Evaluating the effect of LRP/LR antibodies in colorectal carcinoma mouse models and assessing the molecular mechanism of LRP/LR on apoptotic pathways in colorectal carcinoma cells

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dc.contributor.authorVania, Leila
dc.date.accessioned2020-11-16T18:22:12Z
dc.date.available2020-11-16T18:22:12Z
dc.date.issued2020
dc.descriptionThesis submitted in fulfilment of the requirements for the degree Philosophiae Doctor in Molecular and Cell Biology in the Faculty of Science, University of the Witwatersrand, Johannesburg, 2020en_ZA
dc.description.abstractCancer remains one of the leading causes of death around the world with its incidence and mortality rates constantly increasing. Tumourigenic cells are characteristically seen to overexpress the 37kDa/67kDa laminin receptor (LRP/LR) compared to their normal cell counterparts. LRP/LR is involved in promoting tumourigenic processes such as cellular viability maintenance and apoptotic evasion. Thus, the first main aim of this study was to investigate the role of LRP/LR in cellular viability of early (SW-480) and late stage (DLD-1) colorectal carcinoma cells and thereafter assess the molecular mechanism of LRP/LR on apoptotic pathways upon siRNA-mediated down-regulation of LRP/LR in vitro. The data from the study was analysed using a one-way ANOVA, followed by a two-tailed student’s t-test with a confidence interval of 95%. To further validate the data, the Bonferroni post-hoc test was applied, with p-values of less than 0.05 considered to be significant. Results from the in vitro study showed upon siRNA-mediated down-regulation of LRP/LR, the viability of early (SW-480) and late (DLD-1) stage colorectal cancer cells was significantly reduced through increased levels of apoptosis apparent by compromised membrane integrity as determined by Annexin-V/PI assays and confocal microscopy, respectively. Caspase-3 activity assays were performed to confirm apoptosis, where both cell lines exhibited significantly high caspase-3 activity. In addition, down-regulation LRP/LR resulted in a significant increase in caspase-8 and -9 activity in both colorectal carcinoma cell lines. Cell cycle analysis showed that LRP/LR down-regulation resulted in a significant increase in the sub G0/G1 apoptotic phase of DLD-1 cells, when compared to non-transfected cells, confirming the occurrence of apoptosis. To evaluate the mechanistic role of LRP/LR, proteomic analysis of pathways involved in proliferation and apoptosis were further investigated with the MAPK and Apoptotic Proteome Profiler Antibody Arrays™ as well as SWATH-MS (Sequential Window Acquisition of All Theoretical Mass Spectra). Upon siRNA-mediated down-regulation of LRP/LR, various pro-apoptotic proteins including Bax, cleaved caspase-3 and p53 as well as specific proteins in the MAPK cell signalling pathway such as Chk-2 and AMPKα2 were significantly up-regulated in the DLD-1 cells. In addition, several anti-apoptotic proteins were seen to be significantly down-regulated upon siRNA-mediated down-regulation of LRP/LR including xIAP, Claspin and Survivin. The results obtained from the Proteome Profiler Antibody Arrays™ were confirmed through qPCR, by investigating mRNA expression levels of specific proteins. SWATH-MS analysis showed that siRNA-mediated down-regulation of LRP/LR led to significant changes in the proteome of DLD-1 colorectal cancer cells, revealing new roles of the protein as well as confirming its existing roles. Moreover, it showed that LRP/LR may alter components of the MAPK, p53-apoptotic as well as autophagic signalling pathways to aid colorectal cancer cells in continuous growth and survival. Telomerase activity and telomerase-related proteins were also investigated using qPCR and western blotting, respectively. A significant decrease in telomerase activity as well as decreases in phospho-TERT and telomeric repeat-binding factor 2 (TFR-2) protein expression levels were observed in the DLD-1 cells upon down-regulation of LRP/LR. In addition to its role in cell viability and apoptotic evasion, LRP/LR has also been found to be involved in enhancing tumour cell adhesion and invasion, key points of metastasis. However, previous studies revealed that the application of the anti-LRP/LR specific antibody IgG1- iS18 led to a significant reduction in the metastatic potential of colorectal carcinoma cells in vitro. Therefore, the second aim of the present study was to investigate the significance of blocking LRP/LR with the IgG1-iS18 antibody and to determine whether it will be effective in the treatment of colorectal carcinoma in vivo using an MF-1 nude mice model. Results from several in vivo pilot studies performed showed that all mice were able to successfully develop tumours upon intraperitoneal injection of HT-29 colorectal cancer cells. However, it was found that despite various changes in the concentration as well as the route of administration of the IgG1-iS18 antibody, there was no significant effect seen in tumour growth/shrinkage and metastasis. Due to the data obtained from the pilot studies and time constraints, the full study was not performed. Although the mechanism of action of LRP/LR is far from being understood, findings from this study show that LRP/LR is critically implicated in apoptosis and cell viability maintenance. The current study therefore suggests that siRNA-mediated down-regulation of LRP/LR could be a possible therapeutic strategy for the treatment of colorectal canceren_ZA
dc.description.librarianCK2020en_ZA
dc.facultyFaculty of Scienceen_ZA
dc.identifier.urihttps://hdl.handle.net/10539/30185
dc.language.isoenen_ZA
dc.phd.titlePhDen_ZA
dc.titleEvaluating the effect of LRP/LR antibodies in colorectal carcinoma mouse models and assessing the molecular mechanism of LRP/LR on apoptotic pathways in colorectal carcinoma cellsen_ZA
dc.typeThesisen_ZA
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