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.


Determination of upstream kinases involved in the proteasomal degradation of cyclin D1 and CDK4 by PF. a HCT116 cells were pretreated with PD98059 (ERK1/2 inhibitor, 20 μM), SB203580 (p38 inhibitor, 20 μM), SP600125 (JNK inhibitor, 20 μM) or LiCl (GSK3β inhibitor, 20 mM) for 2 h, and then co-treated with PF (25 μg/ml) for 3 h (cyclin D1) or 10 h (CDK4). b HCT116 cells were treated with PF (25 μg/ml) for the indicated times. c HCT116 cells were pretreated with PD98059 (ERK1/2 inhibitor, 20 μM) or LiCl (GSK3β inhibitor, 20 mM) for 2 h, and then co-treated with PF (25 μg/ml) for 1 h. Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against cyclin D1, CDK4, p-ERK1/2, total-ERK1/2, p-GSK3β, total-GSK3β or p-cyclin D1 (Thr286). Actin was used as internal control for Western blot analysis. *P < 0.05 compared to cell without PF treatment
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig5: Determination of upstream kinases involved in the proteasomal degradation of cyclin D1 and CDK4 by PF. a HCT116 cells were pretreated with PD98059 (ERK1/2 inhibitor, 20 μM), SB203580 (p38 inhibitor, 20 μM), SP600125 (JNK inhibitor, 20 μM) or LiCl (GSK3β inhibitor, 20 mM) for 2 h, and then co-treated with PF (25 μg/ml) for 3 h (cyclin D1) or 10 h (CDK4). b HCT116 cells were treated with PF (25 μg/ml) for the indicated times. c HCT116 cells were pretreated with PD98059 (ERK1/2 inhibitor, 20 μM) or LiCl (GSK3β inhibitor, 20 mM) for 2 h, and then co-treated with PF (25 μg/ml) for 1 h. Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against cyclin D1, CDK4, p-ERK1/2, total-ERK1/2, p-GSK3β, total-GSK3β or p-cyclin D1 (Thr286). Actin was used as internal control for Western blot analysis. *P < 0.05 compared to cell without PF treatment

Mentions: To investigate the upstream kinases associated with PF-mediated degradation of cyclin D1 and CDK4 protein, HCT116 cells were pretreated with PD98059 (20 μM, inhibitor of extracellular signal–regulated kinase 1/2 (ERK1/2)), SB203580 (20 μM, p38 inhibitor), SP600125 (20 μM, inhibitor of c-Jun N-terminal kinases (JNK)) or LiCl (20 mM, inhibitor of glycogen synthase kinase 3β (GSK3β)) for 2 h, and then co-treated with PF for 3 h for cyclin D1 or 10 h for CDK4. As shown in Fig. 5a, cyclin D1 degradation by PF was attenuated in presence of the ERK1/2 and GSK3β inhibitor, whereas PF-mediated CDK4 degradation was blocked in presence of GSK3β inhibitor, which suggests that cyclin D1 degradation by PF is dependent on both ERK1/2 and GSK3β, and CDK4 degradation by PF is dependent on GSK3β. Thus, we examined whether PF induces the phosphorylation of these kinase as the active form. As shown in Fig. 5b, the phosphorylation of ERK1/2 and GSK3β was induced at early time points in the presence of PF. In addition, we observed the inhibition of ERK1/2 and GSK3β attenuated PF-mediated Thr-286 phosphorylation of cyclin D1 (Fig. 5c).Fig. 5


Anti-cancer activity of Psoralea fructus through the downregulation of cyclin D1 and CDK4 in human colorectal cancer cells
Determination of upstream kinases involved in the proteasomal degradation of cyclin D1 and CDK4 by PF. a HCT116 cells were pretreated with PD98059 (ERK1/2 inhibitor, 20 μM), SB203580 (p38 inhibitor, 20 μM), SP600125 (JNK inhibitor, 20 μM) or LiCl (GSK3β inhibitor, 20 mM) for 2 h, and then co-treated with PF (25 μg/ml) for 3 h (cyclin D1) or 10 h (CDK4). b HCT116 cells were treated with PF (25 μg/ml) for the indicated times. c HCT116 cells were pretreated with PD98059 (ERK1/2 inhibitor, 20 μM) or LiCl (GSK3β inhibitor, 20 mM) for 2 h, and then co-treated with PF (25 μg/ml) for 1 h. Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against cyclin D1, CDK4, p-ERK1/2, total-ERK1/2, p-GSK3β, total-GSK3β or p-cyclin D1 (Thr286). Actin was used as internal control for Western blot analysis. *P < 0.05 compared to cell without PF treatment
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig5: Determination of upstream kinases involved in the proteasomal degradation of cyclin D1 and CDK4 by PF. a HCT116 cells were pretreated with PD98059 (ERK1/2 inhibitor, 20 μM), SB203580 (p38 inhibitor, 20 μM), SP600125 (JNK inhibitor, 20 μM) or LiCl (GSK3β inhibitor, 20 mM) for 2 h, and then co-treated with PF (25 μg/ml) for 3 h (cyclin D1) or 10 h (CDK4). b HCT116 cells were treated with PF (25 μg/ml) for the indicated times. c HCT116 cells were pretreated with PD98059 (ERK1/2 inhibitor, 20 μM) or LiCl (GSK3β inhibitor, 20 mM) for 2 h, and then co-treated with PF (25 μg/ml) for 1 h. Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against cyclin D1, CDK4, p-ERK1/2, total-ERK1/2, p-GSK3β, total-GSK3β or p-cyclin D1 (Thr286). Actin was used as internal control for Western blot analysis. *P < 0.05 compared to cell without PF treatment
Mentions: To investigate the upstream kinases associated with PF-mediated degradation of cyclin D1 and CDK4 protein, HCT116 cells were pretreated with PD98059 (20 μM, inhibitor of extracellular signal–regulated kinase 1/2 (ERK1/2)), SB203580 (20 μM, p38 inhibitor), SP600125 (20 μM, inhibitor of c-Jun N-terminal kinases (JNK)) or LiCl (20 mM, inhibitor of glycogen synthase kinase 3β (GSK3β)) for 2 h, and then co-treated with PF for 3 h for cyclin D1 or 10 h for CDK4. As shown in Fig. 5a, cyclin D1 degradation by PF was attenuated in presence of the ERK1/2 and GSK3β inhibitor, whereas PF-mediated CDK4 degradation was blocked in presence of GSK3β inhibitor, which suggests that cyclin D1 degradation by PF is dependent on both ERK1/2 and GSK3β, and CDK4 degradation by PF is dependent on GSK3β. Thus, we examined whether PF induces the phosphorylation of these kinase as the active form. As shown in Fig. 5b, the phosphorylation of ERK1/2 and GSK3β was induced at early time points in the presence of PF. In addition, we observed the inhibition of ERK1/2 and GSK3β attenuated PF-mediated Thr-286 phosphorylation of cyclin D1 (Fig. 5c).Fig. 5

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.