1. Effects of metformin and thymoquinone on survival of leukemic cells
Mentor: Assist. Prof. Mirza Suljagić
Candidate: Una Glamočlija, mr.ph.

2. ICGEB grant
Three year project ( ) which receieved grant from International Centre for Genetic Engineering and Biotechnology, Italy.

3. Introduction
Hematological malignancies: generation of resistance to current treatment options
Combined therapy- molecules with low toxicity and which can target pro-survival pathways in cancer cells

4. Chemotherapy sensitizers
Chemicals which are used to improve effectiveness of drugs in treating tumors
They may reduce the concentration of therapeutic agents and eventually prevent cancer cells to acquire resistance

5. Introduction
Metformin and thymoquinone are potential chemotherapy sensitizers with:
Low toxicity
Pro-apoptotic and anti-proliferative effects in various tumor cells

6. Metformin the most prescribed anti-diabetic drug in the world,
has proven safety profile,
has been used for over 40 years,
numerous ongoing clinical trials in cancer patients
In 2005, an epidemiologic study of type 2 diabetes patients demonstrated that treatment with the antidiabetic drug metformin was associated with a significant decrease in cancer incidence

7. Metformin Two main mechanisms of action:
Insulin dependent- senzitizes cells to insulin
Insulin independent- activation of AMPK (AMP dependent kinase)

9. Metformin
effects of metformin on cancer cell metabolism are involved in strong interference with the survival of leukemia cells
Rosilio C, Lounnas N, Nebout M. et al. The metabolic perturbators metformin, phenformin and AICAR interfere with the growth and survival of murine PTEN-deficient T cell lymphomas and human T-ALL/T-LL cancer cells. Cancer Lett Aug 9;336(1):

10. Metformin
human leukemic cells with increased basal Akt phosphorylation were shown to be resistant to metformin-induced apoptosis
metformin’s anticancer effects can be enhanced by combination with Akt and NF-κB inhibitors
Scotland S, Saland E, Skuli N, et al. Mitochondrial energetic and AKT status mediate metabolic effects and apoptosis of metformin in human leukemic cells. Leukemia Nov;27(11):2129

11. Thymoquinone
phytochemical isolated from oil of plant Nigella sativa L.,
has reliable safety profile
inhibits NF-κB pathway through direct interaction with the p65 subunit,
inhibits Akt phosphorylation

12. Thymoquinone
increases antitumor effects of gemcitabine, oxaliplatin, doxorubicine, 5-fluorouracil , cisplatin and ifosfamide
shows nephroprotective effects when combined with ifosfamide, doxorubicine, and cisplatin.

14. Specific aims
To evaluate the proliferation, viability and apoptosis of leukemic cell lines treated with TQ and metformin separately or in combination
To investigate molecular mechanisms of metformin and TQ effects on pathways controlling survival and proliferation
To determine pro-apoptotic effects in cell lines not responding to currently used therapies

15. Experimental design and methods
In order to evaluate the proliferation, viability and apoptosis of leukemic cell lines treated with TQ and metformin separately or in combination
Effects of metformin and TQ on cellular metabolism and determination of IC50 value: WST-1 assay, the combination index (CI) will be calculated
Cell viability rate will be determined by cell counting using the trypan blue exclusion method
Effects on proliferation will be evaluated by BrdU colorimetric assay.
Proliferation will be also assessed by colony formation assay
Effects on apoptosis will be determined by flow cytometry analysis, after double staining with Annexin-V and propidium iodide (PI)

16. WST-1 assay
The stable tetrazolium salt WST-1 is cleaved to a soluble formazan by a complex cellular mechanism that occurs primarily at the cell surface.
largely dependent on the glycolytic production of NAD(P)H in viable cells.
Therefore, the amount of formazan dye formed directly correlates to the number of metabolically active cells in the culture.
Molecular mechanisms of metformin and TQ effects on pathways controlling survival and proliferation

17. WST-1 assay
Molecular mechanisms of metformin and TQ effects on pathways controlling survival and proliferation

19. Trypan blue exclusion method
Molecular mechanisms of metformin and TQ effects on pathways controlling survival and proliferation

20. BrdU colorimetric assay
Molecular mechanisms of metformin and TQ effects on pathways controlling survival and proliferation

21. Colony formation assay
ability of a single cell to grow into a colony (at least 50 cells)
Only a fraction of seeded cells retains the capacity to produce colonies
Colonies are fixed, stained with crystal violet and counted
Molecular mechanisms of metformin and TQ effects on pathways controlling survival and proliferation

22. Colony formation assay
Molecular mechanisms of metformin and TQ effects on pathways controlling survival and proliferation

23. Flow cytometry
Molecular mechanisms of metformin and TQ effects on pathways controlling survival and proliferation

24. Flow cytometry
Molecular mechanisms of metformin and TQ effects on pathways controlling survival and proliferation

25. Flow cytometry
Molecular mechanisms of metformin and TQ effects on pathways controlling survival and proliferation

26. Experimental design and methods
To investigate molecular mechanisms of metformin and TQ effects on pathways controlling survival and proliferation of leukemic cell lines
Western blotting will be used for identification of specific proteins
Molecular mechanisms of metformin and TQ effects on pathways controlling survival and proliferation

27. Western blotting detects specific proteins in a sample
gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide
The proteins are then transferred to a membrane where they are stained with antibodies specific to the target protein
Molecular mechanisms of metformin and TQ effects on pathways controlling survival and proliferation

28. Western blotting
Molecular mechanisms of metformin and TQ effects on pathways controlling survival and proliferation

29. Combinatorial therapy Targeting signaling patways
Thymoquinone and metformin low toxicity towards healthy cells
Combinatorial therapy
Synergistic effects of thymoquinone and metformin
Targeting signaling patways
Overcoming resistance to current treatment options
Succesful therapy