Contributed equally
Grifolin, a secondary metabolic product isolated from the mushroom
Ovarian cancer is a gynecological malignancy with one of the highest mortality rates worldwide (
At present, the use of natural agents for cancer prevention and therapy is attracting increased attention. Grifolin, which originates from the edible fruiting bodies of the mushroom
The present study revealed that grifolin effectively suppressed viability and induced apoptosis of human ovarian cancer A2780 cells. The present study also researched the exact targets and molecular mechanisms underlying grifolin-induced anticancer activity in A2780 cells, and revealed that inactivation of protein kinase B (Akt) and extracellular signal-related kinase (ERK1/2) is necessary for grifolin-induced cell cycle arrest and cell apoptosis. Grifolin may therefore be a promising anti-tumor agent for the treatment of ovarian cancer.
The A2780 cell line was cultured in RPMI-1640 medium with 10% fetal bovine serum. Cells were maintained in a humidified incubator with 5% CO2 at 37°C. The cell culture media was obtained from HyClone (GE Healthcare Life Sciences, Logan, UT, USA), the fetal bovine serum was provided by Cell Signaling Technology, Inc., (Danvers, MA, USA).
Grifolin was provided by the Kunming Institute of Botany; Chinese Academy of Sciences (Kunming, China) with the following structure: 2-trans, trans-farnesyl-5-methylresorcinol (purity, >99%; high performance liquid chromatography analysis). Grifolin was prepared at a concentration of 100 mmol/l stock solution in dimethyl sulfoxide (DMSO) and stored under −20°C so that the final concentration of DMSO was <0.1% in all assays it was used in. DMSO was provided by (Shanghai Biyuntian Bio-Technology Company, Ltd., Shanghai, China).
The effect of grifolin on A2780 cell viability was evaluated by MTT assay. Cells were seeded at a density of 3,000–3,500 cells/well in 96-well plates, which permitted logarithmic growth during the 72 h assay, and cultured overnight at 37°C with 5% CO2. Adherent cells were subsequently exposed to grifolin at varying concentrations (0, 20, 40, 60, 80 and 100 µM) for 24, 48 and 72 h. The cells were then washed with PBS twice and incubated in 20 µl MTT (HyClone; GE Healthcare Life Sciences) for 4 h. The cell suspension was discarded and the cells were supplemented with 100 µl DMSO. At the MTT endpoint, an infinite M200PRO microplate reader (Bio-Rad Laboratories, Hercules, CA, USA) was used to measure cell viability at 490 or 550 nm absorbance. The cell viability trend and the half maximal inhibitory concentrations (IC50) were analyzed using SPSS Statistics 18.0 software (SPSS, Inc., Chicago, IL, USA). Finally, GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA) was used to draw up the bar graph.
A2780 cells (5×102) were seeded on 6-well plates and exposed to grifolin (0, 25, 50 or 75 µM) for 48 h with 5% CO2 at 37°C. The cells were then washed with PBS twice and RPMI-1640 culture medium was added into the plates for ~10 days. The cells were washed with PBS twice, fixed with 4% paraformaldehyde solution for ~15 min, and then stained with 1 ml 0.2% crystal violet (HK Jimei Biology Science and Technology Company, Ltd., Beijing, China) for 30 min. Finally, the numbers of clones were counted and analyzed by using the DP71 fluorescence microscope (Olympus Corporation, Tokyo, Japan) with ×200 magnification 400–600 cells per field of view assessed.
Flow cytometry was used to detect the cell cycle and apoptosis distribution. Cells (1×106) were treated with 0, 25, 50 or 75 µM grifolin for 24 h, collected and washed with ice-cold PBS twice, fixed in 75% ethanol overnight at 4°C and stained with 20 µl Rnase A (Cell Signaling Technology, Inc., Danvers, MA, USA) and 400 µl propidium iodide (PI) at room temperature for 30 min. Apoptosis analysis was also performed using annexin V-fluorescein isothiocyanate and PI, following the manufacturers protocol. The apoptosis kit used in flow cytometry assay was provided by the (#50T; Jiehui Biology Science and Biotechnology Company, Ltd., Beijing, China,). The number of cells was calculated by FACScan flow cytometry (Bio-Rad Laboratories). The data was analyzed by WinMDI v2.9 software (The Scripps Research Institute, San Diego, CA, USA).
A2780 cells at a density of 4×105 were seeded per well and were incubated with grifolin at concentrations of 0, 25, 50 and 75 uM for 24 h, then harvested and homogenized in 100 µl RIPA lysis buffer for ~30 min on ice. The cells were centrifuged for 15 min 4°C at the speed 15,000 × g. The protein samples (20 µg/lane) were separated on 10 or 12% SDS-PAGE, transferred to polyvinylidene difluoride membranes. Following blocking using 5% non-fat milk at room temperature for ~2 h, the membranes were incubated with primary antibodies against phosphorylated (p-) ERK1/2 (#4370; 1:1,000; Cell Signaling Technology, Inc.), ERK1/2 (#4060; 1:1,000; Cell Signaling Technology, Inc., MA, USA), p-Akt (#4691; 1:1,000; Cell Signaling Technology, Inc.), Akt (#4691; 1:1,000; Cell Signaling Technology, Inc.), cleaved-poly (ADP-ribose) polymerase (PARP; #5625; 1:1,000; Cell Signaling Technology, Inc.), BCL2 associated X, apoptosis regulator (Bax; #ab32503; 1:1,000; Abcam, Cambridge, UK), B cell lymphoma-2 (Bcl-2; #ab32124; 1:1,000; Abcam), cleaved-Caspase-3 (#ab2302; 1:1,000; Abcam), CyclinD1 (#2922; 1:1000; Cell Signaling Technology, Inc.), cyclin dependent kinase 4 (CDK4; #12790; 1:1,000; Cell Signaling Technology, Inc.) and β-actin (#8457; 1:10,000; Cell Signaling Technology, Inc.) at 4°C overnight. The blots were washed with TBST (1,000 ml TBST with 1 ml Tween-20) three times and incubated with respective secondary antibodies (Goat anti-rabbit IgG Alexa Fluor® 488; #ab150077; 1:10,000; Cell Signaling Technology, Inc.) at 4°C for 1–2 h. Following three washes in TBST, the ImageQuant LAS 4000 (Immobilon®; EMD Millipore, Billerica, MA, USA) was used to visualize the blots and the expression levels of the proteins were analyzed using the NIH Image J system 1.48v (National Institutes of Health, Bethesda, MD, USA). Statistical significance was calculated using SPSS 18.0 software (SPSS, Inc.) and the histograms were obtained using the Graph Pad system v5.0 (GraphPad Software, Inc.).
A student t-test and one-way analysis of variance was used to compare the means of different treatments, using SPSS 18.0 software (SPSS, Inc.). Date were expressed as the mean ± standard deviation, and P<0.05 was considered to indicate a statistically significant difference.
The effect of grifolin on human ovarian cancer cell viability was analyzed using the MTT assay, clonogenic assay and flow cytometry. For this purpose, A2780 cells were incubated with grifolin (0, 25, 50, 75 and 100 µM) for 24, 48 and 72 h. The results revealed a significant decrease in the viability of A2780 cells, in a dose-dependent and time-dependent manner (P<0.05;
Flow cytometry analysis was used to determine whether grifolin induced apoptosis in A2780 cells. Grifolin-treated A2780 cells were stained with annexin V-FITC and PI following treatment with grifolin (0, 25, 50, and 75 uM) for 24 h. Grifolin-treated A2780 cells were demonstrated to have undergone apoptosis in a dose-dependent manner, and these results corroborated the data from the MTT assay (P<0.05;
Next, the protein expressions of cell cycle proteins (cyclinD1 and CDK4) and apoptosis associated proteins (Bcl-2, Bax, cleaved-caspase-3 and cleaved-PARP) were analyzed to further assess the potential mechanisms underlying grifolin-induced apoptosis and inhibition of cell viability. Western blot analysis was used to assess the expression of cyclinD1 and CDK4, which were visibly decreased in cells treated with grifolin compared with the control (P<0.05;
Activation of phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) pathways has been demonstrated to be associated with the expression of apoptosis-associated proteins. Thus, western blotting was used to evaluate the expression of Akt and ERK1/2 in grifolin-treated A2780 cells, and to determine whether the Akt and ERK1/2 pathways are potential signaling mechanisms underlying grifolin-induced apoptosis. The expression levels of p-Akt and p-ERK1/2 were significantly decreased in a dose-dependent manner in grifolin-treated cells compared with the untreated control (P<0.05;
At present, there has been growing interest in the function of grifolin, in particular concerning its anticancer effects. Grifolin, a secondary metabolite product isolated from the mushroom
Multiple studies have demonstrated that mammalian cell cycle progression is closely associated with cyclins and CDKs (
Members of the caspase family participate in physiological activity through pathways including the mitochondrial and death receptor pathway (
Cellular signaling pathways are known to include diverse proteins, and are complex communication networks that control basic biological functions. Several cellular signaling pathways are known to regulate cell proliferation and apoptosis, including nuclear factor-κB, PI3K/Akt, MAPK and p53 signaling pathways. The aim of the present study was to define the pathways participating in grifolin-induced anti-cancer effects. The majority of previous studies have confirmed that activation of the ERK1/2 or Akt pathway is associated with the anticancer effect of grifolin (
In conclusion, grifolin, which has been known for several decades to demonstrate various pharmacological and physiological effects and to inhibit the growth of multiple cancer cell lines, also had an anticancer effect against the human ovarian cancer cell line, A2780. To the best of our knowledge, the present study is the first report to explore the activity of grifolin on human ovarian cancer cells. The results demonstrated that inactivation of Akt and ERK1/2 was the mechanism underlying grifolin-induced cell cycle arrest and cell apoptosis. Grifolin may therefore be a promising antitumor agent for the treatment of ovarian cancer.
The present study was funded by the National Natural Science Foundation of China (grant nos. 81072121, 81372808 and 81173614), the Science and Technology Development Planning of Shandong (grant nos. 2012G0021823 and 2011GSF12122) and the Science and Technology Development Planning of Jinan (grant no. 201303035).
Effect of grifolin on A2780 human ovarian cancer cell viability. Cells were treated with various concentrations of grifolin and cell viability was analyzed using the MTT assay. The results are representative of three independent experiments. *P<0.05 vs. 0 µM control.
A2780 cells were treated with different concentrations of grifolin, and assessed by clonogenic assay. Data were expressed as the mean ± standard deviation, and the experiments were repeated three times. *P<0.05 vs. 0 µM control.
Effects of grifolin on the cell cycle stage of A2780 human ovarian cancer cells, as assessed by flow cytometry. Data are representative of three independent experiments. *P<0.05 vs. 0 µM control.
Pro-apoptotic activity of grifolin on A2780 human ovarian cancer cells. The proportion of early apoptotic A2780 cells were measured by flow cytometry. Data are representative of three independent experiments. *P<0.05 vs. 0 µM control.
(A) Western blot analysis of cell cycle proteins of A2780 human ovarian cancer cells, following 24 h treatment with 0, 25, 50 or 75 µM grifolin. (B) Western blot analysis of apoptosis-associated proteins following 24 h treatment with 0, 25, 50 or 75 µM grifolin. Equal loading and transfer were demonstrated by repeat probing with β-actin. CDK4, cyclin dependent kinase 4; Bcl-2, B cell lymphoma-2; Bax, Bcl-2 associated X, apoptosis regulator; PARP, poly (ADP-ribose) polymerase. Data are representative of 3 independent experiments. *P<0.05 vs. 0 µM control.
Western blot analysis of p-Akt, Akt, p-ERK1/2 and ERK1/2 expression in A2780 human ovarian cancer cells treated with 0, 25, 50 or 75 µM grifolin for 24 h. Equal loading and transfer were demonstrated by repeat probing with β-actin. p-, phosphorylated; Akt, protein kinase B; ERK1/2, extracellular signal-related kinase. Data are representative of 3 independent experiments. *P<0.05 vs. 0 µM control.