MEKANISME AKSI ANTIKANKER SENYAWA 1,2-EPOKSI-3(3-(3,4-DIMETOKSIFENIL)-4H-1-BENZOPIRAN-4ON)PROPANA PADA KANKER SERVIKS UTERI (Kajian In Vitro dan In silico terhadap Ekspresi MikroRNA-21, -214, - 34, dan Protein Tissue Inhibitors of Metalloproteinase (TIMP-3), p53, serta Bcl-2)

LELLY YUNIARTI, Prof. dr. Sofia Mubarika, M.Med.Sc., Ph.D.; Prof. Dr. Mustofa, M.Kes., Apt.; Prof. Dr. dr. Teguh Aryandono, Sp.B(K)Onk

2018 | Disertasi | DOKTOR ILMU KEDOKTERAN DAN KESEHATAN

Latar Belakang: Tingkat kematian yang tinggi pada penderita kanker serviks uteri terutama di negara berkembang, belum maksimalnya terapi, masih banyaknya efek samping serta harga obat yang mahal mendorong para peneliti untuk mencari obat kanker yang efektif dengan target molekuler yang tepat. Tujuan: Menguji efek antikanker dan ko-kemoterapi 1,2-Epoksi-3(3-(3,4dimetoksifenil)-4H-1-benzopiran-4on)propana (EPI) serta mekanismenya. Metode: Merupakan penelitian eksperimental in vitro pada kultur kanker serviks uteri. Uji sitotoksik dilakukan untuk menentukan IC dan uji kombinasi untuk Indek Kombinasi (IK). Sel HeLa, sebanyak 32 sumuran dibagi menjadi 8 kelompok yaitu kontrol negatif, yang diberi senyawa EPI IC 50, EPI 2IC 50 , doksorubisin (DOX) IC 50 50 , kombinasi EPI+DOX, cisplatin (CIS), kombinasi EPI+CIS. Setelah diberi perlakuan selama 24 jam, diukur ekspresi miR-21, miR214, dan miR-34a menggunakan qRTPCR, sedangkan protein p53, TIMP-3, dan Bcl-2 dengan metode ELISA. Kecocokan target diuji dengan metode in silico. Uji one-way ANOVA dan post hoct Tukey digunakan untuk menguji beda rerata seluruh variabel antar kelompok penelitian, sedangkan untuk mengetahui hubungan ekspresi miRNA dengan konsentrasi protein dilakukan dengan uji korelasi Pearson dan regresi linier sederhana. Sekuensing dilakukan untuk melihat mutasi pada miR-21, dan analisis target menggunakan BLAST. Hasil: IC EPI adalah 33,24 (+-3,01) g/ml, IK EPI+DOX<0,1, dan IK EPI+CIS<0,9. Ekspresi miR-21,-214, dan -34a lebih tinggi pada kelompok perlakuan dibandingkan dengan kelompok kontrol (p<0,05), Ekspresi p53 pada kelompok 6 (1,67 +- 0,31 mcg/ml) dan 8 (1,18 +- 0,18 mcg/ml), ekspresi TIMP-3 kelompok 6 (3,81 +- 0,49 mcg/ml) dan 8 (2,93 +- 0,42 mcg/ml) lebih tinggi dari kelompok kontrol (p<0,05), ekspresi Bcl-2 lebih rendah pada semua kelompok perlakuan (p<0,05), koefisien korelasi antara miR-214 dan Bcl-2 adalah -0,908). Analisis in silico dengan metode docking menunjukkan energi ikatan reseptor p53 dan Bcl-2 dengan EPI lebih tinggi dari energi ikatan dengan ligan nativ. Hasil sekuensing menunjukkan terjadi mutasi pada miR-21, dan uji in silico dengan BLAST menunjukkan terjadi perubahan target pada miR-21 mutan. Simpulan: Efek antikanker EPI bersifat sedang, bersinergi sangat kuat dengan DOX dan bersinergi lemah dengan CIS. Kombinasi EPI+DOX, dan EPI+CIS dapat meningkatkan MiR-214, MiR-34a, p53, dan TIMP-3 dan menurunkan Bcl2, terdapat korelasi negatif sangat kuat antara ekspresi miR-214 dengan Bcl-2. Berdasarkan metode docking protein p53 dan Bcl-2 bukan target yang cocok dari senyawa EPI. Terjadi mutasi pada miR-21dan perubahan target miR-21 mutan.

Background: The insufficiency of treatment for cervical cancer, numerous side effects, and costly drugs encourage researchers to look for effective cancer drugs with accurate molecular target. Aim: This study aims to examine the anticancer and co-chemotherapy effects of 1,2-Epoxy-3(3-(3,4-dimetoxyphenyl)-4H-1-benzopiran-4on) propane (EPI) compounds and their mechanism of EPI compounds on cultures of cervical cancer. Method: This study was an experimental study in vitro on cervical cancer uteri culture. Cytotoxic test was performed to determine IC , while the combination test was conducted to determine the Combination Index (CI). HeLa cells in 32 wells were divided into eight groups as negative control, which were given EPIIC 50 , EPI IC 50 , EPI 2IC 50 , doxorubicin IC 50 50 , combination of EPI+DOX, cisplatin (CIS), and the combination of EPI+CIS respectively. After 24 hours of treatment, HeLa cells expression of miR-21, miR-214, and miR-34a were measured using qRTPCR. Expression of p53, TIMP-3, and Bcl-2 proteins were measured by ELISA method. Suitability of protein target were measured by docking method. One-way ANOVA and post hoc Tukey test was performed to test the mean difference of all variables between the study groups, whereas Pearsons correlation test and simple linear regression were conducted to see the relationship of miRNA expression and its protein concentration. Sequencing is done to see the mutation in miR-21, and miR-21 target were analysed using BLAST. Results: IC for EPI was 33.24 (+-3.01) mcg/ml, EPI+DOX CI was<0.1, and EPI+CIS CI<0.9. The expression of miR-21, -214, and -34a were higher in the treatment group compared to the control group (p<0.05). Expression of p53 in group 6 (1,67+-0.31) mcg/ml and 8 (1.18+-0.18)mcg/ml, TIMP-3 6(3,81 +- 0,49) mcg/ml and 8 (2,93 +- 0,42) mcg/ml were significantly higher compared to the control group (p<0.05), and Bcl-2 expression were significantly lower in all treatment groups (p<0.05). The correlation coefficient between miR-214 and Bcl-2 is -0.908. In silico analysis by docking method showed that EPI bond energy with p53 and Bcl2 receptors was higher than native ligand binding energy with p53 and Bcl-2 receptors. The sequencing results showed mutations in miR-21, and test with BLAST showed a target change in the miR-21 mutant. Conclusion: EPI compounds have a moderate anticancer effect, strong synergy with DOX and weak with CIS. The combination of EPI+DOX and EPI+CIS can increase miR-214, miR-34a, p53 and TIMP-3 expression and decrease Bcl-2 expression. There is a robust negative correlation between the expression of miR214 and Bcl-2. Based on the docking method, proteins p53 and Bcl-2 are unsuitable targets of EPI compounds. There was a mutation in miR-21 and a change in mutant miR-21 target.

Kata Kunci : Kanker serviks, Epoksi, miRNA, Index Kombinasi, miR-21 mutan, Cervical cancer, Epoxy, miRNA, Combination Index, MiR-21mutan.