Ehrlich ascites carcinoma cells (EAC) are a common tumour that is an undifferentiated carcinoma with high transplantable capability, no regression, rapid proliferation, shorter life span, 100% malignancy, and no tumor-specific transplantation antigen (TSTA)1.
Oxidative stress participate in the progression stage of cancer and tumors process2. Because of this, ROS can cause damage or breach to the double chain macromollecules, alterations in Guanin and Thymine bases, and the synthesis of malondialdehyde mutations3. Antioxidants protect the body from the harmful effect of free radicals and ROS4.
Furthermore, the Ehrlich carcinoma model is a breast cancer that is spread from one mouse to another via intraperitoneal passages5,6. In many chemotherapeutic research, Ehrlich models are utilized to create solid tumors by injecting tumour cells subcutaneously or ascetic tumors by injecting tumour cells intraperitoneally7,8.
Despite considerable advancements in cancer treatment, the global prevalence of cancer continues to rise9,10. Cancer prevention using naturally occurring food components, on the other hand, has been deemed a feasible way to reduce the ever-increasing cancer incidence11. Some antioxidants produced in the body (endogens) and others derive from diet (exogenous) are included in these systems12. Vinca alkaloids, taxans, camptothecins and epipodophyllotoxins are some plants which have anticancer properties13. Balanites aegyptiaca contains high level of soluble tannins14, polyphenols15,16 and flavonoids17, which are reactive and excellent antioxidant agents18,19. These reports prompted us to investigate other physiological and pharmacological functions of Balanites aegyptiaca. As an extension of our research on the biological value of neutral products20-25, this study was designed to evaluate anti-angiogenic and renal protective activity of Balanites aegyptiaca seeds extract in Ehrlich ascites carcinoma-bearing mice.
MATERIALS AND METHODS
Materials: EAC cells were obtained from Cairo's Cancer Institute. In Swiss albino mice, the cells were maintained in vivo via intraperitoneal transplantation (2×106 cells per mice) into all groups except the first group26.
Plant material: Balanites aegyptiaca seeds were purchased from the local market in Cairo. Authentication of the plant was carried out by prof. Heba A. Elgizawy, Faculty of Pharmacy, October 6 University.
Preparations of the water extract: Only 0.5 kg of air-dried crushed Balanites aegyptiaca seeds were extracted using hot water (in ratio 1:10). The extraction was carried out at 50̊C for 2 h with stirring at regular intervals. It was then filtered and evaporated to dryness under reduced pressure to yield viscous mass. The extract was kept in airtight containers in a deep freeze maintained at 4°C until the time of further use.
Determination of BASE cytotoxicity on renal carcinoma (RCC-949) cell line: To form a full monolayer sheet, the 96 well tissue culture plate was inoculated with 1×105 cells mL‾1 (100 uL well‾1) and incubated at 37°C for 24 h. The growth medium was decanted from 96 well microtiter plates after forming a confluent sheet of cells and washed two times with washed medium by the cell monolayer. The sample was double diluted in the medium of RPMI with 2% serum (maintenance medium). Only 0.1 mL of each dilution was tested in each well, with 3 wells serving as controls and maintenance. The plate was incubated and checked at 37°C.
Physical symptoms of toxicity, such as partial or total monolayer loss, rounding, shrinkage, or cell granulation, were examined in the cells. The MTT solution (5 mg mL‾1 in PBS) was prepared (BIO BASIC CANADA INC). Each well received 20 uL of MTT solution. MTT was placed on a shaking table for 5 minutes at 150 rpm to thoroughly mix the it into the media. To allow the MTT to be metabolized, incubate for 1-5 h at 37°C and 5% CO2. Excluding the media from the equation. (If possible, dry plate with paper towels to remove residue). In 200 uL DMSO, resuspend formazan (MTT metabolic product). Formazan was placed on a shaking table at 150 rpm for 5 min to thoroughly mix it into the solvent. At 560 nm, read the optical density and deduct the history at 620 nm. The optical density should be proportional to the number of cells.
Mice: The Animal Care and Use Committee of October 6 University developed guidelines for this experiment, which were followed. Adult mice weighing about 25±2 g were purchased from Cairo University, Faculty of Veterinary Medicine. They were housed in air-conditioned cages at a temperature of 22°C, a relative humidity of 60%, and a light period of 8:00 to 20:00. Feed and water was provided ad libitum during the acclimatization period.
Experimental design: The animals were divided into 5 groups consisting of 6 animals, two controls groups and three treatment groups. Description of treatment group is presented in Table 1.
Six mice from each group were dissected on the 31st day, 24 h after the injection, all mice were sacrificed at the end of the experiment. Blood was collected, centrifuged, and plasma urea28, creatinine29, uric acid30 were determined. The tumour mass was removed from each mice in groups (II-V) to estimate its weight. Also, ascites fluid was extracted from the peritoneal cavity and measured using a graduated centrifuge tube27,31.
Renal TBARS32, Nitric Oxide (NOx)33, tumour necrosis factor (TNF-α)34 were determined using enzyme-linked immunoassay (ELISA) (14780 Memorial Drive Suite 216, Houston, Texas). Also, renal GSH35, superoxide dismutase (SOD)36, glutathione peroxidase (GPx)37 and (CAT)38 activities were measured by commercial RANSEL kits (Randox Laboratories, Crumlin, Northern Ireland, UK).
Quantitative real-time PCR: The total RNA was extracted from the renal of the mice, and portions (10-15 μg) of the isolated RNA were subjected to quantitative PCR analysis in real time, using Sepasol-RNA1Super according to instructions of the manufacturer. The two-step RT-PCR gene expression was measured. The level of VEGF-C was quantified with the previously described quantitative real-time PCR. The tests were conducted in 50 mL single-plex reaction mixture. Conditions of reaction were a pre-incubation at 50°C in 2 min, followed by 10 min by 40 cycles at 95°C in 15 s and at 60°C in 1 min, respectively.
The primer sequences were VEGF-C: F 5-AACGTGTCC AAGAAATCAGCC-3, R: 5-AGTCCTCTCCCGCAGTAATCC-3. The internal control used GAPDH-F: 5-CTCAACTACATGGTCTACA TGTTCCA-3 and -R:5 -CCATTCTCGGCCTTGA-CTGT-3’.
Histological assessment: The renal tissue was sliced and parts were fixed in histologic solution of 10% formaldehyde buffered. Only 5 μm thick renal tissue was stained with hematoxylin eosin (HE) and examined by light microscopic according to the method of Bancroft and Steven39.
Ultrasound protocol: Mice were examined at Smart Scan Radiology Center-Cairo, Egypt; all experimental Ethics procedures were achieved. Once placed on the handling platform, each mouse was fixed in a supine recumbent position, the abdominal area was shaved to reduce imaging artifact. A conducting gel was applied to the area and the procedure was done using a multi-frequencies linear transducer (7-12 MHZ).
- The gel helps the transducer makes close contact with the body eliminating air pockets between the transducer and the skin that can block the sound waves to pass into the body. The probe was used on the abdomen and moved back and front over the abdomen until the interested images were captured
- Doppler study was also performed for diagnosis using the same transducer to get more details about the lesions vascularization
- Renal Images were stored on the ultrasound machine including images of all groups
Statistical analysis: Data were analyzed using one-way analysis of variance (ANOVA), followed by the least significant difference (LSD) test using the SPSS 18.0 Statistical Software Program (SPSS, Inc., IBM, Chicago, Illinois, USA). Differences of p<0.05 were considered statistically significant.
Figure 1 shows that the IC50 of BASE against renal carcinoma (RCC-949) cell line was equal to 62.18 µg mL‾1.
Oral administration of BASE (100 and 200 mg kg‾1) and intraperitoneal injection of 5-fluorouracil showed a significant decrease (p< 0.05) in tumour volume and weight compared to the mice bearing EAC (Table 2). The decrease was observed in tumour volume and weight in group of mice fed diet supplemented with BASE compared to 5-fluorouracil.
Table 3, 4 and 5 shows a significant increase in plasma urea, creatinine and uric acid, TNF-α, NOx, and TBARs, and a decrease in CAT, SOD, GPx, and GSH in renal tissue of mice with EAC compared to the standard nonbearing EAC group (p<0.05). Oral administration of BASE (100 and 200 mg kg‾1) and intraperitoneal injection of 5-fluorouracil significantly decreased urea, creatinine and uric acid and increased CAT, SOD and GPx, GSH levels (p<0.05) as compared to mice bearing-EAC.