Tesis

Analisis Potensi Flavonoid sebagai Modulator Autofagi dan Apoptosis Sel MCF-7 melalui Pendekatan Pemodelan Molekular dan In Vitro = Analysis of Flavonoid Potentials as Autophagy and Apoptosis Modulators in MCF-7 Cell Line through Molecular Modeling and In Vitro Study.

Latar belakang: Tujuan penelitian ini adalah untuk menilai potensi flavonoid hasil skrining pemodelan molekular sebagai modulator autofagi sel kanker payudara yang tergambar dari efeknya terhadap inhibisi pertumbuhan, apoptosis dan perubahan ekspresi gen efektor autofagi dan apoptosis. Desain: Desain penelitian ini adalah analisis in silico dengan eksperimen in vitro dengan menggunakan sel MCF-7 (Michigan Cancer Foundation-7) ER+/PR+/HER2- sebagai sampel. Metode: Analisis in silico dilakukan melalui pemodelan farmakofor, penambatan molekular, simulasi dinamika molekul, uji prediksi farmakokinetik dan potensi obat. Untuk uji in vitro, terdapat delapan kelompok perlakuan: kontrol, sel yang diberi 4-hidroksitamoxifen (4-OH-TAM), klorokuin (CQ), hesperidin (Hes), quercitrin (QCI), kombinasi 4-OH-TAM+CQ, 4-OH-TAM+Hes dan 4-OHTAM+QCI. Uji MTT dilakukan untuk menilai sitotoksisitas senyawa terhadap pertumbuhan sel. Flow cytometry dengan penanda Annexin V dan propidium iodida (PI) dilakukan untuk mengetahui status apoptosis. PCR kuantitatif dilakukan untuk menilai perubahan ekspresi gen efektor autofagi (Beclin-1 ) dan gen terkait regulasi apoptosis (Bcl-2, Caspase-3). Hasil: Kajian literatur, skrining berat molekul dan prediksi toksisitas menyeleksi 70 senyawa yang diikutkan dalam pemodelan farmakofor. Strategi pemodelan farmakofor menemukan bahwa optimasi kemiripan deskriptor radial distribution function (RDF) dengan bukti validasi eksperimen interaksi langsung senyawa dengan protein target memberikan performa model terbaik (AUC=0,88, EF=18,4, sensitivitas 91,67% spesifisitas 71 ,49%, akurasi 71 ,86%). Skrining virtual dengan Herbal DB 2.0 dan penambatan molekular mengidentifikasi dua kandidat flavonoid, hesperidin dan quercitrin. Hasil uji MTT menemukan IC50 hesperidin sebesar 16,29 µM yang mendekati IC50 4-OH-TAM pada 14,83 µM. Analisis flow cytometry menemukan bahwa hesperidin menyebabkan lebih banyak apoptosis lanjut dibandingkan quercitrin terutama ketika dikombinasi dengan 4-OH-TAM (7,00% vs 4,99%). Hasil PCR menunjukkan bahwa hesperidin meningkatkan ekspresi gen Beclin-1 namun menginduksi apoptosis melalui penurunan ekspresi gen Bcl-2 dan peningkatan ekspresi Caspase-3. Kesimpulan: Senyawa flavonoid hasil skrining virtual dari pemodelan molekular dan eksperimen in vitro memberikan potensi untuk dikembangkan sebagai modulator autofagi, khususnya hesperidin.
Kata kunci: apoptosis, autofagi, flavonoid, kanker payudara, model farmakofor


Background: The objective of this study is to evaluate the potential of flavonoid compounds, identified through molecular modeling screening, as inhibitors of autophagy in breast cancer cells, as indicated by their effects on growth inhibition, apoptosis, and changes in the expression of autophagy and apoptosis effector genes. Design: This study employs an in silico analysis combined with in vitro experiments using MCF-7 (Michigan Cancer Foundation-7) ER+/PR+/HER2- cells as the sample. Method: The in silico analysis included pharmacophore modeling, molecular docking, molecular dynamics simulation, and predictions of pharmacokinetics and drug-likeness. For the in vitro assays, eight treatment groups were established: control, cells treated with 4-hydroxytamoxifen (4-OH-TAM), chloroquine (CQ), hesperidin (Hes), quercitrin (QCI), a combination of 4-OH-TAM+CQ, 4-OHTAM+Hes and 4-OH-TAM+QCI. The MTT assay was used to assess the cytotoxicity of the compounds and cell proliferation. Flow cytometry with Annexin V and Propidium Iodide (PI) marker was performed to determine apoptosis status. Quantitative PCR was conducted to evaluate changes in the expression of autophagy (Beclin-1) and apoptosis-regulatory genes (Bcl-2, Caspase-3). Result: Literature review, molecular weight screening, and toxicity prediction selected 70 compounds which were included in pharmacophore modeling. The pharmacophore modeling strategy revealed that optimization through radial distribution function (RDF) descriptor similarity along with experimental evidence validation of direct interaction between compounds and target proteins, resulted in the best-performing model (AUC = 0.88, EF = 18.4, sensitivity = 91.67%, specificity = 71.49%, accuracy = 71.86%). Virtual screening using Herbal DB 2.0 and molecular docking identifed two flavonoid candidates: hesperidin and quercitrin. MTT assay results showed an IC50 for hesperidin of 16.29 µM, which is close to the IC50 of 4- OH-TAM at 14.83 µM. Flow cytometry analysis revealed that hesperidin induced more late apoptosis than quercitrin notably in combination with 4-OH-TAM (7.00% vs 4.99%). PCR results indicated that hesperidin stimulated expression of the Beclin- 1 gene, while promoting apoptosis through downregulation ofBcl-2 and upregulation of Caspase-3 expression. Conclusion: The flavonoid compounds identified through virtual screening of molecular modeling and in vitro experiments show potential to be developed as autophagy modulators, particularly hesperidin.
Keywords: apoptosis, autophagy, breast cancer, flavonoids, pharmacophore models