Introduction

Oncology Reports

1021-335X 1791-2431

D.A. Spandidos

10.3892/or.2016.5303

or-37-02-1132

Articles

Apigenin induces both intrinsic and extrinsic pathways of apoptosis in human colon carcinoma HCT-116 cells

Wang

1 2 Zhao

Xin-Huai

1Key Laboratory of Dairy Science, Ministry of Education, Northeast Agricultural University, Harbin, Heilongjiang 150030, P.R. China 2College of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin, Heilongjiang 150040, P.R. China

Correspondence to: Professor Xin-Huai Zhao, Key Laboratory of Dairy Science, Ministry of Education, Northeast Agricultural University, 59 Mucai Street, Harbin, Heilongjiang 150030, P.R. China, E-mail: zhaoxh@neau.edu.cn

012017

08122016

37 2 1132 1140 01072016 24112016

2017

Apigenin is one of the plant-originated flavones with anticancer activities. In this study, apigenin was assessed for its in vitro effects on a human colon carcinoma line (HCT-116 cells) in terms of anti-proliferation, cell cycle progression arrest, apoptosis and intracellular reactive oxygen species (ROS) generation, and then outlined its possible apoptotic mechanism for the cells. Apigenin exerted cytotoxic effect on the cells via inhibiting cell growth in a dose-time-dependent manner and causing morphological changes, arrested cell cycle progression at G₀/G₁ phase, and decreased mitochondrial membrane potential of the treated cells. Apigenin increased respective ROS generation and Ca²⁺ release and thereby, caused ER stress in the treated cells. Apigenin shows apoptosis induction towards the cells, resulting in enhanced portion of apoptotic cells. A mechanism involved ROS generation and endoplasmic reticulum stress was outlined for the apigenin-mediated apoptosis via both intrinsic mitochondrial and extrinsic pathways, based on the assayed mRNA and protein expression levels in the cells. With this mechanism, apigenin resulted in the HCT-116 cells with enhanced intracellular ROS generation and Ca²⁺ release together with damaged mitochondrial membrane, and upregulated protein expression of CHOP, DR5, cleaved BID, Bax, cytochrome c, cleaved caspase-3, cleaved caspase-8 and cleaved caspase-9, which triggered apoptosis of the cells.

apigenin human colon carcinoma cells cytotoxic effect apoptotic mechanism reactive oxygen species endoplasmic reticulum stress

Introduction

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 (1,2), apigenin has received particular interest as a cancer chemopreventive agent for a variety of cancers in recent years. Apigenin inhibits the proliferation of the A2780 ovarian cancer cells in a dose-time-dependent manner through overexpression of ATF3 to suppress Id1 expression (3). Apigenin possesses anti-growth activity on both the prostate (DU-145) and breast (MDA-MB-231) cancer cells through caspase-3 activation mediated by estrogen receptor (ER)-β (4). Apigenin at 60 µM induces apoptosis in the human promyelocytic leukaemia HL-60 cells via mitochondria dysfunction releasing cytochrome c to active caspase-3, caspase-9 and cleavage of poly-(ADP-ribose) polymerase (PARP) (5).

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 (6,7). The endoplasmic reticulum (ER) is the major organelle for protein biosynthesis, folding, maturation and translocation (8). When cells are stimulated, unfolded proteins accumulate in the lumen of ER. Dis-homeostasis of ER results in a pathological response known as ER stress. The organelle has its unique signaling pathways to overcome ER stress, such as i) slowing down translational rate to prevent further accumulation of the mis-folded proteins; ii) upregulating the genes capable of increasing the protein-folding capacity in ER; iii) activating the NFκB to trigger immune and anti-apoptotic responses; and iv) inducing apoptosis (9). When ER organelle is severely impaired, apoptosis is provoked to eliminate the damaged cells notably through upregulating the expression of a sensor protein, C/EBP homologous protein (CHOP) (9). Under non-stressed conditions, CHOP expresses at low level; however, when cells are under adverse conditions (e.g. glucose deprivation, amino acid starvation and ER stress), CHOP will be overexpressed to disturb cell cycle progression (10). CHOP can activate downstream protein expression by upregulating the pro-apoptotic proteins (e.g. Bax and Bak) and by downregulating the anti-apoptotic proteins (e.g. Bcl-2 and Bcl-xl) to induce the intrinsic pathway, and can also upregulate downstream the pro-apoptotic death receptor 5 (DR5) to induce the extrinsic pathway (11).

Intracellular-free radicals such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) are related with ER stress, mitochondria dysfunction and inflammation (12). In general, intracellular ROS levels are relatively low. When cells are exposed to stimuli, intracellular ROS generation increases. Although ROS can promote cell proliferation and differentiation, excessive ROS cause oxidative stress, which is harmful to cells (13). However, in cancer cells, increase of ROS generation is related with apoptosis (14–16). ROS at the same level can kill cancer cells without any significant toxicity to their normal counterparts (17). Increasing evidence shows that overload ROS will cause ER stress. For example, auranofin can induce ROS-mediated ER stress and mitochondrial dysfunction via upregulating CHOP expression and cleaving caspase-3 and PARP in the human gastric cancer cells, BGC-823 and SGC-7901 (18). Sarsasapogenin from a Chinese medical herb Anemarrhena asphodeloides Bunge causes both significant intracellular ROS generation and CHOP expression in the HeLa cells (19). However, if an anti-oxidant N-acetyl cysteine (NAC) is used to treat the cells before sarsasapogenin application, the amounts of ROS (together with ER stress and apoptosis) decrease, implying that sarsasapogenin-induced ER stress and mitochondria dys-function are triggered by ROS generation (19).

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) (20). This indicates that intracellular ROS production is upstream of the ER stress-mediated apoptosis (20). Iso-obtusilactone A isolated from Cinnamomum kotoense can increase intracellular ROS generation, and upregulate CHOP and DR5 protein levels in the hepatoma Hep G2 cells; however, pre-treatment the cells with NAC can decrease ROS generation associated with lower expression levels of the two apoptotic-related proteins (21). It is thus proved that much ROS production is able to provoke ER stress-mediated apoptosis towards cancer cells.

To obtain more evidence on anticancer potential of apigenin, this study aimed to verify its in vitro effects on a human colon carcinoma line (HCT-116 cells), and to outline a possible apoptotic mechanism involving both intracellular ROS generation and ER stress.

Materials and methods <sec> <title>Chemicals and reagents

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).

Cells and antibodies

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).

Assay of cell viability

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 (22).

Morphological observation

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.

Assay of cell cycle progression

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).

Apoptosis analysis by flow cytometry

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.

Detection of mitochondrial membrane potential, intracellular ROS and Ca2+

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 (23).

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 (24).

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 (F_s). The used emission and excitation wavelengths were 510 and 340–380 nm, respectively. The cells treated with 0.1% of Triton X-100 (v/v) were used to determine the maximal fluorescence (F_max), followed by addition of 10 mM EGTA (ethylene glycol tetra-acetic acid, pH 9.0) to determine the minimal fluorescence (F_min). Intracellular Ca²⁺ ([Ca²⁺]_i) was calculated using following equation as [Ca²⁺]_i=k_d (F_s-F_min)/(F_max-F_s), in which k_d has a value of 224 nM (25).

Isolation of mRNAs and quantitative real-time PCR (qRT-PCR)

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 (26). The β-actin housekeeping gene was used as an internal control. The used primers were designed with the sequences below, and synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). i) human β-actin forward, 5′-AACACCCCAGCCATGTACG-3′ and reverse, 5′-ATGTCACGCACGATTTCCC-3′; ii) CHOP forward, 5′-GCCAATGATGTGACCCTCAAT-3′ and reverse, 5′-CCTGGAAATGAAGAGGAAGAA-3′; iii) DR5 forward, 5′-AAGACCCTTGTGCTCGTTGT-3′ and reverse, 5′-GACACATTCGATGTCACTCCA-3′.

Western blot assay

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).

Statistical analysis

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.

Results <sec> <title>Cytotoxic effect of anpigenin on the cells

In vitro effect of apigenin on the HCT-116 cells is described in Fig. 1. Apigenin exerted cytotoxic effect on the cells, and inhibited cell growth (i.e. decreased cell viability) dose- and time-dependently. Apigenin of 40 µM only inhibited cell growth by 9.3–23.1% when the cells were treated for 24–72 h. Thus this dose was not used in other assays except morphological observation. However, higher apigenin dose showed higher cytotoxic effect and thereby resulted in lower cell viability (i.e. greater inhibition). If apigenin was used at 160 µM and the cells were treated for 72 h, growth inhibition reached to a maximum value of 80.5%. The calculated IC₅₀ values of apigenin with treating times of 24, 48 and 72 h were approximately 98.2, 83.3 and 77.9 µM, respectively. These data demonstrated clearly that longer treatment time also resulted in apigenin stronger inhibition (i.e. cytotoxic effect) on the cells.

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 (Fig. 2), especially when apigenin of 160 µM was used. High chromatin condensation and visible formation of apoptotic bodies were found in the treated cells (Fig. 2F). This phenomenon indicated that apigenin was able to induce apoptosis besides growth inhibition on the cells.

G0/G1 phase cell cycle arrest and apoptosis induction of apigenin

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% (Fig. 3). As apigenin dose increased into higher level, the treated cells showed decreasing trend in the portions of both G₂/M and S phases but increasing trend in the portion of G₀/G₁ phase. This demonstrated that apigenin was dose-dependent in arresting the cell cycle at G₀/G₁ phase.

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 Fig. 4 demonstrate that the portion of apoptotic cells (early plus late apoptotic cells) was enhanced with increased apigenin dose. Treatment of the cells with 60–160 µM apigenin caused 5.6, 11.9, 15.5, and 16.7% Annexin V-FITC positive cells (Fig. 4B-E). However, the control cells were detected to only have 3.6% apoptotic cells (Fig. 4A). These results implied that apigenin indeed had apoptosis induction, which was dependent on the used apigenin dose.

Intracellular ROS and Ca2+ as well as MMP loss in response to apigenin treatment

The cells were treated with apigenin of various doses for 24 h, and assayed for their intracellular ROS levels. The results (Fig. 5A) showed that ROS generation in the cells was significantly increased after apigenin exposure. In comparison with the control cells, the cells treated with 160 µM apigenin had increased ROS level up to 301.8%. Other three apigenin doses (60–120 µM) resulted in the cells with increased ROS levels of 160.5, 200.8 and 267.4%. Enhancement of ROS generation depended on the used apigenin doses. In addition, the effect of apigenin on MMP loss of the treated cells was also measured. After 24 h treatment with apigenin of four dose levels, the treated cells showed significant decrease in MMP in comparison with the control cells (Fig. 5B). Apigenin was thus proved able to damage mitochondrial membrane. Moreover, MMP loss of the treated cells was also observed in an apigenin dose-dependent manner. These results pointed out an important fact; that is, apigenin had potential toxic effects on the cells via both mitochondria and ER organelles.

As the results demonstrated in Fig. 6, apigenin at 60–160 µM with treating time of 24 h caused the treated cells increased intracellular Ca²⁺ levels in a dose-dependent manner (P<0.05). Intracellular Ca²⁺ levels of the treated cells were 117.3, 126.7, 135.1 and 147.4% of the control cells. These results of Ca²⁺ release were consistent with those of ROS generation and MMP loss. It is thus verified that higher ROS generation induced ER stress, which consequently resulted in abundant Ca²⁺ release into the cytosol.

Increased mRNA expression of CHOP and DR5 in the treated cells

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 (Fig. 7). Higher apigenin dose clearly resulted in greater upregulation of CHOP and DR5. These results proved that apigenin might induce ER stress and activate the death receptor signaling pathway, which should be underlined to reveal the related mechanisms.

Apoptotic mechanism is involved in ROS generation and ER stress

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 Fig. 8 indicated that apigenin increased protein expression levels of CHOP (1.1–3.5-fold), DR5 (1.2–2.4-fold), cleaved BID (1.1–1.6 fold), cleaved caspase-3 (1.0–1.3-fold), cleaved caspase-8 (1.1–1.6-fold), and cleaved caspase-9 (1.3–1.7-fold) (Fig. 8A), Bax (1.6–3.5-fold), and cytochrome c (1.0–2.2-fold) (Fig. 8B) in the cytosol, but decreased protein expression level of BID (1.0–2.9-fold) (Fig. 8A). Higher apigenin dose led to greater expression changes of these proteins. Based on these results, it is thus verified that apigenin-induced apoptosis was mediated by ROS generation and ER stress, through upregulating CHOP and DR5 and therefore triggering both extrinsic and intrinsic pathways. A molecular mechanism for the apoptosis induction of apigenin on the cells is outlined in Fig. 9. Apigenin conferred the treated cells with ER stress via greater ROS generation, increased CHOP and DR5 mRNA levels, regulated the expression of these pro-apoptotic proteins (cleaved caspase-3, cleaved caspase-8, and cleaved caspase-9, cleaved BID, and Bax), increased cytochrome c release from the mitochondria into the cytosol, and finally initiated apoptosis of the cells.

Discussion

Several studies have reported that apigenin has anticancer activities via anti-proliferation, angiogenesis and apoptosis induction (1,27,28). The involved mechanisms have also been revealed. Apigenin can inhibit the growth of the HT-29 cells dose- and time-dependently, cause DNA fragmentation, and increase mRNA expression levels of CASP3 (late apoptosis, effector) and CASP8 (early apoptosis, initiator) (29). Apigenin inhibits the proliferation of the T-24 cells, and induces apoptosis via the mitochondrial pathway (30). Apigenin can activate PKCδ, which then activate caspase pathway to induce apoptosis in the leukemia THP-1 cells (31). Apigenin is capable of inducing apoptosis via a caspase-dependent pathway in the MDA-MB-453 cells as well as inhibiting the well-known JAK2-STAT3-VEGF signaling pathway (32). Apigenin is also able to upregulate DR5 expression in the human acute lymphoblastic leukemic cell line Jurkat, prostate cancer cell line DU145, and colon cancer cell line DLD-1 to trigger TRAIL-induced apoptosis (33). In this study, apigenin was also observed to inhibit the growth of the HCT-116 cells, to arrest cell cycle at G₀/G₁ phase, to increase both intracellular ROS and Ca²⁺ levels, and to decrease MMP. Finally, both extrinsic and intrinsic pathways were triggered. Clearly, the outlined apoptotic mechanism involved ROS generation and ER stress in this study is different to those reported mechanisms mentioned above.

In normal cells, apigenin processes anti-oxidation. However, in cancer cells, apigenin exhibits pro-oxidation rather than anti-oxidation (34,35). In this study, intracellular ROS level was suggested to play an important role in the ER stress-mediated signaling pathway. This finding is supported by the results from other studies. Apigenin increases ROS level in the human lung cancer A549 cells, which leads to high Bax/Bcl-2 ratio to cause mitochondria dysfunction and to trigger caspase-dependent apoptosis (36). Apigenin can block cell cycle progression at G₂/M stage, increase intracellular ROS production, and therefore cause AKT hypophosphorylation in the triple-negative MDA-MB-468 breast cancer cells (37). In addition, apigenin can trigger intracellular ROS generation, activate mitogen-activated protein kinase (MAPK) ERK1/2 in the human fibroblast-like synoviocyte MH7A cells, and finally cause apoptosis via activation of caspase-3 and −7 (38). These results support that apigenin caused apoptosis induction towards the HCT-116 cells through enhancing ROS generation and resultant ER stress.

The execution of apoptosis is accomplished by caspase family via two major pathways, intrinsic pathway and extrinsic pathway (39). In the intrinsic pathway, cells are stimulated by various toxic compounds, which causes cytochrome c release from the mitochondria into the cytosol. A so-called complex, apoptosome, is thus formed to activate the cleaved caspase-9 as well as downstream caspases (40). The Bcl-2 protein family contains both anti- and pro-apoptotic members. Bcl-2 and its closest Bcl-xL and Bcl-w, can protect cells from apoptosis, whereas other Bcl-2 relatives (e.g. Bax, Bak and Bok) are pro-apoptotic proteins (41). Once the Bax/Bcl-2 ratio increases, apoptosis is induced, resulting in the release of apoptogenic factors from the mitochondria (42). On the contrary, in the extrinsic pathway, the caspases are activated by death receptors such as FAS, TNFR1, DR3, DR4, and DR5 (43). These cell surface proteins recruit adaptor proteins (e.g. FADD) to form a death-inducing signaling complex with procaspase-8 that induces caspase-8 activation. Downstream effector caspases (such as caspase-3, −7 and −9) are thus cleaved (44). However, the extrinsic apoptotic pathway has crosstalk with the intrinsic apoptotic pathway via caspase-8 cleavage of the BID, which then activates the mitochondrial pathway to amplify the apoptotic signal (45).

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 (46). CHOP as protein indicator of ER stress was thus activated to upregulate its downstream protein DR5. DR5 overexpression activated cleaved caspase-8. Cleaved caspase-8 has two roles: to activate cleaved caspase-3 and to cleave BID to generate cleaved BID. Cleaved BID in turn induced Bax activation. Thus, the mitochondrial pathway was amplified by the ER stress signaling pathway.

Other studies have assessed apoptosis induction of some natural products, and shown apoptotic mechanism similar to this study. Hesperidin from Citrus seed can induce apoptosis in the human hepatocellular carcinoma HepG2 cells via both mitochondrial and death receptor pathways, evidenced by the upregulation of Bax and Bak protein levels, downregulation of Bcl-xl protein level, and activation of tBID and caspase-3, −8, and −9 (47). Licochalcone B, isolated from the roots of Chinese licorice, exhibits apoptosis induction towards the oral squamous cell carcinoma HN22 and HSC4 cells via increasing ROS levels, as well as CHOP, DR4 and DR5 protein expression (48). Accompanying by upregulation of these apoptotic proteins, expression levels of anti-apoptotic proteins (e.g. Mcl-1) decrease, but pro-apoptotic proteins (e.g. Bax) level is upregulated (48). Emodin, a natural anthraquinone-type compound from Rheum palmatum L, can induce apoptosis in the human cervical cancer HeLa cells (49). In the emodin-treated cells, protein expression levels of cytochrome c, Apaf-1, FAS, FasL, and FADD are all upregulated whilst those levels of procaspase-3, −8 and −9 are all downregulated (49). These findings state that emodin can induce both intrinsic mitochondrial and extrinsic death receptor apoptotic pathways (49), and therefore provide support to the outlined mechanism of this study.

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.

Acknowledgements

This study was funded by the Key Research Project in Science and Technology of the Education Department of Heilongjiang Province (project no. 11551z018).

Abbreviations

AKT

RAC-α serine/threonine-protein kinase

Apaf-1

apoptotic peptidase activating factor 1

ATF3

activating transcription factor 3

Bak

Bcl-2 antagonist/killer

Bax

Bcl-2 associated × protein

Bcl-2

B-cell cll/lymphoma 2

Bcl-w

Bcl-2-like protein 2

Bcl-xl

B-cell lymphoma-extra large

BID

BH3 interacting domain death agonist

Bok

Bcl-2-related ovarian killer

CCK-8

Cell Counting Kit-8

cDNA

complementary DNA

CHOP

C/EBP homologous protein

COX IV

cytochrome c oxidase IV

DCF-DA

2′,7′-dichloro-fluorescein

DMSO

dimethyl sulphoxide

DPBS

Dulbecco's phosphate-buffered saline

DR3

death receptor 3

DR4

death receptor 4

DR5

death receptor 5

EGTA

ethylene glycol tetraacetic acid

endoplasmic reticulum

FADD

FAS-associating death domain-containing protein

FAS

cell surface death receptor

FasL

fas ligand

5-Fu

5-fluorouracil

Fura-2 Am

Fura-2 pentakis (acetoxymethyl) ester

HEPES

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

Id1

inhibitor of DNA binding 1

JAK2

janus kinase 2

JC-1

5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl benzimidazolocarbocyanine iodide

MAPK

mitogen-activated protein kinase

Mcl-1

myeloid cell leukemia 1

MMP

mitochondrial membrane potential

NAC

N-acetyl cysteine

NFκB

nuclear factor κB subunit

PARP

poly-(ADP-ribose) polymerase

PBS

phosphate buffer saline

propidium iodide

PKCδ

C Protein kinase C δ type

PTP

permeability transition pore

qRT-PCR

quantitative real-time PCR

RIPA

radio immunoprecipitation assay

RNS

reactive nitrogen species

ROS

reactive oxygen species

STAT3

signal transducer and activator of transcription 3

TNFR1

tumor necrosis factor receptor-1

tBID

truncated BID

TRAIL

tumor necrosis factor-related apoptosis-inducing ligand

UPR

unfolded protein response

VEGF

vascular endothelial growth factor A

References 1

Yang

Landau

Huang

Newmark

Inhibition of carcinogenesis by dietary polyphenolic compounds

Annu Rev Nutr213814062001

10.1146/annurev.nutr.21.1.381

11375442

Patel

Shukla

Gupta

Apigenin and cancer chemoprevention: Progress, potential and promise (Review)

Int J Oncol302332452007

17143534

Wang

Fang

Apigenin inhibits proliferation of ovarian cancer A2780 cells through Id1

FEBS Lett583199920032009

10.1016/j.febslet.2009.05.013

19447105

Mak

Leung

Tang

Harwood

Apigenin suppresses cancer cell growth through ERbeta

Neoplasia88969042006

10.1593/neo.06538

17132221

1716010

Wang

Lin-Shiau

Lin

Induction of apoptosis by apigenin and related flavonoids through cytochrome c release and activation of caspase-9 and caspase-3 in leukaemia HL-60 cells

Eur J Cancer35151715251999

10.1016/S0959-8049(99)00168-9

10673981

Bailly-Maitre

Reed

Endoplasmic reticulum stress: Cell life and death decisions

J Clin Invest115265626642005

10.1172/JCI26373

16200199

1236697

Kim

Emi

Tanabe

Murakami

Role of the unfolded protein response in cell death

Apoptosis115132006

10.1007/s10495-005-3088-0

16374548

Malhotra

Kaufman

The endoplasmic reticulum and the unfolded protein response

Semin Cell Dev Biol187167312007

10.1016/j.semcdb.2007.09.003

18023214

2706143

Oyadomari

Mori

Roles of CHOP/GADD153 in endoplasmic reticulum stress

Cell Death Differ113813892004

10.1038/sj.cdd.4401373

14685163

Meares

Mines

Beurel

Eom

Song

Zmijewska

Jope

Glycogen synthase kinase-3 regulates endoplasmic reticulum (ER) stress-induced CHOP expression in neuronal cells

Exp Cell Res317162116282011

10.1016/j.yexcr.2011.02.012

21356208

3103628

Yamaguchi

Wang

CHOP is involved in endoplasmic reticulum stress-induced apoptosis by enhancing DR5 expression in human carcinoma cells

J Biol Chem27945495455022004

10.1074/jbc.M406933200

15322075

Gorman

Healy

Jäger

Samali

Stress management at the ER: Regulators of ER stress-induced apoptosis

Pharmacol Ther1343063162012

10.1016/j.pharmthera.2012.02.003

22387231

Trachootham

Alexandre

Huang

Targeting cancer cells by ROS-mediated mechanisms: A radical therapeutic approach?

Nat Rev Drug Discov85795912009

10.1038/nrd2803

19478820

Fruehauf

Meyskens

Reactive oxygen species: A breath of life or death?

Clin Cancer Res137897942007

10.1158/1078-0432.CCR-06-2082

17289868

Rigas

Sun

Induction of oxidative stress as a mechanism of action of chemopreventive agents against cancer

Br J Cancer98115711602008

10.1038/sj.bjc.6604225

18253125

2359637

Schumacker

Reactive oxygen species in cancer cells: Live by the sword, die by the sword

Cancer Cell101751762006

10.1016/j.ccr.2006.08.015

16959608

Trachootham

Zhou

Zhang

Demizu

Chen

Pelicano

Chiao

Achanta

Arlinghaus

Liu

Selective killing of oncogenically transformed cells through a ROS-mediated mechanism by beta-phenylethyl isothiocyanate

Cancer Cell102412522006

10.1016/j.ccr.2006.08.009

16959615

Zou

Chen

Ying

Zhang

Liu

Yang

Auranofin induces apoptosis by ROS-mediated ER stress and mitochondrial dysfunction and displayed synergistic lethality with piperlongumine in gastric cancer

Oncotarget636505365212015

26431378

4742192

Shen

Zhang

Gong

Sarsasapogenin induces apoptosis via the reactive oxygen species-mediated mitochondrial pathway and ER stress pathway in HeLa cells

Biochem Biophys Res Commun4415195242013

10.1016/j.bbrc.2013.10.101

24383086

Shin

Lee

Koh

Jung

Lim

Lee

Targeting cancer cells via the reactive oxygen species-mediated unfolded protein response with a novel synthetic polyphenol conjugate

Clin Cancer Res20430243132014

10.1158/1078-0432.CCR-14-0424

24938523

Chen

Yiin

Hsu

Wang

Lin

Chern

Isoobtusilactone A sensitizes human hepatoma Hep G2 cells to TRAIL-induced apoptosis via ROS and CHOP-mediated up-regulation of DR5

J Agric Food Chem60353335392012

10.1021/jf2051224

22400995

Johnson

de Mejia

E Gonzalez

Interactions between dietary flavonoids apigenin or luteolin and chemotherapeutic drugs to potentiate anti-proliferative effect on human pancreatic cancer cells, in vitro

Food Chem Toxicol6083912013

10.1016/j.fct.2013.07.036

23871783

Salvioli

Ardizzoni

Franceschi

Cossarizza

JC-1, but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess delta psi changes in intact cells: Implications for studies on mitochondrial functionality during apoptosis

FEBS Lett41177821997

10.1016/S0014-5793(97)00669-8

9247146

Boissy

Trinkle

Nordlund

Separation of pigmented and albino melanocytes and the concomitant evaluation of endogenous peroxide content using flow cytometry

Cytometry107797871989

10.1002/cyto.990100616

2582969

Vega

Chacón

Monteseirín

El Bekay

Alba

Martín-Nieto

Sobrino

Expression of the transcription factor NFAT2 in human neutrophils: IgE-dependent, Ca²⁺- and calcineurin-mediated NFAT2 activation

J Cell Sci120232823372007

10.1242/jcs.000331

17606988

Livak

Schmittgen

Analysis of relative gene expression data using real-time quantitative PCR and the 2^−∆∆CT method

Methods254024082001

10.1006/meth.2001.1262

11846609

Van Dross

Xue

Knudson

Pelling

The chemopreventive bioflavonoid apigenin modulates signal transduction pathways in keratinocyte and colon carcinoma cell lines

J Nutr133(Suppl 1)S3800S38042003

Shukla

Gupta

Dietary agents in the chemoprevention of prostate cancer

Nutr Cancer5318322005

10.1207/s15327914nc5301_3

16351503

Turktekin

Konac

Onen

Alp

Yilmaz

Menevse

Evaluation of the effects of the flavonoid apigenin on apoptotic pathway gene expression on the colon cancer cell line (HT29)

J Med Food14110711172011

10.1089/jmf.2010.0208

21548803

Shi

Shiao

Lee

Shih

Apigenin, a dietary flavonoid, inhibits proliferation of human bladder cancer T-24 cells via blocking cell cycle progression and inducing apoptosis

Cancer Cell Int1533452015

10.1186/s12935-015-0186-0

25859163

4391114

Vargo

Voss

Poustka

Cardounel

Grotewold

Doseff

Apigenin-induced-apoptosis is mediated by the activation of PKCdelta and caspases in leukemia cells

Biochem Pharmacol726816922006

10.1016/j.bcp.2006.06.010

16844095

Seo

Choi

Woo

Jang

Shin

Induction of caspase-dependent apoptosis by apigenin by inhibiting STAT3 signaling in HER2-overexpressing MDA-MB-453 breast cancer cells

Anticancer Res34286928822014

24922650

Horinaka

Yoshida

Shiraishi

Nakata

Wakada

Sakai

The dietary flavonoid apigenin sensitizes malignant tumor cells to tumor necrosis factor-related apoptosis-inducing ligand

Mol Cancer Ther59459512006

10.1158/1535-7163.MCT-05-0431

16648565

Miyoshi

Naniwa

Yamada

Osawa

Nakamura

Dietary flavonoid apigenin is a potential inducer of intracellular oxidative stress: The role in the interruptive apoptotic signal

Arch Biochem Biophys4662742822007

10.1016/j.abb.2007.07.026

17870050

Andueza

García-Garzón

de Galarreta

M Ruiz

Ansorena

Iraburu

López-Zabalza

Martínez-Irujo

Oxidation pathways underlying the pro-oxidant effects of apigenin

Free Radic Biol Med871691802015

10.1016/j.freeradbiomed.2015.06.003

26119779

Chie

Yang

Lee

Yang

Tan

Apigenin induces caspase-dependent apoptosis in human lung cancer A549 cells through Bax- and Bcl-2-triggered mitochondrial pathway

Int J Oncol36147714842010

20428772

Harrison

Coombs

MR Power

Delaney

Hoskin

Exposure of breast cancer cells to a subcytotoxic dose of apigenin causes growth inhibition, oxidative stress, and hypophosphorylation of Akt

Exp Mol Pathol972112172014

10.1016/j.yexmp.2014.07.006

25019465

Shin

Kim

Lee

Cho

Lee

Jeong

Cho

Lee

Apigenin-induced apoptosis is mediated by reactive oxygen species and activation of ERK1/2 in rheumatoid fibroblast-like synoviocytes

Chem Biol Interact18229362009

10.1016/j.cbi.2009.07.016

19647729

Hale

Smith

Sutherland

Stoneman

Longthorne

Culhane

Williams

Apoptosis: Molecular regulation of cell death

Eur J Biochem2361261996

10.1111/j.1432-1033.1996.00001.x

8617251

Wang

Youle

The role of mitochondria in apoptosis

Annu Rev Genet43951182009

10.1146/annurev-genet-102108-134850

19659442

4762029

Inoue

Browne

Melino

Cohen

Ordering of caspases in cells undergoing apoptosis by the intrinsic pathway

Cell Death Differ16105310612009

10.1038/cdd.2009.29

19325570

Cory

Huang

DCS

Adams

The Bcl-2 family: Roles in cell survival and oncogenesis

Oncogene22859086072003

10.1038/sj.onc.1207102

14634621

Ashkenazi

Targeting the extrinsic apoptosis pathway in cancer

Cytokine Growth Factor Rev193253312008

10.1016/j.cytogfr.2008.04.001

18495520

Nagata

Apoptosis by death factor

Cell883553651997

10.1016/S0092-8674(00)81874-7

9039262

Wang

The expanding role of mitochondria in apoptosis

Genes Dev15292229332001

11711427

Berridge

The endoplasmic reticulum: A multifunctional signaling organelle

Cell Calcium322352492002

10.1016/S0143416002001823

12543086

Banjerdpongchai

Wudtiwai

Khaw-On

Rachakhom

Duangnil

Kongtawelert

Hesperidin from Citrus seed induces human hepatocellular carcinoma HepG2 cell apoptosis via both mitochondrial and death receptor pathways

Tumour Biol372272372016

10.1007/s13277-015-3774-7

26194866

4841854

Yoon

Shin

Park

Cho

Lee

Liu

Cho

Chae

Licochalcone B induces apoptosis of human oral squamous cell carcinoma through the extrinsic- and intrinsic-signaling pathways

Int J Oncol48174917572016

26847145

Yaoxian

Hui

Yunyan

Yanqin

Xin

Xiaoke

Emodin induces apoptosis of human cervical cancer heLa cells via intrinsic mitochondrial and extrinsic death receptor pathway

Cancer Cell Int1371782013

10.1186/1475-2867-13-71

23866157

3722080

Figure 1.

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.

Figure 2.

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.

Figure 3.

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.

Figure 4.

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.

Figure 5.

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.

Figure 6.

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).

Figure 7.

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).

Figure 8.

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).

Figure 9.

Suggested mechanism and signaling pathways for the apigenin-induced apoptosis towards the HCT-116 cells. Cyto C, cytochrome c.