Determining the role of the Galectin-3 N-terminus in tumor-specifica poptosis of human glioblastoma cells Público

Abstract

Among malignant cancers, glioblastoma multiforme (GBM) is one of the most aggressive and lethal; patients typically die within a year of diagnosis despite current treatments, which include surgery, chemotherapy and radiation. Because of its treatment-resistant nature, it is important to develop novel, selective therapeutics that can undermine the mechanisms used by this cancer to progress despite an aggressive therapeutic regime. Members of the Van Meir Laboratory at the Winship Cancer Institute of Emory University identified glycoprotein galectin-3 as a candidate for a novel therapeutic option for GBM. A modified form of galectin-3 containing a 37 amino-acid signal peptide sequence at its N-terminus demonstrates powerful, tumor-specific apoptotic ability; notably, against several GBM cell lines. Here we develop additional galectin-3 variants containing distinct N-terminus alterations in an effort to better understand how addition of a signal peptide to galectin-3 enhances its pro-apoptotic ability. More specifically, we aim to understand whether N-terminus elongation or an innate quality of the signal peptide itself enhances the ability of galectin-3 to induce apoptosis of tumor cells. We developed four recombinant galectin-3 variants through PCR-based cloning methods, expression in bacteria and affinity chromatography protein purification. We assessed the effects of each on the cell viability of human GBM cells and found that both full-length-recombinant galectin-3 and a truncated form of the protein lacking several collagen-like domains within the protein's N-terminus led to a slight increase in the GBM cell viability. Elongated galectin-3 had no effect on the viability of the cells and sGal3, engineered with a signal peptide at the N-terminus, led to a drastic decrease in cell viability within 24 hours. These findings suggest that the specific signal peptide addition to sGal3 grants the protein with enhanced apoptotic ability and cannot be mimicked by generic additions to the protein's N-terminus. The variants produced in this work will serve as useful tools for discovering more about the function of the N-terminus of the galectin-3 protein, which may prove useful in further unearthing its roles in cancer.

Table of Contents

Introduction 1

The Clinical Characteristics of Glioblastoma (GBM): Incidence, Survival, Current Treatments, and Future Outlook 2

Pathological Hallmarks of GBM 3

Genetics of GBM 4

Cancer Glycobiology 5

Galectins and Glioma 6

Galectin-3 8

Galectin-3 as a potential therapeutic for GBM 12

Scope of the Thesis 13

Materials and Methods 16

Plasmids and Protein Purification 17

ELISA 18

Cell Culture and Crystal Violet Assay 18

Results 19

Galectin-3 can be purified using a GST purification protocol 20

Secreted Galectin-3 was not successfully purified using a GST purification protocol 21

sGal3 can be purified using a Histidine-tag purification protocol 22

Recombinant Galectin-3 does not reduce the cell viability

of LN229 GBM cells, while recombinant sGal3 does 23

Truncated Galectin-3 was purified using a GST purification method 24

Truncated Galectin-3 (C4-CRD) increases the cell viability of LN229 cells 26

Elongated Galectin-3 (eGal3) can be purified using histidine-tag purification methods 28

Elongated Galectin-3 (eGal3) does not decrease the cell viability of LN229 cells compared to imidazole control 29

Discussion 30 Future Directions 35 References 36 Appendix I: Protocols 40 Appendix II: Plasmids 62

Table of Figures

Figure 1 - Structural Domains of Galectin-3 9

Figure 2 - Classes of Human Galectins 9

Figure 3 - Planned Galectin-3 variants 15

Figure 4 - Galectin-3 can be purified through GST-purification 21

Figure 5 - rGST-sGal3 was not successfully produced in BL21 E. Coli. 22

Figure 6 - sGal3 can be efficiently purified with the His-tag purification method and detected via western blot. 23

Figure 7 - His-tag sGal3 kills GBM cells (LN229) in concentration-dependent manner 24

Figure 8 - Truncated Galectin-3 produced by GST-purification increases LN229 cell proliferation 26

Figure 9 - His-tagged Elongated Galectin-3 has no effect on LN229 cells compared to control 28

Table 1 - Galectin-3 has many different roles in cancer and its function is often dependent on its location. 11

About this Master's Thesis

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Degree	MS
Submission	Master's Thesis
Language	English
Research Field	Biology, General Health Sciences, Oncology Biology, Cell
Palavra-chave	apoptosis galectins galectin-3 cancer cell death cancer biology glioblastoma
Committee Chair / Thesis Advisor	Van Meir, Erwin, Emory University Lee, Sok-Hyong , Emory University
Committee Members	Cummings, Richard, Harvard Medical School Fu, Haian, Emory University

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