Targeting aberrantly activated Notch pathway receptors by using novel combination of Oleanolic acid and β-sitosterol in breast cancer
Abstract
Introduction: The aim of the present study was to explore the synergistic effect of oleanolic acid and β-sitosterol in aberrantly activated notch pathway receptor in breast cancer cells. We were trying to elucidate the probable mechanism of the action of these compounds by which the significant cell death of breast cancer cells was noticed.
Materials and Methods: The cell cytotoxicity was determined using 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and colony formation assay. Apoptotic property has been checked by Hoechst staining and western blot analysis. Moreover, anti-metastatic property was confirmed by wound healing assay, DNA damage was checked by comet assay, microtubule destabilization was checked by Immunofluorescence and cell cycle was checked by FACS, expression of proteins of pathways was checked by western blot analysis.
Results: The MTT assay showed that oleanolic acid and β-sitosterol were able to induce cell cytotoxicity in human breast cancer cell lines MCF-7 and MDA-MB-231 cell lines but compounds showed the maximum effect against MDA-MB-231, so we carried our further studies with MDA-MB-231. After checking the cytotoxicity, we went for colony formation assay and we found that these compounds inhibited colony formation in MDA-MB-231 cells and also showed distinct characteristics of DNA damage which we have checked by comet assay and then we found that these molecules have significant effect on microtubule destabilization, and further leads to apoptosis with enhanced expression of cleaved caspase-3 and cleaved PARP. Interestingly, FACS analysis showed that treated cells are arrest at G2/M stage of cell cycle. For checking the migration ability, we did wound healing assay and which revealed that oleanolic acid and β-sitosterol has anti-metastatic effect against breast cancer cells. All the effect were significantly enhanced when we combine these two compounds. So oleanolic acid and β-sitosterol behave good in a synergistic manner against Breast cancer cells. We did western blot analysis for checking the effect of compounds against Notch and Akt pathways, and noticed the downregulation of the notch pathway receptors which are involved in breast cancer and also noticed the downregulation of Akt expression.
Discussion and Conclusion: First of all, the synergistic role of the oleanolic acid and β-sitosterol on Notch pathway has been reported for the first time. Collectively, the study reveals the therapeutic potential of oleanolic acid and β-sitosterol against breast cancer they are cytotoxic to breast cancer cells and destabilize the microtubules and this action trigger apoptosis by arresting the cells at G2/M phase of cell cycle and eventually causing the cell death of breast cancer cells .In this way it is behaving like microtubule targeting agent (MTA).By this we can also conclude that these compounds are having the potential to target the aberrantly Notch and Akt pathways. So, the synergistic behaviour of oleanolic acid and β-sitosterol might be act as inhibitor of Notch and Akt pathways and may be a probable remedy for breast cancer patients. Further research is needed to explore more potential of these compounds.
