Naidoo, Strinivasen.Botha, Julia Hilary.Mathibe, Lehlohonolo John.2021-06-222021-06-2220172017https://researchspace.ukzn.ac.za/handle/10413/19500Doctoral Degree. University of KwaZulu-Natal, Durban.Introduction According to recent World Health Organisation (WHO) estimates, cancer causes more deaths than coronary heart diseases globally (GLOBOCAN, 2012). While communicable diseases such as HIV/AIDS continue to burden African populations, cancer is increasingly recognised as a critical public and private health problem in Africa (Igene, 2008). It is estimated that by 2030, about 112 921 new cases of cancer will be diagnosed in South Africa (Singh et al., 2015). This would represent a 50% increase of new cancer cases as compared to 2012’s estimates by the WHO. Although there is little doubt about the incidences of cancer, there are, unfortunately, divergent theories in as far as tumourigenesis and the aetiology of cancer. Some researchers hold the view that cancer originates from malignant transformation of normal tissue progenitor and stem cells (Reya et al., 2001). Others believe that cancer is as a result of mature cells that have undergone de-differentiation (Sell, 2004). Notably, latest research has shown that there is a strong association between tissue-specific cancer risk and the lifetime cumulative number of cell divisions of tissue or organ-specific stem cells (Tomasetti & Vogelstein, 2015). Although there are still differing views on the origins of cancer, it is widely accepted that this devastating disease occurs as a result of abnormal cell development and is characterised by uncontrollable cell proliferation. The majority of currently-available cancer treatments target cell proliferation. However, the effectiveness of many cytotoxic drugs, including those that were discovered from plants, is limited by their serious side-effects and cost (Abratt, 2016). Chemotherapeutic agents that were originally discovered from medicinal plants include vinblastine (isolated from Catharanthus roseus), etoposide (isolated from Podophyllum peltatum), paclitaxel (isolated from Taxus brevifolia) and topotecan and camptothecin (isolated from Camptotheca acumenata). Thus, medicinal plants continue to play a critical role in the management of diseases in the world. In Africa, decoctions, which contain extracts from various medicinal plants (Bruneton, 1995; Balunas & Kinghorn, 2005), are widely used for traditional management of many diseases including cancer. However, apart from subjective oral evidence regarding the effectiveness of extracts from various plants, the identity of ingredients, as well as the science and pharmacology of active compounds found in numerous popular concoctions and decoctions are not known. Objectives The main objectives of this study were:  To assess anti-proliferative potential of three plant-derived-compounds, i.e. hypoxoside, ent-Beyer-15-en-19-ol and Z-venusol on human cancer cells, namely DU-145 (prostate), HeLa (cervical) and MCF-7 (breast) in vitro.  To determine the type of cell death, i.e. whether a compound with potential causes apoptotic or necrotic cell death on both human cancer and normal cell lines (such as MCF-12, HMECs and dMVECs).  To investigate how a potential compound exerts its cytotoxicity. Materials and Methods Initially dimethylthiazol-diphenyltetrazolium bromide (MTT) assays were conducted to find the concentrations which may inhibit proliferation in prostate (DU-145), cervical (HeLa) and breast (MCF-7) cancer cells. Normal human cell lines, which were used for control purposes, were the primary human mammary epithelial cells (HMECs), MCF-12 and the dermal microvascular endothelial cells (dMVECs). Initially, cells were exposed for 48 hr to hypoxoside, ent-Beyer-15-en-19-ol and Z-venusol, which were isolated from Hypoxis hemerocallidea, Helichrysum tenax, and Gunnera perpensa, respectively. The concentrations ranged from 2.34 μg/mL to 2400 μg/mL, dissolved in cell specific media. In subsequent experiments, the more sensitive sulforhodamine B (SRB) methodology was used, and cells were exposed to Z-venusol for 24 hr, 48 hr and 72 hr, to much lower concentrations, which ranged from 1.9 μg/mL to 240 μg/mL dissolved in dimethyl sulphoxide (DMSO). To investigate possible pathways of observed cell death, two assays were conducted. These were the fluorescein isothiocyanate (FITC) Annexin V apoptosis detection assay (using the FACS Calibur “JO” E5637 flow cytometer for analysis), and the lactate dehydrogenase (LDH) assay. To explore possible mechanism(s) of action, the activities of interleukin-6 (IL-6) and cyclic adenosine monophosphate (cAMP) were assessed. To investigate the activity of IL-6, cells were exposed for 48 hr to various working concentrations of Z-venusol; that is, 37.5 μg/mL and 75 μg/mL. To investigate the activity of direct cAMP, cells were exposed for 48 hr to various working concentrations of Z-venusol; that is, 37.5 μg/mL, 75 μg/mL, and 150 μg/mL. Absorbance, which is inversely proportional to the concentration of cAMP in both the samples and the standards, was measured using a BioRad (Model 3550) microplate reader. Epinephrine (10 μM) and propranolol (10 μM), were used separately and in combination, added to the highest concentration of Z-venusol for comparison. Main Results & Discussion Hypoxoside resulted in a statistically significant (p < 0.001) 38% and 77% increases in proliferation in MCF-7s at concentrations of hypoxoside 1200 μg/mL and 2400 μg/mL, respectively, after 48 hr exposure. In support of the current findings, Xulu (2013) also reported that hypoxoside, and its active derivative known as rooperol, significantly increases cell proliferation of both cancer and normal mammary cells in vitro (Xulu, 2013). This was considered an undesirable finding with regards to the aim of finding a cure for cancer. Therefore, no further test were carried out on this compound beyond the initial screening stages. The highest concentration (i.e., 2400 μg/mL) of the second compound, that is ent-Beyer-15- en-19-ol, decreased proliferation in prostate cancer cells (DU-145) and in breast cancer cells (MCF-7) by 6% and 19%, respectively. Interestingly, much lower concentrations, i.e. 4.7 μg/mL and 9.4 μg/mL, of ent-beyer-15-en-19-ol significantly (p < 0.05) decreased cell proliferation in cervical cancer cells (HeLa) by 37% and 41%, respectively. The differences in expression of vimentin gene, which is over-expressed in HeLa cells and suppressed in MCF- 7s and DU-145s may explain why this compound showed significant activity only in the cervical cancer cells (Oshima, 2002; Satelli & Li, 2011). More importantly, the ability of this compound to significantly inhibit cell proliferation in the HeLa cell line by almost 50% at lower concentrations offers an opportunity for further studies. The findings with regards to the third compound, i.e. Z-venusol, were the most exciting. Hence investigations on it were developed beyond the screening stages. This compound demonstrated a statistically significant, concentration-dependent, apoptotic inhibitory effect on the proliferation of MCF-7 cells, with an IC50 of 53.7 μg/mL after 72 hr exposure, while the highest concentration (250 μg/mL) resulted in 69% inhibition. Both the FITC Annexin V and LDH results suggested that apoptosis contributed to most of the effects observed. Further, there was non-significant inhibition (20%) of HMEC proliferation observed when the concentration of Z-venusol was increased beyond 16.6 μg/mL. The highest concentration of Z-venusol used in this study resulted in a statistically significant (p < 0.001) 51% inhibition of IL-6 activity in the MCF-7 after 48 hr exposure. None of the Z-venusol concentrations, either alone or in combination with epinephrine, an agonist of the adrenergic receptors, showed any statistically significant effect on the levels of cAMP in the MCF-7s. Surprisingly, there was a significant (p ≤ 0.028) 34% elevation of cAMP levels in cells which were exposed to a combination of Zvenusol and propranolol. If Z-venusol was ever able to be used clinically, there might be a need to increase the dose high enough for the attainment of desired therapeutic effects with minimal cytotoxicity on normal cells, because its potency is much lower than that of cisplatin. Increasing Z-venusol to a therapeutically-effective concentration would be possible as there was no plateauing-off of inhibition of proliferation in MCF-7s. It was only in primary normal human mammary epithelial cells (HMECs) that formation of “plateaus” was observed. Favourably, this selective plateauing-effect might allow the ‘gold-standard’ attainment of the desired cytotoxic effect on cancer cells while preserving normal cells at higher concentrations. There are no studies with which to directly compare the findings of this study. However, reports on effects of the extracts of G. perpensa on various other cancer cell lines provide an opportunity for comparison. For instance, the results of this research support the findings of Simelane and colleagues. They recently reported that G. perpensa extracts caused an inhibition of proliferation of hepatocellular carcinoma cells (HepG2) with an IC50 of 222.33 μg/mL and human embryonic kidney 293 (HEK293) cells, with an IC50 of 279.43 μg/mL both after 48 hr of treatment (Simelane et al., 2012). Conclusion Z-venusol, unlike other compounds studied, has a firm potential to play a role in the treatment of cancer in the future. Its mechanism of action involves IL-6 signaling, which may trigger other downstream mediators and may also involve cAMP “cross-talk”. Recommendations More basic science investigations using other hormone-dependent and highly invasive breast cancer cell lines such as the triple-negative MB-231 cells are needed. In vivo studies, such as using the nude mice model, are needed to confirm the in vitro results and to provide an insight into the benefits of Z-venusol in living systems.enGunnera perpensa.Z-venusol.Cancer.Cytotoxic drugs.Tumourigenesis.Human cancer cells.Apoptotic cells.Thesis