Macrophages activated by lipopolysaccharide and/or phorbol esters exhibited high sensitivity to FWGE , a fermented wheat germ extract. FWGE synergized with lipopolysaccharide and PMA in the induction of the transcription of cytokine genes and release of inflammatory cytokines. At higher concentrations the preparation had a significant negative effect on the proliferation and survival of activated myeloid cell types. FWGE treatment induced the synthesis of ICAM-1 and synergized with the ICAM-inducing effect of TNF, but had no effect on VCAM-1 expression on microvascular endothelial cells. The effect of FWGE on signaling pathways, which are involved in cell activation was studied on HeLa cells as a model system. FWGE treatment increased the activity of stress kinases in a concentration-dependent way, resulting in the activation of AP-1 transcription factor. NF-kappa B–sensitive reporters were also activated by FWGE ; in contrast, no effect of the preparation was observed on PKA-sensitive signaling pathways.

The rejection time of skin graft in thymectomized mice was significantly shortened with FWGE treatment, indicating an immunity-restoring effect.10 As a result of FWGE treatment, the amount of cell surface MHC class I proteins was downregulated by 90 and 69% in Jurkat and Raji B cells, respectively, compared to nonstimulated controls.2 FWGE reduced autoantibody production by inhibiting the Th2 response and it improved clinical manifestations (erythrocyte sedimentation rate [ESR], WBC count, and proteinuria) of the disease in an experimental model of systemic lupus erythematosus.11 FWGE , by its complex mechanism of action, reduced disease progression and the incidence of metastases and consequently significantly improved the survival of patients with colorectal cancer compared to that of patients treated with conventional regimens alone.12 Tumor necrosis factor-alpha (TNF-α), first identified as a serum factor from lipopolysaccharide (LPS)-treated mice, induced hemorrhagic necrosis in tumors. In the human body, TNF is mainly produced by activated macrophages and epithelial cells. In contrast, TNF receptor-1 (TNF-R1) is widely expressed in different cells, which could implicate the manifold effect of TNF in physiologic and/or pathophysiologic processess. In fact, the interaction of TNF with TNF-R1 activates several signal transduction pathways. TNF is a major mediator of apoptosis, inflammation, and immunity; therefore, the role of TNF has been implicated in the pathogenesis of a wide range of human diseases including sepsis, diabetes, inflammatory bowel diseases, osteoporosis, multiple sclerosis, rheumatoid arthritis, and cancer.

13 Because of the importance of TNF in the natural defense against cancer as well as the fact that FWGE induces apoptosis in malignant hematologic and solid tumor cell lines and exhibits immunomodulatory activities, the effect of FWGE  treatment on proinflammatory cytokine production and Ras-mediated cell activation was studied, particularly, TNF-mediated cytotoxicity, TNF production of activated leukocytes, and the proliferation, sensitivity, and survival of TNF-producing and target cells. The molecular mechanisms of FWGE  were also studied using signaling pathway-specific transcription reporters.MATERIAL AND METHODS Cell Line Culture and Reagents Tumor necrosis factor was measured by bioassay on WEHI 164 cells (ECACC No. 87022501). A genetically modified clone (WC1) of this cell line, produced in our laboratory, was also used.14 WC1 cells are several hundred-fold more sensitive to TNF and provide us with a reproducible bioassay. WEHI 164 or WC-1 cells were grown in 96-well plates to a density of 3 × 104 cells/well. After 24 h of incubation at 37°C, serial dilutions of TNF were added to the cells containing 5% fetal calf serum (FCS).

 In WEHI assays, 0.1 µg/ml actinomycin D was also used. After 16-24 h of incubation cell viability was determined using the MTT assay.15 Several human (THP-1, ECACC No. 88081201, MonoMac6, from Dr. D. Wallach, Rehovot) and mouse (P388D and RAW264.7, ECACC Nos. 85011439 and 91062702, respectively) myeloid leukemia cell lines, an Epstein-Barr virus-immortalized human B-cell lymphoma cell line (Raji, ECACC No. 85011429), Sci1, a human B-cell lymphoma (ECACC No. 960905313), and BCL1, a mouse B-cell lymphoma (ECACC No. 90061904) were used to measure TNF production after in vitro activation of the cells by different combinations of FWGE , lipopolysaccharide (LPS), and phorbol-myristyl-acetate (PMA). (The human CDC-HMEC microvascular endothelial cell line was a kind gift of Prof. Hunyadi, Debrecen, Hungary.) WEHI 164, WC/1, and RAW 264.7 cells were grown on DMEM + 5% FCS, Raji was propagated on RPMI-1640 + 10% FCS, and all other cells (including HeLa and CDC-HMEC) were cultured on DMEM + 10% FCS. Media, sera, and reagents were purchased from Sigma Chemical Co. (St. Louis, Mo). Cell viability, proliferation, and cytotoxicity measurements were done using the MTT (3-[4,5-dimethylthiazol2-yl]-2,5-diphenyltetrazolium bromide) assay.15 Human microvascular cells were treated in vitro with TNF and/or FWGE in six plates (35 mm) overnight. For flow cytometry the cells were dislodged with EDTA, rubber policeman, and repeated pipetting. PMA and LPS (Escherichia coli serotype O55:B5) were purchased from SigmaAldrich (Budapest, Hungary). 

FWGE (Biromedicina, Budapest) was freshly dissolved, cleared by centrifugation, and filtered through a 0.1-µ filter just before the experiments. When cells were treated for extended periods, FWGE was replaced every other day. Recombinant human TNF and TNF ELISA were purchased from Genzyme (Wiesbaden-Nordenstadt, Germany). Expression of cell adhesion molecules ICAM1 and VCAM-1 on the surface of CDC-HMEC cells was followed by flow cytometry using monoclonal antibodies from Beckton Dickinson (Soft-Flow, Pécs, Hungary) and a BD Facscalibur apparatus.Analysis of RNA Total cellular RNA was prepared from FWGE -treated macrophages using the guanidium isothyocianate method according to standard protocols, and the integrity of the RNA samples was checked by running 5 µg RNA on formaldehyde gels.The ribonuclease protection assay (RPA) was carried out with the ML-11 multiple probe set using 10 µg of total cellular RNA, essentially as described earlier.16 This set detects transcripts of 10 different murine cytokine genes (lymphotoxin α, TNF, interleukin (IL)-4, IL-5, IL-1α, interferon (IFN)-γ, IL-2, IL-6, IL-1β, and IL3) and the housekeeping L-32 gene used as a control. In each gel, 1/10 part of the total amount of probe and a commonly used mixed spleen sample from LPS-treated mice were always included as a molecular weight marker and a positive contol, respectively.

 TNF mRNA was also detected on Northern blots using 10 µg total RNA for electrophoresis. For these assays a DNA fragment containing the first 885 basepair of the coding region of the murine TNF gene from the pGMTNF-MJ plasmid (a kind gift from Dr. J.-F. Arrighi) was used as the probe. After stripping, blots were rehybridized with a GAPDH probe, pHcGAP (from ATCC, Rockville, MD) for blotting control. RPA gels and Northern blots were visualized and quantified by using a Phosphorimager 445SI (Molecular Dynamics). Plasmids and Transformation All inducible reporter plasmids were purchased from Stratagene; the control, pTK-RL, containing the HSV promoter was from Promega. The reporter plasmid containing the promoter region of the human IL-8 gene (pIL-8-luc) was previously described.17 Constructs coding for the wild-type and mutant Ras proteins, used for the cotransformation experiments, were derivatives of pCDNA3. Cells were transformed by liposomes according to standand protocols, as suggested by the manufacturers of Lipofectamine (GIBCO) and Superfect (Qiagene), respectively. For transient expression, 1–2 × 104 cells were plated into the wells of microtiter plates 1 day before transformation. Next, 500 ng reporter construct and 100 ng control plasmid were mixed with 2.5 µl Superfect or 5 µl Lipofectamine. Measurement of Reporter Activity Activity of the individual promoters was measured by luciferase assay in FWGE -treated cells using the Dual Luciferase Assay kit (Promega). Cells were washed in phosphate-buffered saline solution and lysed in situ in 25 µl of lysis buffer. Ten microliters of this solution was used for measurement. Because TK promoter was not influenced by FWGE treatment, it was used as an internal control during the experiments.

 Luciferase activity was expressed, for example, as a ratio of IL8- luc/TK-Rluc. At least two independent transformations were performed in each experiment, and treatments were repeated at least in three parallels. RESULTS Effect of FWGE on Proliferation and Survival of Different Cell Lines FWGE is used by cancer patients as medical nutriment for prolonged periods. Consequently, experiments were planned to be of long duration (6–7 days) so that the proliferation and survival of the investigated cells in the presence of FWGE could be followed. Cells were grown in the presence of FWGE for 72 h before the experiments, followed by another 24 h during the experiment, and the number of living cells was estimated by MTT methods. FWGE (at a concentration of 8 µg/ml to 10 mg/ml) did not influence the growth rate and surival of any of the investigated, nonmyeloid cell lines (WEHI 164, WC1, Raji, Sci1, BCL1, CDC-HMEC, and HeLa). However, cells of the monocyte-macrophage lineage (MonoMac6, THP-1, P388D, and RAW264.7) were sensitive to the drug, and some lines exhibited highly inhibited growth and limited survival at higher concentrations (0.5 mg/ml and up). This sensitivity was further increased in the presence of macrophage-activating compounds such as LPS or PMA (FIG. 1A, B, and C). FWGE Did Not Modify the TNF Sensitivity of Target Tumor Cells WC1 cells, used for the titration of TNF, were grown in the presence and absence of FWGE .

 In three parallel experiments, FWGE did not modify the TNF sensitivity of target cells significantly. As in FIGURE 1D, slight changes were observed, but in this assay system only a two- to fourfold increase or decrease in TNF sensitivity was judged as significant. Since WC1 is a genetically modified line of WEHI 164, similar experiments were carried out with the parental WEHI 164 line. As expected, WEHI cells, although they were less sensitive to TNF, exhibited similar TNF sensitivities in both the presence and the absence of FWGE (data not shown).FWGE Upregulated AP-1 and NF-
B but Not CRE-Sensitive Reporters in Cervix Carcinoma (HeLa) Cells Using a Signaling Pathway, Which Involves P21ras These results indicate that signaling pathways usually triggered by inflammatory cytokines18 might be involved in the effects of FWGE . To learn more about the molecular events elicited by FWGE , HeLa cells were transiently transfected with signaling pathway-specific reporters and treated with increasing doses of FWGE . As in FIGURE 3B, FWGE activated the stress kinase-sensitive AP-1 luc and NF-κB luc responsive reporters in a dose-dependent manner, whereas it had no effect on the cAMPresponsive CRE luc reporter activity. The possible synergism between FWGE and proinflammatory cytokine-mediated reporter activation was investigated by costimulating HeLa cells with nonsaturating and saturating doses of IL-1 or TNF in the presence or the absence of FWGE . A cytokine–sensitive IL-8 luc reporter, which contains binding sites for NF-κB and AP-1 or an NF-κB luc reporter was transiently transfected and used as a readout signal (FIG. 3C, D, E, and F). 

Except for using IL-1 in a saturating dose on IL-8 luc, FWGE was able to synergize with the cytokine-mediated stimulation. The ability to superinduce proinflammatory cytokine stimulation even in a saturating dose suggests that FWGE -mediated responses might be coupled with other second messenger pathways. This hypothesis was tested using AP-1 luc as a readout and constitutively active or dominant negative p21ras mutants as activators. As in FIG. 3G, FWGE -mediated activation was completely blocked upon transfection with the dominant negative ras mutant. These data indicate that FWGE activates the AP-1 reporter via a signaling pathway that is controlled by p21ras. DISCUSSION Apoptosis is a physiologic process critical for organ development, tissue homeostasis, and elimination of defective or potentially dangerous cells in complex organisms. Apoptosis in mammalian cells can be initiated through two major interrelated pathways, one involving engagement of the TNF family of death receptors and the other involving the release of cytochrome c from mitochondria. Apoptosis and proliferation are intimately coupled. Some cell cycle regulators can influence both cell division and programmed cell death. The linkage of cell cycle and apoptosis has been recognized for c-Myc, p53, pRb, PKA, PKC, Bcl-2, CDK, cyclins, CKI, Ras, and NF-κB.19 It is of importance that both NF-κB and Ras can either induce programmed cell death or protect cells from apoptosis depending on the specific cell type and the type of inducer.20, 21 Experimental data clearly indicate that NF-κB is a major regulator of the inflammatory reaction by controlling the expression of proinflammatory molecules in response to cytokines, oxidative stress, and infectious agents.

.22 Moreover, NF-κB pathway activation occurs during transformation induced by a number of classic oncogenes, including Bcr/abl, Ras, and Rac, and it is necessary for full transforming potential.23 TNF plays a critical role in mediating inflammatory responses by upregulation of genes encoding cell-adhesion molecules required for the recruitment of inflammatory cytokines.24 Because the promoter regions of many of the TNF-regulated genes contain DNA binding sites for NF-κB, activation of NF-κB by TNF is essential to elicit effective immune and inflammatory responses.13 Moreover, the link between NF-κB and cancer stems, in part, by the fact that this transcription factor can induce gene products that control proliferative responses and that suppress apoptotic cascades, such as those induced by TNF.25 That the regulatory cascades of inflammation, immunity, apoptosis, transformation, and cell cycle seem to FWGE , at least partly, the effects of FWGE on TNFmediated cytotoxicity of sensitive tumor cells is somewhat difficult to interpret. On the one hand, FWGE did not exhibit any significant effect on TNF-mediated killing of tumor cells; it neither protected target cells against TNF nor increased their sensitivity to this cytokine. On the other hand, TNF and IL-6 production of activated macrophages was considerably increased when cells were exposed to FWGE for prolonged periods. IL-6 and TNF are typical examples of multifunctional cytokines involved in the regulation of the immune response, hematopoiesis, and inflammation. Their functions are widely overlapping, but each shows its own characteristic properties.26