Sporamin induces apoptosis and inhibits NF-κB activation in human pancreatic cancer cells

Materials

Sporamin was purified from fresh sweet potato tuberous roots, as reported previously. ⁹ Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco-Invitrogen (Carlsbad, CA, USA); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), [³H] thymidine, and dimethyl sulfoxide (DMSO) were bought from Sigma-Aldrich (St. Louis, MO, USA).

Cell culture

PANC-1 and BxPC-3 (American Type Culture Collection (ATCC), Manassas, VA, USA), two human PC cell lines, were cultured in DMEM with 10% heat-inactivated FBS, benzylpenicillin (100 kU/L), and streptomycin (100 mg/L) at 37°C in an incubator containing humidified air with 5% CO₂.

[³H] thymidine incorporation assay

PANC-1 and BxPC-3 cells were plated on 24-well plates and grown to 80% confluency in DMEM with 10% dialyzed and charcoal-stripped FBS. The cell cultures were then rinsed in phenol-free DMEM medium and incubated with respective test substances in phenol-free and serum-free DMEM for 48 h. [³H] thymidine (1.35 × 10⁴ Bq/L) was added. Cells were cultured for another 12 h. The supernatant was aspirated and then washed twice with phosphate-buffered saline (PBS) to remove excess [³H] thymidine. The cells were then dissolved in 0.2 mol/L of NaOH. Radioactivity (incorporation activity) was determined by scintillation counter.

Cell viability assay

PANC-1 and BxPC-3 cells were plated on 96-well plates and grown to 80% confluency. The cell cultures were then rinsed in phenol-free DMEM medium and incubated with respective test substances in phenol-free and serum-free DMEM for 48 h. At the end of this time interval, MTT was added to a final concentration of 0.5 mg/mL. After 1 h of incubation, cultures were removed from the incubator and the formazan crystals were solubilized by adding solubilization solution including 10% (v/v) Triton X-100 and 0.1 mol/L HCl in isopropanol equal to the volume of original culture medium. Colorimetric determination of MTT reduction was made at 570 nm using a microplate enzyme-linked immunosorbent assay (ELISA) reader (Bio-Rad, Hercules, CA, USA).

Morphological determination and quantification of apoptosis

PANC-1 and BxPC-3 cells were cultured on 22 mm × 22 mm glass coverslips on six-well plates at a density of 2 × 10⁵ per well and incubated with or without indicated treatments for the desired time. For nuclear staining assay, cells were fixed with 4% cold paraformaldehyde (pH 7.4) and stained with 0.5 µg/mL 4′,6-diamidino-2-phenylindole (DAPI; Sigma, St. Louis, MO, USA) for 5 min at room temperature. The coverslips were rinsed with PBS and mounted onto slides with a fluorescent mounting medium. The cells were then visualized under a fluorescence microscope (DMI4000B; Olympus, Tokyo, Japan).

Flow cytometry analysis for apoptosis

Apoptosis were measured using the Annexin V-PE/7-AAD Apoptosis Detection Kit (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. Briefly, the cells were incubated with or without indicated treatments for the desired time. Attached and floating cells were pooled, pelleted by centrifugation, washed two times with cold PBS, and then resuspended in 1× binding buffer (10 mM hydroxyethyl piperazine ethanesulfonic acid (HEPES), pH 7.4, 140 mM NaOH, and 2.5 mM CaCl₂) at a concentration of 1 × 10⁶ cells/mL. A volume of 1 × 10⁵ cells were transferred to a 5-mL tube and stained with 5 µL of Annexin V-PE and 5 µL of 7-amino-actinomycin D (7-AAD) for 15 min at room temperature in the dark. Finally, 400 µL of 1× binding buffer was added to each tube and apoptosis was determined using a flow cytometer (BD, Franklin Lakes, NJ, USA).

Western blotting

Cellular total, nuclear, and cytoplasmic protein extractions were carried out using M-Per Mammalian Protein Extraction Reagent (Pierce, Rockford, IL, USA) and Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturers’ instructions. Cell protein extracts were collected and centrifuged at 14,000g in a microcentrifuge for 10 min at 4°C. The supernatants were collected, and the protein concentrations were determined using Bradford’s method. Cell protein extracts were either used immediately or stored at −70°C.

For polyacrylamide gel electrophoresis (PAGE), 20 µg of proteins from each cell extract was loaded into each well of the gel and electrophoresed at 98 V. Proteins on the gel were transferred onto a polyvinylidene difluoride (PVDF) membrane by conventional tank-buffer electrotransfer for 2 h. The membrane was blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline (TBS) containing 0.05% Tween 20 for 1 h. The primary antibodies (all used at a 1:200 dilution) against Bcl-2, Bcl-XL, Bax, IκBα (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phosphor-IκBα (serine 32; Cell Signaling Technology, Danvers, MA, USA), tubulin, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Santa Cruz Biotechnology) were added separately and incubated with the membrane for overnight at 4°C before the membrane was washed three times with TBS/Tween 20. Secondary antibody conjugated to horseradish peroxidase at a 1:2000 dilution was added to the membrane and was incubated at room temperature with gentle agitation. After 1 h, the membrane was washed with four changes of TBS/Tween 20, at 5 min per wash. Immunoreactive bands were visualized using the enhanced chemiluminescence (ECL) Kit (Pierce) and scanned using an LAS-4000 imaging system (Fujifilm, Tokyo, Japan).

Cellular immunofluorescence

Cells were cultured on 22 mm × 22 mm glass coverslips on six-well plates at a density of 2 × 10⁵ per well, allowed to adhere overnight, and treated with indicated concentrations of sporamin or their control vehicles. After 48 h, the cells were washed twice with cold PBS, fixed in 4% cold paraformaldehyde (pH7.4) for 15 min, permeabilized in 0.2% Triton X-100 in double-distilled water for 10 min, blocked with 10% normal non-immune goat serum/5% BSA/PBS at 37°C for 30 min, and then incubated overnight at 4°C with different primary antibody in 5% BSA/PBS solution. Subsequently, fluorescent secondary antibody was added at 37°C for 1 h. Nuclei were counterstained with 0.5 µg/mL DAPI at room temperature for 5 min. The coverslips were rinsed with PBS and mounted onto slides with a fluorescent mounting medium. Images were acquired with the fluorescence microscope (DMI4000B; Olympus).

Electrophoretic mobility shift assay

Electrophoretic mobility shift assay (EMSA) was performed using a gel shift assay system kit (Promega, Madison, WI, USA). The NF-κB oligonucleotide probe (5′-AGT TGA GGG GAC TTT CCC AGG C-3′) was end-labeled with [γ-³²P] adenosine triphosphate (ATP; Free Biotech, Beijing, China) with T4-polynucleotide kinase. Nuclear protein (2 µg) was pre-incubated in binding buffer (10 mM Tris-Cl, pH 7.5; 50 mM NaCl; 1 mM MgCl₂; 0.5 mM ethylenediaminetetraacetic acid (EDTA); 4% glycerol; 0.5 mM dithiothreitol (DTT), and 0.05 g/L of poly (deoxyinosinic-deoxycytidylic acid)) for 15 min at room temperature. After addition of the 1 µL ³²P-labeled oligonucleotide probe, the incubation was continued for 30 min at room temperature. The reaction was stopped by adding 1 µL of gel loading buffer, and the mixture was subjected to non-denaturing 4% polyacrylamide gel electrophoresis in 0.5× Tris/borate/EDTA (TBE) buffer. The gel was vacuum-dried and exposed to X-ray film at −70°C. The specificity of binding was also examined by competing with the unlabeled oligonucleotide. For EMSA supershift assays, nuclear protein extracts prepared from sporamin-treated cells were incubated with antibodies against either NF-κB p50 or NF-κB p65 for 30 min at room temperature before the complex was analyzed by EMSA. Antibodies against pre-immune serum were included as negative controls. The dried gels were visualized by autoradiography using X-ray film.

Statistical analysis

Data are expressed as mean ± standard deviation (SD). Significance of differences between two groups was determined by Scheffé’s modified t test. A p value <0.05 represented a statistically significant difference.

Sporamin inhibits cell viability of PC cells

We tested the ability of sporamin to inhibit the cell viability of two PC cell lines, PANC-1 and BxPC-3, via the MTT assay (Figure 1). PANC-1 and BxPC-3 cells were treated with sporamin at increasing concentrations (0, 12.5, 25, 50, and 100 µM) and for varying times (24, 48, and 72 h). Sporamin was found to inhibit cell viability in a concentration-dependent and time-dependent manner as compared to diluent control.

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Figure 1.

Concentration-dependent and time-dependent effects of sporamin-induced cell viability inhibition of PC cells. PANC-1 and BxPC-3 cells were incubated in the absence or presence of the indicated concentrations of sporamin for different incubation times. Then, the cell viability was determined using the MTT assay (points: mean of four determinations (each in quadruplication)); data are shown as mean ± SD (n = 4 experiments; ^Δp < 0.05; ^ΔΔp < 0.01 vs Control (for the comparison of different time points); *p < 0.05; **p < 0.01 vs Control (for the comparison of different concentration points)).

A more significant decrease in cell viability in a concentration-dependent manner of the PC cells occurred after exposure for 48 h to sporamin compared with the diluent-exposed control cells. Therefore, we treated the PC cells with varying concentrations of sporamin for 48 h for further studies.

Sporamin inhibits cell proliferation activity of PC cells

To test the effects of sporamin on the cell proliferation activity of PANC-1 and BxPC-3 cells, [³H] thymidine incorporation assay was used. As shown in Figure 2, after treatment with sporamin at 25, 50, and 100 µM for 48 h, the [³H] thymidine incorporation activity (Bq/min) of PANC-1 and BxPC-3 cells decreased in a concentration-dependent manner as compared to diluent control.

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Figure 2.

Effects of sporamin on [³H] thymidine incorporation activity of PC cells. PANC-1 and BxPC-3 cells were treated with sporamin for 48 h and their incorporation activity was determined. Data are shown as mean ± SD (n = 4 experiments; *p < 0.05; **p < 0.01 vs Control).

Sporamin induces cell apoptosis in PC cells

To test whether sporamin induces apoptosis in PC cells, sporamin-treated PANC-1 and BxPC-3 cells were examined under fluorescence microscopy after DAPI nuclear staining. The exposure to sporamin (50 µM, 48 h) resulted in a considerable number of PANC-1 and BxPC-3 cells exhibiting condensed chromatin and fragmented nuclei, the typical markers of apoptosis (Figure 3(a)). After sporamin treatment of 25, 50, or 100 µM for 48 h, the number of apoptotic cells (proportion of apoptotic cells (% of total cells)) was increased significantly as compared to diluent control (Figure 3(b)).

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Figure 3.

(a) Effects of sporamin on apoptosis of PC cells. PANC-1 and BxPC-3 cells were treated with or without 50 µM sporamin for 48 h, and then stained with DAPI and photographed under fluorescence microscopy. Condensed and fragmented nuclei appeared in sporamin-treated cells (magnification: 400×). Apoptotic morphological changes in the nucleus were easily distinguishable from intact nuclei and counted, and the percentages were subsequently calculated. Six randomly chosen fields of view were observed with a minimum number of 500 cells scored in each condition. (b) The percentage of apoptotic cells was calculated with total PC cells. (c) The cells were stained with Annexin V-PE/7-AAD and analyzed by flow cytometry (lower left quadrants: viable cells; lower right quadrants: early apoptotic cells (Annexin V+, 7-AAD−); upper right quadrants: non-viable, late apoptotic/necrotic cells (Annexin V+ and 7-AAD+)). The numerical results represent the mean of triplicate plates, and a representative experiment is shown. (d) Bar graphs demonstrated the percentage of apoptotic rates. Data are shown as mean ± SD (n = 4 experiments; *p < 0.05; **p < 0.01 vs Control).

Since sporamin induced marked morphological changes of apoptosis at the concentration of 50 µM for 48 h, PANC-1 and BxPC-3 cells were treated with sporamin (50 µM, 48 h) and stained with Annexin V and 7-AAD to ananlyze apoptosis using flow cytometry. The percentages of apoptotic cells (Annexin V positive) were increased significantly after sporamin treatment (50 µM, 48 h) compared to the control diluent (Figure 3(c) and (d)). These results demonstrate that sporamin can induce cell apoptosis in PC cells.

Sporamin induces downregulation of Bcl-2 and Bcl-XL and upregulation of Bax in PC cells

To address whether Bcl-2, Bcl-XL, and Bax protein expression levels correlate with the apoptosis that occurs after treatment of PANC-1 and BxPC-3 cells with sporamin, the amounts of these proteins in total cell extracts were quantified by western blotting. After sporamin treatment of 25, 50, or 100 µM for 48 h, the Bcl-2 and Bcl-XL expression decreased with evident increases in Bax expression in PANC-1 and BxPC-3 cells (Figure 4). These results indicated that sporamin induced a reduction in heterodimerization of Bcl-2/Bcl-XL with Bax.

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Figure 4.

Effects of sporamin treatment on the expression of Bcl-2, Bcl-XL, and Bax. The protein expression of Bcl-2, Bcl-XL, and Bax in PANC-1 and BxPC-3 cells was detected by western blotting using lysates from PANC-1 and BxPC-3 cells treated with varying concentrations of sporamin for 48 h. Each of the blots shown was demonstrated to have equal protein loading by reprobing with the antibody for GAPDH. GAPDH was used as a loading control for the whole cell extracts. Data are representative of at least two independent experiments with similar results.

Sporamin inhibits constitutive NF-κB activation in PC cells

Because both Bcl-2 and Bcl-XL can be regulated by NF-κB in some cells,^6,11 we postulated that sporamin might downregulate Bcl-2 and Bcl-XL expression by inhibiting NF-κB activation. To investigate the effects of sporamin on constitutive NF-κB activation in PC cells, we prepared the nuclear extracts of PANC-1 and BxPC-3 cells treated with various concentrations of sporamin or control diluent and examined constitutive NF-κB activation by EMSA. As shown in Figure 5(a), a significant decrease of NF-κB activation was observed in PANC-1 and BxPC-3 cells treated with varying concentrations of sporamin (0, 25, 50, and 100 µM).

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Figure 5.

Effects of sporamin on constitutive NF-κB activation in PANC-1 and BxPC-3 cells. (a) Sporamin inhibits constitutive NF-κB activation in a concentration-dependent manner in PANC-1 and BxPC-3 cells. (b) Nuclear extracts from PANC-1 and BxPC-3 cells incubated with 0–100 µM sporamin for 48 h were subjected to EMSA to assess NF-κB DNA-binding activity. The radioactive bands were quantitated using a PhosphorImager using ImageQuant software. The levels of NF-κB DNA-binding activity relative to control are indicated at the top of each band. Supershift assay of 50 µM sporamin-treated PANC-1 and BxPC-3 cells with specific NF-κB antibody. Data are representative of at least two independent experiments with similar results. The NF-κB-specific complex and the free DNA probe are indicated (arrows). (c) NF-κB p50 and NF-κB p65 expression were measured in PANC-1 cells by immunofluorescence. Representative fluorescence micrograph images of NF-κB p50 and NF-κB p65 (red), nuclei (DAPI: blue), and Tubulin (green).

To further confirm the specificity of NF-κB DNA binding, we performed an EMSA supershift assay with antibodies specific for NF-κB p65 and NF-κB p50. For supershift assays, nuclear protein extracts prepared from sporamin (50 µM, 48 h)-treated cells were incubated with antibodies against either NF-κB p50 or NF-κB p65 for 30 min at room temperature before the complex was analyzed by EMSA. As shown in Figure 5(b), a strong supershift in the case of anti-NF-κB p65, but a weaker shift for anti-NF-κB p50 to a higher molecular weight band, suggesting that the observed NF-κB band consisted of these two subunits, NF-κB p50 and NF-κB p65, and sporamin inhibited NF-κB p65 and NF-κB p50 DNA-binding activity in PANC-1 and BxPC-3 cells. Interestingly, we found that sporamin decreased the nuclear expression of NF-κB p50 and NF-κB p65 in PANC-1 cells (Figure 5(c)). These results indicated that sporamin inhibited NF-κB activation.

Sporamin inhibits IκBα phosphorylation and increases total IκBα expression in PC cells

We examined whether the inhibition of NF-κB activation by sporamin was due to decreased degradation of IκBα. As shown in Figure 6, when PANC-1 and BxPC-3 cells were treated with sporamin with varying concentrations (25, 50, or 100 µM), significant concentration-dependent increases in total IκBα expression levels in cytoplasmic fractions were observed in PANC-1 and BxPC-3 cells (Figure 6).

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Figure 6.

Effects of sporamin on the phosphorylation and degradation of IκBα in PC cells. The cytosolic extracts were isolated and subjected to western blot analysis with anti-IκBα antibody. The whole cell extracts were isolated and subjected to western blot analysis using anti-p-IκBα (serine 32) antibody. Each of the blots shown was demonstrated to have equal protein loading by reprobing with the antibody for tubulin or GAPDH. Tubulin and GAPDH were used as loading controls for the cytoplasmic fractions and the whole cell extracts, respectively. Data are representative of at least two independent experiments with similar results.

Because degradation of IκBα normally requires the inhibitor to be phosphorylated, ⁵ it was of interest to examine the extent of IκBα phosphorylation in sporamin-treated PANC-1 and BxPC-3 cells as compared to diluent control. When PANC-1 and BxPC-3 cells were treated with sporamin with varying concentrations (25, 50, or 100 µM) followed by western blotting for the phosphorylated form of IκBα (p-IκBα), concentration-dependent decreases in expression of p-IκBα were observed in total cell extracts of PANC-1 and BxPC-3 cells (Figure 6). These results indicated that sporamin suppressed NF-κB activity might be partly through dephosphorylation of IκBα.