Sains Malaysiana 38(5)(2009): 773–784

Chlorella vulgaris Menunjukkan Kesan Antioksidan dan Antitumor Terhadap Kanser Hepar dalam Kajian in vivo dan in vitro

(Chlorella vulgaris Exhibited Antioxidant and Antitumour Effects against Liver

Cancer in in vivo and in vitro Studies)

Nor Ashikeen Mukti, Suhaniza Sulaiman, Suhana Md Saad, Junaida @ Maimunah Hassan Basari, Mariati Abdul Rahman, Wan Zurinah Wan Ngah, Yasmin Anum Mohd Yusof*

Jabatan Biokimia Fakulti Perubatan

Pusat Perubatan Universiti Kebangsaan Malaysia

Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia

Diserahkan: 7 Oktober 2008 / Diterima: 22 April 2009

ABSTRAK

Chlorella vulgaris (ChV), sejenis alga hijau unisel telah dilaporkan mempunyai khasiat kesihatan pada penyakit tertentu termasuk kanser. Objektif utama kajian ialah untuk mengukur dan menilai kesan antioksidan dan antitumor ekstrak air panas ChV ke atas sel kanser hepar yang dijalankan secara in vivo dan in vitro. Asai DPPH yang dijalankan menunjukkan peratus pengautan ChV yang tinggi. Dalam kajian in vivo, tikus Wistar jantan (200-250 g) dibahagikan kepada lapan kumpulan: tikus kawalan (diet normal), tikus diaruh kanser hepar (diet kurang kolin + 0.1% etionin dalam air minuman) atau singkatannya CDE, tikus diberi rawatan ChV pada tiga dos berbeza (50, 150 dan 300 mg/kg berat badan) dan tikus CDE diberi rawatan ChV pada tiga dos berbeza. Sampel darah dan tisu diambil dari semua kumpulan tikus pada minggu 0, 4, 8 dan 12 untuk penentuan kadar proliferasi dan apoptosis sel untuk melihat kesan antitumor ChV. Peratus pembentukan nodul praneoplasia adalah tinggi pada tikus diaruh kanser hepar (CDE) tetapi ChV pada semua dos berjaya mengurangkannya. Pertambahan jumlah sel kanser semasa hepatokarsinogenesis ditunjukkan dengan peningkatan proliferasi hepatosit yang signifikan (p<0.05) pada tikus CDE berbanding kawalan tetapi ChV pada semua dos berjaya mengurangkan proliferasi secara signifikan (p<0.05). Peratus apoptosis sel didapati meningkat secara signifikan (p<0.05) pada tikus CDE, tetapi peningkatan yang lebih ketara berlaku pada tikus CDE diberi ChV (300 mg/kg berat badan). Dalam kajian in vitro pula, aktiviti antitumor ekstrak air panas ChV telah ditentukan dengan melihat perubahan dalam proliferasi dan apoptosis sel kanser hepar HepG2 yang dikultur di makmal. Ekstrak air panas ChV berjaya menurunkan kadar proliferasi sel HepG2 dengan signifikan secara berkadar terus dengan dos yang digunakan dengan nilai IC50 1.6 mg/ml. Hasil analisis TUNEL pula menunjukkan ekstrak air panas ChV berjaya mengaruh apoptosis dalam sel HepG2. Keputusan ini disokong oleh hasil pemblotan Western dengan peningkatan pengekspresan protein P53 dan protein proapoptosis BAX dan Kaspase-3. Daripada hasil-hasil kajian, dapatlah dicadangkan bahawa ChV berpotensi tinggi sebagai antioksidan serta berupaya memberi kesan antitumor kepada kanser hepar pada kajian in vivo dan in vitro.

Kata kunci: antioksidan; antitumor; apoptosis; Chlorella vulgaris; kanser hepar; proliferasi

ABSTRACT

The unicellular green microalgae Chlorella vulgaris (ChV) has been reported to have beneficial effects on various diseases including cancer. The main objectives of this study were to determine and evaluate the antioxidant and antitumour properties of ChV against liver cancer cells, both in vivo and in vitro. The antioxidant effect of hot water extract of ChV was demonstrated by DPPH assay which exhibited a high percentage of free radical scavenging activity. In in vivo studies, male Wistar rats (200-250 g) were divided into eight groups according to the diet given: control group (normal diet), liver cancer (choline deficient diet + 0.1% ethionine in drinking water to induce liver cancer), abbreviated as CDE group, ChV group with three different doses (50, 150 and 300 mg/kg body weight), and CDE group treated with three different doses of ChV. Rats were killed at 0, 4, 8 and 12 weeks of experiment and blood and tissue samples were taken from the rats in all groups for the determination of cellular proliferation and apoptosis. ChV at all doses reduced the percentage of liver tumour incidence (the preneoplastic nodules) in the CDE (liver cancer) rats when compared to the CDE rats alone. The percentage of hepatocyte proliferation was significantly higher (p<0.05) in CDE rats when compared to control which however was inhibited by ChV at all doses. Meanwhile, the percentage of apoptotic cells in the CDE rats increased significantly (p<0.05) compared to control which was further increased when treated with higher dosage of ChV (300 mg/kg body wt). The antitumour effects of hot water extract of ChV in in vitro studies were determined by observing the changes in the proliferation and apoptosis rate in liver cancer cell line, HepG2. ChV reduced (p<0.05) the proliferation rate of HepG2 cells in dose-dependent manner. The IC50 value was recorded at 1.6 mg/ml. Apoptosis was induced by ChV, as indicated by TUNEL analysis which was supported further by Western blot analysis indicating increased expression of P53 and pro-apoptotic proteins BAX, and Caspase-3. From these findings, it can be suggested that ChV has great potential as an antioxidant with the ability to exert antitumour effects against liver cancer.

Keywords: Antioxidant; antitumour; apoptosis; Chlorella vulgaris; liver cancer; proliferation

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*Pengarang untuk surat-menyurat; email: anum@medic.ukm.my