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Ferrocene-induced lymphocyte activation and anti-tumor activity is mediated by redox-sensitive signaling¹

Felsenstein Medical Research Center, Sackler Faculty of Medicine, Tel Aviv University, Rabin Medical Center, Beilinson Campus, Petah-Tikva, 49100, Israel; and
^* Department of Biochemistry, Weill Medical College of Cornell University, New York, New York, USA

²Correspondence: The Felsenstein Medical Research Center, Rabin Medical Center, Beilinson Campus, Petah Tikva 49100, Israel. E-mail: novog{at}post.tau.ac.il

SPECIFIC AIMS

After our studies of lymphocyte activation associated with redox modification of p21^ras, we examined whether ferrocene, a stable iron-containing compound generating free radicals, could stimulate lymphocytes and macrophages to elicit an anti-tumor effect.

PRINCIPAL FINDINGS

1. Ferrocene-induced immune stimulation
Ferrocene stimulated to a small degree ³H-thymidine incorporation in mouse splenocytes, in vitro. Purified peritoneal macrophages were not stimulated by ferrocene. Ferrocene, however, activated mouse peritoneal macrophages, in vitro, upon their incubation in the presence of lymphocytes and stimulated splenocytes, in vivo. Activation of splenocytes was assessed by an increase of ³H-thymidine incorporation (Fig. 1 A) and that of macrophages by an LPS-induced increase in TNF- and NO production and a rise in oxygen burst (Fig. 1B ).

View larger version (39K): [in this window] [in a new window]   Figure 1. A) Stimulation by ferrocene of 3H-thymidine incorporation in mouse splenocytes, in vivo. Three days after injection (i.p.) of ferrocene, mouse splenocytes were isolated and incorporation of 3H-thymidine was determined (for 30 h, 4 mice per group). Results are expressed as means CPM ± SD of triplicate cultures from each mouse (***P<0.001 ferrocene-treated mice vs. control). B) Activation by ferrocene of mouse peritoneal macrophages, in vivo. Mice were injected (i.p.) with ferrocene (0.05 mg/kg). Three days later, the peritoneal cavity was washed with PBS and macrophage monolayers (106 cells/6 mm plate) were obtained. TNF- and nitric oxide levels were determined in supernatants obtained 6 h after the addition of LPS (10 µg/mL). Oxygen burst (H2O2 production) was determined in the absence and presence of PMA. Four mice were included in each experimental group and the results are expressed as means ± SE (*P<0.05; ***P<0.001 macrophages from ferrocene-treated mice vs. macrophages from PBS-treated mice, both incubated with LPS or PMA). C) Ferrocene induces phosphorylation of ERK1/2 in wild-type Jurkat cells but not in C118S Jurkat cells. Jurkat cells (wild-type and C118S) were incubated (2x105 cells/mL) for 24 h in RPMI 1640 containing 0.5% FCS. Ferrocene and PHA were added as indicated in the figure. After incubation for 5 min at 37°C, cells were centrifuged and lysates were analyzed by Western blot to detect activated (phosphorylated) ERK 1/2.

2. Anti-tumor properties of ferrocene
Ferrocene (administered intraperitoneal, or i.p.) has an anti-tumor effect in mice bearing established lung metastases of B-16 melanoma. Weight of lungs in B-16 inoculated mice vs. controls represents tumor load.

A single administration (i.p.) of ferrocene (0.2 mg/kg) was effective in eliciting an anti-tumor effect. The anti-tumor effect of multiple administration of ferrocene at different doses is depicted in Fig. 2 A. Maximal anti-tumor effect was attained at doses of 0.05–0.2 mg/kg. Lower or higher doses were not effective. Ferrocene, administered via the drinking water, was also effective in eliciting an anti-tumor effect (Fig. 2B, C ). Administration (i.p.) of ferrocene at a dose of 0.05 mg/kg, 3 times each, on days T₁, T₈, and T₁₅ also markedly reduced established lung metastases of Lewis lung carcinoma.

View larger version (36K): [in this window] [in a new window]   Figure 2. A) Anti-tumor effect of ferrocene administered i.p. B-16 melanoma cells were inoculated (T0) via the tail vein. Ferrocene was administered (i.p.) 3 times (T1, T8, and T15). Mice were killed 25 days after tumor inoculation and the lungs were weighed (4–6 mice per group). Results are expressed as the means of the weight of lungs per mouse ± SE (***P<0.001 weight of lungs from tumor-inoculated ferrocene-treated mice vs. tumor-inoculated, control mice). B, C) Effect of ferrocene, administered orally, on established lung metastases in B-16 melanoma bearing mice. Mice inoculated with B-16 melanoma were supplied with drinking water (ad libitum) containing ferrocene at different concentrations for 2 wk. A mouse drinks 5 mL water/day, so the overall doses of ferrocene were 0.125–0.5 mg·kg-1·day-1. Mice were killed 25 days after tumor inoculation and 4–6 mice were included in each group. Results are expressed as the means of the weight of lungs of each mouse ± SE (C) and by direct scoring of lungs’ metastases ± SE (B)..Direct scoring and measuring of metastases determined tumor scoring index of tumor load: conglomerate, 20; large (>5 mm), 3; medium (2–5 mm), 2; small (<2 mm), 1.) .(**P<0.01; ***P<0.001 weight of lungs from tumor-inoculated, ferrocene-treated mice vs. tumor-inoculated control mice). All experiments were repeated 2–3 times with similar results.

3. Adoptive transfer experiments
Based on the hypothesis that ferrocene mediates its effect by immune stimulation, we investigated whether immune cells from ferrocene-treated mice could elicit an anti-tumor effect in otherwise untreated tumor-inoculated mice. Donor mice were inoculated with B-16 melanoma and treated on day T₂ with ferrocene (0.2 mg/kg). Mice were killed on day T₂₅, and splenocytes and peritoneal cells (containing 50% lymphocytes and 50% macrophages) were collected and injected (intravenous on T₁) into B-16 inoculated mice. Data indicated that cells from ferrocene-treated mice were more effective than those from untreated mice in eliciting an anti-tumor effect. Splenocytes from either ferrocene-treated or untreated mice were equally effective in eliciting an anti-tumor effect.

4. Mechanism of action of ferrocene
We postulate that p21^ras is a molecular target of ferrocene by which, along with other molecular targets, it elicits an immune stimulatory effect. p21^ras has been identified as a key molecule in T lymphocyte activation and has been shown to be activated by cellular redox stress. The following findings support our hypothesis.

1) Ferrocene incubated in PBS generated H₂0₂. Ferrocene at concentrations of 5, 50, and 500 nM produced H₂O₂ at concentrations of 8.9, 11, and 11.6 µM, respectively.

2) N-Acetylcysteine (NAC), a free radical scavenger, modulated ferrocene-induced anti-tumor effect in B-16 melanoma-bearing mice. The anti-tumor effect of ferrocene at a low, optimal dose of 0.25 mg/kg was markedly inhibited by NAC. In contrast, NAC enhanced the anti-tumor effect in mice that had been treated with ferrocene at a high, supraoptimal dose of 2.5 mg/kg, which by itself had a negligible anti-tumor effect. These results are in accordance with our finding that the anti-tumor effect of ferrocene resembles a bell-shaped curve: its effect is diminished at supraoptimal doses, which apparently produce extensive oxidation.

3) Ferrocene directly stimulated GTPase activity catalyzed by pure recombinant p21^ras in a bell-shaped pattern. At concentrations of 5, 10, and 50 nM, it stimulated purified p21^ras GTPase to the extent of 121 ± 2, 145 ± 6, and 12 ± 3% of controls, respectively.

4) To establish that p21^ras cysteine 118 is a molecular target site for oxidation by ferrocene, we investigated the stimulatory effect of ferrocene on ERK1/2 (downstream of ras activation) in wild-type Jurkat T cells and Jurkat T cells expressing p21^ras in which cysteine 118 was replaced by a serine residue (C118S). These cells are not susceptible to redox stress modification. As depicted in Fig. 1C , ferrocene (0.05 µM) markedly activated ERK 1/2 in wild-type Jurkat cells. Under similar experimental conditions, ferrocene failed to activate ERK1/2 in Jurkat C118S. PHA, which does not activate p21^ras via redox stress, activated ERK1/2 in both cells. Ferrocene also activated and translocated NFB in human PBM cells, a pathway mediated by ras. Binding NFB to a DNA fragment was demonstrated in an electromobility shift assay.

5. CONCLUSIONS AND SIGNIFICANCE
The immune stimulatory and anti-tumor effects of ferrocene resemble a bell-shaped curve. At high, supraoptimal concentrations, ferrocene is less effective. Its inhibiting effect at high concentrations may be due to oxidation of thiol moieties and other susceptible molecules not relevant to the stimulatory process. The effective dose of ferrocene that elicited an anti-tumor effect is 2000-fold less than its LD₅₀ (440 mg/kg). We postulate that the anti-tumor activity of ferrocene is mediated by immune stimulation. This is supported by our findings that, at low doses, ferrocene activated p21^ras (increased GTPase) and ERK1/2 (downstream of p21^ras activation), stimulated lymphocyte proliferation, and activated peritoneal macrophages. Most important, adoptive transfer experiments demonstrated that mouse peritoneal mononuclear cells from ferrocene-treated mice had an anti-tumor effect in mice that had not been treated with this compound. Ferrocene activated macrophages only in the presence of lymphocytes. It is thus possible that ferrocene-induced macrophage activation is mediated by soluble factors (e.g., interferon-) released from ferrocene-activated lymphocytes. Anti-tumor effects of agents stimulating the immune system have been reported. The clinical use of some of these agents showed limited success in part because of high toxicity. Ferrocene is a stable, small molecule that exhibits immune stimulatory and anti-tumor properties by a distinct mechanism and is effective at low doses upon i.p. and oral administration. As such, it may offer therapeutic advantages over some immune stimulatory agents.

View larger version (20K): [in this window] [in a new window]   Figure 3. Schematic diagram. Proposed mechanism of ferrocene activity.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0558fje; to cite this article, use FASEB J. (January 2, 2003) 10.1096/fj.02-0558fje

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