Benzyl butyl phthalate induces migration, invasion, and angiogenesis of Huh7 hepatocellular carcinoma cells through nongenomic AhR/G-protein signaling

Fluo-4 was purchased from Invitrogen (Carlsbad, CA, USA). BBP, 2-aminoethoxydiphenyl borate (2-APB), and 6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Pd98059 and wortmannin were obtained from Calbiochem-Novabiochem (San Diego, CA, USA).

pEGFP-C1-AhR, a kind gift from Dr. Hsin-yu Lee (Department of Life Science, National Taiwan University), was cloned the AhR gene into pEGFP-C1 (Clontech).

Huh7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies, Grand Island, NY, USA), PLC/PRF/5 and HepG2 cells were cultured in minimum essential medium (MEM) (Life Technologies, Grand Island, NY, USA) and supplemented with 10% fetal bovine serum (Gibco, California,CA, USA), 1% penicillin (100 U/mL), streptomycin (10 μg/mL), and amphotericin-B (250 μg/mL) (Sigma-Aldrich Co, St. Louis, MO). Human umbilical vein endothelial cells (HUVEC) were grown in EGM-2 medium (Lonza, Basel, Switzerland). All cells were cultured at 37°C in 5% CO₂.

For total internal reflection fluorescennce (TIRF) microscopy studies, Huh7 cells were transfected with pEGFP-C1-AhR or pEGFP-C1 as a control using LT1 transfection reagent (Mirus, Madison, WI, USA). After transfection for 24 hours, the cells were harvested and cultured on coverslips for 1 day. Cells were then treated with DMSO as a control or BBP (1 μM) and analyzed by TIRF microscopy (Carl Zeiss, Oberkochen, Germany). GFP intensity was analyzed by Axio Vision Rel. 4.8 software (Carl Zeiss, Oberkochen, Germany).

Calcium imaging was performed using the same method as in a previous study [19] with some modifications. For live cell calcium imaging, Cell-R software was used for microscopy (Olympus, Japan). Huh7 cells were seeded on coverslips and cultured for 24 hours. Cells were incubated with 1 μM Fluo-4, a Ca²⁺-specific dye, at 37°C for 20 minutes in Buffer Salt Saline (BSS) (2 mM CaCl₂, 5.5 mM d-glucose, 130 mM NaCl, 5.4 mM KCl, 20 mM HEPES pH = 7.4, 1 mM MgSO₄) and then washed three times before measuring the relative fluorescence intensity. Cells were pretreated with various concentrations of 2-APB for 10 minutes, and then loaded with 1 μM Fluo-4 for 20 minutes. After washing, cells were maintained in calcium-free medium (5.5 mM d-glucose, 130 mM NaCl, 5.4 mM KCl, 20 mM HEPES (pH = 7.4), and 3 mM MgSO₄) during the experimental periods. The cells were then stimulated by adding BBP (1 μM) after 1 minute. Data were analyzed with Cell-R software.

Huh7 cells were transfected with pEGFP-C1-AhR using LT1 transfection reagent. After overnight transfection, the cells were harvested and cultured on coverslips for 1 day. BBP (1 μM) was added to stimulate the cells before analysis by confocal microscopy. GFP intensity was analyzed by FV10-ASW 3.0 software (Olympus, Japan).

Ultrathin sections of plastic-embedded cells were pretreated with 5% sodium metaperiodate (10 min) by microwave fixation and processing. The grids were incubated with an aliquot of IgG antibodies against AhR or Gα_q/11 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) followed by probing with secondary antimouse IgG gold particles (6 nm) or anti rabbit IgG gold particles (20 nm), respectively. After washing, the sections were blocked by placing the grids on a drop of phosphate-buffered saline (PBS) containing 1% ovalbumin and incubating for 15 minutes. Sections were then stained with uranyl acetate and lead citrate for characterization by transmission electron microscopy (H-700, Hitachi, Japan).

After treatment with BBP (1 μM) or DMSO for the control group, cells were fixed by adding fixation solution (in 3.7% formaldehyde/PBS) at room temperature for 10 minutes. The cells were washed with PBS twice and then permeabilized by adding 70% EtOH at 4°C for 1 hour. Cells were then washed in wash buffer (5 mL 20× saline-sodium citrate (SSC), 5 mL formamide, and nuclease-free water to a final volume of 50 mL) for 5 minutes. Hybridization was performed by mixing 100 μL of hybridization solution (1 g dextran sulfate, 20 × SSC, 1 mL formamide for a 10% final concentration and nuclease-free water to a final volume of 10 mL) with a specific AhR probe (Stellaris™ FISH Probes, Biosearch Technology, Novato, CA, USA) and incubating the mixture overnight at 37°C. Cells were then stained with DAPI (1 μg/mL) for 5 minutes. After washing, a drop of mounting solution was applied. The slides were then covered with the cell-attached cover glasses and sealed with nail polish. Imaging was performed by confocal microscopy.

Huh7 cells (3 × 10⁵) were seeded in six-well plates, cultured for 24 hours, and then incubated overnight in serum free medium. The cells were then treated with BBP (1 μM) for various times intervals. After stimulation, cells were washed twice with PBS. Total RNA was extracted with TRIzol (Invitrogen). The RNA (2 μg) was applied to a Reverse Transcription System (Promega Biosciences, San Luis Obispo, CA, USA) to synthesize cDNA. The cDNA was then amplified by specific primers. The primer pairs were as follows: AhR, forward 5′-TACTCTGCCGCCCAA ACTGG-3′, reverse 5′-GCTCTGCAACCTCCGATTCC-3′; β-actin, forward 5′-CTCGCTGTCCACCTTCCA-3′, reverse 5′-GCTGTCACCTTCACCGTTC-3′. The PCR conditions were 95°C for 5 min, and then 34 cycles of 95°C for 30 sec, 54°C for 30 sec, and 72°C for 1 min, and a final extension at 72°C for 10 min. PCR products were separated on 2% agarose gels and visualized using ethidium bromide.

The following siRNAs were used: scrambled siRNA sense: 5′-GAUCAUACGUGCGAUCAGA-3′, antisense: 5′-UCUGAUCGCACGUAUGAUC-3′ ; AhR siRNA (SASI_Hs02_00332181, Sigma). The following shRNAs were obtained from the National RNAi Core Facility at Academic Sinica: control shRNA, 5′-TACAACAGCCACAACGTCTAT-3′; AhR shRNA (1) (TRCN0000021255), AhR shRNA (2) (TRCN00000245285), NF-κB shRNA (1) (TRCN0000006518), and NF-κB shRNA (2) (TRCN0000006520). Cells were transfected with siRNA (10 nM) or shRNA (2 μg) using LT1 transfection reagent.

Whole cell extracts were prepared in RIPA lysis buffer (Millipore, Temecula, CA, USA) containing 1× protease inhibitor cocktail (Thermo Scientific, Waltham, MA, USA). Protein concentrations were determined using a BCA protein assay kit (Thermo Scientific, Rockford, IL, USA). Equal amounts of protein (50 μg protein) were resolved by sodium dodecyl sulfate-polyacaryamide gel electrophoresis (SDS-PAGE), transferred onto a polyvinylidene difluoride membrane, and blocked with 5% nonfat dry milk for 1 hour at room temperature. After blocking, the membrane was incubated overnight with primary antibodies at 4°C and washed three times with PBST. The horseradish peroxidase conjugated secondary antibodies (Santa Cruz Biotechnology) were incubated for 1 hour at room temperature. The blots were washed three times with PBST and then visualized with an enhanced chemiluminescence kit (Thermo Scientific, Rockford, IL, USA). The primary antibodies were as follows: AhR, Gα_q/11, G_β, PIP2, IP3R, and PI3K from Santa Cruz Biotechnology (1:100); p44/42 MAPK (Erk1/2), Akt, p-Akt (ser473), NFκB, LaminA/C, Histion H3 and α-tubulin from Cell Signaling Technology (1:1000); COX-2 from Abcam (1:1000); β-actin from Sigma (1:1000).

After preclearing for 30 minutes with protein G agarose (Millipore), antibodies specific for Gα_q/11, G_β, or PI3K (1:100; Santa Cruz Biotechnology) or IgG (2 μg, Sigma-Aldrich Co, St. Louis, MO, USA) were added before overnight incubation at 4°C, followed by precipitation for 2 hours with protein G agarose. The beads were washed three times with RIPA lysis buffer, boiled in sample buffer, and the protein were resolved by 8% SDS-PAGE before performing immunoblot analysis of the indicated proteins.

Cell migration assays were performed in 24-well inserts (8-μm pore size; BD Biosciences, Franklin Lakes, NJ, USA) and cell invasion assays were performed in 24-well Matrigel™ Invasion inserts (8-μm pore size; BD Biosciences, Franklin Lakes, NJ, USA). Cells (1 × 10⁴) in serum-free DMEM were seeded in the upper chamber of the insert and DMEM containing 10% fetal bovine serum was added to the lower chamber and followed by incubation for 1 day (for migration) or 2 days (for invasion). The medium and cells were then removed from the top chamber using cotton swabs and PBS. The cells were fixed with 4% paraformaldehyde for 30 minutes, stained with a 0.5% crystal violet solution for 2 hours, and counted under a microscopy.

Huh7 cells were grown in 12-well plates and treated with BBP for 1 day. After treatment, the cells were incubated in fresh medium for 1 day. The media were then collected and centrifuged at 1,000 rpm for 5 minutes to remove cell debris. Vascular endothelial growth factor (VEGF) levels in the conditioned medium were measured with an enzymed-linked immunosorbent assay (ELISA) kit (R&D System, Minneapolis, MN, USA).

HUVEC (2 × 10⁴ cells/mL) were seeded in a 48-well plate pre-coated with Matrigel (BD Biosciences, San Jose, CA, USA). After Huh7 cell-conditioned medium was added to a final volume of 20%, the cells were cultured for 16 hours, stained with calcium-AM (Invitrogen), and visualized under a fluorescence microscope (Olympus). Total tube lengths were measured by MetaMorph software (Leica)

Male 6-week-old nude mice (BALB/cA-nu nu/nu) were purchased from the National Science Council Animal Center (Taiwan). All animal experiments were performed according to a protocol approved by the Institutional Animal Care and Use Committee of Kaohsiung Medical University Hospital (IACUC Approval No: 101060).

Huh7 cells were stably transfected with infrared fluorescent protein [20]. Briefly, 293 T cells were transfected with pCMV-ΔR8.91, pMD.G, and pLKO AS3-reporter gene using LT1 transfection reagent for 3 days, and the supernatant (lentivirus-containing medium) was collected the next day. Huh7 cells (2 × 10⁵) were seeded into six-well plates and incubated for 1 day. The lentivirus-containing medium (200 μL) was mixed with 800 μL of DMEM containing 8 μg/mL polybrene and added to each well, and the cells were incubated for 1 day. A stable clone was selected by puromycin treatment (2 μg/mL) for 14 days. The cells were incubated with 25 μM biliverdin overnight and then purified Huh7-IFP cells by flow cytometry. The hepatocellular carcinoma model of direct intrahepatic injection was performed according to a previous study [21] with some modifications. After a small incision was made in nude mice to access the liver, Huh7-IFP cells (1 × 10⁶) suspended in PBS were slowly injected into the upper left lobe of the liver using a 28-gauge needle. A transparent bleb of cells was formed through the liver capsule after injection. To prevent bleeding, a small piece of sterile gauze was placed, and light pressure was applied on the injection site. After implantation, the mice were placed on a heating pad or below a heating lamp until fully active. The mice were randomly divided into two groups (vehicle or BBP treatment), 18 mice of each. After 3 days, BBP (500 mg/kg) was administered by intraperitoneal (i.p.) injection every 2 days. Previous studies have reported that administration of BBP by i.p. at a dose of 800 mg/kg for 24 weeks results in no significant toxic effects [11, 22], which is a higher dose than that used in this study. Tumor growth was detected by injecting biliverdin (250 nM) into the tail vein 30 minutes before imaging. After 1 month, the mice were sacrificed and the organs (liver, lungs, kidneys, and spleen) were removed and viewed with an Ultra Sensitive Molecular Imaging System (Berthold Technology). The numbers of the organs (lung, kidneys, spleen) that expressed fluorescence, considered as metastasis positive organs were determined.

Liver tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 4-μm thick sections. The sections were deparaffinized in xylene and rehydrated with a graded series of ethanol/water solutions (100% and 95% ethanol) and then water washes. The sections were treated with 10 mM citrate buffer at 95°C to retrieve antigens and blocked with 5% bovine serum albumin. Primary antibodies against PI3K (1:100; Santa Cruz Biotechnology) and NF-κB (1:200; Cell Signaling) were applied to the sections at 4°C overnight, and then the sections were incubated with secondary antibodies and 3,3’-diaminobenzidine. Intensities of PI3K and NF-κB staining were analyzed by Tissue Quest software (Tissue Genomics)

Huh7 cells (3 × 10⁶) were suspended in 150 μL PBS, mixed with 50 μL Matrigel (BD Biosciences), and injected into the flanks of nude mice. BBP (1 μM) was added to the cell suspensions of the treatment groups. After 21 days, the Matrigel plugs were removed. Hemoglobin levels were determined by Drabkin reagent (Sigma), and protein concentrations were normalized to measure blood vessel formation.

Statistical significance was established using the Student’s t-test. A p-value of < 0.05 was considered statistically significant.

The effect of BBP on AhR mRNA expression was examined by RT-PCR. BBP transiently increased AhR mRNA expression until it reached its highest level at 15 minutes after treatment (Figure 1A). Next, we examined the RNA level using a specific AhR mRNA probe. As a result, AhR mRNA expression was increased at 15 minutes after BBP teratment, which was comparable with the RT-PCR results (Figure 1B). Immunoblot analysis of the effect of BBP on AhR expression showed that BBP stimulated AhR expression in a time-dependent manner (Figure 1C).

To investigate whether AhR can be activated at the cell membrane by BBP, Huh7 cells were transfected with pEGFP-C1 as a plasmid control or pEGFP-C1-AhR treated with DMSO as a viechle control or BBP, and then analyzed by TIRF microscopy. AhR-GFP expression peaked at 2 minutes after BBP treatment (Figure 2A). Analysis of AhR movement in Huh7 cells showed that AhR expression near the membrane increased in a time-dependent manner. The experiment was performed by confocal microscopy and analyzed by FlowView 3.0 (Olympus, Japan) (Figure 2B). Stimulation of both Gα_q/11and G_β expression by BBP was analyzed by immunoblotting (Figure 2C). Double immunogold transmission electron microscopy and immunoprecipitation (Figure 2D, 2E) further showed an interaction between AhR and G proteins. The action of AhR was notably nongenomic. To investigate whether the G-protein signaling induced by BBP was AhR dependent, we knocked down AhR using an AhR shRNA. The results showed that BBP-induced Gα_q/11 and G_β expression was suppressed by transfection of the AhR shRNA (Figure 2F).

Downstream signaling triggered by Gα_q/11 was measured by immunoblot analysis of Gα_q/11, PIP2 and IP3R levels. A decrease in PIP2 and an increase in IP3R indicated cleavage of PIP2 to IP3, followed by activation of IP3R (Figure 3A) [23]. The calcium response to BBP was then analyzed with a live-cell calcium imaging system that showed a sharp signal immediately after BBP addition (Figure 3B). To determine whether the calcium was derived from external or internal stores, calcium-free medium was used in a second round of experiments. As a result, calcium release was quickly stimulated by addition of BBP, presumably from internal stores (Figure 3C). The results were then confirmed using 2-APB, an IP3R inhibitor. 2-APB inhibited the internal release of calcium in a dose-dependent manner (Figure 3D). Expression of COX-2 was activated by BBP and inhibited by 2-APB (Figure 3E, 3F). These results suggest that BBP promotes COX-2 expression via AhR/Gα_q/11/calcium signaling.

G_β protein activates PI3K by direct binding [24]. Immunoprecipitation was performed to examine the effect of BBP treatment on interactions between G_β protein and PI3K (Figure 4A). The binding of G_β protein to PI3K was increased after BBP treatment. To further study the downstream pathway triggered by G_β activation, we analyzed PI3K enhancement and Akt phosphorylation by immunoblotting. PI3K and Akt phosphorylation level were increased after BBP treatment (Figure 4B). We also found translocation of NF-κB into the nucleus (Figure 4C). Treatment with a PI3K inhibitor (wortmannin) reduced Akt phosphorylation (Figure 4D) and inhibited NF-κB translocation into the nucleus (Figure 4E). To further confirm whether PI3K/Akt/ NF-κB activation is specifically stimulated by BBP via AhR, we transfected Huh7 cells with two different AhR shRNAs. The increase of PI3K and p-Akt (Figure 4F), and translocation of NF-κB into the nucleus (Figure 4G) induced by BBP were inhibited by transfection of the shRNAs. PI3K/Akt/NF-κB enhances cell migration and invasion [25]. Thus, we further investigated the mechanisms of cell migration and invasion which are induced by BBP. Huh7 cells were transfected with two different AhR shRNAs, NF-κB shRNA, or control shRNA for 48 hours, followed by preparation of cell lysates. The protein levels of AhR and NF-κB markedly decreased after transfection with AhR and NF-κB shRNAs compared with those after transfection with control shRNA (Figure 5A). To further study the effects of BBP on Huh7 cells migration and invasion, transwell migration and invasion assays were performed. AhR and NF-κB shRNAs inhibited cell migration and invasion induced by BBP (Figure 5B). These results suggest that BBP promotes cell migration and invasion by activation of AhR/G_β/PI3K/Akt/NF-κB signaling.

The mouse intrahepatic injection model was established to examine tumor metastasis [26]. Huh7-IFP cells (1 × 10⁶) were suspended in 30 μL PBS and injected into the mouse liver (At 3 days after of implantation, 500 mg/kg BBP was administered by i.p. injection every 2 days. Weekly imaging of the mice confirmed that the tumor sizes increased over time (Figure 5C). The mice were sacrificed after 1 month to compare tumor metastasis between control and BBP treatment groups. The metastasis rates in the lungs, kidneys, and spleen were higher in the BBP treatment group than those in the control group (Figure 5D), suggesting that BBP promotes metastasis. Immunohistochemistry showed that liver PI3K and NF-κB levels were significantly higher in the treatment group than those in the control group (Figure 5E).

To examine the effect of BBP on angiogenesis, the conditioned medium of Huh7 cells that had been treated with BBP was examined for its ability to induce the formation of capillary-like structures by HUVEC (Figure 6A). The levels of VEGF, which promotes angiogenesis [27], were also measured in the conditioned medium (Figure 6A). To explore the associated mechanisms, Huh7 cells were treated with the ERK inhibitor Pd98059 (Figure 6A) or transfected with AhR siRNA (Figure 6B) before collection of the medium. Analyses of the media showed that both Pd98059 and AhR siRNA inhibited tube formation of HUVEC and reduced VEGF induction after BBP treatment. Moreover, we evaluated the phosphorylation levels of ERK (Figure 6C). To further confirm whether activation of ERK was AhR dependent, we trasfected Huh7 cells with two different AhR shRNAs and the results showed inhibition of the phosphorylation levels of ERK induced by BBP (Figure 6D). The angiogenic effects of BBP were then assessed in an in vivo Matrigel plug angiogenesis assay model. Briefly, Huh7 cells were mixed with Matrigel and injected into the flanks of nude mice. After 3 weeks, the mice were sacrificed, and hemoglobin levels in the plug were measured. Hemoglobin levels in the Matrigel plug treated with BBP were significantly higher than those in the control group (Figure 6E).