Anti-cancer activity of Angelica gigas by increasing immune response and stimulating natural killer and natural killer T cells

Angelica gigas Nakai root was obtained from Gangwon province, Korea. The voucher specimen (Y.D. Kim et al. TG-20090258) was deposited at the Herbarium of Hallym University (Chuncheon, Korea). The ISAg was prepared by adding five times greater v/v % water to A. gigas root and extracting twice at 80 °C for 6 h, and then filtered (pore size, 0.45 μm). The resulting extract was concentrated in vacuo and dissolved in 5 to 8 times 70% ethanol at 55 °C for 2 h with stirring. The ethanol-insoluble precipitates were obtained after centrifugation. The phenol-sulfuric acid method was used to measure the total carbohydrate content of the ISAg [20]. Briefly, 200 μl ISAg was mixed with 1 ml 5% phenol; 5 ml H₂SO₄ was then added and mixed well on a vortex mixer. After a 20-min incubation, the color intensity was measured at 490 nm using a Microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). To investigate the constituent sugars, the ISAg was hydrolyzed with H₂SO₄ and subjected to anion-exchange high performance liquid chromatography (ICS-5000, Dionex Co., USA) for quantitative analysis.

Wild-type (WT) C57BL/6 (B6), C3H/HeN (TLR4-WT), and C3H/HeJ (TLR4-mutant) mice were obtained from Jung Ang Lab Animal Inc. (Seoul, Korea). IL-12p40 reporter (Yet40) and IL-12p35 knockout (KO) B6 were provided by Dr. R. Locksley (University of California at San Francisco, CA, USA). All mice used in this study were maintained at Hallym University or Sejong University. The animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at Hallym University (Hallym 2016–34) and Sejong University (SJ-20160705). All experiments were performed blindly and randomly using age- and sex-matched mice. For sacrifice, mice were euthanized by CO₂ asphyxiation. The CpG oligodeoxynucleotides (CpG ODN type B 1826) were manufactured by Bioneer (Daejeon, Korea). LPS was obtained from Sigma-Aldrich (St. Louis, MO, USA). Alpha-galactosylceramide (α-GalCer) was obtained from Enzo Life Sciences (Farmingdale, NY, USA).

Murine macrophage, RAW264.7 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS, Gibco) supplemented with 2 mM glutamine and 100 units/mL penicillin-streptomycin. Cell viability was measured by using CellTiter 96® AQueous assay kit (Promega, Fitchburg, WI, USA). The cultured cells (5 × 10⁴ cells/well) on 96-well plates were treated with serial dilutions of ISAg for 24 h. MTS tetrazolium was added to the plates and incubated at 37 °C for 1 h. Absorbance was measured at 490 nm using a microplate reader.

RAW264.7 cells were incubated with LPS (1 μg/mL) or various amounts of ISAg (0.125–2 μg/mL) at 37 °C for 24 h. The amount of nitrite (NO₂⁻) in the culture supernatant was measured by Griess Reagent System (Promega). The amounts of IL-6, tumor necrosis factor (TNF)-α, and IL-1β secreted to the culture medium were quantified using an ELISA kits (KOMA Biotech, Seoul, Korea).

Total RNA from RAW264.7 cells was isolated with TRIzol reagent (Invitrogen, Waltham, MA, USA). The relative amount of specific mRNA was assessed by RT-PCR using amfiRivert cDNA Synthesis Platinum Master Mix (GenDEPOT, Baker, TX, USA). Quantification of mRNA was performed using qRT-PCR. The AccuPower® 2X GreenStar™ qPCR Master Mix (Bioneer) and the Exicycler™ 96 PCR system (Bioneer) were used according to the manufacturer’s instructions. The sequences of the sense- and antisense-strand primers used for PCR amplification were inducible nitric oxide synthase (iNOS), 5’-GCTACCACATTGAAGAAGCTGGTG-3′, 5’-CCATAGGAAAAGACTGCACCGAAG-3′; cyclooxygenase-2 (COX-2), 5’-GTCTCTCAATGAGTACCGCAAACG-3′, 5’-CTACCATGGTCTCCCCAAAGATAG-3′; IL-6, 5’-GCCAGAGTCCTTCAGAGAGATACA-3′, 5’-ATTGGATGGTCTTGGTCCTTAGCC-3′; IL-1β, 5’-CCTGTGTAATGAAAGACGGCACAC-3′, 5’-CTTGTGAGGTGCTG ATGTACCAGT-3′; TNF-α, 5’-TCTCATCAGTTCTATGGCCCAGAC-3′, 5’-GGCACCA CTAGTTGGTTGTCTTTG-3′. As a control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was also amplified using 5’-GACATCAAGAAGGTGGTGAAGCAG-3′, 5’-CCCTGTTGCTGTAGCCGTATTCAT-3′.

Total cell lysates were extracted using CytoBuster™ Protein Extraction Reagent (Novagen). Equal amounts of protein were separated on 10 to 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels at 300 mA for 20 min. They were then transferred to Immobilon-P polyvinylidene difluoride (PVDF) membranes (Millipore Sigma, Billerica, MA, USA) using a trans-blot SD Semi-Dry Transfer Cell (Bio-Rad, Hercules, CA, USA). After transfer, the membranes were incubated in Tris buffered saline (TBS), 5% dry milk, and 0.2% Tween 20 for 1 h. They were then further incubated in TBS and 0.2% Tween 20 with specific antibodies at 4 °C overnight. After washing, a horseradish peroxidase (HRP)-labeled secondary antibody was applied and the membranes were incubated for 2 h. Immunoblot detection was performed using Immobilon Western HRP Substrate (Millipore Sigma). The following antibodies from Cell Signaling Technology were used: anti-protein kinase B (PBK/Akt), anti-phospho-Akt (Ser473), anti-c-jun N-terminal kinase (JNK), anti-phospho-JNK, anti-p38, anti-phospho-p38, anti-p44/42 extracellular signal-related kinase (ERK), anti-phospho-p44/42 ERK, and anti-GAPDH.

BMDCs were generated from the bone marrow (BM) cells of Yet40 B6 mice, as previously described [21]. Briefly, BM cells were harvested from femurs and tibiae of mice by flushing with Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco). Red blood cells were removed by adding ACK lysis buffer (0.15 M NH₄Cl, 10 mM KHCO₃, and 2 mM EDTA in distilled water), and remaining cells were washed with phosphate buffered saline (PBS) and cultured at a concentration of 1 × 10⁶ cells/well in complete RPMI 1640 medium containing recombinant mouse fms-related tyrosine kinase 3 ligand (Flt3L) (100 ng/ml; R&D Systems, Minneapolis, MN, USA). Fresh cytokine-included culture medium was added on day 5 to generate BMDCs. Five days later, the BMDCs were harvested and stimulated for 16 h with vehicle or ISAg (125 to 2000 ng/ml). The purity of cluster of differentiation (CD)11c⁺ cells of these cultures was > 92%.

The following monoclonal antibodies (mAbs) were obtained from BD Biosciences (San Jose, CA, USA): PE-Cy7-, or allophycocyanin (APC)-conjugated anti-CD11c (clone HL3); phycoerythrin (PE)-, or APC-conjugated anti-NK1.1 (clone PK-136); biotin-conjugated anti-TRAIL; PE-Cy7- or APC-conjugated anti-CD3ε (clone 145-2C11); PE-Cy7-conjugated anti-CD11b (clone M1/70); biotin-conjugated anti-CD45 (clone PC61); PE-conjugated anti-IL-12p40 (clone C15.6); biotin-conjugated CD86 (clone GL1); PE-conjugated anti-TNF-α (clone XP6-XT22); PE-conjugated anti-IgG1 (κ isotype control). The following mAbs from Thermo Fisher Scientific were used: PE-conjugated anti-FasL (clone MFL3); APC-conjugated anti-F4/80 (clone BM8); PE-conjugated anti-perforin (clone eBioOMAK-D); PE-conjugated anti-IFN-γ (clone XMG1.2). Flow cytometric data were acquired using a FACSCalibur (Becton Dickinson Inc., San Jose, CA, USA) and were analyzed with FlowJo analysis tool (Tree Star Inc., Ashland, OR, USA). For surface antibody staining, the cells were collected and washed twice with fluorescence-activated cell sorting (FACS) buffer (PBS containing 0.5% bovine serum albumin). The cells were pre-incubated with purified anti-CD16/CD32 mAbs (BD Bioscience, Bedford, MA, USA) on ice for 10 min for blocking non-specific binding to Fc receptors, and were then stained with fluorescence-labeled mAbs. For intracellular staining, splenocytes were incubated with brefeldin A in RPMI 1640 medium (10 μg/ml) at 37 °C for 2 h. Cells were stained for specific surface markers and fixed with 1% paraformaldehyde. After permeabilization with 0.5% saponin (Sigma-Aldrich), cells were stained with the indicated mAbs (PE-conjugated anti-IL-12p40, anti-TNF-α, anti-IFN-γ, and ant-Perforin; PE-conjugated isotype control rat IgG mAbs) for 30 min. More than 5000 events per sample were acquired using the FACSCalibur.

NK1.1⁺ cells were enriched from total splenocytes isolated from B6 mice using positive selection with anti-APC MACS (Miltenyi Biotec, Bergisch Gladbach, Germany), after staining with APC-conjugated anti-NK1.1 mAb. The cell populations included > 92% NK1.1⁺ cells among all the MACS-purified populations. Splenocytes were prepared as single cell suspensions and cultured in complete RPMI 1640 medium containing 10% FBS supplemented with 5 mM 2-mercaptoethanol, 2 mM L-glutamine, 10 mM HEPES, and 100 units/mL penicillin-streptomycin.

The flow cytometric 7-amino actinomycin D (7-AAD)/carboxyfluorescein succinimidyl ester (CFSE) cytotoxicity assay was used as previously described [22]. NK1.1⁺ cells were isolated as described above and were suspended in complete RPMI 1640 medium. B16 melanoma cells (3 × 10⁶) were stained with CFSE (50 μM) in a 2 ml Hanks’ Balanced Salt Solution at 37 °C for 10 min. NK1.1⁺ cells were incubated with the CFSE-labeled target cells (20,000 cells) at different effector/target (E:T) ratios (27:1, 9:1, 3:1, and 1:1). After a 10-h incubation, the cells were stained with 0.25 μg/ml 7-AAD and were incubated at 37 °C for 10 min. After two washes with PBS containing 1% FBS, cells were resuspended in FACS buffer and their cytotoxicity was evaluated using flow cytometry.

To evaluate the dose-dependent effects of ISAg on innate immune responses, Yet40 B6 mice were given ISAg via oral gavage at doses of 0.5, 1, 2, or 4 mg/mouse, three times per week for 4 weeks. In addition, to measure the time-dependent effects of ISAg on innate immune responses, Yet40 B6 mice were received oral ISAg (4 mg/injection) three times per week for the indicated times (1 to 4 weeks). For experiments to test the involvement of TLR4 signaling in ISAg-mediated innate immune responses, C3H/HeN and C3H/HeJ mice were orally administered ISAg (4 mg/injection) three times per week for 4 weeks. Besides, to investigate whether ISAg-mediated innate immune responses are IL-12 dependent, WT or IL-12p35 KO mice were orally administered ISAg (4 mg/injection) three times per week for 4 weeks. In all the aforementioned experiments, LPS (2 μg/mouse) was used as a positive control to inject mice intraperitoneally (i.p.) once a week for a total of 4 weeks.

To examine in vivo anti-tumor effects of ISAg, Yet40 mice (n = 5/group) received subcutaneous (s.c.) injections of 5 × 10⁵ B16 melanoma cells. One week later, these mice were treated orally using oral gavage with either ISAg (4 mg/injection) or PBS three times per week for the following 2 weeks. As a positive control for anti-tumor immune responses, α-GalCer (2 μg) was injected via the intraperitoneal route two times per week starting 7 days after tumor injection, for a total of 2 weeks. On day 21 after tumor injection, groups of mice were euthanized and tumor tissues were excised for immunological analysis. Tumor-infiltrating leukocytes were isolated using the following procedure: small pieces tumor tissues were digested (15 min, 37 °C) using 2.5 mg/ml collagenase type IV (Sigma-Aldrich) and 1 mg/ml DNase I (Promega). After incubation, the digested tissues were dissociated into single-cell suspensions using a gentleMACS Dissociator and C Tubes (Miltenyi). The cell-containing suspension was passed through a 70-μm pore nylon cell strainer (BD Bioscience, Bedford, MA, USA), and put on ice. The tumor-infiltrating leukocytes were harvested from the interface of a 40/70% Percoll (GE Healthcare, Little Chalfont, UK) gradient after centrifugation at 1000 g for 20 min.

Statistical significance was analyzed using the Excel statistical analysis tool (Microsoft, Redmond, WA, USA). The comparison of two groups was performed by the Student’s t-test. Values of *P < 0.05 and **P < 0.01 were considered to indicate a statistically significant result in the Student’s t-test. VassarStats statistical software (http://​vassarstats.​net/​anova2u.​html) was used for two-way analysis of variance (ANOVA). Values of ^#P < 0.05 and ^##P < 0.01 indicates a statistically significant result in the two-way ANOVA.

A water-soluble polysaccharide fraction of A. gigas, ISAg, was obtained as described in the Methods section. The final product consisted of 63.7% total carbohydrate, 17.4% protein, 0.38% lipid, and inorganic compounds. The maximum content of crude polysaccharide was > 20% of the total extracts. The content of uronic acid, which is a major component of pectin, was 15.1%. The result for the composition of monomeric carbohydrate present in the polysaccharides is presented in Table 1. Glucose was the main polysaccharide component (69.5% of total carbohydrate). The other major sugar constituents were galactose (17.4%) and arabinose (9.0%), Rhamnose, mannose and xylose were also detected as minor components.

To investigate the immuno-stimulatory activity of ISAg, we examined the ISAg-treated mouse macrophage cell line, RAW264.7. ISAg increased NO production of macrophages in a dose-dependent manner, compared with LPS used as a positive control (Fig. 1a). The inducible isoform of nitric oxide synthase (iNOS), which produces NO as an innate immune response defense mechanism, also increased after exposure to ISAg (Fig. 1b). NO stimulates gene expression of COX-2, which converts arachidonic acid to prostaglandin. The transcript level of COX-2 was also increased by the ISAg treatment (Fig. 1c). The high ISAg concentrations (up to 2000 ng/ml) used in all experiments did not induce cytotoxic effects (Fig. 1d).

ISAg also induced macrophages to express the pro-inflammatory cytokine genes IL-6, IL-1β, and TNF-α (Fig. 2). Both the RNA transcripts (Fig. 2a–c) and proteins secreted in the culture medium (Fig. 2d–f) were increased by ISAg treatment. These cytokines activate innate immune responses of pathogen-infected host cells. The immuno-stimulatory effects of ISAg on the production of NO and cytokines might be due to the induction of phosphoinositide 3 kinase (PI3K)/Akt signal transduction pathways and MAPK activity. ISAg significantly induced phosphorylation of Akt and JNK in a dose dependent manner (Fig. 3). Other MAPKs, such as ERK-1/2 and p38, were also phosphorylated after exposure to ISAg. This result was consistent with previously reported results [15].

Angelan stimulation of DC maturation and migration has anti-cancer effects [11, 15]. Little is known about the details of the cytotoxic process or how it affects the tumor cells. To investigate whether ISAg possessed adjuvant-like ability to stimulate DCs, BMDCs derived from Yet40 B6 mice were prepared and subsequently stimulated with ISAg in vitro. Sixteen hours later, both CD86 expression and cytokine production (IL-12p40 and TNF-α) in BMDCs were examined using flow cytometric analysis. ISAg-treated BMDCs significantly increased the expression of the co-stimulatory molecule CD86 and cytokine production (IL-12p40 and TNF-α) (Fig. 4a–d). Taken together, these results indicated that ISAg had a priming effect during activation of the DCs.

Based on these results, we investigated whether in vivo treatment with ISAg might modulate innate immune cells, including DCs and macrophages. To test this hypothesis, we delivered ISAg into Yet40 mice and then examined the expression of IL-12p40 [yellow fluorescent protein (YFP)] using splenic DCs and macrophages isolated from ISAg-treated mice. We found that in vivo treatment with ISAg caused a statistically significant dose-dependent increase in IL-12 secretion (Fig. 5a, b). Next, to examine the kinetics of ISAg-induced IL-12 production in antigen-presenting cells (APCs), we measured IL-12 production in splenic DCs and macrophages from Yet40 B6 mice fed ISAg (4 mg/injection) three times per week for 1 to 4 weeks. We found that a significant increase in IL-12 secretion by DCs and macrophages was saturated after 2 weeks post-ISAg treatment (Fig. 5c). To further understand how ISAg affects IL-12 production in mouse APCs, we used TLR4-mutant C3H/HeJ and TLR4-sufficient C3H/HeN mice to examine whether TLR4 was responsible for ISAg-mediated IL-12 expression. We found that the enhancing effect of ISAg on IL-12 production was not present in APCs from TLR4-mutant C3H/HeJ mice (Fig. 5d). This result suggested that ISAg-induced IL-12 production in APCs was dependent on the TLR4 signaling pathway.

Our results indicated that ISAg had stimulatory effects on APC activation. However, the effects of ISAg on cytotoxic immune cell (e.g., NK1.1⁺ cells including NK and NKT cells) function have not been studied. To examine these effects, we measured the levels of inflammatory cytokine (IFN-γ and TNF-α) production using splenic NK and NKT cells from Yet40 mice given orally administered ISAg (from 0.5 to 4 mg/mouse). In vivo ISAg treatment showed a statistically significant (p < 0.05) increase of NK1.1⁺ cells expressing IFN-γ and TNF-α in Yet40 mice (Fig. 6a, b). We hypothesized that ISAg-associated NK and NKT cell activation is mediated by IL-12. To confirm this, WT or IL-12p35 KO mice were orally administered ISAg and the activation of NK and NKT cells was examined by flow cytometry. The production of inflammatory cytokines in NK and NKT cells of IL-12p35 KO mice was significantly reduced compared to that of WT mice (Fig. 6c, d). These results indicated that IL-12 is required for ISAg-mediated NK and NKT cell activation in vivo. We also examined whether oral administration of ISAg could enhance cytotoxicity and cytokine secretion in NK and NKT cells. NK1.1⁺ cells isolated from PBS- or ISAg-injected WT B6 mice were used as effector cells for the cytotoxicity assay against B16 melanoma cells. We found that in vivo ISAg treatment increased cytotoxicity of NK1.1⁺ cells against the tumor cells (Fig. 6e, f). Based on these results, we propose that NK1.1⁺ cells activated by ISAg treatment can eliminate tumors more efficiently through enhanced effector functions such as cytotoxicity.

Based on the results of the in vitro experiments that examined activation of NK receptor-expressing innate immune cells via IL-12, we hypothesized that in vivo ISAg treatment can inhibit tumor growth in mice. To examine in vivo anti-tumor activity of ISAg, Yet40 mice were inoculated (s.c.) with B16 melanoma. One week later ISAg was orally injected to these mice. ISAg-injected mice had a significant decrease in tumor growth, compared with PBS-injected mice (Fig. 7a, b). To examine whether the increased accumulation of NK and NKT cells was correlated with a decrease in tumor mass, we compared the numbers of NK and NKT cells infiltrated into the tumors between PBS- and ISAg-injected mice. There was a marked increase in accumulation of NK and NKT cells in B16 tumors after ISAg treatment, compared with the PBS-treated mice (Fig. 7c, d). Next, we examined whether there was any change in the expression of cytotoxic molecules such as perforin, TRAIL, or FasL, following ISAg administration. All these molecules were significantly increased in NK and NKT cells after ISAg treatment in vivo (Fig. 8a–c). These results indicated that the cytotoxicity of ISAg treatment against B16 tumor cells in mice was mainly due to the anti-cancer activity of NK and NKT cells.