Methyl alcohol extract of marine green alga Enteromorpha linza (Linnaeus) J. Agardh. induces reactive oxygen species-dependent growth arrest and apoptosis in human hepatocellular carcinoma Hep3B cells

Abstract

BACKGROUND/OBJECTIVES

Although seaweed has recently been attracting attention as an important source for the control of numerous diseases including cancer, studies on the anti-cancer activity of the green alga Enteromorpha linza (Linnaeus) J. Agardh. are still insufficient. The purpose of this study was to investigate the anti-cancer activity of the green alga E. linza in human hepatocellular carcinoma Hep3B cells.

MATERIALS/METHODS

The effect of methyl alcohol extract of E. linza (MEEL) on cell viability and the induction of cell cycle arrest and apoptosis of Hep3B cells was investigated. To evaluate the anti-cancer activity mechanism of MEEL, the production of reactive oxygen species (ROS) and mitochondrial membrane potential were detected. We also investigated changes in the expression of key regulators of cell cycle and apoptosis.

RESULTS

Our results indicated that MEEL-induced inhibition of Hep3B cell proliferation was associated with G1 phase cell cycle arrest, along with the induction of cyclin-dependent kinase (Cdk) inhibitor p21 expression, suppression of cyclin D1 and E expression, and dephosphorylation of retinoblastoma protein (pRB). In addition, MEEL markedly enhanced the complex formation between p21 and Cdk4/6, as well as pRB and the transcription factor E2Fs, respectively. MEEL also induced apoptosis by activation of caspases. Moreover, MEEL interfered with mitochondrial integration by altering the level of Bcl-2 family proteins to increase cytoplasmic release of cytochrome c. Furthermore, MEEL significantly enhanced the generation of ROS, whereas ROS scavenger restored reduced viability by attenuating MEEL-mediated growth arrest and apoptosis.

CONCLUSION

Collectively, the present findings demonstrate that the antiproliferative efficacy of MEEL in Hep3B cells can be achieved through ROS-dependent pathway.

INTRODUCTION

Cancer is one of the most deadly and incurable diseases known to mankind to this day. Among them, hepatocellular carcinoma (HCC), which accounts for about 90% of liver cancers, is the fastest growing and most common type of malignant tumor worldwide, and is the leading cause of cancer death including Korea [1,2]. Environmental and genetic factors such as hepatitis virus infection, excessive alcohol drinking, and ingestion of aflatoxins are involved in the development of HCC due to chronic inflammation of liver cirrhosis, and it also occurs in patients with nonalcoholic fatty liver disease [3,4]. In particular, HCC is a complex multifactorial carcinoma with heterogeneous prognosis, and most patients are diagnosed with HCC at an advanced stage with limited approved systemic chemotherapy [4,5]. The treatment options for HCC include surgical resection, radiation therapy, liver transplantation, chemotherapy, and immunotherapy, but their treatment is very limited in patients with advanced HCC [3,6]. Recently, drugs such as multi-kinase inhibitors have been applied as standard treatments, but serious side effects and drug resistance problems are being raised [7,8]. Therefore, there is a need to develop low-toxic and effective alternative or adjuvant therapies that should improve current treatment modalities.

Recently, the development of drugs based on marine algae has contributed as an important source in the control of numerous diseases, including cancer [9,10]. Because of their health benefits, they have been used as food for a long time and are reported to contain various physiologically active substances, including anti-viral, anti-inflammatory, antioxidant, liver damage protection, neuroprotective, and anti-cancer activity [11,12,13,14,15]. Among them, Enteromorpha linza (Linnaeus) J. Agardh. is a kind of green algae called Ippalae in Korea, belongs to the genus Enteromorpha, and inhabits extensively in the North Pacific region. Several previous studies have indicated that E. linza extract has multiple pharmacological properties, such as anti-bacterial, antioxidant, anti-aging, immunomodulatory and anti-proliferative activity, without side effects [16,17,18,19,20,21]. As an example of its anti-cancer activity, we investigated the anti-proliferative effect of methyl alcohol extract of E. linza (MEEL) on U937 human leukemia cells and suggested that MEEL induces apoptosis of U937 cells in a caspase-dependent manner [22]. To date, green algae such as Ulva lactuca and E. intestinalis have been reported to have potent anti-cancer activity against HCC [19,23], but no further investigation into the anti-cancer activity mechanism of E. linza has been conducted except in our previous study. Therefore, the purpose of this study is to identify additional mechanisms related to the anti-cancer activity of E. linza, and for this purpose, the role of key factors involved in the inhibition of cell proliferation associated with the induction of apoptosis by MEEL was investigated in HCC Hep3B cells.

MATERIALS AND METHODS

Reagents and antibodies

All materials necessary for cell culture were purchased from WelGENE Inc. (Gyeongsan, Korea). Dimethyl sulfoxide (DMSO), N-acetyl-L-cysteine (NAC), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), protein A/G and agarose beads and 2',7'-dichlorofluorescein diacetate (DCF-DA) were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). 4′,6′-diamidino-2-phenylindole (DAPI), propidium iodide (PI) and enhanced chemiluminescence (ECL) kit were obtained from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Mitochondrial fractionation kit and Immun-Blot^® polyvinylidene difluoride (PVDF) membranes were purchased from Active Motif, Inc. (Carlsbad, CA, USA) and Bio-Rad Laboratories, Inc. (Hercules, CA, USA), respectively. Colorimetric caspase activity assay kits and 5,5,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimi-dazoylcarbocyanine iodide (JC-1) were ordered from Abcam, Inc. (Cambridge, MA, UK). Antibodies (Table 1) for immunoblotting were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), Proteintech Group, Inc. (Rosemont, IL, USA), BioWorld MedTech (Dublin, OH, USA) and Cell Signaling Technology, Inc. (Danvers, MA, USA).

Table 1. List of antibodies used for immunoblotting.

Target	Company	Catalogue #	Dilution
Cyclin D1	Santa Cruz Biotechnology	sc-753	1:1,000
Cyclin E	Santa Cruz Biotechnology	sc-198	1:1,000
Cdk4	Santa Cruz Biotechnology	sc-260	1:1,000
Cdk6	Santa Cruz Biotechnology	sc-177	1:1,000
p21	Santa Cruz Biotechnology	sc-6246	1:1,000
p27	Santa Cruz Biotechnology	sc-528	1:1,000
pRB	Cell Signaling Technology	8516S	1:500
E2F-1	Santa Cruz Biotechnology	sc-193	1:1,000
E2F-4	Santa Cruz Biotechnology	sc-866	1:1,000
Caspase-3	Santa Cruz Biotechnology	sc-7272	1:1,000
Caspase-8	Santa Cruz Biotechnology	sc-56070	1:1,000
Caspase-9	Santa Cruz Biotechnology	sc-56076	1:1,000
PARP	Santa Cruz Biotechnology	sc-8007	1:1,000
Bax	Proteintech Group, Inc.	50599-2-Ig	1:1,000
Bcl-2	Santa Cruz Biotechnology	sc-509	1:1,000
Cytochrome c	Cell Signaling Technology	4280S	1:500
COX IV	Santa Cruz Biotechnology	sc-517553	1:1,000
Actin	BioWorld MedTech	BS6007M	1:1,000

Cell culture and MEEL treatment

HCC Hep3B cells were obtained from Korean Cell Line Bank (Seoul, Korea) and were in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C in a humidified atmosphere with 5% CO₂. MEEL was provided by Jeju Bio Resource Extract Bank (Jeju, Korea), and the stock solution was prepared by solubilizing it in DMSO, and then diluted to various concentrations with the culture medium, before treating cells.

Cell viability assay

Hep3B cells were cultured in a medium containing selected concentrations of MEEL for 48 h or pretreated with or without 10 mM NAC for 1 h, and then treated with or without 250 μg/mL MEEL. After treatment, cell viability testing was conducted using the MTT method, according to the previously described method [24]. For examining morphological changes, images of cells with and without MEEL treatment were taken using a phase-contrast microscope (Carl Zeiss, Oberkochen, Germany).

Detection of apoptosis by nuclear morphology change

To evaluate the effects of MEEL on nuclear morphology, DAPI staining was carried out. In brief, cells treated with MEEL in the presence or absence of NAC for 48 h were fixed with 4% paraformaldehyde for 10 min, and then stained with 1 μg/mL DAPI for 10 min. The morphology of DAPI-stained nuclei was examined under fluorescence microscopy (Carl Zeiss).

Cell cycle assay using flow cytometry

For cell cycle analysis, cells were fixed with 70% ethanol, and stored at −20°C overnight, after which cells were stained with PI solution, as previously described [25]. Subsequently, the frequency of cells in each cell cycle phase was analyzed by flow cytometry with Muse™ Cell Analyzer (Millipore Corporation, Hayward, CA, USA).

Protein extraction, co-immunoprecipitation (Co-IP), and immunoblot analysis

As previously described [26], whole cell lysates were prepared from control cells and MEEL-treated cells in the presence or absence of NAC. Mitochondrial and cytoplasmic fractions were extracted using a Mitochondrial Fractionation Kit. In addition, Co-IP assay was performed to evaluate the interactions between specific proteins. Briefly, 1 mg/mL of protein per sample was incubated with protein A/G and agarose beads for 2 h, followed by pre-clarification by washing and centrifugation. The cell lysates were then reacted with the corresponding antibodies together with protein A/G and agarose beads overnight at 4°C with continuous stirring to obtain immune-complexes. Then, the same amount of protein and immune complexes were fractionated by electrophoresis using sodium dodecyl sulfate-polyacrylamide gels, and transferred to PVDF membranes. The membranes were then hybridized with primary antibodies, and then reacted with secondary antibodies conjugated to horseradish peroxidase. The immunoreactive proteins were detected with an ECL kit, according to the manufacturer’s protocol. Cytochrome c oxidase subunit IV (COX IV) and actin were probed as loading controls for mitochondrial and cytosolic proteins.

Analysis of caspase activity

The activity of each caspase was evaluated using caspase activity assay kits, based on the hydrolysis of fluorescent substrate peptides by activated caspases. Briefly, after resuspending the cells in the cell lysis buffer, the supernatants were reacted with each caspase substrate according to the kit instructions. Finally, the concentration of p-nitroaniline released from the substrates was detected by microplate reader, as previously described [27].

Determination of mitochondrial membrane potential (MMP)

The alteration of MMP in Hep3B cells was detected using JC-1 dye. According to the protocol, cells treated with MEEL in the presence or absence of NAC were collected, and immediately stained with 10 μM JC-1 for 30 min. MMP values were then calculated using flow cytometry immediately [28].

Measurement of intracellular reactive oxygen species (ROS) production

The level of ROS in each treatment group was investigated using DCF-DA staining, which could be oxidized into fluorescent DCF by ROS. In brief, MEEL-treated cells with or without NAC were incubated with 10 μM DCF-DA, and then the levels of intracellular ROS were measured using flow cytometry, according to the manufacturer’s procedure [29].

Statistical analysis

Experimental data were statistically analyzed using GraphPad Prism 5.03 software (GraphPad Software Inc., La Jolla, CA, USA), using the unpaired two-tailed Student’s t-test and one-way analysis of variance. All results are presented as the mean ± SD of at least separate experiments. The results were considered statistically significant when P-value was less than 0.05.

RESULTS

MEEL suppressed cell viability and induced apoptosis in Hep3B cells

To investigate the effect of MEEL in Hep3B cell growth using the MTT assay, cells were treated with different concentrations of MEEL for 48 h. As indicated in Fig. 1A, MEEL concentration-dependently suppressed the cell viability of Hep3B cells. As a result of observing the morphological change of cells, the untreated cells maintained their original morphology and were in close contact with each other, but the MEEL-treated cells lost their original shape and the number of suspended cells (dead cells) increased (Fig. 1B). In addition, the results of microscopic fluorescent examination by DAPI staining showed that MEEL-treated cells exhibited chromosomal fragmentation and condensation as characteristics of apoptosis-induced cells in the nucleus (Fig. 1C and D). These data demonstrate that the inhibition of Hep3B cell viability induced by MEEL was due to apoptosis induction.

Fig. 1. MEEL-induced reduction in cell viability in Hep3B cells was associated with induction of cell cycle arrest and apoptosis. (A) Cell viability of Hep3B cells treated with the indicated concentrations of MEEL for 48 h was analyzed by the MTT assay. (B) Cell morphological changes were observed using an inverted microscope (Scale bar: 100 μm). (C) Morphological changes in DAPI-stained nuclei were captured using a fluorescence microscope (Scale bar: 100 μm). Arrows in the image indicate the nuclei of cells in which apoptosis has been induced. (D) The frequencies of apoptotic nuclei are presented as percentage of the total number of cells. (E) After treatment with MEEL for 48 h, flow cytometry was performed to examine the effect of MEEL on cell cycle progression, and the average values of cells corresponding to the cell cycle progression stages were presented. (F) The frequencies of cells belonging to the sub-G1 stage, which refer to the population of cells induced by apoptosis, are shown.

MEEL, methyl alcohol extract of Enteromorpha linza; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DAPI, 4′,6′-diamidino-2-phenylindole.

^*P < 0.05 and ^***P < 0.001 vs. control cells.

MEEL induced cell cycle arrest at G1 phase in Hep3B cells

To verify whether the anti-proliferative efficacy of MEEL in Hep3B cells could be the consequence of cell cycle perturbation, we investigated cell cycle distribution following MEEL treatment. Fig. 1E shows flow cytometry results indicating that the cell frequency in G1 phase increased with a gradual increase in MEEL concentration and a simultaneous decrease in cell frequency in G2/M and S phases. In addition, the frequency of sub-G1 phase cells representing an apoptotic population was increased in a concentration-dependent manner with MEEL treatment (Fig. 1F).

MEEL altered the expression of cell cycle-related regulators in Hep3B cells

Since MEEL arrested the cell cycle in G1 phase, we investigated the expression changes of proteins involved in G1 checkpoint regulation. Fig. 2A shows that MEEL treatment down-regulated the expression of cyclin D1 and cyclin E and up-regulated the expression of p21 in a concentration-dependent manner, but had no significant regulatory effect on cyclin-dependent kinase (Cdk) 4, Cdk6 and p27 expression. Next, we conducted Co-IP analysis to examine the possible interactions between p21 and Cdks and found that the interactions between p21 and Cdk4 and Cdk6 were increased (Fig. 2B). Moreover, as the treatment concentration of MEEL increased, the phosphorylated retinoblastoma protein (pRB) content was significantly reduced, whereas E2F1 and E2F4 protein expression levels remained unaffected by treatment with MEEL (Fig. 2C). In addition, MEEL enhanced the complex formation between pRB and E2Fs (Fig. 2D), indicating that MEEL induced Hep3B cell apoptosis by arresting cell cycle at G1 phase, which was associated with changes in the activity of key regulators of the cell cycle.

Fig. 2. Effect of MEEL on expression of key cell cycle regulators in Hep3B cells. (A, C) After isolation of whole cellular proteins from cells treated with MEEL for 48 h, immunoblot analysis was performed using the indicated antibodies. Equal loading was confirmed with actin. (B, D) Proteins isolated from cells were immunoprecipitated using antibodies against Cdks (B) and E2Fs (D). Immunoblot analysis was then performed using the indicated antibodies. (A, C) The intensity of each protein relative to actin was determined by densitometric analysis and is indicated below each lane. (B, D) The intensities of p21 protein to Cdks and pRB protein to E2Fs were determined by densitometry analysis, respectively, and are shown below each lane.

MEEL, methyl alcohol extract of Enteromorpha linza; Cdk, cyclin-dependent kinase; IP, immunoprecipitation; pRB, retinoblastoma protein.

MEEL modulated the expression of apoptosis-related regulators and induced mitochondrial dysfunction in Hep3B cells

To examine the underlying mechanisms of MEEL-mediated Hep3B cell apoptosis, we investigated changes of apoptosis-related proteins. As shown in Fig. 3, when cells were treated to MEEL, the levels of inactive forms of caspase-3, caspase-8, and caspase-9 were greatly reduced, and their activities were significantly increased, which was associated with cleavage of poly(ADP-ribose) polymerase (PARP) (Fig. 3). In addition, MEEL promoted the expression of the representative pro-apoptotic Bax protein, but reduced the expression of Bcl-2 protein, a member of the anti-apoptotic protein (Fig. 4A). To further explore the role of mitochondria in MEEL-induced apoptosis, it was investigated whether MEEL could alter mitochondrial permeability by inducing a loss of MMP. As indicated in Fig. 4B, flow cytometry results showed that MEEL treatment resulted in disruption of MMP, which was associated with an upregulation of cytochrome c levels in the cytosolic fraction, together with a concomitant downregulation in its mitochondrial level (Fig. 4C). These data demonstrate that MEEL can destroy mitochondrial integrity by regulating Bcl-2 family proteins.

Fig. 3. Activation of caspase by MEEL in HEP3B cells. (A) The expression changes of the indicated protein were investigated using total proteins and the corresponding antibodies. Equal loading was confirmed with actin. The intensities of each pro-caspase and cleaved-PARP relative to actin were determined by densitometry analysis and are indicated below each lane. (B) The activity of each caspase is presented as a relative value of the untreated control group.

MEEL, methyl alcohol extract of Enteromorpha linza; PARP, poly(ADP-ribose) polymerase.

^*P < 0.05, ^**P < 0.01 and ^***P < 0.001 vs. control cells.

Fig. 4. Activation of the mitochondrial apoptotic pathway by MEEL in Hep3B cells. (A) The expression levels of Bcl-2 family proteins in MEEL-treated cells for 48 h were detected by immunoblot analysis using extracted total proteins. Equal loading was confirmed with actin. The intensities of Bax and Bcl-2 proteins relative to actin were determined by densitometric analysis and are indicated below each lane. (B) To measure MMP changes, cells stained with JC-1 were subjected to flow cytometry, and the numbers shown represent the frequency of cells with depolarized mitochondrial membranes. (C) After separation of cytoplasmic and mitochondrial fractions, the expression of cytochrome c was detected by immunoblotting. COX IV and actin were probed as loading controls for each fraction. The intensity of cytochrome c relative to actin and COX IV was measured by densitometric analysis and is shown below each lane.

MEEL, methyl alcohol extract of Enteromorpha linza; JC-1, 5,5,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimi-dazoylcarbocyanine iodide; MMP, mitochondrial membrane potential; COX IV, cytochrome c oxidase subunit IV; C.F., cytoplasmic fraction; M.F., mitochondrial fraction.

^*P < 0.05 and ^***P < 0.001 vs. control cells.

MEEL-induced reduction of MMP was associated with ROS generation in Hep3B cells

DCF-DA staining was performed to determine whether the induction of mitochondrial impairment by MEEL was induced through intracellular ROS generation. Flow cytometry results shown in Fig. 5A show that the accumulation of ROS started to increase within 30 min after MEEL treatment, reached a peak after 1 h, and then decreased. However, MEEL-induced ROS production was significantly abrogated to control levels in the presence of the ROS scavenger, NAC. Furthermore, the mitochondrial impairment after treatment with MEEL was strictly dependent on ROS generation, as evidenced by the recovery of MMP loss found in the pretreatment with NAC (Fig. 5B). In addition, the expression changes of Bax and Bcl-2 by MEEL treatment was not observed in the NAC pretreatment group (Fig. 5C), suggesting that ROS generation may underlie mitochondrial dysfunction induced by MEEL.

Fig. 5. Association of increased ROS production and mitochondrial dysfunction in MEEL-treated Hep3B cells. (A) Cells were incubated for different times in medium containing MEEL (250 μg/mL) or pretreated with NAC (10 mM) for 1 h, and then treated with MEEL (250 μg/mL) for 1 h. After treatment, the extent of ROS production was detected by flow cytometry, and the frequency of DCF-positive cells in each experimental group is shown. (B) To measure MMP changes, cells stained with JC-1 were subjected to flow cytometry, and the numbers shown represent the frequency of cells with depolarized mitochondrial membranes. (C) The expression levels of Bcl-2 family proteins were detected by immunoblot analysis. The intensities of Bax and Bcl-2 proteins relative to actin were determined by densitometric analysis and are indicated below each lane.

MEEL, methyl alcohol extract of Enteromorpha linza; ROS, reactive oxygen species; DCF-DA, 2',7'-dichlorofluorescein diacetate; NAC, N-acetyl-L-cysteine; JC-1, 5,5,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimi-dazoylcarbocyanine iodide; MMP, mitochondrial membrane potential.

^*P < 0.05 and ^***P < 0.001 vs control cells, ^###P < 0.001 vs. MEEL-treated cells.

MEEL induce ROS-dependent anti-cancer activity in Hep3B cells

Finally, we examined the effect of NAC on apoptosis and cell cycle arrest in MEEL-treated Hep3B cells to evaluate whether the generation of ROS acts as a determinant of MEEL-mediated anti-cancer activity. As shown in Fig. 6A and B, NAC pretreatment considerably reversed MEEL-mediated apoptosis, as evidenced by the results of DAPI staining. NAC also significantly lessened the number of cells belonging to the sub-G1 and G1 phases increased by MEEL treatment, while frequencies of cells in S and G2/M phases were maintained at control levels (Fig. 6C and D), which was accompanied by increased cell viability (Fig. 6E). Therefore, these findings indicate that MEEL mediated ROS levels to induce apoptosis associated with G1-phase cell cycle arrest.

Fig. 6. The role of ROS in MEEL-mediated growth arrest and apoptosis in Hep3B cells. Cells were cultured in medium containing MEEL (250 μg/mL) for 48 h, or treated with NAC (10 mM) for 1 h, and further treated with MEEL (250 μg/mL) for 48 h. (A, B) At the end of the treatment time, cells were stained with DAPI, and morphological changes of the nucleus were then observed (Scale bar: 100 μm). Arrows in the image indicate the nuclei of cells in which apoptosis has been induced. (B) The frequencies of apoptotic nuclei are presented as percentage of the total number of cells. (C, D) The frequency of each phase of the cell cycle was investigated using flow cytometry. (C) The average values of cells corresponding to the cell cycle progression stages were presented. (D) The frequencies of sub-G1 phase cells are shown. (E) Cell viability was measured by the MTT assay.

MEEL, methyl alcohol extract of Enteromorpha linza; ROS, reactive oxygen species; NAC, N-acetyl-L-cysteine; DAPI, 4′,6′-diamidino-2-phenylindole; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

^***P < 0.001 vs. control cells, ^###P < 0.001 vs. MEEL-treated cells.

DISCUSSION

In this study, we evaluated the effects and underlying mechanisms of MEEL involved in the induction of growth arrest and apoptosis in HCC Hep3B cells. As a result, it was shown that MEEL significantly inhibited the proliferation of Hep3B cells by inducing G1 cell cycle arrest and apoptosis accompanying ROS generation.

Cell cycle progression and arrest are tightly regulated by the activity of the cyclins and Cdks complexes [30]. In this study, MEEL concentration-dependently inhibited the cell viability and increased the population of cells corresponding to the G1 phase, suggesting that MEEL suppressed the growth of Hep3B cells by enhancing G1 phase arrest. Mechanistically, downregulation of cyclin D1 and cyclin E after MEEL treatment was observed, along with upregulation of Cdk inhibitor p21, without changing the expression of Cdks, E2Fs and p27. MEEL also diminished the phosphorylation of pRB in a concentration-dependent manner. Moreover, MEEL-induced p21 contributed to complex formation with Cdks and dephosphorylated pRB resulted in binding to E2Fs. As is well known, D-type cyclins phosphorylate pRB family proteins by activating Cdk4 and Cdk6, thereby relieving members of the E2F transcription factor from negative repression of pRB to proceed with transcription for the G1 to S phase transition [31,32]. Thereafter, the cyclin E/Cdk2 complex activated by the cyclin D/Cdk4/6 complex further phosphorylates pRB to enter the cell cycle S phase. p21 is one of the downstream genes of the tumor suppressor p53, which can bind and inactivate cyclin/Cdk complexes that mediate G1/S progression [33,34]. However, Hep3B cells used in this study are a p53 null cell line [35], suggesting that the increase in p21 expression by MEEL is independent of p53. Therefore, it can be inferred that MEEL inhibited the activity of the cyclin D/Cdk4/6 axis through induction of p21, thereby suppressing the phosphorylation of pRB to repress the transcriptional activity of E2F, and ultimately blocking entry into the S phase by suppressing the expression of cyclin E.

The apoptosis-inducting pathway is largely divided into an external pathway and an internal pathway according to the initiation signal, and caspases play critical role as implementers of both pathways [36,37]. Among them, the death receptor (DR)-mediated extrinsic pathway is initiated by an increase in caspase-8 activity upon binding of death ligands to DRs, while the mitochondria-mediated intrinsic pathway is mediated by the activation of caspase-9 caused by mitochondrial dysfunction. Since activation of these caspases completes apoptosis by activating effector caspase-3/7, caspase-8 and caspase-9 are commonly classified as initiator caspases, respectively, in both pathways [38,39]. Consistent with the results of our previous study using U937 human leukemia cells [22], MEEL induced the activation of caspase-8 and caspase-9 as well as caspase-3, resulting in degradation of PARP, a DNA repair enzyme that could be cleaved under the action of caspase-3. Among various factors regulating apoptosis, the Bcl-2 family proteins that perform an important function of mitochondrial-mediated apoptosis are composed of anti-apoptotic proteins and pro-apoptotic proteins [37,38]. Downregulation of anti-apoptotic proteins such as Bcl-2 along with upregulation of pro-apoptotic proteins including Bax may increase mitochondrial permeability, resulting in cytoplasmic release of apoptogenic factors such as cytochrome c. Accordingly, activation of the caspase cascade serves as a termination signal for apoptosis [38,39]. Activation of this typical mitochondrial-mediated apoptosis signaling pathway was clearly observed in MEEL-treated Hep3B cells. These results suggest that activation of not only the external pathways but also the internal pathways was in MEEL-induced apoptosis in Hep3B cells.

In normal physiological aspects, a certain level of ROS is required to maintain cellular function, but excessive ROS accumulation can lead to cell damage and death [40,41]. Indeed, many previous studies have suggested that the regulation of ROS is one of the key factors of apoptosis, and that oxidative stress due to intracellular redox imbalance can lead to excessive accumulation of ROS that promotes cell death [42,43]. The major intracellular organelles involved in ROS generation is mitochondria, and since ROS is highly reactive to various macromolecules involved in the electron transport chain, excessive accumulation of ROS is known to be the cause of mitochondrial dysfunction [44,45]. Therefore, we investigated whether the generation of ROS by MEEL is involved in apoptosis in Hep3B cells and found that ROS production peaked within 1 h after MEEL treatment, but was completely offset by the presence of the ROS scavenger, NAC. Furthermore, the mitochondrial dysfunction induced by MEEL exposure was entirely dependent on ROS generation, and the altered expression of Bcl-2 family proteins was also abolished by NAC pretreatment. Consequently, blockade of ROS production could reverse MEEL-induced apoptosis and cycle arrest. These results well support that ROS generation serves as an upstream regulator at least in the anti-cancer activity of MEEL in Hep3B cells. As many previous studies have shown, the active ingredients of seaweed may include polysaccharides, polyphenols, carotenoids, vitamins, minerals, and sterols [46,47]. According to the results of Narasimhan et al. [21], the antioxidant and antiproliferative activities of methanol extracts of seaweeds, including E. linza, were attributed to their high phenol content. Additionally, polysaccharides extracted from this green alga have been reported to have strong antioxidant activity and enhance the proliferation capacity of B and T lymphocytes [18]. Although components belonging to polysaccharides or polyphenols are thought to be the main bioactive components with potent anticancer activity in MEEL, it is currently unclear. Therefore, further research is needed on animal tumor models based on analysis of MEEL components and anti-cancer activity according to the type of cancer cells.

In summary, the results of this study demonstrated that treatment of HCC Hep3B cells with MEEL markedly inhibited cell survival, and triggered cell cycle arrest in the G1 phase (Fig. 7). Down-regulation of cyclin D1 and cyclin E, dephosphorylation of pRB, and up-regulation of p21^WAF1/CIP1 revealed the mechanism of MEEL involving G1 phase arrest. MEEL also induced apoptotic cell death through activation of extrinsic and intrinsic pathways, followed by a sequence of events including activation of caspase cascade, cleavage of PARP, modulation of Bcl-2 family proteins expression, reduction of MMP, and cytosolic release of cytochrome c. Furthermore, MEEL enhanced the generation of ROS. However, such effects of MEEL on growth arrest and apoptosis were markedly attenuated by an ROS scavenger, indicating that MEEL possessed ROS-dependent anti-cancer activity in Hep3B cells.

Fig. 7. Proposed mechanism for cell cycle arrest-mediated apoptosis by MEEL in HCC Hep3B cells.

MEEL, methyl alcohol extract of Enteromorpha linza; NAC, N-acetyl-L-cysteine; MMP, mitochondrial membrane potential; Cdk, cyclin-dependent kinase; pRB, retinoblastoma protein; PARP, poly(ADP-ribose) polymerase.