There is plenty of evidence from epidemiology studies supporting a link between certain components in the human diet and cancer incidence. It is estimated that 3-4 million annual cases of cancer could be prevented worldwide just by changing dietary habits. In parts of the world where vegetables and fruits constitute a large part of the diet, certain cancer incidences are markedly lower compared to Western countries. In particular, consumption of cruciferous vegetables is negatively associated with occurrence of certain cancers. One of the compounds from crucifers that is implicated in chemoprevention, is indole-3-carbinol(I3C), documented to inhibit tumor formation in several tissues in rodents, including the mammary tissue. I3C and is currently being evaluated in several clinical trials as a chemopreventive agent against breast cancer in humans. There are, however, some legitimate concerns regarding the use of Pure I3C since, depending upon conditions of administration, I3C can act as a promoter of hepatocarcinogenesis. Evidence is presented here that dietary I3C can promote and/or enhance liver tumor formation in rainbow trout (supporting earlier reports in literature) and the C57BL/6J mouse (enhancement in short-term pre-initiation exposure through lactational transfer, inhibition in a long-term post-initiation feeding study). I3C is also reported to promote in the rat liver model. A major concern associated with dietary I3C supplementation relates to its estrogenic effects as seen in trout and also its ability to induce certain cytochrome P-450s involved in procarcinogen activation. Biological effects of I3C are attributed to its acid condensation products. It was observed in this study that I3C acts through different mechanisms, including the Ah receptor-mediated and estrogenic pathways. Understanding of the role of I3C derivatives in beneficial and/or hazardous effects resulting from dietary exposure will be crucial in evaluating the promise of I3C as a chemoprevention agent. Questions pertaining to the risk/benefit of the use of dietary I3C supplementation for preventing estrogen-related diseases, without increasing the risk of promotion of hepatocarcinogenesis in humans, may depend on whether the mechanism(s) of action of I3C derivatives in humans is more like the adult mouse or the neonatal mouse, rat and trout. / Graduation date: 1998

Non-small cell lung cancer (NSCLC) causes most of the cancer deaths worldwide. Tyrosine kinase inhibitors (TKIs), like erlotinib and crizotinib, are commonly used as specific treatments targeting epidermal growth factor receptor (EGFR)-mutated and anaplastic lymphoma kinase (ALK)-rearranged NSCLC. Autophagy is a highly conserved cellular process in response to stress. Tumor microenvironment (TME) is composed of both tumor cells and stromal cells. This study aimed to investigate whether autophagy could confer intrinsic and acquired resistance to TKIs in NSCLC, and its role in the presence of TME or in animal models. In the first part of this study, the effect of EGFR TKI or ALK TKI on sensitive NSCLC cells to generate autophagy was investigated, and manipulation of autophagy in these cell lines was performed. Autophagy inhibition was shown to enhance apoptotic effect of TKIs in sensitive NSCLC cells. This part provided strong evidence that TKIs and autophagy inhibitor chloroquine (CQ) work synergistically in sensitive NSCLC cells. Autophagy induction by erlotinib treatment was observed in a HCC827 (lung adenocarcinoma, EGFR exon 19 del) xenograft model, which was in line with the in vitro observation. Correspondingly, the combination of erlotinib (12.5 mg/kg) with CQ (50 mg/kg) in the HCC827 xenograft model achieved greater tumor growth suppression, compared with single drug treatments. In the second part of this study, a model of TME was established to allow study of autophagy under such circumstances. An activated TME with cytokine production, autophagy induction and epithelial-to-mesenchymal transition (EMT) was generated by co-culturing NSCLC cells and human fibroblasts. Sensitivity to TKI under TME was not affected, and combination of chloroquine with TKI under TME remained synergistic compared with single treatments. In the third part of this study, erlotinib-resistant (ER) HCC827 cells were acquired by stepwise exposure to increasing concentrations of erlotinib in cell culture. Common acquired resistance mechanisms to EGFR TKI (EGFR T790M or c-MET amplification) were excluded in this ER HCC827 model, except EMT. Autophagy status in ER HCC827 cells was studied and autophagy manipulation was performed. It was found that CQ and erlotinib worked synergistically to induce cell death even in ER HCC827 cells. In an ER HCC827 xenograft model, significant degree of autophagy and EMT was evident. Interestingly, combining erlotinib (25 mg/kg) with CQ (50 mg/kg) showed better inhibitory effect on tumor growth compared with single treatments. In summary, TKIs induced both apoptosis and autophagy in EGFR-mutated and ALK-rearranged NSCLC cells. Autophagy inhibition by CQ enhanced TKI-induced cell death in sensitive cells. The presence of TME did not confer TKI resistance. Autophagy was highly activated in EGFR-mutated NSCLC cells with acquired resistance to erlotinib. Combination of CQ with erlotinib remained synergistic in the presence of TME and acquired resistance, both in vitro and in vivo. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

No description available.

Epidemiological studies suggest that both retinoid and carotenoid intakes are inversely correlated with the incidence of human cancers. Animal studies show that both retinoids and carotenoids inhibit tumor cell growth. Both retinoids and carotenoids activate the cytotoxicity function of macrophages in animal experiments. The purpose of this study is to evaluate the molecular mechanism for 13-cis retinoic acid (13-cRA) and beta-carotene (BC) induced immunomodulation which could explain their anti-cancer affects. The effects of 13-cRA and BC were studied on various subpopulations of T-lymphocytes both in vitro and in vivo. For in vitro studies, peripheral blood mononuclear cells (PBMC) were incubated with test compounds at clinically achievable concentrations (10⁻⁸M) for three days. Then the cells were stained with monoclonal antibodies followed by the analysis of flow cytometer. For in vivo studies, PBMC were collected from Barrett's esophagus or oral leukoplakia patients during treatment with 13-cRA (1mg/kg/day) or BC (30 mg/day), respectively. Then the cells were analyzed with monoclonal antibodies and flow cytometry. Both compounds showed the capability of stimulating different subpopulations of T-lymphocytes. 13-cRA predominantly increased the number of T-helper cells, their interleukin 2 (IL-2) receptors and their response to mitogens. Whereas, BC elevated the number of Natural Kill (NK) cells, their IL-2 receptors and their cytotoxicity against K562 target cells. Though these immunomodulatory effects appeared to be unaffected by the presence and cytotoxic functions of macrophages, cytokines seemed to have an important role in the retinoid- and carotenoid-induced immunomodulation. Plasma levels of IL-2 and tumor necrosis factor (TNF) measured by ELISA procedures were increased in patients treated for two months with 13-cRA and BC respectively. Anti-IL-2 and anti-TNF antibodies blocked the retinoic- and carotenoid-induced immunomodulation in in vitro studies. These results indicate that 13-cRA, activating T-helper cells with IL-2 production, and BC, activating NK cells with TNF release, induced immunostimulation which might be able to provide the anti-cancer affects in part seen in epidemiological studies.

Because tumours are comprised of "self" cells and antigens, they escape recognition by the immune system, which discriminates between "self" and "non-self". One such antigen is cathepsin B, a lysosomal cysteine proteinase, that has been implicated as one of the proteolytic enzymes involved in tumour invasion and metastasis. Cathepsin B autoantibodies could open possibilities which may be useful in cancer immunotherapy. In this study generation of cathepsin B autoantibodies was attempted by manipulating the immune system into recognising and responding to cathepsin B in complex with a "foreign" protein, bovine serum albumin (BSA). Cathepsin B was isolated from rabbit liver using the three phase partitioning (TPP) method, modified by adding t-butanol in the homogenisation buffer. Isolation of cathepsin Band cathepsin L, using this novel method, minimised the formation of artefacts such as a covalent cathepsin L-stefin B complex and produced higher yields of enzyme. Pure rabbit liver cathepsin B was conjugated to BSA, using glutaraldehyde as coupling agent, and administered intramuscularly into rabbits. Another three inoculation protocols, which functioned as controls were: i) free cathepsin B administered intramuscularly, ii) complexed cathepsin B administered intravenously, and iii) free cathepsin B administered intravenously. IgGs isolated from inoculated rabbits' serum were assayed by a three layer ELISA system, immunoinhibition assays and dot blots. The anti-complex (intramuscular) antibodies showed the highest recognition for cathepsin B and were the only antibodies that were immunoinhibitory. This suggests that the immune system was, to some extend, successfully manipulated into recognising the complexed "self" cathepsin B. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2001.

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

published_or_final_version / Clinical Oncology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Microbiology / Master / Master of Philosophy

No description available.

No description available.