Sitotoksisitas dan efek apoptosis senyawa antikanker dari Eugenia Uniflora L pada sel kanker payudara T47D dan MCF-7

SUPRIYATI, Nita, Dr. Jaka Widada, M.P

2010 | Tesis | S2 Bioteknologi

Kanker payudara merupakan jenis kanker yang paling banyak diderita kaum wanita dan merupakan penyebab kematian kedua setelah kanker paru dan bronkus. Pengobatan kanker dengan bahan alam makin diminati karena sistem pengobatan yang ada belum memberikan hasil yang memuaskan. Tanaman dewandaru (Eugenia uniflora. L) telah dilaporkan memiliki aktifitas antioksidan dan mampu menghambat DNA polymerase pada EBV(Eipstein-Bar Virus), namun penelitian tentang aktifitas antikankernya belum pernah dilaporkan. Penelitian ini bertujuan untuk mengetahui potensi antikanker E. uniflora, mengisolasi senyawa toksiknya dan mempelajari pengaruhnya terhadap sitotoksisitas dan apoptosis pada sel kanker payudara T47D dan MCF-7. Petroleum eter, diklorometan dan metanol digunakan untuk mengekstrak daun E. uniflora. Ekstrak yang dihasilkan kemudian diuji kemampuan sitotoksiknya terhadap sel T47D dengan metode MTT. Pemisahan senyawa toksik dilakukan dengan metode kromatografi kolom vakum dan KLT preparatif. Hasil setiap tahap pemisahan diuji kemampuan sitotoksiknya terhadap sel T47D. Identifikasi golongan kimia senyawa toksik hasil pemisahan dilakukan dengan analisis KLT, spektra UV dan IR. Uji sitotoksisitas , analisis daur sel dan uji morfologi apoptosis senyawa toksik dilakukan terhadap sel T47D dan MCF-7. Analisis daur sel menggunakan flow cytometry dengan pewarnaan propidum iodida, sedangkan uji apoptosis menggunakan pewarnaan ganda etidium bromida/akridin oranye dan diamati dengan mikroskop fluoresens. Ekpresi protein Bcl-2 dan p53 pada sel MCF-7 diamati dengan metode immunositokimia. Hasil penelitian menunjukkan bahwa ekstrak diklorometan E. uniflora memiliki potensi sitotoksik dengan nilai IC50 sebesar 96 μg/ml. Uji sitotoksik senyawa hasil isolasi terhadap sel T47D dan MCF-7 menunjukkan nilai IC50 sebesar 8 dan 10 μg/ml sedangkan pada sel vero sebesar 48 μg/ml. Hasil uji morfologi dan flow cytometry menunjukkan bahwa senyawa toksik mampu menginduksi apoptosis. Hambatan daur sel tampak pada sel MCF-7 dengan akumulasi sel pada fase G1. Peningkatan ekspresi p53 dan penurunan ekspresi Bcl-2 diduga berperan dalam induksi apoptosis sel MCF-7. Senyawa hasil isolasi diduga merupakan triterpenoid.

Breast cancer is a type of cancer most commonly had by women and is the second most frequent cause of death after lung and bronchial cancer. Today, cancer treatment using natural products has attracted more attention as the existing treatments do not provide satisfactory results. Dewandaru (Eugenia uniflora.L), also known as the Surinam Cherry, has been reported to have antioxidant activity and can suppress DNA polymerase of EBV (Eipstein-Bar Virus); however, research on its anticancer activity has not been reported. Therefore, the objective of this research is to study the potential application of E. uniflora as anticancer, isolate the toxic compound and study its impact on cytotoxicity and apoptosis on T47D and MCF-7 breast cancer cells. Petroleum ether, dichlormethane and methanol are used to extract E.uniflora leaves. The extract was tested for its cytotoxic activity on T47D cell line using MTT method. The toxic compounds were separated using vacuum column chromatography and Preparative Thin Layer Chromatography methods. Cytotoxicity test on T47D cells was performed for extracts of each separation stage. Chemical type identification of the toxic compound was performed using TLC, UV and IR spectrophotometry analysis. Cytotoxicity test, cell cycle analysis and apoptosis morphology tests of the toxic compounds were performed on the T47D cells and MCF-7. Flow cytometry assay using propidium iodide staining was used to analyze the cell cycle, while the apoptosis test was conducted using ethidium bromide/acridine orange for observation under fluorescent microscope. Protein expression of Bcl-2 and p53 in MCF-7 cells was observed by immunocytochemistry method. The results showed that dichlormethane extracts of E. uniflora has cytotoxic potential, having IC50 value of 96 μg/ml. The IC50 values of the compounds tested on T47D and MCF-7 cells were 8 and 10 μg/ml, respectively, while the IC50 value for cytotoxic test on vero cells was 48 μg/ml. The results of morphology and flow cytometry tests showed that the toxic compounds are capable of inducing apoptosis. Cell cycle arrest was observed in MCF-7 cells in which cell accumulation occurred in G1 phase. The increase in p53 expression of and the decrease of Bcl-2 expression are predicted to induce the apoptosis of MCF-7 cells. Finally, the isolated compounds are believed to be of triterpenoid group.

Kata Kunci : Eugenia uniflora L, apoptosis, MCF-7, T47D