Aspirin and alterations in DNA repair proteins in the SW480 colorectal cancer cell line

Regular aspirin intake is associated with a reduction in the incidence of colorectal cancer. Aspirin has been shown to be cytotoxic to colorectal cancer cells in vitro. The molecular basis for this cytotoxicity is controversial, with a number of competing hypotheses in circulation. One suggestion is that the protective effect is related to the induction of expression of the DNA mismatch repair (MMR) proteins hMLH1, hMSH2, hMSH6 and hPMS2 in DNA MMR proficient cells. We report that treatment of the DNA MMR competent/p53 mutant colorectal cancer cell line SW480 with 1 mM aspirin for 48 h caused changes in mRNA expression of several key genes involved in DNA damage signalling pathways, including a significant down-regulation in transcription of the genes ATR, BRCA1 and MAPK12. Increases in the transcription of XRCC3 and GADD45alpha genes are also reported. Regulation of these genes could potentially have profound effects on colorectal cancer cells and may play a role in the observed chemo-protective effect of aspirin in vivo. Although a correlation was not seen between transcript and protein levels of ATR, BRCA1 and GADD45alpha, an increase in XRCC3 encoded protein expression upon aspirin treatment in SW480 cells was observed by immunoblotting, immunofluorescence and immunohistochemical analysis. This is the first report of XRCC3 gene transcription and encoded protein expression being susceptible to exposure to the non-steroidal anti-inflammatory drug, aspirin. Furthermore, this study indicates that alterations in gene transcription seen in microarray studies must be verified at the protein level.

Disulfiram/copper complex inhibiting NFκB activity and potentiating cytotoxic effect of gemcitabine on colon and breast cancer cell lines

Most of the gemcitabine (dFdC) resistant cell lines manifested high NF?B activity. The NF?B activity can be induced by dFdC and 5-FU exposure. The chemosensitizing effect of disulfiram (DS), an anti-alcoholism drug and NF?B inhibitor, and copper (Cu) on the chemoresistant cell lines was examined. The DS/Cu complex significantly enhanced the cytotoxicity of dFdC (resistant cells: 12.2–1085-fold) and completely reversed the dFdC resistance in the resitant cell lines. The dFdC-induced NF?B activity was markedly inhibited by DS/Cu complex. The data from this study indicated that DS may be used in clinic to improve the therapeutic effect of dFdC in breast and colon cancer patients.

Anticancer effects of Curcuma C20-dialdehyde against colon and cervical cancer cell lines

Background: Recent attention on chemotherapeutic intervention against cancer has been focused on discovering and developing phytochemicals as anticancer agents with improved efficacy, low drug resistance and toxicity, low cost and limited adverse side effects. In this study, we investigated the effects of Curcuma C20-dialdehyde on growth, apoptosis and cell cycle arrest in colon and cervical cancer cell lines. Materials and Methods: Antiproliferative, apoptosis induction, and cell cycle arrest activities of Curcuma C20-dialdehyde were determined by WST cell proliferation assay, flow cytometric Alexa fluor 488-annexin V/propidium iodide (PI) staining and PI staining, respectively. Results: Curcuma C20 dialdehyde suppressed the proliferation of HCT116, HT29 and HeLa cells, with IC50 values of 65.4±1.74 μg/ml, 58.4±5.20 μg/ml and 72.0±0.03 μg/ml, respectively, with 72 h exposure. Flow cytometric analysis revealed that percentages of early apoptotic cells increased in a dose-dependent manner upon exposure to Curcuma C20-dialdehyde. Furthermore, exposure to lower concentrations of this compound significantly induced cell cycle arrest at G1 phase for both HCT116 and HT29 cells, while higher concentrations increased sub-G1 populations. However, the concentrations used in this study could not induce cell cycle arrest but rather induced apoptotic cell death in HeLa cells. Conclusions: Our findings suggest that the phytochemical Curcuma C20-dialdehyde may be a potential antineoplastic agent for colon and cervical cancer chemotherapy and/or chemoprevention. Further studies are needed to characterize the drug target or mode of action of the Curcuma C20-dialdehyde as an anticancer agent.

An aqueous extract of Fagonia cretica induces DNA damage, cell cycle arrest and apoptosis in breast cancer cells via FOXO3a and p53 expression

Background - Plants have proved to be an important source of anti-cancer drugs. Here we have investigated the cytotoxic action of an aqueous extract of Fagonia cretica, used widely as a herbal tea-based treatment for breast cancer. Methodology/Principal Findings - Using flow cytometric analysis of cells labeled with cyclin A, annexin V and propidium iodide, we describe a time and dose-dependent arrest of the cell cycle in G0/G1 phase of the cell cycle and apoptosis following extract treatment in MCF-7 (WT-p53) and MDA-MB-231 (mutant-p53) human breast cancer cell lines with a markedly reduced effect on primary human mammary epithelial cells. Analysis of p53 protein expression and of its downstream transcription targets, p21 and BAX, revealed a p53 associated growth arrest within 5 hours of extract treatment and apoptosis within 24 hours. DNA double strand breaks measured as ?-H2AX were detected early in both MCF-7 and MDA-MB-231 cells. However, loss of cell viability was only partly due to a p53-driven response; as MDA-MB-231 and p53-knockdown MCF-7 cells both underwent cell cycle arrest and death following extract treatment. p53-independent growth arrest and cytotoxicity following DNA damage has been previously ascribed to FOXO3a expression. Here, in MCF-7 and MDA-MB-231 cells, FOXO3a expression was increased significantly within 3 hours of extract treatment and FOXO3 siRNA reduced the extract-induced loss of cell viability in both cell lines. Conclusions/Significance - Our results demonstrate for the first time that an aqueous extract of Fagonia cretica can induce cell cycle arrest and apoptosis via p53-dependent and independent mechanisms, with activation of the DNA damage response. We also show that FOXO3a is required for activity in the absence of p53. Our findings indicate that Fagonia cretica aqueous extract contains potential anti-cancer agents acting either singly or in combination against breast cancer cell proliferation via DNA damage-induced FOXO3a and p53 expression.

Cytotoxicity of 3,4-dihalogenated 2(5H)-furanones

Mucohalogen acids have been used for the preparation of a variety of 3,4-clihalogenated 2(5H)-furanones. In one synthetic step the carbarnates 2a-c and the pseudoanhydrides 4a-e were prepared using isocyanates and acid anhydrides. A series of 5-alkoxylated 3,4-dichloro-2(5H)-furanones 5a-o have been synthesized with a wide range of lipophilicity, using the hydroxy-form of mucohalogen acids 1a and 1b. The 5-allyl-3,4-dichloro-2(5H)-furanone 5f was derived into the dihydro-isoxazol 6 and the oxirane 7. The methyl ester 5a was converted with ammonia into the tetramic acid chloride 11. The pseudo acid chloride 3 was reacted further into the bis aziricline 8. Reduction of the mucochloric acid 1a furnished the trichlorofuranone 3. The cytotoxicity of these simple and bis-cyclic butenolides have been evaluated in tissue culture on MAC13 and MAC16 cancer cell lines using the MTT cytotoxicity assay. The ester 5g, the acetate 4b and the carbamate 2b displayed a cytotoxicity in the low micromolar range. Further, an IC50 (50% inhibitory concentration) of 50 nM and 30 nm was determined forthe epoxide 7 and the aziridine 18. © 2004 The Authors Recieved.

Synthesis and evaluation of 5-arylated 2(5H)-furanones and 2-arylated pyridazin-3(2H)-ones as anti-cancer agents

Bis-cyclic butenolides, 5-arylated 2(5H)-furanones 6a-c, 7a, b and the 3(2H)-pyridazones 9a-d were prepared by using the aldehyde form of muco halogen acids in electrophilic substitution reactions and in an aldol-like condensation reaction. The cytotoxicity of these simple and bis-cyclic butenolides have been evaluated in tissue culture studies on MAC 13 and MAC 16 murine colon cancer cell lines. The butyl furanone 3 displayed the highest cytotoxicity of 3 μM, as one selected example of a series of dichlorinated pseudoesters. The 5-arylated 2(5H)-furanones 6 and 7 did not show a structure-activity relationship (SAR) depending on the substitution pattern of the aromatic system. An IC50 (concentration inhibiting growth by 50%) was found within a range of 30-50 and 40-50 μM for the MAC 13 and MAC 16 cell lines, respectively. The pyridazine series 9 showed a maximum in-vitro activity for the p-methoxydrivative 9b, having an IC50 of 17 in MAC 13 and 11 μM in MAC 16 cell lines. Selected examples of each series and further novel 2(5H)-furanones such as the hydrazone 5 and the hydantoin 8 have been screened in-vivo in mice and the data are presented. For the pyridazines 9a-d, the in-vitro cytotoxicity correlated with an in-vivo inhibition of tumour growth. The ring expansion of the 5-membered 2(5H)-furanone ring system such as 6a into the 6-membered 3(2H)-pyridazone 9b led to an agent with improved antineoplastic properties. On the resistant MAC 16 cell line the pyridazone 9b displayed 52% tumour inhibition in mice at a dose of 50 mg kg-1 compared with 27% for the 5-FU standard.

Cytotoxic activity of Fagonia cretica against human breast cancer cells

In many parts of the world, plants are directly utilised for their medicinal properties. Traditional medicine from Pakistan, India and the Far East is well documented and its history is embedded in folklore. It has been documented that an aqueous extract of the desert shrub, Fagonia cretica, is a popular treatment for breast cancer in Pakistan. The administration of an aqueous extract of Fagonia cretica is reported effective at reducing tumour size and improving the quality of life of breast cancer patients, is well tolerated and does not exhibit adverse effects like vomiting, diarrhoea or alopecia which are common side effects of standard cytotoxic therapy. In the past, many pharmacologically active and chemotherapeutic compounds have been isolated from plants which subsequently have proven to be successful in clinical trials and been used as primary compounds in therapeutic regimes. Fagonia cretica has historical use as a treatment for breast cancer, yet there is little scientific evidence which shows chemotherapeutic potential towards breast tumours. Preparation and analysis of an aqueous extract of Fagonia cretica may reveal novel chemotherapeutic agents that can be used to effectively target cancer cells. An understanding of the mechanism of any activity may improve our understanding of cancer cell biology and reveal novel therapeutic targets. This thesis describes for the first time that an aqueous extract of Fagonia cretica shows potent in vitro cytotoxic activity towards breast cancer epithelial cell lines which was not seen towards normal mammary epithelial cells. Elucidation and characterisation of the cytotoxic mechanism was undertaken by analysing DNA damage, cell cycle status, apoptosis, metabolic state and expression of transcription factors and their targets. Finally, methods for the isolation and identification of active compound(s) were developed using various chromatographic techniques. An aqueous extract of Fagonia cretica was able to reduce cell viability significantly in two phenotypically different breast cancer cell lines (MCF-7 and MDA-MB-231). This activity was markedly reduced in normal mammary epithelial cells (HMEpC). Further investigation into the mode of action revealed that extract treatment induced cell cycle arrest and apoptosis in both MCF-7 and MDA-MB-231 cell lines. This coincided with the formation of DNA double stranded breaks and the DNA repair marker ?-H2AX. In MCF-7 cells, ATM/ATR activation resulted in increased p53 expression and of its transcriptional targets p21 and bax, suggesting a role for a p53-mediated response. Furthermore, inhibition of extract-induced p53 expression with siRNA reduced the cytotoxic effect against MCF-7 cells. Extract treatment was also associated with increased FOXO3a expression in MCF-7 and MDA-MB-231 cells. In the absence of functional p53, siRNA knockdown of extract-induced FOXO3a expression was completely abrogated, suggesting that FOXO3a plays a vital role in extract-induced cytotoxicity. Isolation and characterisation of the active compound(s) within the extract was attempted using liquid chromatography and mass spectrometry in conjunction with a cell viability assay. Multiple fractionations generated an active fraction that contained four major compounds as detected by mass spectrometry. However, none of these compounds were identified structurally or chemically due to constraints within the methodology.

S100P dissociates myosin IIA filaments and focal adhesion sites to reduce cell adhesion and enhance cell migration

S100 proteins promote cancer cell migration and metastasis. To investigate their roles in the process of migration we have constructed inducible systems for S100P in rat mammary and human HeLa cells that show a linear relationship between its intracellular levels and cell migration. S100P, like S100A4, differentially interacts with the isoforms of nonmuscle myosin II (NMIIA, K(d) = 0.5 µm; IIB, K(d) = 8 µm; IIC, K(d) = 1.0 µm). Accordingly, S100P dissociates NMIIA and IIC filaments but not IIB in vitro. NMIIA knockdown increases migration in non-induced cells and there is no further increase upon induction of S100P, whereas NMIIB knockdown reduces cell migration whether or not S100P is induced. NMIIC knockdown does not affect S100P-enhanced cell migration. Further study shows that NMIIA physically interacts with S100P in living cells. In the cytoplasm, S100P occurs in discrete nodules along NMIIA-containing filaments. Induction of S100P causes more peripheral distribution of NMIIA filaments. This change is paralleled by a significant drop in vinculin-containing, actin-terminating focal adhesion sites (FAS) per cell. The induction of S100P, consequently, causes significant reduction in cellular adhesion. Addition of a focal adhesion kinase (FAK) inhibitor reduces disassembly of FAS and thereby suppresses S100P-enhanced cell migration. In conclusion, this work has demonstrated a mechanism whereby the S100P-induced dissociation of NMIIA filaments leads to a weakening of FAS, reduced cell adhesion, and enhanced cell migration, the first major step in the metastatic cascade.

Synthesis and cytotoxity of oxysterols:studies on the A,B ring polyoxygenated sterols

Oxysterols (OS), the polyoxygenated sterols, represent a class of potent regulatory molecules for important biological actions. Cytotoxicity of OS is one of the most important aspects in studies of OS bioactivities. However, studies, the structure-activity relationship (SAR) study in particular, have been hampered by the limited availability of structurally diverse OS in numbers and amounts. The aim of this project was to develop robust synthetic methods for the preparation of polyhydroxyl sterols, thereof, evaluate their cytotoxicity and establish structure-activity relationship. First, we found hydrophobicity of the side chain is essential for 7-HC's cytotoxicity, and a limited number of hydroxyl groups and a desired configuration on the A, B ring are required for a potent cytotoxicity of an OS, after syntheses and tests of a number of 7-HC's analogues against cancer cell lines. Then polyoxygenation of cholesterol A, B rings was explored. A preparative method for the synthesis of four diastereomerically pure cholest-4-en-3,6-diols was developed. Epoxidation on these cholest-4-en-3,6-diols showed that an allyl group exerts an auxiliary role in producing products with desired configuration in syntheses of the eight diastereomerically pure 45-epoxycholestane-3,6-diols. Reduction of the eight 45-epoxycholestane-3,6-diols produced all eight isomers of the cytotoxic 5α-acholestane 3β,5,6β-triol (CT) for the first time. Epoxide ring opening with protic or Lewis acids on the eight 45-epoxycholestane-3,6-diols are carefully studied. The results demonstrated a combination of an acid and a solvent affected the outcomes of a reaction dramatically. Acyl group participation and migration play an important role with numbers of substrates under certain conditions. All the eight 4,5-trans cholestane- 3,4,5,6-tetrols were synthesised through manipulation of acyl participation. Furthermore these reaction conditions were tested when a number of cholestane-3,4, 5,6,7-pentols and other C3-C7 oxygenated sterols were synthesised for the first time. Introduction of an oxygenated functional group through cholest-2-ene derivatives was studied. The elimination of 3-(4-toluenesulfonate) esters showed the interaction between the existing hydroxyls or acyls with the reaction centre often resulted in different products. The allyl oxidation, epoxidation and Epoxide ring opening reactions are investigated with these cholest-2-enes.

Studies on the mechanism of action on antitumour imidazotetrazinones

The imidazotetrazinones are clinically active antitumour agents, temozolomide currently proving successful in the treatment of melanomas and gliomas. The exact nature of the biological processes underlying response are as yet unclear.This thesis attempts to identify the cellular targets important to the cytotoxicity of imidazotetrazinones, to elucidate the pathways by which this damage leads to cell death, and to identify mechanisms by which tumour cells may circumvent this action. The levels of the DNA repair enzymes O6-alkylguanine-DNA-alkyltransferase (O6-AGAT) and 3-methyladenine-DNA-glycosylase (3MAG) have been examined in a range of murine and human cell lines with differential sensitivity to temozolomide. All the cell lines were proficient in 3MAG despite there being 40-fold difference in sensitivity to temozolomide. This suggests that while 3-methyladenine is a major product of temozolomide alkylation of DNA it is unlikely to be a cytotoxic lesion. In contrast, there was a 20-fold variation in O6-AGAT levels and the concentration of this repair enzyme correlated with variations in cytotoxicity. Furthermore, depletion of this enzyme in a resistant, O6-AGAT proficient cell line (Raji), by pre-treatment with the free base O6-methylguanine resulted in 54% sensitisation to the effects of temozolomide. These observations have been extended to 3 glioma cell lines; results that support the view that the cytotoxicity of temozolomide is related to alkylation at the O6-position of guanine and that resistance to this drug is determined by efficient repair of this lesion. It is clear, however, the other factors may influence tumour response since temozolomide showed little differential activity towards 3 established solid murine tumours in vivo, despite different tumour O6-AGAT levels. Unlike mitozolomide, temozolomide is incapable of cross-linking DNA and a mechanism by which O6-methylguanine may exert lethality is unclear. The cytotoxicity of the methyl group may be due to its disruption of DNA-protein interactions, or alternatively cell death may not be a direct result of the alkyl group itself, but manifested by DNA single-strand breaks. Enhanced alkaline elution rates were found for the DNA of Raji cells treated with temozolomide following alkyltransferase depletion, suggesting a relationship between O6-methylguanine and the induction single-strand breaks. Such breaks can activate poly(ADP-ribose) synthetase (ADPRT) an enzyme capable of rapid and lethal depletion of cellular NAD levels. However, at concentrations of temozolomlde relevant in vivo little change in adenine nucleotides was detected in cell lines, although this enzyme would appear important in modulating DNA repair since inhibition of ADPRT potentiated temozolomide cytotoxicity in Raji cells but not O6-AGAT deficient GM892A cells. Cell lines have been reported that are O6-AGAT deficient yet resistant to methylating agents. Thus, resistance to temozolomide may arise not only by removal of the methyl group from the O6-position of guanine, but also from another mechanism involving caffeine-sensitive post-replication repair or mismatch repair activity. A modification of the standard Maxam Gilbert sequencing technique was used to determine the sequence specificity of guanine-N7 alkylation. Temozolomide preferentially alkylated runs of guanines with the intensity of reaction increasing with the number of adjacent guanines in the DNA sequence. Comparable results were obtained with a polymerase-stop assay, although neither technique elucidates the sequence specificity of O6-guanine alkylation. The importance of such specificity to cytotoxicity is uncertain, although guanine-rich sequences are common to the promoter regions of oncogenes. Expression of a plasmid reporter gene under the control of the Ha-ras proto~oncogene promoter was inhibited by alkylation with temozolomide when transfected into cancer cell lines, However, this inhibition did not appear to be related to O6~guanine alkylation and therefore would seem unimportant to the chemotherapeutic activity of temozolomide.

Intracellar signalling in the HGT-1 gastric cell line and in rat isolated parietal cells

The work presented in this thesis was undertaken to increase understanding of the intracellular mechanisms regulating acid secretion by gastric parietal cells. Investigation of the effects of protein kinase C on secretory activity induced by a variety of agents was a major objective. A further aim was to establish the sites at which epidermal growth factor (EGF) acts to stimulate prostaglandin E2 (PGE2) production and to inhibit acid secretion. These investigations were carried out by using the HGT-1 human gastric cancer cell line and freshly isolated rat parietal cells. In HGT-1 cells, the cyclic AMP response to histamine and to truncated glucagon-like peptide 1 (TGLP-1) was reduced when protein kinase C was activated by 12-0-tetradecanoylphorbol 13-acetate (TPA). Receptor-binding studies and experiments in which cyclic AMP production in HGT-1 cells was stimulated by gastric inhibitory polypeptide, cholera toxin and forskolin suggested that the effect of TPA was mediated by uncoupling of the histamine H2 receptor from the guanine nucleotide regulatory protein Gs, possibly by phosphorylation of the receptor. An involvement of protein kinase C α in this effect was suggested because an antibody to this isoform specifically prevented the inhibitory effects of TPA on histamine-stimulated adenylate cyclase activity in a membrane fraction prepared from HGT-1 cells. Carbachol-stimulated secretory activity in parietal cells was specifically inhibited by Ro 31-8220, a bisindolylmaleimide inhibitor of protein kinase C. Thus protein kinase C may play a role in the activation of the secretory response to carbachol. In parietal cells prelabelled with [3H]-arachidonic acid or [3H]myristic acid, EGF did not affect [3H]-fatty acid or [3H] - diacylglycerol content. No evidence for effects of EGF on phosphatidylinositol glycan-specific phospholipase C, phospholipase A2 or on low Km cyclic AMP phosphodiesterase activities were found.

A novel role for the adipokine visfatin/pre-B cell colony-enhancing factor 1 in prostate carcinogenesis

Adipose tissue is now well established as an endocrine organ and multiple hormones termed ‘adipokines’ are released from it. With the rapidly increasing obese population and the increased risk mortality from prostate cancer within the obese population we looked to investigate the role of the adipokine visfatin in LNCaP and PC3 prostate cancer cell lines. Using immunohistochemistry and immunocytochemistry we demonstrate visfatin expression in LNCaP (androgen-sensitive) and PC3 (androgen-insensitive) human prostate cancer cell lines as well as human prostate cancer tissue. Additionally, we show that visfatin increases PC3 cell proliferation and demonstrate the activation of the MAPKs ERK-1/2 and p38. Moreover we also demonstrate that visfatin promotes the expression and activity of MMP-2/9 which are important proteases involved in the breakdown of the extracellular matrix, suggesting a possible role for visfatin in prostate cancer metastases. These data suggest a contributory and multifunctional role for visfatin in prostate cancer progression, with particular relevance and emphasis in an obese population.

The role of TG2 in regulating S100A4-mediated mammary tumour cell migration

The importance of S100A4, a Ca2+-binding protein, in mediating tumour cell migration, both intracellularly and extracellularly, is well documented. Tissue transglutaminase (TG2) a Ca2+-dependent protein crosslinking enzyme, has also been shown to enhance cell migration. Here by using the well characterised non-metastatic rat mammary R37 cells (transfected with empty vector) and highly metastatic KP1 cells (R37 cells transfected with S100A4), we demonstrate that inhibition of TG2 either by TG2 inhibitors or transfection of cells with TG2 shRNA block S100A4-accelerated cell migration in the KP1cells and in R37 cells treated with exogenous S100A4. Cell migration was also blocked by the treatment with the non-cell permeabilizing TG2 inhibitor R294, in the human breast cancer cell line MDA-MB-231 (Clone 16, which has a high level of TG2 expression). Inhibition was paralleled by a decrease in S100A4 polymer formation. co-immunoprecipitation and Far Western blotting assays and cross-linking assays showed not only the direct interaction between TG2 and S100A4, but also confirmed S100A4 as a substrate for TG2. Using specific functional blocking antibodies, a targeting peptide and a recombinant protein as a competitive treatment, we revealed the involvement of syndecan-4 and a5ß1 integrin co-signalling pathways linked by activation of PKCa in this TG2 and S100A4-mediated cell migration. We propose a mechanism for TG2-regulated S100A4-related mediated cell migration, which is dependent on TG2 crosslinking.

Tissue Transglutaminase (TG2) is a potential therapeutic target in the treatment of chemoresistant breast cancer

Tissue transglutaminase (TG2) has been suggested to be a key player in the progression and metastasis of chemoresistant breast cancer. One of the foremost survival signalling pathways implicated in causing drug resistance in breast cancer is the constitutive activation of NFκB (Nuclear Factor -kappa B) induced by TG2. This study provides a mechanism by which TG2 constitutively activates NFκB which in turn confers chemoresistance to breast cancer cells against doxorubicin. Breast cancer cell lines with varying expression levels of TG2 as well as TG2 null breast cancer cells transfected with TG2 were used as the major cell models for this study. This study made use of cell permeable and impermeable TG2 inhibitors, specific TG2 and Rel A/ p65 targeting siRNA, TG2 functional blocking antibodies, IKK inhibitors and a specific targeting peptide against Rel A/p65 to investigate the pathway of activation involved in the constitutive activation of NFκB by TG2 leading to drug resistance. Crucial to the activation of Rel A/p65 and drug resistance in the breast cancer cells is the interaction between the complex of IκBα and Rel A/p65 with TG2 which results in the dimerization of Rel A/p65 and polymerization of IκBα. The association of TG2 with the IκBα-NFκB complex was determined to be independent of IKKα/β function. The polymerized IκBα is degraded in the cytoplasm by the μ-calpain pathway which allows the cross linked Rel A/ p65 dimers to translocate into the nucleus. Using R283 and ZDON (cell permeable TG2 activity inhibitors) and specific TG2 targeting siRNA, the Rel A/ p65 dimer formation could be inhibited. Co-immunoprecipitation studies confirmed that the phosphorylation of the Rel A/p65 dimers at the Ser536 residue by IKKε took place in the cell nucleus. Importantly, this study also investigated the transcriptional regulation of the TGM2 gene by the pSer536 Rel A/ p65 dimer and the importance of this TG2-NFκB feedback loop in conferring drug resistance to breast cancer cells. This data provides evidence that TG2 could be a key therapeutic target in the treatment of chemoresistant breast cancer.

The Role of Aquaporin 3 (AQP3) in Breast Cancer

The increasing prevalence of breast cancer (BC) in different parts of the world, particularly in the UK, highlights the importance of research into the aetiology and pathology of the disease. BC is the most common malignancy affecting women worldwide. Aquaporins (AQPs) are membrane protein channels that regulate cellular water flow. Recently, studies have demonstrated that expression of AQP3 is up-regulated in cancerous breast tissue. The present study examines the role of AQP3 in BC cell biology. Examination of clinical cases of BC showed higher AQP3 gene and protein expression in cancer tissues compared to healthy border tissues. In distinct clinicopathological groups however there were no differences observed with regards to AQP3 expression, suggesting that AQP3 expression may not be a predictor of lymph node infiltration or tumour grade. shRNA technology was used to knockdown gene expression of AQP3 in the invasive MDA-MB-231 BC cellular model. Cellular proliferation, migration, invasion, adhesion and response to the 5- fluorouracil (5-FU) based chemotherapy treatment were investigated in parental and knockdown cell line. AQP3 knockdown cells showed reduction in cellular proliferation, migration, invasion and increase in cell sensitivity to 5-FU compared with wild type (WT) or scrambled control (SC) cells. The effects of AQP3 knockdown on cellular glycolytic ability and ATP cellular content were quantified. Indirect glucose uptake was also measured by quantifying reconditioned media. AQP3 knockdown cells showed significantly lower levels of glucose uptake as compared to WT or SC. However there was no difference in the glycolytic ability and ATP content of the cells suggesting AQP3 has no role in cancer cell energetics. These data collectively suggest AQP3 expression is associated with the BC disease clinically and plays a role in multiple important aspects of BC pathophysiology, thus AQP3 represents a novel target for therapeutic intervention.