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most sites. Moreover, many publications attributed the Sampling and detection of EAC-volume and cell number
[9]
anti-carcinogenic properties of garlic principally to allyl At the end of the experimental period, animals were
sulfides, among them DADS, which was shown to suppress sacrificed under diethyl ether anesthesia and 0.2 mL saline
growth and facilitate the death of different tumor cells. [2,6-10] was intraperitoneally injected. One minute later, EAC-aliquot
was aspirated by a sterile syringe into a test tube. The volume
This study was designed to assess the anti-proliferative of EAC-fluid for each mouse was measured. The number of
and anti-tumor cytotoxic efficacies against Ehrlich alive and dead EAC-cells were determined using trypan blue
ascites carcinoma (EAC) (which corresponds to mammary exclusion assay. Alive and dead EAC-cells were counted by
[13]
adenocarcinoma in female mice) in vivo and in vitro. In Neubauer hemocytometer. Briefly, 40 μL of EAC-aliquot was
addition, the apoptogenic effect of DADS was assessed via added to 4 mL 2% trypan blue (dissolved in 0.9% saline) and
following the changes in the anti-apoptotic protein, Bcl-2 the mixture was left for 5 min. One drop from the mixture
and apoptotic markers, p53 and terminal deoxynucleotidyl was taken on Neubauer hemocytometer and the number of
transferase (TdT) by immunohistochemical methods, imaging stained cells (dead cells) and nonstained cells (viable or alive
and semi-quantitative analysis. The experimental study was
approved by review board of Beni-Suef University. cells) were counted. Total number of EAC-cells and percent
of dead EAC-cells were calculated for each EAC-bearing
mouse. This procedure was adopted from the methods
METHODS
of Freitas et al. and Chandru et al. [15]
[14]
Tested agent (DADS)
DADS (C H S ) was obtained from Fluka Chemie Blood samples were obtained from carotid artery of each
6 10 2
GmbH, Buchs, Switzerland (lot and filling code mouse into ethylenediaminetetraacetic acid (15%)-containing

427432/1 55004115). DADS was dissolved in dimethyl tubes under mild diethyl ether anesthesia and were centrifuged
sulfoxide (DMSO) for in vivo and in vitro studies and was at 3,000 g for 15 min. The plasma was aspirated into Eppendorf
administered orally by gastric gavage at dose level of tubes and kept in deep freezer at -30 °C until used for plasma
100 mg/kg body weight (b.w.)/day [11] for 2 weeks. Its sialic acid determination. One milliliter of EAC fluid from
structure is CH =CH-CH -S-S-CH -CH=CH as indicated each mouse was homogenized in 2 mL saline (0.9% NaCl)
2
2
2
2
in the previous publication. [12] and centrifuged at 3,000 g for 15 min. The supernatant was
aspirated into Eppendorf tubes and kept in deep freezer
Animals and EAC-bearing model at -30 °C until used for ascites sialic acid determination.
Normal albino mice were obtained from Animal House,
Institute of Ophthalmology, Giza, Egypt. EAC-bearing stock Part of EAC-aliquot from each tumor-bearing mouse was
mice were obtained from Cancer Biology Department, centrifuged at 3,000 g for 15 min and the precipitated
National Cancer Institute, Cairo University, Egypt. To EAC-cells were fixed in neutral buffered formalin for
induce EAC in mice for the experimental study, 0.2 mL EAC histological and immunohistochemical studies.
aliquot aspirated from stock mice was added to 9.8 mL
saline (dilution is 1:50) and 0.2 mL of this diluted EAC was Determination of plasma and ascites sialic acid
intraperitoneally administered by syringe into each mice. concentration
Plasma and ascites sialic acid level was determined
Animal grouping according to the method of Warren. In this method,
[16]
The EAC-injected mice were divided into two groups each
containing 12 animals. Mice in group 1 (control group) were sialic acid is oxidized into formylpyruvic acid which reacts
administered 10% DMSO as a vehicle in a volume equivalent with thiobarbituric acid to form a pink color product. The
to that given to treated animals. Group 2 was treated with color intensity measured at 549 nm is proportional to the
DADS, dissolved in 10% DMSO, at a dose of 100 mg/kg b.w. concentration of sialic acid in the sample.
The treatments were orally applied by gastric intubation
between 10 and 12 AM daily for 2 weeks beginning from the Histological and immunohistochemical investigations
1st day of EAC-intraperitoneal inoculation. The fixed samples were transferred to the Department
of Pathology, National Cancer Institute for processing,
Animal survival blocking, sectioning and staining with hematoxylin and
The number of animals survived in each group was determined eosin or mounting on positive slides for immunhistochemical
at the end of the experiment and the survival percent in each investigations. Sections mounted onto positive-charged
group was calculated as follows: survival percent = (number slides (Fisher Scientific, Pittsburgh, PA, USA) were used to
of survived animals/total number of animals) ×100. detect the Bcl-2 and p53 reactivity or apoptotic cells using

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