2.7.2. Sample Collection:
At the 21st day of the study, blood samples were
collected from the orbital sinus (retro-orbital plexus), under light
ether anesthesia, by a clean sterile capillary tube that was inserted at
the inner canthus of the eye. The blood was left to be clotted for 20
minutes then centrifuged at 4000 rpm for 15 min. Serum samples were
separated, collected and stored at (-20ºC) for determination of liver
enzymes, urea, creatinine, and vascular endothelial growth factor
(VEGF). RNA was also isolated from stored serum to assess the expression
of miRNA 122. After cervical dislocation, tumor discs were separated,
weighed and fixed in 10% neutral buffered formalin for
immunohistochemical investigations.
- Methods of analysis:
- Determination of liver and kidney functions:
Serum activity of aspartate transaminase (AST) and alanine transaminase
(ALT) enzymes were measured by enzymatic colorimetric method
(Biodiagnostic, Egypt). Serum urea and creatinine levels were measured
calorimetrically (Diamond, Egypt). The procedures were done according to
the manufacturer’s instructions.
Determination of vascular endothelial growth factor (VEGF):
Serum VEGF levels were determined using Quantikine® ELISA kit purchased
from R&D systems (USA). The procedures were done according to the
manufacturer’s protocol, and the concentration was expressed as pg/g
tissue.
Determination of miRNA-122 expression by quantitative real
time polymerase chain reaction (qPCR):
Total RNA, including small RNA, was isolated from serum samples using
Qiagen miRNeasy Mini kit (Cat. No. 217004) (Qiagen, Germany) following
the protocol supplied by the manufacturer. RNA concentration was
determined by using NanoDrop spectrophotometer (Thermo Fisher Scientific
Inc., USA).). MicroRNA-122 expression was quantified by qRT-PCR. RNA was
converted to cDNA by TaqMan® microRNA reverse transcriptase kit (Cat.
No. 4366596) and Taqman assays hsa-miRNA-122 (5×) and RNU6B (5×) (Cat.
No. 4427975) (Applied Biosystems, USA). MiRNA-122 expression was
assessed against RNU6B as an endogenous control where
ΔCT and fold change were calculated. Assays used for
amplification were has-miR- 122 (20×) and RNU6B (20×) (Cat. No. 4427975)
(Applied Biosystems, USA). The PCR reactions were performed in 20 μL
total volume, including 10 μL TaqMan® Universal Master Mix (Cat. No.
4440043) (Applied Biosystems, USA), 1 μL from each assay, 4 μL
complementary DNA (cDNA), and 5 μL RNase- free water. PCR conditions
consisted of 1 min at 95 °C, followed by 40 cycles of 95 °C for 15 s and
60 °C for 1 min and a final extension at 72 °C for 5 min. Real-time PCR
was conducted using StepOnePlus Real-Time PCR thermal cycler (Applied
Biosystems, USA).
Immunohistochemistry
Tumor discs for immunohistochemical examination were fixed in 10%
neutral buffered formalin overnight and then embedded in paraffin. When
analyzed, all paraffin-embedded tissues were sectioned at 4-μm,
deparaffinized in xylene, and hydrated through graded ethyl alcohol
series in decreasing concentrations (100%, 90%, 80% and 70%).
Antigen retrieval was performed according to Tris/EDTA buffer (pH=9)
antigen retrieval protocol. Tumor discs were stained applying the
EnVision™ FLEX HRP labeled, High pH, method according to the
manufacturer’s staining protocol (Dako). Endogenous peroxidase activity
was quenched by incubating the specimen for 10 min with phosphate buffer
containing 3% hydrogen peroxide. Sections were incubated with
corresponding primary antibodies at 4°C overnight. After conjugation
with dextran coupled with peroxidase molecules and goat secondary
antibody molecules against rabbit immunoglobulins, sections were
incubated in DAB substrate chromogen for 2 minutes. Finally, Mayer’s
hematoxylin was used for counter staining.
Evaluation of antibodies expression
ImageJ was used for the semi-quantitative analysis of the
immunohistochemical reactions. The images were captured by an optical
microscope with a 40X objectives (Optika B-352A, Italia) coupled to a
camera (HDCE30C) using its software and quantified using the ImageJ
MacBiophotonics (National Institutes of Health, USA) software package
developed by McMaster University (Ontario, Canada). The expression of
VEGFR2 & CD34 were assessed and percentage of stained area was measured
Statistical analysis:
All data were presented as mean ± standard deviation (SD). Differences
among groups was assessed using unpaired Student’s t test and one-way
ANOVA. P value less than 0.05 was considered to be statistically
significant. Calculations were performed with the SPSS 21.0 statistical
software package (IBM, USA).
- RESULTS
- Effect on tumor weight:
Tumor discs were removed and weighed on the 21st day of treatment. All
treatment groups showed a significant decrease in the tumor weight
compared to the EAC control group (p< 0.01) except for the
group treated with benzoimidazoquinazoline (5 mg/kg) where the average
tumor weight was significantly higher than that observed in the standard
cisplatin group ((p< 0.01) (Fig. 2 ).
Effect on liver and kidney functions:
ALT and AST levels were increased in the EAC control mice and in all
treatment, groups compared to the normal group (Table 1 ).
However, the observed increase cannot be considered as an indication of
liver toxicity. Creatinine and urea levels were also increased in the
control mice compared to the normal group. Expectedly, creatinine and
urea levels increased significantly in the cisplatin treated group
compared to the control group (p<0.01). Treatment with the
investigated compounds, benzoimidazoquinazoline (C1) and
benzimidazotriazine (C2), in both doses (5 mg/kg and 10 mg/kg) showed
significantly lower levels of creatinine and urea compared to the
cisplatin treated group (p< 0.05).
Effect on serum levels of VEGF:
VEGF serum level was evaluated in the current study as a marker for
angiogenesis. VEGF levels were significantly higher in the EAC control
group versus the normal mice (p<0.001). The serum levels of
VEGF decreased significantly on treatment with cisplatin, the higher
dose of benzoimidazoquinazoline, and both doses of benzimidazotriazine
(p<0.05), while the lower dose of benzoimidazoquinazoline had
no effect on VEGF levels (Fig. 3) .