Culture medium RPMI 1640, fetal bovine serum (FBS), HEPES and L-glutamine were purchased from Life Technologies (Grand Island, NY, USA). Trypan blue, MTT were obtained from Sigma Aldrich (St. Louis, MO, USA). Annexin-V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) were from BD Biosciences (San Jose, CA, USA), and 2,7-dichlorodihydrofluorescein diacetate (H2-DCFDA) was from Molecular Probes/Invitrogen (Eugene, OR, USA). Caspase-2, caspase-3 and caspase-9 activities were evaluated by using commercial available kits from R&D Systems (Minneapolis, MN, USA). For evaluation of hepatic enzymes such as aspartate amino transferase (AST), alanine amino transferase (ALT), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) commercial kits were used (Span Diagnostics Ltd., Vadodara, Gujarat, India).
Collection and extraction of EEGE
Fresh algae of G. edulis were collected from the regional sea shore during the month of December in the Mandapam region, Tamil Nadu. Alcoholic extract of the algae was prepared as described earlier and the presence of biologically active components including alkaloids, flavonoids, sterols, terpenoids, proteins, saponins, phenols, coumarins, tannins and glycosides was documented using spectrophotometric analysis . No specific permission was required for the collection of these algae as these were collected from regional sea shore, not covered by any regulatory body and private land. This study does not involve any endangered or protected species. A voucher specimen of this algae was matched with the local herbarium authentic specimen (Herbarium no. AC.126.96.36.199) housed at Central Marine Fischeries Research Institute, Cochin, Kerala, India and was deposited in the herbarium.
Animals and mouse tumor model
Adult swiss albino mice weighing between 25–30 g were procured from Tamilnadu Veterinary and animal Science University, Chennai. The animals were kept in well-ventilated cages and fed with commercial food and water ad libitum and raised under specific pathogen-free conditions. The study was conducted with necessary ethical clearance from Institutional Animal Ethics Committee (IAEC) of Srimad Andavan Arts & Science College. EAT cells were provided as courtesy sample by Amala Cancer Research Center, Thrissur, India. Ascitic tumor cells were counted by trypan blue dye exclusion method and always found to be 95% or more viable. Cells were maintained in mice in ascites form by successive transplantation of 6×106 cells/mouse in a volume of 0.2 ml in PBS .
In vitro EAT cell culture
Following inoculation of EAT cells in mice abdominal cavity, after ten days the cells were collected by needle aspiration, washed in saline and erythrocytes were removed by washing in
35 mM NaCl. Cells were cultured in RPMI 1640 supplemented with HEPES (25 mM), L-glutamine (2 mM), sodium bicarbonate (25 mM), 10% FBS, 2-mercaptoethanol (50 μM) and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin) at 37°C in 5% of CO2 incubator. Viability and cell density were determined by the trypan blue dye exclusion test.
Evaluation of EEGE cytotoxicity in EAT cells
In a 96 well plate, EAT cells (3×105/ml) in RPMI 1640 with 10% FBS were seeded in quadruplicate. EEGE was dissolved in PBS which final concentration was adjusted to less than 0.1% (v/v) of the solvent in culture medium. The cells were treated with EEGE while control samples were treated with the corresponding volume of culture medium containing PBS. All samples were incubated in 5% CO2 incubator for 72 hours at 37°C in a 100% humidity atmosphere. Cell proliferation was determined using the standard MTT assay  and the phosphatase activity assay .
Leukocyte culture and evaluation of EEGE cytotoxicity
Peripheral human blood was obtained from healthy adult volunteer with prior ethical approval and diluted with an equal volume of RPMI 1640 medium. Mononuclear cell was isolated using Ficoll-Hypaque density gradient separation solution, washed twice in RPMI1640 medium. Cells were suspended in RPMI1640 medium supplemented with 2 mM glutamine, antibiotics and 10% FBS. Leukocytes at a density of 1 × 106 plating cells/ml were cultured with 5 μg/ml of phytohemagglutinin in 96-well microtiter plates. Cells were incubated with EEGE in a 5% CO2 incubator for 72 h at 37°C. Control samples were treated with the corresponding volume of culture medium containing less than 0.1% PBS. After treatment, cell proliferation was determined using the MTT reduction assay .
EAT cells (5 × 106) were treated with various concentrations of EEGE including 0, 25, 50 and 100 μg/ml for 72 hours were washed with PBS. Total and reduced glutathione concentration in the cells was estimated by Glutathione Assay Kit from Sigma. The cells were processed as per kit protocol. The sample is first deproteinized with the 5% 5-sulfosalicylic acid solution. Glutathione content of the sample is then assayed using a kinetic assay in which catalytic amounts of glutathione cause a continuous reduction of 5,5′-dithiobis-(2-nitrobenzoic) acid (DTNB) to TNB. The oxidized glutathione formed is recycled by glutathione reductase and NADPH. The product, TNB, is assayed colorimetrically at 412 nm.
Reactive oxygen species (ROS) measurement
EAT cells (5 × 106) were treated with EEGE (50 μg/ml) for 8, 12 and 24 hours in a 96-well plate followed by analysis of intracellular ROS using the oxidation-sensitive fluorescent probe 2,7-dichlorofluorescein diacetate (DCFH-DA). DCFH-DA enters cells and is hydrolyzed to membrane-impermeant dichlorofluorescein, which reacts with ROS to form the highly fluorescent dichlorofluorescein. Briefly, EAT cells were loaded with 5 μM DCFH-DA for the last 30 min of EEGE and the fluorescence of the generated DCF was measured in a fluorimeter plate reader at 490 nm excitation and 538 nm emission. Corrected values according to the cell number estimated by the trypan blue assay and the amount of ROS formed was expressed relative to the control [21, 22].
DNA fragmentation was evaluated by using protocol described by McGahon et al. (1995) with modification. EAT cells (5 × 106) were incubated with the EEGE at different concentrions (0, 25, 50, 100 μg/ml) for 48 hours to estimate the DNA fragmentation at 37°C. After 48 hours, cell suspension containing 4-6×105 cells in a microcentrifuge tube was centrifuged for 5 min at 2000 × g, 4°C. The cell pellet was processed to isolate the DNA as per the protocol followed by addition of 10 pg/ml RNase (Boehringer Mannheim, Indianapolis, IN) and were incubated at 50°C for 1 hour. DNA was purified using DNA purification kit from Qiagen as per manufactures protocol. Extracted DNA was dissolved in 50 μL TE buffer, and electrophoresis was performed on a 1.8% agarose gel containing ethidium bromide  and densitometric analysis of bands was done by ImageJ Software (NIH, USA).
Determination of caspases activities
EAT cells (5 × 106) were incubated with EEGE (0, 25, 50, 100 μg/ml) for 72 hours and followed by measurement of caspase-2, caspase-3 and caspase-9 activities using colorimetric protease kits as per the manufacturer’s protocol. To prepare total cellular protein, cells were pelleted by centrifugation and lysed on ice and total protein concentration in the lysate was measured. With each X-pNA substrate (200 μM final concentration) 200 μg of proteins were incubated at 37°C for 4 hours in a 96 well plate. The absorbance of the samples was measured at 405 nm and the increase in the caspase activity of treated cells was determined by comparing the results with the untreated cells and standard drug after background correction.
Annexin V-FITC/PI analysis
Detection of apoptosis was performed using the Annexin V-FITC/PI apoptosis detection kit according to manufacturer’s protocol. Briefly, both EEGE treated and untreated EAT cells were washed in 1× PBS and stained with annexin V-FITC conjugate and PI. Cells were then analyzed by flow cytometry (BD FACSCalibur, USA) using BD CellQuest acquisition and analysis software.
The antitumor activity of EEGE was evaluated by measuring survival time and tumor growth inhibition. Mice were inoculated with 6×106 EAT cells by i.p. route. After 24 h, EEGE was administered by i.p. injections of 0.2 ml per mouse. Endpoint of experiments was determined by spontaneous death of animals. The ascitic fluid from the peritoneal cavity of tumor bearing mice was quantitatively isolated by peritoneal lavage after death. The total number of tumor cells was counted by the trypan blue exclusion method. EEGE solutions were prepared in PBS containing 10% Tween 80. Control mice received the vehicle control as i.p. injection of 10% Tween 80 in PBS for the same time period.
In vivo toxicological studies
An extended 35-day toxicity study of EEGE was conducted in adult swiss albino mice with daily doses of 300 mg/kg. Two groups of six animals each were used for toxicity study where animals had free access to water and food. The first group was served as vehicle control and second group was given 300 mg/kg of EEGE in PBS, containing 10% Tween 80, by i.p. injections of 0.2 ml per mouse, once daily. Every day morning clinical signs of gross toxicity, behavioral changes and mortality were observed. Body weight of individual animal was recorded before and at the end of experiment. After 24 hours of the administration of the last dose, the animals were euthanized by anesthetizing with ketamine hydrochloride and xylazine hydrochloride administered . Whole blood was then sampled from the retro-orbital sinus with suitable hematological tube. For hematology assays 150 μl was retained and the remaining volume was used for serum biochemistry. The blood for serum biochemistry was allowed to clot at room temperature and was centrifuged at 3000 rpm for 10 min for serum separation. After blood collection, all mice were killed by cervical dislocation and liver and kidneys were collected, washed in PBS, fixed with 10% formalin and stored for histopathological examination.
Hematological and biochemical analyses
Whole blood was immediately analyzed for complete blood count with differential and platelet count using the fully automated analyzer (Hitachi, Tokyo, Japan). Serum samples were analyzed for AST, ALT activities, and also ALP and LDH levels by commercial kits as per manufacturer’s instruction.
Routine histological processes were employed for paraffin inclusion, sectioning and H/E staining of liver and kidney from mice treated with EEGE and vehicle control. A histopathologist performed a complete examination of the tissues.
All in vitro experiments were performed in triplicate and results are represented as means ± SD. Significant differences among groups was performed by ANOVA followed by Tukey test. The survival of mice was demonstrated using the Kaplan–Meier method and the logrank (Cox–Mantel) statistical test was applied to compare the curves for non-parametric procedures. Values where p < 0.05, differences were considered significant at representing two-sided test of statistical significance.