Transcript ahmed-m-kabel-taif-university-ksa

Targeting Sphingosine Kinase 1 and
Apoptosis by Metformin to Decrease Tumor
Resistance to Adriamycin
By
Dr. Ahmed Mohamed Kabel
Pharmacology Department, Faculty of Medicine, Tanta University, Egypt
Pharmacology Department, College of Pharmacy, Taif University, KSA
• Adriamycin (ADR, Doxorubicin) is an
anthracycline antibiotic that is frequently
used as a treatment for many types of
cancer such as leukemia, lymphoma, breast,
ovarian and lung cancer.
• It damages DNA by intercalation into DNA
and inhibition of topoisomerase II resulting
in DNA strand breaks.
• The major limiting factor for the use of ADR in
cancer therapy is the development of resistance.
• The mechanisms of this resistance may include
increased activity of sphingosine kinase-1 (SphK1)
enzyme which inhibits penetration and
intercalation of ADR into DNA and significantly
abolishing the anticancer effect of ADR.
• This makes it essential to combine ADR with
agents that inhibit these mechanisms to decrease
the incidence of resistance of cancer cells to ADR.
• Metformin is an antidiabetic agent that decreases
intestinal absorption of glucose, increases its
anaerobic metabolism and improves insulin
sensitivity.
• Studies on animal models had demonstrated that
metformin prevents tumor development and
inhibits cell proliferation. This effect may be
mediated through its regulatory role on the
hormonal, metabolic and immune functions.
• The main molecular target of metformin is AMPactivated protein kinase (AMPK) signaling that
plays a crucial role in the control of cell division
and proliferation.
• Moreover, metformin had been shown to
improve
endothelial
function,
decrease
inflammatory response, and regulate immune
functions which have a major role in the
pathogenesis of cancer.
• Also, metformin was proven to inhibit SphK1
activity which was one of the main factors
contributing to resistance of cancer cells to
chemotherapy.
So, The aim of this work was to study
the effect of targeting sphingosine kinase 1
and apoptosis by metformin on tumor
resistance
to
adriamycin
using
transplantable tumor model in mice.
a
• In this study, we used a model of solid Ehrlich
carcinoma (SEC), where Ehrlich carcinoma cells
(ECCs) were implanted subcutaneously into the
right thigh of the hind limb of mice. A palpable
solid tumor mass (about 100 mm3) was
developed within 10 days.
Ehrlich Ascites Carcinoma
Used for induction of SEC
SEC
Control
One hundred BALB/C mice were divided into
5 equal groups of twenty mice each as follows:
Group (1): the Control untreated group
Group (2): ECCs were implanted subcutaneously
into the right thigh of the hind limb of mice.
Group (3): Adriamycin was given by intra-tumoral
injection on days 10, 15, 20, 25, 30 and 35 after
implantation of ECCs.
Group (4): Metformin was given to mice orally
starting 10 days after implantation of ECCs and
continued for 32 days.
Group (5): Adriamycin and metformin were given
together starting 10 days after implantation of
ECCs and continued for 32 days by the above
regimens.
• Tumor volume was measured on days 15, 20, 25, 30,
35 and 40 after implantation of ECCs.
• In the 42nd day after implantation of ECCs, the
animals were killed and the tumor was excised and
divided into two portions: one for homogenization
and the other for histopathological examination.
• The tumor tissue was homogenized for
determination of tissue catalase, glutathione
reductase, malondialdehyde, sphingosine kinase 1
activity, caspase 3 activity and tumor necrosis factor
alpha.
1400
Tumor volume (mm3)
1200
1000
a
800
600
a
abc
SEC
SEC+ADR
SEC+Met
SEC+ADR+Met
400
200
0
Day 15 Day 20 Day 25 Day 30 Day 35 Day 40
Effect of different treatments on tumor volume
a Significant compared to SEC group (P < 0.05).
b Significant compared to SEC+ADR group (P < 0.05).
c Significant compared to SEC+Metformin group (P < 0.05).
300
35
250
Tissue MDA umol/gm
Tissue catalase U/mg tissue
30
200
25
20
150
15
100
10
50
5
0
0
Control
SEC
ADR+SEC
Met+SEC
Control
ADR+Met+SEC
SEC
ADR+SEC
Met+SEC ADR+Met+SEC
1000
Tissue glutathione reductase
U/g
900
800
700
600
500
400
300
200
100
0
Control
SEC
ADR+SEC
Met+SEC ADR+Met+SEC
Effect of different treatments on tissue antioxidant parameters
1600
Tissue TNF alpha pg/g
1400
1200
1000
800
600
400
200
0
Control
SEC
ADR+SEC
Met+SEC ADR+Met+SEC
Effect of different treatments on tissue TNF-α
Tissue caspase 3 activity
nmol/mg protein/min
10
9
8
7
6
5
4
3
2
1
0
Control
SEC
ADR+SEC
Met+SEC ADR+Met+SEC
Effect of different treatments on tissue caspase 3 activity
Tissue SphK 1 activity
pmol/min/mg protein
70
60
50
40
30
20
10
0
Control
SEC
ADR+SEC
Met+SEC ADR+Met+SEC
Effect of different treatments on tissue SphK1 activity
Apoptotic index %
7
6
5
4
3
2
1
0
Control
SEC
ADR+SEC
Met+SEC ADR+Met+SEC
Effect of different treatments on the apoptotic index
H&E stained sections of a) SEC showing sheets of small tumor cells
representing cell proliferation surrounding areas of necrosis;
b) ADR group showing focal necrosis with sheets of malignant cells;
c) Metformin showing focal necrosis;
d) ADR/metformin combination group showing extensive necrosis.
A photomicrograph of a) SEC group showing negative staining for p53;
b) ADR group showing positive (+++) p53 expression;
c) Metformin group showing positive (++) p53 expression;
d) ADR/metformin group showing positive (++++) p53 expression.
Conclusions
• The combination of ADR and metformin had a
better effect than each of these drugs alone
against transplantable tumor model in mice.
• This might be due to the combined antioxidant
and anti-inflammatory properties of both drugs
together with their ability to induce apoptosis of
cancer cells.
• Moreover, metformin was able to decrease
SphK1 enzyme activity which potentiates the
effect and decreases resistance of cancer cells to
ADR.
•
So, it is recommended to add metformin to
the anti-cancer regimens containing ADR to
decrease resistance of cancer cells to ADR.