Supervisors Amal Mohamed El gayar Nehal Mohsen Elsherbiny

MANSOURA UNIVERSITY
FACULTY OF
PHARMACY
Department of Biochemistry
Presented BY
Hala Said Mohammed Mahmoud Bayomi
B. Pharm. Sci., (2010)
Supervisors
Prof
Amal Mohamed El gayar
Professor of Biochemistry
Faculty of Pharmacy-Mansoura University
Dr.
Nehal Mohsen Elsherbiny
Lecturer of Biochemistry
Faculty of Pharmacy - Mansoura University
Thesis submitted as a Partial Fulfillment for
Master Degree in Pharmaceutical Sciences (Biochemistry)
2015
Summary and conclusion
Summary and conclusion
The present study aimed to evaluate the anti-tumor activity of SB-431542, as well as
their impact on cisplatin (CP)-evoked chemo preventive effects, in EAC model in mice.
In this study, we evaluated TGF-β and TGF-βR1 levels in response to treatment with
CP or SB-435142 or their combinations. In addition, correlations were made between TGFβR1 and the dissociation of TGF precursor molecule in response to treatment with SB-435142
mainly.
CP is a commonly used cytotoxic agent in the treatment of numerous solid tumors in
various clinical settings.However, the clinical usefulness of CP is limited by the development
of serious nephrotoxicity, a side effect that is also reproducible in various animal models. The
ability of SB-435142 to protect rats against CP-induced nephrotoxicity was also evaluated.
A:Evaluation of the anti-tumor profiles of SB-431542 in Ehrlich ascites carcinoma
(EAC) model in mice:
42 Swiss albino mice were divided into 7 groups,6 mice each .All mices (except
those of control group) were injected i.pwith 1 mL of EAC cells containing 1x106 cells for
tumor induction. Cisplatin and SB-431542 were injected i.p along 3 weeks(the time of whole
experiment).
Mice were divided into the following groups:
1.Normal control group:received 0.2 ml of phosphate buffer saline (PBS, 10 mM, pH 7.4)
i.pserve as control group.
2.EAC-bearing mice non-treated control group: animals were left without any treatment
until sacrification.
3.EAC-bearing mice cisplatin (5mg/kg)treated group: animals were injected cis solution
i.p. at a dose of 5 mg /kg body weight ,single dose on the first day.
4. EAC-bearing mice SB-431542 (1 mg /kg)treated group: animals were injected i.p. with
SB solution (1 mg /kg body weight), twice a week.
5. EAC-bearing mice SB-431542 (5 mg /kg)treated group: animals were injected i.p. with
SB solution (5 mg /kg body weight), twice a week.
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Study of TGF-β1 pathway in Ehrlich ascites carcinoma model
6. EAC-bearing mice cis(5 mg/kg) and SB (1 mg /kg)treated group:
animals were injected i.p. with cis solution( 5 mg/kg body weight) ,single dose on the first
day and also were injected i.p. with SB solution (1 mg /kg body weight), twice a week.
7. EAC-bearing mice cis(5 mg/kg) and SB (5 mg /kg)treated group:
animals were injected i.p. with cis solution( 5 mg/kg body weight) ,single dose on the first
day and also were injected i.p. with SB solution (5 mg /kg body weight), twice a week.
After the end of the specified period of the experiment (3weeks) , animals were
anesthetized, ascitic fluids were withdrawn from tumor-bearing mice by needle aspiration
from the peritoneal cavity under aseptic conditions where ascetic tumor volume (ml) is
measured.
Fluid was washed three times with normal saline by centrifugation at 1000 r.p.m at
room temperature. EAC cells were then tested for viability using trypan blue. The cells were
examined microscopically using a hemocytometer. Following the viability test, cells were
counted under the microscope. Blood samples from different groups of mice were also
collected and complete blood count is estimated:
a. Red blood cells
b. White blood cells
c. Hemoglobin
d. Hematocrit.
Sera from different groups is separated and the following parameters are estimated:
1. Liver functions measured by two parameters :
a. Albumin level
b. Serum glutamic-pyruvic transaminase (sGPT )level
5. Kidney function measured by creatinine
Finally, the animals were sacrificed and kidneys and livers were removed, then were
used to make a 10% (w/v) homogenate in phosphate buffer saline (PBS, pH 7.4). The
homogenate was centrifuged at 3000 rpm for 10 min at 4oC and the supernatant was removed
and stored on ice or stored at –80oC for further assay and the following parameters are
evaluated:
1. Oxidative stress biomarkers:
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Summary and conclusion
a. Superoxide dismutase (SOD).
b. Malondialdehyde (MDA).
2. Expression level of biomarkers involved in TGF-β1 signaling including:
a. Transforming growth factor beta1 receptor (TGF-βR1).
b. Transforming growth factor beta1(TGF-β1).
c.Latency Associated Peptide (LAP)
3. Expression level ofsoluble cluster of differentiation (sCD93) as an inflammatory marker.
4. Expression level ofCaspase-3 as a biomarker for apoptosis.
The following results were obtained:
1. The increase in mean survival time and percent increase in life span confirmed the antitumor activity of the drug. Tumor size and total viable cell count, measured in all groups,
showed a significant reduction in groups treated with SB-431542. The degree of inhibition
increased when the doses of this compound increased,indicating a pronounced dosedependent anti-tumor activity.Furthermore, the combination of SB-431542 with CP enhanced
the chemoprevention obtained beyond their individual effects.
2. Treatment with SB-431542 restored haemoglobin content and maintained normal values of
RBC and hematocrit indicating its haematopoietic protecting activity.
3. SB-431542 improved liver functions as it showed a dose dependent increase in albumin
level and also caused significant decrease in ALT level as compared to EAC group.
4. SB-431542 improved kidney functions as it caused significant decrease in creatinine level.
5.The EAC-control group recorded a marked increase in oxidative stress, where it showed
significant increase inhepatic and renal MDA and significant decrease in SOD level.
Treatment with SB-431542 reduced hepatic and renal MDA level and increased SOD activity,
as compared to the EAC-control group..
6. SB-431542 decreased TGF-β1 and TGF-βR1 levels significantly as compared to EAC
group and that caused feedback mechanism between the receptor and the dissociation of TGF
precursor molecule and that was demonstrated by the decrease in the measured level of LAP.
7. SB-435142 showed significant decrease in both hepatic and renal sCD93 level compared to
EAC group and that indicate the anti inflammatory effect of the drug while the lower dose
showed non significant decrease.
8. SB-431542 partially work by apoptosis in contrast tocisplatin which exert its anti tumor
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Study of TGF-β1 pathway in Ehrlich ascites carcinoma model
action through induction of apoptosis.
B:Evaluation of the anti-tumor profiles of SB-431542 in Ehrlich ascites carcinoma
(EAC) tissue culture:
EAC cells were established in the Netherlands Cancer Institute. The Ehrlich-tumor
cells were maintained in female Swiss albino mice by serial IP passage at 7–10 day intervals.
The mice were killed by cervical dislocation 7 days after inoculation, and the ascites fluid
collected. EAC cells were grown in 96-well flat bottom plate with growth medium RPMI
(Roswell Park Memorial Institute) 1640 and 10% FCS (Fetal calf serum) for 24 hours at 37°C
in a 5% CO2 incubator to allow the cells to grow. Cells were transferred to serum free
medium and exposed to different concentrations of cisplatin and SB-431542 (5, 10 and 20
µM) and placed in the humidified 5% CO2 incubator for 48 h. Cells incubated in culture
medium alone served as a control(untreated wells).
The following parameters were measured:
1.Cell viability using MTT assay.
2. Cytotoxicity with LDH.
3. Oxidative stress biomarkers:
a. Myeloperovidase (MPO).
b.Malondialdehyde (MDA).
4.Expression level of important biomarkers involved in TGF-β1 signaling including:
a. Transforming growth factor beta1 receptor (TGF-βR1).
b. Transforming growth factor beta1(TGF-β1).
5. Expression level ofCaspase-3 as a biomarker for apoptosis.
The following results were obtained:
1. Treatment with SB-431542 decreased EAC cellssurvival by more than 50 % by reaching 20
µM. Combination between cisplatin and SB-431542 decreased EAC cellssurvival by about 80
%.
2. Treatment with SB-431542 increased cell cytotoxicity by about 1.5 fold. Cisplatin and SB431542 combination increased cell cytotoxicity by about 2.5 fold.
3. Treatment with SB-431542 was found to decrease MDA level by about 40 % at 20 µM.
Cisplatin in combination with SB-431542 decreased MDA level by about 50 %.In addition,
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Summary and conclusion
about 2.2-fold increase in MPO level was observed after treatment with SB-431542.
However, after combination between the two drugs, the level of MPO increased by about 1.9fold
4. Treatment with SB-431542 decreased TGF-βR1 level by about 50 % after reaching the
maximum concentration (20 µM). Treatment with a combination of cisplatin and SB-431542
decreased TGF-βR1 level about 40 %.
5. SB-431542 decreased TGF-β1 level by about 30 % by reaching its maximum
concentration. Using combination of cisplatin and SB-431542 decreased TGF-β1 level by
about 20 %.
6. SB-431542 noticed to have little effect on caspase-3 activity. By combining CP and SB431542 drugs, caspase-3 activity increased by about 2.5 fold by reaching maximum mixture
concentration.
C: Evaluation of the possible protective effects of the SB-435142 against CP-induced
nephrotoxicity in rats:
Nephrotoxicity was induced in all groups except control and treated control groups by
injection of CP (10 mg/kg, IP) as a single dose in the 4th day of the experiment. Serum
samples were collected after 7 and/or 10 days from the start of the experiment to assay the
levels of urea and creatinine.
50 Male rats were divided into the following groups:
Group (1): received 0.2 ml of phosphate buffer saline (PBS, 10 mM, pH 7.4) i.p. in the 1st,
4th and 7th day of the treatment to serve as control group.
Group (2): received SB-431542 (1 mg/kg, i.p.), in the 1st, 4th and 7th day of the treatment to
serve asthe treated control group.
Group (3): received cisplatin(10 mg/kg, i.p.., on the 4th day of the experiment) 1 h prior to
the injection of 0.2 ml of PBS, i.p. The animals received 0.2 ml of PBS, i.p., in the 1st, 4th
and 7th day of the treatment.
Group (4): received cisplatin (10 mg/kg, i.p., on the 4th day of the experiment) 1 h prior to
the injection of SB-431542 (1 mg/kg, i.p.). The animals received SB-431542, in the 1st, 4th
and 7th day ofthe treatment.
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Study of TGF-β1 pathway in Ehrlich ascites carcinoma model
Group (5): received cisplatin (10 mg/kg, i.p., on the 4th day of the experiment) 1 h prior to
the injection of SB-431542 (3 mg/kg, i.p.). The animals received SB-431542 in the 1st, 4th
and 7th day of treatment.
After the end of the specified period of the experiment (7 or 10 days), animals were
anesthetized, blood samples were collected and sera were separated in which the following
parameters were measured:
Kidney function measured by:
a. creatinine
b.urea (discussed later).
Finally, the animals were sacrificed and kidneys were removed. The right kidney was
fixed in 10% buffered formalin for subsequent morphological analysis and the left one was
used to make a 10% (w/v) homogenate in phosphate buffer saline (PBS, pH 7.4). The
homogenate was centrifuged at 3000 rpm for 10 min at 4oC and the supernatant was removed
and stored on ice or stored at –80oC for further assay in which the following parameters were
measured:
1. Oxidative stress biomarkers:
a. Superoxide dismutase (SOD).
b.Malondialdehyde (MDA).
2. Expression level of important biomarkers involved in TGF-β1 signaling including:
a. Transforming growth factor beta1 receptor (TGF-βR1).
b. Transforming growth factor beta1(TGF-β1).
c. Latency Associated Peptide (LAP)
3. Expression level of sCD93 as an inflammatory marker.
4. Expression level ofCaspase-3 as a biomarker for apoptosis.
The following results were obtained:
1.Cisplatin reduced survival rate up to 60% and 20% after 7 and 10 days, respectively.
Treatment with 1 mg/kg SB-431542 increased survival rate to 40% after 10 days with no
remarkable change after 7 days. Treatment with 3 mg/kg SB-431542 increased survival rate to
90% after 7 days and 70% after 10 days treatment.
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Summary and conclusion
2.Cisplatin caused 3.6-fold increase in serum creatinine level after 7 days treatment and 8-fold
increase after 10 days treatment as compared to control group. Treatment with SB-431542 for
7 and 10 days showed dose dependent reduction in serum creatinine level.
3.Cisplatin increased serum urea level 3.8-fold after 7 days treatment and 13.7-fold after 10
days treatment as compared to control group.Treatment with SB-431542 showed dose
dependent reduction in serum urea level in cisplatin group.
4. Cisplatin treated rats for 7 days and 10 days showed 2.2- and 2.95-fold increase in renal
MDA level as compared to control group. SB-431542 treatment showed dose dependent
decrease in renal MDA level as compared to cisplatin group.
5. Cisplatin resulted in 23% and 44% reduction in renal SOD activity after 7 and 10 days,
respectively. SB-431542 (1 and 3 mg/kg) treated rats showed significant increase in renal
SOD activity as compared to cisplatin group.
6. Cisplatin increased renal TGF-βR1 1.7- and 2.3-fold of control group after 7 and 10
daystreatment, respectively. Treatment with SB-431542 for 7 and 10 days showed dose
dependentreduction in renal TGF-βR1 as compared to control group.
7. Cisplatin increased renal TGF-β1 level 1.6- and 2.1-fold after 7 and 10 days,
respectively.Treatment with SB-431542 (1 and 3 mg/kg) cause marked decrease in renal
TGF-β level after 7and 10 days in cisplatin.
8. Cisplatin resulted in 1.7- and 2.6-fold increase in renal sCD93 level after 7 and 10
days,respectively. Treatment with SB-431542 (1 and 3 mg/kg) caused significant reduction of
renalsCD93 as compared to cisplatin.
9. Cisplatin increased renal caspase-3 level 1.8- and 2-fold after 7 and 10 days, respectively.
Treatment with SB-431542 caused dose dependent significant reduction of caspase-3 level
after 7 and 10 days in cisplatingroup.
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Study of TGF-β1 pathway in Ehrlich ascites carcinoma model
Conclusion:
This is the first study to declare the cytotoxic effect of SB-431542 on EAC
models.SB-431542 was previously studied as a potent antitumor agent for different human
cancers.In this study we could conclude that SB-431542 induced cytotoxic effects on Ehrlich
ascites carcinoma cells by several mechanisms including antioxidant and TGF-β1 inhibition
mechanisms.
In addition, we also could conclude that blocking TGF-βR1 by SB-431542 results in a
dose dependent amelioration of the impairment of renal tissues function and structure in rats
treated with cisplatin via multiple mechanisms including: (1) reducing cisplatin-induced
oxidative stress, as indicated by reducing renal MDA levels and restoring the activity of renal
SOD; (2) blocking cisplatin-induced elevation of renal inflammatory cytokines such as
sCD93; (3) reducing cisplatin-induced elevation of renal fibrosis markers as TGF-1β; and (4)
inhibiting cisplatin-induced activation of renalcaspase-3.
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