Browsing by Author "De Kock, M."
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Item Comparative in vitro study of the anti-cancer effect of apricot and peach kernel extracts on human colon cancer cells(University of the Western Cape, 2015) Cassiem, Wagheda; De Kock, M.; Mohammed, A.Amygdalin, a controversial anti-cancer agent, is a cyanogenic glycoside plant compound found in apricot and peach kernels. Both amygdalin and its patented form, Laetrile®, have been promoted and sold as "vitamin B-17", although neither compound is a vitamin. No consensus on the efficacy of amygdalin regarding the treatment of different cancers has been reached. Cancer is now the third leading cause of death worldwide. More than 7.6 million deaths were estimated to have occurred in 2007 and by 2030 it is projected to increase to 17 million cancer deaths per year. Cancers of the lung, breast, colon/rectum, liver and prostate are no longer largely confined to Western industrialized countries but are among the most common cancers worldwide (Thun et al. 2010). In South Africa it is estimated that one in every four males and one in every five females will be affected by a cancer diagnosis in their lifetime. The most common cancers in males are prostrate, lung, oesophagus, bladder and colorectal and in females they are cervix, breast, colorectal, oesophagus and lung (Haggar & Boushey 2009). Colon cancer is one of the most prevalent cancers worldwide, especially in western societies and is nutrition dependent (Klenow et al. 2009). It is one of the leading causes of death in both men and women in industrialised western countries. Colon cancer development involves both hereditary factors and lifestyle factors which include absence of physical exercise, unbalanced nutrition and long term smoking (Forman et al. 2004; Heavey et al. 2004). Colon cancer is traditionally treated by the resection of the colon, chemotherapy, radium therapy, and pharmaceutical hormonal drugs (Willson et al. 1987; Padussis et al. 2004)). Epidemiological studies supports evidence that colon cancer is preventable by adjusting the diet (Forman et al. 2004) and a protective effect is attributable to polyphenols and foods such as fruits and vegetables (Araújo et al. 2011). It was reported by Ruan et al. (2006) that the addition of Chinese Herbal Medicine in conjunction with chemotherapy notonly raised the efficacy of the chemotherapeutic drug, but also reduced the toxic side-effects. The aim of this research was to carry out a comparative in vitro study of the anti-tumour effect of the Chinese , South African and Turkish apricot (Xing ren / Armeniacea Semen) and Chinese and South African peach (Tao ren / Persica Semen) kernel extracts on the HT-29 colon cancer cell line.All the extracts significantly reduced cell viability and inhibited proliferation in the HT-29 cancer cells after 24 hours with the lipophilic and total fractions of CAK being the most effective. After 72 hours, it is clear that the inhibitory effects have been abolished and replaced by a stimulatory effect as the cell viability is higher in the treated cultures than the untreated controls. Results show that the total and the hydrophilic fractions of all the kernels increased cell viability more than the lipophilic fractions. It cannot be said with certainty that it was the amygdalin metabolite cyanide that affected the cell viability or induced apoptosis on its own. If hydrolysis of amygdalin indeed happened and cyanide was produced, it would affect the cells by shutting down aerobic respiration. Since cancer cells have more β- glucosidases and less rhodanese than normal cells, it is a possibility that the HT-29 cancer cells had some rhodanese to convert cyanide into a relatively harmless compound thiocyanate. It could be that in vitro this conversion, in light of the low enzyme levels in the HT-29 cancer cells, happened slowly and that the effect was only seen after 48 hour. However, this does not explain the overall inhibition even by the lipophilic fractions that should not contain any amygdalin or the eventual stimulatory effect, observed from 48 hour onwards.The S phase block observed, was mostly seen after 24 hour exposure to organic extractions, with the SAK showing 86% of cells in the S phase in contrast to the aqueous extractions which only slightly increased the S phase fraction. This could indicate that synergistic and/or additive effects between polyphenolic compounds may also be responsible for the reduction of cell viability, proliferation and apoptosis. All the kernels and the various fractions affected cell viability and to an extent cell cycle progression, but more studies is needed to establish the most effective kernel and specific fraction or signature active component. Inhibition of cell viability and proliferation and the induction of apoptosis could be an important preventive approach in chemoprevention. Understanding how dietary components regulate proliferation and cell survival could play a critical role in development of new enriched agents that can prevent and treat cancer with reduced risk of toxicity.Item The effect of maternal nicotine exposure on the alveolar wall composition during the phases of lung development(University of the Western Cape, 2015) Adonis, Jihaan; Maritz, G.S.; De Kock, M.Cigarette smoking is one of the foremost causes of chronic obstructive pulmonary diseases such as emphysema and chronic bronchitis, and although it is the most preventable causes of death, it accounts for approximately 6 million deaths worldwide each year. Cigarette smoking during pregnancy and lactation remains one of the primary modifiable risk factors for undesirable fetal, obstetrical, and developmental outcomes. Consequently, the offspring of the smoking mother is exposed to nicotine via the blood and the milk of the mother. As a result, nicotine interacts with the developing offspring and therefore interferes with normal fetal lung development. Maternal smoking during gestation and lactation has been associated with both short and long term health risks ranging from intrauterine growth restriction to physiological abnormalities. Maternal smoking has also been strongly linked to an increased risk for pulmonary diseases and respiratory morbidity in the offspring of the smoking mother. The main objectives of this study were to determine the effects of maternal nicotine exposure during gestation and lactation on the alveolar wall composition during lung development in the offspring; if maternal nicotine exposure during gestation and lactation induces premature cellular senescence in the lungs of the offspring; to clarify the role of pulmonary fibroblasts in premature senescence; and to establish whether tomato juice supplementation will prevent premature aging in the lungs of rats that were exposed to nicotine via the placenta and mother’s milk. From the data generated in this study it was evident that maternal nicotine exposure during gestation and lactation compromises the gas exchange function of the lungs of the F1 offspring. This was prevented by supplementing the mother’s diet with tomato juice which is then received by the offspring via the placenta and mother’s milk. This is conceivably achieved by maintaining the oxidant-anti-oxidant ratio of the mother and of the developing fetus and neonate, thereby averting premature senescence caused by nicotine exposure. Moreover, the present study also demonstrates that a decrease in fibroblast density is associated with emphysematous-like lesions in the lungs of the nicotine exposed F1 progeny. Since pulmonary fibroblasts are chief contributors to the extracellular matrix of the lungs, involved in alveolar multiplication and regeneration; premature aging or cessation of the metabolically active fibroblasts largely contributes to diminished lung structure and function.Item The interaction between 6 MV X-rays and p(66)/Be neutrons with spherical gold nanoparticles to induce cellular damage(University of the Western Cape, 2016) Engelbrecht, Monique; De Kock, M.; Slabbert, J. P.Despite the advances in therapies such as chemotherapy and radiotherapy, tumours have been shown to be resistant to the treatments. Gold nanoparticles (AuNPs) have been recognized as effective radiosensitizers of low energy (e.g. 200–500 kV) X-rays, leading to the emission of Auger electrons that cause highly localised ionizing damage to cells. Spherical AuNPs were synthesised via the reduction of the chloroaurate ions by sodium citrate. Characterisation of AuNPs involved UV-visible spectrophotometry, zeta (Z) potential, dynamic light scattering (DLS) and polydispersity index (PDI) measurements for determination of surface plasmon resonance (SPR), surface charge and stability, as well as transmission electron microscopy (TEM) for hydrodynamic core sizes, size distribution width and shape of AuNPs. Both the 5 and 10 nm AuNPs were found to be anionic with λmax absorbance of 525 nm and uniform size distribution. DLS measurement at 38.12 nm and 48.50 nm, respectively for 5 nm and 10 nm AuNPs, points to aggregation of the AuNPs. However, TEM measurements confirmed the core size of the 10 nm AuNPs. Non-malignant Chinese hamster ovary (CHO-K1), brain endothelial (BEnd5), breast (MCF-10A), isolated human lymphocytes and malignant breast (MCF-7) cell lines were treated with 50 μg/ml of AuNPs, and irradiated with either 1, 2 or 4 Gy X-rays or 1 or 2 Gy p(66)/Be neutron radiation. The γ-H2AX foci assay, cytokinesis-block micronucleus assay, MTT assay and fluorescence-activated cell sorting (FACS) was used to determine that amount of double stranded breaks (DSBs) in isolated lymphocytes, the presence and number of micronuclei (MNi) within binucleated cells (BNCs), cell viability and cell cycle progression, respectively. Preliminary experiments that established the reliability of the study regarding the induction of DNA damage after the bombardment of AuNPs by scattered low kV X-rays, were carried out on lymphocytes. Combined treatment (AuNPs and radiation) resulted in more endogenous foci in comparison to lymphocytes that were treated with AuNPs only. The CHO-K1 and MCF-7 cells showed higher MNi frequencies after the combination treatment of AuNPs and radiation compared to the number of MNi in samples exposed to AuNPs and radiation separately. The AuNPs alone influenced the cellular kinetics of all cell types. Interaction indices, which is the enhancement factor of AuNPs in combination with radiation, for AuNPs and 6 MV 2 Gy X-rays of 1.6 to 1.7 and 1.3 to 1.4 have respectively been determined for CHO-K1 and MCF-7 cells, whilst that for the other cell types used in the study were not different from Unity. As expected, the interaction indices between AuNPs and p(66)/Be neutrons was lower than the interaction indices after 2 Gy X-rays, as p(66)/Be neutrons interact only with the nuclei of the AuNP's atoms and the X-ray photons interact with the orbital electrons of the atoms of the AuNPs leading to Auger electron emission. The cell viability assay showed that 50 μg/ml of AuNPs had an inhibitory effect on cellular proliferation, in all four cell linnes whereas the lower concentrations (2.5, 5 and 10 μg/ml) had no effect. Results in this study, revealed an increase in the accumulation of CHO-K1 an MCF-7 cells in the G₂/M phase of the cell cycle after being treated with AuNPs followed by X-ray radiation, suggesting that the cells have possibly been sensitised to the damaging effects of radiation. Further studies are required to quantify internalised AuNPs and to then link the possible concentration differences of the AuNPs to differences in radiation damage effects observed for the different cell types.Item The possible effect of Hypoxis hemerocalledia (African potato) on blood glucose levels : an in vitro study(University of the Western Cape, 2015) Swayeb, Amel Ahmed; Dietrich, Daneel; De Kock, M.The plant Hypoxis hemerocallidea, also known as the African potato, is commonly used as a traditional medicine to treat diabetes in South Africa. The mechanism by which it lowers blood glucose levels is not known. The main aim of this research was to study the possible hypoglycemic effect of HH using RIN-5 F pancreatic tumor cells. To accomplish this, the study was divided into three parts: (1) to test whether exposure of RIN-5F cells to glucose and HH extract affect the cell proliferation and cell viability, (2) to test whether the HH extract have an effect on insulin secretion, and (3) to test whether the HH extract has an effect on alpha amylase and alpha glucosiadase enzyme activity. The RIN-5F cells were exposed to different concentrations of glucose (5, 10, 20, 37.5, 50, 55, 74, and 92.3 mM) for different times (1, 3, 6 and 24 hours). The RIN-5F cells were also exposed to different concentrations of HH (50, 100, 150, 200 and 500 μ/ml) for different times (1, 3, 6 and 24 hours). Cell proliferation was evaluated using crystal violet staining and cell viability was evaluated using the XTT assay. To evaluate the effect of glucose and HH on RIN-5 F cell insulin secretion the cells were exposed to HH (100 μg/ml or 500 μg/ml) and / or glucose (2 mM or 50 mM) for 30 or 90 minutes. Insulin, α-amylase activity and α-glucosidase activity were evaluated by using commercially available colorimetric assays. Enzymatic activity in the presence of HH was compared with positive controls for α-amylase activity or α-glucosidase activity. Results are expressed as means ± SEM or median. Statistical differences among groups were analyzed by analyses of variance. P < 0.05 was considered as significant. An increase in the cell viability and cell proliferation was found when RIN-5 F cells were exposed to high glucose concentrations and a high dose of HH extract for a short time period (1, 3 and 6 hours). When the cells were exposed to the HH extract over 24 hours, HH did not affect cell viability significantly. Insulin secretion from RIN-5 F cells was increased when exposed to low glucose (2 mM) or high glucose (50 mM) for 30 minutes. Insulin secretion was increased from RIN5F cells after exposure to low HH (100 μg/ml) or high HH (500 μg/ml) for 30 minutes. Exposure of RIN5-F cells to HH for 90 minutes caused a further increase in insulin secretion from (4.3±0.17 mIU/mg protein; P ≤ 0.01) in 100 μg/ml, to (7.87±0.17 mIU/mg protein; P ≤ 0.001) in 500 μg/ml. At both 30 minutes and 90 minutes, insulin secretion was significantly higher when cells where exposed to 500 μg/ml HH compared to 100 μg/ml HH. Insulin secretion by cells exposed to 2 mM glucose + 100 μg/ml HH (4.69±0.16 mIU/mg protein; P ≤ 0.001) was significantly higher than when exposed to 2 mM glucose only (2.27±0.17 mIU/mg protein), while the insulin secretion in 2 mM glucose + 500 μg/ml HH (2.56±0.17 mIU/mg protein; P > 0.05) was not significantly different from that in 2 mM glucose treated cells (2.27±0.17 mIU/mg protein). Similar results are obtained after 90 minutes. In the presence of high-glucose (50 mM), at both 30 minutes and 90 minutes, insulin secretion was significantly decreased when cells where exposed to low concentration of HH (100 μg/ml) and high concentration of HH (500 μg/ml). The HH extract produced α-amylase enzyme inhibition. The maximum inhibition was at a concentration of 10μg/ml (922±117U/ml; P ≤ 0.01). The 5 μg/ml concentrations failed to produce significant inhibition. The HH extract had significant α- glucosidase inhibitory activity at a concentration of 5μg/ml (0.12±0.3U/ml; P ≤ 0.001) or 10μg/ml (0.13±0.3U/ml; P ≤ 0.001). In conclusion, based on its ability to inhibit α-amylase and α- glucosidase activity HH has the potential to be used in control of blood glucose levels. The HH aqueous extract increased insulin secretion under our basic experimental conditions and in the presence of low glucose levels, but not at high (50 mM) glucose concentrations. Insulin secretion in the presence of different glucose concentrations, in the presence of HH, needs further investigation. It is recommended that the ability of HH to stimulate insulin secretion be evaluated at 15-20 mM glucose.Item Radiosensitization effect of gold nanoparticles in proton therapy(Frontiers Media, 2021) Cunningham, C.; De Kock, M.; Engelbrecht, M.; Miles, X.The number of proton therapy facilities and the clinical usage of high energy proton beams for cancer treatment has substantially increased over the last decade. This is mainly due to the superior dose distribution of proton beams resulting in a reduction of side effects and a lower integral dose compared to conventional X-ray radiotherapy. More recently, the usage of metallic nanoparticles as radiosensitizers to enhance radiotherapy is receiving growing attention. While this strategy was originally intended for X-ray radiotherapy, there is currently a small number of experimental studies indicating promising results for proton therapy. However, most of these studies used low proton energies, which are less applicable to clinical practice; and very small gold nanoparticles (AuNPs). Therefore, this proof of principle study evaluates the radiosensitization effect of larger AuNPs in combination with a 200 MeV proton beam. CHO-K1 cells were exposed to a concentration of 10 μg/ml of 50 nm AuNPs for 4 hours before irradiation with a clinical proton beam at NRF iThemba LABS. AuNP internalization was confirmed by inductively coupled mass spectrometry and transmission electron microscopy, showing a random distribution of AuNPs throughout the cytoplasm of the cells and even some close localization to the nuclear membrane. The combined exposure to AuNPs and protons resulted in an increase in cell killing, which was 27.1% at 2 Gy and 43.8% at 6 Gy, compared to proton irradiation alone, illustrating the radiosensitizing potential of AuNPs. Additionally, cells were irradiated at different positions along the proton depth-dose curve to investigate the LET-dependence of AuNP radiosensitization. An increase in cytogenetic damage was observed at all depths for the combined treatment compared to protons alone, but no incremental increase with LET could be determined. In conclusion, this study confirms the potential of 50 nm AuNPs to increase the therapeutic efficacy of proton therapy.