
During the last decades, the incidence and mortality of colorectal cancer (CRC) have gradually increased and among all cancers worldwide, CRC is now ranked third (Maharjan
The aberrant activation of canonical Wnt/β-catenin signaling pathways is frequently found in CRC patients with a relatively poorer clinical outcome (Fodde
In our continuous study to explore the anticancer agents from natural products, the extract of
Roswell Park Memorial Institute (RPMI) 1640 medium, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), sodium pyruvate, L-glutamine, antibiotic/antimycotic solution, TRI reagent, Lipofectamine 2000 and trypsin-EDTA were purchased from Invitrogen (Carlsbad, CA, USA). Dimethyl sulfoxide (DMSO), sulforhodamine B (SRB), trichloroacetic acid (TCA), bovine serum albumin (BSA), isoprophanol and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). The luciferase reporter plasmids (TOPflash and FOPflash), Renilla luciferase reporter vectors, and pcCDNA β-catenin- and TCF4-expression vectors were obtained from Upstate Biotechnology (Lake Placid, NY, USA). The Reverse Transcription Kit was purchased from Toyobo (Osaka, Japan). The Dual Luciferase Reporter Assay System was purchased from Promega (Madison, WI, USA). The iQ SYBR Green Supermix was obtained from Bio-Rad Laboratories (Hercules, CA, USA). For real-time PCR, the gene-specific primers were designed by Roche (Basel, Switzerland) and synthesized by Bioneer (Dajeon, Korea). The goat anti-rabbit IgG-HRP, anti-mouse IgG-HRP, anti-β actin, anti-TCF4, anti-cyclin B1, anti-cyclin A, anti-CDK2, anti-CDC2, anti-PARP, anti-p21 and anti-p27 were purchased from Santa Cruz Biotechnology, Inc (Dallas, TX, USA). Anti-β-catenin, anti-survivin, anti-Axin1, anti-Axin2, anti-c-Myc, anti-cyclin D, anti-GSK-3β, anti-cleaved caspase3 and anti-cleaved caspase9 were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-cleaved poly (ADP-ribose) polymerase (PARP) and Annexin V-Fluorescein isothiocyanate (Annexin V-FITC) Apoptosis Detection Kit I were purchased from BD Bioscience (Franklin Lakes, NJ, USA).
Dried whole plant materials (1,150 g) were pulverized and then extracted with MeOH (twice 10 L each) in room temperature (RT), yielding a crude extract of 123.2 g. The crude extract (119.8 g) was dissolved in deionized water (500 mL) and then partitioned with EtOAc (500 mL×2), furnishing 35 g of EtOAc soluble extract. A portion of the EtOAc-soluble extract (32 g) was subjected to silica gel column chromatography (620 g) eluted with a gradient mixture of chloroform and MeOH (100:0 to 0:100) and gave 11 subfractions (JaE1-JaE11). JaE3 (10.4 g) was subjected to silica gel column chromatography (200 g), eluting with a gradient mixture of chloroform and MeOH (40:1 to 0:1), and furnished 16 subfractions (JaE3-1 to JaE3-16). From JaE3-14, Nodosin (23.2 mg, Fig. 1A) was precipitated. The structure of Nodosin was confirmed by comparing the measured values with the published values (Yan
Human embryonic kidney cells (HEK293), human normal lung fibroblast cell (MRC-5), human colorectal cancer cells (HCT116), human breast cancer cells (MDA-MB-231), human lung cancer cells (A549), human liver cancer cells (SK-HEP-1), and human gastric cancer cells (SNU-638) were purchased from the American Type Culture Collection (Manassas, VA, USA). HEK293, MRC5, MDA-MB-231 and SK-HEP-1 were maintained in DMEM, while HCT116, A549 and SNU-638 were cultured in RPMI-1640 media supplemented with 10% FBS and antibiotics-antimycotics (PSF: 100 units/mL sodium penicillin G, 100 μg/mL streptomycin, and 250 ng/mL amphotericin B) (Thermo-Scientific, MA, USA) in a humidified incubator containing 5% CO2 at 37°C.
Cells were seeded in 96-well plates and then treated with samples for 24, 48, or 72 h. After incubation, the cells were fixed with 10% TCA for 30 min, dried overnight, and stained with 0.4% SRB in 1% acetic acid for 2 h. The unbound dye was washed out using 1% acetic acid, after which the stained cells were dried and then were resuspended in 10 mM Tris (PH 10.0) (Kang
Transient transfections were conducted using Lipofectamine 2000 (Invitrogen). HEK293 and HCT116 cells were seeded in 48-well plates, and then the cells were transfected with 0.1 µg of a luciferase reporter plasmid (TOPflash or FOPflash) and 0.005 µg of the Renilla luciferase vector for normalization. HEK293 cells were also co-transfected with 0.02 µg of the pcDNA β-catenin expression vector and 0.004 µg of the TCF4 expression vector to activate the Wnt pathway (Kang
Total RNA was extracted from cells using TRI reagent (Invitrogen). RNA extracts were reverse-transcribed using the Toyobo Reverse Transcription System (Toyobo). Real-time PCR was performed using iQ SYBR Green Supermix (Bio-Rad Laboratories) according to the manufacturer’s instructions. The primer sequences were as follows:
Total cell lysates were mixed with lysis buffer, and boiled for 10-20 min at 100°C. The protein concentrations of the cell lysates were determined by the BCA protein assay (Byun
The cells were seeded at a density of 2×106 cells in 100 mm cell culture dish. After 24 h incubation, the cells were treated with Nodosin and washed with PBS, and then the cell pellets were fixed with 70% ethanol overnight at –20°C. Fixed cells were pelleted and washed with PBS. The cells were resuspended in 100 μg/mL RNase A and 50 μg/mL of propidium iodide (PI) in the dark for 30 min at room temperature. The DNA content of the fluorescence binding cells was analyzed using a flow cytometry FACScalibur flowcytometer (BD Bioscience, Franklin Lakes, NJ, USA). The distribution of cell contents was measured with 5,000 cells in each group and the results were represented as histograms of the DNA content (Jung
The cells were treated with Nodosin for 48 h and then stained with Annexin V-FITC and PI using an annexin V-FITC apoptosis detection kit (BD Bioscience) according to the manufacturer’s instruction. Briefly, the incubated cells were harvested and resuspended with 1×binding buffer. Annexin V-FITC and PI (5 μL) were added to cell suspensions and further incubated in the dark for 15 min at room temperature. Stained cells were immediately analyzed with 1× binding buffer using a flow cytometer (Kim
Data are presented as the mean values ± standard deviation for the indicated number of independently performed experiments. All data are representative of the results of at least three independent experiments. Statistical significance (*
To evaluate whether Nodosin inhibits the growth of human cancer cells, a cell proliferation assay was conducted in a panel of cancer cell lines. Nodosin effectively inhibited the growth of various solid cancer cell lines. Among the cancer cell lines tested, the HCT116 colon cancer cells were shown to be the most sensitive in the growth-inhibitory activity of Nodosin with the IC50 values of 4.05 μM after 72 h incubation (Table 1). In addition, Nodosin did not significantly inhibit the growth of MRC5, a normal lung fibroblast cell line (IC50 value >20 μM), suggesting that Nodosin exhibits a relatively selective growth inhibition against cancer cells compared to a normal cell. We further extended to determine the growth inhibitory activity of Nodosin in the HCT116 cells after incubation of 24 h and 48 h treatment. Nodosin showed growth inhibition of the cells in a time- and concentration-dependent manner (Fig. 1B). The morphological change of the cancer cells treated with Nodosin was also observed under phase-contrast microscopy after 24 h treatment. The treatment of Nodosin (30 μM) evoked morphological changes with rounded shapes and the cells were floated in culture media (Fig. 1C). Moreover, the effects of Nodosin on colony formation were evaluated in the HCT116 cells. The cells were treated with Nodosin for 72 h, and then media were replaced two or three times per week for approximately 2 weeks. As shown in Fig. 1D, Nodosin significantly suppressed colony formation in the HCT116 cells. These data suggest that Nodosin effectively inhibits cancer cell proliferation and colony formation in human colorectal cancer cells.
Table 1 . Anti-proliferative activity of Nodosin in a panel of human cancer cell lines
IC50 (μM) | SNU638 | SK-HEP-1 | A549 | HCT116 | MDA-MB-231 | MRC-5 |
---|---|---|---|---|---|---|
Nodosin | 7.53 | 5.33 | 13.73 | 4.05 | 7.12 | >20 |
Etoposide | 0.39 | 0.77 | 0.25 | 0.21 | 7.1 | >20 |
aCancer cell lines: SNU638 (stomach), SK-HEP-1 (liver), A549 (lung), HCT116 (colon), MDA-MB-231 (breast), MRC-5 (normal lung epithelial cell); bEtoposide was used as a positive control.
To further confirm whether the growth-inhibitory activity of Nodosin against HCT116 cells is associated with the suppression of the Wnt signaling pathways, the effect of Nodosin on Wnt/β-catenin-mediated transcriptional activity was primarily evaluated. Using the T-cell factor (TCF) reporter gene (TOPflash), luciferase reporter gene assay was conducted in HCT116 cells and HEK293 cells. Nodosin significantly inhibited TOPflash activity in the TCF/β-catenin-stimulated luciferase activity in HEK293 cells (Fig. 2A). In addition, Nodosin also effectively suppressed overactivated TOPflash activity in the intrinsic β-catenin-mutated HCT116 colon cancer cells (Fig. 2B). We further detected the effect of Nodosin on the nuclear localization of β-catenin in HCT116 cells. As shown in Fig. 2C, immunocytochemical analysis revealed that overexpression of β-catenin in the nuclear fractions was significantly suppressed by the treatment of Nodosin (30 μM) in HCT116 cells. Real-time PCR analysis also suggested that the suppression of Wnt/β-catenin signaling by Nodosin is involved in the down-regulation of mRNA expression of Wnt target genes, such as CCND1 (cyclin D1), AXIN2, BIRC5 (survivin), and CTNNB1 (β-catenin), which are key regulators of cancer cell proliferation, survival, and progression (Fig. 2D). Furthermore, Nodosin also significantly suppressed Wnt/β-catenin target protein expressions including β-catenin, p-GSK-3β, c-Myc, and Survivin in HCT116 cells (Fig. 2E). Collectively, these data indicated that the growth-inhibitory activity of Nodosin against colorectal cancer cells may be in part associated with the effective suppression of endogenously activated Wnt signaling pathways in HCT116 colorectal cancer cells.
The Wnt signaling pathway plays a pivotal role in cell proliferation and survival. To further clarify the involvement of anti-proliferative activity of Nodosin with the modulation of the Wnt signaling pathway, we primarily determined cell cycle distribution with the treatment of Nodosin in HCT116 cells. Flow cytometry analysis revealed that the treatment of Nodosin for 24 h in cancer cells significantly induced G2/M phase cell cycle arrest in a concentration-dependent manner. In particular, the cell populations of G2/M phase were manifestly increased from 13.87% in the vehicle-treated control cells, to 45.88% in the Nodosin (30 μM)-treated cells (Fig. 3A). The cell cycle is finely controlled by the regulation of cell cycle checkpoint proteins. In the G2/M phase, the SCF E3 ubiquitin ligase consisting of S phase kinase-associated protein 1 (Skp1) and cullin-1 (Cul-1), and a member of the F-box protein, are actively involved in the cell cycle progression (Willems
To further evaluate whether Nodosin is able to induce apoptosis for prolonged exposure in HCT116 cells, the cells were treated with Nodosin for 48 h. Flow cytometric analysis was performed after double staining with Annexin V-FITC/PI. As shown in Fig. 4A, the populations of apoptotic cells including early and late apoptosis were induced in a concentration-dependent manner. In particular, Nodosin significantly exhibited the population of apoptotic cell death with up to 61.3% at the treatment of 30 μM Nodosin in HCT116 cells. In addition, the induction of apoptosis was in part correlated with the down-regulation of the anti-apoptotic proteins Bcl-2 and Bid expression, and up-regulation of pro-apoptotic proteins such as cleaved caspase-8, cleaved caspase-9 and cleaved PARP through the suppression of caspase-9 and PARP expression, respectively (Fig. 4B). These data suggest that Nodosin is able to induce apoptotic cell death via mitochondria-dependent pathway in HCT116 cells.
A body of evidence suggests that the constitutive activation of Wnt signaling is essential for the growth and survival of colorectal cancer cells (Zhan
In summary, the present study for the first time demonstrates that Nodosin, a natural diterpenoid from
This study was supported by a Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry (IPET) through the Agricultural Biotechnology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (No. 114071-3).
The authors declare there is no conflict of interest.
![]() |
![]() |