimethylthiazolyl-2) 2,5-diphenyltetrazolium bromide (MTT, 5 mg/mL, Sigma-Aldrich) was added to each well and incubated for 4 h at 37 °C. The formazan granules were dissolved in 150 μL dimethyl sulfoxide (DMSO) for 10 min. Optical density (OD) was then measured at a wavelength of 490 nm. Each MTT assay was performed in quadruplicate and repeated three times.

In order to observe the presence of condensed chromatin and apoptotic bodies, cells were stained with Hoechst 33258 dye. Cells seeded in 96-well plates were fixed in 3:1 methanol/acetic acid for 10 min at room temperature, washed in PBS (phosphate buffered saline) and stained for 30 min in PBS with 40% paraformaldehyde and 10 μg/mL Hoechst 33258. After washing in PBS for several times, nuclear morphology was observed under a fluorescence microscope (Zeiss, Germany).

To further verify the apoptotic phenotype, cell cultures were also analyzed with an Annexin V-FITC/propidium iodide (PI) kit (Roche, Manheim, Germany), according to the manufacturer’s instructions. Annexin-V immuno-cytofluorescence was detected by flow cytometry. After various treatments, cells were collected and centrifuged. The cell pellet was washed in PBS and centrifuged again. The pellet was resuspended in Annexin-V and PI according to the manufacturer’s protocol. Cells were analyzed on a Beckman flow cytometer (Beckman Coulter, Brea, CA, United States).

Cells were plated in 100-mm² tissue culture dishes at 60% confluence and incubated overnight. After treatment, cell lysates were obtained using cold radioimmunoprecipitation assay buffer [RIPA buffer contain: 20 mmol/L Tris-HCl (pH 8.0), 100 mmol/L NaCl, 10% glycerol, 1% NP-40, and 0.5% sodium deoxycholate]. Proteins (20 mg) were separated on precasted Bis-Tris NuPAGE gels (Bio-Rad, Hercules, CA, United States), electroblotted to polyvinylidene difluoride membranes (Millipore) and then blocked for 1 h at room temperature in TBS-T [50 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, and 0.1% Tween 20] containing 5% nonfat milk. Membranes were then incubated overnight at 4 °C or for 1 h at room temperature with the indicated primary antibodies: Notch1 (1:1000), SNAIL1 (1:500), SNAIL2 (1:1,000), E-cadherin (1:1000), IL-24 (1:1000), XIAP (1:500), VEGF (1:1000) and GAPDH (1:5000). Anti-mouse or anti-rabbit secondary antibody conjugated to horseradish peroxidase (HRP) was used to visualize the stained bands with an ECL (enhanced chemiluminescence, Santa Cruz, CA, United States) kit.

HepG2 cells were seeded in 6-well plates and cultured until confluence. A wound was then created by manually scraping the cell monolayer with a 200-microliter pipette tip. The cultures were washed twice with serum free medium to remove floating cells. The cells were then incubated in DMEM supplemented with 1% FBS. Cell migration into the wound was observed at 12 h in eight randomly selected microscopic fields for each condition and time point. Images were acquired with a Nikon DS-5M Camera System mounted on a phase-contrast Leitz microscope.

Scrambled (siCON) and specific siRNAs targeting Notch1 were obtained from Santa Cruz Biotechnology. Transfection of HepG2 cells was performed using the Amaxa system (Amaxa, Cologne, Germany) following their specifications. Firstly, 10⁶ cells in 100 μL of medium was mixed with 3 μg siRNA and then transferred to an Amaxa-certified cuvette. For transfection, we used the program V-01. Transfection efficiency was between 75% and 85% (data not shown), as checked by flow cytometry, using a fluorescein-labeled non-targeted siRNA control (Cell Signaling). After 4 h cells were treated with IL-24 as indicated. Cells were examined for gene downregulation and other properties 48 h after transfection.

The caspase-3/7 activity was detected with the Apo-One Homogeneous caspase-3/7 assay kit (Promega Corporation, Madison, WI). Briefly, cells were seeded into 96-well plates at a density of 2 x 10⁴ cells/well, incubated overnight and subsequently treated with siCON, siNotch1 with or without IL-24. For the caspase assay, the substrate was added after treatment for 48 h (as indicated in the text) and plates were read 2 h later using a Tecan Plate Reader (Tecan, Group. Ltd., Switzerland). All treatments were done in triplicate. Background absorbance was determined by the incubating media with substrate alone or subtracting the values from wells containing cells.

Each experiment was repeated at least three times. All the experimental data are presented as mean ± SD. The differences among means were statistically analyzed by a t-test. All statistical analyses were performed using SPSS13.0 software (Chicago, IL, United States). P < 0.05 was considered statistically significant.

The antiproliferative effects of GSI-I in combination with IL-24 were first examined in two hepatocellular carcinoma cell lines. Using as a single agent or in combination, the dose-response curves of the effects exerted by the compounds on cell viability are showed in Figure 1A. GSI-I at doses of 0-1 μmol/L for 24 h was almost ineffective in the cell lines. GSI-I at a dose of 2.5 μmol/L caused a remarkable reduction in cell viability of about 38% in HepG2 cells. The addition of IL-24 (at a concentration of 50 ng/mL) clearly potentiated the effects of GSI-I on HepG2 cells. IL-24 in combination with GSI-I (1 or 2.5 μmol/L) reduced cell viability of about 30% and 15%, respectively. However, treatment of cells with IL-24 alone did not induce any cytotoxic effect in the HCC cell lines. The addition of IL-24 to GSI-I treated SMMC7721 cells induced modest effects and the reduction of cell viability was only about 25%.

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Figure 1 γ-secretase inhibitor-I/interleukin-24 combined treatment induces apoptotic cell death in hepatocellular carcinoma cells. A: Cytotoxic effects exerted on SMMC7721 cells by γ-secretase inhibitor-I (GSI-I) employed alone or in combination with recombinant interleukin (IL)-24. After treatment for 24 h cell viability was evaluated by MTT assay as reported in Materials and Methods; B: Cytotoxic effects exerted on HepG2 cells by GSI-I employed alone or in combination with recombinant IL-24. After treatment for 24 h cell viability was evaluated by MTT assay as reported in Materials and Methods. The data represent mean ± SD; ^aP < 0.05 vs control cells.

We next examined the effects of the two compounds IL-24 and GSI-I on cell morphology by means of light microscopy. As shown in Figure 2A (top panel), after treatment with the GSI-I/IL-24 combination for 24 h cells appeared rounded, fragmented and floated in the culture medium, while the two drugs, employed separately, did not induce any significant remarkable effect (Figure 2A). In the same experimental conditions, the nuclei stained with Hoechst 33258 and the fluorescence microscopy analysis of nuclei stained with Hoechst 33258 evidenced a significant increase in the number of cells with condensed and fragmented nuclei, which is a typical apoptotic feature (Figure 2A).

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Figure 2 γ-secretase inhibitor-I/interleukin-24 combined treatment induces apoptotic cell death in hepatocellular carcinoma cells. A: Apoptotic effects induced by treatment of HepG2 cells with GSI-I and/or IL-24 for 24 h. Apoptotic morphology was evaluated by staining of the cells with Hoechst 33258 (magnification × 200); B: Annexin V positive cells were quantified by flow cytometric analysis after double staining of cells with Annexin V and propidium iodide at 6 h of treatment. Results are representative of three independent experiments. GSI-I: γ-secretase inhibitor-I; IL: Interleukin.

We analyzed the externalisation of phosphatidylserine on cell plasma membranes by Annexin V/PI staining to quantify early apoptotic effects. Following GSI-I/IL-24 combined treatment for 6 h, 47.2% of HepG2 cells were Annexin V positive/PI negative, while no significant effect was detected in cells treated with the compounds employed separately (Figure 2B).

Since Notch1 expression was associated with HCC metastasis as previously reported, we further investigated the link between Notch1 expression and the epithelial-mesenchymal transition (EMT) in HCC cells. HepG2 cell line was used for a comparison of the target protein expression profile of Notch1- and EMT-related genes. Decreased expression of Notch1 and its associated proteins SNAIL1 and SNAIL2 was detected in metastatic HCC cells. Increased E-cadherin protein expression was noted in the presence of IL-24 and GSI-I (Figure 3A). Loss of E-cadherin expression is a remarkable feature of EMT. As we all know, the level of E-cadherin is inversely associated with the tumor stage. Programmed cell death can be regulated by certain factors with either pro- or anti-apoptotic action in human cells. The upregulation of the former and/or the downregulation of the latter can be considered as important events to induce cell apoptosis[24]. To this purpose, we examined the expression levels of MMP-2 and IAP family members in HepG2 cells treated with GSI-I and/or IL-24. Furthermore, we proved that increased GSI-I and IL-24 in HepG2 cancer cells were associated with downregulation of MMP-2, XIAP and VEGF (Figure 3B).

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Figure 3 Cytotoxic effects of interleukin-24 and γ-secretase inhibitor-I assessed by Western blot analysis. A, B: HepG2 cells were treated for 48 h in the presence of GSI-I and/or IL-24. The levels of the proteins were determined by Western blot analysis using specific antibodies as indicated. GAPDH blots were included to show equal protein loading for all of the samples. Results are representative of three independent experiments. GSI-I: γ-secretase inhibitor-I; IL: Interleukin.

Wound healing assay is widely used to analyze cell proliferations and cell migration. As previously reported, for HepG2 cells, the cell invasion has a great corresponding relationship with cell migration and wound healing assay. The invasion properties of cancer cells were detected by the wound closure in these assays. In our study HepG2 cells were treated with IL-24 and/or GSI-I, and the wound closure was analyzed by microscopy. In the absence of treatment, HepG2 cells could migrate into the scratched space in 24 h (Figure 4A). With the treatment of IL-24 or GSI-I, the wound was still open after 24 h, indicating that IL-24 or GSI-I has the ability of inhibiting the HepG2 cell migration or invasion (Figure 4B and C). To further confirm the inhibition effectiveness, we loaded the cells with IL-24 and GSI-I. The distance of the wound closure significantly correlated with the concentrations of IL-24 and GSI-I (Figure 4).

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Figure 4 Wound healing in HepG2 cells treated with γ-secretase inhibitor-I and/or interleukin-24. Representative wounds after scratch wounding and after healing were recorded with a microscope (A-D). A: Control HepG2 cells; B: HepG2 cells treated with GSI-I; C: HepG2 cells treated with IL-24; D: HepG2 cells treated with GSI-I and IL-24. GSI-I: γ-secretase inhibitor-I; IL: Interleukin.

It has been showed that GSIs can act through different biochemical pathways, and we next investigated whether the effects induced by GSI-I/IL-24 combination were related to the specific inhibition of Notch signaling caused by GSI-I. We downregulated the Notch gene and evaluated the biological effects of IL-24 addition. Notch-1, as demonstrated by the authors, is upstream of Notch-4. After confirming the downregulation of Notch-1 level (Figure 5A, upper panel), we analyzed the biological effect of IL-24 addition on HepG2 cell viability. MMP-2 and poly (ADP)-ribose polymerase (PARP) proteins are the key proteins in detecting cell apoptosis. Figure 5A demonstrated that treatment of Notch-1 silenced HepG2 cells with 50 ng/mL IL-24 alone for 48 h induced cytotoxic effects quite similar to those observed in non-silenced cells treated with GSI-I/IL-24 combination.

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Figure 5 RNA interfering against Notch-1 in combination with interleukin-24 treatment in HepG2 cells. HepG2 cells were transfected for 48 h with siNotch-1 or siScr. Cells were treated with IL-24 for 48 h. A: Western blot analysis of the levels of Notch-1, MMP-2 and PARP was performed after siCON or siNotch1 treatment as reported in Materials and Methods; B: Caspase 3/7 activity was estimated by luminescent assay. ^aP < 0.05 vs siCON in HepG2 cells.

To investigate the mechanism of apoptosis, we analyzed HepG2 cells treated with siNotch1 or siCON plus IL-24 or not for 48 h by caspase-3/7 activity analysis. Caspase-3/7 activity was increased in the addition of siNotch1 plus IL-24 treatment, indicating that siNotch1 plus IL-24 could sensitize HCC cells to the cytotoxic agents (Figure 5B)

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide and half of these cases were estimated to occur in China. The major reason for the poor prognosis is that HCC often relapses due to intrahepatic and distant metastases. Thus, the discovery and research of new molecular targets of blocking metastasis are the primary goal for HCC therapy.

Notch signaling pathway plays an important role in HCC metastasis. Notch1, one of the Notch pathway receptors, is significantly higher in HCC than in adjacent non-tumor liver tissue. Abnormal Notch1 expression has been shown to be strongly associated with HCC metastasis, which may be mediated through the Notch1/Snail1/E-cadherin pathway. Interleukin (IL)-24 mediates the inhibition of adhesion and invasion of HepG2 and BEL-7402 cells by increasing the expression of E-cadherin and p-ERK.

To date, there is no study regarding synergistic effect of Notch1 down-regulation and IL-24 on apoptosis, invasion and migration of HCC cells. In this study, the authors found that the down-regulation of Notch1 by γ-secretase inhibitor-I (GSI-I) or siRNA combined with IL-24 can sensitize apoptosis and decrease the invasion and migration capabilities of HepG2 cells. Furthermore, the authors researched the mechanism and found that besides the inhibiting effects on SNAIL1 and SNAIL2, treatment with GSI-I/IL-24 combination also induced a clear up-regulation of E-cadherin and down-regulation of MMP-2.

In understanding the role and mechanism of Notch1/Snail1/E-cadherin pathways in inhibiting invasion and migration of HCC cell lines, this study indicate, for the first time, that GSI-I/IL-24 combination might represent a novel and potentially effective tool for HCC treatment.

All Notch receptors are activated by γ-secretase, and the inhibitors of this enzyme (GSIs) have attracted increasing interest. GSIs were firstly employed in the treatment of Alzheimer’s disease to prevent amyloid precursor protein cleavage and the consequent release of amyloid β-peptide. More recently, it has been observed that GSIs also possess the ability to induce growth arrest and/or apoptosis in some tumor cell lines while other tumor cells were resistant to the molecules.

In this paper the role of Notch 1 deregulation in HCC has been evaluated. It was observed a decrease in HCC cell invasion and migration by Notch1 down-regulation. Notch 1 inhibition by GSI-I increased HCC cell apoptosis induced by IL-24. The down-regulation of Notch1 by specific siRNA or GSI-I increased E-cadherin expression and inhibited HCC cell invasion and migration. It is concluded that a combination therapy of GSI-I/IL-24 may be a novel and potentially effective treatment for HCC.