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the TdT-mediated dUTP nick end labeling (TUNEL) assay. as mean ± standard error. Values with P > 0.05 are not
[17]
The TUNEL assay was performed using a kit (in situ cell death significantly different while values with P < 0.05 and P < 0.01
detection kit, Roche Molecular Biochemicals, Manheim, are significantly and highly significantly different respectively.
Germany) according to the protocol provided by the F-probability expresses the general effect between groups.
manufacturer, while Bcl-2 and p53 reactivity were determined
following method of Gao and Zhou. Briefly, before the RESULTS
[18]
incubation with antibodies, endogenous peroxidase activity
was quenched, slides washed and then incubated in a Effects on animal survival and mortality
blocking solution of hydrogen peroxide 1% in methanol, in After 2 weeks of EAC-intraperitoneal transplantation, 8 of
darkness for 15 min. Antigen retrieval occurred in citrate 12 mice administered 10% DMSO as a vehicle survived; the
buffer 10 mmol/L, pH = 6.0. After cooling, sections were survival percentage was 66.66%. The treatment of EAC-bearing
rinsed in tap water and then phosphate buffer saline (PBS). mice with DADS markedly increased the survival percentage
Primary antibodies for either Bcl-2 (DakoCytomation, USA) to reach 83.33% at the end of the experiment [Table 1].
or p53 (Lab Vision Corporation, 47777 Warm Springs Blvd,
Fremont, CA, USA), diluted 1:150 and 1:100, respectively in Effects on EAC-fl uid volume and cell number in vivo
PBS, were applied for 1 h at 37 °C. Secondary biotinylated The inhibitory effect of DADS on EAC-cells in vivo was tested
antibody diluted 1:100 and 1:200 in PBS was applied for a in terms of EAC-fluid volume, number of total and alive
period of 30 min at 37 °C. Streptavidin-biotin or avidin-biotin EAC-cells, and number and percent of EAC-dead cells. The
complex with horseradish peroxidase (ABC/HRP) was applied daily treatment of EAC-bearing mice with DADS for 2 weeks
for 10 min at room temperature. Bound antibody complex after EAC-cells intraperitoneal transplantation induced a
was visualized by the reaction of 3, 3’-diaminobenzidine (DAB) significant decrease (LSD; P < 0.05) of EAC-fluid volume,
substrate and counterstained with hematoxylin. Secondary number of total and alive EAC-cells. On the other hand, the
biotinylated antibody, ABC/HRP and DAB were obtained from number and percent of dead EAC-cells exhibited a potential
Zymed Laboratories (Invitrogen Immunoprotection, 561 increase (LSD; P < 0.01) as compared to EAC-bearing
Eccles Avenue, South San Francisco, CA, USA). Hematoxylin control counterparts. The ratio of EAC-fluid volume to total
was obtained from Sigma Chemical Company, USA. EAC-number was also highly significantly (LSD; P < 0.01)
increased in EAC-bearing mice treated with DADS as
Imaging and semi-quantitative analysis of Bcl-2, p53 and compared with control mice [Table 1].
terminal deoxynucleotidyl transferase
The yellowish brown colored stained area were analyzed With regards to one-way ANOVA, it was found that the
in pixels; percent area and intensity were detected effect between groups on number of total and alive
by ImageJ software, US National Institutes of Health, EAC-cells and percent of dead cells was very highly
Bethesda, Maryland, USA (http://imagej.nih.gov/ij/). significant (F-probability; P < 0.001), while the effect on
EAC-volume and number of dead cells was only highly
Anti-tumor cytotoxicity against EAC-cells in vitro significant (P < 0.01) [Table 1].
The viability of cells as a result of 6 different concentrations
(0, 6.25, 12.5, 25, 50, and 100 μg/mL) of DADS was tested Effect on plasma and ascites sialic acid level in vivo
by trypan blue exclusion assay according to the method of Plasma sialic acid level in EAC-bearing mice was highly
Ahmed and Ahmed. Briefly, EAC-cells at concentration significantly (P < 0.01; -35.05%) decreased as a result of
[19]
2.5 × 10 cells/mL suspended in PBS were incubated at 37 °C treatment with DADS. The ascites sialic acid concentration
5
for 2 h in the presence of different concentrations of DADS was also highly significantly (P < 0.01; -28.49%) decreased
dissolved in dimethyl sulfoxide. At the end of incubation in EAC-bearing mice treated with DADS as compared with
period, equal volume of trypan blue solution was added to EAC-bearing control [Table 2].
sample cells, then the stained cells (dead cells) and unstained
cells (alive cells) were counted using Neubauer hemocytometer. Histological and immunohistochemical effects on EAC-cells
The percent of dead cells for each test was calculated. As indicated by low and high magnification, EAC cells of
EAC-bearing control female mice [Figure 1a and c] were
Statistical analysis characterized with abundant basophilic and dark stained
Data were analyzed by one-way analysis of variance (ANOVA) cytoplasm as well as moderate sized nuclei. As a result of
followed by a least significance difference (LSD) test to treatment with DADS [Figure 1b and d], EAC-cells decreased
compare the groups with each other. Data were expressed in number and size and appeared with a narrow rim of
[20]

Hepatoma Research | Volume 1 | Issue 2 | July 15, 2015 69