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Induction of apoptotic death of human hepatocellular carcinoma (HepG2) cells by ethanolic extract from litchi (Litchi chinensis Sonn.) flower
Journal of Functional Foods 19 (2015) 100–109
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Induction of apoptotic death of human hepatocellular carcinoma (HepG2) cells by ethanolic extract from litchi (Litchi chinensis Sonn.) flower Jau-Tien Lin a,b, Yuan-Yen Chang b,c, Yi-Chen Chen d, Chao-Chin Hu a,b, Yu-Pei Chang e,f, Shih-Han Hsu e,f, Deng-Jye Yang e,f,* a
School of Medical Applied Chemistry, Chung Shan Medical University, 110, Jianguo N. Road, Sec. 1, Taichung 402, Taiwan Department of Medical Education, Chung Shan Medical University Hospital, 110, Jianguo N. Road, Sec. 1, Taichung 402, Taiwan c Department of Microbiology and Immunology, School of Medicine, Chung Shan Medical University, 110, Jianguo N. Road, Sec. 1, Taichung 402, Taiwan d Department of Animal Science and Technology, National Taiwan University, 1, Roosevelt Road, Sec. 4, Taipei, 106, Taiwan e School of Health Diet and Industry Management, Chung Shan Medical University, 110, Jianguo N. Road, Sec. 1, Taichung 402, Taiwan f Department of Nutrition, Chung Shan Medical University Hospital, 110, Jianguo N. Road, Sec. 1, Taichung 402, Taiwan b
A R T I C L E
I N F O
A B S T R A C T
Article history:
The cytotoxic activity of litchi (Litchi chinensis Sonn.) flower ethanolic extract (LFEE) against
Received 13 April 2015
A549 (human lung adenocarcinoma), KB (human nasopharyngeal carcinoma), MCF-7 (human
Received in revised form 19 August
breast adenocarcinoma) and HepG2 (human hepatocellular carcinoma) cells was evaluated.
2015
LFEE exhibited better anti-proliferative action against HepG2 cells, which induced G2/M phase
Accepted 24 August 2015
arrest and apoptosis in HepG2 cells. HepG2 cells treated with LFEE (0.3 mg/mL) enhanced
Available online
expressions of p53, tBid, Bad and Bax, along with decreased expressions of Bcl-2 and Bcl-xL, and induced the release of cytochrome c from mitochondria to cytosol. That also accom-
Keywords:
panied by activation of caspases-3, -8 and -9 and cleavage of poly (adenosine diphosphate
Litchi (Litchi chinensis Sonn.) flower
(ADP)-ribose) polymerase (PARP); furthermore, down-regulations of phosphoinositide 3 kinase
Apoptosis
(PI3K), Akt, Akt phosphorylation and Bad phosphorylation could also be observed. Five fla-
Hep G2 cell
vonoids (total amount, 101.07 mg/g dried extract (g DE)), nine phenolic acids (total amount,
Caspase
55.07 mg/ g DE) and proanthocyanidin A2 (81.20 mg/ g DE) could be determined in LFEE.
Bcl-2 family proteins
© 2015 Elsevier Ltd. All rights reserved.
PI3K/Akt
1.
Introduction
Litchi (Litchi chinensis Sonn.) originating in Southeast Asian (Rivera-López, Ordorica-Falomir, & Wesche-Ebeling, 1999) is an
important economic crop in Taiwan. The tree blooms in late March and fruit matures in late June. In addition to normal eating, the fruit is extensively used to produce many products, such as jelly, juice, vinegar and wine (Salunke & Desai, 1984). Notwithstanding litchi flower is generally considered an
* Corresponding author. School of Health Diet and Industry Management, Chung Shan Medical University, and Department of Nutrition, Chung Shan Medical University Hospital, 110, Jianguo N. Road, Sec. 1, Taichung City 402, Taiwan. Tel.: +886 4 24730022 ext. 11868; fax: +886 4 2324188. E-mail address: [email protected] (D.-J. Yang). http://dx.doi.org/10.1016/j.jff.2015.08.023 1756-4646/© 2015 Elsevier Ltd. All rights reserved.
Journal of Functional Foods 19 (2015) 100–109
agricultural by-product; the flower with favourable flavour is also collected to manufacture tea for drinking in Taiwan. Previous investigations had demonstrated that litchi flower containing remarkable amount of phenolic compounds had many significant bioactive attributes, such as antioxidant (Chen, Lin, Liu, Lu, & Yang, 2011; Liu, Lin, Wang, Chen, & Yang, 2009), anti-inflammation (Yang et al., 2014), cardiovascular protection (Yang et al., 2010), hepatoprotection (Hwang et al., 2013) and anti-obesity (Wu et al., 2013). Anti-carcinogenic activities have been demonstrated in many flower extracts. Hsu et al. (2010) indicated that longan flower aqueous extract containing rich amount of polyphenols could induce cell-cycle arrest and apoptosis in colorectal cancer cells. Moravcˇíková, Kuceková, Mlcˇek, Rop, and Humpolícˇek (2012) demonstrated that methanolic extracts of edible flowers, including Ag yoncha, Wild chive, Meadow salsify and Garden sorrel, had higher level of polyphenols, which should be responsible for inhibition of human hepatocellular carcinoma cell line (HepG2) proliferation. Polyphenols could arrest cancer cell cycle (Lin, Liang, & Lin-Shiau, 1999), induce cancer cell apoptosis (Nichenametla, Taruscio, Barney, & Exon, 2006), and interfere in cancer initiation, promotion and progression (Link, Balaguer, & Goel, 2010). However, the anti-cancer effect of litchi flower extract has not been explored yet. In the study, we evaluated the cytotoxic activity of litchi flower ethanolic extract (LFEE) against A549 (human lung adenocarcinoma), KB (human nasopharyngeal carcinoma), MCF-7 (human breast adenocarcinoma) and HepG2 (human hepatocellular carcinoma) cell lines first. Through the comparative experiments with Clone 9 cell line (an epithelial cell line isolated from normal liver taken from a young male rat) and two anti-cancer chemotherapy drugs (fluorouracil, 5-FU and methotrexate, MTX), the better LFEE concentration was exploded to investigate the pathway of LFEE-induced apoptotic death in HepG2 cells.
2-mercaptoethanol, potassium chloride, monobasic potassium phosphate, Tween 20 and Tris base were obtained from Sigma Co. (St. Louis, MO, USA). Minimum Essential Medium (MEM), Dulbecco’s Modified Eagle’s Medium (DMEM), RPMI 1640 Medium, Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12), foetal bovine serum (FBS), penicillinstreptomycin (PS), penicillin-streptomycin-amphotericin (PSA) and lyophilised trypsin-EDTA were purchased from Gibco/ Invitrogen Co. (Carlsbad, CA, USA). Sodium dodecyl sulfate (SDS) was purchased from Merck (Darmstadt, Germany). Antibody against α-tubulin and the monoclonal antibody against cytochrome c, PARP, PI3K and caspases-3, and -8, were purchased from GeneTex, Inc. (Alton Pkwy., Irvine, CA, USA). Antibody against Bid, Bax, Bad, phosphorylated Bad (phospho-Bad (Ser136)), Bcl-xL, Bcl-2, α-tubulin, Akt and phosphorylated Akt (phospho-Akt) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibody against caspase-9, and Bid were purchased from Cell Signaling Technology, Inc (Danvers, MA, USA). Trypan blue was purchased from (MP Biomedicals, Santa Ana, CA, USA). The horseradish peroxidaseconjugated anti-goat or anti-rabbit IgG were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). Ammonium persulphate (APS), N, N′-methylenebisacrylamide (acrylamide), bromophenol blue, N, N, N′, N′-tetramethylethylenediamine (TEMED) and Triton X-100 were purchased from (Amresco Inc., Solon, OH, USA).
2.2.
Materials and methods
2.1.
Chemicals and reagents
Distilled deionised water (dd H 2 O) was prepared by an UltrapureTM water purification system (Lotun Co., Ltd., Taipei, Taiwan). Acetonitrile, methanol, ethanol, phosphoric acid and acetic acid were purchased from Merck Co. (Darmstadt, Germany). Standards of flavonoids including apigenin, catechin, daidzein, diosmin, epicatechin, eriodictyol, genistein, hesperetin, hesperidin, isorhamnetin, kamempferol, luteolin, morin, myricetin, narigenin, naringin, neohesperidin, quercetin, quercitrin and rutin, and phenolic acids including p-anisic, caffeic, chlorogenic, p-coumaric, ferulic, gallic, gentisic, p-hydroxybenzoic, rosmarinic, sinapic, syringic and vanillic acids were purchased from Sigma Co. (St. Louis, MO, USA). Proanthocyanidin A2 was isolated according to our previous study (Yang et al., 2014). Sulphanilamide, N-(1-Naphthyl) ethylenediamine dihydrochloride, phosphate-buffered saline (PBS), bovine serum albumin (BSA), ethylenediaminetetraacetic acid disodium salt dihydrate (Na 2 EDTA), glycine, sodium fluoride, sodium pyrophosphate, phenylmethanesulfonyl fluoride, propidium iodide, di-sodium hydrogen phosphate,
Preparation of LFEE
Fresh litchi flowers gathered in March 2014 from a farm in Taichung, Taiwan were lyophilised and then extracted with 95% ethanol (1/20, g/mL) in the dark for 24 h. The filtrate was evaporated to dryness in a rotary vacuum evaporator (Panchun Scientific Co., Kaohsiung, Taiwan). LFEE in an airtight bottle was stored at −80 °C prior to experiment.
2.3.
2.
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Cell line
Cell lines for MCF-7 (ATCC No. HTB-22) maintained in MEM medium with 10% heat-inactivated FBS and 1% PSA, Hep G2 (ATCC No. HB-8065) maintained in MEM medium with 10% heatinactivated FBS and 1% PS, A549 (ATCC No. CCL-185) maintained in DMEM with 10% heat-inactivated FBS and 1% PS, KB (ATCC No. CCL-17) maintained in RPMI-1640 medium with 10% heat-inactivated FBS and 1% PSA, and Clone-9 (ATCC No. CRL1439) maintained in DMEM/F-12 medium with 10% heatinactivated FBS and 1% PSA obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). These cell lines were incubated in a Thermo Scientific Forma Direct Heat CO2 Incubator (Thermo Fisher Scientific Inc., Mississauga, ON, Canada) (37 °C, 5% CO2).
2.4.
Cell viability assay
WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4disulphophenyl)-2H-tetrazolium, monosodium salt) in Cell Counting Kit-8 (CCK-8) could be reduced by dehydrogenases in the cell and produced a tissue culture medium soluble orange coloured formazan. It was used to assay the cell viability. The generated formazan level is proportional to the number of living
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Journal of Functional Foods 19 (2015) 100–109
cells directly (Ishiyama, Miyazono, Sasamoto, Ohkura, & Ueno, 1997). Each cell line seeded in each well of 24-well plates at a density of 2 × 105 cells/well was cultured in medium with various concentrations (0, 0.1, 0.2, 0.3, 0.4 and 0.5 mg/mL) of samples dissolved in ethanol (ethanol in each well was 0.0475% v/v) at 37 °C for 24 h in the CO2 incubator. The CCK-8 solution (10 µL) was then added into each well and incubated at 37 °C for 2 hr. The absorbance at 450/595 nm was measured with a microplate reader (Multiskan Spectrum, Thermo Co., Vantaa, Finland). The absorption for the control cells was regarded as 100% cell viability.
2.5.
Cell cycle analysis
Cells seeded in 10 cm dishes (1 × 106 cells/mL) were cultured in medium without or with 0.3 mg/mL of LFEE dissolved in ethanol at 37 °C for 12 or 24 h in the CO2 incubator. Ethanol concentration was 0.0475% (v/v). The cells were then fixed by 80% ethanol overnight at 4 °C. After washing with PBS, the cells were mixed with 0.1 mg/mL RNase, and stained with propidium iodide (40 µg/mL). The stained cells were analysed by a FACS Calibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Data collection and treatment were performed through the CellQuest and ModFit LT software (Becton Dickinson).
2.6.
DNA fragmentation analysis
HepG2 cells seeded in 10 cm dishes (1 × 106 cells/mL) were cultured in medium without or with 0.3 mg/mL of LFEE dissolved in ethanol at 37 °C for 24 h in the CO2 incubator. Ethanol concentration was 0.0475% (v/v). Cells were washed twice with phosphate-buffered saline (PBS) (pH 7.4), and DNA fragmentation analysis was carried out with DNA Laddering Kit (Trevigen, Inc., Gaithersburg, MA, USA) according to the manufacturer’s instructions. The eluants containing DNA pellets were electrophoresed (1.5 h at 80 V/30 mA) on a 1% agarose gel containing 0.5 mg/mL ethidium bromide. The gel was detected and photographed by ultraviolet gel documentation system.
2.7.
Immunoblotting analysis
Cells were lysed and protein extraction was carried out. The protein level was determined by a protein assay kit (Bio-Rad, Laboratories, Inc., Hercules, CA, USA). Cell proteins were separated in 12% of SDS-polyacrylamide gel, which was done at 110 V for 80 min with a standard running buffer (24 mM TrisHCl, 190 mM glycine, 0.5% SDS, pH8.3) and electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane at 200 mA for 90 min in transfer buffer (20 mM Tris-HCl, 150 mM glycine, 10% methanol, and 0.01% SDS). The membranes were then blocked with 5% non-fat milk, washed, and subsequently incubated with a specific antibody. After washing, the membrane was incubated with horseradish peroxidaseconjugated goat anti-mouse antibody or goat anti-rabbit antibody (Bio-Rad, Hercules, CA, USA). The protein bands were visualised with an enhanced chemiluminescence (ECL) system (Amersham Biosciences, Piscataway, NJ, USA). The protein band densities were quantified through the Alpha Imager 2200 software (Alpha Innotech Co., San Leandro, CA, USA). The amount of α-tubulin (57 kDa) in each lane was also detected as a control.
2.8.
Determination of phytochemicals in LFEE
A PrimeLine™ Gradient Model 500G HPLC pump system (Analytical Scientific Instruments, Inc., El Sobrante, CA, USA), a S-3210 photodiode-array (PDA) detector (Schambeck SFD GmbH, Bad Honnef, Germany), and a Thermo Finnigan LCQ classic quadrupole ion trap HPLC-mass (MS) system (Gentech, Arcade, NY, USA) equipped with electrospray ionisation (ESI) source (positive ion mode) were used for sample analysis. The HPLC-MS (conditions: nebuliser pressure, 70 psi; capillary temperature, 200 °C; dry gas flow, 11 L/min; electrospray voltage of the ion source, 3000 V; capillary exit, -159 V; skimmer, 40 V) was used to get mass spectra recording from m/z 50 to 1000. Flavonoids and phenolic acids in LFEE dissolved in methanol (0.25 mg/mL) were determined by high performance liquid chromatography (HPLC) according to the conditions of Chen et al. (2011): stationary phase, Inspire C18 column (250 × 4.6 mm, 5 µm; Dikma Technologies Inc., Lake Forest, CA, USA); mobile phase, methanol (solvent A) and 9% acetic acid solution prepared with dd H2O (solvent B) (conditions: 5–17% A from 0 to 5 min and kept at 17% A from 5 to 25 min; 17–31% A from 25 to 40 min and kept at 31% A from 40 to 76 min; 31–40 % A from 76 to 80 min and kept at 40% A from 80 to 120 min); flow rate, 0.8 mL/min; detection, 210–650 nm. Proanthocyanidins were analysed based on the report of Hwang et al. (2013): stationary phase, Inspire C18 column (250 mm × 4.6 mm i.d., 5 µm); mobile phase, acetonitrile (solvent A) and 2% acetic acid solution prepared with dd H2O with (solvent B) (conditions:10– 20% A from 0 to 10 min; 20–30% A from 10 to 13 min; 30–100 % A from 10 to 13 min; 30–100 % A from 13 to 15 min and kept at 100% A from 15 to 20 min); flow rate, 1 mL/min.
2.9.
Statistical analysis
All assays were performed in triplicate and the mean values were calculated. The data were subjected to analysis of variance (ANOVA), and Duncan’s multiple range tests were used to assess differences between means. Significant differences were concluded at a level of p < 0.05.
3.
Results
3.1. Flavonoids, phenolic acids and proanthocyanidin A2 in LFEE Through HPLC analysis, we can see that LFEE had five flavonoids, including epicatechin (79.23 ± 3.62 mg/ g of dried extract (mg/g DE)), rutin (17.25 ± 1.12 mg/g DE), naringin (0.52 ± 0.03 mg/g DE), quercitrin (2.06 ± 0.09 mg/gDE) and neohesperidin (2.01 ± 0.17 mg/gDE); the total amount of these flavonoids was 101.07 ± 5.03 mg/g DE). Moreover, nine phenolic acids, including gallic acid (0.21 ± 0.01 mg/g DE), gentisic acid (30.26 ± 1.98 mg/g DE), chlorogenic acid (2.65 ± 0.11 mg/g DE), vanillic acid (0.47 ± 0.02 mg/g DE), p-coumaric acid (0.19 ± 0.01 mg/g DE), ferulic acid (15.12 ± 1.07 mg/g DE), sinapic acid (4.20 ± 0.26 mg/g DE), syringic acid (0.75 ± 0.03 mg/g DE) and p-anisic acid (1.22 ± 0.03 mg/g DE) could also be found in LFEE, resulting in 55.07 ± 3.52 mg/g DE for the total amount of phenolic acids.
Journal of Functional Foods 19 (2015) 100–109
Proanthocyanidin A2, the major proanthocyanidin in LFEE, was 81.20 ± 3.17 mg/g DE. LFEE solution was prepared by ethanol to estimate its cytotoxicity against cancer cells.
3.2.
Cytotoxicity of LFEE against cancer cells
Through cytotoxicity assays for LFEE against A549, KB, MCF-7 and HepG2 cells, LFEE might be more effective against HepG2
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cell (IC50 = 0.33 ± 0.02 mg/mL) than other types of cancer cells. The higher the LFEE level was used, the lower the HepG2 cell viability was observed (Fig. 1). In order to know the effect of LFEE on the growth of normal liver cell, Clone 9 cell isolated from normal rat liver was used for comparison. Though the viability of Clone 9 cell treated with LFEE was also decreased in a concentration-dependent manner, the cytotoxicity of LFEE to Clone 9 cell (IC50 = 0.45 ± 0.02 mg/mL) was relatively lower
Fig. 1 – Effect of LFEE on viability of A549, MCF-7, KB, Hep G2 and Clone 9 cells. The cells were incubated with various concentrations (0.1, 0.2, 0.3, 0.4, and 0.5 mg/mL) of LFEE dissolved in ethanol for 24 h. Ethanol concentration in media was 0.0475% (v/v) except mock. Cell survival (% control) was measured by CCK-8 assay. Values are presented as mean ± SD of six independent experiments. Values with different letters are significantly different at the level of p < 0.05. C: control (without LFEE); M: mock (without LFEE and ethanol).
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Journal of Functional Foods 19 (2015) 100–109
Fig. 2 – Effect of anti-cancer drugs on viability of Hep G2 and Clone 9 cells. The cells were incubated with various concentrations (0.1, 0.2, 0.3 and 0.4 mg/mL) of fluorouracil (5-FU) or methotrexate (MTX) dissolved in ethanol for 24 h. Ethanol concentration in media was 0.0475% (v/v) except mock. Cell survival (% control) was measured by CCK-8 assay. Values are presented as mean ± SD of six independent experiments. Values with different letters (capital for Clone 9; lowercase for Hep G2) are significantly different at the level of p < 0.05. C: control (without drug); M: mock (without drug and ethanol).
than that to HepG2 cell (Fig. 1). Fig. 2 shows that fluorouracil (5-FU) and methotrexate (MTX) could suppress not only HepG2 cell growth but also Clone 9 cell growth. LFEE and the two anticancer chemotherapy drugs seemed to show the similar antiproliferative effects on HepG2 and Clone 9 cells at 0.3 mg/mL (Figs. 1 and 2). Therefore, this concentration was used to evaluate whether the cytotoxic effect of LFEE on HepG2 cells was due to apoptotic death. Because epicatechin is a major flavonoid in LFEE and has been demonstrated to induce apoptosis in human colon cancer cell (Kim, Mollah, & Kim, 2012), the compound was used for comparison.
3.3.
LFEE induced apoptosis in HepG2 cells
The cell cycle distribution of HepG2 cells treated with 0.3 mg/mL of LFEE was determined by flow cytometry at 12 and 24 h. Fig. 3A shows that Sub-G1 phase level for HepG2 cells clearly increased as compared with control (HepG2 cells without LFEE). It suggested that LFEE could cause DNA fragmentation of the cells. Fig. 3A also indicates a remarkable HepG2 cell population in G2/M phase following 12 h treatment with LFEE. It illustrated that HepG2 cell cycle was arrested in G2/M phase; the cells entering into next cycle and executing division were
Journal of Functional Foods 19 (2015) 100–109
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Fig. 3 – Effects of LFEE on cell cycle progression (A) and DNA fragmentation (B) in HepG2 cells. For cell cycle analysis, HepG2 cells were treated with or without (control) 0.3 mg/mL of LFEE for 12 and 24 h, respectively. The cells were then harvested and fixed by 70% alcohol. The cell cycle progression was analysed by flow cytometry after the cells were stained with propidium iodide. For DNA fragmentation, HepG2 cells treated with or without (control, C) 0.3 mg/mL of LFEE or epicatechin (EC) for 24 h were assayed with DNA Laddering Kit and electrophoresed on a 1% agarose gel. EC was used for comparison. M: marker.
moderated. However, the levels of G2/M phase and Sub-G1 phase for the cells incubated with LFEE for 24 h were greatly decreased and increased, respectively.The results might attribute to substantial cell apoptosis. Agarose gel electrophoresis showed that HepG2 cells incubated with LFEE for 24 h also induced internucleosomal DNA fragmentation (Fig. 3B), which is a biochemical hallmark of apoptosis (Nagata, 2000). Epicatechin could also induce internucleosomal DNA fragmentation in HepG2 cells.
3.4. LFEE activated caspase cascade in HepG2 cells for apoptosis HepG2 cells incubated with 0.3 mg/mL of LFEE for 24 h and caspase cascade were investigated by Western blotting. Results showed that procaspase-8 was decreased remarkably, whereas its cleaved form (active caspase-8) was significantly increased (Fig. 4). In addition, LFEE or epicatechin treatment led to procaspase-9 cleavage; its cleaved form (active caspase-9)
was significantly increased as well (Fig. 4). Similar results are also shown in Fig. 4 for procaspase-3 and its cleaved form (active caspase-3). Because PARP is one of the downstream substrates of activated caspase-3 (O’Brien, Moravec, & Riss, 2001), the degraded product of PARP (89 kDa) upon LFEE or epicatechin treatment was examined in the investigation. Fig. 4 shows that the cleavage of PARP activated by caspase-3 was significantly increased. These results implied that LFEE could stimulate the activation of caspases-8, -9, and -3 and cleavage of PARP to induce apoptosis in HepG2 cells. HepG2 cells treated with epicatechin also showed similar results.
3.5. LFEE modulated mitochondrial pathways and up-regulated p53 expression in HepG2 cells for apoptosis Fig. 5 shows that Bid expression was significantly decreased after HepG2 cells treated with 0.3 mg/mL of LFEE for 24 h, whereas its cleaved form (truncated Bid, tBid) expression was
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Journal of Functional Foods 19 (2015) 100–109
C
LFEE
EC
C Pro-Casepase 3 (35 kDa)
Cleaved (active) form (17 kDa) Fold
c
1
a
Fold
1
a
LFEE
EC b
0.3±0.01 0.5±0.02b Bid (22 kDa) Cleaved form (tBid, 15 kDa)
Fold
1c
4.2±0.3a 3.2±0.2b
Fold
1a
0.2±0.01c 0.5±0.03b
b
3.4±0.4 2.3±0.3
Pro-Casepase 8 (55 kDa)
Cleaved (active) form (18 kDa) Fold
c
1
a
3.1±0.2 1.8±0.1
Bcl-2 (29 kDa) Fold
1c
0.2±0.01a 0.4±0.01b
b
Bcl-xL (32 kDa)
Pro-Casepase 9 (47 kDa)
Fold
1c
3.1±0.3a 2.6±0.2b
Cleaved (active) form (37 kDa) Fold
c
1
a
Bax (23 kDa)
b
3.6±0.3 2.7±0.2
Fold
1a
0.3±0.02a 0.4±0.03b Mitochondrial Cytochrome c (15 kDa)
PARP (116 kDa) Cleaved form (89 kDa) Fold
1c
2.7±0.2a 2.2±0.2b
Fold
1c
3.1±0.2a 2.5±0.2b Cytosolic Cytochrome c (15 kDa)
a-Tubulin (57 kDa)
Fig. 4 – Expressions of caspases-3, -8 and -9 and PARP in LFEE-treated HepG2 cells. HepG2 cells were treated with or without (control, C) 0.3 mg/mL of LFEE or epicatechin (EC) for 24 h. Protein extracts were prepared for Western blot assay using indicated antibodies, and α-tubulin was used for loading control. Each value (fold) is expressed as means ± SD (n = 3); means with different letters are significantly different at the level of p < 0.05. EC was used for comparison.
significantly increased. It indicated that LFEE treatment resulted in the cleavage of cytosolic Bid to tBid, which promotes apoptosis at the mitochondria (Lutter, Perkins, & Wang, 2001). Moreover, the expressions of anti-apoptotic proteins Bcl-2 and BcL-xL were apparently decreased, whereas the expression of pro-apoptotic protein Bax was significantly increased. In addition, the levels of mitochondrial and cytosolic cytochrome c were decreased and increased, respectively (Fig. 5). It indicated the release of cytochrome c from mitochondria to the cytosol, which caused the activation of caspase family of protease and triggered the onset of apoptosis (Liu, Kim, Yang, Jemmerson, & Wang, 1996). Because p53 plays a role in apoptosis (Mahfudh & Lope Pihie, 2008), the effect of LFEE on p53 expression was also estimated. LFEE treatment significantly enhanced p53 expression in HepG2 cells (Fig. 5). Our study demonstrated that LFEE induced apoptosis in HepG2 cells through mitochondrial pathway and up-regulation of p53 expression. Similar results also showed in HepG2 cells treated with epicatechin.
Fold
1c
2.8±0.2b 3.2±0.2a p53 (53 kDa) α-Tubulin (57 kDa)
Fig. 5 – Expressions of Bid, Bcl-2, Bcl-xL, Bax, cytochrome c and P53 in LFEE-treated HepG2 cells. HepG2 cells were treated with or without (control, C) 0.3 mg/mL of LFEE or epicatechin (EC) for 24 h. Protein extracts were prepared for Western blot assay using indicated antibodies, and α-tubulin was used for loading control. Each value (fold) is expressed as means ± SD (n = 3); means with different letters are significantly different at the level of p < 0.05. EC was used for comparison.
Results showed that PI3K protein expression in HepG2 cells treated with 0.3 mg/mL of LFEE for 24 h was reduced significantly (Fig. 6). Furthermore, the levels of Akt protein as well as phospho-Akt protein were lowered significantly (Fig. 6). Bad is one of the pro-apoptotic proteins, whereas phosphorylation of Bad is anti-apoptotic. Akt activation enhances Bad phosphorylation that enhances survival of cancer cells (Zundel & Giaccia, 1998). In the study, Bad expression was significantly enhanced, whereas Bad phosphorylation was significantly down-regulated in HepG2 cells treated with LFEE, respectively (Fig. 6). These results illustrated that LFEE induced apoptosis in HepG2 cells through the suppression of the activation of PI3K-Akt-Bad pathway. HepG2 cells treated with epicatechin also showed similar results.
3.6. LFEE down-regulated PI3K expression, Akt activation and Bad phosphorylation in HepG2 cells for apoptosis
4.
This study further examined whether LFEE would influence the expressions of PI3K, Akt and phospho-Akt in HepG2 cells.
Natural products containing rich sources of compounds can be used for anticancer drug discovery; their induction of cell
Discussion
Journal of Functional Foods 19 (2015) 100–109
C Fold
LFEE
1c
EC
0.7±0.04a 0.8±0.03b PI3K (23 kDa)
Fold
1a
0.3±0.02c 0.5±0.02b Phospho-Akt (23 kDa)
Fold
1a
0.6±0.05b 0.4±0.02c Akt (23 kDa)
Fold
1c
1.7±0.1a 1.4±0.1b Bad (23 kDa)
Fold
1a
0.3±0.02a 0.5±0.03b Phospho-Bad (ser 136) (23 kDa) a-Tubulin (57 kDa)
Fig. 6 – Expression of PI3K, Akt, phospho-Akt, Bad and phospho-Bad in LFEE-treated HepG2 cells. HepG2 cells were treated with or without (control, C) 0.3 mg/mL of LFEE or epicatechin (EC) for 24 h. Protein extracts were prepared for Western blot assay using indicated antibodies, and α-tubulin was used for loading control. Each value (fold) is expressed as means ± SD (n = 3); means with different letters are significantly different at the level of p < 0.05. EC was used for comparison.
death with a study of mechanism is valuable to design more effective chemotherapy agents on tumour cells (Gordaliza, 2007). Nichenametla et al. (2006) indicated that fruits, vegetables, leaves, nuts, seeds, flowers, and barks with rich sources of phenolic compounds exhibiting antioxidant activity were reported to inhibit mutagenesis and carcinogenesis. Abnormal activation of proliferative pathways takes place in many types of cancer. Inhibition of apoptosis might play a role in the carcinogenic process (Kaufmann & Gores, 2000). Induction of apoptosis is considered as one of the important mechanisms for the targeted therapy of various cancers (Constantini, Jocotot, Decaudin, & Kroemer, 2000). Choedon, Shukla, and Kumar (2010) demonstrated that aqueous extract of flowers of Butea monosperma with high level of flavonoids exhibited a strong anti-cancer activity, including growth inhibition, cell cycle arrest and pro-apoptotic activity in Huh7 human hepatoma cells. Hsu et al. (2010) illustrated that longan (Dimocarpus Longan Lour.) flower contained notable amount of polyphenolics, including flavonoids and proanthocyanidins. Its aqueous extract could inhibit proliferation of Colo 320DM colorectal cancer cells; an apoptotic mechanism induced by the extract involving a loss of mitochondrial membrane potential and caspase-3 activation could be observed. Litchi belongs to Sapindaceae family as longan. To the best of our knowledge, there was no related report regarding the anti-cancer activity of litchi flower. In the study, we found that ethanolic extract of the flower (LFEE) containing rich flavonoids,
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phenolic acids and proanthocyanidins exhibited higher antiproliferative effect on HepG2 cells as compared to that on A549, KB and MCF-7 cells. LFEE also had the lower cytotoxicity for Clone 9 normal liver cell than that for HepG2 cell (Fig. 1). HepG2 cells treated with LFEE imposed growth arrest in G2 phase and triggered pro-apoptotic death (Fig. 3). Kaufmann and Gores (2000) indicated shrinkage and fragmentation of the cells and nuclei and degradation of the chromosomal DNA into nucleosomal units during apoptotic process. Internucleosomal DNA cleavage has been one of the main biochemical criteria used to distinguish apoptosis from necrosis. Several investigations have detected large DNA fragments of 50–200 kbp during apoptosis (Nagata, 2000). Our results showed that LFEE induced largescale DNA (50-200 kbp) degradation in HepG2 cell (Fig. 3). It illustrated that LFEE induced HepG2 cell death through apoptosis. Gupta (2001) indicated that induction of tumour cell apoptosis may be characterised by the cell shrinkage, chromatin condensation, DNA fragmentation and many biochemical characteristics, including the activation of death receptor pathway, mitochondrial pathway and/or caspases cascades and so on. Ashe and Berry (2003) indicated that caspases are critical for the execution of apoptosis, which are synthesised as procaspases and then proteolytically processed to their active forms. Caspase-3 interacted with caspase-8 and caspase-9 is the one ultimately responsible for the generality of the apoptotic effects. It is activated in the apoptotic cell both by extrinsic (death receptor) and intrinsic (mitochondrial) pathways. The death receptor pathway initially activates procaspase-8, whereas the mitochondrial pathway initially involves procaspase-9 (Kaufmann & Gores, 2000). In our investigation, activation of caspases-8 and -9 (initiator caspases) as well as caspase-3 (effector caspase) could be observed in HepG2 cells treated with LFEE (Fig. 4). Boucher, Gobeil, Samejima, Earnshaw, and Poirier (2001) reported that caspase-3 leads to downstream cleavage of various cytoplasmic or nuclear substrates, including PARP; cleavage of PARP is one of the hallmark events of apoptosis. LFEE treatment could also significantly induce PARP cleavage in HepG2 cells (Fig. 4). The results might illustrate that LFEE induced apoptosis in HepG2 cells through extrinsic and intrinsic pathways. Wei et al. (2000) revealed that Bid (an inactive precursor) localised in the cytosolic fraction of cells is a pro-apoptotic member of the Bcl-2 family. Bid is cleaved by caspase-8 to generate tBid, which translocates to the mitochondrial outer membrane and triggers cytochrome c release from the mitochondria, leading to complex formation with Apaf-1 and caspase-9 and resulting in the activation of caspase-9. Furthermore, tBid interacts with both the anti-apoptotic family member Bcl-2-Bcl-xL and the pro-apoptotic protein Bax. Our data show that LFEE treatment increased tBid production along with increase of Bax and Bad (pro-apoptotic proteins) and decrease of Bcl-2 and Bcl-xL (anti-apoptotic proteins) in HepG2 cells (Figs. 5 and 6). Moreover, induction of release of cytochrome c from mitochondria to cytosol could also be found clearly (Fig. 5). The results could further clarify that LFEE induced apoptosis in HepG2 cells through mitochondrial pathway. Mahfudh and Lope Pihie (2008) presented that p53, a tumour suppressor protein, was linked to apoptosis. Expression of p53 responds to various stresses, such as DNA damage.
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Journal of Functional Foods 19 (2015) 100–109
Death factor Receptor
Growth factor Receptor
Fas R
LFEE
DNA Damage
RTK
Genotoxic Stress FADD
PI3K AKT
Procaspase-8
p53 Bcl-2
Bcl-xL
Bid
Caspase-8
Bax Bad tBid Apaf-1
Cyt C
Procaspase-9 Cyt C Caspase-9 Procaspase-3 LFEE
Caspase-3
PARP
Cleaved PARP
Apoptosis
Fig. 7 – Possible role of LFEE in the induction of HepG2 cell apoptosis.
Up-regulation of p53 was followed by the increase of proapoptotic Bax and decrease of anti-apoptotic Bcl-2 (Amaral, Xavier, Steer, & Rodrigues, 2010). In HepG2 cells, a clear increase in p53 protein over the basal expression could be observed in our work (Fig. 5). Therefore, the increase of Bax/ Bcl-2 expression ratio via increasing p53 expression might result in LFEE-induced apoptosis in HepG2 cells. Bad, a pro-apoptotic protein, can couple death signals to mitochondria and promote apoptosis by suppressing the protective action of Bcl-xL. Nevertheless, phosphorylation of Bad has been demonstrated to disrupt its pro-apoptotic effect (Jin, Gao, Flagg, & Deng, 2004). Jeong et al. (2008) expressed that the PI3K and Akt signalling pathways play an important role in regulating cell cycle progression and cell survival. Zundel and Giaccia (1998) indicated that PI3K can act as an anti-apoptotic agent in cells. Akt, a major mediator of cell survival, can be activated by PI3K, which can suppress the induction of apoptosis through the phosphorylation of Bad. Akt is also linked to inhibition of p53 and cell survival. Our results clearly showed down-regulation of PIK by LFEE treatment and resulted in suppression of Akt activation and decrease of Bad phosphorylation in HepG2 cells (Fig. 6). Therefore, inhibition of the activation of PI3K-AktBad pathway should play an important role for LFEE-induced apoptosis in HepG2 cells.
To the best of our knowledge, no investigation regarding the anti-proliferative effect of litchi flower against the growth of cancer cells have been reported. Our results suggest that LFEE exhibited a notable effect for growth inhibition of HepG2 cells. Besides cell cycle arrest, LFEE could trigger pro-apoptotic death of HepG2 cells through activation of caspase cascade, modulation of mitochondrial pathways, up-regulation of p53 expression, and suppression of the activation of PI3K-Akt-Bad pathway. The results are summarised in Fig. 7. Consequently, the beneficial effects of LFEE could play an inspired role for exploiting novel therapeutics against liver cancer.
Acknowledgement This research was supported by the Ministry of Science and Technology, Taiwan (Project No. NSC 101-2313-B-040-009 -MY3).
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Induction of apoptotic death of human hepatocellular carcinoma (HepG2) cells by ethanolic extract from litchi (Litchi chinensis Sonn.) flower Jau-Tien Lin a,b, Yuan-Yen Chang b,c, Yi-Chen Chen d, Chao-Chin Hu a,b, Yu-Pei Chang e,f, Shih-Han Hsu e,f, Deng-Jye Yang e,f,* a
School of Medical Applied Chemistry, Chung Shan Medical University, 110, Jianguo N. Road, Sec. 1, Taichung 402, Taiwan Department of Medical Education, Chung Shan Medical University Hospital, 110, Jianguo N. Road, Sec. 1, Taichung 402, Taiwan c Department of Microbiology and Immunology, School of Medicine, Chung Shan Medical University, 110, Jianguo N. Road, Sec. 1, Taichung 402, Taiwan d Department of Animal Science and Technology, National Taiwan University, 1, Roosevelt Road, Sec. 4, Taipei, 106, Taiwan e School of Health Diet and Industry Management, Chung Shan Medical University, 110, Jianguo N. Road, Sec. 1, Taichung 402, Taiwan f Department of Nutrition, Chung Shan Medical University Hospital, 110, Jianguo N. Road, Sec. 1, Taichung 402, Taiwan b
A R T I C L E
I N F O
A B S T R A C T
Article history:
The cytotoxic activity of litchi (Litchi chinensis Sonn.) flower ethanolic extract (LFEE) against
Received 13 April 2015
A549 (human lung adenocarcinoma), KB (human nasopharyngeal carcinoma), MCF-7 (human
Received in revised form 19 August
breast adenocarcinoma) and HepG2 (human hepatocellular carcinoma) cells was evaluated.
2015
LFEE exhibited better anti-proliferative action against HepG2 cells, which induced G2/M phase
Accepted 24 August 2015
arrest and apoptosis in HepG2 cells. HepG2 cells treated with LFEE (0.3 mg/mL) enhanced
Available online
expressions of p53, tBid, Bad and Bax, along with decreased expressions of Bcl-2 and Bcl-xL, and induced the release of cytochrome c from mitochondria to cytosol. That also accom-
Keywords:
panied by activation of caspases-3, -8 and -9 and cleavage of poly (adenosine diphosphate
Litchi (Litchi chinensis Sonn.) flower
(ADP)-ribose) polymerase (PARP); furthermore, down-regulations of phosphoinositide 3 kinase
Apoptosis
(PI3K), Akt, Akt phosphorylation and Bad phosphorylation could also be observed. Five fla-
Hep G2 cell
vonoids (total amount, 101.07 mg/g dried extract (g DE)), nine phenolic acids (total amount,
Caspase
55.07 mg/ g DE) and proanthocyanidin A2 (81.20 mg/ g DE) could be determined in LFEE.
Bcl-2 family proteins
© 2015 Elsevier Ltd. All rights reserved.
PI3K/Akt
1.
Introduction
Litchi (Litchi chinensis Sonn.) originating in Southeast Asian (Rivera-López, Ordorica-Falomir, & Wesche-Ebeling, 1999) is an
important economic crop in Taiwan. The tree blooms in late March and fruit matures in late June. In addition to normal eating, the fruit is extensively used to produce many products, such as jelly, juice, vinegar and wine (Salunke & Desai, 1984). Notwithstanding litchi flower is generally considered an
* Corresponding author. School of Health Diet and Industry Management, Chung Shan Medical University, and Department of Nutrition, Chung Shan Medical University Hospital, 110, Jianguo N. Road, Sec. 1, Taichung City 402, Taiwan. Tel.: +886 4 24730022 ext. 11868; fax: +886 4 2324188. E-mail address: [email protected] (D.-J. Yang). http://dx.doi.org/10.1016/j.jff.2015.08.023 1756-4646/© 2015 Elsevier Ltd. All rights reserved.
Journal of Functional Foods 19 (2015) 100–109
agricultural by-product; the flower with favourable flavour is also collected to manufacture tea for drinking in Taiwan. Previous investigations had demonstrated that litchi flower containing remarkable amount of phenolic compounds had many significant bioactive attributes, such as antioxidant (Chen, Lin, Liu, Lu, & Yang, 2011; Liu, Lin, Wang, Chen, & Yang, 2009), anti-inflammation (Yang et al., 2014), cardiovascular protection (Yang et al., 2010), hepatoprotection (Hwang et al., 2013) and anti-obesity (Wu et al., 2013). Anti-carcinogenic activities have been demonstrated in many flower extracts. Hsu et al. (2010) indicated that longan flower aqueous extract containing rich amount of polyphenols could induce cell-cycle arrest and apoptosis in colorectal cancer cells. Moravcˇíková, Kuceková, Mlcˇek, Rop, and Humpolícˇek (2012) demonstrated that methanolic extracts of edible flowers, including Ag yoncha, Wild chive, Meadow salsify and Garden sorrel, had higher level of polyphenols, which should be responsible for inhibition of human hepatocellular carcinoma cell line (HepG2) proliferation. Polyphenols could arrest cancer cell cycle (Lin, Liang, & Lin-Shiau, 1999), induce cancer cell apoptosis (Nichenametla, Taruscio, Barney, & Exon, 2006), and interfere in cancer initiation, promotion and progression (Link, Balaguer, & Goel, 2010). However, the anti-cancer effect of litchi flower extract has not been explored yet. In the study, we evaluated the cytotoxic activity of litchi flower ethanolic extract (LFEE) against A549 (human lung adenocarcinoma), KB (human nasopharyngeal carcinoma), MCF-7 (human breast adenocarcinoma) and HepG2 (human hepatocellular carcinoma) cell lines first. Through the comparative experiments with Clone 9 cell line (an epithelial cell line isolated from normal liver taken from a young male rat) and two anti-cancer chemotherapy drugs (fluorouracil, 5-FU and methotrexate, MTX), the better LFEE concentration was exploded to investigate the pathway of LFEE-induced apoptotic death in HepG2 cells.
2-mercaptoethanol, potassium chloride, monobasic potassium phosphate, Tween 20 and Tris base were obtained from Sigma Co. (St. Louis, MO, USA). Minimum Essential Medium (MEM), Dulbecco’s Modified Eagle’s Medium (DMEM), RPMI 1640 Medium, Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12), foetal bovine serum (FBS), penicillinstreptomycin (PS), penicillin-streptomycin-amphotericin (PSA) and lyophilised trypsin-EDTA were purchased from Gibco/ Invitrogen Co. (Carlsbad, CA, USA). Sodium dodecyl sulfate (SDS) was purchased from Merck (Darmstadt, Germany). Antibody against α-tubulin and the monoclonal antibody against cytochrome c, PARP, PI3K and caspases-3, and -8, were purchased from GeneTex, Inc. (Alton Pkwy., Irvine, CA, USA). Antibody against Bid, Bax, Bad, phosphorylated Bad (phospho-Bad (Ser136)), Bcl-xL, Bcl-2, α-tubulin, Akt and phosphorylated Akt (phospho-Akt) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibody against caspase-9, and Bid were purchased from Cell Signaling Technology, Inc (Danvers, MA, USA). Trypan blue was purchased from (MP Biomedicals, Santa Ana, CA, USA). The horseradish peroxidaseconjugated anti-goat or anti-rabbit IgG were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). Ammonium persulphate (APS), N, N′-methylenebisacrylamide (acrylamide), bromophenol blue, N, N, N′, N′-tetramethylethylenediamine (TEMED) and Triton X-100 were purchased from (Amresco Inc., Solon, OH, USA).
2.2.
Materials and methods
2.1.
Chemicals and reagents
Distilled deionised water (dd H 2 O) was prepared by an UltrapureTM water purification system (Lotun Co., Ltd., Taipei, Taiwan). Acetonitrile, methanol, ethanol, phosphoric acid and acetic acid were purchased from Merck Co. (Darmstadt, Germany). Standards of flavonoids including apigenin, catechin, daidzein, diosmin, epicatechin, eriodictyol, genistein, hesperetin, hesperidin, isorhamnetin, kamempferol, luteolin, morin, myricetin, narigenin, naringin, neohesperidin, quercetin, quercitrin and rutin, and phenolic acids including p-anisic, caffeic, chlorogenic, p-coumaric, ferulic, gallic, gentisic, p-hydroxybenzoic, rosmarinic, sinapic, syringic and vanillic acids were purchased from Sigma Co. (St. Louis, MO, USA). Proanthocyanidin A2 was isolated according to our previous study (Yang et al., 2014). Sulphanilamide, N-(1-Naphthyl) ethylenediamine dihydrochloride, phosphate-buffered saline (PBS), bovine serum albumin (BSA), ethylenediaminetetraacetic acid disodium salt dihydrate (Na 2 EDTA), glycine, sodium fluoride, sodium pyrophosphate, phenylmethanesulfonyl fluoride, propidium iodide, di-sodium hydrogen phosphate,
Preparation of LFEE
Fresh litchi flowers gathered in March 2014 from a farm in Taichung, Taiwan were lyophilised and then extracted with 95% ethanol (1/20, g/mL) in the dark for 24 h. The filtrate was evaporated to dryness in a rotary vacuum evaporator (Panchun Scientific Co., Kaohsiung, Taiwan). LFEE in an airtight bottle was stored at −80 °C prior to experiment.
2.3.
2.
101
Cell line
Cell lines for MCF-7 (ATCC No. HTB-22) maintained in MEM medium with 10% heat-inactivated FBS and 1% PSA, Hep G2 (ATCC No. HB-8065) maintained in MEM medium with 10% heatinactivated FBS and 1% PS, A549 (ATCC No. CCL-185) maintained in DMEM with 10% heat-inactivated FBS and 1% PS, KB (ATCC No. CCL-17) maintained in RPMI-1640 medium with 10% heat-inactivated FBS and 1% PSA, and Clone-9 (ATCC No. CRL1439) maintained in DMEM/F-12 medium with 10% heatinactivated FBS and 1% PSA obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). These cell lines were incubated in a Thermo Scientific Forma Direct Heat CO2 Incubator (Thermo Fisher Scientific Inc., Mississauga, ON, Canada) (37 °C, 5% CO2).
2.4.
Cell viability assay
WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4disulphophenyl)-2H-tetrazolium, monosodium salt) in Cell Counting Kit-8 (CCK-8) could be reduced by dehydrogenases in the cell and produced a tissue culture medium soluble orange coloured formazan. It was used to assay the cell viability. The generated formazan level is proportional to the number of living
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Journal of Functional Foods 19 (2015) 100–109
cells directly (Ishiyama, Miyazono, Sasamoto, Ohkura, & Ueno, 1997). Each cell line seeded in each well of 24-well plates at a density of 2 × 105 cells/well was cultured in medium with various concentrations (0, 0.1, 0.2, 0.3, 0.4 and 0.5 mg/mL) of samples dissolved in ethanol (ethanol in each well was 0.0475% v/v) at 37 °C for 24 h in the CO2 incubator. The CCK-8 solution (10 µL) was then added into each well and incubated at 37 °C for 2 hr. The absorbance at 450/595 nm was measured with a microplate reader (Multiskan Spectrum, Thermo Co., Vantaa, Finland). The absorption for the control cells was regarded as 100% cell viability.
2.5.
Cell cycle analysis
Cells seeded in 10 cm dishes (1 × 106 cells/mL) were cultured in medium without or with 0.3 mg/mL of LFEE dissolved in ethanol at 37 °C for 12 or 24 h in the CO2 incubator. Ethanol concentration was 0.0475% (v/v). The cells were then fixed by 80% ethanol overnight at 4 °C. After washing with PBS, the cells were mixed with 0.1 mg/mL RNase, and stained with propidium iodide (40 µg/mL). The stained cells were analysed by a FACS Calibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Data collection and treatment were performed through the CellQuest and ModFit LT software (Becton Dickinson).
2.6.
DNA fragmentation analysis
HepG2 cells seeded in 10 cm dishes (1 × 106 cells/mL) were cultured in medium without or with 0.3 mg/mL of LFEE dissolved in ethanol at 37 °C for 24 h in the CO2 incubator. Ethanol concentration was 0.0475% (v/v). Cells were washed twice with phosphate-buffered saline (PBS) (pH 7.4), and DNA fragmentation analysis was carried out with DNA Laddering Kit (Trevigen, Inc., Gaithersburg, MA, USA) according to the manufacturer’s instructions. The eluants containing DNA pellets were electrophoresed (1.5 h at 80 V/30 mA) on a 1% agarose gel containing 0.5 mg/mL ethidium bromide. The gel was detected and photographed by ultraviolet gel documentation system.
2.7.
Immunoblotting analysis
Cells were lysed and protein extraction was carried out. The protein level was determined by a protein assay kit (Bio-Rad, Laboratories, Inc., Hercules, CA, USA). Cell proteins were separated in 12% of SDS-polyacrylamide gel, which was done at 110 V for 80 min with a standard running buffer (24 mM TrisHCl, 190 mM glycine, 0.5% SDS, pH8.3) and electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane at 200 mA for 90 min in transfer buffer (20 mM Tris-HCl, 150 mM glycine, 10% methanol, and 0.01% SDS). The membranes were then blocked with 5% non-fat milk, washed, and subsequently incubated with a specific antibody. After washing, the membrane was incubated with horseradish peroxidaseconjugated goat anti-mouse antibody or goat anti-rabbit antibody (Bio-Rad, Hercules, CA, USA). The protein bands were visualised with an enhanced chemiluminescence (ECL) system (Amersham Biosciences, Piscataway, NJ, USA). The protein band densities were quantified through the Alpha Imager 2200 software (Alpha Innotech Co., San Leandro, CA, USA). The amount of α-tubulin (57 kDa) in each lane was also detected as a control.
2.8.
Determination of phytochemicals in LFEE
A PrimeLine™ Gradient Model 500G HPLC pump system (Analytical Scientific Instruments, Inc., El Sobrante, CA, USA), a S-3210 photodiode-array (PDA) detector (Schambeck SFD GmbH, Bad Honnef, Germany), and a Thermo Finnigan LCQ classic quadrupole ion trap HPLC-mass (MS) system (Gentech, Arcade, NY, USA) equipped with electrospray ionisation (ESI) source (positive ion mode) were used for sample analysis. The HPLC-MS (conditions: nebuliser pressure, 70 psi; capillary temperature, 200 °C; dry gas flow, 11 L/min; electrospray voltage of the ion source, 3000 V; capillary exit, -159 V; skimmer, 40 V) was used to get mass spectra recording from m/z 50 to 1000. Flavonoids and phenolic acids in LFEE dissolved in methanol (0.25 mg/mL) were determined by high performance liquid chromatography (HPLC) according to the conditions of Chen et al. (2011): stationary phase, Inspire C18 column (250 × 4.6 mm, 5 µm; Dikma Technologies Inc., Lake Forest, CA, USA); mobile phase, methanol (solvent A) and 9% acetic acid solution prepared with dd H2O (solvent B) (conditions: 5–17% A from 0 to 5 min and kept at 17% A from 5 to 25 min; 17–31% A from 25 to 40 min and kept at 31% A from 40 to 76 min; 31–40 % A from 76 to 80 min and kept at 40% A from 80 to 120 min); flow rate, 0.8 mL/min; detection, 210–650 nm. Proanthocyanidins were analysed based on the report of Hwang et al. (2013): stationary phase, Inspire C18 column (250 mm × 4.6 mm i.d., 5 µm); mobile phase, acetonitrile (solvent A) and 2% acetic acid solution prepared with dd H2O with (solvent B) (conditions:10– 20% A from 0 to 10 min; 20–30% A from 10 to 13 min; 30–100 % A from 10 to 13 min; 30–100 % A from 13 to 15 min and kept at 100% A from 15 to 20 min); flow rate, 1 mL/min.
2.9.
Statistical analysis
All assays were performed in triplicate and the mean values were calculated. The data were subjected to analysis of variance (ANOVA), and Duncan’s multiple range tests were used to assess differences between means. Significant differences were concluded at a level of p < 0.05.
3.
Results
3.1. Flavonoids, phenolic acids and proanthocyanidin A2 in LFEE Through HPLC analysis, we can see that LFEE had five flavonoids, including epicatechin (79.23 ± 3.62 mg/ g of dried extract (mg/g DE)), rutin (17.25 ± 1.12 mg/g DE), naringin (0.52 ± 0.03 mg/g DE), quercitrin (2.06 ± 0.09 mg/gDE) and neohesperidin (2.01 ± 0.17 mg/gDE); the total amount of these flavonoids was 101.07 ± 5.03 mg/g DE). Moreover, nine phenolic acids, including gallic acid (0.21 ± 0.01 mg/g DE), gentisic acid (30.26 ± 1.98 mg/g DE), chlorogenic acid (2.65 ± 0.11 mg/g DE), vanillic acid (0.47 ± 0.02 mg/g DE), p-coumaric acid (0.19 ± 0.01 mg/g DE), ferulic acid (15.12 ± 1.07 mg/g DE), sinapic acid (4.20 ± 0.26 mg/g DE), syringic acid (0.75 ± 0.03 mg/g DE) and p-anisic acid (1.22 ± 0.03 mg/g DE) could also be found in LFEE, resulting in 55.07 ± 3.52 mg/g DE for the total amount of phenolic acids.
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Proanthocyanidin A2, the major proanthocyanidin in LFEE, was 81.20 ± 3.17 mg/g DE. LFEE solution was prepared by ethanol to estimate its cytotoxicity against cancer cells.
3.2.
Cytotoxicity of LFEE against cancer cells
Through cytotoxicity assays for LFEE against A549, KB, MCF-7 and HepG2 cells, LFEE might be more effective against HepG2
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cell (IC50 = 0.33 ± 0.02 mg/mL) than other types of cancer cells. The higher the LFEE level was used, the lower the HepG2 cell viability was observed (Fig. 1). In order to know the effect of LFEE on the growth of normal liver cell, Clone 9 cell isolated from normal rat liver was used for comparison. Though the viability of Clone 9 cell treated with LFEE was also decreased in a concentration-dependent manner, the cytotoxicity of LFEE to Clone 9 cell (IC50 = 0.45 ± 0.02 mg/mL) was relatively lower
Fig. 1 – Effect of LFEE on viability of A549, MCF-7, KB, Hep G2 and Clone 9 cells. The cells were incubated with various concentrations (0.1, 0.2, 0.3, 0.4, and 0.5 mg/mL) of LFEE dissolved in ethanol for 24 h. Ethanol concentration in media was 0.0475% (v/v) except mock. Cell survival (% control) was measured by CCK-8 assay. Values are presented as mean ± SD of six independent experiments. Values with different letters are significantly different at the level of p < 0.05. C: control (without LFEE); M: mock (without LFEE and ethanol).
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Fig. 2 – Effect of anti-cancer drugs on viability of Hep G2 and Clone 9 cells. The cells were incubated with various concentrations (0.1, 0.2, 0.3 and 0.4 mg/mL) of fluorouracil (5-FU) or methotrexate (MTX) dissolved in ethanol for 24 h. Ethanol concentration in media was 0.0475% (v/v) except mock. Cell survival (% control) was measured by CCK-8 assay. Values are presented as mean ± SD of six independent experiments. Values with different letters (capital for Clone 9; lowercase for Hep G2) are significantly different at the level of p < 0.05. C: control (without drug); M: mock (without drug and ethanol).
than that to HepG2 cell (Fig. 1). Fig. 2 shows that fluorouracil (5-FU) and methotrexate (MTX) could suppress not only HepG2 cell growth but also Clone 9 cell growth. LFEE and the two anticancer chemotherapy drugs seemed to show the similar antiproliferative effects on HepG2 and Clone 9 cells at 0.3 mg/mL (Figs. 1 and 2). Therefore, this concentration was used to evaluate whether the cytotoxic effect of LFEE on HepG2 cells was due to apoptotic death. Because epicatechin is a major flavonoid in LFEE and has been demonstrated to induce apoptosis in human colon cancer cell (Kim, Mollah, & Kim, 2012), the compound was used for comparison.
3.3.
LFEE induced apoptosis in HepG2 cells
The cell cycle distribution of HepG2 cells treated with 0.3 mg/mL of LFEE was determined by flow cytometry at 12 and 24 h. Fig. 3A shows that Sub-G1 phase level for HepG2 cells clearly increased as compared with control (HepG2 cells without LFEE). It suggested that LFEE could cause DNA fragmentation of the cells. Fig. 3A also indicates a remarkable HepG2 cell population in G2/M phase following 12 h treatment with LFEE. It illustrated that HepG2 cell cycle was arrested in G2/M phase; the cells entering into next cycle and executing division were
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Fig. 3 – Effects of LFEE on cell cycle progression (A) and DNA fragmentation (B) in HepG2 cells. For cell cycle analysis, HepG2 cells were treated with or without (control) 0.3 mg/mL of LFEE for 12 and 24 h, respectively. The cells were then harvested and fixed by 70% alcohol. The cell cycle progression was analysed by flow cytometry after the cells were stained with propidium iodide. For DNA fragmentation, HepG2 cells treated with or without (control, C) 0.3 mg/mL of LFEE or epicatechin (EC) for 24 h were assayed with DNA Laddering Kit and electrophoresed on a 1% agarose gel. EC was used for comparison. M: marker.
moderated. However, the levels of G2/M phase and Sub-G1 phase for the cells incubated with LFEE for 24 h were greatly decreased and increased, respectively.The results might attribute to substantial cell apoptosis. Agarose gel electrophoresis showed that HepG2 cells incubated with LFEE for 24 h also induced internucleosomal DNA fragmentation (Fig. 3B), which is a biochemical hallmark of apoptosis (Nagata, 2000). Epicatechin could also induce internucleosomal DNA fragmentation in HepG2 cells.
3.4. LFEE activated caspase cascade in HepG2 cells for apoptosis HepG2 cells incubated with 0.3 mg/mL of LFEE for 24 h and caspase cascade were investigated by Western blotting. Results showed that procaspase-8 was decreased remarkably, whereas its cleaved form (active caspase-8) was significantly increased (Fig. 4). In addition, LFEE or epicatechin treatment led to procaspase-9 cleavage; its cleaved form (active caspase-9)
was significantly increased as well (Fig. 4). Similar results are also shown in Fig. 4 for procaspase-3 and its cleaved form (active caspase-3). Because PARP is one of the downstream substrates of activated caspase-3 (O’Brien, Moravec, & Riss, 2001), the degraded product of PARP (89 kDa) upon LFEE or epicatechin treatment was examined in the investigation. Fig. 4 shows that the cleavage of PARP activated by caspase-3 was significantly increased. These results implied that LFEE could stimulate the activation of caspases-8, -9, and -3 and cleavage of PARP to induce apoptosis in HepG2 cells. HepG2 cells treated with epicatechin also showed similar results.
3.5. LFEE modulated mitochondrial pathways and up-regulated p53 expression in HepG2 cells for apoptosis Fig. 5 shows that Bid expression was significantly decreased after HepG2 cells treated with 0.3 mg/mL of LFEE for 24 h, whereas its cleaved form (truncated Bid, tBid) expression was
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C
LFEE
EC
C Pro-Casepase 3 (35 kDa)
Cleaved (active) form (17 kDa) Fold
c
1
a
Fold
1
a
LFEE
EC b
0.3±0.01 0.5±0.02b Bid (22 kDa) Cleaved form (tBid, 15 kDa)
Fold
1c
4.2±0.3a 3.2±0.2b
Fold
1a
0.2±0.01c 0.5±0.03b
b
3.4±0.4 2.3±0.3
Pro-Casepase 8 (55 kDa)
Cleaved (active) form (18 kDa) Fold
c
1
a
3.1±0.2 1.8±0.1
Bcl-2 (29 kDa) Fold
1c
0.2±0.01a 0.4±0.01b
b
Bcl-xL (32 kDa)
Pro-Casepase 9 (47 kDa)
Fold
1c
3.1±0.3a 2.6±0.2b
Cleaved (active) form (37 kDa) Fold
c
1
a
Bax (23 kDa)
b
3.6±0.3 2.7±0.2
Fold
1a
0.3±0.02a 0.4±0.03b Mitochondrial Cytochrome c (15 kDa)
PARP (116 kDa) Cleaved form (89 kDa) Fold
1c
2.7±0.2a 2.2±0.2b
Fold
1c
3.1±0.2a 2.5±0.2b Cytosolic Cytochrome c (15 kDa)
a-Tubulin (57 kDa)
Fig. 4 – Expressions of caspases-3, -8 and -9 and PARP in LFEE-treated HepG2 cells. HepG2 cells were treated with or without (control, C) 0.3 mg/mL of LFEE or epicatechin (EC) for 24 h. Protein extracts were prepared for Western blot assay using indicated antibodies, and α-tubulin was used for loading control. Each value (fold) is expressed as means ± SD (n = 3); means with different letters are significantly different at the level of p < 0.05. EC was used for comparison.
significantly increased. It indicated that LFEE treatment resulted in the cleavage of cytosolic Bid to tBid, which promotes apoptosis at the mitochondria (Lutter, Perkins, & Wang, 2001). Moreover, the expressions of anti-apoptotic proteins Bcl-2 and BcL-xL were apparently decreased, whereas the expression of pro-apoptotic protein Bax was significantly increased. In addition, the levels of mitochondrial and cytosolic cytochrome c were decreased and increased, respectively (Fig. 5). It indicated the release of cytochrome c from mitochondria to the cytosol, which caused the activation of caspase family of protease and triggered the onset of apoptosis (Liu, Kim, Yang, Jemmerson, & Wang, 1996). Because p53 plays a role in apoptosis (Mahfudh & Lope Pihie, 2008), the effect of LFEE on p53 expression was also estimated. LFEE treatment significantly enhanced p53 expression in HepG2 cells (Fig. 5). Our study demonstrated that LFEE induced apoptosis in HepG2 cells through mitochondrial pathway and up-regulation of p53 expression. Similar results also showed in HepG2 cells treated with epicatechin.
Fold
1c
2.8±0.2b 3.2±0.2a p53 (53 kDa) α-Tubulin (57 kDa)
Fig. 5 – Expressions of Bid, Bcl-2, Bcl-xL, Bax, cytochrome c and P53 in LFEE-treated HepG2 cells. HepG2 cells were treated with or without (control, C) 0.3 mg/mL of LFEE or epicatechin (EC) for 24 h. Protein extracts were prepared for Western blot assay using indicated antibodies, and α-tubulin was used for loading control. Each value (fold) is expressed as means ± SD (n = 3); means with different letters are significantly different at the level of p < 0.05. EC was used for comparison.
Results showed that PI3K protein expression in HepG2 cells treated with 0.3 mg/mL of LFEE for 24 h was reduced significantly (Fig. 6). Furthermore, the levels of Akt protein as well as phospho-Akt protein were lowered significantly (Fig. 6). Bad is one of the pro-apoptotic proteins, whereas phosphorylation of Bad is anti-apoptotic. Akt activation enhances Bad phosphorylation that enhances survival of cancer cells (Zundel & Giaccia, 1998). In the study, Bad expression was significantly enhanced, whereas Bad phosphorylation was significantly down-regulated in HepG2 cells treated with LFEE, respectively (Fig. 6). These results illustrated that LFEE induced apoptosis in HepG2 cells through the suppression of the activation of PI3K-Akt-Bad pathway. HepG2 cells treated with epicatechin also showed similar results.
3.6. LFEE down-regulated PI3K expression, Akt activation and Bad phosphorylation in HepG2 cells for apoptosis
4.
This study further examined whether LFEE would influence the expressions of PI3K, Akt and phospho-Akt in HepG2 cells.
Natural products containing rich sources of compounds can be used for anticancer drug discovery; their induction of cell
Discussion
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C Fold
LFEE
1c
EC
0.7±0.04a 0.8±0.03b PI3K (23 kDa)
Fold
1a
0.3±0.02c 0.5±0.02b Phospho-Akt (23 kDa)
Fold
1a
0.6±0.05b 0.4±0.02c Akt (23 kDa)
Fold
1c
1.7±0.1a 1.4±0.1b Bad (23 kDa)
Fold
1a
0.3±0.02a 0.5±0.03b Phospho-Bad (ser 136) (23 kDa) a-Tubulin (57 kDa)
Fig. 6 – Expression of PI3K, Akt, phospho-Akt, Bad and phospho-Bad in LFEE-treated HepG2 cells. HepG2 cells were treated with or without (control, C) 0.3 mg/mL of LFEE or epicatechin (EC) for 24 h. Protein extracts were prepared for Western blot assay using indicated antibodies, and α-tubulin was used for loading control. Each value (fold) is expressed as means ± SD (n = 3); means with different letters are significantly different at the level of p < 0.05. EC was used for comparison.
death with a study of mechanism is valuable to design more effective chemotherapy agents on tumour cells (Gordaliza, 2007). Nichenametla et al. (2006) indicated that fruits, vegetables, leaves, nuts, seeds, flowers, and barks with rich sources of phenolic compounds exhibiting antioxidant activity were reported to inhibit mutagenesis and carcinogenesis. Abnormal activation of proliferative pathways takes place in many types of cancer. Inhibition of apoptosis might play a role in the carcinogenic process (Kaufmann & Gores, 2000). Induction of apoptosis is considered as one of the important mechanisms for the targeted therapy of various cancers (Constantini, Jocotot, Decaudin, & Kroemer, 2000). Choedon, Shukla, and Kumar (2010) demonstrated that aqueous extract of flowers of Butea monosperma with high level of flavonoids exhibited a strong anti-cancer activity, including growth inhibition, cell cycle arrest and pro-apoptotic activity in Huh7 human hepatoma cells. Hsu et al. (2010) illustrated that longan (Dimocarpus Longan Lour.) flower contained notable amount of polyphenolics, including flavonoids and proanthocyanidins. Its aqueous extract could inhibit proliferation of Colo 320DM colorectal cancer cells; an apoptotic mechanism induced by the extract involving a loss of mitochondrial membrane potential and caspase-3 activation could be observed. Litchi belongs to Sapindaceae family as longan. To the best of our knowledge, there was no related report regarding the anti-cancer activity of litchi flower. In the study, we found that ethanolic extract of the flower (LFEE) containing rich flavonoids,
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phenolic acids and proanthocyanidins exhibited higher antiproliferative effect on HepG2 cells as compared to that on A549, KB and MCF-7 cells. LFEE also had the lower cytotoxicity for Clone 9 normal liver cell than that for HepG2 cell (Fig. 1). HepG2 cells treated with LFEE imposed growth arrest in G2 phase and triggered pro-apoptotic death (Fig. 3). Kaufmann and Gores (2000) indicated shrinkage and fragmentation of the cells and nuclei and degradation of the chromosomal DNA into nucleosomal units during apoptotic process. Internucleosomal DNA cleavage has been one of the main biochemical criteria used to distinguish apoptosis from necrosis. Several investigations have detected large DNA fragments of 50–200 kbp during apoptosis (Nagata, 2000). Our results showed that LFEE induced largescale DNA (50-200 kbp) degradation in HepG2 cell (Fig. 3). It illustrated that LFEE induced HepG2 cell death through apoptosis. Gupta (2001) indicated that induction of tumour cell apoptosis may be characterised by the cell shrinkage, chromatin condensation, DNA fragmentation and many biochemical characteristics, including the activation of death receptor pathway, mitochondrial pathway and/or caspases cascades and so on. Ashe and Berry (2003) indicated that caspases are critical for the execution of apoptosis, which are synthesised as procaspases and then proteolytically processed to their active forms. Caspase-3 interacted with caspase-8 and caspase-9 is the one ultimately responsible for the generality of the apoptotic effects. It is activated in the apoptotic cell both by extrinsic (death receptor) and intrinsic (mitochondrial) pathways. The death receptor pathway initially activates procaspase-8, whereas the mitochondrial pathway initially involves procaspase-9 (Kaufmann & Gores, 2000). In our investigation, activation of caspases-8 and -9 (initiator caspases) as well as caspase-3 (effector caspase) could be observed in HepG2 cells treated with LFEE (Fig. 4). Boucher, Gobeil, Samejima, Earnshaw, and Poirier (2001) reported that caspase-3 leads to downstream cleavage of various cytoplasmic or nuclear substrates, including PARP; cleavage of PARP is one of the hallmark events of apoptosis. LFEE treatment could also significantly induce PARP cleavage in HepG2 cells (Fig. 4). The results might illustrate that LFEE induced apoptosis in HepG2 cells through extrinsic and intrinsic pathways. Wei et al. (2000) revealed that Bid (an inactive precursor) localised in the cytosolic fraction of cells is a pro-apoptotic member of the Bcl-2 family. Bid is cleaved by caspase-8 to generate tBid, which translocates to the mitochondrial outer membrane and triggers cytochrome c release from the mitochondria, leading to complex formation with Apaf-1 and caspase-9 and resulting in the activation of caspase-9. Furthermore, tBid interacts with both the anti-apoptotic family member Bcl-2-Bcl-xL and the pro-apoptotic protein Bax. Our data show that LFEE treatment increased tBid production along with increase of Bax and Bad (pro-apoptotic proteins) and decrease of Bcl-2 and Bcl-xL (anti-apoptotic proteins) in HepG2 cells (Figs. 5 and 6). Moreover, induction of release of cytochrome c from mitochondria to cytosol could also be found clearly (Fig. 5). The results could further clarify that LFEE induced apoptosis in HepG2 cells through mitochondrial pathway. Mahfudh and Lope Pihie (2008) presented that p53, a tumour suppressor protein, was linked to apoptosis. Expression of p53 responds to various stresses, such as DNA damage.
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Death factor Receptor
Growth factor Receptor
Fas R
LFEE
DNA Damage
RTK
Genotoxic Stress FADD
PI3K AKT
Procaspase-8
p53 Bcl-2
Bcl-xL
Bid
Caspase-8
Bax Bad tBid Apaf-1
Cyt C
Procaspase-9 Cyt C Caspase-9 Procaspase-3 LFEE
Caspase-3
PARP
Cleaved PARP
Apoptosis
Fig. 7 – Possible role of LFEE in the induction of HepG2 cell apoptosis.
Up-regulation of p53 was followed by the increase of proapoptotic Bax and decrease of anti-apoptotic Bcl-2 (Amaral, Xavier, Steer, & Rodrigues, 2010). In HepG2 cells, a clear increase in p53 protein over the basal expression could be observed in our work (Fig. 5). Therefore, the increase of Bax/ Bcl-2 expression ratio via increasing p53 expression might result in LFEE-induced apoptosis in HepG2 cells. Bad, a pro-apoptotic protein, can couple death signals to mitochondria and promote apoptosis by suppressing the protective action of Bcl-xL. Nevertheless, phosphorylation of Bad has been demonstrated to disrupt its pro-apoptotic effect (Jin, Gao, Flagg, & Deng, 2004). Jeong et al. (2008) expressed that the PI3K and Akt signalling pathways play an important role in regulating cell cycle progression and cell survival. Zundel and Giaccia (1998) indicated that PI3K can act as an anti-apoptotic agent in cells. Akt, a major mediator of cell survival, can be activated by PI3K, which can suppress the induction of apoptosis through the phosphorylation of Bad. Akt is also linked to inhibition of p53 and cell survival. Our results clearly showed down-regulation of PIK by LFEE treatment and resulted in suppression of Akt activation and decrease of Bad phosphorylation in HepG2 cells (Fig. 6). Therefore, inhibition of the activation of PI3K-AktBad pathway should play an important role for LFEE-induced apoptosis in HepG2 cells.
To the best of our knowledge, no investigation regarding the anti-proliferative effect of litchi flower against the growth of cancer cells have been reported. Our results suggest that LFEE exhibited a notable effect for growth inhibition of HepG2 cells. Besides cell cycle arrest, LFEE could trigger pro-apoptotic death of HepG2 cells through activation of caspase cascade, modulation of mitochondrial pathways, up-regulation of p53 expression, and suppression of the activation of PI3K-Akt-Bad pathway. The results are summarised in Fig. 7. Consequently, the beneficial effects of LFEE could play an inspired role for exploiting novel therapeutics against liver cancer.
Acknowledgement This research was supported by the Ministry of Science and Technology, Taiwan (Project No. NSC 101-2313-B-040-009 -MY3).
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