Casticin, a polymethoxyflavone, has been reported to exert anticancer activities. The objectives of this study were to examine the molecular mechanisms by which casticin induces the growth inhibition and cell cycle arrest in human hepatocellular carcinoma (HCC) cells. The HCC cell lines Hep G2 and PLC/PRF/5 were cultured
Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the third most common cause of cancer-related mortality, resulting in approximately 500,000 deaths per annum. Most HCC cases occur in either Eastern Asia (particularly in China) or sub-Saharan Africa (
Casticin is one of the main components of the fruits of
Forkhead box class O (FOXO) subfamily of forkhead transcription factors include FOXO1a/FKHR, FOXO3a/FKHRL1 and FOXO4/AFX (
In the present study, we demonstrated that casticin induced FOXO3a dephosphorylation and FOXM1 inactivation, leading to growth inhibition and cell cycle arrest in HCC cells. These results suggest that forkhead transcription factor FOXM1 is a downstream cellular target and a potential novel marker for casticin action and that casticin activates FOXO3a to suppress FOXM1 expression in HCC cells.
Casticin was purchased from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China). It has a molecular weight of 374.3 ku, appears as yellow crystals and has a purity of 98.0%. Casticin was prepared in dimethyl sulfoxide (DMSO) as a 10 mmol/l stock solution and diluted in medium to the indicated concentration before use. The following items were purchased from Hunan Clonetimes Biotech Co., Ltd. (Changsha, China): RPMI-1640 medium (Invitrogen Life Technologies, Carlsbad, CA, USA), fetal bovine serum (Invitrogen Life Technologies), propidium iodide (PI; Sigma-Aldrich, St. Louis, MO, USA), antibodies against FOXO3a, phospho-FOXO3a-Thr32 (Millipore), FoxM1, cyclin dependent kinase (CDK1), cyclin B, p27KIP1, cdc25B and β-actin (Santa Cruz Biotechnology, Inc.). Lipofectamine 2000 was purchased from Invitrogen Life Technologies. Protease inhibitor cocktail and all other chemicals were obtained from Sigma.
Hep G2 (p53 wild-type) and PLC/PRF/5 (p53 mutant) cells were obtained from American Type Culture Collection (Rockville, MD, USA). They were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen Life Technologies) in an incubator containing 50 ml/l CO2 at 37˚C.
Cells were plated in 24-well plates at a density of 300 cells/well for 24 h, prior to the addition of various concentrations of casticin (2.5, 5.0 and 10.0 μmol/l). After 24 h of treatment, the drug-containing medium was removed and replaced with complete growth medium. Medium was changed every three days for 10 days until visible colonies formed. Colonies were simultaneously fixed and stained with Wright-Giemsa solution in methanol and manually counted. Individually stained colonies in each well were counted. The colony formation fraction was calculated as follows: Colony number/(number of cells seeded × plating efficiency), where plating efficiency was equivalent to the colony number divided by the number of cells seeded in the drug-free medium.
Cell cycle analysis was performed using PI staining as described previously (
A control non-specific small interfering RNA (siRNA) (UUCUCCGAACGUGUCACGUdTdT) was purchased from Qiagen. FOXO3a siRNA (ACUCCGGGUC CAGCUCCAC) was synthesized by Shanghai GenePharma Co. (Shanghai, China). Transfection of siRNA was carried out with Lipofectamine 2000 (Invitrogen Life Technologies) according to the procedure recommended by the manufacturer. Twenty-four hours after transfection, the cells were treated with DMSO (control) or casticin at the indicated concentrations for 24 h. The cells were then collected and processed for western blotting and clonogenic assay.
Desired cells (1×106) were seeded in 100-mm culture dishes, allowed to attach by overnight incubation and treated with DMSO (control) or 2.5, 5.0 and 10.0 μmol/l casticin for specified time periods. The cell lysates were prepared as described by us previously (
The database was set up with the SPSS 15.0 software package (SPSS Inc., Chicago, IL, USA) to be analyzed. Data are represented as means ± SD. The means of multiple groups were compared with one-way ANOVA, after the equal assessment of variance. The comparisons among the means were performed using the LSD method. Statistical comparison was also performed with the two-tailed t-test when appropriate. A P<0.05 was considered to indicate a statistically significant result.
Since the previous study demonstrated that casticin inhibited the viability of HCC cells using an MTT assay (
We next sought to evaluate the effects of casticin treatment on the phase distribution of the cell cycle using FCM after PI staining. As shown in
It has been previously reported that the loss of FOXM1 expression induces the growth inhibition and cell cycle arrest in HCC cells (
To further confirm the effect of casticin on FOXM1 functional regulation, we assessed the expression of downstream target genes of FOXM1 in HCC cells after casticin treatment. It is well known that FOXM1 has several downstream target genes, such as CDK1, CDC25B, cyclin B1 and p27KIP1, for the regulation of growth and cell cycle arrest in cells. Western blot analysis results showed that casticin inhibited the expression of CDK1, CDC25B, cyclin B1 and increased p27KIP1 in Hep G2 (
Since FOXO3a is the upstream regulator of the FOXM1 transcription factor (
In order to confirm the relevance of the FOXO3a factor in the cellular growth inhibition response to casticin, we decided to perform gene silencing experiments. To this end, cells were generated in which FOXO3a protein expression was abrogated using siRNA technology (
Abnormal cell proliferation is an important characteristic of malignant tumors including HCC. This suggests that cell cycle arrest could be an effective method in the treatment of malignant tumors. We previously showed that casticin inhibits the viability of HCC cells (
FOXM1, one member of the forkhead box transcription factor family, is a critical regulator of cell cycle progression and functions in cell proliferation, organogenesis, aging and carcinogenesis (
The above findings indicate that FOXM1 is a potential specific target for HCC therapy. Notably, a recent study showed that lower expression of FOXM1 in HCC was associated with prolonged disease-free survival after curative liver resection and validated FOXM1 as a prognostic marker for HCC (
FOXO3a is a member of the forkhead box class O (FOXO) transcription factor family and an important regulator of FOXM1 activity and function (
In conclusion, these results showed that downregulation of the expression levels of phosphorylated FOXO3a and FOXM1 in HCC cells by casticin decreased the colony formation ability and induced G2/M phase cell cycle arrest. Furthermore, a decrease in the FOXM1 expression level resulted in downregulation of CDK1, CDC25B and cyclins B1 along with upregulation of p27. The depletion of FOXO3a also reduced the effects of casticin. Our study provides clearly evidence that the FOXO3a/FOXM1 signaling pathway may serve as a new target for the natural flavonoid casticin in HCC therapy.
This study was supported by grants from the National Natural Science Foundation (81172375) and the Municipal Bureau of Science and Technology of Changsha, Hunan, China (K1104060-31).
Casticin induces growth inhibition and cell cycle arrest in the G2/M phase in HCC cells. Cells were treated with the indicated concentrations of casticin for 24 h. (A and B) Casticin (CAS) decreased the colony number in the Hep G2 and PLC/PRF/5 cell lines. (C and D) Casticin increased the cell population in the G2/M phase in the Hep G2 and PLC/PRF/5 cell lines. Data and error bars are presented as means ± SD. *P<0.05, **P<0.01, ***P<0.001 when comparing these treatments with DMSO. #P<0.05 when comparing these treatments with 2.5 μM casticin.
Downregulation of FOXM1 expression at the mRNA and protein levels by casticin in HCC cells. Cells were treated with the indicated concentrations of casticin for 24 h. (A) Casticin downregulated the expression of FOXM1 at the mRNA level (RT-PCR) and protein level (western blotting; WB) in the HepG2 cell line. (B) Casticin downregulated the expression of FOXM1 at the mRNA level (RT-PCR) and at the protein level (WB) in the PLC/PRF/5 cell line.
Decrease in the protein expression of FOXM1 downstream targets CDK1, CDC25B and cyclin B1 and increase in the protein expression of p27 by casticin in HCC cells. Cells were treated with the indicated concentrations of casticin for 24 h. (A) The expression of CDK1, CDC25B, cyclin B1 and p27 proteins was analyzed using western blotting in the Hep G2 cell line. β-actin was used as a loading control. (b) The expression of CDK1, CDC25B, cyclin B1 and p27 proteins was analyzed using western blotting in the PLC/PRF/5 cell line. β-actin was used as a loading control.
Reduction in the protein expression of phosphorylated FOXO3a by casticin in HCC cells. Cells were treated with the indicated concentrations of casticin for 24 h. (A) The protein expression of phosphorylated FOXO3a was analyzed using western blotting in the Hep G2 cell line. Total FOXO3a was used as a loading control. (B) The protein expression of phosphorylated FOXO3a was analyzed using western blotting in the PLC/PRF/5 cell line. Total FOXO3a was used as a loading control.
Depletion of FOXO3a by siRNA increased protein expression of FOXM1 and attenuated the inhibitory effects of casticin on colony formation in HCC cells. (A) Cells were transiently transfected with a control non-specific siRNA or a FOXO3a-targeted siRNA for 24 h. The expression of FOXO3a and FOXM1 proteins was analyzed using western blotting in the Hep G2 cell line. β-actin was used as a loading control. (B) Cells were transiently transfected with a control non-specific siRNA or a FOXO3a-targeted siRNA for 24 h. The expression of FOXO3a and FOXM1 proteins was analyzed using western blotting in the PLC/PRF/5 cell line. β-actin was used as a loading control. (C) Hep G2 cells were transiently transfected with the non-specific siRNA and the FOXO3a-targeted siRNA, respectively. Reduction in the colony numbers by casticin was attenuated by FOXO3a siRNA transfection. (D) PLC/PRF/5 cells were transiently transfected with the non-specific siRNA and the FOXO3a-targeted siRNA, respectively. Reduction in the colony numbers by casticin was attenuated by FOXO3a siRNA transfection. Data and error bars are presented as means ± SD. *P<0.05 versus treatment with DMSO in transfected cells using the non-specific siRNA. #P<0.05 versus treatment with casticin at the same concentrations in transfected cells using the non-specific siRNA.