Specific Inhibition of GSK-3β by Tideglusib: Potential Therapeutic Target for Neuroblastoma and Glioblastoma Cancer Stem Cells
American University of Beirut
Department of Anatomy, Cell Biology, and Physiological Sciences, Faculty of Medicine
Prof. Dr. Elie Al-Chaer
Assoc. Prof. Dr. Wassim Abou-Kheir
4 weeks
Cities/Months Jan Feb Mar Apr May Jun Jul Augt Sep Oct Nov Dec
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Type of Research Project
- Basic science
What is the background of the project?
Nervous system tumors are highly aggressive cancers in both children and adults, particularly neuroblastoma and glioblastoma. Previous investigations conducted in our lab addressed both: • The potency of Metformin, an AMP-activated protein kinase (AMPK) drug, and 9-β-d-Arabinofuranosyl Adenine Ara-a in suppressing the proliferation, viability, and invasive potential of the glioblastoma and neuroblastoma cell lines. • Triciribine and Rapamycin (mTOR) in inhibiting two different points of the Akt/mTOR pathway, a signaling pathway designated as a master regulator for cancer, in vitro on glioblastoma and neuroblastoma human cell lines and their cancer stem cells. Glycogen synthase kinase 3 (GSK3), a serine/threonine protein kinase, is a potential therapeutic target in many diseases, including cancer. We aimed to assess the anti-tumor effects of an irreversible GSK-3β inhibitor, Tideglusib (TDG), on human neuroblastoma and glioblastoma cell lines.
What is the aim of the project?
1. Evaluate the expression status of GSK-3β in human neuroblastoma and glioblastoma 2. Assess the potency of a GSK-3β inhibitor, Tideglusib (TDG), in suppressing the proliferation, viability, and migration of human neuroblastoma and glioblastoma tumor cells in vitro 3. Investigate the effect of TDG on the cancer stem cell (CSC) population of human neuroblastoma and glioblastoma cell lines
What techniques and methods are used?
1. Assessing the mRNA expression levels of GSK-3β by surveying a publicly available array data set comprised of human pan-tumor samples 2. Collecting normal and tumor human brain tissue samples and staining them for GSK-3β using immunohistochemistry and comparing between them. 3. Do western blotting analyses on different human neuroblastoma and glioblastoma cell lines for expression of GSK-3β. 4. Do MTT and Trypan blue assays to assess the effect of Tideglusib (TDG), GSK-3β inhibitor, on cell proliferation and viability in vitro 5. Investigate the effect of TDG on cell migration of SH-SY5Y and U-251 MG cells using wound-healing assay 6. Assess the potential inhibitory effect of TDG as an anti-cancerous drug by targeting the CSC population of neuroblastoma and glioblastoma tumors, which is deciphered by the ability of cells to form neurospheres in vitro.
What is the role of the student?
- The student will observe the practical experiments but will be highly involved in the analysis of the results
- The tasks of the student will be performed on his/her own
- The tasks will be done under supervision
What are the tasks expected to be accomplished by the student?
Students will have the opportunity to shadow and learn from lab members performing the following experiments. 1. Histology and Immunohistochemistry (IHC): Immunohistochemical analysis will be used to examine for the expression of GSK-3β in human NB and GBM tissue specimens obtained from the American University of Beirut Medical Center (AUBMC). Protocols for staining of tissues will be followed (H&E staining and IHC). 2. Cell Culture and Treatments: SK-N-SH (human neuroblastoma), U118 and A172 (human GBM) cells (ATCC, USA) will be cultured and maintained in DMEM Ham’s F-12 media with 10% FBS, 1% P/S, and Plasmocin Prophylactic in a humidified incubator (37°C; 5% CO2). The cells will be treated with different concentrations of TDG drug reconstituted in DMSO. 3. MTT/Cell Viability and Trypan Blue Assays: for the assessment of the anti-proliferative effect of TDG in vitro. 4. Wound-Healing Migration Assay: to assess the effect of TDG on migration of cells in vitro. 5. Trans-well Invasion Assay: to assess the effect of TDG on invasion of cells in vitro. 6. qRT-PCR and Western Blot Analyses: to study the gene and protein expression levels of GSK-3β and other different molecular pathway targets (p53, E-cadherin, Vimentin, Cyclin-D1, and others) in NB and GBM cells (untreated vs. treated). RNA and protein samples will be extracted and specific protocols will be followed. Bands will be then visualized using chemiluminescence. 7. 3D Culture and Sphere-Formation Assay: to investigate the effect of TDG on the CSC population of NB and GBM cells. In addition, it is expected that students will run their own experiments under the supervision of lab members.
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
Yes, literature review and power point presentations.
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- The student will prepare a poster
- The student will prepare a presentation
- The student will prepare a scientific report
- The student will prepare an abstract
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
Basic Cell Culturing Techniques plus Time and Work commitment
Are there any legal limitations in the student’s involvement
Type of students accepted
This project accepts:
- Medical students
- Graduated students (less than 6 months)
- Pre-Medical students from the American-British system
- Students in biomedical fields
- Bahmad; H. F.; Mouhieddine; T. H.; Chalhoub; R. M.; Assi; S.; Araji; T.; Chamaa; F.; ... & Abou-Kheir; W. (2018). The Akt/mTOR pathway in cancer stem/progenitor cells is a potential therapeutic target for glioblastoma and neuroblastoma. Oncotarget; 9(71); 33549.
- Mouhieddine; T. H.; Nokkari; A.; Itani; M. M.; Chamaa; F.; Bahmad; H.; Monzer; A.; ... & Abou-Kheir; W. (2015). Metformin and ara-a effectively suppress brain cancer by targeting cancer stem/progenitor cells. Frontiers in neuroscience; 9; 442.