Apigenin is one of the plant-originated flavones with anticancer activities. In this study, apigenin was assessed for its
Apigenin (4′,5,7-trihydroxy-flavone) is a plant-derived flavonoid compound, and is ubiquitously found in both fruits and vegetables. Besides its anti-inflammatory, anti-oxidant and anticancer properties (
In the eukaryotic cells, protein molecules spontaneously fold during or after biosynthesis to form biologically functional conformation. Failure to fold into native three-dimensional structure generally leads to inactive proteins (
Intracellular-free radicals such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) are related with ER stress, mitochondria dysfunction and inflammation (
Pre-treatment of the MIA PaCa-2 cells with NAC will markedly decrease intracellular ROS generation induced by a synthesized polyphenol conjugate (E)-3-(3,5-dimethoxyphenyl)-1-(2-methoxyphenyl)prop-2-en-1-one (DPP-23), along with reduced expression of the UPR (unfolded protein response) proteins (GRP78/BiP, IRE1a, and CHOP) (
To obtain more evidence on anticancer potential of apigenin, this study aimed to verify its
Apigenin with purity >99% was purchased from Shanghai Yousi Biotechnology Co. Ltd. (Shanghai, China). Cell Counting Kit-8 (CCK-8), cell cycle analysis kit, Annexin V-FITC apoptosis detection kit, Hoechst 33258, ROS assay kit, mitochondrial membrane potential assay kit with JC-1, Fura-2 pentakis (acetoxymethyl) ester (Fura-2 Am), radio immunoprecipitation assay (RIPA) lysis buffer and BCA protein assay kit were purchased from Beyotime Institute of Biotechnology (Shanghai, China). TRNzol Universal Reagent, TIANScript RT kit and Real Master Mix (SYBR Green) were purchased form Tiangen Biotech, Co. Ltd. (Beijing, China). Other chemicals used were of analytical grade. Water used was generated from Milli-Q Plus system (Millipore, New York, NY, USA).
The cell line (HCT-116) used in this study was obtained from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). The cells were cultured in McCoy's 5A medium (Sigma-Aldrich, Co. St. Louis, MO, USA) supplemented with 10% of fetal bovine serum (Hyclone, Logan, UT, USA) at 37°C in 5% CO2 atmosphere, as recommended by the cell supplier.
Primary antibodies CHOP, DR5, cytochrome c oxidase IV (COX IV), cytochrome c, Bax, BID, cleaved caspase-3, −8, and −9, as well as secondary antibodies were provided by Cell Signaling Technology (Shanghai) Biological Reagents Co., Ltd. (Shanghai, China).
The cells were seeded at a density of 1×104 cells per 100 µl per well onto the 96-well plates. After cell attachment, the medium was discarded. Dimethyl sulphoxide (DMSO, negative control) of 0.1%, 5-fluorouracil (5-Fu, positive control) of 100 µM, and apigenin of 40–160 µM were added to treat the cells for 24, 48 and 72 h, respectively. After that, the solutions were discarded, and the cells were washed twice by a phosphate buffer saline (PBS, 0.01 µM, pH 7.0). CCK-8 solution of 10 µl was added into each well to make a final concentration of 10%. The plates were then incubated at 37°C for another 4 h. A microplate reader (Bio-Rad Laboratories, Hercules, CA, USA) was used to measure the absorbencies at 570 nm. The vehicle-treated cells were taken as 100% viable. The growth inhibition of the cells was thus calculated as previously described (
The cells (1×104 cells per 100 µl per well) were seeded onto 6-well plates for attachment, treated by 0.1% DMSO, 40–160 µM apigenin for 24 h, rinsed with the PBS twice, followed by the treatment of 0.5 ml of paraformaldehyde (4%) in the PBS at 4°C overnight to fix the cells. The cells were washed with the PBS twice, stained with Hoechst 33258 of 0.5 ml for 5 min at room temperature in the dark, and then rinsed with the PBS twice. The stained cells were observed and photographed under a fluorescence microscope (Olympus, Tokyo, Japan) with respective excitation and emission wavelengths of 350 and 460 nm.
After 24 h of treatment with 0.1% DMSO or 60–160 µM apigenin, the cells were harvested and washed with the PBS. Ice-cold 70% ethanol was added to fix the cells at 4°C overnight. After that, the cells were rinsed with the ice-cold PBS, and incubated with 25 µl propidium iodide (PI, 50 µg/ml) and 10 µl RNase (100 µg/ml) for 30 min at 37°C in the dark. Flow cytometric cell analysis was performed using a BD FACSort flow cytometry (Becton Dickson Immunocytometry-Systems, San Jose, CA, USA). CellQuest software (ModFit software, Verity Software House, Inc., Topsham, ME, USA) was used to determine the portions of the cells in different cell stages of cell cycle progression (G0/G1, S, and G2/M phases).
The cells treated with 0.1% DMSO or 60–160 µM apigenin in the 6-well plates were harvested after 24 h, and washed twice with the ice-cold PBS. Double staining with FITC-Annexin V and PI was carried out using the Annexin V-FITC Apoptosis Detection kit according to the manufacturer's protocol. Briefly, the cells were incubated with 5 µl of the Annexin V-FITC and 10 µl of PI (20 µg/ml) at room temperature for 20 min in the dark. The cells were then discriminated into viable, necrotic, early apoptotic, and late apoptotic cells, using flow cytometry and CellQuest software as above.
The cells (1×106 per chamber) were treated with DMSO (0.1%, control) and apigenin of 60–160 µM for 24 h. Afterward, the cells were re-suspended in fresh medium, and incubated with the JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl benzimidazolocarbocyanine iodide) of 1 ml at 37°C for 20 min. The cells were rinsed with Dulbecco's phosphate-buffered saline (DPBS) twice, and re-suspended in 2 ml medium. The loss of mitochondrial membrane potential (MMP) was evaluated by the flow cytometry with respective excitation and emission wavelengths of 485 and 590 nm as previously described (
For the assay of intracellular ROS, the cells were treated with 0.1% DMSO or 60–160 µM apigenin for 24 h at 37°C, followed by two washes with the PBS. Then, 1 ml of DCF-DA (2′,7′-dichlorofluorescein, 10 µM) were added into each well, and the cells were re-incubated at 37°C for another 20 min. The cells were rinsed with the fresh medium three times. Fluorescence intensities were detected by a fluorescence spectrophotometer (F-4500, Hitachi, Tokyo, Japan) at 488/525 nm with a 525 nm cut-off (
The cells were seeded overnight and then applied with 0.1% DMSO or 60–160 µM apigenin at 37°C for 24 h. The cells were collected and rinsed with the Krebs-Ringer buffer (pH 7.4), which contained 137 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.5 mM CaCl2, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 25 mM D-glucose. The cells were collected and incubated with the Fura-2 AM (5 µM) at 37°C for 60 min. After that, the cells were washed twice and re-suspended in the Krebs-Ringer buffer, and measured for fluorescence (
Total RNA of the HCT-116 cells was extracted with the TRNzol Universal Reagent (Tiangen Biotech, Co. Ltd.), and complementary DNA (cDNA) was then reverse transcribed using the TIANScript RT kit and the protocol provided by the kit manufacturer. The qRT-PCR was performed using a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). cDNA of 1 µl was added to 9 µl of 2.5X Real Master Mix (20X SYBR Green) containing 5 µl of each of the corresponding primer pairs to make a final system volume of 20 µl for each well. Thermo-cycling conditions were used as follows: initial activation for 1 min at 95°C, followed by 40 cycles of denaturation at 95°C for 15 sec, annealing for 20 sec at 60°C and extension for 32 sec at 68°C. The fluorescence was measured during the extension step. Relative expression levels of the target genes were determined using the 2−∆∆Ct method (
After 24 h treatment with 0.1% DMSO or 60–160 µM apigenin at 37°C for 24 h, the cells were harvested and lysed with the RIPA lysis buffer, followed by a centrifugation at 14,000 × g at 4°C for 10 min. The supernatants were collected after boiling for 5 min. Protein contents were measured using the BCA Protein Assay kit. Proteins (50 µg) were separated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and electro-transferred onto nitrocellulose membranes. The membranes were blocked in 5% non-fat milk at 37°C for 1 h, and then incubated with the primary antibodies at 4°C overnight. Afterward, the membranes were washed with the TBST buffer (containing 10 mM Tris-HCl, pH 7.6, 150 mM NaCl, and 0.1% Tween-20) three times, and incubated with the secondary antibodies at 37°C for 1 h. Images of the blots were captured, and densitometric analysis was performed using an ImageQuant LAS 500 (Fujifilm, Tokyo, Japan).
All values are expressed as mean values or mean values ± standard derivations from three independent experiments and analyses. Statistical significance between different groups was analyzed by one-way analysis of variance (ANOVA) with Duncan's multiple range tests using the SPSS version 13.0 (SPSS Inc., Chicago, IL, USA). Statistical significance was defined at P<0.05.
In addition, when the HCT-116 cells were treated with apigenin of various doses, nuclei morphological changes were observed in the treated cells after Hoechst 33258 staining (
To understand if apigenin had effect on cell cycle progression of the HCT-116 cells, the distribution of the cells in different cell cycle phases was assessed using flow cytometry. Consistent changes in the cell cycle at 24 h were observed along with increased apigenin doses (60–160 µM). In the control cells, the respective portions of S, G0/G1 and G2/M phases were 52.2, 25.3 and 21.4% (
After 24 h exposure to apigenin of 60–160 µM, the cells were collected and detected to show potential apoptosis induction of apigenin. The results shown in
The cells were treated with apigenin of various doses for 24 h, and assayed for their intracellular ROS levels. The results (
As the results demonstrated in
Real-time RT-PCR results showed that apgenin at dose levels of 60, 120, and 160 µM could upregulate both CHOP (1.2-, 2.3-, and 3.4-fold) and DR5 (1.1-, 1.5-, and 2.1-fold) mRNA expression in the treated cells (
To reveal the underlying mechanism responsible for apigenin-induced apoptosis in the HCT-116 cells, expression levels of these associated proteins were evaluated. The results given in
Several studies have reported that apigenin has anticancer activities via anti-proliferation, angiogenesis and apoptosis induction (
In normal cells, apigenin processes anti-oxidation. However, in cancer cells, apigenin exhibits pro-oxidation rather than anti-oxidation (
The execution of apoptosis is accomplished by caspase family via two major pathways, intrinsic pathway and extrinsic pathway (
When the HCT-116 cells were treated with apigenin, they showed decreased MMP and upregulated Bax protein. As a consequence, cytochrome c was released from the mitochondria to the cytosol, which activated cleaved caspase-9 and cleaved caspase-3 to induce apoptosis. In addition, accumulation of intracellular ROS led to continuous release of Ca2+ from the ER lumen to the cytosol, which consequently caused ER stress. Overload Ca2+ levels in the cytoplasm accelerated Ca2+ influx into the mitochondria, which resulted in greater ROS generation and finally caused the opening of the permeability transition pore. Increased ROS generation within the mitochondria was thus released, and as a feedback to simulate the Ca2+ release channels on the ER (
Other studies have assessed apoptosis induction of some natural products, and shown apoptotic mechanism similar to this study. Hesperidin from
Based on the present results, it is concluded that apigenin is a promising anticancer compound capable of inhibiting proliferation of the colon carcinoma HCT-116 cells, disturbing cell cycle progression to arrest the cells at G0/G1 phase, causing abundant intracellular ROS generation and Ca2+ release, destroying mitochondrial membrane, and inducing apoptosis. Apigenin can upregulate the expression of CHOP, DR5, cleaved caspase-3, cleaved caspase-8, and cleaved caspase-9, cleaved BID, and Bax, and can also enhance cytochrome c release. The outlined apoptotic mechanism is thus, for the first time, suggested to involve ROS-induced and ER stress-mediated intrinsic and extrinsic pathways.
This study was funded by the Key Research Project in Science and Technology of the Education Department of Heilongjiang Province (project no. 11551z018).
RAC-α serine/threonine-protein kinase
apoptotic peptidase activating factor 1
activating transcription factor 3
Bcl-2 antagonist/killer
Bcl-2 associated × protein
B-cell cll/lymphoma 2
Bcl-2-like protein 2
B-cell lymphoma-extra large
BH3 interacting domain death agonist
Bcl-2-related ovarian killer
Cell Counting Kit-8
complementary DNA
C/EBP homologous protein
cytochrome c oxidase IV
2′,7′-dichloro-fluorescein
dimethyl sulphoxide
Dulbecco's phosphate-buffered saline
death receptor 3
death receptor 4
death receptor 5
ethylene glycol tetraacetic acid
endoplasmic reticulum
FAS-associating death domain-containing protein
cell surface death receptor
fas ligand
5-fluorouracil
Fura-2 pentakis (acetoxymethyl) ester
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
inhibitor of DNA binding 1
janus kinase 2
5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl benzimidazolocarbocyanine iodide
mitogen-activated protein kinase
myeloid cell leukemia 1
mitochondrial membrane potential
N-acetyl cysteine
nuclear factor κB subunit
poly-(ADP-ribose) polymerase
phosphate buffer saline
propidium iodide
C Protein kinase C δ type
permeability transition pore
quantitative real-time PCR
radio immunoprecipitation assay
reactive nitrogen species
reactive oxygen species
signal transducer and activator of transcription 3
tumor necrosis factor receptor-1
truncated BID
tumor necrosis factor-related apoptosis-inducing ligand
unfolded protein response
vascular endothelial growth factor A
Effects of apigenin doses on the growth of the HCT-116 cells. The cells were exposed to 0.1% DMSO (negative control), 40–160 µM apigenin, and 100 µM 5-fluorouracil (positive control) for 24–72 h, respectively.
Morphological changes of the HCT-116 cells exposed to apigenin. Apoptotic-like cells show shrinkage, condensed domain and apoptotic bodies. (A-F) represents the control cells, and the cells treated by apigenin of 40, 60, 80, 120, and 160 µM, respectively.
Apigenin disturbed the HCT-116 cells cycle arrest at G0/G1 phase in a dose-dependent manner. (A-E) represent the control cells, and the cells treated by apigenin at 40, 60, 80, 120, and 160 µM, respectively.
Apoptosis induction of apigenin towards the HCT-116 cells. The cells were exposed to 0.1% DMSO (A) and apigenin at 60 µM (B); 80 µM (C); 120 µM (D); and 160 µM (E), for 24 h, and then the number of cells undergoing apoptosis was detected using Annexin V staining and flow cytometry.
Effects of apigenin doses on intracellular ROS generation (A) and mitochondrial membrane potential loss (B) of the HCT-116 cells. The cells were treated with apigenin for 24 h. Different lowercase letters above the columns indicate significant data differences between different groups (P<0.05). ∆Ψm, mitochondrial membrane potential loss.
Effects of apigenin doses on intracellular Ca2+ of the HCT-116 cells. The cells were treated with apigenin for 24 h, and the data were expressed as fluorescence ratio of 340/380 nm (F340/F380). Different lowercase letters above the columns indicate significant data differences between the different groups (P<0.05).
Effects of apigenin doses on mRNA expression of CHOP and DR5 in the HCT-116 cells. The cells were exposed to 0.1% DMSO (as control), and 60, 120, and 160 µM agpigenin for 24 h. Different lowercase letters above the columns indicate significant data differences between the different groups (P<0.05).
Western blotting results for expression levels of the related proteins in the HCT-116 cells exposed to three apigenin doses. The cells were treated with 0.1% DMSO (control) and 60–160 µM apigenin for 24 h. Apigenin promoted upregulation of CHOP, DR5, cleaved caspase-8, cleaved caspase-9, cleaved caspase-3, cleaved BID, Bax, and cytochrome c (Cyto C).
Suggested mechanism and signaling pathways for the apigenin-induced apoptosis towards the HCT-116 cells. Cyto C, cytochrome c.