Cervical cancer is the most common malignancy after breast cancer in women worldwide. It accounts for 83% of all new cases and 85% cancer death in developing countries. In South Africa, cervical cancer is the common cancer in women with an annual crude incidence rate of 30.2 per 100,000 women and the highest rate were found to be in women between the ages of 66-69 years. Approximately 6800 women in S.A face new case of cervical cancer while accounting for 3700 cancer death annually. Because of unequal access to the health facilities, socio-economic differences, HPV and HIV infection, the rate of cervical cancer in black women is higher (42.1%) compared to the low rate in white women. Because of the name “cancer bush’ given to Sutherlandia Frutescence(S.F) plant by the traditional healers as well as Xhosas, Zulu, Sotho and cape Dutch for its anti-cancer activity, the plant was in this study to confirm its cytotoxic effect on the cervical cancer cell lines. Aim of the study: to evaluate the methanolic extracts of Sutherlandia Frutescens on cervical cancer cell lines. Materials and Methods: The MTT assay was performed to evaluate SiHa cell lines treated with methanolic extract of S.F (50μg/ml, 100μg/ml, 150μg/ml and 200μg/ml). The three compounds (Canavanine, GABA and Pinitol) were also evaluated for its anti-tumour activity. The cell growth was then showed in real time using Xcilligence. Flow cytometry was employed to determine the mode of action. Caspase 3/7 assay was performed to confirm if cell death was via caspase-dependent or independent and ATP was done to assess the ATP level in S.F treated cells. Results: MTT shows a significant decline in cells treated with 50μg/ml, 100μg/ml and 200μg/ml of the extract and 50μg/ml was concluded to be the concentration at which 50% of the cells die. The ATP results are inconsistent with MTT result; the ATP level increased in S.F treated cells. Cell index which represents the quantitative measure of cell growth in real time decline upon treatment with 50μg/ml. Flow cytometry showed cells are dying by apoptosis and the cell cycle arrest is mostly in the S phase. The cell death was caspase-dependent as it shows an increased luminescence which is proportional to the number of caspase. The concentrations of the compounds used, Canavanine (1000μM, 1500μM and 2500μM), GABA (100μM, 300μM and 500μM) and Pinitol (30μM, 90μM and 120μM) induce cell death and cell death shows to decrease after the maximum concentration. Conclusion: Sutherlandia Frutescence has proven with number of research that it induces cell death via apoptosis. After evaluating its cytotoxicity, the plant shows to be a promising anti-tumor agent that needs to be clinically proven. / Life Sciences / M. Sc. (Life Science)

Thesis (MSc (Physiological Sciences))--University of Stellenbosch, 2011. / Includes bibliography. / ENGLISH ABSTRACT: The hypothalamic-pituitary-adrenal-(HPA) axis has long been closely associated with psychological stress-induced activation of the adrenal cortex and subsequent glucocorticoid production. Another, less known peripheral limb of the psychological stress response, is the sympatho adrenal medullary pathway. We hypothesized that the sympatho-adrenal medullary system constitutes the primary response to acute psychological stress, with the HPA-axis functioning as a secondary response. We tested our hypothesis by manipulating a model of acute mild psychological stress (restraint) by blocking IL-6, a valuable constituent of the sympatho-adrenal medullary system. Serum corticosterone concentration increased in response to stress (7 ± 3 vs. 57 ± 4 ng/ml; P<0.0001), a response attenuated when IL-6 was blocked (17 ± 7 ng/ml). Stress increased pituitary mass only when IL-6 was blocked (38 ± 3 vs. 65 ± 6 mg; P <0.001). Stress increased left adrenal mass only in the presence of IL-6 (34 ± 1 vs. 73 ± 8 mg; P <0.00001). Stress did not influence the circulating levels of TNF-α, IL-1β or IL-6 significantly. IL-1β and TNF-α concentrations in the unstressed rats were lower when IL-6 was blocked. We then manipulated the stress model by administering S. frutescens extract to elucidate both the central and peripheral effects of acute S. frutescens administration on the psychological stress response. Restraint caused decreases in hippocampal GR levels when compared to respective controls. S. frutescens administration and exposure to restraint synergistically decreased hippocampal GABAAR levels. In addition, exposure to both stress and S. frutescens led to a noteworthy increase in pituitary mass (P = 0.078), as well as pituitary ACTH levels (P < 0.01). Similarly, differences in circulating ACTH levels showed an effect of stress on ACTH secretion only in the presence S. frutescens (P < 0.05). Adrenal mass was significantly increased in S. frutescenstreated animals that were also exposed to restraint (P < 0.05). Adrenal levels of ACTH showed a reciprocal trend to pituitary and circulating ACTH levels. No statistically significant differences were seen in adrenal IL-6 content. However, marked increases in IL-6 levels were seen at this level with administration of S. frutescens stress exposure and a cumulative increase seen with both S. frutescens-treatment and stress exposure. Hippocampal GABAAR, pituitary mass, pituitary ACTH and circulating ACTH levels showed a similar trend towards a synergistic effect of S. frutescens and restraint in activation of the psychological stress response, while adrenal ACTH levels showed an inverse trend. Hippocampal GR did not show any effect of stress or S. frutescens-treatment. The results from these two experiments indicate that the sympatho-adrenal medullary system constitutes the primary response to acute mild psychological stress and that the HPA-axis is only activated during an exacerbated stress response or when the sympatho-adrenal medullary contribution is inadequate. Furthermore, the acute administration of S. frutescens possibly led to a functional shift in GABAergic function, resulting in activation of the stress response. The anecdotal reports of a “docile” effect of S. frutescens most likely results from activation of the mesolimbic dopaminergic system by the hippocampus and amygdala. These results have dramatic consequence in GABA-based anxiety-treatments. / AFRIKAANSE OPSOMMING: Die hipotalamo-pituïtêre-adrenale (HPA)-as is lank bekend as ‘n primêre rolspeler in die respons op emosionele stres en daaropvolgende glukokortikoïed produksie. ‘n Ander, minder bekende arm van die sielkundige stres respons is die simpatiese bynier-medulla-sisteem. Ons hipotese was dat die laasgenoemde simpatiese bynier-medulla-sisteem die primêre respons tot sielkundige stres behartig terwyl die HPA-as ‘n sekondêre respons bied. Ons het ons hipotese getoets deur die manupilering van ‘n beproefde stres model waar ons IL-6, ‘n waardevolle rolspeler in die simpatiese bynier-medulla-sisteem, onderdruk het. In respons op stress, het serum kortikosteroon konsentrasies toegeneem slegs in die teenwoordigheid van IL-6 (7 ± 3 vs. 57 ± 4 ng/ml; P<0.0001), maar nie wanneer IL-6 onderdruk is nie (17 ± 7 ng/ml). Stres het ‘n verhoging in hipofise massa teweeggebring slegs tydens die onderdrukking van IL-6 (38 ± 3 vs. 65 ± 6 mg; P <0.001). Stres het ook linker-byniermassa verhoog slegs wanneer voldoende IL-6 beskikbaar was (34 ± 1 vs. 73 ± 8 mg; P <0.00001). Stres alleen het geen invloed gehad op serum IL-1β, IL-6 of TNF-α nie, maar die onderdrukking van IL-6 het wel ‘n inhiberende effek op basale IL-1β en TNF-α gehad. Daarna het ons weer eens die stresmodel manipuleer deur die rotte ‘n S. frutescens ekstrak te gee in ‘n poging om beide die sentrale en perifere effekte daarvan op die sielkundige stres respons te evalueer. Stres alleen het gelei tot ‘n afname in GR terwyl ‘n kombinasie van stres en S. frutescens administrasie tot ‘n afname in GABAARα1 in die hippokampus gelei het. Hierdie kombinasie het ook tot ‘n merkwaardige toename in hipofise massa (P = 0.078) sowel as ACTH-inhoud van die hipofise (P < 0.01) gelei. ‘n Soortgelyke patroon is waargeneem betreffende sirkulerende ACTH en byniermassa met P < 0.05 vir elk. Bynier ACTH inhoud, aan die ander kant, het ‘n omgekeerd eweredige verhouding met ACTH in die hipofise en in sirkulasie getoon. Bynier IL- 6 inhoud het geen statisties beduidende verskille getoon nie, maar ‘n merkwaardige verhoging is weereens gesien met ‘n kombinasie van stres en S. frutescens administrasie. Die soortgelyke tendens wat waargeneem word in GABAAR in die hippokampus, asook hipofise- en sirkulerende ACTH vlakke, en dui op ‘n samewerkende rol van stres en S. frutescens in die aktivering van die sielkundige stres respons. GR in die hippokampus toon geen veranderinge nie. Die resultate van die twee eksperimente dui op ‘n primêre rol van die simpatiese bynier-medulla-sisteem in die respons op ‘n akute stressor en dat die HPA-as net geaktiveer word tydens ‘n ooreiste stres reaksie of indien die simpatiese bynier-medulla-sisteem onderdruk word. Die waargenome “verdowings”-effek van S. frutescens word moontlik deur aktivering van die mesolimbiese dopamien pad deur die hippokampus en amigdala bewerkstellig. Die resultate mag ook lei tot die heroorweging van GABA-gebaseerde angs medikasies.