Graduation date: 2006

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

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.

Colorectal cancer is estimated to cause approximately 50,000 deaths each year in the United States. Epidemiological studies have demonstrated an inverse association between sunlight exposure, which stimulates the formation of vitamin D in the skin, and colorectal carcinoma. Laboratory studies report that metabolites of vitamin D, acting through the vitamin D receptor (VDR), regulate cellular proliferation, differentiation and apoptosis. In addition, VDR contains a polymorphic variant, FokI, which results in two different isoforms of VDR. We have demonstrated a differential suppression of β-catenin transcriptional activity by these isoforms in the presence of 1,25(OH)₂D₃ (1,25D). Epidemiological evaluation of metachronous colorectal adenoma formation indicates that VDR includes several single nucleotide polymorphisms (SNPs) which influence the odds of developing colorectal adenoma. In addition, we have found full length Adenomatous Polyposis Coli (APC), a frequently mutated tumor suppressor gene in colorectal cancer, augments both the interaction of VDR and β-catenin as well as the suppression of β-catenin transcriptional activity in the presence of 1,25D. We have also demonstrated in epidemiological studies that the presence of a T-A haplotype in APC codons 486 and 1822, respectively, reduces the odds of any metachronous adenoma by 27% [odds ratio (OR), 0.73; 95% confidence interval (95% CI), 0.59 – 0.91]. Taken together, these data support not only a protective role for vitamin D acting through the VDR, but also for an important role of heritable polymorphic variation in VDR and APC in carcinogenesis.

Sulforafan är en isotiacyanat som beskrivs ha effektiva cancerpreventativa egenskaper. Kemikalien görs tillgänglig för människan genom konsumtion av korsblommiga grönsaker så som broccoli och grönkål. Cancer är vanligt, och i Sverige räknar man med att var tredje människa kommer att drabbas under sin livstid. I följande litteraturstudie var syftet att sammanställa information om på vilket sätt sulforafan påverkar koloncancerceller, samt söka evidens för att konsumtion av sulforafanrika grönsaker bidrar till minskad risk att drabbas av koloncancer. Sulforafan har cancerpreventativa egenskaper i cellkultur så som inhibering av histondeacetylas-aktivitet, inducering av cellcykelarrest och apoptos och minskad proliferation hos cancercellerna. Det minskar även uttryck av gener som är inblandade i angiogenes.Det finns inte nog med evidens om broccolikonsumtion, på grund av sitt höga innehåll av sulforafan, skulle vara cancerpreventativt hos människan. / Sulforaphane is an isothiocyanate that is described as having chemopreventative effects. The phytochemical is made available to humans by dietary consumption of cruciferous vegetables such as broccoli and kale. Cancer is a common disease, and in Sweden it is estimated that one in three will be diagnosed with cancer during their lifetime. This review study aims to summarize the effect of sulforaphane on human colon cancer cells, and seek evidence that consumption of cruciferous vegetables reduces the risk of developing colon cancer. Sulforaphane is considered chemopreventative in vitro through inhibition of histone deacetylas activity, induction of cell cycle arrest and apoptosis and through reduction of cell proliferation. It has also been shown to reduces expression of genes involved in angiogenesis.There is not enough evidence to confirm that dietary broccoli consumption, through its high content of sulforaphane, would be chemopreventative.

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

<p>Breast cancer is currently the most common non-skin cancer and the second leading cause of cancer-related death in women here in the United States. X-ray mammography is currently the standard clinical imaging modality for breast cancer screening and diagnosis due to its high sensitivity and resolution at a low patient dose. With the advancement of breast imaging from analog to digital, quantitative measurements rather than qualitative assessments can be made from these images. One such measurement, mammographic breast density (i.e. the percentage of the entire breast volume that is taken up by dense glandular tissue), has been shown to be a biomarker well correlated with cancer risk. However, a digital mammogram still suffers from its projective nature. The resulting overlap of normal breast tissue can obscure lesions, limit quantitative measurement accuracy, and present false alarms leading to unnecessary recall studies. To address this key limitation, several 3D imaging techniques have been developed such as breast magnetic resonance imaging (MRI), dedicated breast computed tomography (CT), and digital breast tomosynthesis (tomo). Perhaps the most recently developed modality is tomo, which is a limited-angle cone-beam CT of the breast compressed in the same geometry as mammography. Because tomo retains all the aforementioned advantages of mammography but adds depth information and can be built based on an existing digital mammography device, measuring breast density and extracting other quantitative features from tomo images was a major focus of this study.</p><p>Before attempting to measure breast density and other features from reconstructed tomo image volumes, the quantitative potential of this imaging modality was assessed. First, we explored a slice-by-slice technique that measures tissue density using only the information from a single slice from the reconstructed tomosynthesis volume with geometrically simple tissue-equivalent phantoms. Once this task has been satisfactorily performed, we studied a probabilistic approach toward quantitation of the entire 3D volume. Some work has been done previously in the realm of 2D hidden Markov random fields (HMRFs) to categorize mammograms according to their Wolf pattern, detect mammographic lesions, and segment satellite and mixed media (text/photograph) images. For this project, a 3D hidden Markov model (HMM) method was developed and applied to tomo images under the simplified assumption that the possible tissue type of each tomo voxel is either adipose (fatty) or glandular (dense). Because adipose and glandular tissue is easily distinguished in MR images, patient breast MRIs were used to train, validate, and finally to assess the accuracy of our HMM segmentation algorithm when applied to tomo images by comparing the volumetric breast density to the MRI breast density for the same patient. Because they are so often studied conjunctively, several image texture features were calculated and compared between MRI and tomo as well.</p><p>Another aim of our study was to investigate whether changes in macroscopic 3D imaging features (texture and density) can accurately predict the chemoprevention response that was measured with Random Periareolar Fine Needle Aspiration (RPFNA) cytology for a uniquely young high-risk cohort of women. This aim to investigate the potential of combining multi-modality macroscopic 3D imaging information with a cytological measure of risk and then investigating how response to tamoxifen and other chemoprevention treatment affects each of these risk biomarkers in young, high-risk women is completely novel in the fields of medical physics and biomedical engineering.</p> / Dissertation

Flavonoids, present in fruits, vegetables and beverages derived from plants, have been described as health-promoting, disease-preventing dietary supplements, and have activity as cancer preventive agents. The cancer protective effects of flavonoids have been attributed to a wide variety of mechanisms, including modulating enzyme activities resulting in the decreased carcinogenicity of xenobiotics. Cytochrome P4501A1 (CYP1A1) is a Phase I enzyme which is known to be involved in the activation of procarcinogens and Glutathione S-Transferase (GST) is a Phase II enzyme which is largely responsible for the detoxification of carcinogens. In this study, it was aimed to investigate the mechanisms of inhibition of CYP1A1 and GST activities of fish by phenolic compounds/flavonoids. Leaping mullet (Liza saliens), captured from highly polluted sites of izmir Bay, expressing high levels of CYP1A, were used in order to investigate these effects. It was demonstrated that all of the phenolic compounds/flavonoids used, exert an inhibitory effect on both CYP1A1 associated 7-Ethoxyresorufin-O-deethylase (EROD) activity and GST activities of fish, although the degree of inhibition was varied with the flavonoid used. Of the flavonoids tested, the most potent inhibitor of CYP1A1 associated EROD activity was found to be quercetin. The potency of the phenolic compounds/flavonoids to inhibit CYP1A1 associated EROD activity follow the sequence of quercetin &gt / resveratrol &gt / naringenin &gt / hesperidin &gt / rutin with IC50 values of 1.32 &micro / M, 3.59 &micro / M, 9.78 &micro / M, 98.5 &micro / M and 0.64 mM respectively. Quercetin, resveratrol, hesperidin and rutin were found to inhibit EROD activity in a competitive manner, on the other hand, naringenin was found to inhibit EROD activity in a non-competitive manner. Inhibition constant (Ki) values of quercetin, resveratrol, naringenin, hesperidin and rutin were calculated from Dixon plots as 0.12 &micro / M, 0.67 &micro / M, 2.63 &micro / M, 18 &micro / M and 0.1 mM, respectively. In the case of GST enzyme, it was demonstrated that all of the phenolic compounds/flavonoids used, exert an inhibitory effect on both total GST and GST-Mu activities of fish. Of the flavonoids tested, the most effective inhibitor of total GST activity was found to be resveratrol. The potency of the phenolic compounds/flavonoids to inhibit total GST activity follow the sequence of resveratrol &gt / quercetin &gt / rutin &gt / naringenin &gt / hesperidin with IC50 values of 7.1 &micro / M, 24.5 &micro / M, 89 &micro / M, 116 &micro / M and 118 &micro / M respectively. Resveratrol, quercetin and hesperidin were found to inhibit total GST activity in a competitive manner, on the other hand, rutin and naringenin were found to inhibit GST activity in a mixed type manner. Ki values of resveratrol, quercetin, hesperidin, naringenin and rutin were calculated from Dixon plots as 3.2 &micro / M, 12.5 &micro / M, 45 &micro / M, 128 &micro / M and 150 &micro / M respectively. In the case of GST-Mu activity, the most potent inhibitor was found to be rutin. The potency of the phenolic compounds/flavonoids to inhibit GST-Mu activity follow the sequence of rutin &gt / resveratrol &gt / quercetin &gt / naringenin &gt / hesperidin with IC50 values of 66.5 &micro / M, 72.3 &micro / M, 113.5 &micro / M, 135.5 &micro / M and 196 &micro / M, respectively. In conclusion, this study indicated that flavonoids were the strong inhibitors of CYP1A1 associated EROD activity and GST activities of mullet liver. The modulation of drug-metabolizing enzymes by flavonoids is important in terms of human health, since these enzymes can activate or inactivate carcinogens. The potential role of xenobiotic metabolizers CYP1 family in the activation of carcinogens and inactivation of chemotherapeutics suggests a potential therapeutic benefits in inhibiting these enzymes. The results of the present study support the hypothesis that flavonoids may be involved in the prevention of malignant transformation, by reducing the formation of carcinogens through inhibition of enzymes such as CYP1A1 which is known to be involved in carcinogen activation.

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.