This study with experimental animals was conducted in accordance with the decision No. 14/30 taken on February 12, 2014 from the Erciyes University Local Ethics Committee of the Department of Animal Experiments. Male BALB/c mice weighing 25-30 g and 8-10 weeks were used throughout the study. Eight groups were identified in the study. The was designed to have 10 mice in each group. Unlike these groups, 4 animals were used to create a stock animal group. During the study period, the mice used in the experiment were maintained at a stable temperature of 21ºC in specially set and air-conditioned place with a day cycle of 12 h and a night cycle of 12 h. The created stock mice were arranged at the initial stage in such a way to get enough EAT cells before the formation of experimental groups. Stock mice's EAT cells were used both solid and liquid tumor formation in vivo for in vitro cell culture.
Sterilization and dissolution of the rhamnetin extract
Rhamnetin (Sigma Aldrich) was purchased as a powder. Solution prepared freshly before the experiment, for each experimental group rhamnetin extract was dissolved in methanol to provide the desired concentrations.
Stock mice formation
When creating stock animals, EAT cells were stored at -80 oC. After the cells were thawed at standard laboratory conditions (25 oc), 0.1 ml was injected intraperitoneally (IP) into the stock animals from the joint of the abdomen and left hind leg. An acid tumor in an animal was expected to form within about 1 week. Cells in ascitic fluid from 1x10℃ stock animal were injected intraperitoneally into mice in PBS buffer (0.1 ml) to form a liquid tumor. To form a solid tumor, it was again applied subcutaneously (SC) to the neck areas at pbs(0.1 ml).
Formation of the experimental groups
Experimental Groups
Groups of ascites tumor: s In this group, the subjects were divided into groups of 4. Cancer was not established in the negative control group. Animals were included in the study by giving 0.1ml of physiological saline (PS) instead of acid liquid. Mice in the positive control group were given 0.1-ml acid liquid with 1x10⁶ EAT cells intraperitoneally. Liquid tumor + 100µg/kg the study was started by intraperitoneally administering 0.1-ml acid liquid with 1x10⁶ EAT cells to mice in the rhamnetin group. 24 h after the start of the experiment, the animals were injected intraperitoneally with rhamnetin 100µg/kg/day in 0.1ml intraperitoneally for 10 days. Liquid tumor + 200µg/kg the study was started by intraperitoneally administering 0.1-ml acid liquid with 1x10⁶ EAT cells to mice in the rhamnetin group. 24 h after the start of the experiment, animals were intraperitoneally injected with 0.1ml of 200µg/kg/day rhamnetin intraperitoneally for 10 days. 11th day in the treatment groups, intraperitoneal acid fluid was withdrawn on the day and a cell count was performed. Subjects in all groups, 11th day animals were sedated with ketamine-xylacine (50 mg/kg,15 mg/kg). After this procedure, intra-abdominal organs were removed and tissues were prepared for histological examinations.
Groups of solid tumor: s cancer was not created in the negative control group. Instead of acid liquid, 0.1ml of physiological saline (PS) was administered subcutaneously from the back of the neck to the animals. 0.1 ml intraperitoneally to animals for 15 days 24 h after the start of the experiment saline was continued to be given physiologically. The study was started by giving 0.1ml of acid liquid subcutaneously from the back of the neck region to the mice in the positive control group with 1x10⁶ EAT cells. Solid tumor + 100µg/kg of 0.1ml treatment groups, acid liquid with 1x10⁶ EAT cells was injected subcutaneously from the back of the neck to mice in the rhamnetin group and the study was started. 24 h after the start of the experiment, animals were intraperitoneally injected with 0.1ml of 100µg/kg/day rhamnetin intraperitoneally for 15 days. Solid tumor + 200µg/kg of 0.1ml treatment groups, acid liquid with 1x10⁶ EAT cells was administered subcutaneously from the back of the neck to mice in the rhamnetin group and the study was initiated. 24 h after the start of the experiment, animals were intraperitoneally injected with 0.1ml of 200µg/kg/day rhamnetin intraperitoneally for 15 days. Subjects in all experimental groups, 16th day animals were sedated with ketamine-xylacine (50 mg/kg,15 mg/kg). The tumor tissue formed in the back of the neck region was removed and prepared for histopathological examination. Solid tumor volumes developed in animals over 15 days were measured with an electronic compass (Tumor Volume (mm³) = Width²XLengthX0,52)
Immunohistochemical practices
TUNEL labelling (“Terminal deoxynucleotyl transferase mediated dUTP Nick-end Labeling”) technique, which is the most sensitive and fastest method of apoptosis tunnel staining, was used in tumor tissue sections of the experimental and control groups (each group includes 10 mice). It is used for DNA strand breaks in apoptotic cells in situ detection. Briefly, 5 µm pieces placed on Poly-L-Lysine coated laminates were deparaffinized in xylene. In the next step, it was added to alcohol in decreasing concentrations and incubated in pbs buffer for 5 minutes at standart laboratory temperature. Fragments incubated with proteinase K (15 minutes) were washed with pure water and then treated with hydrogen peroxide (30%) for the endogenous peroxidase activity suppression. In the next steps, immunofluorescence staining was performed according to the staining technique included in the kit as given by the manufacturer(“Millipore, S7110, Apoptaq In Situ Cell Death Detection Kit”). In these experimental steps, the pieces washed with PBS buffer were incubated with TdT enzyme for 60 minutes at 37 °C in a humid and dark environment. Fractions washed with PBS began to react again with the anti-digoxigenin conjugate at standart laboratory temperature. All incubation steps were performed in a humid environment. An immunofluorescent microscope (Olympus BX 51) was used to get images from the slides. Apoptotic cells were detected in the images obtained after 4',6-diamidino-2-phenylindole (DAPI) and fluorescence isothiocyanate (FITCH).
Factor VIII staining
Factor VIII staining is a method used to detect increased angiogenesis in tumor tissue. Therefore, we used this technique in this study. The biotin- avidin -peroxidase technique was applied to solid tumor tissues obtained from mice for the determination expression of the factor VIII. In this study, Tumor tissue sections placed on laminate with polylysine from all mice in groups were placed in trays/cassettes and incubated overnight (60 °C). It was then treated with a series of xylenes, and then with decreasing degrees of alcohol, which was then rehydrated. It was washed 3 times with pure water for 2 minutes. Next, for antigen retrieval, 10% citrate buffer was heat exposed in a microwave oven at 600 W (5 minute) and then cooled to room temperature (10 minute). Sections washed with PBS buffer were treated with hydrogen peroxide (3%) for 12 min and washed once more with PBS to inhibit endogenous peroxidase activity. A staining kit (Thermo Scientific) was used in the next steps.
Afterward the repeated washing steps, the peroxidase substrate with diaminobenzidine (DAB) (Thermo Scientific) in the kit was treated for 1.5 min to make the immunoreactivity visible. Contrast stained sections were washed several times with gill haematoxylin and pure water. Lastly, the parts that were dehydrated with alcohol and xylene were sealed with a sealing solution (Entellan® , Merck). Samples were investigated under an Olympus BX51 microscope (Tokyo, Japan, Olympus BX51).
In the vitro experimental group
The doses of rhamnetin effects of 1, 2.5, and 5 μg/ml on EAT cells were investigated on the cell culture. Rhamnetin was dissolved within methanol (1%). The cell culture medium was prepared along with 80% Dulbecco Middle Eagle Medium 20% fetal bovine serum, and 1 ml penicillin/streptomycin solution:10 mg/ml streptomycin and 10,000 unit/ml penicillin. The plates with 96 sections were divided into 4 groups as the tumor control group, 1, 2.5 and 5 μg/ml rhamnetin treatment groups in the way that there would be 24 wells in each group. In each well 104.000 EAT cells were cultured. After these steps, the different doses of rhamnetin effects were examined by performing dead and vital cell counts 3 and 24 h later.
The cell count
In the form of suspension, the MEAL cells in the medium were tube-laced with indoor swimming pools; and 100-ml trypan was added. 50 µl of cell solution was pipetted on the Thomas slide. After keeping the Thoma slide on the microscope for 15-20 seconds, the cells were counted at 40x zoom.
Statistical analysis
Q-Q graphs and histograms were examined than Shapiro-Wilk test was applied to evaluate the normality of the data. ANOVA test was used for the comparison between the groups. Tukey test was used for multicomponent comparisons. One-way analysis of variance (ANOVA) for continuous variables or Kruskal–Wallis test was performed to compare the differences between groups. Levene's test was used to test the homogeneity of variance. Kruskal–Wallis analysis was used for comparisons between groups. Tukey and Dunn-Bonferroni tests were used for multiple comparisons. Dunn-Bonferroni test was used for multiple comparisons. Kruskal–Wallis and ANOVA were used for apoptosis and factor VIII between groups. In the in vitro experimental groups, Kruskal–Wallis test was used for dose comparison. Analysis was performed using the IBM SPSS Statistics 22 programmes. A P-value of less than 5% was considered statistically significant.