Limits...
Anti-cancer activity of Psoralea fructus through the downregulation of cyclin D1 and CDK4 in human colorectal cancer cells

View Article: PubMed Central - PubMed

ABSTRACT

Background: Psoralea Fructus (PF), the dried and ripe fruit of Psoralea corylifolia exhibits an anti-cancer activity. However, the molecular mechanisms by which PF inhibits the proliferation of cancer cells have not been elucidated in detail. Cyclin D1 and CDK4 are important regulatory proteins in cell growth and are overexpressed in many cancer cells. In this study, we investigated the molecular mechanism of PF on the downregulation of cyclin D1 and CDK4 level.

Methods: Cell growth was evaluated by MTT assay. The effect of PF on cyclin D1 and CDK4 expression was evaluated by Western blot or RT-PCR.

Results: PF suppressed the proliferation of human colorectal cancer cell lines such as HCT116 (IC50: 45.3 ± 1.2 μg/ml), SW480 (IC50: 37.9 ± 1.6 μg/ml), LoVo (IC50: 23.3 ± 1.9 μg/ml μg/ml) HT-29 (IC50 value: 40.7 ± 1.5 μg/ml). PF induced decrease in the protein expression of cyclin D1 and CDK4. However, the mRNA expression of cyclin D1 and CDK4 did not be changed by PF; rather it suppressed the expression of cyclin D1 and CDK4 via the proteasomal degradation. In cyclin D1 degradation, we found that T286 of cyclin D1 play a pivotal role in PF-mediated cyclin D1 degradation. Subsequent experiments with several kinase inhibitors suggest that PF-mediated degradation of cyclin D1 and CDK4 is dependent on ERK1/2 and/or GSK3β.

Conclusions: Our results suggest that PF has potential to be a candidate for the development of chemoprevention or therapeutic agents for human colorectal cancer.

No MeSH data available.


T286 phosphorylation of cyclin D1 by PF contributes to the proteasomal degradation. a HCT116 cells were transfected with wild type HA-tagged cyclin D1 or HA-tagged T286A cyclin D1 expression vector, and then treated with PF (25 μg/ml). *P < 0.05 compared to cell without PF treatment. b HCT116 cells were treated with PF (25 μg/ml) for the indicated times. Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against HA-cyclin D1 and p-cyclin D1 (Thr286). Actin was used as internal control for Western blot analysis
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC5037586&req=5

Fig4: T286 phosphorylation of cyclin D1 by PF contributes to the proteasomal degradation. a HCT116 cells were transfected with wild type HA-tagged cyclin D1 or HA-tagged T286A cyclin D1 expression vector, and then treated with PF (25 μg/ml). *P < 0.05 compared to cell without PF treatment. b HCT116 cells were treated with PF (25 μg/ml) for the indicated times. Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against HA-cyclin D1 and p-cyclin D1 (Thr286). Actin was used as internal control for Western blot analysis

Mentions: Threonine-286 (Thr-286) phosphorylation of cyclin D and subsequent ubiquitination pathway has been reported to be the main cyclin D1 degradation pathway [15]. To investigate whether Thr-286 phosphorylation is critical in PF-mediated cyclin D1 degradation, HCT116 cells were transfected with HA-tagged wild type cyclin D1 and HA-tagged T286A cyclin D1. As shown in Fig. 4a, exogenous wild type cyclin D1 was decreased by PF, whereas the degradation of T286A cyclin D1 was suppressed. In addition, we observed that PF phosphorylated Thr-286 of cyclin D1 (Fig. 4b). These results suggest that Thr-286 site plays an important role in PF-induced cyclin D1 degradation.Fig. 4


Anti-cancer activity of Psoralea fructus through the downregulation of cyclin D1 and CDK4 in human colorectal cancer cells
T286 phosphorylation of cyclin D1 by PF contributes to the proteasomal degradation. a HCT116 cells were transfected with wild type HA-tagged cyclin D1 or HA-tagged T286A cyclin D1 expression vector, and then treated with PF (25 μg/ml). *P < 0.05 compared to cell without PF treatment. b HCT116 cells were treated with PF (25 μg/ml) for the indicated times. Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against HA-cyclin D1 and p-cyclin D1 (Thr286). Actin was used as internal control for Western blot analysis
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5037586&req=5

Fig4: T286 phosphorylation of cyclin D1 by PF contributes to the proteasomal degradation. a HCT116 cells were transfected with wild type HA-tagged cyclin D1 or HA-tagged T286A cyclin D1 expression vector, and then treated with PF (25 μg/ml). *P < 0.05 compared to cell without PF treatment. b HCT116 cells were treated with PF (25 μg/ml) for the indicated times. Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against HA-cyclin D1 and p-cyclin D1 (Thr286). Actin was used as internal control for Western blot analysis
Mentions: Threonine-286 (Thr-286) phosphorylation of cyclin D and subsequent ubiquitination pathway has been reported to be the main cyclin D1 degradation pathway [15]. To investigate whether Thr-286 phosphorylation is critical in PF-mediated cyclin D1 degradation, HCT116 cells were transfected with HA-tagged wild type cyclin D1 and HA-tagged T286A cyclin D1. As shown in Fig. 4a, exogenous wild type cyclin D1 was decreased by PF, whereas the degradation of T286A cyclin D1 was suppressed. In addition, we observed that PF phosphorylated Thr-286 of cyclin D1 (Fig. 4b). These results suggest that Thr-286 site plays an important role in PF-induced cyclin D1 degradation.Fig. 4

View Article: PubMed Central - PubMed

ABSTRACT

Background: Psoralea Fructus (PF), the dried and ripe fruit of Psoralea corylifolia exhibits an anti-cancer activity. However, the molecular mechanisms by which PF inhibits the proliferation of cancer cells have not been elucidated in detail. Cyclin D1 and CDK4 are important regulatory proteins in cell growth and are overexpressed in many cancer cells. In this study, we investigated the molecular mechanism of PF on the downregulation of cyclin D1 and CDK4 level.

Methods: Cell growth was evaluated by MTT assay. The effect of PF on cyclin D1 and CDK4 expression was evaluated by Western blot or RT-PCR.

Results: PF suppressed the proliferation of human colorectal cancer cell lines such as HCT116 (IC50: 45.3&thinsp;&plusmn;&thinsp;1.2&nbsp;&mu;g/ml), SW480 (IC50: 37.9&thinsp;&plusmn;&thinsp;1.6&nbsp;&mu;g/ml), LoVo (IC50: 23.3&thinsp;&plusmn;&thinsp;1.9&nbsp;&mu;g/ml&nbsp;&mu;g/ml) HT-29 (IC50 value: 40.7&thinsp;&plusmn;&thinsp;1.5&nbsp;&mu;g/ml). PF induced decrease in the protein expression of cyclin D1 and CDK4. However, the mRNA expression of cyclin D1 and CDK4 did not be changed by PF; rather it suppressed the expression of cyclin D1 and CDK4 via the proteasomal degradation. In cyclin D1 degradation, we found that T286 of cyclin D1 play a pivotal role in PF-mediated cyclin D1 degradation. Subsequent experiments with several kinase inhibitors suggest that PF-mediated degradation of cyclin D1 and CDK4 is dependent on ERK1/2 and/or GSK3&beta;.

Conclusions: Our results suggest that PF has potential to be a candidate for the development of chemoprevention or therapeutic agents for human colorectal cancer.

No MeSH data available.