Pharmacy

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Alterations in Colorectal Cancer Cell-extracellular Matrix Interactions Upon Acquisition of Chemotherapy Resistance.
(University of Waterloo, 2015-12-16) Berg, Spencer; Blay, Jonathan
Adjuvant chemotherapy is an essential component in the treatment of advanced colorectal cancer (CRC). Unfortunately, many patients experience recurrence with aggressive, chemotherapy-resistant disease for which the prognosis is poor and treatment options are limited. Understanding the changes that cancer cells undergo during the acquisition of a drug-resistant phenotype is therefore of critical importance in improving CRC patient outcomes. We chose to study resistance to the CRC chemotherapy agent, irinotecan, by first creating a CRC cell line that is highly resistant to its active metabolite (SN-38). In particular, we were interested in how SN-38 resistant CRC cells (HT-29S) were altered from parental, drug-sensitive CRC cells (HT-29) in their relationship with a feature of their microenvironment, the extracellular matrix (ECM). We found that HT-29S cells form a matrix composed of the ECM glycoprotein fibronectin when cultured as 3-dimensional spheroids, whereas HT-29 cells do not. The increase in fibronectin matrix deposition coincided with an increased fibronectin adhesive capacity of the SN-38-resistant cells, likely due to an increased expression of the integrin α5 subunit, which together with integrin β1 forms the primary fibronectin receptor. Furthermore, we demonstrated an activation of a phosphoinositide-3-kinase (PI3K)/protein kinase B (Akt) pro-survival signalling pathway downstream of integrin α5β1 ligation to fibronectin. Inhibition of this signalling pathway with the PI3K inhibitor LY294002 sensitized HT-29S cells to SN-38, but did not alter the response of the parental cell line to the chemotherapy agent. Finally, we have determined that HT-29S cells appear to undergo an epithelial to mesenchymal transition during the acquisition of chemotherapy resistance, and that this transition may be responsible for the upregulation of integrin α5 in the resistant population.
Analysis of plasma chemokines and circulating tumour cells in colorectal and breast cancer patient peripheral blood
(University of Waterloo, 2020-01-22) Patel, Deep; Blay, Jonathan
Circulating tumour cells (CTCs) provide a prognostic value in solid tumours including colorectal and breast. Enumeration of tumour cells from blood is becoming a common practice in informing prognosis and may guide therapy decisions. Enumeration alone does not capture heterogeneity of tumours and varying functional abilities of the CTCs to interact with the secondary microenvironment. Characterizing the isolated CTCs and assessing their functional abilities can track molecular changes in the disease progress. As a step toward identifying functional features of CTCs that could aid in clinical decisions, this study was aimed at analyzing chemokine release profile in drug resistance and developing a CTC isolation technique based on extracellular matrix interactions. Cancer cells release different chemokines and express chemokine receptors which together work to direct cell infiltrates in the tumour microenvironment. This work examined changes in the profile of chemokine release using a model of drug resistance based on the colorectal cancer cell line HT29 and its counterpart HT29-R that is resistant to the late-stage chemotherapy drug irinotecan (SN-38). Following an initial screening of mRNA expression through PCR and qPCR, five of the chemokines (CCL2, CCL15, CXCL8, CXCL12, and CCL20) were analyzed further for their release patterns amongst cell lines and peripheral blood of healthy volunteers and stage IV colorectal and breast cancer patients. The release pattern of chemokines in patient samples differed from the results of the in vitro drug-resistance model. Specific tumour location, previous therapies, and genetic variability are all examples of the factors that may provide unique patterns and complicate modelling for chemokine release in late-stage cancer. A detailed analysis revealed an upward trend for midkine (NEGF2) when baseline and 12 months plasma samples were compared. Migration studies may further reveal the consequences of this expression profile. Migration assays were carried out with Transwell® chambers and HepG2 cells to partially mimic the hepatic microenvironment. Such studies can guide future functional studies for isolated CTCs. We next sought to investigate extracellular matrix protein interactions, which might depend on a changing chemokine milieu. We utilized cancer cells’ ability to adhere to extracellular matrix and created a platform to isolate CTCs from the peripheral blood samples. A total of 14 colorectal and 7 breast cancer patients donated blood samples. Adhesion assays were performed with a range of different ECM proteins. We identified an optimal ECM substratum composed of collagen and fibronectin at a mass coating ratio of 2:1. The isolated CTCs were identified through immunofluorescence with epithelial marker antibodies (EpCAM and pan-cytokeratin). Identification of CTCs was further confirmed by exclusion with a hematopoietic origin marker CD45. The captured number of cells ranged from 0 to 296, whereas the mean number was 26 and the median was 22 per patient sample (~8mL). This technique not only allows enumeration, but also isolates cells based on a functional approach. The isolated cells are successful in adhering to extracellular matrix proteins and can be further characterized through functional markers. Overall, this study addresses two unique functional features of CTCs – their expression of certain chemokines and their ability to interact with both fibronectin and collagen - that form the basis to provide future clinical utility. Such an approach will help to inform clinicians about the aggressive nature of an individual tumour and guide treatment decisions toward best prognostic outcomes.
Cell Density-Dependent Changes in the Localization of the CD26 Protein in Colorectal Cancer Cells in Response to Flavonoid Treatment
(University of Waterloo, 2019-05-24) Diaconu, Bogdan; Blay, Jonathan
Colorectal cancer is the 3rd most common cancer worldwide and this rate of incidence is largely attributable to lifestyle factors such as the diet. A group of plant secondary metabolites called flavonoids has been found to exert various anticancer activities in colorectal cancer cell lines and is indeed thought to function similarly in vivo. The cell-surface enzymes CD26, CD38, and CD73 are present in colorectal cancers and their presence and activities in this context have the potential to be modulated by extracellular factors such as dietary flavonoids. The levels of CD26 protein in particular have been previously found to increase at the cell surface in HT-29 colorectal cancer cells following treatment with the flavonoid apigenin. Another extracellular factor which may alter the response of cancer cells is cell density. In this work, the role of apigenin was investigated first on the mRNA transcription of CD26, CD38, and CD73 in HT-29 cells cultured to increasing degrees of confluence. CD26 was identified as the most promising target because it revealed the greatest degree of mRNA variability. Next, the amount of CD26 mRNA and protein was quantified in HT-29 cells treated with apigenin and the related flavonoids genistein, kaempferol, and luteolin. Finally, the localization of CD26 protein was observed in these cells following flavonoid treatment. This investigation showed no consistent changes in either mRNA expression or whole cell protein abundance for CD26. However, there was a distinct change in cellular localization of CD26 in response to apigenin and genistein and this was seen particularly in colorectal cancer cells at low levels of confluence. The relocation of the CD26 protein may depend on particular features of the flavonoid structure. Furthermore this effect appears be modulated by a change in cell confluence. Therefore this study provides new insights with respect to the role of flavonoids in regulating CD26 in colorectal cancer cells.
Characterising changes in adhesion and enzyme activity related to drug resistance in colon cancer cells
(University of Waterloo, 2018-12-21) Dekker, Heather; Blay, Jonathan
Metastatic colorectal cancer is often fatal, and drug resistance to chemotherapeutic agents is one of the primary contributing factors to this lethality. Drug resistance arises from exposure to chemotherapies, and it can be mediated through a variety of mechanisms. One of these mechanisms is alteration of enzymes within the cancer cells to affect the processing or removal of the drug. Carboxylesterase is an example of an enzyme that converts irinotecan, a drug used in metastatic colorectal cancer treatments, into the active metabolite SN-38. Carboxylesterase enzymes are found in high quantities in both the liver and intestinal cells. The presence of carboxylesterase in intestinal and liver cells is an important consideration in the processing of colorectal cancer treatments. Glutathione S-transferase is another enzyme that has been implicated in drug resistance because of its ability to conjugate reduced glutathione to xenobiotic substances, facilitating their removal. Additionally, drug resistance can affect the behaviours of cells. Drug-resistant cells can exhibit changes in their motility and aggressiveness compared to drug-sensitive cells. In this study I investigated cellular behavioural changes in SN-38-resistant colon cancer cells compared to their SN-38-sensitive counterparts. In addition to behavioural changes, I also sought to determine if elevations in carboxylesterase and glutathione S-transferase enzymes were contributing to the drug resistance in these colon cancer cells.
Characterization of Bacteriophage λ Displaying Epidermal Growth Factor in the Uptake, Infiltration and Formation of HT29 Spheroids
(University of Waterloo, 2019-07-31) Huh, Haein; Slavcev, Roderick; Blay, Jonathan
Solid tumours are characterized by a complex structure comprised of extracellular matrix, neoplastic cells and stromal cells, each presenting a barrier to conventional anticancer chemotherapy as well as carrier-mediated drug delivery. Poor penetration of therapeutics into the interstitial tumour microenvironment remains a challenge, with drugs accumulating primarily in the regions of tumours that are situated closer to blood vessels. Bacteriophages do not infect eukaryotic cells, yet they have been shown to penetrate mucosal barriers, including multiple layers of epithelial cells and endothelium. In this work, we used NIH3T3 fibroblast and HT29 colon adenocarcinoma multicellular spheroids to represent the stroma and parenchyma of solid tumours and then to examine infiltration of bacteriophages as a means of delivering therapeutic cargo. By using phage display technology to decorate the surface of λ bacteriophage with epidermal growth factor (EGF), we compared the cell-phage interactions between wildtype phages and EGF-displaying phages, including the assessment of phages’ capacity to traverse the tumour interstitium, to be subjected to cell internalization and their effects on spheroid growth. Both wildtype and EGF-displaying λ phages were observed to adhere to the HT29 and NIH3T3 spheroids and to internalize into cells as early as 30 min following administration. EGF-phage treatment also slowed HT29 spheroid growth – demonstrating a delay in initial aggregation and formation of loosely-organized structures that led to much smaller spheroid formation in the latter stages of growth. These results support the potential for the therapeutic employment of bacteriophages as nanocarriers for targeted delivery.
Characterizing Changes in Dipeptidyl Peptidase IV/CD26 on Colon Carcinoma Cells in Response to Prostaglandin Treatment
(University of Waterloo, 2018-08-01) Durocher, Alexandra; Blay, Jonathan
Dipeptidyl peptidase IV (DPPIV) – also known as CD26 – is a multifunctional transmembrane protease found on the surface of most human cells, and shows variable expression between different types of cancer. DPPIV activity may play a role in inhibiting cancer progression by interacting with components of the tumour microenvironment. DPPIV degrades several signalling molecules including the chemokine CXCL12. CXCL12 has been shown to facilitate tumour development when associating with its cell-surface receptor CXCR4. The cell-surface expressions of CXCR4 and DPPIV/CD26 exhibit an inverse relationship; an increase in DPPIV/CD26 is accompanied by a decrease in CXCR4, and vice versa. J-series prostaglandins (i.e. PGD2, PGJ2 and 15d-PGJ2) have been shown to downregulate CXCR4 expression on colorectal carcinoma cells, which in turn may upregulate the expression and activity of DPPIV/CD26. Therefore, I investigated whether J-series prostaglandins altered DPPIV/CD26 expression and activity on colorectal cancer cells. Results showed that these prostaglandins did not upregulate DPPIV/CD26 mRNA expression, nor did they increase its whole-cell or cell-surface protein expression. J-series prostaglandins did increase the mean DPPIV/CD26 dipeptidase activity, however this increase was not statistically significant due to substantial inter-experimental variability. 15d-PGJ2 treatment also significantly decreased the rate of cell migration on both CRC cells and normal fibroblasts. These findings suggest that J-series prostaglandins may promote DPPIV/CD26 specific activity in colorectal carcinoma. However, 15d-PGJ2 was found to be significantly unstable at high concentrations in serum-supplemented culture media and this instability in culture medium may explain the inconsistent cell response in vitro.

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Browsing Pharmacy by Author "Blay, Jonathan"