Ethanol extracts from the branch of Taxillus yadoriki parasitic to Neolitsea sericea induces cyclin D1 proteasomal degradation through cyclin D1 nuclear export

Dulbecco’s Modified Eagle medium (DMEM)/F-12 1:1 Modified medium (DMEM/F-12) for the cell culture was purchased from Lonza (Walkersville, MD, USA). LiCl, MG132, PD98059, SB230580, SP600125, LY294002, BAY 11–7280, leptomycin B (LMB) and 3-(4,5-dimethylthizaol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and N-acetyl-L-cysteine (NAC) were purchased from Sigma Aldrich (St. Louis, MO, USA). Antibodies against cyclin D1, phospho-cyclin D1 (Thr286), HA-tag, CRM1 and β-actin were purchased from Cell Signaling (Bervely, MA, USA). All chemicals were purchased from Fisher Scientific, unless otherwise specified.

Taxillus yadoriki (TY) parasitic to Cryptomeria japonica (CJ), Neolitsea sericea (NS), Prunus serrulata (PS), Cinnamomum camphora (CC) and Quercus acutissima (QA), respectively, was collected from Jeju island, Korea and formally identified by Ho Jun Son as a researcher of Forest Medicinal Resources Research Center, Korea. Twenty gram of the branches (B) or leaves (L) from TY-CJ, TY-NS, TY-PS, TY-CC and TY-QA was extracted with 400 ml of 70% ethanol with shaking for 72 h. After 72 h, the ethanol-soluble fraction was filtered and concentrated to approximately 120 ml volume using a vacuum evaporator and then freeze-dried. The ethanol extracts was kept in a refrigerator until use.

Human colorectal cancer cell lines (HCT116 and SW480), human breast cancer cell line (MDA-MB-231), human pancreatic cancer cell line (AsPC-1), human non-small cell lung cancer cell line (A549) and human prostate cancer cell line (PC-3) were purchased from Korean Cell Line Bank (Seoul, Korea) and grown in DMEM/F-12 supplemented with 10% fatal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin. The cells were maintained at 37 °C under a humidified atmosphere of 5% CO₂. The test samples were dissolved in dimethyl sulfoxide (DMSO) and treated to cells. DMSO was used as a vehicle and the final DMSO concentration did not exceed 0.1% (v/v).

Cell proliferation was evaluated by MTT assay. Briefly, cells were plated at a density of 3 × 10⁴ cells/well in 96-well plate and incubated for 24 h. The cells were treated with the test sample at the indicated concentrations for 24 h. Then, the cells were incubated with 50 μl of MTT solution (1 mg/ml) for an additional 2 h. The resulting crystals were dissolved in DMSO. The formation of formazan was measured by reading absorbance at a wavelength of 570 nm using UV/Visible spectrophotometer (Human Cop., Xma-3000PC, Seoul, Korea).

HCT116 cells were plated at a density of 1 × 10⁶ cells/well in 6-well plate and incubated for 24 h. The cells were treated with TY-NS-B for 24 h. After then, the cells were dissociated with trypsin, washed in cold PBS and fixed with 70% cold ethanol on ice for 30 min. The suspensions were centrifuged at 1500 rpm for 5 min. The pellets were resuspended in a solution containing 50 μg/ml propidium iodide, 1 mg/ml sodium citrate, 0.3 ml nonidet P-40 and 5 μg/ml RNase A and stayed on ice atleast 40 min. Then the pellets were analyzed by a flow cytometer.

Cytosol and nuclear fractions of cells were prepared using a nuclear extract kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer’s protocols. Briefly, the cells after treatment were harvested with 1 × cold hypotonic buffer and incubated at 4 °C for 15 min. After adding detergent and vortexing for 10 s, the cells were centrifuged at 14,000 g for 1 min at 4 °C and the supernatants (cytoplasmic fraction) were collected and stored at − 80 °C for further analysis. The cell pellets were used for nuclear fraction collection. Cell pellets were re-suspended with complete lysis buffer by pipetting up and down, and incubated at 4 °C for 30 min under shaking. After 30 min, nuclear suspensions were centrifuged at 14,000 g for 10 min at 4 °C, and the supernatants (nuclear fraction) were stored at − 80 °C for further analysis.

Cells were plated at a density of 2 × 10⁶ cells/well in 6-well plate and grown to 80% confluence. After treatment, the cells were washed with 1 × phosphate-buffered saline (PBS), and lysed in radioimmunoprecipitation assay (RIPA) buffer (Boston Bio Products, Ashland, MA, USA) supplemented with protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitor cocktail (Sigma-Aldrich), and centrifuged at 15,000 × rpm for 10 min at 4 °C. Protein concentration was determined by the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA). The proteins were separated on SDS-PAGE and transferred to PVDF membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were blocked for non-specific binding with 5% non-fat dry milk in Tris-buffered saline containing 0.05% Tween 20 (TBS-T) for 1 h at room temperature and then incubated with specific primary antibodies in 5% non-fat dry milk at 4 °C overnight. After three washes with TBS-T, the blots were incubated with horse radish peroxidase (HRP)-conjugated immunoglobulin G (IgG) for 1 h at room temperature and chemiluminescence was detected with ECL Western blotting substrate (Amersham Biosciences, Piscataway, NJ, USA) and visualized in Polaroid film.

After treatment, total RNA was prepared using a RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and total RNA (1 μg) was reverse-transcribed using a Verso cDNA Kit (Thermo Scientific, Pittsburgh, PA, USA) according to the manufacturer’s protocol for cDNA synthesis. PCR was carried out using PCR Master Mix Kit (Promega, Madison, WI, USA) with human primers for cyclin D1 and GAPDH as followed: cyclin D1: forward 5′-aactacctggaccgcttcct-3′ and reverse 5′-ccacttgagcttgttcacca-3′, GAPDH: forward 5′-acccagaagactgtggatgg-3′ and reverse 5′-ttctagacggcaggtcaggt-3′. The following PCR reaction conditions were used: 1 cycle of (3 min at 94 °C for denaturation), 25 cycles of (30 s at 94 °C for denaturation, 30 s at 60 °C for annealing, and 30 s at 72 °C for elongation), and 1 cycle of (5 min for extension at 72 °C).

HA-tagged wild type cyclin D1 and HA-tagged T286A cyclin D1 were provided from Addgene (Cambridge, MA, USA). Transient transfection of the vectors was performed using the PolyJet DNA transfection reagent (SignaGen Laboratories, Ijamsville, MD, USA) according to the manufacturers’ instruction.

Since Taxillus yadoriki (TY) as one of the mistletoes is parasitic to various host trees, we hypothesized that TY’s anti-cancer activity may be different according to the host tree species. Thus, we performed the comparative study of TY on anti-proliferation according to the host tree species. As shown in Fig. 1, the anti-proliferative effect of TY parasitic to Neolitsea sericea (NS) was highest in the host tree species such as Cryptomeria japonica (CJ), Neolitsea sericea (NS), Prunus serrulata (PS), Cinnamomum camphora (CC) and Quercus acutissima (QA). In addition, we observed that the branch (B) of TY is higher than leaves (L) in anti-proliferative effect. Thus, we selected the branch of Taxillus yadoriki parasitic to Neolitsea sericea (TY-NS-B) for the further study.

To investigate whether TY-NS-B affects cancer cells growth, human colorectal cancer cell lines, HCT116 and SW480, were treated with TY-NS-B at the indicated concentrations for 24 h. As shown in Fig. 2a, TY-NS-B dose-dependently induced the cell growth arrest. Because cyclin D1 protein has been regarded as one of the factor associated with the regulation of the cell growth [17], we evaluated that TN-NS-B downregulates cyclin D1 expression. As a result, TN-NS-S decreased cyclin D1 expression in both mRNA and protein level (Fig. 2b). Because cyclin D1 regulates G1/S transition in the cell cycle, we investigated whether the downregulation of cyclin D1 by TY-NS-B contributes to the accumulation of cells in G1 phase. As shown in Fig. 2c, the majority of HCT116 cells without TY-NS-B treatment were in S phase. However, TY-NS-B dose-dependently induced the accumulation of G0/G1 phase in HCT116 cells. In addition, we tested whether TY-NS-B suppresses cell proliferation and cyclin D1 expression in other cancer cells such as MDA-MB-231 (human breast cancer cell line), AsPC-1 (human pancreatic cancer cell line), A549 (human non-small cell lung cancer cell line) and PC-3 (human prostate cancer cell line). As shown in Fig. 3a, TY-NS-B dose-dependently inhibited the growth of MDA-MB-231, AsPC-1, A549 and PC-3 cells. Furthermore, we observed that cyclin D1 protein level was decreased by TY-NS-B treatment in MDA-MB-231, AsPC-1, A549 and PC-3 cells (Fig. 3b).

We observed that the level change of cyclin D1 protein by TY-NS-B is more dramatic than that of cyclin D1 mRNA (Fig. 2a), which indicates that TY-NS-B may affect cyclin D1 protein stability. To elucidate that the induction of cyclin D1 proteasomal degradation by TY-NS-B, HCT116 and SW480 cells were pretreated with MG132 and then co-treated with TY-NS-B. As shown in Fig. 4, the treatment of TY-NS-B alone downregulated cyclin D1 protein level. However, the presence of MG132 blocked cyclin D1 downregulation mediated by TY-NS-B. In addition, we investigated whether the inhibition of cyclin D1 proteasomal degradation by MG132 affects anti-proliferative effect of TY-NS-B. As shown in Fig. 4b, MG132 treatment attenuated TY-NS-B-mediated inhibition of cell proliferation in HCT116 and SW480 cells. These results indicate that cyclin D1 proteasomal degradation by TY-NS-B may contribute to cyclin D1 downregulation, and subsequently inhibit the cell proliferation.

Threonine-286 (T286) phosphorylation of cyclin D1 has been known to be involved in cyclin D1 proteasomal degradation [18]. Thus, we investigated whether TY-NS-B mediates T286 phosphorylation of cyclin D1. As shown in Fig. 5, T286 phosphorylation of cyclin D1 was increased at 1 h after TY-NS-B treatment in both HCT116 and SW480 cells. However, cyclin D1 protein level was significantly decreased at 6 h and 3 h after TY-NS-B treatment in HCT116 and SW480, respectively. These results indicate that T286 phosphorylation of cyclin D1 by TY-NS-B may contribute to cyclin D1 degradation. To confirm whether T286 phosphorylation of cyclin D1 by TY-NS-B is essential to the induction of cyclin D1 proteasomal degradation, HCT116 and SW480 cells were transfected with HA-tagged wild type-cyclin D1 or T286A-cyclin D1, and then treated with TY-NS-B. In this experiment, we observed that TY-NS-B decreases HA-cyclin D1 in the cells transfected with wild type-cyclin D1 at 24 h after treatment, while did not affect the change of HA-cyclin D1 level in the cells transfected with T286A-cyclin D1 (Fig. 6). These findings indicate that cyclin D1 proteasomal degradation by TY-NS-B is dependent on threonine-286 phosphorylation of cyclin D1.

T286 phosphorylation and subsequent proteasomal degradation of cyclin D1 has been reported to be regulated by a variety of kinases. To determine the upstream kinases involved in cyclin D1 proteasomal degradation by TY-NS-B, the specific inhibitors such as PD98059 (ERK1/2 inhibitor), SB203580 (p38 inhibitor), SP600125 (JNK inhibitor), LiCl (GSK3β inhibitor), LY294002 (PI3K inhibitor) and BAY 11–7082 (IκK inhibitor) were used. In addition, NAC (ROS scavenger) was used because reactive oxygen species (ROS) has been known to be associated with cyclin D1 degradation [19]. In this study, we observed that TY-NS-B decreases cyclin D1 protein level in both absence and presence of each inhibitor (Fig. 7a-e). Among the upstream kinases involved in cyclin D1 degradation, GSK-3β is reported to be required for cyclin D1 degradation to induce cell cycle arrest [19]. Thus, we evaluated that LiCl as a GSK-3β inhibitor affects the protein and phosphorylation level of β-catenin. As shown in Fig. 7b (left panel), LiCl increased β-catenin protein level by attenuating β-catenin phosphorylation. These results indicate that cyclin D1 proteasomal degradation by TY-NS-B is independent on ERK1/2, p38, JNK, GSK3β, PI3K, IκK and ROS.

Although we failed to determine the upstream kinases involved in cyclin D1 proteasomal degradation by TY-NS-B, we observed that the treatment of LMB as a nuclear export inhibitor attenuated the downregulation of cyclin D1 by TY-NS-B (Fig. 8a). Indeed, there is growing evidence that T286 phosphorylation of cyclin D1 results in cyclin D1 nuclear export to cytoplasm, which is associated with cyclin D1 proteasomal degradation [20]. Thus, we investigated whether T286 phosphorylation of cyclin D1 by TY-NS-B affects cyclin D1 nuclear export to cytoplasm. As shown in Fig. 8b, cyclin D1 level of the cells transfected with wild type-cyclin D1 was increased in the cytoplasm and decreased in the nucleus by TY-NS-B. However, the transfection of T286A-cyclin D1 blocked cyclin D1 nuclear export by TY-NS-B, resulting in the nuclear accumulation of cyclin D1 level. In addition, we investigated whether TY-NS-B affects the expression of CRM1 as a nuclear exportin. As a result, TY-NS-B dose-dependently increased CRM1 expression. These findings indicate that the increased CRM1 protein by TY-NS-B may export the phosphorylated cyclin D1 at T286 from the nucleus to cytoplasm, which results in cyclin D1 degradation.