Petros Beyene (Professor)Tuasha Nigatu2020-10-142023-11-092020-10-142023-11-092020-07-07http://etd.aau.edu.et/handle/123456789/22715The incidence and mortality of breast cancer in women is increasing worldwide. Breast cancer has high intertumoral and intratumoral heterogeneity. Cancer stem cells (CSCs) are one of the many subpopulations of cancer cells and are involved in tumor initiation, progression, evolution and metastasis, and are believed to be responsible for chemotherapeutic resistance. The present study investigated in vitro anticancer activities of extracts from seven medicinal plants traditionally used to treat breast cancer in Ethiopia. Chemical compounds were also isolated from the most potent bioactive fractions. The study determined cytotoxicity of the crude methanol extracts (80 %, v/v) and that of solvent fractions (1:1, v/v) of the most cytotoxic ones. Furthermore, the anti-CSCs potency of the bioactive solvent fractions and the isolated compounds were also investigated. Vacuum liquid chromatography, preparative thin layer chromatography, column chromatography and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) were employed for the phytochemical characterization. Cytotoxicity of the phytochemicals against the human breast cancer cell lines, JIMT-1, HCC1937 and MCF-7 and the normal-like breast epithelial cell line, MCF-10A, was determined by a spectrophotometric MTT assay. To investigate specific activity against CSCs, flow cytometric determination of the aldehyde dehydrogenase (ALDH) positivity and determination of CD44+/CD24- CSC subpopulations as well as the colony forming efficiency (CFE) in a serum-free soft agar iv were performed. The effects of phytochemical treatment on tumor necrosis factor-α (TNF-ɑ)-induced translocation of the nuclear factor-kappa B (NF-κB) to the nucleus and on cell migration in a wound healing assay were also investigated. The half maximal inhibitory concentrations (IC50) were determined for the crude methanol extract of Sideroxylon oxyacanthum (0.09 μg/mL, MCF-7), Clematis simensis (0.17 μg/mL, HCC1937) and Dovyalis abyssinica (0.21 μg/mL, MCF-7). Ethyl acetate solvent fraction of V. leopoldi was the most cytotoxic against JIMT-1 cells (IC50 = 0.87 μg/mL). The bioactive solvent fractions of V. leopoldi, S. oxyacanthum and C. simensis showed dose dependent reduction in the ALDH+ subpopulation of JIMT-1 cells. The chloroform fraction of C. simensis (80 μg/mL) completely blocked CFE in JIMT-1 cells. The bioactive solvent fractions of V. leopoldi, S. oxyacanthum and C. simensis significantly reduced cell migration (P<0.05). The ethyl acetate fraction of V. leopoldi (0.87 μg/mL) significantly reduced the number of NF-κB positive nuclei (P<0.001). Seven compounds - four sesquiterpene lactones and three flavonoids, were isolated from V. leopoldi. Among the sesquiterpene lactones, one was a novel compound (“Ndb21a2-P”, C20H26O8, M. Wt. = 394.42), which was highly cytotoxic (IC50 = 1.6 μM) to JIMT-1 cells. Whereas all sesquiterpene lactones significantly reduced cell migration (P<0.05), the compound Vernomenin was most effective (P<0.005) in reducing the ALDH+ CSC subpopulation. Also, another isolate, 11ß,13- dihydrovernodalin, resulted in significant reduction in NF-κB positive nuclei (P<0.05). The study showed V. leopoldi to be the most cytotoxic medicinal plant to cancer cells and the sesquiterpene lactones isolated from it had desirable CSCs-specific activities. The study further showed the importance of systematic selection of traditional medicinal plants for anticancer experimental studies. In conclusion, it is recommended that an in-depth investigation into the chemotherapeutic potentials of the sesquiterpene v lactones from V. leopoldi and their structural analogues, be considered as this could augment the global breast cancer drug discovery research.enBioactivity-Guided FractionationBreast CancerCancer Stem CellsCytotoxicityIn VitroMTTSesquiterpene LactonesTraditional MedicineThesis