Aktivitas Senyawa Semisintetik Kuasinoid dari Buah Makasar (Brucea javanica [L.] Merr) sebagai Antikanker dengan Target Protein P53, Bcl-2, Kaspase-3, COX-2 dan c-Myc

Sonlimar Mangunsong, Drs.,Apt.,M.Kes., Prof. dr. Iwan Dwiprahasto, M.MedSc.

2013 | Disertasi | S3 Kedokteran Umum

Pada saat ini, insidensi kanker serviks telah menempati urutan kedua teratas pada wanita penderita kanker di negara berkembang termasuk di Indonesia. Sehingga pencarian obat yang lebih tepat masih diperlukan. Model sel kanker serviks yang dapat digunakan dalam uji in vitro adalah sel HeLa. Telah ditemukan gen yang berperan dalam pembentukan kanker antara lain P53, Bcl-2, c-Myc, dan kaspase-3. Perbaikan DNA banyak dipengaruhi oleh kompleks p53 dengan protein-protein terkait menuju apoptosis. Kanker juga terkait erat dengan inflamasi yang dipicu oleh adanya COX-2. Brucea javanica (L.) Merr adalah salah satu tumbuhan yang telah digunakan sebagai obat untuk antiinflamasi, antibabesial, antivirus, antiplasmodial serta antikanker. Penelitian ini dimulai dari isolasi senyawa brusein A dari biji buah makasar (B. javanica). Dilanjutkan dengan semisintesis masing-masing dengan senyawa pereaksi: klorobenzoil klorida, benzoilklorida. Senyawa yang diperoleh adalah brusein A (0,0035%), 3-O-klorobenzoilbrusein rendemen (38%), dan 3-O-benzoilbrusein rendemen (40%). Uji aktivitas sitotoksik dilakukan terhadap sel HeLa dan uji toksisitas terhadap sel Vero. Hasil isolasi dan hasil semisintesis dielusidasi strukturnya dengan UV/vis, IR, H-NMR, C-NMR, HRESIMS dan LC-MS. Uji mekanisme molekulernya dengan flow sitometri dan teknik Immunocytochemistry (ICC) melalui pemacuan apoptosis P53 dan peningkatan level kaspase-3, antiproliferasi dengan peningkatan ekspresi P53, penurunan level Bcl-2, dan c-Myc, serta antiinflamasi dengan penurunan COX-2. Hasil uji sitotoksik terhadap sel HeLa memberikan nilai IC50 brusein A 87,45±13 μg/mL; 3-Oklorobenzoilbrusein 41,5 ± 4,79 μg/mL dan 3-O-benzoilbrusein 119,12±2,65 μg/mL dengan kontrol positif doksorubisin 5,66±1,22 μg/mL dan vinblastin 8,77±2,32 μg/mL. Hasil uji toksisitas terhadap sel Vero memberikan nilai IC50 dari brusein A, 3-O-klorobenzoilbrusein, 3-Obenzoilbrusein, doksorubisin dan vinblastin berturut-turut adalah 1366,55±53,43 μg/mL; 1385,15±81,57 μg/mL; 870,61±4,00 μg /mL; 61,46±28,37 μg /mL; dan 102,43±16,86 μg /mL. Pemberian ketiga bahan uji dengan kadar 100, 50 dan 25 μg/mL mempunyai aktivitas terhadap peningkatan ekspresi P53 (93,93%-45,13%), penurunan ekspresi protein Bcl-2 sebesar (81,19%- 18,99%); peningkatan ekspresi kaspase-3 (cleaved) sebesar (96,81% -31,06%), penurunan ekspresi c-Myc (23,08%-70,93%) serta penurunan ekspresi COX-2 (19,60%-9,01%). Berdasarkan hasil penelitian dapat disimpulkan bahwa brusein A, 3-O-klorobenzoilbrusein A dan 3-Obenzoilbrusein A berperan sebagai antikanker serviks yang selektif, melalui mekanisme pemacuan apoptosis, penghambatan proliferasi dan penurunan efek inflamasi.

The Incidence of cervical cancer recently had a leading caused of cancer in woman mostly in developing country including Indonesia. Therefore the pursuit of that cancer drugs is still needed. Trial in vitro models of cervical cancer by using HeLa cell has been approved. It has been known the role of several gens in cancer development which include P53, Bcl-2, c-Myc and Caspase-3. Due to apoptosis DNA repaired was influenced by P53 proteins complex. Cancer development also depends on inflammatory effect contributed by COX-2. Brucea javanica (L.) Merr as a herbal plant has been used to treat several diseases as antiinflamatory, antibabesial, antivirus, antiplasmodial as well as anticancer. In this study it was performed how to isolate bruceine A from the seed of “buah makasar”. Following had semi-synthesis of that compound with each of chlorobenzoyl chloride, and benzoyl chloride. As a result bruceine A have been isolated (0.0035%), 3-O-chlorobenzoylbruceine (38%) and 3-O-benzoyl bruceine (40%). The cytotoxic activity and its toxicity were done both in HeLa and Vero cells. That isolated and semisynthesis products confirmed by structural elucidation with UV/vis, FT-IR, H-NMR, CNMR, HPLC, HRESI-MS and LC-MS. Molecular activity was tested by flow cytometry and immunocytochemistry assay through induction apoptosis by increasing P53 and caspase-3 levels, antiproliferation through the increasing of P53 expression and the lowering levels among of Bcl-2, c-Myc, as well as COX-2. The IC50 value of bruceine A on HeLa cells as cytotoxicity effect were 87.45±13.0 μg/mL; 3-O-chlorobenzoylbruceine 41.5±4.79 μg/mL and 3-O-benzoylbruceine 119.12±2.65 μg/mL with doxorubicin 5.66±1.22 μg/mL and vinblastin 8.77±2.32 μg/mL as positive control. The IC50 value of bruceine A, 3-O-chlorobenzoylbruceine, 3-O-benzoylbruceine, doxorubicin and vinblastin on Vero cells tested were more selective than HeLa cells. Their cytotoxicity effect were 1366.55±53.43 μg/mL; 1385.15±81.57 μg/mL; 870.61±4.00 μg/mL; 61.46±28.37 μg/mL; and 102.43±16.86 μg/mL respectively. That isolated and semisynthesis compounds gave in 100, 50, 25 ìg/mL serial concentration have increased the P53 expression (93.93%-45.13%), decreased Bcl-2 expression (81.19%-18.99%); increased Caspase-3 (cleaved) (96.81%-31.06%) and decreased the c-Myc expression (23.08%-70.93%); decreased COX-2 expression (19,60%- 9,01%). This study comes to conclusions that bruceine A, 3-O-chlorobenzoylbruceine, and 3-Obenzoylbruceine showed anticancer cervix activity via apoptotic inductions, inhibiting of their proliferation and inflammatory effects.

Kata Kunci : Brucea javanica, Semisintesis, Brusein A, 3-O-Klorobenzoilbrusein, 3-OBenzoilbrusein, Bcl-2, Kaspase-3, P53, c-Myc, COX-2, Immunositokimia