Abstract
Cell membranes of all organisms are decorated with a dense layer of glycan structures linked to proteins and lipids, which play essential roles in the control of multiple biological processes, including cell adhesion, recognition, and signaling. Unlike protein synthesis, synthesis of glycans is not a template-driven process; instead, it is determined by the coordinated activities of multiple enzymes. Changes in activities of enzymes that constitute the glycosylation machinery lead to changes in glycan structures, resulting in function modification of glycosylated molecules. Cumulative evidence has demonstrated that cell surface glycans are important players in the regulation of pro-survival and programmed cell death signaling pathways. Glycans that are present on the cell surface could mediate their effects either directly, through changes in function of underlying extracellular signaling molecules, or indirectly, mediated by lectin-glycans interactions. This thesis addresses the role of extracellular membrane-associated glycans in the induction of programmed cell death via activation of multiple signaling pathways.
Altered glycosylation is considered a major hallmark of cancer and is also associated with high metastatic potential and poor prognosis in patients. Tumor-associated glycans have been described as prospective targets for drug delivery in cancer and development of diagnostic tools to differentiate cancer from healthy cells. Furthermore, the use of lectins that demonstrate cytotoxic properties upon binding with cognate sugar structures showed high potential in development of lectin-based anti-cancer therapies. To better characterize the mechanism of lectin-mediated programmed cell death, cytotoxic properties of several lectins of plant and fungal origin were evaluated in murine adenocarcinoma cells. While targeting different sets of glycans, Maackia amurensis lectin I, wheat germ agglutinin, and Aleuria aurantia lectin activated caspase- independent cell death with the involvement of several cell death signaling pathways. Moreover, a role of sialic acid-containing glycans in induction of programmed cell death mediated by some lectins has been demonstrated in this work.
Considering that cell surface glycans play important roles in regulation of cell survival and programmed cell death, various cell surface glycoengineering approaches, which involve either modification of endogenous glycans or incorporation of glycans with desired structure into the cell membrane, represent another interesting tool that could be used to increase the efficiency of anti-cancer agents. Here, a way to induce programmed cell death upon cell coating with sialyllactose using cyclic carbamate technology has been demonstrated, and several methods to characterize molecular mechanisms of the given mode of cell death have been employed. The use of pharmacological inhibitors targeting key apoptotic, pyroptotic, and necroptotic molecules revealed that sialyllactose-dependent mode of programmed cell death partially relies on activation of several caspases involved in the execution of apoptosis and pyroptosis. Deeper characterization of molecular mechanisms of sialyllactose-dependent cell death using a genome- wide CRISPR-Cas9 genetic screen appeared to be inefficient to provide a reliable list of candidate genes involved in this mode of programmed cell death, which could be explained by the activation of sialyllactose-dependent programmed cell death via parallel activation of several signaling pathways.
In conclusion, this work confirmed the role of cell membrane-glycans as key players in the activation of multiple modes of programmed cell death, which could be used in the development of effective anti-cancer therapies. Moreover, the potential of Maackia amurensis lectin I, wheat germ agglutinin, Aleuria aurantia lectin, and Erythrina cristagalli lectin to trigger programmed cell death was evaluated. Additionally, this thesis demonstrates a cell sialylation-dependent mode of programmed cell death and discusses available methods that could be employed to characterize this mode of programmed cells death. Overall, the data that have been presented and discussed in this thesis expand the knowledge about roles of cell surface glycosylation and provide new ideas that could be applied in the development of glycosylation-dependent approaches to treat cancer.
Parshenkov, Aleksei