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.
  1. Methods of analysis:
  2. 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).
  1. RESULTS
  2. 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) .