KAJIAN POTENSI DAN MEKANISME AKSI p-HIDROKSI-m-METOKSI-KALKON SEBAGAI AGEN ANTIKANKER DAN KO-KEMOTERAPI DOKSORUBISIN PADA SEL KANKER PAYUDARA T47D DAN MCF-7

RETNO ARIANINGRUM, Prof. Dr. Retno Sunarminingsih, M.Sc., Apt;Prof. dr. Sofia Mubarika, M.Med.Sc., Ph.D;Prof. Dr. Edy Meiyanto, M.Si, Apt.

2017 | Disertasi | S3 Bioteknologi

Terapi tertaget kanker payudara belum mengatasi kanker jenis triple negative breast cancer (TNBC) karena mempunyai tingkat proliferasi yang tinggi, rekurensi dan resisten agen kemoterapi, sehingga pengobatan ko-kemoterapi perlu dikembangkan. Studi ini meneliti bahan alam sintetik derivat kalkon, p-Hidroksi m-Metoksi-Kalkon (pHmMK) atau 3-(4'-hidroksi-3'-metoksifenil)-1-fenil-2-propen-1-on, baik tunggal dan kombinasinya dengan Doksorubisin (DOX) untuk diketahui potensi dan mekanisme aksinya sebagai agen anti kanker payudara pada sel T47D dan MCF-7 meliputi sitotoksisitas, penghambatan daur sel, pemacuan apoptosis dan penghambatan resistensi. Uji sitotoksisitas pHmMK dan kombinasinya dengan DOX menggunakan MTT [3-(4,5-dimethylthiazol-2-il)-2.5-dipheniltetrazolium bromide] assay untuk menentukan potensi sitotoksik berdasar IC50-nya, serta nilai combination index (CI)-nya. Penetapan hambatan daur sel, pacuan apoptosis dan ekspresi P-gp dianalisis dengan flow cytometry sedangkan ekspresi protein regulator daur sel, apoptosis, dan resistensi diamati dengan immunositokimia menggunakan antibodi monoklonal masing-masing. Hasil penelitian menunjukkan bahwa pHmMK bersifat sitotoksik pada sel T47D dan MCF-7 dengan IC50 berturut-turut 48 dan 40 mikromolar, sedangkan DOX menunjukkan nilai IC50 sebesar 84 nM pada sel T47D dan 6 mikromolar pada MCF-7. Kombinasi pHmMK-DOX menunjukkan efek sinergi kuat pada sel T47D dan sinergi pada MCF-7. Pemberian pHmMK menyebabkan akumulasi sel pada fase G2/M pada sel T47D, serta G2/M dan S pada sel MCF-7. Kombinasi pHmMK-DOX menyebabkan akumulasi G2/M dan S pada sel T47D serta G2/M pada MCF-7. pHmMK dan kombinasi pHmMK-DOX menurunkan ekspresi cyclin B. Peningkatan apoptosis akibat pemberian pHmMK dan kombinasi pHmMK-DOX berupa penurunan ekspresi Bcl-2, peningkatan ekspresi Bax, c-Caspase-3 (sel T47D), c-Caspase-9 (sel MCF-7) dan c-PARP. pHmMK juga terbukti menurunkan resistensi DOX berdasarkan penurunan ekspresi P-gp pada sel MCF-7. Kesimpulan dari penelitian ini bahwa pHmMK berpotensi sebagai anti kanker payudara; dan berpotensi memberikan efek sinergi dengan DOX pada hambatan proliferasi, hambatan daur sel pada fase G2/M dan S; serta peningkatan apoptosis dan menurunkan resistensi DOX.

Targeted therapy has been unsuccesfulled to treat breast cancer of which triple negative breast cancer (TNBC)-type that has a high rate of proliferation, recurrence and chemotherapeutic resistance. Therefore co-chemotherapeutic treatment needs to be developed. This study explored the potencies as well as the mechanisms of action of a synthetic natural substance of chalcone derivate of p-Hydroxy-m-Methoxy-Chalcone (pHmMC) or 3- (4'-hydroxy-3'-methoxyphenyl) -1-phenyl-2-propen-1-one, either as a single treatment or a combination with Doxorubicin (DOX) against breast cancer on T47D and MCF-7 cells covering the cytotoxicity, the proliferative cell inhibition, apoptotic induction and resistance inhibition. The MTT [3-(4,5-dimethylthiazol-2-il)-2.5-dipheniltetrazolium bromide] assay was carried out to determine the cytotoxic potencies (IC50) and the combination index (CI) of the pHmMK and its combination. The cell cycle inhibition, apoptotic induction and expression of P-gp were observed using flow cytometry; while the expressions of regulator proteins of cell cycle, apoptosis, and resistance were observed by immunocytochemistry using respective monoclonal antibodies. The results showed that pHmMC performed cytotoxic effect to T47D and MCF-7 cells with IC50 values of 48 and 40 micromolar respectively, whereas DOX gave IC50 values of 84 nM in T47D cells and 6 micromolar in MCF-7 cells. The combination of pHmMC-DOX gave a strong synergistic effect on T47D cells and synergistic effect on MCF-7 cells. pHmMC caused cell accumulation on the G2/M phase in T47D cells, and on the G2/M and S phases in MCF-7 cells. The combination of pHmMC-DOX caused cell accumulation on the G2/M and S phases in T47D cells, and on the G2/M phase in MCF-7 cells. The pHmMC and pHmMC-DOX decreased the cyclin B expression. Due to the pHmMC and its combination treatments, an apoptosis induction was observed through decreasing expression of Bcl-2, increasing Bax, c-Caspase-3 (T47D cells), c-Caspase-9 (MCF-7 cells), and c-PARP expressions. It was showed that pHmMC decreased DOX resistance through the decreasing of P-gP expression in MCF-7 cells. It was concluded that pHmMC has a potency as anti-breast cancer; and it gives potential synergistic effects with DOX on proliferative inhibiton, cell cycle arrest at G2/M and S, as well as apoptotic induction; and decreasing of DOX resistance.

Kata Kunci : p-hidroksi-m-metoksi-kalkon, antikanker, mekanisme aksi, ko-kemoterapi, T47D dan MCF-7