Antiproliferative Activity of Microalgal Extracts on ...

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Malaysian Jo~mralof Science 27 (2): 19 - 3 1 (2008)

Antiproliferative Activity of Microalgal Extracts on Nasopharyngeal Carcinoma (NPC) Cells P. J. LaiIg4;W. L. chu2*; R. ~ a i d u " A. ~ ; S. B. ~ h o o Y. ~ ;Y. Kok I; Shar, M. M?; S. N. Ling2; J. W. ~ a k ' ;K. C. Lim4; P. Balraj4 and S. M. Phang5 I

Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia International Medical University, 126 Jalan 19/155B, 57000 Kuala Lumpur, Malaysia * [email protected] Telephone: 6 03 86567228 Facsimile: 6 03 86567229 (corresponding author) 3 School of Medicine and Health Sciences, Monash University Sunway Campus, Jalan Lagoon Selatan, 46150 Selangor, Malaysia 4 Molecular Pathology Unit, Institute for Medical Research, Jalan Pahang, 50588 Kuala Lumpur, Malaysia 5 Institute of Biological Sciences and Institute of Ocean and Earth Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia Received 11"' April 2008, accepted in revised form lothSeptember 2008. 2

ABSTRACT Nasopharyngeal carcinoma (NPC) is a type of epithelial neoplasms arising from the nasopharynx. The disease is frequently encountered in southern China, Taiwan and Hong Kong. Algae are known to be a potential source of bioactive compounds with antiproliferative activity on cancer cells. The objective of this study was to screen extracts of microalgae from the University of Malaya Algae Culture Collection (UMACC) for antiproliferative activity against NPC cell lines. Solvent and aqueous extracts of 19 microalgae from the UMACC were screened for antiproliferative activity on four NPC cell lines, namely CNE1, HONEI, TWOl and TW04. The methanol extracts from two microalgae, namely Ankistrodesmus convolutus UMACC 101 and Synechococcus elongatus UMACC 105 showed antiproliferative activity on the NPC cell lines, with ICS0values ranging from 95.7 to 98.OpglmL. The methanol extract from Synechococcus elongatus was subjected to fractionation by column and thin layer chromatography (TLC) using a combination of hexane and acetone at different ratios. Fraction 7' obtained from the pooled eluents of 6:4, 5 5 , 4:5 and 3:7 hexane-acetone was the most active, with IC50 values of 10.2 and 4.8 pg/mZ, on CNEl and TWO4 cells respectively. The cytotoxic effect of Fraction 7'was evident against CNEl and TWO4 cells after 24 and 48h respectively at 30.0 pg/mL. The mechanism of action of Fraction 7' could be through induction of apoptosis as indicated by the increased percentage of cells in the Sub-GO phase in cell cycle analysis by flow cytometry. The active compound in Fraction 7' could be a derivative product of chlorophyll a, as indicated by the blackish green spot after separation by TLC. Further studies are warranted to elucidate the structure of the active principle in Fraction 7'. ABSTRAK Nasofarin Karsinoma (NPC) merupakan neoplasm yang timbul daripada epithelium nasofarin. Insiden penyakit tersebut adalah kerap di kalangan penduduk di Selatan China, Taiwan dan Hong Kong. Alga merupakan sumber bahan bioaktif dengan aktiviti antiproliferatif terhadap sel kanser. Objektif kajian ini adalah untuk menyaring extrak daripada koleksi mikroalga dalam University Malaya Algae Culture Collection (UMACC) untuk aktiviti antiproliferatif terhadap sel NPC. Aktiviti antiproliferatif ekstrak daripada 19 mikroalga dalam koleksi tersebut terhadap empat jenis sel NPC berikut telah disaring : CNEI, HONEI, TWOl dan TW04. Ekstrak metanol daripada tiga mikroalga, iaitu Ankistrodesmus convolz~tusUMACC 105 dan Synechococcz~selongattls UMACC 105 menunjukkan aktiviti antiproliferatif terhadap sel NPC, dengan nilai ICso dalam julat 95.7 hingga 98.0 pglmL. Pemisahan ekstrak metanol daripada Synechococcus elongatus kepada beberapa fiaksi dengan kromatografi kolurnn dan lapisan nipis (TLC) meng'gunakan kombinasi hexan dan aseton juga dilakukan. Fraksi 7' yang diperolehi daripada kombinasi eluen 6:4, 5 5 , 4:6 dan 3:7 hexan-aseton adalah paling aktif, dengan ICS0sebanyak 10.2 dan 4.8 pg/rnL terhadap sel CNEl dan TW04. Kesan sitotoksik Fraksi 7' terhadap CNEl dan TWO4 adalah ketara selepas 24 dan 48 jam pada 30.0 pg/mL. Mekanisme tindakan Fraksi 7' adalah melalui kesan apoptosis memandangkan peratus sel dalam fasa Sub-GO

meningkat dalam analisa flow sitometri. Kompound aktif dalam Fraksi 7' besar kemungkinan adalah produk deriviatif klorofil a, seperti yang ditunjukkan oleh tompokan hijau gelap selepas pemisahan dengan TLC. Kajian lanjut adalah wajar untuk mengenalpasti struktur kompoun aktif dalam Fraksi 7'. (Microalgae, nasopharyngeal carcinoma (NPC), antiproliferative activity, Ankisti-odesmus convolutus, Syneclzococci~seloi~gatus)

INTRODUCTION Nasopharyngeal carcinoma (NPC) is a type of epithelial neoplasms arising from any part of the nasopharynx [I]. Although NPC is rare in most parts of the world, it is endemic in a few welldefined populations [2]. The disease is frequently encountered in southern China, Taiwan,'Hong Kong, among Eskimos of the Arctic region, and to a lesser extent in Southeast Asia. In Malaysia, the ethnic group of Bidayuh in Sarawak has been shown to have a high risk of NPC [3]. The aetiology of NPC is multifactorial involving virological, genetic and environmental factors. Epstein-Barr virus (EBV), a type of gamma herpesvirus, is strongly linked to the development of NPC. Environmental factors such as the consumption of Cantonese-style salted fish and other preserved foods containing volatile nitrosamines are important carcinogenic factor of NPC 121. NPC patients are managed using a combination of surgery, radiotherapy and chemotherapy based on the stage of the disease [I]. However, both radiotherapy and chemotherapy can cause severe side effects to the patient. In view of this, there is a need for more effective drugs with fewer side effects. Diugs developed from natural products have often been sought after for the treatment of cancer. Algae are a potential source of bioactive compounds ranging from fatty acids, carotenoids, steroids, polysaccharides, lectins, halogenated compounds to polyketides [4]. A major effort to screen more than 1000 strains of cynaobacteria (blue-green algae) for antineoplastic compounds was carried out in the 90's [5]. Since then, there have been many reports on extracts and compounds from algae which show antiproliferative activity on cancer cells. For instance, both the methanol extract and the active compound caulerpenyne from the marine alga

CaziIerpa 7-acemosa have significant antiproliferative effect on neuroblastoma cell lines [6]. The cytotoxic activity of purified caulerpenyne is comparable to the commercial anticancer drug etoposide. The extracellular polysaccharide from the marine microalga Gymnoclinium inhibits growth of human myeloid leukaemia cells by . acting on DNA topoisomerases [7]. The polysaccharide also shows significant antiproliferative activity on various cancer cell lines, inbluding breast, colon, lung, ovary, kidney and stomach cancers. Recently, Teruya et al. (2007) [8] demonstrated that oversulfated fi~coidan(polysaccharide) from the brown alga Cladosiphon okanzurarzus has antiproliferative activity on human leukemia cells by inducing apoptosis. Other compounds from algae which show antiproliferative activity on cancer cells include phycocyanin and fatty acids. For instance, recombinant C-phycocyanin from Anabaena has been shown to inhibit proliferation of cancer cells by inducing apoptosis [9]. Palrnitic acid from the red alga Amplziroa zonnta shows cytotoxic activity on human leukemic cells [lo]. The antiproliferative activity of algae extracts may also be due to their antioxidant properties. For instance, the antiproliferative activity of extracts from Laminai-in and Porphym on HeLa cells are positively correlated with their total polyphenol contents [ll]. .Many cancer cell lines have been used for the screening of algal extracts and compounds for anitproliferative activity. However, there have been no reports on the effect of algal extracts and compounds on NPC cells. The algae of Malaysia are a potential biological resource that is yet to be fully explored. A collection of microalgae isolated from various habitats in Malaysia 5as been established at the University of Malaya Algae Culture Collection (UMACC) [12, 131. Some of the strains have been shown to produce appreciable amounts of high-value chemicals such as polyunsaturated

'

fatty acids and carotenoids [14, 151. Here, we report on the results of our first screening of 19 microalgal strains from the collection for antiproliferative activity against NPC cell lines. The methanol extract which showed biological activity was subjected to bioassay-guided fractionation using chromatographic techniques. The effect of the active fraction on the cell cycle was investigated using flow cytometry. MATERIALS AND METHODS Cell lines Four EBV-negative NPC cell lines, CNE1, HONE1, TWO1 and TWO4 were used in this study. The cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum and 500 unitslml penicillin and 500' p g l d streptomycin. The cultures were maintained at 37OC in a humidified atmosphere with 5% C02.

Microalgae A total of 19 strains of microalgae from the University of Malaya Algae Culture Collection (UMACC), consisting of four marine and 15 freshwater strains were screened (Table 1). The freshwater microalgae were grown in Bold Basal Medium (BBM) [16] while Spirulirza platensis UMACC 161 and Synechococcus elongatus UMACC 105 were grown in Kosaric Medium [12]. The marine microalgae were grown in Provasoli Medium [ 171. The cultures were placed on illuminated shelves (42 pmol/m2/s-I, 12:12 h light-dark cycle) in an air-conditioned room (28'C). Upon attaining stationary phase, the cells were harvested by centrifugation (3,000 rpm, 15 min) and the cell pellets were dried at 37OC before extraction.

List of microalgae from the University of Malaya Algae Culture Collection (UMACC) Table 1. screened for antiproliferative activity on nasopharyngeal carcinoma (NPC) cell lines STRAIN ISOLATE NUMBER HABITAT Cyanophyta (Blue-green algae) Synechococcus elorzgattrs UMACC 105 Freshwater Spir-ulinn platensis UMACC 159 . Brackish, alkaline waters Spirzllina platensis UMACC 160 Brackish, alkaline waters Spii-trliizaplatensis UMACC 161 Brackish, alkaline waters Spirulina platensis UMACC 162 Brackish, alkaline waters Spil-tilinaplatensis UMACC 199 Brackish, alkaline waters Spirulina platensis UMACC 200 Brackish, alkaline waters Oscillatorin sp. UMACC 2 16 Marine A~zacystissp. UMACC 244 Marine Chlorophyta (Green algae) Chlorella vulgaris UMACC 001 Freshwater Chlorella sp. UMACC 245 Freshwater Chlorococcunz sp. UMACC 074 Freshwater Arzkistrodesnztrs convoluf~rs UMACC 101 Freshwater Prasinococcus sp. UMACC 168 Marine Stichococcus sp. UMACC 246 Freshwater Klebso~r7zidiumsp. UMACC 247 Freshwater Bacillariophyta (Diatoms) Arnphiprora sp. UMACC 239 Marine Pyrrhophyta (Dinoflagellates) Synzbiodiniurn sp. UMACC 188 Marine, symbiont Rhodophyta (Red algae) Porpl?vridiun cruentunz UMACC 196 Marine Preparation of extracts from microalgae The following solvents were used to prepare the extracts from the microalgae: methanol, chloroform, hexane and water. In addition, sequential extraction using chloroform followed by methanol, hexane followed by chloroform and

hexane followed by methanol was carried out. The dried biomass (25 - 30 mg) was mixed with the solvent (5 rnL) and homogenised using a tissue grinder. The mixture was centrifuged (3000 ipm) and the supernatant was transferred to a clean tube, before being evaporated using a

rotary evaporator (50°C). The dried extract was dissolved again in a small volume of solvent and transferred into a small vial before use for the cytotoxicity assay. For the sequential extraction, the algal biomass was mashed with the first solvent, and the residue was extracted with the second solvent. Fractionation of crude extracts The crude methanol extract of Synechococc~~s elongatus was subjected to fractionation using column and thin layer chromatography (TLC). This alga was chosen as it grew faster and produced higher biomass than Ankistrodesrnus con~~oluttu.The crude methanol extract (0.5 g)

was loaded onto silica gel (Merck, Germany) column and eluted with n-hexane-acetone at ratios ranging from 9:l to 3:7. The remaining sample was eluted with methanol. With this fractionation scheme, a total of 40 fractions were obtained. The fractions from the column chromatography (F1 - F40) were further analysed by TLC and grouped into eight fractions (F'l F'8). Details of the fractionation are given in Table 2. The pooled fractions based on the separation by TLC (designated as F'l to F'8) were used for cytotoxicity testing. The fractions were concentrated by vacuum concentrator and stored at 4OC until use for the testing.

Table 2. Fractions from the methanol extract of Sy~zechococcuselongatus used for cytotoxicity testing SOLVENT COLUMN THIN .LAYER

9: 1 hexane-acetone

8:2 hexane-acetone

I

FRACTIONS (F) F1 F2 - F4 F5 F6 - F7 A

7:3 hexane-acetone 6:4 hexane-acetone 5:5 hexane-acetone 4:6 hexane-acetone 3:7 hexane-acetone Methanol

I I I

FRACTIONS (F') ~ " 1 F'2 F'3

-

a"

F l l -F13 F14 - F15 F16 F17 - F20 ~21-~25 F26 - F30 F31 -F35 F36 - F40

Cytotoxicity testing Colorimetric assay based on 3-(4,5dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT ) was used for the cytotoxicity testing [la]. The MTT assay is based on the ability of the viable cells to reduce tetrazolium based compound to blue formazan product in their mitochondria. CNE1, TWO1, TWO4 and HONE1 cells were seeded at a concentration of 2 x 10' cellslml in 96-well plates. The extracts and fractions were dissolved in dimethyl sulfoxide (DMSO) before being added to the cell cultures. After overnight incubation, the crude microalgal extracts (0, 7.5, 15.0, 30.0 and 60.0 pglml) were added to the four NPC cell lines. For those crude microalgae extracts which showed a positive effect on the morphology of the NPC cells, hrther screening was carried out using higher concentrations, ranging from 30, 40, 50, 60, 70, 80, 90 to 100 pglrnl. The fractions of crude

F'6 -

F'7

F'8 methanol extract from Synechococcus elongatus were tested on two NF'C cell lines, CNEl and TWO4 at concentrations ranging from 0, 7.5, 15.0, 30.0 and 60.0 pglml. After 72 h incubation, the viability of the cells was assessed using MTT assay [la]. The assay was based on the. amounts of formazan product formed, which was determined by measuring the absorbance at 570 and 630 nm (background). The percentage of cell viability was determined as follows: [ODtreated cultures

OD untreated cultures 1 X loo

The concentration of the extracts and fractions that killed 50% of the cells (ICSO)was determined based on the percentage cell viability curves. The experiment was repeated at least three times for each testing.

Mdaysian Jollrrlal of Science 27 (2): 19 - 3 1 (2008)

The MTT assay for CNEl and TWO4 was repeated using Fraction 7' of the methanol elorzgatzrs at extract fsom Synechoccus concentrations ranging fsom 0, 3.75, 7.50, 15.00 to 30.00 pg/mL. The cells were harvested for the assay after 0, 24,48 and 72 11.

Cell Cycle Analysis The effect of the active fraction from the methanol extract of Syrzechococc~iselongattu on the cell cycle of CNEl and TWO4 cells was investigated using flow cytometry. The CNEl and TWO4 cells (3 mL) at 1.5 x 10' cell/mL were plated in six well plates and incubated for 24 h before being added with the active fraction at concentrations of IC50 defermined from the cytotoxicity testing. The cells were hawested by trypsinisation at 6, 12, 24, 48 and 72 h, together with the floating cells in the medium. After incubation, the cells were harvested by centrifugation (1000X g) and washed with Phosphate Buffered Saline (PBS). The cell pellets were fixed with 500 pL iced cold 80% ethanol and kept at -20°C for 2 h. The cells were washed twice with PBS before being stained with PBS containing 0.1% triton X-100, 10 mM EDTA, 50pg/ml RNase and 2pglml propidium iodide.

The cells were then incubated for 30 min at 4 OC before being analyzed by a flow cytometer (Beckman Coulter, USA) within 24 h. The cell cycle kinetics was acquired on 10,000 cells using the EX 32 v1.28 analysis software.

Statistical analysis One-way ANOVA followed by Duncan's Multiple Range test was used to compare the experimental data from the cell cycle analysis. '

RESULTS

Cytotoxicity testing A total of 133 solvent extracts from 19 strains of microalgae (nine cynobacteria, seven chlorophytes, one diatom, one dinoflagellate and one rhodophye) were screened for cytotoxic effect on four NPC cell lines (CNEl, HONE1, TWOl and TW04). In the preliminary screening, all the solvent extracts did not reduce the viability of the cell lines tested even at 60 pg/mL. However, the methanol extracts from Ankistrodesmuc convolutus, Syrzechococcus elolzgatus and Spirulina platensis caused morphological changes in cells exposed to high concentrations of the extracts (Figure 1).

a) Control b) Treated with methanol extracts Figure 1. Morphological changes in TWO4 cells after being incubated with 60 pg/mL of methanol extract fsom Ankistrodesmw co~zvolutwfor 72 h. The treated cells were rounded up (Arrow 1) with large space between the cells (Asrow 2). Scale bar = 50 pm. Further testing was carried out using methanol extracts of the t h e e microalgae at increased concentrations up to 100 pg/mL (Figure 2). Ankistrodesnzus Methanol extracts from col~volur~~s and Syrzeclzococcus elorzgatta, but not

Spirulinn platensis showed antiproliferative activity against CNE1, TWO4 and TWOl cells, giving ICso values ranging from 95.7 to 98.0 pg/mL (Table 3). In contrast, methanol extract from Spirulirza platerzsis was not active against

Malaysiart Joztrtral of Science 27 ( 2 ) : 19 - 3 1 (2008)

the NPC cell lines (ICS0> 100.0 p g l d ) . Of the cell lines tested, HONEl seemed to be the most resistant. Fractionation of the methanol extract from elongntus was carried out for hrther testing. There was marked increase in bioactivity against CNEl and TWO4 cells after fractionation of the extract (Figure 3). Fraction 7' was the most active fraction, with IC50values of 10.2 and 4.8 against CNEl and TWO4 cells respectively. In comparison, the ICSOvalues of cisplastin were 5.3 and 5.0 pg/mL respectively. The cytotoxic activity of Fraction 7'on the normal cell line NIH 3T3 (mouse fibroblast) was much lower than NPC cells, with ICS0 value of 40.0 pg1mL.

S'nechococc~is

Further tests were conducted on CNEl and TWO cells using Fraction 7' from Syi~eckococcus

elongnhls on a time-course manner. The cytotoxic effect was evident against CNEl and TWO4 cells after 24 and 48 h respectively at 30 pg/mL (Figure 4). Cell cycle analysis The percentage of cells at the different phases of the cell cycle was determined based on the DNA content analysed using flow cytometry. The percentage of cells in the Sub-GO phase for both CNE 1 and TWO4 increased with time when treated with Fraction 7' of the extract from Synechococcus elongatus (Figure 5). There was a significant decrease (p < 0.05) in the percentage of cells in the GOIG1 phase for CNE 1 after 24 h (Figure 3a). For TWO4 cells, there was a significant decrease @ < 0.05) in the percentage of cells in the S and G2lM phase after 24 h (Figure 3b).

Figure 2a. Percentage of cell viability of CNE1, TWO1, TWO4 and HONEl treated with methanol extracts from Ankistrodesmus convolutes. The cell viability was assessed by MTT assay after incubation with the extract for 72 h. Vertical bars show standard deviations from three replicates.

Malaysian Jorlrt~alof Scietlce 27 (2): 19 - 3 1 (2008)

160 143 n

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120

& 3 L% u

loo 80

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$ 0

60

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20 0 0

20

' 4 0

60 Comenlmth (119,ml)

+CNE1 *TVJ04

-TWO1

80

-x

100

120

- HONE1

Figure 2b. Percentage of cell viability of CNE1, TWOl, TWO4 and HONEl treated with methanol extracts from Syizeclzococcus elongatus. The cell viability was assessed by MTT assay after incubation with the extract for 72 h. Vertical bars show standard deviations from three replicates. 140

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Figure 2c. Percentage of cell viability of CNE1, TWOl, TWO4 and HONEl treated with methanol extracts from Spilwlina platensis. The cell viability was assessed by MTT assay after incubation with the extract for 72 h. Vertical bars show standard deviations from three replicates.

I

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Concentration +@nd)

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a) CNEl cells

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Concentration (Wn12)

+ -lF -'

-6-.F'2--+-F'3+

F74~F'5-m-F'6-m-F'7

:.:

F'8-e,ckp~in

b) TWO4 cells Figure 3. Percentage cell viability of TWO4 and CNEl cells after being treated with fractions from methanol extract of Synecl~ococcwelongatus and cisplatin. The cell viability was assessed by MTT assay after incubation with the extract or drug for 72 h. Vertical bars show standard deviations from thee replicates.

Mala)~siailJorrrtial o f Science 27 (2): I9 - 3 1 (2008)

24

Hour

48

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I-+-~ontml+3.75p&d~7.5~gfd+15~d&-)30~ddl

a) CNEl cells

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0.2

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72

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Control -3.75

pglrnl

7.5 pglml -nk--

15 pgfml -30

pglrnl

b) TWO4 cells Figure 4. Changes in absorbance at 570 nrn based on MTT assay of the a) CNEl and b) TWO4 cells after incubation with Fraction 7' from the methanol extract of Synechococczis elongatus. The control consisted of cells without any extract.

Table 3. Concentrations of methanol extracts (pg/rnL) from three microalgae which inhibit 50% of viability (ICSo) of the NPC cell lines based on MTT assay. Data are presented as mean standard deviation (n = 3).

+

MICROALGAL EXTRACT Aizkistr-oclesmus convolurt~s UMACC 101 Sy~zechococcuseloizgatt~s UMACC 10.5 Spii.ulirza plcltensis UMACC 161

CNEl 98.0 & 1.4

NPC CELL LINE TWO1 TWO4 95.7 =k 2.9 96.3 =k 1.7

HONE 1 100

95.7 & 2.9

> 100

96.7 Lt 1.9

100

> 100

> 100

> 100

100

12

61-

24

48

Time (h)

I

I

a) CNE cells

U

6

12

a

24

T i m (h) [ a ~ u b UGoK31 ~ o 0 5 IG2M

72

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b) TWO4 cells Figure 5. Percentage of cells at different phases of the cell cycle for a) CNEl and b) TWO4 cells after being treated with Fraction 7' from the methanol extract of Syneckococcus elongatus. The cells were incubated for different durations with the extract at concentrations of ICjo determined from the cytotoxicity testing. After incubation, the cells were harvested for cell cycle analysis by flow cytonletry. Different alphabets on the bar charts sllowillg percentage of cells at a particular phase indicate significant differences at p < 0.05 (n = 3).

DISCUSSION :

In the present study, we have demonstrated that of the 19 strains of microalgae screened, only the methanol extracts from three species showed antiproliferative activity on the NPC cell lines.

This is the first report on the antiproliferative activity of rnicroalgal extracts on NPC cells although there have been many studies on such effect on other cancer cell lines [6, 71. Both Ankistrodesnzus convolutus and Syrzeclzococcus elongntus have previously been shown to produce

Ma1n)~siartJ o ~ ~ r rof ~ aScierzcc l 27 (2): 19 - 3 1 (2008)

a variety of pigments and other useful compounds. For instance, Anltistrodesn~trs convolutus is known to produce appreciable amounts of pigments such as lutein and violaxanthin [14].

Spii-tllii~aplateizsis is a well-known commercial alga that is currently consumed as a health food. It has been shown to have a wide range of bioactivity including antiviral and anticancer activity [19]. For instance, phycocyanin from Spii.uliizn plateizsis is known to inhibit proliferation of HeLa cells by affecting the expression of CD59 gene [20]. The extract of Spir-ulina also inhibits the proliferation of oral cancer cells in hamsters [21]. However, the present study showed that extract ffom this alga was the least active among the three species tested. Of the four cell lines tested, TWO4 and TWO1 were more sensitive to the extracts of Syneclzococct~s elongatus and Ankistrodesmtu convolt~tuscompared to CNEl and HONEI. This could be due to the different structural properties of the cell lines as they originated from different sources. Both TWO1 and TWO4 cell lines were from patients from Taiwan with keratinizing squamous cell carcinoma [22]. In comparison, CNE 1 was from a well-differentiated carcinoma established from the biopsy specimen of an NPC patient in China [23] while HONE1 was established from poorly differentiated squamous cell carcinoma of a patient in Hunan, China [24]. The cell lines might be different in terms of stsucture, especially cytoskeletal properties and this might account for their different response to the extracts. The activity of the extract from Syizecococcza elongattls increased after fractionation. This could be due to the increased concentration of the cytotoxic compounds in those active fractions. The enhancement of cytotoxic activity after fractionation has been demonstrated using extracts from seaweeds against melanoma cells [25]. It was reported that acetone extract from Lobophor-a vnriegata does not show any activity, but semi-purified fractions inhibit the growth of melanoma cells. However, there are also studies which showed that methanol extract may have higher antiproliferative activity compared to pure compounds. For instance, methanol extract from Cat~lerparacenzosa has stronger antiproliferative activity than the purified caulerpenyne because

the extract contains more toxic transformation products [6]. The antiproliferative effect of Fraction 7' from Synechococcus elongattls might be due to its apoptotic effect on both TWO4 and CNEl cells, as indicated by the increased population of cells at Sub Go phase. The enhancement of apoptosis in cancer cells has been I-eported for other algal extracts and compounds. For instance, methanol extract from Catilerpa racernosa enhances apoptosis in neuroblastoma cells [6]. An extracellular polysaccharide from Gymnodiiziztin induces apoptosis in human myeloid leukemia cells by inhibiting DNA topoisomerases I and I1 [7]. Similarly, palmitic acid from the extract of the red alga Amphiroa zonata shows cytotoxic effect against human leukemic cells by inducing apoptosis [lo]. In addition, phycocyanin from Anabaeiza has been shown to induce apoptosis by depolyrnerisation of microtubules and actinfilaments in cancer cells [9]. Compounds from algae may induce apoptosis in cancer cells through several biochemical pathways. For instance, fucoidan from the seaweed Cladosiphoiz okamtlranus induces apoptosis in human leukemic cells via caspase-3 and -7 activationdependent pathway [8] while a polysaccharide from Capsosiphoiz fulvescens acts by inhibiting the insulin-like growth factor-I receptor [26]. The induction of apoptosis by the algal extracts should be fi~rther confirmed with other investigations, such as direct morphological observations using fluorescence technique [6] or cell death detection by ELISA [9]. Other apoptotic assays such as DNA-laddering, annexin-V staining and caspase-3 activity should also be used. The effect of the extract from Synechococcus elongatus on the cell cycle differs from other algal compounds and extracts which all-est the cells at a particular phase. For instance, fucoxanthin from brown seaweeds arrests the cell cycle of hepatic carcinoma at GOIG1 phase by down-regulating the cyclin D activity [27] while methanol extract from the seaweed Gloiopeltis ftlrcata induces the arrest of the cell cycle of hepatocarcinoma cells at G2/M phase [28]. The active fraction from the methanol extract of Synechococcus elongattls is likely to be pigment component as indicated by the TLC profile. Algal pigments such as hcoxanthin are known to have antiproliferative activity [27]. However, Fraction

-

7' is unlikely to be a carotenoid as the TLC spot appeared blackish green. It could be a derivative product of chlorophyll a. The chlorophyll derivative phaeophorbide a has been shown to induce apoptosis in human hepatocellular carcinoma after photoactivation [29]. In addition, a chlorophyll-related compound, phaeophytin a has been shown to be the active principle from the extract of Enteronzorpha prolifera suppressing skin tumorigenesis in mouse [30]. Further studies are to elucidate the structure of the active compound in Fraction 7' are worthwhile. Antineoplastic activity of this fraction on cell lines may not be extrapolated to actual NPC. tumour cells. Thus, further studies should focus on the effect of this fraction on in vivo model using animals.

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ACKNOWLEDGEMENTS This research was generously supported by the Ministry of Science, Technology and Innovation (MOSTI), Malaysia. The original source of the NPC cell lines was from Professor George Tsao from the University of Hong Kong. The NPC cell lines were kindly given to the authors by Professor Sam Choon Kook from the University of Malaya. Professors Tsao and Sam are gratefully acknowledged for the cell lines. The authors thank the Director of the Institute for Medical Research for pelmission to'publish the findings.

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10. REFERENCES 1. Chan, A. T. C., Teo, P. M. L. and Johnson, P. J. (2002). Nasophalyngeal carcinoma. Annal. Otzcol. 13:1007 - 1015. 2. Chang, E. T. and Adami, H. (2006). The enigmatic epidemiology of nasopharyngeal carcinoma. Cancer Epidenziol. Biomarlcers Prev. 15 (10): 1765 - 1777. 3. Devi, B. C. R., Pisani, P., Tang, T. S. and Parkin, D. M. (2004). High incidence of nasopharyngeal carcinoma in native people of Sarawak, Borneo Island. Cancer Epidenziol. Bionzarke~sPrev. 13 (3): 482 486. 4. Cardozo, K. H. M, Guaratini, T., Barros, M. P., Falcao, V. R., Tonon, A. P., Lopez, N. P., Campos, S., Torres, M. A., Souza, A. O., Colepicolo, P. and Pinto, E. (2007). Metabolites from algae with economical

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