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Introduction

Hepatocellular carcinoma (HCC), the predominant form of primary liver cancer, is one of the most prevalent malignancies and the third leading cause of cancer-related mortality worldwide with ~700,000 deaths each year (1). The incidence of HCC is dramatically increasing, and to date there are no effective chemoprevention or systemic treatments available. Therefore, it is of utmost importance to elucidate the molecular mechanisms of HCC. An increasing number of studies have indicated that chronic inflammation plays a crucial role in the occurrence and development of HCC (2–4), yet the precise mechanisms are still unclear.

Prostaglandin E2 (PGE2), a bioactive lipid which is produced predominantly from arachidonic acid by cyclooxygenases (COXs) and prostaglandin E synthases (PGES), is generally considered to be a potent pro-inflammatory mediator (5). Furthermore, substantial evidence has shown that PGE2 is also associated with several serious human diseases, including malignant tumors (6,7). PGE2 may promote cancer cell growth, adhesion, invasion, metastasis and angiogenesis (8), and thus participate in the tumorigenesis and progression of numerous human cancers, such as HCC (9–13), breast (14,15), gastrointestinal (16) and prostate cancer (17). PGE2 exerts diverse biological effects through four cognate E prostanoid receptors (EP receptor) from EP1 to EP4 (5,18), among which, the EP4 receptor is believed to be closely associated with cancer cell proliferation and invasion in many types of human cancer (19–23).

Moreover, the activation or overexpression of protooncogenes is involved in HCC (24). The c-myc proto-oncogene is the human cellular homologue of avian myelocytomatosis viral oncogene (v-myc), encoding a transcription factor c-Myc protein that upregulates the expression of many target genes and thus promotes cell proliferation and tumorigenesis. In addition, c-myc expression itself is regulated at multiple levels including transcription, post-transcription and post-translation (25,26). In HCC, the activation or overexpression of the c-myc proto-oncogene has been well documented (27,28). In addition, research supports a central role for c-Myc in human hepatocarcinogenesis (29). Thus, it is vital to explore the detailed molecular mechanisms of c-myc activation in HCC.

Given our previous results showing that PGE2 could notably enhance the cell growth and invasive ability of HCC cells (10–13), and the involvement of c-myc activation in hepatocarcinogenesis, the present study was designed to evaluate our hypothesis that PGE2 may promote the cell growth and invasion of HCC cells through upregulation of c-Myc protein expression. Data from the present study revealed that PGE2 significantly upregulated the expression of c-Myc at the mRNA and protein levels both via the EP4 receptor and the coupled GS/AC/cAMP/PKA/CREB signaling pathway, thus promoting cell growth and invasiveness in HCC cells.

Materials and methods

Materials

The human HCC cell line Huh-7 was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Dulbecco’s modified Eagle’s medium (DMEM) and Lipofectamine 2000 were from Life Technologies (Grand Island, NY, USA). PGE2, PGE1 alcohol, GW627368X and the cyclic AMP EIA kit were from Cayman Chemical Co. (Ann Arbor, MI, USA). SQ22536, forskolin, H89, actinomycin D (Act D) and cycloheximide (CHX) were from Sigma-Aldrich (St. Louis, MO, USA). Cell Counting Kit-8 (CCK-8) was from Dojindo Laboratories (Kumamoto, Japan). High Pure RNA isolation kit was from Roche (Mannheim, Germany). PrimeScript RT reagent kit was from Takara (Dalian, China). Anti-c-Myc (no. 5605), anti-CREB (no. 9104), anti-phosphorylated CREB (no. 9198, Ser133) antibodies were from Cell Signaling Technology (Danvers, MA, USA); anti-β-actin mouse monoclonal antibody (BM0627) was from Boster (Wuhan, China), and anti-β-tubulin antibody (no. 21335) was from SAB (Signalway Antibody; College Park, MD, USA). The protein assay dye reagent was from Bio-Rad (Hercules, CA, USA). ECL Prime Western Blotting Detection reagent was from GE Healthcare (Piscataway, NJ, USA). The Transwell unit was from Corning (Cambridge, MA, USA). Matrigel was from BD Biosciences (San Jose, CA, USA).

Cell line and culture

Human HCC Huh-7 cells were cultured in DMEM with 10% fetal calf serum (FCS) at 37°C in a humidified 5% CO2 incubator. The experiments were performed when cells reached 80% confluency and were conducted in serum-free medium.

Cell proliferation assay

Cell proliferation was determined using the CCK-8 kit from Dojindo Laboratories. This kit contains the WST-8 reagent, a tetrazolium salt that can be reduced by mitochondrial dehydrogenases in viable cells to produce an orange colored formazan dye. Briefly, 600 μl of cell suspension (1×105 cells) was plated in each well of 24-well plates. After a 24-h culture to allow reattachment, the cells were then incubated with different treatments at the indicated concentrations and time periods. Cell proliferation reagent, WST-8 (60 μl), was subsequently added to each well. The incubation was continued from 30 min to 4 h at 37°C and absorbance at 450 nm was measured using an automatic ELISA plate reader.

Cell invasion assays

Cell invasion assays were performed in Matrigel-coated 24-well Transwell units. Cells (5×104) were added to the upper Transwell chamber and media with 10% FCS were added to the lower Transwell chamber. The serumfree media plus pharmacological agents were added in the upper Transwell chamber. After 24 h of incubation at 37°C, the cells were fixed with ethanol and then stained by 0.1% crystal violet for 30 min at room temperature. After washing the wells with PBS, the cells on the upper surface of the filter were removed with a cotton swab. The invaded cells on the lower surface of the membrane were solubilized with 10% acetic acid for 10 min and quantified by measuring the absorbance at 550 nm.

Western blotting

Cells were treated with different pharmacological agents at 37°C for various times, as indicated in the experiments. The cells were collected into lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% sodium deoxycholate, 1% Nonidet P-40, 0.1% SDS, 1 mM PMSF, sodium orthovanadate, sodium fluoride and aprotinin) and placed on ice for 30 min. Cell lysates were sonicated on ice for at least 30 sec and then cleared by centrifugation at 15,000 × g for 30 min at 4°C. Protein concentrations of lysates were measured using Bio-Rad protein assay dye reagent. Equal amounts of proteins (40 μg) were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. Membranes were blocked with 5% non-fat milk-TBST buffer for 1 h at room temperature and incubated with the corresponding primary antibodies overnight at 4°C with gentle shaking. Then, the membranes were washed by TBST and incubated for 2 h with the peroxidase-conjugated secondary anti-rabbit or anti-mouse antibodies at room temperature. The signals were detected using enhanced chemiluminescent reagent (ECL) and analyzed using Image Lab 4.0 analysis software from Bio-Rad.

RNA isolation and real-time PCR

Total RNA from the cultured cells was isolated using the High Pure RNA isolation kit according to the manufacturer’s instructions. Reverse transcription was carried out with the PrimeScript RT reagent kit according to the standard protocol. For quantification of mRNA expression, real-time PCR was performed using the following primer pairs: c-Myc, 5′-AGGCTATTCTGCCCATTT-3′ (forward) and 5′-TCGTAGTCGAGGTCATAGTTC-3′ (reverse); EP4, 5′-CATCTTACTCATTGCCACCT-3′ (forward) and 5′-TACTGAGCACTGTCTTTCTC-3′ (reverse); GAPDH, 5′-TTCCAGGAGCGAGATCCCT-3′ (forward), and 5′-CACCCATGACGAACATGGG-3′ (reverse). Real-time PCR analysis was performed on Roche LightCycler Nano instrument using FastStart Essential DNA Green Master Mix from Roche, and GAPDH was used as the endogenous control. PCR conditions were pre-incubation at 95°C for 10 min (1 cycle) followed by 40 cycles of 95°C for 20 sec, 60°C for 20 sec and 72°C for 20 sec. All treatments and conditions were performed in triplicate to calculate the statistical significance.

siRNA interference

The siRNA targeting human EP4 receptor (siRNA ID: s11455) was purchased from Life Technologies. The siRNA reagents specific to c-Myc (no. 6341) and CREB (no. 6588) were from Cell Signaling Technology. Huh-7 cells (2×105) were plated in 6-well plates for 24 h, resulting in a 30–50% confluent cell monolayer. The cells were then transfected with the targeting siRNA or the negative control siRNA (N.C. siRNA) from GenePharma (Shanghai, China) using Lipofectamine 2000. After transfection, depletion of target protein was confirmed by western blotting or real-time PCR analysis, and the cells were subsequently used for further experiments.

cAMP assay

Intracellular cAMP levels were measured using an enzyme immunoassay kit. Briefly, Huh-7 cells were cultured in 35-mm dishes until they reached 80% confluency and were then treated with PGE1 alcohol or vehicle at 37°C for different times. The cells were harvested in 0.1 M HCl solution, and incubated for 20 min at room temperature. After centrifugation at 1,000 × g for 10 min, the supernatant (50 μl) was analyzed for cAMP content according to the manufacturer’s instructions.

Statistical analysis

Data are presented as means ± SD. P-values were calculated using the Student’s t-test for unpaired samples with MS Excel software. The results were considered significantly different at P<0.05.

Results

PGE2 upregulates c-Myc mRNA and protein expression in HCC cells

To determine the direct effect of PGE2 on c-Myc expression in HCC cells, Huh-7 cells were treated with PGE2 at various doses or for different times, and then the levels of mRNA and protein expression of c-Myc were analyzed by real-time PCR and western blotting, respectively. Results from the western blotting experiments showed that 3 μM PGE2 treatment significantly increased c-Myc protein expression from 0.5 to 2 h, and at 2 h it reached a maximum value which was 183% fold of the value at 0 h (Fig. 1A). Moreover, the expression levels of c-Myc protein were all upregulated by 2 h PGE2 treatment with a dose from 0.1 to 10 μM (Fig. 1B). On the other hand, the mRNA level of c-Myc was obviously elevated by 3 μM PGE2 from 0.5 h, and it peaked at 1 h which was 5.16-fold of that at 0 h and then declined to the basal level at 2 h (Fig. 1C). In addition, treatment of cells with 0.1 to 10 μM PGE2 for 1 h markedly increased the mRNA level of c-Myc in the Huh-7 cells (Fig. 1D). These data indicate that PGE2 upregulates c-Myc expression at the mRNA and protein levels in the HCC cells.

Figure 1 PGE2 upregulates c-Myc expression at the mRNA and protein levels in Huh-7 cells. (A) Time course of the effects of PGE2 on c-Myc protein expression. Huh-7 cells were treated with 3 μM PGE2 for the indicated times, and protein expression of c-Myc was determined by western blotting. (B) Dose effect of PGE2 on c-Myc protein expression. Huh-7 cells were incubated with PGE2 at the indicated doses for 2 h, and protein expression of c-Myc was determined by western blotting. Quantitative analysis of c-Myc protein expression was carried out by calculating the ratio between c-Myc protein and β-actin expression levels from three different experiments. (C) Time course of the effects of PGE2 on c-Myc mRNA expression. Huh-7 cells were treated with 3 μM PGE2 for the indicated times, and mRNA expression of c-Myc was examined by real-time PCR. (D) Dose effect of PGE2 on c-Myc mRNA expression. Huh-7 cells were incubated with PGE2 at the indicated doses for 1 h, and mRNA expression of c-Myc was examined by real-time PCR. Results are presented as the means ± SD of three independent experiments. *P<0.05, **P<0.01 compared with the control. PGE2, prostaglandin E2.

Knockdown of c-Myc blocks PGE2-induced HCC cell growth and invasion

Our previous results demonstrated that PGE2 promotes cell growth and invasion in HCC cells (10–13). Now, we revealed that PGE2 directly upregulated c-Myc protein expression in the HCC cells, a key transcription factor with cancer-promoting effects. Thus, the role of c-Myc upregulation in PGE2-induced HCC cell growth and invasion need to be further investigated. As shown in Fig. 2A, c-Myc siRNA significantly lowered the basal proliferation and blocked PGE2-induced proliferation in the HCC Huh-7 cells. In addition, c-Myc siRNA showed a similar inhibitory effect on PGE2-induced HCC cell invasion (Fig. 2B). Depletion of c-Myc protein by siRNA transfection was confirmed by western blot analysis (Fig. 2C). These results showed that the upregulation of c-Myc protein plays an important role in PGE2-induced HCC cell growth and invasion.

Figure 2 Knockdown of c-Myc blocks PGE2-induced HCC cell growth and invasion. (A) Knockdown of c-Myc blocked PGE2-induced HCC cell growth. Huh-7 cells were transfected with c-Myc siRNA or N.C. siRNA (siRNA concentration 80 nM) for 24 h and then stimulated with PGE2 (10 μM) in serum-free medium for 24 h, and cell proliferation was measured using the CCK-8 kit. (B) Knockdown of c-Myc blocked PGE2-induced HCC cell invasion. Huh-7 cells were transfected with c-Myc siRNA or N.C. siRNA (siRNA concentration 80 nM), and 24 h post-transfection cell suspensions were prepared. Serum-free medium (100 μl) containing 5×104 cells were seeded in the upper chamber in the presence or absence of 10 μM PGE2, and regular medium containing 10% FCS was added to the lower chamber. The cell invasion assay was conducted after 24 h. Representative images of invaded cells from the different groups are shown in the upper panel. Quantitative analysis of invaded cells is shown in the lower panel. (C) RNAi efficiency of c-Myc siRNA in Huh-7 cells. After transfection of Huh-7 cells with c-Myc siRNA or N.C. siRNA (siRNA concentration 80 nM) for 24 h, the protein expression of c-Myc was determined by western blotting. Quantitative analysis of c-Myc protein expression was carried out by calculating the ratio between c-Myc protein and β-actin expression levels from three different experiments. Results are presented as the means ± SD of three independent experiments. *P<0.05, **P<0.01 compared with the control; #P<0.05; ##P<0.01 compared with PGE2 treatment. PGE2, prostaglandin E2; N.C., negative control.

EP4 receptor is involved in PGE2-induced c-Myc expression in HCC cells

EP4 receptor is closely associated with cancer cell growth and invasion in various human cancers (19–23). Thus, we investigated whether the EP4 receptor is also involved in PGE2-induced c-Myc expression in HCC cells. As shown in Fig. 3A and B, treatment of Huh-7 cells with PGE1 alcohol, the EP4 receptor selective agonist, significantly increased the expression level of c-Myc protein in a time- and dose-dependent manner. In addition, the mRNA expression level of c-Myc was also upregulated by PGE1 alcohol in Huh-7 cells in a dose-dependent manner (Fig. 3C and D). These results suggest a key role of the EP4 receptor in the regulation of c-Myc expression in HCC cells.

Figure 3 The EP4 receptor selective agonist PGE1 alcohol mimics the upregulatory effect of PGE2 on c-Myc expression in Huh-7 cells. (A and B) Time effect and dose effect of PGE1 alcohol on c-Myc protein expression. Huh-7 cells were treated with PGE1 alcohol (3 μM) for the indicated times (A) or PGE1 alcohol at the indicated doses for 2 h (B), and protein expression of c-Myc was determined by western blotting. Quantitative analysis of c-Myc protein expression was carried out by calculating the ratio between c-Myc protein and β-actin expression levels from three different experiments. (C and D) Time effect and dose effect of PGE1 alcohol on c-Myc mRNA expression. Huh-7 cells were treated with PGE1 alcohol (3 μM) for the indicated times (C) or PGE1 alcohol at the indicated doses for 1 h (D), and mRNA expression of c-Myc was examined by real-time PCR. Results are presented as the means ± SD of three independent experiments. *P<0.05, **P<0.01 compared with the control. PGE1, prostaglandin E1; PGE2, prostaglandin E2.

To further confirm the involvement of the EP4 receptor in PGE2-induced c-Myc expression, we examined the effects of an EP4 receptor selective antagonist or EP4 receptor siRNA on PGE2-induced c-Myc expression in HCC cells. As shown in Fig. 4A and B, pretreatment of Huh-7 cells with GW627368X, the EP4 receptor selective antagonist, markedly reduced PGE2-or PGE1 alcohol-induced c-Myc expression at the protein and mRNA levels. In addition, EP4 receptor siRNA also blocked the upregulation of c-Myc protein expression by PGE2 or PGE1 alcohol in the Huh-7 cells (Fig. 4C). The interference efficacy of EP4 receptor siRNA was verified by real-time PCR analysis, showing that EP4 siRNA significantly lowered the mRNA expression of the EP4 receptor in the Huh-7 cells (Fig. 4D). These observations indicate that the EP4 receptor is involved in PGE2-induced c-Myc expression in HCC cells.

Figure 4 EP4 receptor antagonism or siRNA interference attenuates PGE2-induced c-Myc expression in Huh-7 cells. (A and B) Effect of the EP4 receptor selective antagonist GW627368X on PGE2-induced c-Myc expression. Huh-7 cells were pretreated for 1 h with GW627368X (4 μM) followed by stimulation with PGE2 (3 μM) or PGE1 alcohol (3 μM), and protein expression of c-Myc was determined by western blotting after 2 h (A) and mRNA expression of c-Myc was examined by real-time PCR after 1 h (B). (C) Effect of EP4 receptor siRNA on PGE2-induced c-Myc protein expression. Huh-7 cells were transfected with EP4 siRNA or N.C. siRNA for 72 h and then stimulated with PGE2 (3 μM) or PGE1 alcohol (3 μM) in serum-free medium for 2 h, and protein expression of c-Myc was determined by western blotting. Quantitative analysis of c-Myc protein expression was carried out by calculating the ratio between c-Myc protein and β-actin expression levels from three different experiments. (D) RNAi efficiency of EP4 siRNA in Huh-7 cells. After transfection of Huh-7 cells with EP4 siRNA or N.C. siRNA for 72 h, the mRNA expression of the EP4 receptor was examined by real-time PCR. Results are presented as the means ± SD of three independent experiments. *P<0.05, **P<0.01 compared with the control; §P<0.05, §§P<0.01 compared with the PGE2 treatment; #P<0.05, ##P<0.01 compared with the PGE1 alcohol treatment. GW, GW627368X. PGE2, prostaglandin E2; PGE1, prostaglandin E1; N.C., negative control.

EP4 receptor-mediated c-Myc protein upregulation depends on de novo biosynthesis of c-Myc mRNA and its protein in HCC cells

As a key protein in cells, c-Myc expression is tightly regulated at multiple levels, including transcription, post-transcription and post-translation (25,26). To elucidate the mechanisms of EP4 receptor-mediated c-Myc protein upregulation, Act D and CHX, inhibitors of de novo RNA synthesis and de novo protein synthesis, respectively, were used. As shown in Fig. 5A, pretreatment with Act D or CHX significantly reduced EP4 receptor-mediated c-Myc protein upregulation in the Huh-7 cells, suggesting a crucial role of de novo biosynthesis of c-Myc mRNA and its protein in EP4 receptor-mediated c-Myc protein upregulation. The pharmacological effects of Act D or CHX on the mRNA level of c-Myc were further examined by real-time PCR experiment, and the results showed that Act D markedly lowered the basal and EP4 receptor-mediated c-Myc mRNA expression, while CHX, the inhibitor of de novo protein synthesis, had no such effect (Fig. 5B). These findings indicate that EP4 receptor-mediated c-Myc protein upregulation greatly depends on the de novo biosynthesis of c-Myc mRNA and its protein in HCC cells.

Figure 5 Effects of Act D and CHX, de novo biosynthesis inhibitors of RNA and protein, respectively, on EP4 receptor-mediated c-Myc protein upregulation in Huh-7 cells. Huh-7 cells were pretreated for 1 h with Act D (5 μg/ml) or CHX (50 μg/ml) followed by stimulation with 3 μM PGE1 alcohol, and protein expression of c-Myc was determined by western blotting after 2 h (A) and mRNA expression of c-Myc was examined by real-time PCR after 1 h (B). Quantitative analysis of c-Myc protein expression was carried out by calculating the ratio between c-Myc protein and β-actin expression levels from three different experiments. Results are presented as the means ± SD of three independent experiments. *P<0.05, **P<0.01 compared with the control; #P<0.05, ##P<0.01 compared with the PGE1 alcohol treatment. PGE2, prostaglandin E2. Act D, actinomycin D; CHX, cycloheximide; PGE1, prostaglandin E1.

The GS/AC/cAMP signaling pathway is involved in EP4 receptor-mediated c-Myc upregulation in HCC cells

The EP4 receptor, a G protein coupled receptor, is usually coupled with GS protein to activate adenylate cyclase (AC) and elevate intracellular cAMP levels (5,18). To confirm the downstream signaling pathway of the EP4 receptor in HCC cells, we measured the direct effect of the EP4 receptor agonist on the intracellular cAMP level in Huh-7 cells. As shown in Fig. 6, stimulation of cells with PGE1 alcohol notably increased the intracellular cAMP level in a time-dependent manner, and at around 30–45 min the cAMP level reached a maximum value which was ~15-fold of the control. By contrast, the vehicle DMSO had no effect on the cAMP level. These data demonstrated that the EP4 receptor is coupled to the GS/AC/cAMP signaling pathway in HCC cells.

Figure 6 EP4 receptor agonist PGE1 alcohol elevates the intracellular cAMP level in Huh-7 cells. Huh-7 cells were incubated with PGE1 alcohol (3 μM) or the vehicle DMSO for the indicated times, and the intracellular cAMP level was measured by EIA assay. Results are presented as the means ± SD of three independent experiments. PGE1, prostaglandin E1.

To determine the role of the GS/AC/cAMP signaling pathway in EP4 receptor-mediated c-Myc upregulation, Huh-7 cells were treated with a specific activator or inhibitor of AC, and then their effects on c-Myc expression in HCC cells were examined. As shown in Fig. 7A and B, treatment of Huh-7 cells with forskolin, the specific activator of AC, resulted in a dose-dependent increase in c-Myc expression at the protein and mRNA levels. Pretreatment of cells with SQ22536, the specific inhibitor of AC, markedly reduced EP4 receptor-mediated c-Myc upregulation at the protein level and partly at the mRNA level (Fig. 7C and D). These results revealed that the GS/AC/cAMP signaling pathway is involved in EP4 receptor-mediated c-Myc upregulation in HCC cells.

Figure 7 Involvement of the GS/AC/cAMP signaling pathway in EP4 receptor-mediated c-Myc upregulation in Huh-7 cells. (A and B) The specific AC activator forskolin induced c-Myc expression at the mRNA and protein levels. Huh-7 cells were treated with forskolin at the indicated doses, and protein expression of c-Myc was determined after 2 h by western blotting (A) and mRNA expression of c-Myc was examined after 1 h by real-time PCR (B). (C and D) The specific AC inhibitor SQ22536 reduced EP4 receptor-mediated c-Myc upregulation. Huh-7 cells were pretreated for 1 h with SQ22536 (200 μM) followed by stimulation with 3 μM PGE1 alcohol, and protein expression of c-Myc was determined after 2 h (C) and mRNA expression of c-Myc was examined after 1 h (D). Quantitative analysis of c-Myc protein expression was carried out by calculating the ratio between c-Myc protein and β-actin expression levels from three different experiments. Results are presented as the means ± SD of three independent experiments. *P<0.05, **P<0.01 compared with the control; #P<0.05, ##P<0.01 compared with the PGE1 alcohol treatment. AC, adenylate cyclase; PGE1, prostaglandin E1.

Involvement of the PKA/CREB pathway in EP4 receptor-mediated c-Myc upregulation in HCC cells

An elevated intracellular cAMP level leads to protein kinase A (PKA) activation, while activated PKA could transfer into the cell nucleus and phosphorylate transcription factor CREB protein, thus regulating gene expression (30). Firstly, we investigated the involvement of PKA activation in EP4 receptor-mediated c-Myc upregulation in HCC cells by using the PKA-specific inhibitor H89. As shown in Fig. 8A and B, pretreatment of Huh-7 cells with H89 significantly reduced EP4 receptor-mediated c-Myc upregulation at the protein and mRNA levels, suggesting an important role of PKA in EP4 receptor-mediated c-Myc upregulation. Secondly, the role of CREB in EP4 receptor-mediated c-Myc upregulation was also examined. Stimulation of Huh-7 cells with the EP4 receptor agonist PGE1 alcohol led to a significant increase in the phosphorylation of CREB at Ser133 (Fig. 9A), a crucial event for transcriptional activation by CREB. Meanwhile, the CREB siRNA markedly reduced EP4 receptor-mediated c-Myc protein upregulation in Huh-7 cells (Fig. 9B). The downregulation of CREB protein by siRNA transfection in the Huh-7 cells was confirmed by western blotting (Fig. 9C). Based on these findings, the PKA/CREB pathway is also involved in EP4 receptor-mediated c-Myc upregulation in HCC cells.

Figure 8 Involvement of PKA in EP4 receptor-mediated c-Myc upregulation in Huh-7 cells. Huh-7 cells were pretreated for 1 h with the PKA specific inhibitor H89 (10 μM) followed by stimulation with 3 μM PGE1 alcohol, and protein expression of c-Myc was determined after 2 h by western blotting (A) and mRNA expression of c-Myc was examined after 1 h by real-time PCR (B). Quantitative analysis of c-Myc protein expression was carried out by calculating the ratio between c-Myc protein and β-actin expression levels from three different experiments. Results are presented as the means ± SD of three independent experiments. **P<0.01 compared with the control; #P<0.05, ##P<0.01 compared with the PGE1 alcohol treatment. PKA, protein kinase A; PGE1, prostaglandin E1.

Figure 9 Involvement of CREB in EP4 receptor-mediated c-Myc upregulation in Huh-7 cells. (A) CREB phosphorylation at Ser133 induced by PGE1 alcohol. Huh-7 cells were treated with PGE1 alcohol (3 μM) for indicated times, and the phosphorylated CREB and total CREB protein expression were determined by western blotting. Quantitative analysis of CREB phosphorylation was carried out by calculating the ratio between the CREB protein and phosphorylation levels from three different experiments. (B) CREB siRNA reduced EP4 receptor-mediated c-Myc protein upregulation. Huh-7 cells were transfected with CREB siRNA or N.C. siRNA (siRNA concentration 80 nM) for 48 h and then stimulated with PGE1 alcohol (3 μM) in serum-free medium for 2 h, and protein expression of c-Myc was determined by western blotting. Quantitative analysis of c-Myc protein expression was carried out by calculating the ratio between c-Myc protein and β-actin expression levels from three different experiments. (C) RNAi efficiency of CREB siRNA in Huh-7 cells. After transfection of Huh-7 cells with CREB siRNA or N.C. siRNA (siRNA concentration 80 nM) for 48 h, the protein expression of CREB was determined by western blotting. Quantitative analysis of CREB protein expression was carried out by calculating the ratio between CREB protein and β-tubulin expression levels from three different experiments. Results are presented as the means ± SD of three independent experiments. *P<0.05, **P<0.01 compared with the control; #P<0.05 compared with the PGE1 alcohol treatment. PGE1, prostaglandin E1; N.C., negative control.

Discussion

Hepatocellular carcinoma (HCC) represents a major health problem worldwide. To date, our understanding of the molecular mechanisms of this disease remains rudimentary. A large body of studies support a pivotal role of chronic inflammation in the pathogenesis of HCC (2–4). Yet, the underlying mechanisms are not well understood.

PGE2, an inflammatory mediator and the predominant product of COX-2, has been shown to be involved in various human cancers, including HCC. Our previous results revealed that PGE2 significantly enhanced the cell growth, migration and invasion in HCC cells (10–13). Although several signaling pathways have been identified such as transactivation of EGFR receptor (31), phosphorylation of FAK kinase (12,13), the detailed mechanisms of PGE2 in HCC remain to be further studied.

c-Myc protein encoded by the proto-oncogene c-myc functions as a transcription factor. After dimerizing with its partner protein Max, c-Myc binds to E box sequence elements (5′-CACGTG-3′) to activate the transcription of many target genes, and thus promotes cell proliferation and tumorigenesis (26). In addition, substantial studies confirmed that c-myc is involved in human hepatocarcinogenesis. Approximately 30% of human HCC samples exhibit gene amplification of c-myc (27,28), and the major etiological factors of HCC including hepatitis C or B virus infection, and aflatoxin could induce overexpression of c-Myc protein (32–34). However, the exact mechanisms of c-myc activation in HCC are largely unknown.

Since PGE2 and c-Myc are both involved in HCC, and have the potential for causing tumorigenesis, the internal relationship between PGE2 and c-Myc in HCC is of particular interest to us. In the present study, we found that PGE2 directly upregulated c-Myc expression at the mRNA and protein levels, and knockdown of c-Myc protein greatly suppressed PGE2-induced HCC cell growth and invasion in Huh-7 cells. These findings firmly confirm that c-Myc is a critical regulator in PGE2-induced HCC cell growth and invasion, suggesting a probable molecular mechanism for inflammation-induced hepatocarcinogenesis.

PGE2 exerts biological effects through four types of EP receptors on the cell surface membrane, designated as EP1, EP2, EP3 and EP4 (5,18). Studies indicate that the EP4 receptor plays a crucial role in PGE2-mediated tumorigenesis in many types of cancer. For example, PGE2 promotes renal cancer cell invasion via the EP4 receptor and small GTPase Rap signaling (19). In colon cancer cells, PGE2 induces S100p expression to enhance cancer cell growth and migration via the EP4 receptor signaling (20). In lung cancer cells, PGE2 promotes cell migration via EP4-β Arrestin1-c-Src signal-some (21). Likewise, we found that PGE2 induced c-Myc expression to promote cell growth and invasion in HCC cells mainly through the EP4 receptor.

Usually, c-Myc expression is tightly controlled by a number of mechanisms at the transcriptional, post-transcriptional and post-translational levels. Many transcription factors such as TCF, FBP, CNBP, NF-κB and AP-1 can bind to the promoter of the human c-myc gene, and upregulate its expression at the transcriptional level (25,41). In addition, the c-Myc protein itself can be post-translational modified at multiple sites by phosphorylation or ubiquitination. Two key phosphorylation sites of Ser62 and Thr58 located within the N terminus are important for the regulation of c-Myc protein stabilization, yet have opposite function. Ser62 phosphorylation stabilizes c-Myc protein, while T58 phosphorylation leads to its protein degradation (35). In human esophageal squamous cell carcinoma cells, PGE2 was shown to upregulate c-Myc protein expression by stimulating Ser62 phosphorylation and then stabilizing its protein at the post-translational level (36). However, the present study revealed that in HCC cells the EP4 receptor-mediated c-Myc protein upregulation largely depended on de novo biosynthesis of c-Myc mRNA and its protein.

As a G protein coupled receptor, the EP4 receptor is believed to be coupled with GS protein to activate AC and elevate intracellular cAMP levels (5,18). Our results confirmed that the EP4 receptor activated the GS/AC/cAMP signaling pathway in HCC cells. In addition, we demonstrated that this canonical pathway is involved in EP4 receptor-mediated c-Myc upregulation in HCC cells. This finding is similar to a study in human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs), which showed that PGE2 upregulated the expression of c-Myc and VEGF via EP2/cAMP signaling and thus promoted cell proliferation of hUCB-MSCs (37).

In cells, an elevated cAMP level subsequently activates three main targets including protein kinase A (PKA), the exchange protein activated by cAMP (Epac) and the cyclicnucleotide- gated ion channels (CNGCs) (30). Among them, PKA has been shown to regulate many aspects of cell functions, such as metabolism, signal transduction and gene expression (38). The effect of PKA on gene transcription is mainly achieved by direct phosphorylation of the transcription factor CREB at the site of Ser133 following stimulation of gene transcription by activated CREB (39,40). By using a specific inhibitor and siRNA, we demonstrated that the signaling pathway of PKA/CREB is also involved in the EP4 receptor-mediated c-Myc upregulation in HCC cells.

CREB, one member of the bZIP (basic domain/leucine zipper) transcription factor family, can activate numerous target genes including c-fos and JunD through cAMP response elements (CREs). Full CRE is an 8 bp palindrome (5′-TGACGTCA-3′), and the half motif (CGTCA) is also active for CREB binding and cAMP responsiveness. It is reported that of 105 genes with functional CREs identified in the literature, approximately half contain a full palindrome, with the other half containing a single CGTCA motif (40). In the present study, we found that CREB is involved in the upregulation of c-Myc expression in HCC cells. Yet, there are no data or study which shows that the promoter of the human c-myc gene contains a or more full CRE (41). Based on sequence analysis, we found that two half CGTCA motifs locate in the upstream of transcription start site (TSS) of the human c-myc gene, at −3,005 and −3,759 bp, respectively. Therefore, we speculated that CREB upregulates the expression of c-myc by following possible mechanisms: by binding to these two half CGTCA motifs; indirect action, by stimulation of the expression of other transcription factors such as AP-1 and then activation of c-Myc expression or other unknown mechanisms. Thus, the exact mechanisms involved in c-Myc upregulation by CREB need to be further investigated.

In summary, the present study revealed that PGE2 directly upregulated c-Myc expression via the EP4R/GS/AC/cAMP/PKA/CREB signaling pathway to promote cell growth and invasion in human HCC cells. This finding provides a further insight into the mechanisms by which PGE2 enhances HCC cell growth and invasion. Targeting of the PGE2/EP4R/c-Myc pathway may be a new therapeutic strategy to prevent and cure human HCC.

Acknowledgements

The present study was supported by the National Natural Science Foundation of China (nos. 30871015 and 81172003), and by a project funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions.

References