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ABSTRACT Cassava is a known source of linamarin, but difficulties associated with its isolation have prevented it from being exploited as a source. A batch adsorption process using activated carbon at the appropriate contact time proved successful in its isolation with ultrafiltration playing a pivotal role in the purification process. Result revealed that optimum purification was obtained with increasing amount of crude cassava extract (CCE) purified. 60g of CCE took 32 mins, 80 g, 34 mins while 100 g took 36 mins of contact time, where 1.7 g, 2.0 g and 2.5 g of purified product were obtained, respectively. The purification process in batch mode was also carried out at different temperatures ranging from 25 to 65oC. Results showed that purification increases with increase in temperature. In a bid to ascertain the moles of linamarin adsorbed per pore volume of activated carbon used, the composite isotherm was found to represent the measured adsorption data quite well. The adsorption of linamarin was used to study the goodness of fit criteria (R2) for the entire process. Results showed that R2 value was best with decreasing amount of CCE purified (R2=1 for 60 g) at the temperature of 45oC. Compound elucidation of purified product by Picrate paper test, IR and 1HNMR confirmed the structure of linamarin. Cytotoxic effects of linamarin on MCF-7, HT-29, and HL-60 cells were determined using the 3 - (4, 5 – dimethylthiazol-2-yl) – 2, 5 – diphenyltetrazolium bromide (MTT) assay. Cytotoxic effects were significantly increased in the presence of linamarase, which catalysed the hydrolysis of linamarin to hydrogen cyanide. A 10–fold decrease in the IC50 values obtained for linamarin or crude extract in the presence of linamarase was determined for HL-60 cells. This study thus describes a method for the isolation and purification of linamarin from cassava, as well as the potential of this compound as an anticancer agent.

C1 inhibitor (C1 INH) is a member of the serine protease inhibitor (serpin) superfamily. It is the only physiological inhibitor of protease C1r and C1s in the complement system. C1 INH is a single chain glycoprotein with apparent molecular weight of 105 KDa, consisting of 478 amino acids. C1 INH N-terminal domain includes first 98 amino acids with 10 definite and 7 potential glycosylation site. Various of carbohydrates are present on the cell surface and component of ECM (extracellular matrix) in every eukaryotic cell, including both cancer cells and cells that are important for tumur survival. Carbohydrates on the cancer cell surface have been shown to be important in many aspects of cancer cell physiological processes, involved in cell growth and cell adhesion. Carbohydrates are also able to bind and interact with growth factors and other proteins that trigger signal transduction. Interfere carbohydrates maybe offer a useful therapeutic approach for treating cancers. In order to understand whether the C1 INH NT98 polypeptides can influences cancer or not, we amplified a DNA fragment encoding C1 INH N-terminal domain 98 residues (C1 INH NT98) by PCR, and transfer to the plasmid pGEX-2T, than use E.coli (BL21 strain) to express the non-glycosylated polypeptides, and further analyze the influence of the effective roles exhibited by the polypeptides non-glycosylated on breast cancer cell MDA-MB-435s. Proliferation and migration assays in our experiment showed that non-glycosylated C1 INH NT98 can inhibited breast cancer cell growth and migration, and the mechanism needed to be clarified clearly through extensive research.

Aryl hydrocarbon receptor nuclear translocator (ARNT) is an important player in xenobiotic and hypoxic responses. In addition to this, my mentor has shown that ARNT is an integral cofactor of NF-kB signaling. However, these initial observations of ARNT-mediated NF-kB modulation were based on simultaneous suppression of the two ARNT isoforms, isoform 1 and 3, and therefore precluded the isolated examination of each isoform’s function. We show here that lymphoid malignancies exhibit higher levels of ARNT isoform 1 compared to ARNT isoform 3. However, normal T and B lymphocytes are seen to harbor equal levels of ARNT isoform 1 and 3. We hypothesize that the increase in ARNT isoform 1 is necessary for the growth of these cancer cells as suppression of isoform 1 resulted in S-phase cell cycle arrest. These findings reveal that ARNT isoform 1 potentiates cell growth by antagonizing a p53 cell cycle inhibitory mechanism and this further suggests that ARNT targeted therapies would benefit chemotherapy regimens. / text

Timely and spatially controlled endosomal trafficking and signaling is important for cell proliferation, directed cell migration, and cell invasion, which are frequently misregulated in cancer cells. Cdc42-interacting protein-4 (CIP4) family adaptors promote endocytosis by inducing membrane invaginations via their Fer/CIP4 Homology-Bin/Amphyphysin/Rvs (F-BAR) domains, coupled with activation of the actin assembly machinery to promote vesicle fusion or motility. My thesis focuses on defining the roles of CIP4, and a related protein, Transducer of Cdc42-mediated actin assembly-1 (Toca-1), in regulating Epidermal Growth Factor Receptor (EGFR) endocytosis, EGFR trafficking, cancer cell motility, and invasion. In Chapter 2, I show that CIP4 and Toca-1 localize to early endosomes and promote EGFR trafficking from early endosomes to lysosomes for degradation, thus limiting extracellular signal-regulated kinase signaling from early endosomes and proliferation of A431 carcinoma cells. In Chapter 3, I provide novel evidence that depletion of Toca-1 results in defects in actin-based lamellipodial protrusions that are required for cell motility. The cause of these defects may relate to altered recruitment of the Abelson-interactor-1 and its effector Wiskott-Aldrich syndrome protein family verprolin-homologous protein to the lamellipodia in A431 cells depleted of Toca-1. Results in Chapter 4 identify CIP4 as a negative regulator of breast cancer invasiveness downstream of Src protein-tyrosine kinase. Src is a potent inducer of extracellular matrix (ECM)-degrading structures called invadopodia that function in tissue invasion by cancer cells. I found that CIP4 is a Src substrate that localizes to Src-induced invadopodia in MDA-MB-231 breast cancer cells. Interestingly, depletion of CIP4 results in enhanced ECM degradation, invadopodia formation, and invasiveness compared to control cells. Thus, CIP4 and Toca-1 are multifaceted regulators of EGFR downregulation, EGF-induced cell motility, and Src-induced cell invasion. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2010-08-25 11:44:46.934

Oesophageal adenocarcinoma (OAC) is a leading cause of cancer death in the UK and current systemic therapies are ineffective for the majority of patients. The central aim of this work was to explore strategies to identify novel therapeutic targets. Research has failed, thus far, to identify a dominant oncogene in OAC, although the tumour suppressor p53 is frequently mutated. Inhibiting the mitotic kinase, polo-like kinase 1 (PLK-1), was proposed as a synthetic lethal strategy. PLK-1 was demonstrated to be over-expressed in both verified OAC cell lines and human OAC tissue compared to non-transformed cells and epithelium. Mutation of p53 was associated with over-expression of PLK-1 in both OAC and ovarian cancer tissue. Using a carefully validated viability assay, both an established and novel PLK-1 inhibitor were demonstrated to induce a G2/M arrest and reduce OAC cell proliferation. Relative selectivity was demonstrated for OAC compared to non-transformed cells. This therapeutic window could be enhanced with the induction of cancer cell cytotoxicity by pulsed administration of a short half-life inhibitor. Immunotherapeutics offer potential tumour-selectivity but no OAC-specific proteins have been defined. A comparative proteomic approach was employed to identify OAC-specific proteins as potential therapeutic targets. A tissue resource was established and methods to lyse fresh frozen biopsies optimised. An isobaric quantitative proteomic workflow was applied to OAC and matched normal biopsies and quantitative accuracy confirmed for 6 candidate proteins by immunohistochemistry. Proteome coverage and quantitative dynamic range were compared between isobaric and label-free systematic sequencing proteomic strategies applied to further patients’ tissues. The challenges of combining incomplete datasets were approached with a Bayesian framework to estimate the probability that a protein was missed during an experiment compared to not being present in the sample. This method was applied to generate a complete set of protein identifications and relative tissue expression. To gain insight into the dysregulated cellular processes in human OAC tissue, a network analysis was applied to the quantitative proteomic data. Enriched functional clusters were identified suggesting deranged glucose metabolism, potentially due to the Warburg effect. These findings were duplicated and candidate tumour-specific proteins identified in a further set of biopsies using the optimised quantitative proteomic method. The combined quantitative oesophageal proteomic dataset represents the largest in OAC to date. This thesis demonstrates a hypothesis-driven, synthetic lethal approach can yield cancer-selective therapeutic effects. Novel candidate therapeutic targets are also revealed through the development of quantitative proteomic methods and the application of network analysis.

Orientador: Gisele Fabrino Machado / Banca: Felipe Augusto Ruiz Sueiro / Banca: Antonio Carlos Alessi / Resumo: A causa dos tumores da glândula perianal é desconhecida, entretanto eles podem ser modulados por influência de hormônios gonadais, basicamente a testosterona, uma vez que há uma alta prevalência (até 95%) de regressão da forma benigna dessa neoplasia após a castração dos machos. A proteína Ki-67 foi descrita pela primeira vez em 1983 é uma proteína nãohistona, que não é expressa em células na fase G0, mas que pode ser detectada nas fases ativas do ciclo celular, G1, S, G2 e mitose Verificou-se que o Ki-67 está associado com a proliferação celular, estando a população de células tumorais positivas ao Ki-67 correlacionada com a evolução clínica da doença. Foram estudados 42 casos de neoplasias da glândula perineal, sendo 15 adenomas, 15 epiteliomas, 12 carcinomas. Foram utilizadas 13 amostras de tecido de glândula perianal normal, como controle. De cada animal foram obtidas informações como: sexo, idade, raça, presença de outras neoplasias, se castrado ou não, se em caso de fêmea havia o uso de anticoncepcional, tempo de evolução, recidiva e tempo de sobrevida. Dos 42 casos de neoplasias de glândulas perianais, 34 (80,95%) foram provenientes de cães machos e 8 (19,05%) de fêmeas. A média de idade dos machos com a neoplasia foi de 9,8 anos, e das fêmeas 7,4 anos, sendo que para os dois sexos a idade variou de 5 a 15 anos. Em relação às raças, 11 (26,19%) eram S.R.D. (sem raça definida), 9 (21,42%) Poodle, 4 (9,52%) Cocker Spaniel e Husky Siberiano. O histórico de diagnóstico de outras neoplasias, foi constatado em 23,80% dos animais deste estudo. Em relação à castração, 32 (94,11%) dos machos não eram castrados e apenas 8 (19,05%) fêmeas eram. O tempo de evolução da neoplasia apresentou média de 1,2 anos. Histórico de recidiva da neoplasia foram relatados em 14 animais, sendo maior... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The cause of perianal gland tumors is unknown, though they may be modulated by the influence of gonadal hormones, primarily testosterone, since there is a high prevalence (up to 95%) of regression of the benign tumor after males castration. Protein Ki-67 was first described in 1983, and it is a nonhistone protein, which is not expressed in G0 phase cells, but can be detected in the active phases of the cell cycle, G1, S, G2 and mitosis. It was found that Ki-67 is associated with cell proliferation, and the population of tumor cells positive for Ki-67 correlated with clinical outcome. In this present work we studied 42 cases of perineal gland neoplasms, being 15 adenomas, 15 epitheliomas and 12 carcinomas. We used 13 tissue samples of normal perianal gland as control. From each animal we obtained information such as gender, age, race, presence of other malignancies, neutering status, whether if there were female contraceptive use, duration, recurrence and survival time. Of the 42 cases of perianal gland neoplasms, 34 (80.95%) were from males and eight dogs (19.05%) females. The males average age with cancer was 9.8 years and 7.4 years for females and for both sexes aged between 5 and 15 years. In regard to race, 11 (26.19%) were no breed, 9 (21.42%) were Poodle, and 4 (9.52%) were Siberian Husky and Cocker Spaniels. The history of diagnosis of other cancers, was found in 23.80% of the animals in this study. In relation to castration, 32 (94.11%) of males were not castrated and only 8 (19.05%) were females. The progression of the tumor showed an average of 1.2 years. History of recurrence of cancer were reported in 14 animals, and was higher (66.66%) for carcinomas. Ki-67 staining revealed that the carcinomas showed higher proliferation rate (9.87%) compared to groups of epitheliomas... (Complete abstract click electronic access below) / Mestre

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3), the most active metabolite of vitamin D, inhibits the proliferation of a variety of cell types including adenocarcinoma of the prostate. The primary mechanism for the antiproliferative effects of 1,25-(OH)2D3 in prostate cancer cells is inhibition of G1 to S phase cell cycle progression. While 1,25-(OH)2D3-mediated growth inhibition requires the vitamin D receptor (VDR), a ligand activated transcription factor, expression of functional VDR is not sufficient. To define target genes that might participate in the antiproliferative actions of 1,25-(OH)2D3, we developed a derivative of the human prostate cancer cell line, LNCaP, which retains transcriptionally active VDRs but unlike parental LNCaP cells, is not growth inhibited by 1,25-(OH)2D3. Gene expression profiling of these resistant cells (termed VitD.R) compared to control LNCaP cells revealed two novel 1,25-(OH)2D3-inducible genes, GADD45G and MIG6. The expression of GADD45G and MIG6 genes was induced by 1,25-(OH)2D3 in LNCaP but not in the resistant VitD.R or in ALVA31 cells, human prostate cancer cells that exhibit natural resistance to growth inhibition by 1,25-(OH)2D3 despite expression of functional VDR. Ectopic expression of GADD45G but not MIG6 in either LNCaP or ALVA31 cells resulted in accumulation of cells in G1 and inhibition of proliferation equal to or greater than that caused by 1,25-(OH)2D3 treatment. While GADD45G is induced by androgens in prostate cancer cells, up-regulation of GADD45G by 1,25-(OH)2D3 was not dependent on androgen receptor signaling further refuting a requirement for androgens/androgen receptor in vitamin D-mediated growth inhibition in prostate cancer cells. These data introduce two novel 1,25-(OH)2D3-regulated genes and establish GADD45G as a growth inhibitory protein in prostate cancer. Further, defects in vitamin D-mediated induction of GADD45G may underlie vitamin D resistance in prostate cancer cells. We previously demonstrated that the antiproliferative actions of 1,25-(OH)2D3 in prostate cancer cells are associated with decreased CDK2 activity and increased stability of the cyclin dependent kinase inhibitor (CKI) p27KIP1. We defined a novel mechanism that may underlie these antiproliferative effects, 1,25-(OH)2D3 -mediated cytoplasmic relocalization of CDK2, which would provide a unifying mechanism for the observed effects of 1,25-(OH)2D3 on CDK2 and p27. In the present study, we investigated the role of CDK2 cytoplasmic relocalization in the antiproliferative effects of 1,25-(OH)2D3. CDK2 was found to be necessary for prostate cancer cell proliferation. In contrast, while p27KIP1 is induced by 1,25-(OH)2D3, this CKI was completely dispensable for 1,25-(OH)2D3-mediated growth inhibition. Reduction of CDK2 activity by 1,25-(OH)2D3 was associated with decreased T160 phosphorylation, a residue whose phosphorylation in the nucleus is essential for CDK2 activity. Since cyclin E is important for nuclear translocation of CDK2, we investigated cyclin E effects on 1,25D-mediated growth inhibition. Ectopic expression of cyclin E blocked 1,25-(OH)2D3-mediated cytoplasmic relocalization of CDK2 and all antiproliferative effects of 1,25-(OH)2D3, yet endogenous levels of cyclin E or binding to CDK2 were not affected by 1,25-(OH)2D3. Similarly, knockdown of the CDK2 substrate retinoblastoma (Rb), which causes cyclin E up-regulation, resulted in resistance to 1,25-(OH)2D3 mediated growth inhibition. VitD.R cells did not exhibit 1,25-(OH)2D3-mediated cytoplasmic relocalization of CDK2. Importantly, targeting CDK2 to the nucleus of LNCaP cells blocked G1 accumulation and growth inhibition by 1,25-(OH)2D3. These data establish central roles for CDK2 nuclear-cytoplasmic trafficking and uncoupling of VDR in the regulation of antiproliferative target genes in the mechanisms of 1,25-(OH)2D3-mediated growth inhibition in prostate cancer cells. Since 1,25-(OH)2D3 continues to be evaluated for its chemotherapeutic and chemopreventative potential, elucidating the mechanism of 1,25-(OH)2D3 antiproliferative effects is critical in the determination of 1,25-(OH)2D3 responsiveness and the design of individualized treatment strategies.

Micro (mi)RNAs have recently been found to play an important role in cancer biology. In order to further understand how miRNAs affect prostate tumour progression, we evaluated miRNA expression in two invasive prostate tumour lines, PC3 and DU145. We then focused our evaluation on a novel miRNA, miR-105, whose levels were significantly decreased in both tumour cell lines as compared to normal prostate epithelial cells. As miR-105 levels were reduced in prostate tumour cell lines, we restored its expression following transfection of cells with mimic constructs to over-express miR-105 in both cell lines, in order to determine its effect on various tumourigenic properties. Over-expression caused decreased tumour cell proliferation, anchorage-independent growth and invasion in vitro and inhibited tumour growth in vivo. We further identified CDK6 as a putative target of miR-105, which likely contributed to its inhibition of tumour cell growth. Our results suggest that miR-105 inhibits tumour cell proliferation and may be an interesting target to regulate tumour growth or potentially used as a biomarker to differentiate between less and more aggressive tumours in patients.

C1-esterase inhibitor (C1-inh) can inhibitor the first complement protein as C1s and C1r activity to reach adjust classical pathway, avoid excessive activation of the complement system to cause disease. C1-inhibitor protein composed of 478 amino acids with two domains: C terminal domain (serpin domain) and N terminal domain. The early focus has been to angioedema associated with cancer found so far. So the purpose of the study was to investigate whether the C1-inh cause for the breast cancer cell proliferation and migration. We use recombinant gene transform Escherichia coli strain BL21(DE3) and expression. Recombinant protein was purified using affinity column. Influence of proliferation and migration on breast cancer cells were tested by purified recombinant C1-inh. In breast cancer cell proliferation results showed, C1-inh significant proliferation of breast cancer, and when the higher concentration, the longer the incubation time, the remarkable effect of promoting proliferation is even more obvious. The results in breast cancer cell migration is also significant in the C1-inh to promote breast cancer cell migration, and when the higher concentration of the longer incubation time, the significant increased migration is more effective. Therefore, this study does note C1-inh to promote breast cancer cell proliferation and migration.