INVESTIGATION OF THE EFFECT OF BIBR1532 / RAPAMISIN COMBINATION ON BREAST CANCER STEM CELLS AND TELOMERASE ENZYME IN 3D AND 2D CELL CULTURE SYSTEM
Project no. 107 · Status: Kapanmış · 2017
Summary
Cancer stem cells (CSC) have been known to be responsible for tumor growth, metastasis and cancer resistance. Telomerase activity has been shown high in breast cancer, especially breast cancer stem cells in many studies. BIBR1532 is a selective anti-cancer agent that inhibits telomerase enzyme at the protein level. PI3K/Akt/mTOR pathway contributing malignant progression via aberrant signalization in breast cancer has a causative role in the self-renewal of CSC and drug resistance. Rapamycin, known as the cornerstone of rapalog and used in the treatment of breast cancer, suppresses mTOR activity and displays an effective profile on breast cancer stem cells (BCSC). It has been supported by many studies that mTOR regulated telomerase activity at the translational and post-translational levels. In this study, it was aimed to compare the effects of the combination of telomerase inhibitor BIBR1532 and rapamycin on breast cancer stem cells by the 2D and 3D culture medium. Cytotoxic effect of BIBR1532, rapamycin and their combinations on BCSCs were assessed via the WST-1 method, drug interactions in BCSCs by isobologram analysis, the apoptotic effect of dose groups by Annexin V method, the changes in gene expression regarding PI3K/AKT/mTOR pathway by using the qRT-PCR method. IC50 values of BIBR1532 and Rapamycin were found as 29.91 µM and 20.51 µM in BCSCs cell incubated in 2D culture medium, respectively. In 3D culture, IC50 values of BIBR1532 and Rapamycin on BCSCs cells were 50.08 µM and 37.22 µM, respectively. When two active compounds were combined, it was found that with the synergistic effect of the combination in 2D culture medium, BIBR1532 decreased to 23 µM with 1.30 DRI and rapamycin to 7.57 µM with 2.60 DRI. In BCSCs incubated in 3D culture, ED50 value of BIBR1532 decreased to 20.52 µM with 2.44 DRI and rapamycin to 9.45 µM with 3.93 DRI. Apoptosis was detected to induce 2.27-fold in BIBR1532-treated-2D-BCSCs, 4.79-fold in Comb-treated-2D-BCSCs, 3.58-fold in in BIBR1532-treated-3D-BCSCs and 7.44-fold in Comb-treated-3D-BCSCs. When the changes in gene expression were examined, it was determined that the expression of genes inhibiting cancer development such as TP53, PTEN, DDIT4, DDIT4L decreased, and the expression of genes such as hTERT, mTOR, RHOA, AKT, HIF1A, PRKAA supporting PI3K/AKT/mTOR-mediated cancer progression decreased. As a result, in this study, with the combination of BIBR1532 and rapamycin, two different malignant aberrations were simultaneously targeted in breast cancer stem cells and promising results were obtained. We anticipate that this study may provide a different perspective on further studies.