Coix lacryma-jobi var. ma-yuen Stapf sprout extract has anti-metastatic activity in colon cancer cells in vitro

CLSE was manufactured in the herbarium of the Herbal Crop Research Institute (Eumseong, Republic of Korea).

Deferoxamine (DFO), Phorbol 12-myristate 13-acetate (PMA), and SC79 were obtained from Sigma-Aldrich (St. Louis, MO, USA). CLSE and DFO were dissolved in water. PMA and SC79 were dissolved in the solvent dimethyl sulfoxide (DMSO).

Coix cultivars were obtained from the National Institute of Crop Science (Miryang, Republic of Korea). Coix were germinated in a modified commercial soil bed (0.7–1.0 mg/m³ soil bulk density, 450–650 mg/L available phosphate, 800–1000 mg/kg nitrogen) (Punong Bed Soil, Gyeongju, Republic of Korea). The germinated Coix was grown at 22–23 °C with humidity of 60% in a 900–1000 lx environment. Between 15 and 22 d after germination, young barley leaves about 8–13-cm long were harvested and freeze-dried [10]. We used a water extraction method because most traditional Oriental herbs are decocted in boiling water. In addition, some components are more soluble in water than in organic solvents. Crushed plant materials (200 g each) were extracted three times under reflux with distilled water. The water extracts were combined and lyophilized. The yield was 25% (wt/wt) of the dried Coix sprouts. Extracts were stored at −20 °C until usage. A voucher specimen (HPR-208) was deposited in the herbarium of Herbal Crop Research Institute (Eumseong, Republic of Korea).

HCT116 and CCD-18Co cells were obtained from the Korean Cell Line Bank (Seoul, Republic of Korea). Human umbilical vein endothelial cells (HUVECs) were obtained from the Lonza (San Diego, CA, USA). HCT116 cells were cultured in McCoy’s medium (Gibco Cell Culture, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin-streptomycin (Gibco). CCD-18Co cells were cultured in MEM (Gibco) with 10% FBS (Gibco) and 1% penicillin-streptomycin (Gibco), and were used between passages 5 and 6. HUVECs were grown in EBM-2 (Lonza) supplemented with an EGM™-2 SingleQuots™ kit (Lonza), and used between passages 2 and 4 for experiments. Cells were incubated at 37 °C in a humidified atmosphere with 5% CO₂. A hypoxia incubator (New Brunswick Scientific, Edison, NJ, USA) containing 1% O₂, 5% CO₂, and 94% N₂ was used to create hypoxic conditions.

Cells were seeded into 96-well plates and exposed to various concentrations of CLSE for 24–72 h prior to the addition of 10 μL CCK-8 solution (Dojindo Molecular Technologies, Inc., Rockville, MD, USA) containing 2-(2-methoxy-4-nitrophenyl0–3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H–tetrazolium, monosodium salt (WST-8) to each well. After 1 h of incubation at 37 °C in a humidified atmosphere with 5% CO₂, the absorbance was determined at 450 nm.

Cells were seeded in 60-mm plates and then scratched using a pipette tip after 24-h incubation. After washing with phosphate buffered saline (PBS), cells were incubated in medium containing CLSE and/or 100 μM DFO for 24 h. Images were then obtained at 0 and 24 h with an Olympus CFX41 microscope (Hamburg, Germany) at 40× magnification. The area of cell migration was quantified using ImageJ software (https://​imagej.​nih.​gov/​ij/​) and the percentage of wound closure was calculated as described previously [11].

The experiment was performed as described previously [11]. Briefly, cells in CLSE-containing serum-free medium were seeded into the inner chamber with the lower surface coated with 0.2% gelatin. The cells were incubated in normoxic or hypoxic conditions for 24 h. 20% FBS-containing medium in the bottom chamber was used as a chemoattractant. Cells on the upper membrane were wiped off using wet cotton swabs after fixation with methanol and crystal violet staining. Cells on the lower surface were mounted using mounting solution (Vectashield®; Vector Laboratories Burlingame, CA, USA). Stained cells were counted under a light microscope (DP72; Olympus, Hamburg, Germany) and images were taken at 200× magnification. All of the experiments were independently repeated in triplicate.

The matrigel invasion assay was performed as previously described [11]. Briefly, the lower surface were coated with 0.2% gelatin, and then the upper surfaces were coated with Matrigel® (BD Biosciences, San Jose, CA, USA) at 37 °C for 2 h. Cells in CLSE-containing serum-free medium were plated into the inner chamber and incubated in normoxic or hypoxic conditions for 48 h with 20% FBS in the bottom chamber as the chemoattractant. The cells were processed as described for the transwell migration assay. All of the experiments were independently repeated in triplicate.

Cells were treated with or without CLSE and/or DFO for 24 h, suspended in serum-free McCoy’s medium, and then seeded in a 96-well plate that had been pre-coated with Matrigel® (BD Biosciences). After incubation at 37 °C for 90 min, the cells were washed with PBS and treated with 0.5 mg/mL 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The absorbance of formazan crystals dissolved in 100 μL DMSO was determined at 570 nm using a microplate reader. The experiments were independently performed in triplicate.

Cells were harvested with lysis buffer [50 mM Tris-HCl (pH 8.0), 0.5% sodium deoxycholate, 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), and 1% NP-40]. Antibodies to p65, phospho-p65, extracellular signal-regulated kinase (ERK) 1/2, phospho-ERK1/2, protein kinase B (AKT), phospho-AKT, c-Jun N-terminal kinase (JNK), phospho-JNK, p38, phospho-p38, Signal transducer and activator of transcription 3 (STAT3), and phospho-STAT3 were all purchased from Cell Signaling Technology (Beverly, MA, USA). β-actin antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Immunoblot bands were quantified as a ratio of phosphorylated protein/total protein using ImageJ software.

HCT116 cells were treated with or without CLSE (0.5, 1 mg/mL) and/or DFO (100 μM) for 24 h in complete medium, and then the supernatant was saved. The supernatant, called conditioned medium, was filtered and stored at −70 °C.

HUVECs were diluted in conditioned medium and seeded in a 48-well plate that had been pre-coated with Matrigel®. After a 12-h incubation period, images of formed tubules were taken at 40× magnification and quantified by determining the number of branching points. The experiments were independently performed in triplicate.

All data were analyzed with GraphPad Prism® software using a two-tailed Student t test. P values less than 0.05 were significantly considered.

To examine effects on the viability of colon cancer cells, HCT116, and of normal cells, CCD-18Co, caused by CLSE treatment, we performed a CCK-8 assay. As shown in Fig. 1, CLSE strongly decreased the viability of HCT116 cells compared to CCD-18Co cells. Next, we performed scratch-wound healing assays to determine if CLSE had an effect on the migratory potency of HCT116 cells. For this assay, we used DFO, a reagent used to simulate hypoxic conditions. As shown in Fig. 2a and b, DFO enhanced the migration of colon cancer cells (p < 0.05). However, HCT116 cells co-treated with DFO and CLSE had 47–87% less healing ability than cells treated with DFO only (p < 0.05). Furthermore, to confirm the inhibitory effect of CLSE on HCT116 cell migration, we plated CLSE-treated HCT116 cells in a Transwell® chamber and incubated the cells for 24 h under hypoxic conditions. The results showed that CLSE inhibited the migration of hypoxic HCT116 cells in the Transwell® chamber by 48–80% compared with the hypoxia control group (p < 0.01) (Fig. 2c, d). These results imply that CLSE has the potential to inhibit HCT116 cell migration under hypoxic conditions.

In cancer cell metastasis, invasion is as important as migration. To investigate the effects of CLSE on colon cancer cell invasion, we performed an invasion assay using a Transwell® coated with Matrigel®. As we showed, hypoxia promoted the invasion of HCT116 cells (p < 0.01). However, CLSE-treated cells under hypoxic conditions showed a 21–54% reduction in invasion compared with the hypoxia control group (p < 0.05) (Fig. 3a, b).

Metastatic cancer cells have high adhesion potency to migrate to secondary sites for new tumor growth. Therefore, cell adhesion assays are used to examine the metastatic potency of cancer cells. As shown in Fig. 3c, DFO-treated cells had increased adhesion compared with untreated cells (p < 0.05); however, cells co-treated with DFO and CLSE showed a 19–50% reduction in adhesive ability compared with cells treated with DFO only (p < 0.001, p < 0.01). Taken together, these results suggest that CLSE regulates the invasion and adhesion of HCT116 cells under hypoxic conditions.

To identify the cellular signaling pathways regulated by CLSE during colon cancer cell metastasis under hypoxic conditions, we examined the expression of signaling markers using Western blot analysis. Among the various signaling markers investigated, hypoxia-induced activation of ERK1/2 and AKT were downregulated by 1 mg/mL CLSE (Fig. 4a). To confirm whether the activation of ERK1/2 and AKT were repressed by CLSE in HCT116 cells, we performed western blot and scratch-wound healing assay at earlier stage in the presence of the ERK1/2 and AKT activators, PMA and SC79, respectively. As shown in Fig. 4b, inhibition of the ERK1/2 and AKT pathways by CLSE was reversed in CLSE-treated HCT116 cells given PMA or SC79. However, activation of other signaling proteins (p38, p65, STAT3, JNK) was not affected by CLSE treatment at an earlier stage (Fig. 4c). In addition, in the scratch-wound healing assay, PMA and SC79 could compromise the inhibitory effect of CLSE on the migration of HCT116 cells under hypoxic conditions by 57–71% and 64–77%, respectively (p < 0.05, p < 0.01) (Fig. 4d, e). Therefore, our results suggest that CLSE represses HCT116 cell migration through inactivation of the ERK1/2 and AKT pathways under hypoxic conditions.

The formation of new blood vessels (angiogenesis) is crucial for invasive tumor growth and metastasis. To demonstrate that CLSE affects angiogenesis, conditioned media, obtained from growing HCT116 cells in media supplemented with CLSE and/or DFO, was used to treat HUVEC cells. The media obtained from DFO-treated HCT116 cells augmented tube formation by HUVECs (p < 0.01), whereas conditioned media, obtained from CLSE and DFO-treated HCT116 cells, decreased tube formation by 55–91% (p < 0.001). These results suggest that CLSE may mediate its anti-angiogenic effect by regulating the secretion of angiogenic factors by colon cancer cells (Fig. 5).