FWGE ® (Biromedicina Co., Budapest, Hungary), a wheat germ preparation with immunomodulant and antimetastatic activity, was applied simultaneously with cytostatic drugs of different modes of action, in vitro and in vivo, in order to find out whether this simultaneous administration exerts an antagonistic or a synergistic effect on the viability of cell cultures, tumor growth, and survival of animals, inoculated with a transplantable mouse tumor (3LL-HH). In vitro, FWGE did not influence the effect on the viability of MCF-7, HepG2, or Vero cells, exerted by Dacarbazine, 5-fluorouracyl, or Adriblastina. In vivo, FWGE , combined with Endoxan, Navelbine, and doxorubicin, did not prevent the tumor growth inhibitory effect of the cytostatic drugs. The combination of FWGE with the cytostatic drugs did not increase the toxicity of the cytostatic compounds, and did not exert any toxic effect. FWGE may be administered together with cytostatic drugs, without the risk of increasing toxicity or decreasing antiproliferative activity.

Recently, the antimetastatic effect of FWGE has been confirmed on human colon cancer.6,7 MATERIALS AND METHODS Chemicals MTT reagent was purchased from Sigma (St. Louis, MS), fetal calf serum from GIBCO (Invitrogen Life Technologies, Paisley, Scotland), 5-fluorouracyl from TEVA Pharmaceutical Industries Ltd., Netanya, Israel, Adriblastina® (doxorubicin) from Farmitalia, Milan, Italy, Endoxan® (cyclophosphamide) from Asta Medica Ltd., Budapest, Hungary, Dacarbazine (DTIC) from PlivaLachema, Beno, Czech Republic, Navelbine® (vinorelbine) from Pierre Fabre Medicament, Boulogne, France. FWGE is a wheat germ extract dispersed in maltodextrine, fructose, and siliciumdioxide to prevent adherence. FWGE was provided by Biromedicina Co. (Budapest, Hungary). A 40 mg/mL stock solution of FWGE was prepared in Dulbecco’s modified Eagle’s medium (DMEM), filter sterilized, and serial dilutions were made. Cell cultures MCF-7 (ECACC 86012803) and Vero (ECACC 84113001) cell cultures were purchased from ECACC (European Collection of Cell Cultures, Salisbury, UK), HepG2 (ATCC HB-8065) from ATCC (American Type Culture Collection, Manassas, VA), and cultured in DMEM without phenol red (GIBCO, Meckenheim, Germany), using plastic culture dishes and microwell plates (Nunc A/S, Roskilde, Denmark). DMEM medium was supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, and antibiotics: 100 units/mL penicillin and 100 g/mL streptomycin (Sigma, St. Louis, MS). Cells growing as a monolayer were kept in an isolated 37°C, 5% CO2 tissue incubator compartment. 

Treatment Cytotoxicity testing of FWGE (at 24 hours) was performed in the concentration range of 156 g/mL and 5 mg/mL using two estrogen receptor (ER) positive (MCF-7 and HepG2) and an ER negative (Vero) cell lines. The noncytotoxic concentration (500 g/mL) of FWGE was used in the experiments. FWGE and the cytostatics were administered to MCF-7 cell cultures 24 hours after plating. Control cultures were maintained in DMEM similar to the treated cultures. Four samples of cells were cultured and treated in a volume of 100 l in 96-well tissue culture plates for a further 24 hours for the MTT assay. 5 FU was applied in a dose range between 5 and 2000 g/mL; DTIC (Dacarbazine) between 5 and 1200 g/mL; Adriblastina between 1 and 400 g/mL. MTT Assay Cytotoxic effects on the viability of cells were determined using tetrazolium dye (MTT; 3[ 4,5- dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, as described by Horiuchi et al. 3 103 cells/well were plated in 96-microwell plates. An MTT solution was prepared at 5 mg/mL in PBS, filter sterilized and stored in the dark at 4°C for a maximum of 1 month. 20 l of MTT reagent was added to each 100 L of culture. After incubation for 3 hours at 37°C, the formazan crystals were dissolved by the addition of 100 l of propanol to the culture wells. The plates were further incubated for 20 minutes at room temperature, and optical density (O.D.) of the wells was determined using an Anthos 2020 (Salzburg, Austria) ELISA microplate reader at a test wavelength of 570 nM and a reference wavelength of 690 nM. Each plate contained “blank” background control wells holding an appropriate volume of media, but no cells.

 All experiments were performed three times, with 4 wells for each concentration of the tested agents. The control cells were grown under the same conditions, without the addition of the test compounds. Cell survival (% of control) was calculated relative to untreated control cells. In Vivo Studies Experimental Animals In all experiments, C57BL inbred mice were used. The animals were 8–10 weeks old and weighed 20–22g. They were kept in plastic cages (5 animals per cage) and were fed with rodent pellet (Charles River Hungary Ltd., Gödöllö, Hungary) and tap water ad libitum. The room temperature was 20°C–22°C, with a relative humidity of 55%  5%. Tumor Model The transplantable, highly metastatic variant of Lewis lung carcinoma (3LL-HH) tumor line grown on mice was used in the experiments. 3LLHH cells were inoculated intramuscularly into the muscles of the left hind leg of the mice. The inoculated tumor cell number was 2 105 per animal. Treatment FWGE , and also cytostatic treatment, was started 24 hours after tumor implantation. FWGE was dissolved in water and administered by means of a gastric tube. The daily dose was 3g/kg body weight per os administered in 0.1 mL of water. Control animals received tap water daily (0.1 mL), also by means of a gastric tube. The dose of Endoxan was 250 mg/kg body weight, intraperitoneally, as a single injection, 24 hours after tumor inoculation. The dose of doxorubicin was 0.2 mg per mouse, as a single injection, 24 hours after tumor inoculation. 

The dose of Navelbine was 4–6–16 mg/kg body weight, respectively, as intraperitoneal injections, 24 hours and 8 days after tumor inoculation. The following groups were formed: (1) Solvent-treated control (inoculated with 3LL-HH tumor)—10 animals for each experiment, except in the case of doxorubicin, where 5 animals were used. (2) Endoxan treatment—10 animals (3) Endoxan treatment—10 animals (4) Adriblastina treatment—5 animals (5) FWGE + FWGE treatment—5 animals (6) Navelbine treatment—10 animals (7) Navelbine + FWGE treatment—10 animals A group of 10 mice was established in each experiment, treated only with FWGE . Overall clinical status of the experimental mice, tumor growth, and spontaneous death of the tumor-bearing animals was followed. The usual tumorous cachexia and the toxic signs caused by cytostatics (such as anemia, spotted hemorrhages of the mucous membranes in a few cases) were observed in the case of single treatment and also in the case of combination therapy. No difference could be observed between these signs shown by the groups which received Endoxan, Adriblastina, or Navelbine treatment or a combination of one of the drugs and FWGE . The death of the animals was the result of tumor growth in each case. Mice treated with Endoxan and Adriblastina were followed up until the spontaneous death of the animals. Mice treated with Navelbine were sacrificed on day 13 after tumor inoculation. Tumor weight was measured in the case of Navelbine-treated animals. Statistical Analysis Statistical analyses were performed with the Student’s paired t test and p values 0.05 were considered to be significant. 

RESULTS In Vitro Studies DITC, Adriblastina, and 5FU treatment resulted in a dose-dependent decrease in viability of the 347 cell cultures, depending also on the estrogen receptor positivity or negativity of the cell lines. The 500 g/mL dose of FWGE , which proved to be noncytotoxic in our previous experiments, did not increase or decrease the viability of any of the cell cultures treated also with DITC, Adriblastina, or 5FU. This effect was not influenced by the dose of the cytostatics. The results are documented in Figs. 1, 2, and 3. In Vivo Studies All tumor-bearing control and treated mice of the experimental groups 2, 3, 4, 5 and the FWGE group died because of the growth and propagation of the tumor. The mice treated with Endoxan and those which received combination therapy (Endoxan and FWGE ) showed significant survival compared to the control group (Table 1). Adriblastina and Adriblastina plus FWGE treatment did not cause any increase or decrease in survival (Table 2). FWGE treatment did not influence the effect of Navelbine on the weight of 3LL-HH tumors (Table 3). 

In addition to the three prototypes of cytostatic drugs (5FU, DTIC, doxorubicin) applied in vitro, in the in vivo experiments the classical alkylating agent Endoxan and Navelbine, a new member of agents causing mitotic arrest, were also involved. Simultaneous administration of FWGE with these two compounds concluded with the same result observed in the case of the three other cytostatic drugs (i.e., the effect of these compounds on primary tumor growth and death of animals caused by tumor growth was not influenced by simultaneous FWGE treatment). Our results suggest that FWGE , a new immunomodulant with antimetastatic activity, may be administered together with cytostatic drugs, without increasing toxicity or decreasing the antiproliferative effect of the cytostatics. ACKNOWLEDGMENTS This work was supported from grant NKFP 1/016/2001. Z.L.M. is a recipient of the grant OTKA T030799. We thank Katalin Molnar for her excellent technical help.