Anti-invasion and apoptosis induction of chlorella (Chlorella sorokiniana) in Hep G2 human hepatocellular carcinoma cells

Anti-invasion and apoptosis induction of chlorella (Chlorella

sorokiniana) in Hep G2 human hepatocellular carcinoma cells

Jing-Gung Chung

, Hsin-Yi Peng

, Yu-Chan Chu

, You-Miin Hsieh

, San-Der Wang

,

Su-Tze Chou

a_{Department of Biological Science and Technology, China Medical University, No. 91 Hsueh-Shih Road, Taichung 404, Taiwan, ROC} b_{Department of Food and Nutrition, Providence University, No. 200 Chung-Chi Road, Shalu, Taichung 433, Taiwan, ROC}

c_{Division of Gastroenterology, Department of Medicine, Taipei Veterans General Hospital, No. 201, Sec. 2, Shipai Road, Beitou District,}

Taipei 112, Taiwan, ROC

A R T I C L E I N F O Article history:

Received 22 September 2011 Received in revised form 9 December 2011

Accepted 20 December 2011 Available online 16 January 2012 Keywords:

Chlorella Apoptosis Invasion

Human hepatocellular carcinoma cells

Hep G2 cells

A B S T R A C T

The effects of 80% ethanolic extract derived from commercial granule chlorella (GPE) on cell viability, invasion capacity and apoptosis in human hepatoma cell line (Hep G2 cells) were investigated. The results demonstrated that GPE decreased cell viability, induced apoptosis and showed invasion inhibitory effects in the Hep G2 cells. GPE-triggered apoptosis was confirmed by 40_{-6-diamidino-2-phenyindole (DAPI) staining and comet assay. GPE}

pro-moted an increase of reactive oxygen species (ROS) and Ca2+_{, and loss of mitochondrial}

membrane potential (DWm) accompanied by cytochrome c release that was due to the decrease of Bcl-2 in the Hep G2 cells. GPE also induced the protein levels of apoptosis-inducing factor (AIF), increased the levels of caspase-3, -8 and -9, and stimulated the levels of fatty acid synthase (Fas) and Fas ligand (FasL) in the Hep G2 cells. Additionally GPE inhib-ited invasion of Hep G2 cells by down-regulation of the expression of matrix metallopro-teinase (MMP)-2 and -9. Furthermore, cellular glutathione content and superoxide dismutases (SOD) activities were significantly reduced and thiobarbituric acid-reactive sub-stances (TBARS) levels were significantly increased after GPE treatment. These results sug-gest that GPE can induce cytotoxicity on Hep G2 cells and inhibit the invasive capacity of malignant cells.

2011 Elsevier Ltd. All rights reserved.

1.

Introduction

Hepatocellular carcinoma is one of the most common cancers in the world and it is a multifactorial disease, caused by smoking, alcohol, toxins and the human hepatitis virus. Inducing cell apoptosis is an important strategy for killing cancer cells. Apoptosis is a programmed cell death that leads to elimination of unwanted, damaged or infected cells. Cells undergo apoptosis through distinct pathways including fatty acid synthase (Fas) and Fas ligand (FasL) which results in

the activation of the caspase-8, mitochondria-dependent pathway and the caspase-3-dependent pathway triggering the cytoplasmic release of apoptotic mitochondrial pro-teins before leading to apoptosis (Eva, Una, & Afshin, 2003). Chlorella, unicellular green algae, has been found to contain highly nutritious substances with various biological effects. Glycoproteins derived from Chlorella vulgaris exhibited antitu-mour (Kuniaki et al., 1998) and immunomodulatory effects (Takashi et al., 2000). (Sulaiman, Shamaan, Ngah, & Yusof, 2006) showed that C. vulgaris has a chemopreventive effect

1756-4646/$ - see front matter  2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jff.2011.12.008

* Corresponding author: Tel.: +886 4 2632 8001; fax: +886 4 2653 0027. E-mail address:[email protected](S.-T. Chou).

A v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m

on liver cancer induced by ethionine in rats. Several reports supported that the bioactive component polysaccharides from Chlorella pyrenoidosa were responsible for antitumor po-tential and immunomodulatory activities (Jianchun et al., 2007; Yang, Shi, Sheng, & Hu, 2006). Furthermore, chlorella contains various pigments such as carotenoids and chloro-phylls which have been attributed to the biological properties including antioxidative, antilipidemic, antiatherosclerotic and antitumour activities (Cha et al., 2010). Researches have demonstrated that the bioactive compounds and biological activities of chlorella depend on the extraction solvents and extraction techniques used (Cha et al., 2010; Plaza et al., in press). Plaza et al. (in press) pointed out that ethanol as the most appropriate solvent to extract compounds with biological activities from chlorella not only for the higher yields and chemical composition obtained but also for the GRAS (generally recognized as safe) consideration. A recent study has shown that water extract of chlorella inhibits growth of human hepatoma Hep G2 cells (Wu, Ho, Shieh, & Lu, 2005), information on its antitumor properties and cellular mechanisms remain limited. Because it was ob-served in our pre-study that 80% ethanolic extract (GPE) showed a higher growth inhibitory effect on Hep G2 cells than aqueous or 50% ethanolic extract derived from com-mercial granule chlorella. Therefore, in this study, we inves-tigated the effects of GPE on cell viability, invasion capacity and apoptosis in human hepatoma cell line (Hep G2 cells). The pigments, b-carotene, chlorophyll a, chlorophyll b, and lutein will also be determined by using HPLC equipped with a photodiode array detector.

2.

Materials and methods

2.1. Preparation of 80% ethanolic extract of chlorella (GPE) Dried, powdered chlorella (Chlorella sorokiniana) was pur-chased from Taiwan Chlorella Manufacturing Co. Ltd. (Taipei, Taiwan, ROC). Chlorella was extracted with 80% ethanol (50-fold) by stirring for 2 h. The decoction was filtered, evaporated and dried by a vacuum freeze-dryer. The yield of the dried GPE was 14.0%. The extract was sealed in plastic bottles and stored at 70 C. For the present experiments, GPE was dis-solved in DMSO before adding it to cell cultures.

2.2. Chemical and reagents

b-Carotene, chlorophyll a, chlorophyll b, 40

-6-diamidino-2-phenyindole (DAPI), dimethyl sulphoxide (DMSO), ethidium bromide (EtBr), lutein, monobromobimane (MbBr), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), phenylmethanesulphonyl fluoride (PMSF), 1,1,3,3-tet-raethoxypropane (TEP), triton X-100, tris–HCl and ribonucle-ase-A (RNase A) were obtained from the Sigma Chemical Co. (St. Louis, MO, USA). Dulbecco’s modified eagle medium (DMEM), fetal bovine serum (FBS), penicillin–streptomycin and trypsin–ethylenediaminetetraacetic acid were obtained from Invitrogen Life Technologies (Carlsbad, CA, USA). All other chemicals were at least reagent grade.

2.3. HPLC analysis

The pigments composition including b-carotene, chlorophyll a, chlorophyll b and lutein of the GPE were analyzed accord-ing to a method described by Gouveia et al. (2006). The GPE was dissolved in the mobile phase and filtered through a 0.45 lm filter before HPLC analysis. Reversed-phase analysis of pigments in GPE were performed on a HPLC (LCP 4100, ECOM, Praha 2, Czech Republic) with a Polaris C18 cartridge column (Varian, CA, USA, 250 mm · 4.6 mm i.d., 5 lm particle size) and a photodiode array detector (DAD 230, ChromTech, Apple valley, MN, USA) with acetonitrile:methanol:water (65:35:2) as eluent and with a flow rate 1.0 ml/min. Injection volume was 20 ll. Finally, chromatographic data were ana-lyzed using WorkDAD HPLC system manager software (Chrom Tech, MN, USA).

2.4. Human hepatoma cell line and culture condition Human hepatoma cell line (Hep G2) was obtained from the Food Industry Research and Development Institution (Shinchu, Taiwan). The Hep G2 cells were cultured in DMEM supple-mented with 10% FBS, 2 mML-glutamine, and 1% penicillin– streptomycin (100 U/ml penicillin and 100 lg/ml streptomycin). The cells were maintained in a humidified 5% CO2incubator

at 37 C and the cells were sub-cultured every 3–4 days to maintain logarithmic growth and were allowed to grow for 24 h before treatments were applied.

2.5. Morphological changes and cell viability analysis The Hep G2 cells were plated in 12-well plates at a density of 1 · 105_{cells/ml. The cells were treated with different}

concen-trations (31.2, 62.5, 125.0, 250.0 and 500.0 lg/ml) of GPE or PBS as a solvent control and grown at 37 C, 5% CO2and 95% air

for 24 or 48 h. To determine morphological changes, the cells were photographed by a phase-contrast microscope and the MTT assay (Dariusz, Sarah, Richard, & Michael, 1993) was used to determine cell viability.

2.6. DAPI staining

The cells were incubated with different concentrations (31.2, 62.5, 125.0, 250.0 and 500.0 lg/ml) of GPE for 48 h. The cells were washed by PBS and stained by DAPI staining before being photographed (Su, Chen, Lin, Wu, & Chung, 2006). 2.7. Single cell gel electrophoresis (Comet assay)

The cells were incubated with different concentrations (31.2, 62.5, 125.0, 250.0 and 500.0 lg/ml) of GPE for 48 h. The cells were then harvested by centrifugation, the DNA was isolated and was then gel electrophoresis according to the method described by Su et al. (2006). The DNA damage in the GPE-treated cells was quantified as comet moment (the DNA product in the tail and the mean migration distance in the tail) were compared with untreated cells.

2.8. Measurement of reactive oxygen species (ROS) and superoxide anion production, mitochondrial membrane potential (DWm) and Ca2+_release

The Hep G2 cells were incubated with 500.0 lg/ml of GPE for 1, 3, 6 or 24 h. The cells were harvested and washed twice with PBS then re-suspended with specific fluorochromes. For ROS analysis, the cells were re-suspended in 500 ll 10 lM of 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA); for DWm analysis, the cells were re-suspended in 500 ll 1 lM of 3,30

-dihexyloxcarbocyanine (DiOC6), and for Ca2+ _{detection, the}

cells were re-suspended in 3 lg/ml Indo 1/AM, respectively and analyzed by flow cytometry as previously described (Wu et al., 2006). The superoxide anion measurement, the cells were incubated with 500.0 lg/ml of GPE for 2, 4, 6, 12, 24 and 48 h, based on the NBT assays, was carried out according to the method ofFreire et al. (2003).

2.9. In vitro invasion assay

Cell invasion was determined by using Matrigel-coated trans-well cell culture chambers (8 m pore size; Millipore Corp., Bille-rica, MA, USA) according to the method ofChen et al. (2010). 2.10. Western blotting

The total proteins were collected from Hep G2 cells after treat-ment with 500.0 lg/ml of GPE over 0, 6, 12, 24 and 48 h, then each protein was determined individually such as Bcl-2, cytochrome c, AIF, Fas, FasL, caspase-3, -8, -9, MMP-2, -9 and b-actin were measured by sodium dodecylsulphate polyacryl-amide gel electrophoresis (SDS-PAGE) and Western blotting as described bySu et al. (2006).

2.11. Measurement of lipid peroxide levels, superoxide dismutase (SOD) activity and glutathione (GSH) levels in Hep G2 cells

The Hep G2 cells after treatment with 500.0 lg/ml of GPE over 48 h and harvested, and then sonicated with 1 mM PMSF buf-fer in order to obtain the cell homogenate. The thiobarbituric acid reactive substances (TBARS) method was used to esti-mate cellular TBARS level spectrophotometrically at 535 nm (Botsoglou et al., 1994). TEP was used as a standard. Cell GSH was reduced by dithiothreitol/phosphate solution and derivatized with MbBr prior to HPLC analysis (Yanga, Chou, Liu, Tsaia, & Kuo, 1995). SOD activity was determined spectro-photometrically at 325 nm based on the SOD-mediated de-crease in the rate of pyrogallol autoxidation under alkali conditions (Stefan & Gudrun, 1974).

The protein content of cell homogenate was determined based on the Biuret reaction (Smith et al., 1985) of the BCA kit using BSA standards. The TBARS and GSH levels in cells were expressed as nanomoles/mg protein and the specific activity of the SOD was expressed as unit/mg protein. 2.12. Statistical analysis

The data were analyzed using Student’s t-test to make a sta-tistical comparison between control and GPE-treated cells.

The results were expressed as mean ± SD of three indepen-dent experiments in replicate for both control and GPE-trea-ted groups. Values of P < 0.05 were taken as being significant.

3.

Results

3.1. The carotenoids and chlorophylls in GPE

Identification of carotenoids and chlorophylls in GPE by HPLC-DAD. Fig. 1 showed the HPLC chromatogram of the lutein, chlorophyll b and chlorophyll a in GPE. The identification of carotenoids and chlorophylls were primarily based on reten-tion time and absorpreten-tion spectra by comparison with four known standards. The retention time of lutein, chlorophyll b, chlorophyll a and b-carotene were 7.41, 15.72, 24.58 and 41.48 min, respectively and the absorption maximum wave-length (kmax) was 442.5, 464.7, 430.2 and 449.1 nm,

respec-tively. The regression equations based on the standard curves for lutein, chlorophyll b, chlorophyll a and b-carotene were y = 165.27x + 159.46, y = 41.03x + 87.78, y = 46.72x + 41.95, and y = 4.04x + 23.95, respectively, with correlation coeffi-cients all being higher than 0.99. Based on HPLC analysis, the contents of lutein, chlorophyll a, and chlorophyll b were 3.8, 42.9 and 15.1 mg/g in GPE. However, there was no observed amount of b-carotene in GPE.

3.2. The GPE on morphological changes, cell viability and apoptosis of Hep G2 cells

After the Hep G2 cells were exposed to different concentra-tions of GPE over 48 h, the results from phase-contrast microscope examination indicated that the cells were mor-phologically-changed by GPE treatment (data not shown). Effects of GPE on the growth of Hep G2 cells were investigated by the MTT method. As shown inFig. 2A, the growth inhibi-tory effect of GPE was observed in a dose and time-dependent manner. At 48 h, the maximal effect on proliferation inhibi-tion was observed with 500 lg/ml GPE, which inhibited prolif-eration in 36.2% of Hep G2 cells.

Fig. 1 – HPLC-DAD chromatogram of carotenoids and chlorophylls in GPE. HPLC-DAD conditions were described in the text.

To investigate the effect of GPE on the occurrence of apop-tosis from Hep G2 cells, we isolated cells after 48 h then stained using DAPI and photographed by fluorescence micro-scope. As shown inFig. 2B, the percentage of cells stained by DAPI was significantly different between GPE- treated and un-treated cells. Apparently, the effects of GPE induced apoptosis were in a concentration-dependent manner. A separate experiment was conducted to re-confirm the influence of GPE on DNA damage. As shown inFig. 2C, treatment of Hep G2 cells with GPE caused elevation in the DNA damage in a linear dose dependent manner (R2_{= 0.977) measured as}

comet moment.

3.3. The GPE on the levels of ROS, superoxide anion, Ca2+ and mitochondrial membrane potential (DWm) in Hep G2 cells The ROS levels were stimulated quite early and time-depen-dently when the Hep G2 cells were incubated with 500.0 lg/ ml GPE (Fig. 3A). The percentage of ROS was significantly dif-ferent between GPE treated and untreated cells. As shown in Fig. 3B, superoxide anion was induced in Hep G2 cells at 12 h after GPE treatment and was of a time-dependent manner. Furthermore, the addition of GPE caused significant increase levels of superoxide anion in Hep G2 cells over 24 and 48 h. Flow cytometric analysis indicated that the DWm levels signif-icantly decreased in the GPE-treated cells as compared with the control cells (Fig. 3C). Also, it can be seen inFig. 3D that the cytoplasmic Ca2+ _{were significantly increased as}

com-pared with the control cells and were in a time-dependently manner.

3.4. The GPE on the expressions of apoptosis-associated specific proteins from Hep G2 cells

The data demonstrated that 500.0 lg/ml GPE for 24 and 48 h can significantly decrease the expression of Bcl-2 (Fig. 4A) and increase the expressions of cytochrome c, AIF (Fig. 4A), caspases-3, -9 (Fig. 4B), Fas and FasL and caspase-8 (Fig. 4C) which may contribute to the occurrence of apoptosis in the examined cells.

3.5. Anti-invasion effect of GPE in Hep G2 cells

Effects of GPE on cell invasion were investigated using a reconstructed basement membrane and results were shown in Fig. 5A. After 48 h incubation, significantly fewer cells had invaded and migrated through the artificial membrane in the GPE-treated cells than in the control cells. The quanti-fication of cells in the lower chamber fromFig. 5B indicated that GPE significantly inhibited Hep G2 cell invasion, the per-centage of inhibition ratio is 24–75% and this effect was in a concentration-dependent manner. Furthermore, protein lev-els of MMP-2 and -9 significantly decreased following 500.0 lg/ml of GPE treatment in the Hep G2 cells (Fig. 5C). 3.6. The GPE on the redox status of HepG2 cells

Treatment with GPE (500 lg/ml) for 48 h was associated with a significant decrease in cellular GSH levels and SOD activities by 46.2% and 75.9% respectively as compared with the control cells (Table 1). Meanwhile, TBARS levels in Hep G2 cells in-creased significantly after GPE treatment by 66.8% when com-pared with the control cells.

4.

Discussion

It is well known that hepatocellular carcinoma is one of the major diseases causing death throughout the world and many investigators are working to discover a new agent for hepa-toma therapy. Many studies have reported that natural plants and/or their naturally occurring compounds, acacetin (Hsu, Kuo, & Lin, 2004) and apigenin (Chianga, Ng, Lin, Kuo, & Lin,

(A)

(B)

(C)

Time (h)

V

iability (%

)

* * *

*

*

*

* *

*

*

Comet moment

-Fig. 2 – Effect of GPE on Hep G2 cells survival determined by MTT assay (A), apoptosis examined by DAPI staining (B) and comet assay (C). Each value represents the mean ± S.D. of three experiments. An asterisk indicates significant difference (P < 0.05) between GPE-treated cells and control as analyzed by Student’s t-test.

2006), common flavonoids in fruits and vegetables; curcumin, a natural product present in turmeric (Lo´pez-La´zaro, 2008); Euchresta formosana Radix (Hsu et al., 2007), Piper methysticum Forster (Lude et al., 2008), Physalis angulata and Physalis peruvi-ana (Wu et al., 2004), traditional herbal medicines; are poten-tial inhibitors of tumor cell proliferation and apoptotic inducers in hepatoma cells. In fact, many plant derivatives have been used as anticancer agents in clinical patients (Katz, 2001). Chlorella has been found to contain a great variety of nutrients that are essential for human health and it is also widely used as a food supplement (Jianchun et al., 2007). The results of this study demonstrated that in vitro treatment of Hep G2 cells with GPE over 48 h induced morphological changes associated with the decrease of the percentage of cell viability in dose- and time-dependent manners. Our DAPI staining and comet assay resulted also indicated that GPE in-duced DNA damage in the Hep G2 cells. The anti-apoptotic protein, Bcl-2, play an important role in the induction of apoptosis (Willis, Day, Hinds, & Huang, 2003). Our data showed that GPE treatment decreased the levels of Bcl-2 and mitochondrial membrane potential (DWm) and promoted the levels of cytochrome c release from mitochondria in Hep G2 cells. The data also showed that GPE induced AIF expres-sion before leading to apoptosis in Hep G2 cells. AIF is another possible protein that can be liberated from mitochondria to nuclei to trigger DNA fragmentation and participates in the induction of apoptosis in a caspase-independent manner (Susin et al., 1999). We also demonstrated a significant induc-tion of the execuinduc-tion protease of apoptosis, caspase-3 and caspase-9. This is in agreement with many reports that re-leased cytochrome c from mitochondria activates initiator caspase-9, in turn, activates a sequential cascade of caspases,

GPE 500μg/ml 1.0 0.64 0.44 0.53 0.43 1.0 1.98 1.69 2.13 1.67 1.0 0.74 0.74 1.21 1.46 (h) 0 6 12 24 48 Bcl-2 cytochrome c AIF β-actin

(A)

(B)

(C)

Fig. 4 – The expression of Bcl-2, cytochrome c and AIF (A), caspase-3 and caspase-9 (B), and Fas, FasL and caspase-8 (C) in Hep G2 cells treated with GPE (500 lg/ml). The protein levels were determined by Western blot analysis. b-Actin was used as the protein loading control.

ROS pr oduction (% ) -10 0 10 20 30 40 50 Time (h) 24 0 1 3 6

*

*

*

*

(A)

(B)

Relative level of super

o xide anion 0.6 0.8 1.0 1.2 1.4 1.6 1.8

*

*

*

(C)

*

*

*

*

*

(D)

Fig. 3 – Effect of GPE on intracellular ROS (A), superoxide anion production (B), mitochondria membrane potential (C) and intracellular calcium levels (D) in Hep G2 cells. The result is expressed as the mean ± S.D. of three experiments. An asterisk indicates significance (P < 0.05) from the 0 h.

(A)

(B)

(C)

Concentration (

μg/ml)

Relative of invasion (%

)

0

20

40

60

80

100

120

0

31.2

62.5

125

250

500

*

*

*

*

*

GPE 500

μg/ml

1.0

0.80

0.83

0.73

0.34

1.0

0.21

0.26

0.31

0.33

(h)

0

6

12

24

48

MMP-2

β-actin

MMP-9

Fig. 5 – Effects of GPE on the invasion of Hep G2 cells in vitro. (A) Cells that penetrated through the matrigel to the lower surface of the filter were stained with crystal violet and shown under a light microscope at 100·. (B) Quantification of cells in the lower chambers, which was done by counting at 100·. (C) The expression of MMP-2 and MMP-9 in Hep G2 cells treated with GPE (500 lg/ml). An asterisk indicates significant difference (P < 0.05) between GPE-treated cells and the control group as analyzed by Student’s t-test.

Table 1 – Effect of GPE on redox system of Hep G2 cells over 48 h incubation. The result is expressed as the mean ± S.D. of three experiments. An asterisk indicates significance (P < 0.05) from the control group.

Control group GPE-treated group

TBARS (nmole/mg protein) 75.52 ± 3.92 125.98 ± 13.13*

SOD (unit/mg protein) 7.17 ± 0.80 1.73 ± 0.60*

GSH (nmole/mg protein) 315.76 ± 7.51 170.03 ± 13.17*

The result is expressed as the mean ± S.D. of three experiments. An asterisk indicates significance (P < 0.05) from the control group.

especially caspase-3, resulting in the proteolysis of death sub-strates and subsequent DNA degradation and apoptotic death (Cory, Vaux, Strasser, Harris, & Adams, 1999; Wang, 2001).

ROS are recognized as mediators of the apoptotic signaling pathway (Li et al., 2003). Higher levels of ROS are known to in-duce not only cell death (Wulf, 2002), but also DNA damage and genomic instability (Cerutti, 1994). The redox state of the cell is also known to regulate its growth behavior ( El-Mis-siry & El-Aziz, 2000). Other studies have indicated that cancer chemo-preventive agents induce apoptosis in part with the generation of ROS and the disruption of redox homeostasis (Lin, Fujii, & Hou, 2003; Xia, Lundgren, Bergstrand, DePierre, & Na¨ssberger, 1999). In this study, we found that GPE in-creased the production of ROS, especially superoxide anion and H2O2, in short periods of time. Also, the treatment with

GPE over 48 h was associated with a significant decrease in cellular GSH levels and SOD activities, and with an increase in TBARS levels in Hep G2 cells. Cellular glutathione is a major component of the intracellular reducing factor and a critical determinant for proper apoptotic signaling cascade and for the correct cell dismantling during apoptosis (Armstrong et al., 2002). Evidence suggests that severe intracellular GSH depletion can impair cell’s defense against toxic compounds and may result in cell injury and death (Reed, 1990). Recently, it has been suggested that lipid peroxidation, determined by TBARS levels may impair a variety of intra- and extra-mito-chondrial membrane transport systems that contribute to apoptosis (Kristal, Park, & Yu, 1996). The present data indi-cated that GPE may be a modulator of the cellular redox sta-tus and exert pro-oxidant effects in Hep G2 cells exposed to oxidative stress. It is well known that ROS may lead to endo-plasmic reticulum stress and in turn lead to Ca2+_{release. Our}

experiment demonstrated that cells treated with GPE for as little as one hour increased the levels of Ca2+in Hep G2 cells. However, the detailed mechanisms of GPE on endoplasmic reticulum stress remain to be deciphered. The Fas/FasL sys-tem is another major mechanism for apoptosis. When Fas receptor proteins are activated by binding to FasL, the activa-tion of caspase 8 occurs, in turn, execute cell apoptotic death (Rao et al., 2004). We observed the enhanced expression of Fas, FasL and caspase 8 suggesting that at least part of GPE-induced Hep G2 cells’ apoptosis might be due to Fas-mediated death.

Cancer development involves multi-step process as which cancer eventually spreads from one area of the body to other organs or tissues during the late metastasic stage. It is well known that gelatinases such as MMP-2 and MMP-9 are di-rectly involved in metastasis and that the suppression of gel-atinase and will greatly contribute to the control of metastasis (Zeng, Cohen, & Guillem, 1999). Also, MMP-2 and MMP-9 are considered to be particularly important targets for the devel-opment of anticancer drugs because they are associated with aggressive, advanced, invasive or metastatic tumor pheno-type (Birkedal-Hansen et al., 1993). In the present study, we demonstrated that GPE treatment can inhibit the levels of MMP-2 and MMP-9 coinciding with the inhibition of invasion of the Hep G2 cells after treatment with GPE. Our study provides additional information on the antimetastatic poten-tial of GPE beyond its antitumor activity.

Recently, the field of available natural sources has been further increased by also including some algae and, even more interestingly, microalgae. These microorganisms are a potentially great source of natural compounds that could be used as functional ingredients (Plaza, Herrero, Cifuentes, & Iban~ez, 2009). Chlorophylls and carotenoids are present abundantly in green plants and possess important biological activities including antioxidant (Tsai, Wu, & Chen, 2010), anti-tumour (Tsai et al., 2010) and anti-invasion (Kozuki, Miura, & Yagasaki, 2000). According toWu, Wu, and Shi (2007)Chlorella contained 2–4 mg/g dry cell weight of lutein. Lutein is not only an important natural food dye and additive but also a strong antioxidant that may be useful in reducing the incidence of cancer (Park, Chew, & Wong, 1998).Cha, Koo, and Lee (2008) reported that the bioactive carotenoid, xanthophylls of Chlo-rella ellipsoidea exerted strong antiproliferative effects on hu-man colon cancer cells. Tsai et al. (2010) reported that carotenoids and chlorophylls isolated from Gynostemma penta-phyllum had antiproliferative effect on hepatoma cells. In this study, GPE contain lutein, chlorophyll a, and chlorophyll b and they may have contributed, in a substantial part at least, to the antitumour and anti-invasion activities of GPE.

In conclusion, for the first time, we found that the molec-ular mechanism of GPE induced cytotoxicity on Hep G2 cells. GPE may be a modulator of the cellular redox status and can exhibit pro-oxidant activity in Hep G2 cells. In addition, GPE induced ROS and Ca2+_{production, decreased the Bcl-2 levels,}

changed the DWm before it triggered the release of cyto-chrome c and subsequently induced the processing of procas-pase-9 and procaspase-3 which led to the cleavage of DNA fragmentation. GPE also induced AIF and Fas/FasL pathways before leading to apoptosis in Hep G2 cells. Furthermore, we show for the first time that GPE can inhibit the invasion of Hep G2 cells in vitro. Chlorophylls and lutein may be responsi-ble for the functional ingredients in GPE. Taken together these findings provide new insights into the possible pathways of chlorella-induced apoptosis and anti-invasion potential in human hepatoma G2 cells.

Acknowledgments

This research was supported by National Science Council, Taiwan (NSC 94-2745-B-126-004-URD, NSC 95-2745-B-126-004-URD and NSC 96-2745-B-126-95-2745-B-126-004-URD) and its financial support was greatly appreciated. We thank Prof. Phoency Lai of the Carbohydrate Laboratory, Providence University, for invaluable assistance with the HPLC analysis.

R E F E R E N C E S

Armstrong, J. S., Teinauer, K. K. S., Hornung, B., Irish, J. M., Lecane, P., Birrell, G. W., Peehl, D. M., & Knox, S. J. (2002). Role of glutathione depletion and reactive oxygen species generation in apoptotic signaling in a human B lymphoma cell line. Cell Death and Differentiation, 9(3), 252–263.

Birkedal-Hansen, H., Moore, W. G., Bodden, M. K., Windsor, L. J., Birkedal-Hansen, B., DeCarlo, A., & Engler, J. A. (1993). Matrix

metalloproteinases: A review. Critical Reviews in Oral Biology and Medicine, 4(2), 197–250.

Botsoglou, N. A., Fletouris, D. J., Papageorgiou, G. E., Vassilopoulos, V. N., Mantis, A. J., & Trakatellis, A. G. (1994). Rapid, sensitive, and specific thiobarbituric acid method for measuring lipid peroxidation in animal tissue, food, and feedstuff samples. Journal of Agricultural and Food Chemistry, 42(9), 1931–1937. Cerutti, P. A. (1994). Oxy-radicals and cancer. Lancet, 344(8926),

862–863.

Cha, K. H., Kang, S. W., Kim, C. Y., Um, B. H., Na, Y. R., & Pan, C. H. (2010). Effect of pressurized liquids on extraction of

antioxidants from Chlorella vulgaris. Journal of Agricultural and Food Chemistry, 58(8), 4756–4761.

Cha, K. H., Koo, S. Y., & Lee, D. U. (2008). Antiproliferative effects of carotenoids extracted from Chlorella ellipsoidea and Chlorella vulgaris on human colon cancer cells. Journal of Agricultural and Food Chemistry, 56(22), 10521–10526.

Chen, Y. Y., Chiang, S. Y., Lin, J. G., Ma, Y. S., Liao, C. L., Weng, S. W., Weng, S. W., Weng, S. W., & Weng, S. W. (2010). Emodin, aloe-emodin and rhein inhibit migration and invasion in human tongue cancer SCC-4 cells through the inhibition of gene expression of matrix metalloproteinase-9. International Journal of Oncology, 36(5), 1113–1120.

Chianga, L. C., Ng, L. T., Lin, I. C., Kuo, P. L., & Lin, C. C. (2006). Anti-proliferative effect of apigenin and its apoptotic induction in human Hep G2 cells. Cancer Letters, 237(2), 207–214.

Cory, S., Vaux, D. L., Strasser, A., Harris, A. W., & Adams, J. M. (1999). Insights from Bcl-2 and Myc: Malignancy involves abrogation of apoptosis as well as sustained proliferation. Cancer Research, 59(7), 1685s–1692s.

Dariusz, S., Sarah, J. S., Richard, H. C., & Michael, B. (1993). An improved MTT assay. Journal of Immunological Methods, 157(1–2), 203–207.

El-Missiry, M. A., & El-Aziz, A. F. A. (2000). Influence of melatonin on proliferation and antioxidant system in Ehrlich ascites carcinoma cells. Cancer Letters, 151(2), 119–125.

Eva, S., Una, F., & Afshin, S. (2003). Caspase-12 and ER-stress-mediated apoptosis: The story so far. Annals of the New York Academy of Sciences, 1010, 186–194.

Freire, A. C. G., de Assis, C. F., Frick, A. O., da Silva Meloa, P., Hauna, M., Aoyamaa, H., Dura´nb, N., Sauerc, M. M., & Kalla´sc, E´. G. (2003). Influence of protein phosphatase inhibitors on HL60 cells death induction by dehydrocrotonin. Leukemia Research, 27(9), 823–829.

Gouveia, L., Veloso, V., Reis, A., Fernandes, H., Novais, J., & Empis, J. (2006). Evolution of pigment composition in Chlorella vulgaris. Bioresource Technology, 57(2), 157–163.

Hsu, Y. L., Kuo, P. L., & Lin, C. C. (2004). Acacetin inhibits the proliferation of Hep G2 by blocking cell cycle progression and inducing apoptosis. Biochemical Pharmacology, 67(5), 823–829. Hsu, S. C., Kuo, C. L., Lin, J. P., Lee, J. H., Lin, C. C., Su, C. C., Yang, M.

D., & Chung, J. G. (2007). Crude extracts of Euchresta formosana radix inhibit invasion and migration of human hepatocellular carcinoma cells. Anticancer Research, 27(4B), 2377–2384. Jianchun, S., Fang, Y., Zhihong, X., Liyan, Z., Xiaojun, Z., & Qiuhui,

H. (2007). Preparation, identification and their antitumor activities in vitro of polysaccharides from Chlorella pyrenoidosa. Food of Chemistry, 105(2), 533–539.

Katz, S. (2001). Beneficial uses of plant pathogens: Anticancer and drug agents derived from plant pathogens. Canadian Journal of Plant Pathology, 24(1), 10–13.

Kozuki, Y., Miura, Y., & Yagasaki, K. (2000). Inhibitory effects of carotenoids on the invasion of rat ascites hepatoma cells in culture. Cancer Letters, 151, 111–115.

Kristal, B. S., Park, B. K., & Yu, B. P. (1996). 4-Hydroxyhexenal is a potent inducer of the mitochondrial permeability transition. The Journal of Biological Chemistry, 271(11), 6033–6038.

Kuniaki, T., Akira, Y., Kiyoshi, N., Takashi, H., Masao, O., Yukihiro, S., & Kikuo, N. (1998). A novel glycoprotein obtained from Chlorella vulgaris strain CK22 shows antimetastatic

immunopotentiation. Cancer Immunology, Immunotherapy, 45(6), 313–320.

Li, N., Ragheb, K., Lawler, G., Sturgis, J., Rajwa, B., Melendez, J. A., & Robinson, J. P. (2003). DPI induces mitochondrial superoxide-mediated apoptosis. Free Radical Biology and Medicine, 34(4), 465–477.

Lin, S., Fujii, M., & Hou, D. X. (2003). Rhein induces apoptosis in HL-60 cells via reactive oxygen species-independent mitochondrial death pathway. Archives of Biochemistry and Biophysics, 418(2), 99–107.

Lude, S., Torok, M., Dieterle, S., Jaggi, R., Buter, K. B., &

Krahenbuhl, S. (2008). Hepatocellular toxicity of kava leaf and root extracts. Phytomedicine, 15(1–2), 120–131.

Lo´pez-La´zaro, M. (2008). Anticancer and carcinogenic properties of curcumin: Considerations for its clinical development as a cancer chemopreventive and chemotherapeutic agent. Molecular Nutrition & Food Research, 1, S103–S127.

Park, J. S., Chew, B. P., & Wong, T. S. (1998). Dietary lutein from marigold extract inhibits mammary tumor development in BALB/c mice. The Journal of Nutrition, 128, 1650–1656. Plaza, M., Herrero, M., Cifuentes, A., & Iban~ez, E. (2009).

Innovative natural functional ingredients from microalgae. Journal of Agricultural and Food Chemistry, 57(16), 7159–7170. Plaza, M., Santoyo, S., Jaime, L., Avalo, B., Cifuentes, A., Reglero, G.,

Reina, G. G. B., Sen˜ora´ns, F. J., Iba´n˜ez, E. (in press).

Comprehensive characterization of the functional activities of pressurized liquid and ultrasound-assisted extracts from Chlorella vulgaris. LWT – Food Science and Technology. doi:10.1016/j.lwt.2011.09.024.

Rao, R. V., Hermel, E., Castro-Obregon, S., Rio, G. D., Ellerby, L. M., Ellerby, H. M., & Bredesen, D. E. (2004). Coupling endoplasmic reticulum stress to the cell death program. Cell Death and Differentiation, 11(4), 372–380.

Reed, D. J. (1990). Glutathione: Toxicological implications. Annual Review of Pharmacology and Toxicology, 30, 603–631.

Smith, P., Krohn, R., Hermanson, G., Mallia, A., Gartner, F., Provenzano, M., Fujimoto, E., Goeke, N., Olson, B., & Klenk, D. (1985). Measurement of protein using bicinchoninic acid. Analytical Biochemistry, 150(1), 76–85.

Stefan, M., & Gudrun, M. (1974). Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. European Journal of Biochemistry, 47(3), 469–474.

Su, C. C., Chen, G. W., Lin, J. G., Wu, L. T., & Chung, J. G. (2006). Curcumin inhibits cell migration of human colon cancer colo 205 cells through the inhibition of nuclear factor kappa B/p65 and down-regulates cyclooxygenase-2 and matrix

metalloproteinase-2 expressions. Anticancer Research, 26(2A), 1281–1288.

Sulaiman, S., Shamaan, N. A., Ngah, W. Z. W., & Yusof, Y. A. M. (2006). Chemopreventive effect of Chlorella vulgaris in choline deficient diet and ethionine induced liver carcinogenesis in rats. International Journal of Cancer Research, 2(3), 234–241. Susin, S. A., Lorenzo, H. K., Zamzami, N., Marzo, I., Snow, B. E.,

Brothers, G. M., Greg, M., & Mangion, J. (1999). Molecular characterization of mitochondrial apoptosis-inducing factor. Nature, 397(6718), 441–446.

Takashi, H., Kiyoshi, N., Shoichiro, K., Yotaro, A., Akira, Y., & Yasunobu, Y. (2000). Chlorella vulgaris culture supernatant (CVS) reduces psychological stress-induced apoptosis in thymocytes of mice. International Journal of

Immunopharmacology, 22(11), 877–885.

Tsai, Y. C., Wu, W. B., & Chen, B. H. (2010). Preparation of carotenoids and chlorophylls from Gynostemma pentaphyllum

(Thunb.) Makino and their antiproliferation effect on hepatoma cell. Journal of Medicinal Food, 13(6), 1431–1442. Wang, X. (2001). The expanding role of mitochondria in apoptosis.

Genes and Development, 15(22), 2922–2933.

Willis, S., Day, C. L., Hinds, M. G., & Huang, D. C. S. (2003). The Bcl-2 regulated apoptotic pathway. Journal of Cell Science, 116(Bcl-20), 4053–4056.

Wu, L. C., Ho, J. A. A., Shieh, M. C., & Lu, I. W. (2005). Antioxidant and antiproliferative activities of Spirulina and Chlorella water extracts. Journal of Agricultural and Food Chemistry, 53(10), 4207–4212.

Wu, C. C., Lin, J. P., Yang, J. S., Chou, S. T., Chen, S. C., Lin, Y. T., Lin, H. L., & Chung, J. G. (2006). Capsaicin induced cell cycle arrest and apoptosis in human esophagus epidermoid carcinoma CE 81T/VGH cells through the elevation of intracellular reactive oxygen species and Ca2+_{productions and caspase-3}

activation. Mutation Research, 601, 71–82.

Wu, S. J., Ng, L. T., Chen, C. H., Lin, D. L., Wang, S. S., & Lin, C. C. (2004). Antihepatoma activity of Physalis angulata and P. peruviana extracts and their effects on apoptosis in human Hep G2 cells. Life Sciences, 74(16), 2061–2073.

Wu, Z., Wu, S., & Shi, X. (2007). Supercritical fluid extraction and determination of lutein in heterotrophycally cultivated Chlorella pyrenoidosa. Journal of Food Process Engineering, 30(2), 174–185.

Wulf, D. (2002). Free radicals in the physiological control of cell function. Physiological Reviews, 82(1), 47–95.

Xia, Z., Lundgren, B., Bergstrand, A., DePierre, J. W., & Na¨ssberger, L. (1999). Changes in the generation of reactive oxygen species and in mitochondrial membrane potential during apoptosis induced by the antidepressants imipramine, clomipramine, and citalopram and the effects on these changes by Bcl-2 and Bcl-XL. Biochemical Pharmacology, 57(10), 1199–1208.

Yang, F., Shi, Y., Sheng, J., & Hu, Q. (2006). In vivo

immunomodulatory activity of polysaccharides derived from Chlorella pyrenoidosa. European Food Research and Technology, 224(2), 225–228.

Yanga, C. S., Chou, S. T., Liu, L., Tsaia, P. J., & Kuo, J. S. (1995). Effect of ageing on human plasma glutathione concentrations as determined by high-performance liquid chromatography with fluorimetric detection. Journal of Chromatography B: Biomedical Sciences and Applications, 674(1), 23–30.

Zeng, Z. S., Cohen, A. M., & Guillem, J. G. (1999). Loss of basement membrane type IV collagen is associated with increased expression of metalloproteinases 2 and 9 (MMP-2 and MMP-9) during human colorectal tumorigenesis. Carcinogenesis, 20(5), 749–755.