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

Abstract

Background: Although the inhibitory effect of mistletoe on cancer cell growth has been reported, the underlying mechanisms to explain its anti-proliferative activity are not fully studied. Thus, we elucidated the potential molecular mechanism of the branch from Taxillus yadoriki (TY) parasitic to Neolitsea sericea (NS) (TY-NS-B) for the anti-proliferative effect.

Methods: Anti-cell proliferative effect was evaluated by MTT assay. The change of cyclin D1 protein or mRNA level was evaluated by Western blot and RT-RCR, respectively.

Results: In comparison of anti-proliferative effect of TY from the host trees such as Cryptomeria japonica (CJ), Neolitsea sericea (NS), Prunus serrulata (PS), Cinnamomum camphora (CC) and Quercus acutissima (QA), TY-NS showed higher anti-cell proliferative effect than TY-CJ, TY-PS, TY-CC or TY-QA. In addition, the anti-proliferative effect of branch from TY from all host trees was better than leaves. Thus, we selected the branch from Taxillus yadoriki parasitic to Neolitsea sericea (TY-NS-B) for the further study. TY-NS-B inhibited the cell proliferation in the various cancer cells and downregulated cyclin D1 protein level. MG132 treatment attenuated cyclin D1 downregulation of cyclin D1 protein level by TY-NS-B. In addition, TY-NS-B increased threonine-286 (T286) phosphorylation of cyclin D1, and the mutation of T286 to alanine (T286A) blocked cyclin D1 proteasomal degradation by TY-NS-B. But the upstream factors related to cyclin D1 degradation such as ERK1/2, p38, JNK, GSK3β, PI3K, IκK or ROS did not affect cyclin D1 degradation by TY-NS-B. However, LMB treatment was observed to inhibit cyclin D1 degradation by TY-NS-B, and T286A blocked cyclin D1 degradation through suppressing cyclin D1 redistribution from nucleus to cytoplasm by TY-NS-B. In addition, TY-NS-B activated CRM1 expression.

Conclusions: Our results suggest that TY-NS-B may suppress cell proliferation by downregulating cyclin D1 protein level through proteasomal degradation via T286 phosphorylation-dependent cyclin D1 nuclear export. These findings will provide the evidence that TY-NS-B has potential to be a candidate for the development of chemoprevention or therapeutic agents for human cancer.

Keywords: Anticancer; Cell growth arrest; Cyclin D1; Mistletoe; Taxillus yadoriki.

Fig. 1

Comparison of anti-proliferative effect of TY from the host trees and plant parts. HCT116 and SW480 cells were plated overnight and then treated with the samples for 24 h. Cell growth was measured using MTT assay. *P < 0.05 compared to cell without the sample treatment

Fig. 2

The effect of TY-NS-B on the cell growth and cyclin D1 expression in human colorectal cancer cells. a HCT116 and SW480 cells were plated overnight and then treated with TY-NS-B at the indicated concentrations for 24 h. Cell growth was measured using MTT assay. Data represent mean ± SD for three independent experiments. *P < 0.05 compared to cell without TY-NS-B. b HCT116 and SW480 cells were plated overnight and then treated with TY-NS-B at the indicated concentrations for 24 h. For Western blot analysis, cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibody against cyclin D1. Actin was used as internal control for Western blot analysis. For RT-PCR analysis of the gene expression of cyclin D1, total RNA was prepared. GAPDH was used as internal control for RP-PCR. Relative density for Western blot and RT-PCR was measured using the software Un-SCAN-IT gel Version 5.1 (Silk Scientific, Inc., Orem, UT, USA). Data represent mean ± SD for three independent experiments. *P < 0.05 compared to cell without TY-NS-B. c HCT116 cells were plated overnight and then treated with TY-NS-B at the indicated concentrations for 24 h. Cell cycle progression was analyzed by flow cytometer

Fig. 3

The effect of TY-NS-B on the cell growth and cyclin D1 expression in other cancer cells. a Human breast cancer cells (MDA-MB-231), human pancreatic cancer cells (AsPC-1), human non-small cell lung cancer cells (A549) and human prostate cancer cells (PC-3) were plated overnight and then treated with TY-NS-B at the indicated concentrations for 24 h. Cell growth was measured using MTT assay. Data represent mean ± SD for three independent experiments. *P < 0.05 compared to cell without TY-NS-B. b MDA-MB-231, AsPC-1, A549 and PC-3 were plated overnight and then treated with TY-NS-B at the indicated concentrations for 24 h. Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibody against cyclin D1. Actin was used as internal control for Western blot analysis

Fig. 4

The effect of TY-NS-B on cyclin D1 degradation. a HCT116 and SW480 cells were pretreated with MG132 (10 and 20 μM) and then co-treated with TY-NS-B (50 μg/ml). Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibody against cyclin D1. Actin was used as internal control for Western blot analysis. Relative density for Western blot was measured using the software Un-SCAN-IT gel Version 5.1 (Silk Scientific, Inc). Data represent mean ± SD for three independent experiments. *P < 0.05 compared to cell without TY-NS-B. b HCT116 and SW480 cells were pretreated with MG132 (20 μM) and then co-treated with TY-NS-B (50 μg/ml) for 24 h. Cell growth was measured using MTT assay. Data represent mean ± SD for three independent experiments. *P < 0.05 compared to cell without TY-NS-B

Fig. 5

Phosphorylation of T286 of cyclin D1 by TY-NS-B. HCT116 and SW480 cells were plated overnight and then treated with TY-NS-B (50 μg/ml) at the indicated times. Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibody against p-cyclin D1 (T286) and cyclin D1. Actin was used as internal control for Western blot analysis. Relative density for Western blot was measured using the software Un-SCAN-IT gel Version 5.1 (Silk Scientific, Inc). Data represent mean ± SD for three independent experiments. *P < 0.05 compared to cell without TY-NS-B

Fig. 6

Cyclin D1 degradation by TY-NS-B is dependent on T286 phosphorylation. HCT116 and SW480 cells were transfected with HA-tagged wild type-cyclin D1 or T286A-cyclin D1, and then co-treated with TY-NS-B (50 μg/ml) for the indicated times. Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibody against HA-cyclin D1. Actin was used as internal control for Western blot analysis. Relative density for Western blot was measured using the software Un-SCAN-IT gel Version 5.1 (Silk Scientific, Inc). Data represent mean ± SD for three independent experiments. *P < 0.05 compared to cell without TY-NS-B

Fig. 7

Determination of factors involved in cyclin D1 degradation by TY-NS-B. a HCT116 cells were pretreated with 20 μM of PD98059, SB203580 or SP600125. b Right panel: HCT116 cells were pretreated with 20 mM of LiCl. Left panel: HCT116 cells were treated with 20 mM of LiCl (c) HCT116 cells were pretreated with 20 μM of LY294002. d HCT116 cells were pretreated with 5 and10 μM of BAY 11–7082. e HCT116 cells were pretreated with 10 mM of NAC. After pretreatment of each inhibitor, HCT116 cells were co-treated with TY-NS-B (50 μg/ml). Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibody against cyclin D1. Actin was used as internal control for Western blot analysis. Relative density for Western blot was measured using the software Un-SCAN-IT gel Version 5.1 (Silk Scientific, Inc). Data represent mean ± SD for three independent experiments. *P < 0.05 compared to cell without TY-NS-B

Fig. 8

Cyclin D1 nuclear export contributes to cyclin D1 degradation by TY-NS-B. a HCT116 cells were pretreated with 50 nM of LMB and then co-treated with TY-NS-B (50 μg/ml). b HCT116 cells were transfected with HA-tagged wild type-cyclin D1 or T286A-cyclin D1, and then co-treated with TY-NS-B (50 μg/ml). After treatment, cytoplasm and nucleus were prepared. c HCT116 and SW480 cells were treated with TY-NS-B. Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibody against cyclin D1, HA-cyclin D1 and CRM1. Actin was used as internal control for Western blot analysis. Relative density for Western blot was measured using the software Un-SCAN-IT gel Version 5.1 (Silk Scientific, Inc). Data represent mean ± SD for three independent experiments. *P < 0.05 compared to cell without TY-NS-B