STRENGTHENING OF CHEMOTHERAPY AGENTS cytotoxicity of doxorubicin BY FRACTION BRAZILEIN cup (Caesalpinia sappan L.) THROUGH INDUCED APOPTOSIS IN BREAST CANCER CELLS T47D
ABSTRACT: Doxorubicin is a chemotherapeutic agent that is commonly used for breast cancer therapy. The problems come when the use of doxorubicin cause side effects and there are cases of resistance in clinical applications. One of the strategy to solve doxorubicin problems is the application of co-chemoterapeutic agent using brazilein contained fraction. Therefore, this study aimed to determine the potency of the brazilein contained fraction to increases cytotoxicity of doxorubicin, and its combination can induce apoptosis in T47D cells, as well as explore the mechanisms of apoptosis through interaction prediction of brazilin and brazilein to caspase 3 based on molecular docking. Brazilein contained fraction (BCF) is a collection sample of CCRC Pharmacy UGM obtained from methanolic extract of Caesalpinia sappan L. bark and then fractionated using column chromatography. Thin layer chromatography test was used to verify the brazilein compound. MTT assay was used to evaluate cytotoxicity of single and combination application with parameters of IC50 for evaluation of a single treatment, and the value of CI, DRI, and isobologram profile as combinations parameter. The apoptotic induction test used flow cytometry with parameters percentage of apoptotic cell death. Exploration of brazilein and brazilin compounds interaction on caspase 3 was performed with molecular docking using PLANTS software with parameter of docking scores and visualization interaction at binding site. The results of this study indicate that doxorubicin has cytotoxic effect on T47D cells with IC50 values of 403 nM. From the cytotoxicity test showed the combination of 1/6 IC50, 1/4 IC50, 1/3 IC50, dan ½ IC50 of brazilein contained fraction and doxorubicin gave synergistic combination based on CI values and have DRI>1 showed the combination could reduce toxicity. In observation of the apoptosis induction test on the combination treatment of ½ IC50 brazilein contained fraction and ½ IC50 doxorubicin increased the number of apoptosis cells compared with single treatment of doxorubicin and brazilein contained fraction. Brazilein and brazilin had docking score of -72.46 and -74.96 and could interact on native ligand binding site at caspase 3 which is on amino acid residu of leu of 219, Phe 215, and Ile 259. Overall the results of this study indicate that BCF has a potency to be developed as a co-chemotherapy agent of breast cancer.
