The production of NO(X), MDA,CAT,SOD,GSH , GPx, ALT,AST,GGT (as liver function test), urea, and creatinine (as kidney function test) were evaluated by colorimetric assays, also, histopathological examination of liver and tumor tissue and characterization of cell death within tumor tissue was evaluated. In vitro results showed treatment of EAC cells with different concentrations of FWGE (0.21-85 mg/ml) showed cytotoxicity with IC50 at concentration of 0.8mg /ml , and in case of FWGE-nano-Se, showed cytotoxicity with IC50 at concentration of 0.8 mg /ml FWGE +0.75 µg /ml nano-Se. Also, in vivo studies results of FWGE-nano-Se mixture treated group showed significant reduction in the tumor volume compared to positive control group and FWGE treated group. Moreover, results of antioxidant parameters showed significant increase in SOD, GSH, GPx and CAT and significant decrease NO(X)and MDA and improvement in liver and kidney function tests. Apoptosis and histopathological examination revealed that FWGE-nano-Se mixture has antimetastatic effect and induced apoptosis in Ehrlich carcinoma cells. We concluded that the anti-tumor mechanisms of FWGE-nano-Se may be mediated by preventing oxidative damage, improved liver and kidney function, decrease metastases of cancer cells and increae apoptosis. So FWGE-nano-Se might be a potential alternative agent for cancer therapy. Clinical trials will be needed to spur the development of FWGE-nano-Se as cancer therapeutic agents. 

 The production of NO(X), MDA,CAT,SOD,GSH , GPx, ALT,AST,GGT (as liver function test), urea, and creatinine (as kidney function test) were evaluated by colorimetric assays, also, histopathological examination of liver and tumor tissue and characterization of cell death within tumor tissue was evaluated. In vitro results showed treatment of EAC cells with different concentrations of FWGE (0.21-85 mg/ml) showed cytotoxicity with IC50 at concentration of 0.8mg /ml , and in case of FWGE-nano-Se, showed cytotoxicity with IC50 at concentration of 0.8 mg /ml FWGE +0.75 µg /ml nano-Se. Also, in vivo studies results of FWGE-nano-Se mixture treated group showed significant reduction in the tumor volume compared to positive control group and FWGE treated group. Moreover, results of antioxidant parameters showed significant increase in SOD, GSH, GPx and CAT and significant decrease NO(X)and MDA and improvement in liver and kidney function tests. Apoptosis and histopathological examination revealed that FWGE-nano-Se mixture has antimetastatic effect and induced apoptosis in Ehrlich carcinoma cells. We concluded that the anti-tumor mechanisms of FWGE-nano-Se may be mediated by preventing oxidative damage, improved liver and kidney function, decrease metastases of cancer cells and increae apoptosis. So FWGE-nano-Se might be a potential alternative agent for cancer therapy. Clinical trials will be needed to spur the development of FWGE-nano-Se as cancer therapeutic agents. 

INTRODUCTION 

Nanotechology holds promise for medication and nutrition because materials at the nanometer dimension exhibit novel properties different to those of both isolated atoms and bulk material [1]. Nanoparticles are designed to carry anti-cancer drugs and bring that medication all the way to the diseased cells in a person’s body without harming the healthy cells [2]. Selenium, as one of the essential elements for the health of mammalian animals, has key functions in the balancing of the redox system, the proper functions of the immune system, and anticarcinogenetic effects [3]. Nano-Se can serve as an antioxidant with reduced risk of selenium toxicity and as a potential chemopreventive agent [4]. The role of seleno compounds as chemopreventive and chemotherapeutic agents has been supported by a large number of epidemiological, preclinical and clinical studies [5]. Fermented wheat germ extract (FWGE) is a concentrated extract of wheat germ derived from the germ of the wheat plant and differs from ordinary wheat germ in that it is fermented with yeast to concentrate biologically-active benzoquinones. It contains two quinones, 2-methoxy benzoquinone and 2,6-dimethoxybenzquinone that likely play a significant role in exerting several of its biological properties [6]. Preclinical in vitro and in vivo data suggested antiproliferative, antimetastatic and immunological effects of FWGE [7, 8].

 FWGE is not a drug, nor an alternative to standard anticancer drugs or standard therapies: FWGE is a dietary supplement to be given to cancer patients to help drugs to work better [9]. MATERIALS AND METHODS Animals Female Swiss albino mice weighing 20–25 g were obtained from the Egyptian Organization for Biological Products and Vaccines (VACSERA, Giza, Egypt). Animals were kept under standard conditions and were allowed free access to a standard requirement diet and water ad. Libitum. Animals were kept under a controlled lighting condition (light: dark, 12 h:12 h). Ehrlich Ascites Carcinoma Cells: A line of Ehrlich Ascites Carcinoma (EAC) cells was supplied from National Cancer Institute, Cancer Biology Department.Egypt. Tumor induction: Solid tumors were produced by intramuscular inoculation with 0.2 ml of EAC, which contained 2.5 x 106 viable EAC cells, in the right thigh of the lower limb of each mouse. Mice with a palpable solid tumor, its diameter was 10mm³, that developed within 10 days after inoculation were used in this study. Chemicals: Wheat germ was obtained from local market and stored in sealed plastic bags at 4oC before use. Selenium and all chemical and kits purchased from Sigma (USA). Production of FWGE and FWGE-nano-Se mixture for bioevaluation of antitumor activity as anticancer was prepared as follows: Preparation of FWGE: Thirty grams of active Saccharomyces cerevisiae cells were suspended in 270ml dist. water and mixed with 90g of wheat germ.The mixture was then fermented at 37ºC for 48hs in incubator. 

The suspension was centrifuged at 3000rpm for 10min and the supernatant was freeze dried by (LyoTrap (NCRRT) USA) and the resulted powder was kept in sealed vial. Preparation of FWGE-nano-Se mixture: To 100ml deionized water add 1 ml selenious acid (0.04mM), 4ml of 0.2mM GSH solution containing 200mg of bovine serum albumin with stirring to initiate the reaction. The pH of the mixture was adjusted to 7.2 with 1.0 M sodium hydroxide, during which the red elemental Se and oxidized glutathione (GSSG) formed. The reaction lasted 1hour under sonication. The red solution was dialyzed against doubly distilled water for 96 h with the water changing every 24 h to separate GSSG from Nano-Se. Centrifugation at 20000rpm(Hettich cooling centrifug; type Werk Nr. Made in Germany).The pellets were mixed with fermented wheat germ extract under sonication conditions for 1 hour to form mixture. Cell viability assay: EAC viable cells were counted by trypan blue exclusion method where, 10μl trypan blue (0.05%) was mixed with 10μl of the cell suspensions. Within 5 minutes, the mixture was spread onto haemocytometer, covered with a cover slip and then cells were examined under microscope. Dead cells are blue stained but viable cells are not [10]. Experimental design: Sixty female Swiss albino mice were divided into 4 groups each contain 15 mice as follows:

 Group (1): Served as negative control and orally received saline served as negative control group (NTBM: Non-tumor bearing mice). Group (2): Tumor bearing mice without any treatment served as positive control group (TBM) for 6 weeks.Group (3): Tumor bearing mice received FWGE at dose of 3gm /Kg body weight/day (TBM(FWGE)) for 6weeks. Group (4): Tumor bearing mice received FWGE-nanoSe at a dose of 2.125 g (dry weight)/kg body weight/ day for FWGE and 2 mg/kg body weight for nano-Se (TBM (FWGE-nanoSe)) for 6 weeks. Blood and tissue sampling: Directly, after animals were sacrificed, blood was collected after 2 and 6 weeks. liver and tumor were dissected out every 2, and 6 weeks from the beginning of treatment, part of them was homogenated and samples (N.B. muscle tissue of negative control group was dissected to be compared with tumor bearing group) were prepared in ice-cold phosphate buffer which used for determination of antioxidant parameters and the other portions of tumor and liver at the end of experiment (after 6 weeks of treatments) were dissected and kept in 10% formalin for histopathological examination and apoptosis detection (in tumor tissue). Tumor volume determination After 10 days from inoculation of Ehrlich carcinoma, tumor volume was measured twice a week using a Vernier caliper and determined by applying the following equation according to Jensen et al. [11]: Tumor volume = 1/2(length × width2 ) Where length is the greatest longitudinal diameter and width is the greatest transverse diameter. Estimation of Malondialdhyde (MDA) level: Lipid peroxidation is measured colorimetrically according to the method of Yoshioka et al. 

[12] based on measurement of Malondialdhyde (MDA) as one of the main end products of lipid peroxidation by thiobarbituric acid test. Estimation of catalase (CAT) activity Catalase activity was measured in plasma and 10% liver homogenate according to the method of Sinha [13]. The dichromate/ acetic acid reagent can be thought of as a "stop bath" for catalase activity. As soon as enzyme reaction mixture hits the acetic acid, its activity is inhibited, any hydrogen peroxide, which has not been split by catalase will react with dichromate to give a blue precipitate of perchromic acid. This unstable precipitate was then decomposed by heating to give the green color solution which was measured spectrophotometery at 570 nm. Estimation of glutathione content (GSH): Glutathione was measured according to the colorimetric method of Beutler et al. [14]. This method is based on spectrophotometrically measurement of the yellow color of 2-nitro-5- thiobenzoic acid which was produced as one product of this reaction: Glutathione + 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) 2-nitro-5-thiobenzoic acid + glutathione disulfide (GSSG).

 Estimation of superoxide dismutase activity (SOD): SOD activity is measured in blood and 10% tissue homogenate according to the method of Minami & Yoshikawa [15].SOD catalyzes the dismutation of the superoxide radical (O- ) into hydrogen peroxide (H2O2) and elemental oxygen (O2).Superoxide ions, generated from auto-oxidation of pyrogallol, convert the nitro blue tetrazolium chloride (NBT) to NBT-diformazan which absorbs light at 550 nm. SOD reduces the superoxide ion concentration thereby lowering the rate of NBT-diformazan formation. The extent of reduction in the appearance of NBT-diformazan is a measure of SOD activity present in samples. Estimation of nitrate/nitrite (NO(x)): Nitric oxide was determined according to the method described by Miranda et al. [16]. Nitric oxide is relatively unstable in the presences of molecular oxygen, with an apparent half life approximately 3-5 seconds and is rapidly oxidized to nitrate and nitrite totally designated as NOx. A high correlation between endogenous nitric oxide production and nitrite/nitrate (NOx) levels has been established. The measurement of these levels provides a reliable and quantitative estimate of nitric oxide output in vivo.The assay determines total nitrite/nitrate level based on the reduction of any nitrate to nitrite by vanadium followed by the detection of total nitrite by Griess reagent.

While, the liver of tumor bearing mice treated with FWGE-nano-Se mixture showed distribution of neoplastic cells among the hepatic cords (Fig2d). In muscle tissue: Histopathological examination of muscle sections of positive control group showed extensive invasion of neoplastic cell and necrosis of muscular tissue (Fig 3a). But in TBM(FWGE) showing focal aggregation of neoplastic cells surrounded by muscular tissue capsule (Fig.3b). While, in TBM(FWGE-nano-Se) showing small aggregated foci of neoplastic cells (Fig.3c). Characterization of cell death within the tumor (Apoptosis): Analysis of our photographs revealed normal mice tumor cells stained green with the presence of some level of apoptotic cells as expected (stained orange) presented in figure (4a,b).Also, analysis of the results revealed induction of apoptosis under the effect of FWGE treatment with the presence of bright green cells in the middle area of section in Fig.(4c,d). Also, Fig.(4 e,f) revealed that combination of FWGE with Se nanoparticles reply more antitumor efficacy by induction of more apoptosis Discussion: Application of FWGE and FWGE-nano-Se mixture on EAC cells table (1&2) showed cytotoxicity with maximum cell mortality (98.22% and 98.78%)respectively at 85mg/ml FWGE and 80 µg /ml nano-Se after 1 hour incubation. Our results are in agreement with TomoskoziFarkas & Daood [24] who stated that, some benzoquinones such as 2,6-dimehoxy benzoquinone (2,6-DMBQ) have been proved to exhibit cytotoxic effect in EATC, and thereby inhibit tumor propagation and metastases. Also, Hedvegi et al.

 [25] reported that 2,6-DMBQ and 2-MBQ (benzoquinones presents in FWGE) are cytotoxic for malignant tumor cells. FWGE induced apoptosis and exerted significant antiproliferative activity in a broad spectrum of tumor cell lines. FWGE and FWGE -nanoSe mixture fig (1) showed marked regression in tumor growth that were observed by the significant reduction in tumor volume and tumor weight when compared with untreated group. These observations are in agreement with those recorded by Hidvegi et al. [26] who concluded that, growth inhibition of EAC tumor can be achieved by treatment of tumor bearing mice with a mixture of 2,6-DMBQ and ascorbic acid. One of the largest known natural source for 2,6-dimethoxy-benzoquinone (DMBQ) and 2- methoxy-benzoquinone is wheat germ as glycosides; yeast glycosidase activity present during fermentation leads to release of the benzoquinones as aglycones [6]. The biological activity of quinine is connected with their participation in redoxy-cycles in the form of free reactive radicals. Their ability to produce aryl-nucleophil compounds, particularly by reaction with thiol and amino groups may explain the extreme activity of these compounds [26]. Results of antioxidant parameters of TBM group (significant increase in MDA and NO(X) and significant decrease in SOD, GPx, GSH and CAT) are in agreement with Kumaraguruparan et al. [27] who found that the presence of tumor caused disequilibria of the antioxidant defense system. Moreover, Hayat [28] demonstrated that, lipid peroxidation level was significantly increased in blood, liver and tumor tissues of EAC mice when compared with control group. In contrary, Cheeseman et al. [29] who suggested that, there is a decrease rate of lipid peroxidation in liver tumor cell than normal liver cells.