Effects of metformin and thymoquinone
on survival of leukemic cells
Mentor: Assist. Prof. Mirza Suljagić
Candidate: Una Glamočlija, mr.ph.
ICGEB grant
• Three year project (20162019) which receieved
grant from International
Centre for Genetic
Engineering and
Biotechnology, Italy.
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
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
Introduction
• Metformin and thymoquinone are
potential chemotherapy sensitizers with:
– Low toxicity
– Pro-apoptotic and anti-proliferative effects
in various tumor cells
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
Metformin
• Two main mechanisms of action:
– Insulin dependent- senzitizes cells to insulin
– Insulin independent- activation of AMPK
(AMP dependent kinase)
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. 2013 Aug 9;336(1):114-26.
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. 2013 Nov;27(11):2129
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
Thymoquinone
• increases antitumor effects of
gemcitabine, oxaliplatin, doxorubicine, 5fluorouracil , cisplatin and ifosfamide
• shows nephroprotective effects when
combined with ifosfamide, doxorubicine,
and cisplatin.
Cell membrane
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
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)
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.
WST-1 assay
Trypan blue exclusion method
BrdU colorimetric assay
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
Colony formation assay
Flow cytometry
Flow cytometry
Flow cytometry
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
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
Western blotting
• Thymoquinone
and metformin
low toxicity
towards
healthy cells
Targeting
signaling patways
• Synergistic
effects of
thymoquinone
and metformin
• Overcoming
resistance to
current
treatment
options
Combinatorial therapy
Succesful therapy