Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Glioblastoma-Specific Anticancer Activity of Pheophorbide a from the Edible Red Seaweed Grateloupia elliptica

  • Cho, MyoungLae (East Sea Research Institute, Korea Institute of Ocean Science and Technology) ;
  • Park, Gab-Man (Department of Parasitology, Kwandong University, College of Medicine) ;
  • Kim, Su-Nam (KIST Gangneung Institute) ;
  • Amna, Touseef (Department of Microbiology, Kwandong University, College of Medicine) ;
  • Lee, Seokjoon (Department of Pharmacology, Kwandong University, College of Medicine) ;
  • Shin, Woon-Seob (Department of Microbiology, Kwandong University, College of Medicine)
  • Received : 2013.08.30
  • Accepted : 2013.11.28
  • Published : 2014.03.28
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Abstract

The chlorophyll-related compound pheophorbide a (Pa) was successively purified from an edible red seaweed, Grateloupia elliptica, using silica, octadecyl silica column chromatography and reversed phase-high-performance liquid chromatography, as well as the cell cycle inhibitory and apoptotic effects of Pa being investigated in U87MG glioblastoma cells. The Pa exhibited strong anticancer effects in the absence of direct photo-irradiation against various cancer cell lines, including U87MG, SK-OV-3, and HeLa cells. Among the cancer cells, the strongest anticancer activity of Pa exhibited on U87MG cells with $IC_{50}$ values of 2.8 ${\mu}g/ml$. In addition, Pa specifically had cytostatic activity on glioblastoma cells rather than human umbilical vein endothelial cells. Analysis of the cell cycle distribution showed that Pa induced G0/G1 arrest of U87 MG cells. In addition, arrested cells induced late apoptosis and DNA degradation under dark condition. These results suggest that Pa isolated from G. elliptica is a potential glioblastoma-specific anticancer agent without side effects on normal cells.

Keywords

Introduction

Glioblastoma multiforme (GBM) is the most common type of brain tumors and is a rapidly progressive and biologically aggressive disease with a high mortality rate, with patients usually surviving less than 12 months following initial diagnosis [29, 36]. GBM patients are normally treated with surgery followed by radiation and chemotherapy [1, 34]. However, several anti-GBM agents used as chemotherapeutics have displayed serious side effects in patients, owing to nonspecific cytotoxic effects on both tumor and normal cells [14]. Therefore, the development of safe GBM therapeutic agents without side effects has become an urgent issue, and many studies have been conducted to identify cytotoxic effects selective against only cancer cells [23, 31]. Recently, cell cycle inhibition and/or induction of apoptosis in glioblastoma cells have come to be appreciated as targets for the management of GBM [11]. Regulation of the cell cycle machinery may induce arrest in different phases of the cell cycle and thereby reduce the growth and proliferation of cancer cells [12]. Additionally, cell cycle arrest of cancer cells may be related to apoptosis, as well as the magnitude of DNA damage [6].

Natural products derived from marine resources are attractive sources for the development of new medicinal and therapeutic agents, and more than 3,000 new anticancer compounds have been identified from marine organisms, such as tunicates, microorganisms, sponges, and sea hares [28, 30]. In a recent study, marine seaweedderived natural anticancer compounds were isolated from brown algae, such as fucoxanthin from Ishige okamurae [20] and eckol from Ecklonia cava [16], as well as red algae, including elatol from Laurencia microcladia [10]. However, despite extensive research on potential anticancer compounds from various types of marine seaweeds, few studies have specifically evaluated the selective anticancer compounds in red seaweeds. In the current study, during in vitroscreening of various marine seaweeds for potential anticancer agents, we observed that methanol extracts of Grateloupia elliptica effectively inhibited U87MG glioblastoma cells. The red seaweed G. elliptica is one of the most popular edible seaweeds in Northeast Asian countries, where it is also commonly used for making glue. Although the potent α-glucosidase inhibitory activity of bromophenols from G. elliptica has been previously established [21], the isolation of pheophorbide a (Pa) from G. elliptica, and its selective anticancer activity on glioblastoma cells in the absence of direct photo-irradiation, have not been reported. Therefore, in this study, we isolated Pa from G. elliptica, verified by the results of nuclear magnetic resonance (NMR) and other analytical data, and the glioblastoma-specific anticancer activity of the Pa was investigated.

 

Materials and Methods

Sample and Materials

The edible red seaweed G. elliptica was collected at a nearby seashore area in Jangho, Samcheck, Korea in March 2010. The raw sample was immediately frozen and stored at -70℃ until further use.

Phosphate-buffered saline (PBS), trypsin, L-glutamine, fetal bovine serum, and Trizol reagent were purchased from Gibco-Invitrogen Co. (Paisley, UK). RPMI-1640 medium was purchased from Lonza Inc. (Walkersville, MD, USA). Agarose, propidium iodide, and 100-base-pair DNA ladder were obtained from Promega (Madison, WI, USA). MTT (3-(4,5-dimethylthiazol-2-yl)2,5-dipheneyl tetrazolium bromide) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Reversed-phase gel (ODS-AQ, YMC, Japan) and silica gel 60 (Merck, Germany) were used for column chromatography. All other chemicals and reagents used in this work were of analytical grade.

Purification and Identification of Pa from G. elliptica

The raw samples (G. elliptica, 10 kg) were extracted twice with methanol (20 L) at room temperature for 24 h. After filtration, the crude extracts were evaporated to dryness under a rotary evaporator and a vacuum drier at 30℃. The crude extracts (150.3 g) were then dissolved in distilled water and partitioned sequentially in three different solvents, n-hexane (HX), chloroform (CF), and ethyl acetate (EA), to fractionate polar and nonpolar compounds. The EA fraction (7.82 g) was then further fractionated using a silica gel column (Kieselgel 60, 70-230 mesh; Merck) with the following solvent conditions: HX/EA (2:1, 200 ml) and HX/EA/MeOH (2:1:0.2, 600 ml). The strongly active fractions (750 mg) were collected and further purified with C18 reversed-phase column chromatography (ODS-AQ, YMC, Japan) by step-gradient elution with each of the different percentages of methanol (80-100%, 100 ml). In addition, the active compound (57.2 mg) was purified by reversed-phase high-performance liquid chromatography (RPHPLC) using a Waters HPLC system (breeze 1525; NY, USA) equipped with a Waters 2489 UV/Vis detector and a C18 analytical HPLC column (Chromolith performance RP-18e; Merck) at eluting conditions with a flow rate of 1 ml/min, an eluent of 90% acetonitrile, and a monitoring wavelength of 220 nm. Finally, the active compound (10.5 mg) was identified by comparing its 1H and 13C NMR data with the literature [17].

Cell Culture

Human glioblastoma cells (U87MG), mouse melanoma cells (B16-BL6), human epithelial carcinoma cells (HeLa), human cervical cancer cells (SiHa), and human ovarian cancer cells (SKOV-3) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). The cancer cell lines were cultured in DMEM with 10% fetal bovine serum, 100 μg/ml streptomycin, 100 U/ml penicillin, and 3.7 mg/ml NaHCO3. Human umbilical vein endothelial cells (HUVEC) were obtained from human umbilical cord veins, essentially as described by Jaffe et al. [18]. The HUVEC were cultured in M199 with EGM-2 medium containing growth factor (Cambrex, Walkersville, MD, USA) supplemented with 100 μg/ml streptomycin, 100 U/ml penicillin, and 3.7 mg/ml NaHCO3. The various cancer cells and HUVEC were incubated in an atmosphere of 5% CO2 at 37℃ and were subcultured every 3 days.

Anticancer Activity Assays

The anticancer activity of samples on various cancer cells was determined by MTT assay. Cancer cell lines (5 × 103 cells/well) were inoculated into a 96-well plate in triplicate, incubated overnight at 37℃ in the presence of 5% CO2, treated with Pa, and further incubated for 48 h. Twenty microliter of MTT solution (5 mg/ml) was added to each well, and the plates were incubated at 37℃ for 4 h. The supernatant was aspirated, and the MTTformazan crystals generated metabolically viable cells were dissolved in 200 μl of DMSO. The absorbance at 570 nm was determined using a microplate reader (SpectraMax250; Molecular Devices, CA, USA). For the various cancer cell lines, 50% suppression of viability was calculated relative to the control (blank) treatment and expressed as IC50.

The cytototoxic effect of Pa was determined using a viability assay of confluent cells (2 × 104 cells/well) as a resting cell after U87MG cells were treated with 50 μg/ml of Pa during 48 h under dark condition. In addition, the cytotostatic effect of Pa was determined by cell growth assay at the same condition to the above cytotoxic assay except for the initial cell concentration (5 × 103 cells/well).

Cell Cycle Analysis

For analysis of cell cycle phase distribution and apoptosis, U87MG cells (5 × 105 cells/well) were inoculated in a 6-well plate and incubated for 24 h. Subsequently, cells were incubated with Pa (50 μg/ml) and harvested at various time points. For flow cytometric analysis, cells were washed with PBS and fixed with 70% ethanol for 60 min at room temperature. After washing, the cells were centrifuged, rehydrated with PBS, and stained with 1.5 μM of propidium iodide (PI) solution. Stained cells were then analyzed by a flow-cytometer (Becton & Dickinson Biosciences, CA, USA).

Detection of Morphological Change and DNA Fragmentation in U87MG cells

U87MG cells (5 × 105 cells/well) were seeded in a 6-well plate and incubated for 60 h with Pa (50 μg/ml). After cultivation, the cell morphological change was observed under a phase-contrast microscope during 0, 12, 24, and 48 h. Subsequently, cells were trypsinized, harvested by centrifugation at 1,500 ×g for 5 min, and washed twice with 100 μl of PBS buffer. The cells were then resuspended in 400 μl of DNA lysis buffer (200 mM Tris-HCl, 100 mM EDTA, 1% SDS, pH 8.3), and DNA was extracted using Trizol reagent. The extracted DNA (40 μg) was resuspended in 20 μl of Tris-acetate EDTA buffer supplemented with 2 μl of sample buffer (0.25% bromophenol blue and 30% glyceric acid). The fragmentation pattern of the extracted genomic DNA was visualized by electrophoresis in a 2% agarose gel containing 1 μg/ml ethidium bromide and photographed under ultraviolet transillumination.

Statistical Analysis

The selective anticancer activity and cell viability assays were performed in triplicate, and the cell cycle and DNA fragmentation tests were performed in duplicate. The data are presented as the mean values ± standard deviations. The comparison of quantitative variables was performed using analysis of variance (ANOVA), and the differences were calculated using Tukey’s test (p < 0.05). The IC50 values were determined with GraphPad Prism software (ver. 3.0; GraphPad Software, San Diego, CA, USA).

 

Results

Purification and Identification of Pa from G. elliptica

Among the solvent-partitioned fractions tested in the current study, the EA fraction exhibited the highest anticancer activity on U87MG cells, and was subjected to silica gel and C18 reversed-phase chromatography. Ultimately, the fraction with the strongest anticancer activity toward U87MG cells was isolated via C18 reversed-phase HPLC, and the structure and molecular weight of the active compound identified it as Pa (Fig. 1) based on a comparison of the NMR and QTOF-MS data with previous literatures [9, 17].

Pheophorbide a: Dark green amorphous solid; QTOF-MS: m/z 593.3062 [M+H]+ (C35H36N4O5); 1H-NMR (CDCl3, 500MHz): δ 9.51 (s, 10-H), 9.36 (s, 5-H), 7.88 (dd, J = 17.5, 11.0 Hz, 31-H), 6.12 (d, J = 11.0 Hz, 32-H), 6.22 (s, 132-H), 4.22 (brd, J = 7.5 Hz, 17-H), 4.46 (q, J = 7.0 Hz, 18-H), 3.67 (q, J = 7.5 Hz, 81-H), 3.68 (s, 121-Me), 3.21 (s, 21-Me), 3.39 (s, 71-Me), 3.87 (s, 152-CO2Me), 2.63 (m, 171-CH2), 2.28 (m, 172-CH2), 1.68 (t, J = 7.5 Hz, 82-Me), 1.82 (d, J = 7.5 Hz, 181-Me), -1.69 (NH); 13C-NMR (CDCl3, 125MHz): 11.2 (C-71), 12.1 (C-21), 12.1 (C-121), 17.3 (C-82), 19.4 (C-81), 23.1 (C-181), 29.5 (C-172), 30.6 (C-171), 50.0 (C-18), 50.9 (C-17), 52.9 (C-132, CO2Me), 64.6 (C-132), 92.5 (C-20), 97.5 (C-5), 104.4 (C-10), 104.8 (C-15), 122.9 (C-32), 128.9 (C-13), 128.9 (C-31), 128.9 (C-12), 131.9 (C-2), 136.2 (C-3), 136.5 (C-4), 137.8 (C-11), 142.1 (C-1), 145.1 (C-8), 148.5 (C-9), 151.0 (C-14), 155.4 (C-6), 160.5 (C-16), 169.6 (C-132, CO2), 172.2 (C-19), 177.3 (C-172, CO2), 189.6 (C-131).

Fig. 1.Chemical structure of Pa isolated from G. elliptica.

Anticancer Activity of Pa

The IC50 values of Pa on the various cancer cell lines (U87MG, B16-BL6, HeLa, SiHa, and SK-OV-3) and HUVEC ranged from 2.8 to 18.3 μg/ml (Table 1), with the U87MG cells exhibiting the strongest anticancer effect (IC50 = 2.8 μg/ml) followed by the SK-OV-3 (7.0 μg/ml) and HeLa (9.5 μg/ml) cell lines. In addition, a cytotoxic effect of Pa was not observed on normal endothelial HUVEC (IC50 > 50 μg/ml), although the positive control, paclitaxel, exhibited a significantly stronger cytotoxic activity (IC50 = 0.3 μg/ml). However, in the B16-BL6 and SiHa cell lines, Pa exhibited a markedly weak anticancer effect. These results imply that Pa obtained from G. elliptica has a selective anticancer effect on glioblastoma cells, with no toxic effects on normal endothelial cells. Therefore, we decided to further investigate the effects of Pa on the growth and viability of U87MG cells.

Table 1.The IC50 value was defined as a 50% reduction in absorbance at a wavelength of 570 nm. Various cancer cell lines were inoculated in 96-well plates at a density of 5 × 103 cells/well. In addition, HUVEC were inoculated in a 48-well plate at a density of 1× 104 cells/well.

Cytostatic Activity of Pa on U87MG cells

The inhibitory effect of Pa on cell growth was determined using U87MG and HUVEC in dark condition. As shown in Fig. 2A, Pa (0-20 μg/ml) significantly reduced U87MG cell growth in a dose-dependent manner in the absence of direct photo-irradiation, indicating that Pa exerted an inhibitory effect on glioblastoma cells under photoindependent conditions. In contrast, even the highest treatment concentration of Pa (20 μg/ml) was not observed to have a growth inhibitory effect on HUVEC. These results suggest that Pa may have strong growth inhibitory effects specific only to the human glioblastoma cell line U87MG. Consequently, in order to know whether Pa has a cytostatic or cytotoxic effect, we investigated the effects of Pa and paclitaxel on cell survival in fully confluent U87MG cells. The results revealed that Pa treatment of the U87MG cells at a concentration of 50 μg/ml for 48 h did not decrease cell viability, whereas paclitaxel at the concentration of 10 μg/ml exhibited a strong cytotoxic effect on U87MG cells from 12 h onwards (Fig. 2B). These cell survival and viability data clearly indicated that Pa specifically inhibits growth of U87MG cells, instead of HUVEC as normal cells.

Fig. 2.Growth inhibitory activity of Pa on U87MG cells and HUVEC (A), and cell viability effects of Pa and paclitaxel on U87MG cells (B). Cell growth and survival were measured using the MTT method. The values are expressed as the mean ± SD of triplicate experiments. The cell viability was measured every 12 h during a 2 day incubation.

Analysis of Cell Cycle and Apoptosis Effects on U87MG Cells

Cancer cell growth may be inhibited by targeting multiple different pathways, including cell cycle inhibition, necrosis, and apoptosis. In the present study, we investigated the cell cycle inhibitory and apoptotic effects of Pa on U87MG cells in dark condition. Cell cycle analysis of U87MG cells revealed that the population in G0/G1 phase increased (12.5%) and the population in G2/M phase was decreased (10%) after treatment with Pa (50 μg/ml) for 16 h compared with the control (Fig. 3A). Moreover, the sub-G1 fraction of 87MG cells increased significantly (19.2%) compared with the control (Fig. 3B).

Apoptosis was confirmed by the presence of apoptotic bodies and nuclear condensation [20]. Observations with an inverted microscope showed that Pa induced morphologic change of the cell from a spindle type to a round type when U87MG cells were treated with 50 μg/ml of Pa without photoactivation. In addition, U87MG cell apoptosis and accumulation of cell debris were also observed after Pa treatment for 60 h (Fig. 4A). Treatment of U87MG cells with Pa for 60 h significantly induced DNA degradation and fragmentation (Fig. 4B). These results are consistent with our earlier results relating to increased sub-G1 phase arrest for apoptosis of U87MG cells. We observed evidence for apoptosis, including cell debris and DNA fragmentation. Previous studies have reported that photoactivated Pa is capable of inducing apoptosis in cancer cells [3, 15, 33]. However, in the present study, Pa isolated from G. elliptica showed U87MG cell-specific cytostatic activity, and induced G0/G1 phase arrest and late apoptosis on U87MG cells in the absence of direct photoactivation.

Fig. 3.Effects of Pa (50 μg/ml) on cell cycle distribution (A) and the population in sub-G1 phase (B) in U87MG cells. Cells were harvested at each time interval and stained with 1.5 μM propidium iodide solution. Cell cycle and sub-G1 fractions of the U87MG cells were analyzed by a flow-cytometer according to the procedure in Material and Methods.

Fig. 4.Effects of Pa on cellular morphology (A) and DNA fragmentation (B) in U87MG cells. Changes in the cellular morphology of U87MG cells treated with Pa (50 μg/ml) were photographed under a phase-contrast inverted microscope at 100× (A-E) or 200× (F); A (Con 0 h), B (Con 24 h), C (Con 48 h), D (Sam 24 h), E (Sam 48 h), and F (Sam 60 h). The arrows indicate cell debris and apoptotic body. The DNA fragmentation of U87MG cells was analyzed by treatment with 50 μg/ml of Pa for 60 h, after which cells were harvested at each time interval, and DNA was extracted with Trizol-reagent, and DNA in the loading buffer was loaded on 2% agarose gel containing 1 μg/ml of ethidium bromide. After electrophoresis, the gel was photographed under ultraviolet transillumination.

 

Discussion

Red seaweeds are very popular edible seaweeds that are consumed as health foods by many people in Japan, China, Thailand, and South of Korea. In the current study, Pa isolated from the edible red seaweed G. elliptica showed specific anticancer activity toward glioblastoma (Table 1). Pa is the product of chlorophyll breakdown and contains a porphyrin ring, which has been reported to be important for photoactivation assays and photodynamic therapy (PDT) [5, 7, 32]. However, chlorophylls did not affect on both U87MG and HUVEC growth in the absence of direct photoactivation (data not shown), because chlorophylls are a photosynthetic pigment and chemoprevention compound [27]. PDT is used therapeutically in a variety of cancers, such as skin, gastric, lung, and prostate cancers [2, 19, 26, 37]. The reaction mechanism of PDT can be explained in several steps: PDT compounds penetration and fixation in cellular membrane; photosensitized formation of high reactive singlet oxygen; and damage to essential cellular components such as nucleic acid, proteins, and lipids [8]. It was previously reported that Pa isolated from the land plants Carpinus betulus and Neptunia oleracea exhibited strong anticancer activities on various cancer cell lines [6, 25], and in vivo studies of Pa have also shown strong therapeutic efficacy in various cancers in the photoirradiation condition [4, 13]. These studies demonstrated that Pa is a photosensitizer that can inhibit the growth of various cancer cells with photoactivation. However, widespread use of PDT to treat solid tumors is difficult owing to the cost, complexity, and availability of the laser and light systems [24]. Glioblastoma cells have shown resistance to various pro-apoptotic stimuli, and as a result, glioma in patients has dismal prognoses [22]. Therefore, the development of safe glioblastoma-specific anticancer agents has become an important issue.

Recently, Islam et al. [17] reported on the anti-inflammatory activity of Pa in the absence of direct photoactivation. Pa isolated from a brown seaweed, Saccharina japonica, inhibited LPS-induced nitric oxide production and inducible nitric oxide synthase. Thus, it is possible that Pa also has biological activity under photo-independent condition. In the current study, we investigated the glioblastoma-specific anticancer activity of Pa isolated from G. elliptica and analyzed its cell cycle inhibitory effect on U87MG cells (Table 1, Fig. 3). The data clearly demonstrated that the Pa indeed displayed glioblastoma-specific anticancer activity, G0/G1 phase arrest, and apoptotic effects on U87MG cells, even in the absence of direct photo-irradiation. These results suggest that Pa affects the G1/S checkpoint instead of the G2/M checkpoint in the U87MG cell cycle, although the mechanism of G0/G1 arrest is not known. Moreover, Pa may affect checkpoint inhibitors in the G2/M phase. Therefore, Pa specifically inhibited only growing U87MG cells but not resting U87MG (Fig. 2). In addition, the growth inhibition (G0/G1 phase arrest) of U87MG cells induced by Pa treatment was associated with apoptosis and nuclear damage. Consistent with our data, it has been reported that, in Hep3B cells, Pa isolated from Scutellaria barbata induced apoptosis, degraded genomic DNA, and induced sub-G1 cell cycle arrest with non-toxicity on normal human liver cells under photo-activation [6]. Necrosis is a pathological process that occurs when cells are exposed to a severe physical or chemical injury. In contrast, apoptosis is one of the most fundamental biological processes, and is the process of cell death by activation of suicide mechanisms [35].

Cancer-cell-specific anticancer activity is very important for anticancer drug development. Therefore, selective inhibitors of glioblastoma may provide clinically effective anti-GBM therapies, resulting in a marked reduction in malignant primary brain tumor burden. In the present study, Pa inhibited only the growth of glioblastoma cells (U87MG) despite not being under photoactivation. Thus, the glioblastoma-specific anticancer effect of Pa isolated from G. elliptica may be due to different reactions than other photoactivation anticancer activities. Further investigations are necessary to determine the intracellular signal pathways affected by Pa treatment in the absence of photoactivation. Additionally, the glioblastoma-specific anticancer activity of Pa should be explored using an in vivo experiment to provide a better understanding of its side effects on normal organs.

In conclusion, the glioblastoma-specific anticancer compound Pa was purified from the red seaweed G. elliptica by bioactivity-guided isolation, and the cell cycle and apoptotic effects were investigated using U87MG cells. Despite the lack of photo-irradiation, Pa showed strong anticancer effects on U87MG cells (IC50 = 2.8 μg/ml), whereas nontoxic effects were observed on resting U87MG cells or normal HUVEC. The glioblastoma growth inhibitory activity of Pa was associated with cell cycle arrest in the G0/G1 phase and apoptosis, with degradation of genomic DNA. Hence, these results suggest that Pa isolated from G. elliptica could be a good source for glioblastoma-specific therapy with no clinical side effects.

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