Anti-proliferative activity of A. Oxyphylla and its bioactive constituent nootkatone in colorectal cancer cells

A. oxyphylla was purchased from Kyung-Dong Market in Seoul, Korea. The authenticity was confirmed at least twice through morphological analysis by Dr. Jaeyoon Cha, Department of Food Science and Nutrition, Dong-A University, Busan, Republic of Korea. A voucher specimen (No. EHNP-H8) has been deposited in the R&D Center, EastHill Corporation, Suwon, Gyeonggi-do, Republic of Korea. The plants were washed and ground using a laboratory mill to a particle size of 100 mesh. Ethanol (70%) was added to the ground plants and extracted at 70 °C for 48 h with stirring at 500 rpm. The extract was filtered using Toyo No. 4 filter paper and concentrated using a vacuum evaporator. Finally, the concentrate was diluted in dimethyl sulfoxide to obtain a final concentration 100 mg/mL. Nootkatone was purchased from Tokyo Chemical Industry (Tokyo, Japan). Epoxomicin and Puromycin (P8833–10) were purchased from Sigma Aldrich (St. Louis, MO, USA) and MG132 was purchased from AdooQ® Bioscience (Irvine, CA, USA). Antibodies for Cyclin D1 (sc-753), HRP conjugated β-actin (sc-47,778), and p53 (sc-126) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibody for NAG-1 was previously described [13].

All cells used in this study were purchased from American Type Culture Collection (ATCC). Cells were tested by ATCC for post-freeze viability, growth properties, morphology, mycoplasma contamination, species determination (cytochrome c oxidase I assay and short tandem repeat analysis), sterility test and human pathogenic virus testing. Upon arrival, cell lines were straightaway resuscitated and frozen in aliquots in liquid nitrogen. HCT-116 (Human colorectal carcinoma) and HT-29 (Human colorectal adenocarcinoma) cells were cultured using McCoy’s 5A media (Gibco life technologies, Carlsbad, CA, USA). SW480 (Human colorectal adenocarcinoma), DLD-1 (Human colorectal adenocarcinoma) were cultured using RPMI-1640 media (GIBCO). Both McCoy’s 5A media and RPMI-1640 media contained 10% Fetal bovine serum (FBS; GIBCO) and 1% penicillin/streptomycin (GIBCO). All cells were maintained at 37 °C and 5% CO_2.

The promoter-luciferase constructs pNAG1–1086/+ 41, pNAG1–474/+ 41, and pNAG1–133/+ 41 were previously described [12]. The expression vector pcDNA-EGR-1 has also been described [19]. The luciferase and pRL-null plasmids were transfected into cells using PolyJet™ In Vitro DNA Transfection reagent (SignaGen, Frederick, MD, USA) according to manufacturer’s instructions. Luciferase activity was measured using Dual-Luciferase® Reporter Assay kit (Promega, WI, USA) as previously described [20].

Both HCT-116 and SW480 cells were seeded at a density of 1 × 10⁴ cells/well in 6-well culture plates. Cells were treated with nootkatone at various doses (10 μM, 50 μM, or 100 μM) for 9 days. The culture media containing indicated concentrations of nootkatone were changed every 3 days. After treatment, the plate was washed with phosphate-buffered saline, and cells were fixed with 4% paraformaldehyde (Biosesang, Gyeonggi-do, Korea), followed by staining with 1% crystal violet solution (V5265, Sigma Aldrich). The number of colonies was counted using Image J software 1.52a (National Institutes of Health, MD, USA).

Western blot analysis was conducted as previously described [21]. Briefly, 50 μg proteins were separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (GVS filter technology, Zola Predosa BO, Italy). The blotted membrane was then blocked with 5% skim milk for 1 hr at room temperature and incubated overnight with specific antibodies at 4 °C. After incubation with HRP conjugated secondary antibody in 5% skim milk for 1 hr at room temperature, the blotted membranes were visualized using the Alliance Q9 mini imaging system (Cambridge, UK) and quantified using ImageJ software 1.52a (National Institutes of Health, MD, USA).

Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Five hundred nanograms of total RNA was used to synthesize cDNA using Verso cDNA Synthesis kit (Thermo scientific, Waltham, MA, USA). PCR products were amplified using the following primer pairs: cyclin D1 (F: 5′-CAA TGA CCC CGC ACG ATT TC-3′, R: 5′-AAG TTG TTG GGG CTC CTC AG-3′), NAG-1 (F: 5′-CTC CAG ATT CCG AGA CTT GC-3′, R:5′-AGA CAT ACG CAG GTG CAG GT-3′), GAPDH (F: 5′-GAC CAC AGT CCA TGC CAT CAC T-3′, R: TCC ACC ACC CTG TTG CTG TAG-3′). Thermal cycling conditions for NAG-1 were as follows: initial denaturation at 95 °C for 2 min, followed by 25–35 cycles of 94 °C for 30 s, 53.2 °C for 30 s, and 72 °C for 1 min, and final elongation at 72 °C for 5 min. For cyclin D1 and GAPDH, amplification and annealing temperatures were set to 52.5 °C and 60 °C respectively. PCR products were electrophoresed on a 1.5% agarose gel and photographed using the Alliance Q9 mini imaging system.

HCT-116 and SW480 were seeded in 96-well plates (1 × 10³ cells/well for HCT-116 cells and 2 × 10³ cells/well for SW480 cells) and incubated for 24 h with 100 μL of complete medium. Different dose of A. oxyphylla was treated for the indicated time. Cell proliferation assays were then performed using CellTiter 96® AQueous One Solution (Promega, WI, USA) according to the manufacturer’s instructions. After indicated time of culture, 20 μL of One Solution reagent was added to each well and cells were incubated for 1 h at 37 °C. Cell viability was estimated by measuring the absorbance at 492 nm using Multiskan FC spectrophotometer (Thermo Fisher Scientific, Waltham, MA).

The 2,2-diphenyl-1-picrylhydrazyl (DPPH, #14805, Cayman Chemical, MI, USA) and 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS, #A1888, Sigma Aldrich) were used for the radical scavenging assay, as previously described [22]. The absorbance was measured using a Multiskan™ FC microplate photometer (Thermo Fisher Scientific, Waltham, MA). L-ascorbic acid (#A0537, TCI, Tokyo, Japan) was used as a reference standard in both assays. Determination of the percentage of radical scavenging effect was considered using the following equation:

The VCEAC for ABTS assay and the IC50 value were calculated as half the concentration of the sample that can scavenge 50% of the DPPH free radical.

Seven hundred and fifty HCT-116 cells were seeded in an ultra-low attachment round bottom 96-well plate (Coster, Kennebunk, ME, USA), and cultured for 4 days. After spheroids were formed, half of the media was replaced with complete media containing 2 times the required dosage of nootkatone, and spheroids were incubated for 3 days. Spheroid viability was measured by the CellTiter-Glo® 3D Cell Viability Assay (Promega, Madison, WI, USA) in accordance with the manufacturer’s instuctions. Spheroid volume was calculated using the following formula: 0.5 × Length × Width².

A. oxyphylla has been used as a traditional Chinese medicine for many years. However, the molecular mechanism of A. oxyphylla extract as an anticancer agent has not been elucidated. In the current study, ethanol extracts of A. oxyphylla have been obtained and examined for potential antiproliferative activity in two human colorectal cancer cell lines. Treatment with A. oxyphylla extract affected HCT-116 and SW480 cell growth in a dose- and time-dependent manner with IC50 values of 89.3 μg/ml and > 100 μg/ml, respectively. (Fig. 1a–b). At 48 h and 96 h, A. oxyphylla significantly inhibited cell growth both in p53 wild type (HCT-116) and p53 mutant colorectal cancer cells (SW480), at a concentration of 100 μg/ml.

Antioxidant activity of natural compounds has been shown to be highly related to anticancer effects [23]. Thus, the antioxidant activity of A. oxyphylla extract has been investigated. DPPH and ABTS radical scavenging activity assays were performed to determine the antioxidant capacity of A. oxyphylla extract at various concentrations. The effects of A. oxyphylla extract and ascorbic acid on the ABTS radical compound are shown in Fig. 2a. The ABTS scavenging activities increased in correlation with increasing concentrations of A. oxyphylla extract. As a positive control, L-ascorbic acid displayed high antioxidant activity with decreased ABTS radical at a low concentration. Similarly, the DPPH radical scavenging activity of A. oxyphylla extract is presented in Fig. 2b. Scavenging activity increased in a dose-dependent manner up to 1000 ng/mL; with a similar trend observed in the ABTS assay. Antioxidant activity was also measured using a luciferase construct containing the antioxidant response element (ARE). After transfection into HCT-116 cells, luciferase activity was increased in the presence of A. oxyphylla, suggesting that A. oxyphylla may activate NRF2 which binds ARE sites (Fig. 2c). In addition, Heme oxygenase-1 (HO-1) protein levels were analyzed by western blot as a marker for antioxidant activity to confirm the antioxidant effect of the extract. Results showed that HO-1 protein levels increased in a dose-dependent manner (Fig. 2d). Overall, our results indicate that A. oxyphylla possesses antioxidant activity.

To elucidate the molecular mechanism by which A. oxyphylla affects anticancer activity in colorectal cancer cells, expression of NAG-1 and cyclin D1 have been determined. An increase in NAG-1 expression was observed, whereas cyclin D1 expression level was decreased in all four colorectal cancer cell lines treated with A. oxyphylla (Fig. 3a–d). These results suggest that A. oxyphylla extract may regulate colorectal cancer cell growth by elevating NAG-1 protein expression and decreasing cyclin D1 protein expression.

One of the bioactive compounds found in A. oxyphylla is nootkatone. (Fig. 4a) [6]. To determine whether nootkatone can account for the antiproliferative effect of A. oxyphylla, we analyzed cell growth both by counting cells and by performing colony and spheroid formation assays. Colorectal cancer cells were treated with nootkatone at concentrations of 10 μM, 50 μM and 100 μM, wherein nootkatone treatment resulted in cell growth inhibition in a dose and time dependent manner (Fig. 4b), similar to the trend observed following A. oxyphylla extract treatment (Fig. 1). Furthermore, in the colony formation assay, nootkatone showed a dose-dependent inhibition of colony formation in two colorectal cancer cell lines (Fig. 4c). In the spheroid formation assay, nootkatone decreased spheroid formation (viability and volume) in HCT-116 cells (Fig. 4d), indicating that nootkatone possesses anti-tumorigenic activity not only in a 2D culture system, but also in a 3D culture system.

We examined whether nootkatone may decrease cyclin D1 or increase NAG-1 expression at the transcription level. An increased RNA level of NAG-1 was observed in the presence of nootkatone treatment, whereas cyclin D1 RNA levels did not change in cells treated with nootkatone (Fig. 5a). Protein levels of NAG-1 and cyclin D1 were also analyzed revealing that both NAG-1 and cyclin D1 were altered by nootkatone treatment at the protein level (Fig. 5b). Taken together, our results indicate that nootkatone may affect cyclin D1 at the protein level and NAG-1 at the transcriptional level.

To clarify the molecular mechanism by which nootkatone decreases cyclin D1 protein levels, a protein stability assay was performed wherein HCT-116 and SW480 cell lines were treated with puromycin and proteosomal inhibitors. In both cell lines, cyclin D1 protein expression was dramatically decreased by nootkatone treatment compared to the control, indicating that nootkatone may affect the stability of the cyclin D1 protein in the cells (Fig. 6a). Furthermore, proteosomal inhibitors epoxomicin and MG132 were combined with nootkatone to determine whether this would rescue the protein-destabilizing effect of cyclin D1 by nootkatone. Results showed that epoxomicin and MG132 partially restored cyclin D1 protein levels, indicating that nootkatone may affect cyclin D1 degradation through a proteosomal degradation pathway-related mechanism (Fig. 6b).

To examine the direct effect of nootkatone on NAG-1 expression, a NAG-1 promoter-luciferase construct containing 1086 bp was transfected into HCT-116 cells and luciferase activity was measured following nootkatone treatment. NAG-1 promoter activity was increased in a dose-dependent manner, with the highest expression corresponding to nootkatone treatment at a concentration of 100 μM (Fig. 7a). To identify the response element region in the NAG-1 promoter which responded to nootkatone, three deletion mutant clone plasmids were designed and analyzed for nootkatone-inducing activity following transfection. The response element position which was the most affected by nootkatone seems to be located within the 133 bp of the NAG-1 promoter (Fig. 6b). It is known that EGR-1 plays a role in transcriptional regulation of NAG-1 in this promoter region [19]. To confirm whether EGR-1 plays a role in nootkatone-induced NAG-1 expression, an EGR-1 luciferase vector was co-transfected with the 133 bp NAG-1 luciferase construct. The results indicated that EGR-1 indeed increased luciferase activity and activated the 133 bp region of the NAG-1 promoter (Fig. 7c). Subsequently, we measured whether EGR-1 protein was increased by the nootkatone treatment. Nootkatone led to increased EGR-1 expression in both HCT-116 and SW480 cells in a dose-dependent manner (Fig. 7d). Finally, we determined whether nootkatone affects EGR-1 expression at the transcriptional level. The result showed EGR-1 promoter activity was increased in the presence of nootkatone in a dose-dependent manner (Fig. 7e), indicating that nootkatone induces EGR-1 at the transcriptional level ultimately leading to the induction of NAG-1 promoter activity.