Mekanisme molekuler aktivitas analog kurkumin pentagamavunon terhadap sel kanker payudara (T74D)
DA'I, Muhammad, Promotor Prof. Dr. Umar Anggara Jenie, M.Sc. Apt
2008 | Disertasi |Upaya penemuan obat antikanker perlu terus dilakukan untuk mendapat senyawa baru yang aman dan selektif dalam menghambat pertumbuhan sel kanker. Berdasarkan penelitian sebelumnya analog kurkumin pentagamavunon (PGV) memiliki potensi aktivitas sebagai antikanker. Penelitian ini diarahkan untuk menentukan mekanisme molekuler aktivitas antikanker turunan PGV: PGV-0 dan PGV-1. Penelitian ditujukan untuk menentukan kemampuan menginduksi apoptosis dan menghambat daur sel senyawa PGV terhadap sel kanker payudara T47D. Penelitian diawali dengan menentukan kemurnian senyawa uji dan struktur senyawa uji dengan menggunakan analisis HPLC dan elusidasi struktur dengan spektroskopi, IR, 1H-NMR, 13C-NMR dan MS serta difraksi sinar X. Penelitian dilanjutkan dengan uji sitotoksik dan analisis profil kinetika pertumbuhan sel dengan metode MTT. Kemampuan menginduksi apoptosis pada sel T47D ditentukan melalui pengamatan morfologi DNA inti sel dengan metode pengecatan menggunakan akridin oranye-etidium bromida dan dengan DAPI. Efek modulasi senyawa uji pada daur sel ditentukan dengan metode analisis flowcytometric. Analisis mekanisme molekuler aktivitas antikanker senyawa uji ditentukan dengan metode Western blot dan analisis immunositokimia. Senyawa PGV-0 dan PGV-1 memiliki struktur isomer geometrik entgagenentgagen (E-E). Nilai IC50 hasil percobaan terhadap sel T47D senyawa kurkumin, PGV-0 dan PGV-1 berturut-turut adalah: 24,97; 9,39 dan 1,74 μM. Analisis profil kinetika pertumbuhan menunjukkan PGV-1 (2,5 μM) mampu menghambat pertumbuhan sel lebih baik dibanding PGV-0 (5,0 μM) maupun kurkumin (10,0 μM). Senyawa PGV-1 2,5 μM mampu menginduksi akumulasi sel pada fase G2-M pada pengamatan jam ke-12 dan diikuti akumulasi hiperploidi sel. Senyawa PGV-1 mampu menginduksi kenaikan ekspresi p21 dan aktivasi Cdc-2 pada analisis Western blot. Kurkumin dan analognya mampu menginduksi apoptosis sel pada pengamatan morfologi DNA. Analisis molekuler menunjukkan ketiga senyawa mampu menginduksi cleavage PARP dan aktivasi Caspase-3 dan Caspase-7. Apoptosis terjadi melalui mekanisme tidak tergantung p53. Pengamatan immunositokimia menunjukkan PGV-1 menyebabkan terjadinya kondensasi kromatin. Secara keseluruhan hasil penelitian membuktikan analog kurkumin khususnya PGV-1 mampu menghambat daur sel, menyebabkan akumulasi hiperploid sel dan memacu terjadinya apoptosis. Hasil tersebut mengindikasikan analog kurkumin PGV-1 memiliki aktivitas sebagai senyawa antimikrotubul.
Anticancer research requires continuous effort to get new compounds which are safe and selective to inhibit cancer cell growth. Previous experiments indicated curcumin analogues pentagamavunone (PGV) had potency as anticancer. The present experiment was carried out to determine the molecular mechanism of PGV as anticancer especially in cell cycle modulation and apoptosis induction. The research was conducted by firstly determining the test compounds PGV-0 and PGV-1 purity by HPLC. Structure elucidation of the test compounds was carried out by LC-MS, NMR spectroscopy and xray diffraction analysis. To determine potential activity of the test compounds, a cytotoxicity test was carried out using MTT method followed by observation on the kinetics of T47D breast cancer cells growth treated with the compounds. This test was compared with curcumin as the lead compound. Determination of the influence of treatment with the test compounds against cell proliferation in various phases of cell cycle was carried out by flowcytometric analysis. Molecular observation on the proteins influencing the regulation of cell cycle was carried out by Western blot and immunocytochemistry analysis. Apoptosis induction by the test compounds was examined using a staining method with acridine orange and ethidium bromide. Molecular mechanism was examined by Western blot method against proteins that involved in apoptosis and cell cycle regulation. The experiment showed that PGV-0 and PGV-1 have geometrical isomers entgagen-entgagen (E-E). Curcumin analogues had stronger potency to inhibit breast cancer cells than curcumin. PGV-1 had the smallest IC50, i.e. 1,74 μM compared with PGV-0 (9.39 μM) and curcumin (24.97 μM). Flowcytometric analysis of T47D cells showed that PGV-1 inhibited breast cancer cell growth at G2-M phase after 12 hours incubation and cell hyperploidy after 24 hours incubation. PGV-1 treatment increased the expression of p21 protein and induced the activation of Cdc-2 on T47D cells. Analysis of DNA morphology indicated Curcumin and PGV induced apoptosis on T47D cells. The apoptotic effect of the test compounds (PGV-0, PGV-1 and curcumin) on T47D cells was confirmed by the occurance of PARP cleavage and Caspase-3 and Caspase-7 activation. Immunocytochemistry analysis indicated PGV-1 induced chromatin condensation. Taken together, all of those results indicate that PGV-1 inhibits cell cycle progression of T47D cells, induce cell accumulation in hyperploidy and then induce apoptosis to the cells. The results indicate that PGV-1 acts as antimicrotubule compounds.
Kata Kunci : Obat anti-kanker,Kurkumin pentagamavunon,Kanker payudara
