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

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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.


PF induces the proteasomal degradation of cyclin D1 and CDK4. a-d The cells were treated with the indicated concentrations of PF for 24 h. For RT-PCR analysis of the gene expression of cyclin D1 and CDK4, total RNA was prepared after PF treatment for 24 h. GAPDH was used as internal control for RP-PCR. e HCT116 cells were pretreated with MG132 for 2 h, and then co-treated with PF (25 μg/ml) for 3 h (cyclin D1) or 10 h (CDK4). Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against cyclin D1 and CDK4. Actin was used as internal control for Western blot analysis. *P < 0.05 compared to cell without PF treatment
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Fig3: PF induces the proteasomal degradation of cyclin D1 and CDK4. a-d The cells were treated with the indicated concentrations of PF for 24 h. For RT-PCR analysis of the gene expression of cyclin D1 and CDK4, total RNA was prepared after PF treatment for 24 h. GAPDH was used as internal control for RP-PCR. e HCT116 cells were pretreated with MG132 for 2 h, and then co-treated with PF (25 μg/ml) for 3 h (cyclin D1) or 10 h (CDK4). Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against cyclin D1 and CDK4. Actin was used as internal control for Western blot analysis. *P < 0.05 compared to cell without PF treatment

Mentions: To elucidate the potential mechanism by which PF down-regulates the protein level of cyclin D1 and CDK4, the cells were treated with PF, and then mRNA levels of cyclin D1 and CDK4 were evaluated using RT-PCR. As shown in Fig. 3a-d, the mRNA levels of cyclin D1 and CDK4 were not significantly changed in the presence of PF. This result indicates that PF may regulate the protein level of cyclin D1 and CDK4 through proteasomal-dependent degradation. To investigate whether PF affects the modification of cyclin D1 and CDK4 protein, HCT116 cells were pretreated with MG132 for 2 h, and then co-treated with PF for 3 h for cyclin D1 or 10 h for CDK4. As shown in Fig. 3e, the degradation of cyclin D1 and CDK4 protein was restored in presence of the proteasomal inhibitor, MG132, which suggests that PF down-regulates cyclin D1 and CDK4 protein at the post-translational level via the proteasomal pathway.Fig. 3


Anti-cancer activity of Psoralea fructus through the downregulation of cyclin D1 and CDK4 in human colorectal cancer cells
PF induces the proteasomal degradation of cyclin D1 and CDK4. a-d The cells were treated with the indicated concentrations of PF for 24 h. For RT-PCR analysis of the gene expression of cyclin D1 and CDK4, total RNA was prepared after PF treatment for 24 h. GAPDH was used as internal control for RP-PCR. e HCT116 cells were pretreated with MG132 for 2 h, and then co-treated with PF (25 μg/ml) for 3 h (cyclin D1) or 10 h (CDK4). Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against cyclin D1 and CDK4. Actin was used as internal control for Western blot analysis. *P < 0.05 compared to cell without PF treatment
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Fig3: PF induces the proteasomal degradation of cyclin D1 and CDK4. a-d The cells were treated with the indicated concentrations of PF for 24 h. For RT-PCR analysis of the gene expression of cyclin D1 and CDK4, total RNA was prepared after PF treatment for 24 h. GAPDH was used as internal control for RP-PCR. e HCT116 cells were pretreated with MG132 for 2 h, and then co-treated with PF (25 μg/ml) for 3 h (cyclin D1) or 10 h (CDK4). Cell lysates were subjected to SDS-PAGE and the Western blot was performed using antibodies against cyclin D1 and CDK4. Actin was used as internal control for Western blot analysis. *P < 0.05 compared to cell without PF treatment
Mentions: To elucidate the potential mechanism by which PF down-regulates the protein level of cyclin D1 and CDK4, the cells were treated with PF, and then mRNA levels of cyclin D1 and CDK4 were evaluated using RT-PCR. As shown in Fig. 3a-d, the mRNA levels of cyclin D1 and CDK4 were not significantly changed in the presence of PF. This result indicates that PF may regulate the protein level of cyclin D1 and CDK4 through proteasomal-dependent degradation. To investigate whether PF affects the modification of cyclin D1 and CDK4 protein, HCT116 cells were pretreated with MG132 for 2 h, and then co-treated with PF for 3 h for cyclin D1 or 10 h for CDK4. As shown in Fig. 3e, the degradation of cyclin D1 and CDK4 protein was restored in presence of the proteasomal inhibitor, MG132, which suggests that PF down-regulates cyclin D1 and CDK4 protein at the post-translational level via the proteasomal pathway.Fig. 3

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.