APOPTOSIS INDUCTION IN HUMAN BREAST CANCER MCF-7 CELL LINE AND IN SILICO DOCKING ANALYSIS OF (S)-RUGULACTONE FROM CRYPTOCARYA PULCHRINERVIA TO APOPTOTIC–RELATED PROTEIN

• Main Author: Fransisca, Mathea
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/50762
• Institution: Institut Teknologi Bandung

Cancer is a disease in which some cells in the body continuously divide abnormally. This abnormal cell division occurs due to mutations in certain genes in those cells. One of the most frequent cancer cases that happens in Indonesia is breast cancer. Nowadays, research on how to treat cancer are still conducted, one of them is by using natural-based drugs. (S)-rugulactone compound is a natural compound which has cytotoxic effect against cancer cells. This compound has been isolated from leaves of Cryptocarya pulchrinervia (Lauraceae) plant, which is an Indonesian endemic plant from Sumatera Island. In the previous research, it has been reported that the stereoisomer of (S)-rugulactone, namely (R)-rugulactone from C. rugulosa, could cause apoptotic cell death in breast cancer cells by inhibiting NF-?B activation pathway. Therefore, the objectives of this research are to analyze the effect of (S)-rugulactone in inducing cell death in human breast cancer MCF-7 cells and also to predict protein target of (S)-rugulactone and the docking location related to cell death induction. Cell death was observed using Hoechst staining and RealTime-GloTM Annexin V Apoptosis and Necrosis Assay (Promega) kit. Treatment groups in this research were negative control, positive control (Paclitaxel 0.2 ?M), DMSO 0.1 %, (S)-rugulactone IC25 (0.675 ?g/mL), IC37.5 (1.215 ?g/mL), IC50 (2.025 ?g/mL), IC62.5 (3.375 ?g/mL), and IC75 (6.075 ?g/mL). The data was collected from one repetition experiment with total samples per treatment group as much as 2 well each for Hoechst staining and 3 well each for Annexin V Apoptosis and Necrosis Assay. Meanwhile, in silico molecular docking analysis and interaction visualization were conducted using AutoDock Vina and LigPlot+ program. Proteins that were analyzed are proteins involved in NF-?B activation pathway, which are IRAK-4, IRAK-1, TAK-1, IKKB, and RIP-1. Hoechst staining result showed that (S)-rugulactone exposure caused apoptotic cell death in IC37.5, IC50, IC62.5, and IC75 treatment groups, which was characterized by nuclei fragmentation and apoptotic bodies formation. These results were also supported by the result from RealTime-GloTM kit. In this part, apoptotic cell death determined by the rise of luminescence signal followed by fluorescence signal in several hours later. Besides apoptosis, non-apoptotic cell death was also found in the IC75 treatment group. This results could be observed both by Hoechst staining and RealTime-GloTM kit. (S)-rugulactone was expected to induce apoptosis by inhibiting NF-?B activation pathway, same as (R)-rugulactone. This presumption was confirmed through molecular docking analysis. Based on this analysis, it was predicted that (S)-rugulactone could interact with IRAK-4, IRAK-1, and TAK-1 protein in their ATP-binding pocket by forming some specific interactions in a form of hydrogen bonds. (R)-rugulactone was also predicted to interact with these proteins in the same protein region. These interactions were predicted to inhibit some protein phosphorylation process, which was crucial for NF-?B activation signalling pathway. Furthermore, it might lead to the downregulation of some gene expression, which are important for maintaining cell survival and cell cycle process. From the results obtained, it could be concluded that (S)-rugulactone could induce apoptosis of MCF-7 cells in dose dependent manner. However, (S)-rugulactone with a high concentration (IC75) could induce apoptosis as well as non-apoptotic cell death. Based on in silico analysis it was known that (S)-rugulactone could interact with IRAK-4, IRAK-1, and TAK-1 protein in their ATP-binding pocket, same as (R)-rugulactone.