published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Graduation date: 1994

No description available.

Graduation date: 2006

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Targeting the epigenome, including the use of histone deacetylase (HDAC) inhibitors, is a novel strategy for cancer chemoprevention. Sulforaphane (SFN), a compound found at high levels in broccoli and broccoli sprouts, is a potent inducer of Phase 2 detoxification enzymes and inhibits tumorigenesis in animal models. SFN also has a marked effect on cell cycle checkpoint controls and cell survival/apoptosis in various cancer cells, through mechanisms that are poorly understood. Based on the structure of known histone deacetylase inhibitors, it was hypothesized that SFN may possess HDAC inhibitory properties. Initial studies confirmed that, indeed, at physiologically-relevant concentrations, SFN inhibited HDAC activity in human colorectal cancer cells, with a concomitant increase in acetylated histones H3 and H4, induction of p21 expression, and increased acetylated histone H4 associated with the P21 promoter. A metabolite of SFN, SFN-Cysteine, was found to be the active HDAC inhibitor. Furthermore, in BPH-1, LnCaP, and PC-3 human prostate epithelial cells, SFN inhibited HDAC activity and increased acetylation of histones. SFN also induced p21 expression, with an increase in acetylated histone H4 associated with the P21 promoter in BPH-1 cells. The downstream effects of HDAC inhibition by SFN included induction of pro-apoptotic proteins and repression of anti-apoptotic proteins, and an increase in multi-caspase activity. Dietary SFN suppressed the growth of human prostate cancer PC-3 xenografts and inhibited HDAC activity in the xenografts, peripheral blood mononuclear cells (PBMC), and prostates. In time-course studies, a single oral dose of SFN induced histone acetylation at 6 and 24 h in mouse colonic mucosa, and long-term dietary SFN treatment increased histone acetylation in the ileum, colon, PBMC, and prostates. Moreover, dietary SFN suppressed intestinal tumorigenesis significantly in Apc[superscrip min] mice, with an increase in acetylated histones detected in the normal-looking ileum and polyps and polyps from the colon. Overall, the data presented in this thesis support a novel mechanism for chemoprevention by SFN in vivo, through inhibition of histone deacetylase. The findings also imply that SFN will offer significant protection against at least two of the major cancer killers in the US, namely colon and prostate cancer. / Graduation date: 2005

Indole-3-carbinol (I3C) is a naturally occurring plant alkaloid, found in significant concentrations in cruciferous vegetables such as broccoli and Brussels sprouts. I3C is an unstable compound that undergoes rapid oligomerization in an acidic environment to form higher order condensation products (I3C-ACPs), such as 3-3'-diindolylmethane (DIM). Both I3C and DIM are marketed as dietary supplements and are under investigation as potential chemopreventive agents, despite limited data on the effects of chronic exposure. Previous studies have demonstrated that the chemopreventive potential of I3C and DIM in animal studies is dependent on species, strain, tissue and timing of treatment relative to carcinogen exposure, and long-term post-initiation exposure can even promote tumors. The majority of biological effects from I3C are the result of the abilities DIM and other I3C-ACPs to bind to the aryl hydrocarbon receptor and the subsequent induction of phase I and phase II enzymes. Phase I and phase II enzyme induction in many cases leads to protection from carcinogens by increasing the rate of metabolism and excretion but in some cases enhances carcinogenicity by increasing the rate of bioactivation. It has been demonstrated that modulation of enzyme levels can also result in altered metabolism of compounds that could affect efficacy and toxicity of pharmaceuticals and xenobiotics. The current work utilizes chronic dietary I3C and DIM exposures in rodent models to further elucidate the effect these compounds might have on health, drug metabolism and carcinogenesis. The reduced weight of Fischer 344 rats treated with 2500 ppm I3C for 1 year may be indicative of adverse effects but toxicity was not confirmed by blood chemistry or histopathological examination. Furthermore, no toxicity was observed after a comparable treatment of Sprague-Dawley rats. As observed after acute and sub-chronic exposures to I3C and DIM, we documented significant induction of cytochrome P450 enzymes and a related modification to drug metabolism in liver slice incubations. Evidence is also provided that may suggest that tumor modulation in mice may occur through an estrogenic mechanism. Further studies should be completed to determine the potential for similar responses in humans. / Graduation date: 2004

Chemoprevention may be defined as the inhibition, delay or reversal of carcinogenesis by dietary compounds or their derivatives. "Imifino" is a collective name for many wild plants used predominantly by rural people as herbs in cooking. Many of these herbs possess medicinal properties. As the rural population is at higher risk of exposure to dietary carcinogens, such as mycotoxins, this pilot study was undertaken to determine whether the Amaranthus lividus plant held potential for use in chemopreventive strategies. The plant leaves were extracted to obtain individual solvent fractions. Cytotoxic profiling of the fractions using the SNO oesophageal adenocarcinoma cell line and normal human lymphocytes was achieved using the methylthiazol tetrazolium salt bioreduction assay. The SNO cell line, the A549 lung adenocarcinoma cell line and normal human lymphocytes were utilised for the evaluation of the anti-mycotoxigenic potential of the plant fractions in combination with two important dietary carcinogens, aflatoxin B1 and fumonisin B1. A specific biomarker assay (the induction of reduced glutathione) was employed using the SNO cell line. Flow cytometry was also conducted to determine the apoptotic properties of the acetone fraction on normal human lymphocytes. The results of the anti-mycotoxigenic study showed that certain fractions did have protective effects against both of the carcinogens tested. In addition, these effects were noted in the two cancerous cell lines, which were of different tissue origin. None of the fractions tested were toxic towards the normal human lymphocytes. The glutathione assay indicated that certain acetone fraction dilutions were inducive to reduced glutathione production. This plant is a promising candidate for further investigation concerning chemoprevention and the rural community could be educated on the possible benefits of this herb. / Thesis (M.Med.)-University of KwaZulu-Natal, 2005.

In order to gain insights into the underlying mechanisms, several methods including, flow cytometric, western blot and quantitative real-time PCR analyses were employed. AITC-induced cell growth inhibition in SW620 cells was mainly caused by G2/M arrest, which was accompanied by regulatory proteins modifications. Results of western blot and quantitative real-time PCR analysis showed clear downregulation of pivotal phosphatases Cdc25B and Cdc25C at both transcriptional and post-translational levels in AITC-treated cells. Subsequently, accumulation of inhibitory phosphorylation of Cdc2 on Thr14 and Tyr15 were resulted. Furthermore, an AITC induced apoptosis after prolonged exposure was observed. It was a caspase-mediated apoptosis as evidenced by the activation of initiator caspases (-8 and -9), effector caspases (-3 and -7) and cleavage of Poly (ADP-ribose) polymerase (PARP). Besides in vitro studies, the antitumor activity of AITC was further illustrated by a nude mice xenografts experiment. Treatment with 10 micromol AITC could effectively suppress the growth of SW620 xenografts in vivo. Taken together, our results suggest that AITC is an attractive candidate for future research in chemotherapy and chemoprevention. / Many epidemiological studies indicate that a high intake of cruciferous vegetables, such as cabbage, broccoli and Brussels sprouts, may reduce the risk of certain types of cancer. Glucosinolates in cruciferous vegetables and their digested products are suggested to play an important role in such chemoprevention. When plant tissue is physically damaged, glucosidic bonds are cleaved by endogenous myrosinase to produce various products. Among these products, isothiocyanates (ITCs) draw most of the attention because of their potent antitumor activities. But the molecular mechanism leading to such effects has not yet been defined. / The objective of this study was to investigate the chemotherapeutic potential of allyl isothiocyanate (AITC) towards human colorectal adenocarcinoma cells. Another commonly founded ITC, phenylethyl isothiocyanate (PEITC) was employed as a reference sample. The growth inhibitory effects of ITCs on different colorectal adenocarcinoma cells were investigated using in vitro cell models. Both AITC and PEITC were found to inhibit the growth and proliferation of Caco-2, COLO 201 and SW620 cells in a time- and dose-dependent manner. Based on sensitivity, the most vulnerable SW620 cells were chosen for further studies. In the following BrdU assay, IC50 values for 24-h AITC and PEITC treatments were determined to be 30.2 and 9.21 microM, respectively. At the same time, the effects of ITCs on human normal skin fibroblast Hs68 cells were also investigated. It was found that the survival of Hs68 cells was not affected by the treatments of AITC. However, the survival of Hs68 cells was greatly affected by PEITC-treatments in a dose- and time-dependent manner. / Lau, Wing Sze. / Adviser: Wong Yum Shing. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 115-128). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The present study employed a two-phased approach to investigate the possible mechanisms involved in the chemopreventive properties of rooibos (Aspalathus linearis) and different honeybush species (Cyclopia spp.) in vitro. In the first phase, the effect of unfermented methanol and aqueous herbal tea extracts against the growth parameters (cell viability, proliferation and apoptosis) of normal (CRL 7761); premalignant (HaCaT); and malignant (CRL 7762) skin cells was evaluated and compared to green tea extracts. The predictive potential of polyphenol content (total polyphenol and flavanol/proanthocyanidins) and antioxidant properties (ABTS; ORAC; FRAP and LPO) in the biological activity of extracts in cells was also assessed. Of the herbal teas, the methanol extract of rooibos was the most active and it inhibited the growth of skin cells presumably by inducing mitochondrial dysfunction via membrane depolarisation. At lower concentrations, this activity was associated with inhibition of cell proliferation that was selective for cancer cells whilst higher concentrations induced apoptosis that was more prominent in premalignant cells. The strong antioxidant properties of the extracts implicated the role of pro-oxidative polyphenol/iron interactions involving monomeric flavonoids and polymeric proanthocyanidins in the cytotoxic effects of rooibos. The strong relationship between total polyphenolic and flavanol/proanthocyanidins content, antioxidant properties and reduction of cell viability indicated that these parameters (polyphenols and antioxidant properties) can serve as predictive tools for the cytotoxic effects of rooibos in vitro. The aqueous extracts of honeybush species, although weaker, displayed similar effects to rooibos extracts in cells with C. genistoides being the most effective at selectively inhibiting the proliferation of cancer cells whilst the pro-apoptotic activity of C. subternata and C. intermedia was more prominent in premalignant cells. The underlying mechanisms are also likely to result from pro-oxidative mechanisms resulting from polyphenol/iron interactions that mainly involve polymeric flavanol-like proanthocyanidin compounds in honeybush. In contrast, the methanol extracts exhibited weaker cytotoxic effects and protected cancer cells from going into apoptosis. The cytoprotective effects of honeybush species are possibly mediated by the major monomeric compounds such as mangiferin and hesperidin through antioxidant mechanisms that result in reduction of oxidative stress. Due to the possible dual role of the monomeric and polymeric compounds in the honeybush extracts, the total polyphenolic content of these herbal teas may not be a good indicator of biological activity in vitro. However, as aqueous extracts displayed high flavanol/proanthocyanidins content and exceptional activity in the ABTS assay, these parameters may be considered as indicators of cytotoxicity. On the other hand, methanol extracts, particularly from the xanthone-rich species (C. genistoides and C. longifolia) which exhibited the weakest cytotoxic effects, were more active in the ORAC thus this assay may be a useful predictor for cytoprotective activity. In the second phase, an in vitro UVB/HaCaT model which used IL-1α as a biomarker for early inflammation was developed and validated with known anti-inflammatory compounds, dexamethasone and ibuprofen. It was used to determine the specific mechanisms involved in the modulatory effects of the herbal tea extracts against inflammation. Rooibos extracts and the aqueous extract of honeybush enhanced the cytotoxic effects of UVB in the model and exhibited indirect anti-inflammatory effects as they removed icIL-1α containing cells via apoptosis. In contrast, methanol extracts of honeybush exacerbated icIL-1α by protecting UVB stimulated cells from undergoing apoptosis. In conclusion, methanol extract of rooibos and aqueous extracts of honeybush species may be useful in protecting the skin after UVB exposure. These herbal tea extracts may block initiation and delay the promotion stage during skin carcinogenesis by removing premalignant cells via apoptosis and preventing onset of inflammation. In contrast, due to their cytoprotective effects, methanol extracts of honeybush may be more effective at preventing oxidative stress in skin before UVB exposure. Future studies should focus on the effects of extracts and polyphenolic fractions on the oxidative status of the cells and development of biomarkers of chemoprevention that can be utilised in vivo and in human skin. / AFRIKAANSE OPSOMMING: In hierdie studie word moontlike velkankerwerende eienskappe van rooibos (Aspalathus linearis) en ‘n aantal heuningbos (Cyclopia spp.) spesies deur twee afsonderlike benaderings bestudeer. Die eerste benadering ondersoek die effek van die kruietee op groeiparameters van velselle [lewensvatbaarheid, groei en dood van normale selle (CRL 7761), vroeë kankerselle (HaCaT) en kankerselle (CRL 7762)]. Tydens eksperimente is die moontlikheid om polifenoolinhoud (totale polifenole, en flavanol/proantosianidiene verhouding) en antioksidant-eienskappe te gebruik om die biologiese funksies van die ekstrakte in die selle te voorspel, geevalueer. Die metanolekstrak van rooibos het die groei van selle die effektiefste gestop, moontlik deur depolarisasie van die mitokondriale membraan. By lae konsentrasies van die ekstrak is die groei van kankerselle selektief gestop, terwyl vroeë kankerselle die sensitiefste by hoër konsentrasies was. Die hoë antioksidant-aktiwiteit van die rooibosekstrak kan moontlik ‘n rol speel in die indusering van sitotoksiese effekte in die selle en kan toegeskryf word aan die pro-antioksidant aktiwiteit van die polifenole weens hul interaksie met yster. ‘n Spesifieke funksie word vir die monomeriese flavonoïede en die polimeriese proantosianidiene geïmpliseer. Die sterk verwantskap tussen die totale polifenoolinhoud, flavanol/proantosianidien inhoud en antioksidant aktiwiteit met die verlaging in selgroei, maak hul relevante parameters van die voorspellingsmodel. Die waterekstrakte van heuningbos induseer ook soortgelyke maar swakker effekte met die induksie van kankersel dood, met C. genistoides die selektiefste en C. subternata en C. intermedia die aktiefste spesies wat die groei van die vroeë kanker selle inhibeer. Die onderliggende meganismes betrokke blyk ook aan ‘n pro-oksidant effek toe geskryf te wees, waartydens spesifieke polifenool/yster interaksies betrokke is. In teenstelling met rooibos, beskerm die metanolekstrak van heuningbos kankerselle teen seldood, wat moontlik verband hou met die antioksidant-eienskappe van die hoof monomeriese polifenole, mangiferien/isomangiferien en hesperidien. Vanweë die dubbele rol van die monomeriese polifenole en polimeriese verbindings in heuninghbosekstrakte is die totale polifenol inhoud nie ‘n goeie indikator van die biologiese aktiwiteit in vitro nie. Daarenteen is die flavanol/proantosianien inhoud en die hoë aktiwiteit in die ABTS antioksidanttoets goeie indikators om seldood te voorspel. In teenstelling hiermee het die metanolekstrakte van die xantoon-ryke spesies (C. genistoides en C. longifolia) ‘n baie lae effek op seldood, maar ‘n hoë aktiwitiet in die ORAC toets getoon, wat ‘n goeie rigtingwyser is om die beskermende effek in selle te voorspel. Met die tweede benadering is die anti-inflammatoriese eienskappe en die onderliggende meganismes van die kruietee ondersoek in ‘n UVB/HaCaT selmodel. Intrasellulêre interleukin 1α (IL-1α) is as merker gebruik en die model is geëvalueer deur bekende anti-inflammatoriese verbindings soos dexamethasone en ibuprofin te gebruik. Die metanolekstrak van rooibos en die waterekstrak van heuningbos het die toksiese effek van UVB in die model verhoog deur selle met verhoogde vlakke,van icIL-1α te verwyder deur middel van die induksie van seldood. Die metanolekstrak beskerm die selle teen die oksidatiewe skade wat deur UVB geïnduseer word en verwyder nie selle met hoë IL-1α vlakke nie. Ter opsomming blyk dit dat die metanolekstrak van rooibos en die waterekstrak van heuningbos moontlik gebuik kan word om die vel te beskerm teen die induksie van icIL-1α en sodoende die inisiëring van kanker te blokkeer en ook die promosie van kanker te vertraag. Die beskermende effek van die metanolekstrak kan moontlik aangewend word om die oksidatiewe skade wat deur UVB veroorsaak word teen te werk deur dit aan te wend voordat blootstelling plaasvind. Toekomstige studies behoort verdere karakterisering van die polifenoolsamestelling van die ekstrakte in te sluit en hul effek op die oksidatiewe status en anti-inflammoriese effekte van selle te bepaal ten einde sekere merkers te identifiseer vir vel studies in vivo.