Selektivitas dan mekanisme antikanker 5Alfa-Oleandrin dan 16,17-Dehidrodeasetil-5Alfa-Oleandrin hasil isolasi dari daun Jure (Nerium indicum Mill.)

WAHYUNINGSIH, Mae Sri Hartati, Promotor Prof.Dr. Ibnu Gholib Gandjar, DEA.,Apt

2006 | Disertasi | S3 MIPA (Ilmu Farmasi)

Kebutuhan obat baru antikanker tidak pernah berhenti sampai kapanpun karena obat ideal antikanker yang bebas efek samping belum ditemukan sampai sekarang. Daun Jure (Nerium indicum Mill.) merupakan tanaman yang secara tradisional telah digunakan untuk pengobatan kanker. Melalui metode Bioassay (Brine Shrimp Lethality Test=BST) guided solvent extraction and isolation, tiga senyawa (NiO1, NiO2 dan NiO3) berhasil diisolasi dari fraksi aktif ekstrak CHCl3 daun N. indicum. Pemeriksaan lebih lanjut terhadap NiO2 menunjukan bahwa NiO2 masih terdiri dari 4 senyawa, sehingga perlu dilakukan isolasi lanjut untuk mendapatkan senyawa murni yang berpotensi antikanker. Penelitian ini bertujuan untuk menentukan senyawa berpotensi antikanker, efek sitotoksik, identifikasi struktur serta penelusuran mekanisme kerja antikanker senyawa terpilih yaitu senyawa yang mempunyai efek sitotoksik tertinggi pada sel kanker dan terendah pada sel normal. Penentuan selektivitas senyawa (Ni01, Ni02A-D dan Ni03) dilakukan dengan uji sitotoksik terhadap berbagai sel kanker (MCF7, EVSA-T, T47D, H226, IGROV, A498, WiDR, M19, HeLa) dan sel normal (Vero) pada satu seri kadar yang sama (0,6 -12500 ng/ml) dengan metode MTT. Identifikasi struktur senyawa terpilih dilakukan secara spektroskopi (UV, IR, MS, NMR); NiO2D diidentifikasi sebagai 5a-oleandrin (C32H48O9), dan NiO2C diidentifikasi sebagai 16,17-dehidrodeasetil-5a-oleandrin (C30H44O7). Penelusuran mekanisme kerja dilakukan dengan pengecatan Hoechst 33342, gel elektroforesis, ekspresi protein (Bcl-2 dan Bax) dengan Western Blotting, ekspresi protein p53 dengan immunohistokimia, dan penghitungan persentase apoptosis sel dengan metode FITC-Annexin V. Hasil uji menunjukkan 5a-oleandrin toksik pada sel Hela (IC50=8,38x10-6 mM), dan pada dosis (3,47x10-4mM, inkubasi 24 jam) menyebabkan terjadinya fragmen DNA (pita smear sekitar 200 bp), kondensasi kromatin (biru muda terang), penurunan ekspresi protein Bcl-2 dan peningkatan ekspresi Bax. Senyawa 16,17-dehidrodeasetil-5a-oleandrin toksik pada sel A498 (IC50=1,43x10-5mM), dan pada dosis (3,88x10-4mM, inkubasi 48 jam), menyebabkan fragmentasi DNA, dan peningkatan ekspresi protein p53 dibandingkan dengan kontrol. Perhitungan persentase apoptosis sel A498 setelah pemberian 16,17-dehidrodeasetil-5aoleandrin dilakukan dengan Flow cytometry dan menghasilkan % kematian sel yang meningkat seiring dengan lama waktu inkubasi. Penelitian ini dapat disimpulkan bahwa 5a-oleandrin selektif terhadap sel HeLa (kanker serviks) dan dapat memacu apoptosis melalui penurunan ekspresi protein Bcl-2 dan peningkatan ekspresi protein Bax. Senyawa 16,17- dehidrodeasetil-5a-oleandrin selektif terhadap sel A498 (kanker ginjal) dengan mekanisme kerja meningkatkan ekspresi protein p53.

Searching of new anticancer drugs never ends as side-effect free anticancer drugs have not been found till today. The leaves of Nerium indicum Mill. are used traditionally to treat cancers. By using Bioassay (BST = Brine Shrimp Lethality Test) guided extraction and isolation method, three compounds (Ni01, Ni02 and Ni03) were isolated from active fraction separated from the CHCl3 extract of N. indicum leaves. Compound Ni02 was separated further by chromatographic method to give 4 compounds. The aims of this study were to determine cytotoxic effect of NiO1, NiO2A-D, NiO3 on several human cancer cells as well as normal cells in vitro, to identify the structure of the selected compounds (toxic to cancer cells but not on normal cells), and to determine their mechanism of action at molecular level. The compounds (Ni01, Ni02A-D and Ni03) were tested for their cytotoxic effect on several human cancer cells (MCF7, EVSA-T, T47D, H226, IGROV, A498, WIDR, M19, HeLa) and normal cells (Vero) using MTT method at the same series of concentrations (0.6-12500 ng/ml). Molecular structures of the selected compounds were identified using spectroscopic (UV, IR, MS, NMR) method as 5a-oleandrin (C32H48O9) for NiO2D, and 16,17-dehidrodeacetil-5aoleandrin (C30H44O7) for Ni02C. Their mechanism of action was analyzed by staining the cells with Hoechst 33342, agarose gel electrophoresis, Western Blotting, immunohistochemistry, and quantification of apoptotic cells counted by FITC-Labeled Annexin V. The 5a-oleandrin was cytotoxic on HeLa cells (IC50=8,38x10-6 mM). Upon incubation of HeLa cells with 5a-oleandrin (3,47x10-4 mM) for 24 hours followed by gel electrophoresis, a smear band at about 200 bp was observed and staining with Hoechst 33342, a broken up light blue color of nucleus was seen (compared to intensive color of untreated control). The 5a-oleandrin treated cells decreased the Bcl-2 protein expression and increased the Bax protein expression. The 16,17-dehydrodeacetyl-5a-oleandrin was cytotoxic on A498 cells (IC50= 1,43x10-5 mM) in vitro. Upon incubation of A498 cells with 16,17- dehydrodeacetyl-5a-oleandrin (3,88x10-4mM) for 48 hours followed by staining with Ethidium bromide, clearly orange to bright red color of DNA fragments were observed. The percentage of apoptotic cells increased when the cells was treated with 16,17-dehydrodeacetyl-5a-oleandrin (3,88x10-4 mM) parallel to time consuming, and the p53 protein expression was also increased. Based on those data, it is concluded that 5a-oleandrin compound is selective to HeLa cells, and this compound induces apoptosis by reducing the Bcl- 2 protein expression and increasing the Bax protein expression. The 16,17- dehydrodeacetyl-5a-oleandrin is selective to A498 cells, and this compound increases p53 protein expression.

Kata Kunci : Obat Antikanker,Daun Jure,5 Alfa,Oleandrin