Contributed equally
Gastric cancer is the second most common cancer worldwide next to lung cancer (
Over the past few years, flavonoids from dietary sources have attracted interest in preventing cancer with low toxicity. Flavonoids are abundantly present in fresh fruits and vegetables and have various health benefits (
Apoptosis is a critical cell death mechanism with a distinctive phenotype and plays an important role in the mechanism of chemotherapies against various types of carcinoma (
Based on the above evidence, in the present study, we investigated the anticancer activity and the related mechanism of FCP in AGS cells. The present study provides new insight for understanding the mechanism of the anticancer effects of FCP in AGS cells.
The fruit of
RPMI-1640 medium, fetal bovine serum (FBS) and antibiotics (penicillin/streptomycin) were purchased from Gibco (BRL Life Technologies, Grand Island, NY, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Antibodies to Bcl-xL, Bax, caspase-3, -6, -8 and -9, cleaved caspase-3, poly(AdP ribose) polymerase (PARP), cleaved PARP, p-Akt, JNK, p-JNK, p38, p-p38, ERK1/2 and p-ERK1/2 were purchased from Cell Signaling Technology (Danvers, MA, USA). The Akt and β-actin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Millipore (Billerica, MA, USA), respectively. Horseradish peroxidase (HRP)-coupled goat anti-mouse IgG and anti-rabbit IgG were purchased from Enzo Life Sciences. Muse™ Cell Cycle kit and Annexin V and Dead Cell kit were purchased from Millipore. Materials and chemicals used for electrophoresis were obtained from Bio-Rad (Hercules, CA, USA).
Human gastric cancer AGS cells were obtained from the Korean Cell Line Bank (Seoul, Korea). The AGS cells were cultured in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated FBS and 1% penicillin/streptomycin in a humidified atmosphere with 5% CO2 at 37°C. To assess the effect of FCP on AGS cell growth, the cells were seeded at 10×104 cells/ml in a 12-well plate and were treated with FCP at various concentrations (25, 50, 75, 100, 125 and 150
The AGS cells were incubated without or with FCP at concentrations of 75 and 150
The AGS cells were treated with the indicated concentrations of FCP for 24 h at 37°C, and the cells were washed with cold PBS and fixed with 37% formaldehyde (1:4 dilution with 95% ethanol) for 10 min at room temperature. The fixed cells were washed with PBS and stained with a 4′,6-diamidino-2-phenylindole (DAPI, Vectashield H-1500; Vector Laboratories, Inc., Burlingame, CA, USA). The nuclear morphology of the cells was examined by fluorescence microscopy(x400 magnification; Leica, Germany).
For the western blot analysis, the AGS cells were treated with the indicated concentrations of FCP for the indicated times at 37°C and the cells were lysed in ice-cold RIPA buffer [1% (w/w) NP-40, 1% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 0.15 M NaCl, 0.01 M sodium phosphate buffer, pH 7.2, 2 mM EDTA, and 50 mM NaF (as phosphatase inhibitor) and protease inhibitors]. The protein concentrations were determined using a Bradford assay (Bio-Rad) method (
The statistical analysis was calculated by the Student's t-test, using SPSS version 10.0 for Windows (SPSS, Chicago, IL, USA). The results are expressed as the mean ± standard deviation (SD) of at least three independent experiments. The statistical significance was accepted as P< 0.05.
The flavonoids were isolated from the fruit of
To determine the appropriate inhibitory concentrations of FCP, firstly AGS cells were treated with various concentrations (0–150
Next, flow cytometry was performed to determine cell cycle distribution and the population of cell death in the FCP-treated AGS cells. FCP treatment increased the percentage of the sub-G1 cells (apoptotic cell population) by 18, 41 (P<0.01) and 45% (P<0.01) at 0, 75 and 150
Western blotting was performed to determine whether FCP-induced cell death was caspase-dependent. In addition, we examined the expression of apoptosis-related proteins, such as Bax and Bcl-2, in the FCP-treated AGS cells. The results showed that the expression of procaspase-3, -6, -8 and -9 was significantly decreased while cleaved caspase-3 and cleaved PARP were significantly increased in a dose-dependent manner (
The PI3K/AKT and MAPK signaling pathways play an important role in regulating cell proliferation and apoptosis. Since the activity of AKT is regulated by phosphorylation, we examined the phosphorylation status of PI3K/AKT and MAPKs by immunoblotting during the FCP-induced apoptosis in AGS cells. FCP significantly dephosphorylated AKT at 75 and 150
The present study was designed to determine whether FCP induces cell death and to further investigate the underlying mechanisms of the FCP-induced apoptosis of AGS cells. Flavonoids are naturally occurring botanical polyphenols present in plant foods and can safely modulate the physiological function and enhance the anticancer activity against various human cancer cell lines (
Firstly, FCP significantly suppressed the growth of AGS cells in a dose-dependent manner. Evidence suggests that apoptosis (type I programmed cell death) is the most popular underlying mechanism by which various anticancer and chemopreventive agents including natural compounds exert anticancer effects (
For the evaluation of their underlying mechanisms, immunoblotting was performed. The results showed that the expression of procaspase -3, -6, -8 and -9 was significantly downregulated in a dose-dependent manner. Caspase-3 is a crucial executioner caspase that activates cleavage of PARP which results in apoptosis. In our study, the increased expression of cleaved caspase-3 simultaneously induced PARP cleavage (
We further examined the phosphorylation status of PI3K/AKT and MAPKs by immunoblotting to elucidate the molecular mechanism and pathways involved in FCP-induced apoptosis. We demonstrated that FCP inhibited the constitutive level of PI3K and its downstream target AKT (
In conclusion, we demonstrated that FCP suppressed cell viability and induced caspase-dependent cell death in the AGS cells. The induction of apoptosis triggered in the FCP-treated AGS cells was modulated by the PI3K/AKT and MAPK signaling pathways (
The present study was supported by a grant from the National Research Foundation (NRF) of Korea funded by the Ministry of Science, ICT and Future Planning (nos. 2012M3A9B8019303 and 2012R1A2A2A06045015) and the National R&D Program for Cancer Control, Ministry for Health, Welfare and Family Affairs, Republic of Korea (no. 0820050).
HPLC chromatogram patterns of
Inhibitory effects of FCP on AGS cells. The AGS cells were treated with the indicated concentrations of FCP for 24 h. (A) Cell viability was determined by an MTT assay. The data are expressed as the mean ± standard deviation (SD) of at least three independent experiments (*P<0.05, **P<0.01 compared to the control). (B) Morphology of the cells was examined under light microscopy (x400 magnification).
Regulatory effect of FCP on cell cycle progression of AGS cells. The AGS cells were treated with the indicated concentrations of FCP for 24 h. (A and B) Cell cycle distribution was determined by using the Muse Cell Cycle kit, and stained samples were analyzed with the Muse™ Mini FACS machine. The data are expressed as the mean ± standard deviation (SD) of at least three independent experiments (*P<0.05, **P<0.01 compared to the control).
Regulatory effect of FCP on the apoptosis of AGS cells. The AGS cells were treated with the indicated concentrations of FCP for 24 h. (A and B) Apoptosis was assessed by Annexin V-PI double staining using the Muse™ Mini FACS machine. The data are expressed as the mean ± standard deviation (SD) of at least three independent experiments (*P<0.05, **P<0.01 compared to the control). (C) The cells were stained with DAPI and analyzed by fluorescence microscopy (white arrows indicate fragmented or condensed nuclei).
Caspase activation and subsequent cleavage of PARP in the FCP-treated AGS cells. The AGS cells were treated with the indicated concentrations of FCP for 24 h. The cell lysates were subjected to SDS-PAGE and analyzed by immunoblotting. Densitometric analyses of procaspase-3, -6, -8, and -9, cleaved caspase-3, PARP and cleaved PARP and the Bax/Bcl-xL ratio are expressed as mean ± SD of three independent experiments (*P<0.05, **P<0.01 compared with the control).
FCP modulates the PI3K/AKT and MAPK signaling pathways in the AGS cells. The AGS cells were treated with the indicated concentrations of FCP for 24 h. The cell lysates were subjected to SDS-PAGE and analyzed by immunoblotting. Densitometric analyses of AKT, p-AKT, ERK1/2, p-ERK1/2, JNK, p-JNK, p38, and p-p38 MAPK are expressed as mean ± SD of three independent experiments (*P<0.05, **P<0.01 compared with the control).
Schematic diagram representing the anticancer mechanism of the FCP-induced apoptosis in AGS cells. FCP induced mitochondrial-dependent apoptosis by upregulation of the Bax/Bcl-xL ratio, caspase-3 activation and subsequent cleavage of PARP. Furthermore, FCP activates PI3K/AKT and MAPK signaling pathways in AGS cells. Taken together, the PI3K/AKT and MAPK pathways are involved in FCP-induced apoptosis in AGS cells, and FCP may have chemotherapeutic potential for the treatment of gastric cancer (→ indicates activation, ⊥ indicates inhibition, — indicates indirect or multiple pathways).
List of identified flavonoids from
Compound | RT (min) | [M-H]−/[M-H]+ | MS/MS | Mean ± SD | |
---|---|---|---|---|---|
1 | Naringin | 16.93 | 579/− | 459, 313, 271, 193, 151 | 2,483.5±1.6 |
2 | Hesperidin | 18.45 | 609/− | 608, 325, 301 | 1,163.2±1.6 |
3 | Hydroxypentamethoxyflavone | 39.88 | /389 | 374, 359, 341, 165 | 2,785.2±10.9 |
4 | Hydroxypentamethoxyflavone | 39.88 | /389 | 374, 359, 341, 165 | 393.4±2.3 |
5 | Sinensetin | 42.57 | −/373 | 373, 358, 343, 339, 329, 320, 312, 283, 181, 151 | 384.7±4.2 |
6 | Pectolinarigenin | 43.84 | /313 | 313, 285, 181, 156, 153, 135 | 525.8±13.2 |
7 | Dihydroxytetramethoxyflavone | 44.76 | 375 | 375, 360, 345, 342, 314, 302, 299, 285, 271, 227, 212, 197, 169, 166, 149 | 370.2±4.2 |
8 | Nobiletin | 45.32 | −/403 | 388, 373, 355, 327, 211, 165 | 3,911.9±5.5 |
9 | Heptamethoxyflavone | 46.15 | /433 | 418, 403, 385, 211, 165 | 674.5±4.4 |
10 | Tetramethyl-O-isoscutellarein | 47.99 | −/343 | 343, 328, 313, 299, 285, 211, 181, 135, 133 | 3,417.4±11.8 |
11 | Hydroxypentamethoxyflavone | 49.35 | /389 | 374, 359, 341, 165 | 1,258.3±7.7 |
12 | Hydroxyhexatamethoxyflavone | 50.56 | /419 | 404, 389, 373, 361, 343, 328, 315, 283, 227, 165 | 154.5±3.5 |
13 | Hydroxypentamethoxyflavone | 52.67 | 359 | 359, 344, 329, 311, 298, 286, 241, 224, 227, 211, 197, 183, 179, 135 | 258.5±1.7 |