A Possible Mechanism of Cisplatin-induced Tumor Necrosis Factor (tnf)-α Production in Murine Macrophages

Cisplatin has been used for the treatment of various solid cancers or sarcomas; however, it can induce severe adverse effects. Among these adverse effects, nephrotoxicity, which has the potential to be a dose-limiting factor of this agent, develops due to the secretion of tumor necrosis factor-α (TNF-α) from macrophages; however, the precise mechanisms are still unclear. To elucidate possible mechanisms, we investigated the involvement of mitogen-activated protein kinases (MAPK) and reactive oxygen species (ROS) in cisplatin-induced TNF-α mRNA expression and protein production in the mouse macrophage-like cell line, RAW 264. Cisplatin (1 μM) significantly increased TNF-α mRNA expression and protein production. Extracellular-regulated kinase (ERK) and p38 MAPK, but not c-Jun N-terminal kinase (JNK), phosphorylation increased in response to cisplatin. Although an ERK inhibitor (PD98059) suppressed both cis-platin-induced TNF-α mRNA expression and its protein production, a p38 MAPK inhibitor (SB203580) decreased TNF-α protein production only. A JNK inhibitor (SP600125) had no effect on cisplatin-induced TNF-α mRNA expression. Furthermore, a scavenger of ROS, N,N'-dimethylthiourea, suppressed both ERK activation and TNF-α mRNA expression. These results suggest that the phosphorylation of ERK by ROS is involved in cisplatin-induced TNF-α mRNA expression and that the signaling pathway of p38 MAPK is related to TNF-α protein production.


Introduction
Cisplatin has been used for the treatment of various solid cancers or sarcomas; however, it can cause severe adverse effects including renal and cochlear injuries [1,2].Nephrotoxicity, which is caused by the accumulation of cisplatin in the proximal tubule, has the potential to be a dose-limiting factor of this therapy.Previous studies reported that proximal tubular injury was caused due to increases in macrophage infiltration by cisplatin [3,4] and subsequent overproduction of the pro-inflammatory cytokine, tumor necrosis factor (TNF)-α, in tubule cells [5][6][7].Thus, inhibiting the production and secretion of TNF-α in macrophages may be advantageous in reducing cisplatin-induced renal injury.The production of proinflammatory cytokines including TNF-α is mediated by the activation of mitogen-activated protein kinase (MAPK) family members [6].MAPK pathways are cascades of serine/threonine kinases comprising three families members; extracellular signal-regulated kinase (ERK), p38 MAPK, and c-Jun N-terminal kinase (JNK), that are activated by chemical stresses and reactive oxygen species (ROS) [8][9][10].The ERK signaling module is involved in cell growth, proliferation, and survival.On the other hand, the activation of p38 MAPK and JNK generally results in inflammation [8,9,11].Ramesh et al. reported that cisplatin induced TNF-α mRNA expression via activation of the p38 MAPK pathway in renal proximal tubular cells [12].However, the possible mechanism of cisplatin-induced TNF-α expression in macrophages has not been fully elucidated.In this study, we investigated the mechanism of cisplatin-induced TNF-α production in macrophages, with a focus on MAPK pathways, using the mouse macrophage-like cell line, RAW 264.

TNF-α Protein Assay
Cells were harvested in 12 well plates (1 × 10 5 cells/well) and treated with PD98059 (1 μM) or SB203580 (1 μM) between 0.5 h and 72 h after cisplatin (1 μM) stimulation.TNF-α protein levels in the supernatant were then determined using an ELISA assay kit (Mouse TNF-α Quantikine ELISA Kit, R&D Systems, Minneapolis, MN, USA) according to the manufacturer's protocol.The absorbance of each well was read at 450nm using a microplate reader.

Cell Viability
Cells were cultured in varying concentrations of cisplatin between 0.01 μM and 10 μM for 24 h and cytotoxicity was evaluated by trypan blue staining (Gibco, Grand Island, NY, USA).

Statistical Analysis
Data were expressed as mean ± S.E.M. and analyzed statistically using a one-way analysis of variance followed by the Dunnett's Multiple Comparison Test.Values of p < 0.05 were determined to be significant.

Involvement of the MAPK Pathway on Cisplatin-Induced TNF-α mRNA Expression and Protein Production
Cisplatin dose-dependently increased TNF-α mRNA expression.A marked increase was observed with the administration of 1 μM cisplatin (Figure 1(A)).We ob-served no significant suppression in cell viability when cells were stimulated with cisplatin at a concentration of 1 μM (data not shown).PD98059, SB203580, or SP600125 alone did not affect TNF-α mRNA expression in RAW264 cells.PD98059 at a dose of 1 μM and 10 μM significantly inhibited cisplatin-induced TNF-α mRNA expression, but any dose of SB203580 and SP600125 did not inhibit cisplatin induced TNF-α mRNA expression (Fig- As shown in Figure 2, cisplatin significantly increased ERK and p38 MAPK to 1.4 and 1.7-fold higher than that of controls, respectively; however, a similar response was not observed for JNK phosphorylation.Pretreatment with PD98059 and SB203580 significantly suppressed cisplatin-induced TNF-α protein production and levels in PD98059-treated cells and returned them to control levels (control: 142.6 ± 2.0 pg/ml, cisplatin: 231.7 ± 8.5 pg/ml, cisplatin + PD98059: 143.3 ± 10.9 pg/ml, cisplatin + SB203580: 177.4 ± 17.9 pg/ml) (Figure 3).

The Effect of a Radical Scavenger on TNF-α mRNA Expression and MAPK Phosphorylation
Although DMTU alone did not affect TNF-α mRNA expression, it significantly reduced cisplatin-induced TNF-α mRNA expression to the level of controls (Figure 4(A)).Furthermore, DMTU significantly decreased cisplatin-induced phosphorylation of ERK, but not that of p38 MAPK (Figure 4(B)).

Discussion
In this study, we demonstrated that cisplatin significantly increased TNF-α mRNA expression (Figure 1(A)).Using specific inhibitors of MAP kinase family members, we found that the transcription of TNF-α mRNA is dependent on ERK, but not p38 MAPK or JNK (Figures 1(B)-(D)).Several studies have shown that p38 MAPK and JNK are involved in inflammation and that their activation results in the production of inflammatory cytokines including TNF-α [9,13].Our results suggest that p38 MAPK and JNK did not suppress the expression of cisplatin-induced TNF-α mRNA (Figures 1(C) and (D)).
Previous reports have revealed that p38 MAPK is involved in the nucleocytoplasmic transport of TNF-α mRNA [14] and its phosphorylation causes TNF-α mRNA stabilization; thus, this activation is associated with an increase in the production of TNF-α protein.
We actually found that cisplatin induced p38 MAPK phosphorylation under the condition of increased TNF-α mRNA expression (Figure 2), and its inhibitor, SB203580, suppressed cisplatin-induced TNF-α protein production without affecting TNF-α mRNA expression (Figure 3).These findings suggested that p38 MAPK pathways are   also closely related to the process of cisplatin-induced TNF-α protein synthesis rather than its mRNA expression.
On the other hand, although Swantek et al. reported that the JNK pathway mainly regulates the translation of TNF-α mRNA [15], we confirmed that cisplatin was not involved in the phosphorylation of JNK and SP600125 did not affect cisplatin-induced expression of TNF-α mRNA in RAW264 cells 24 h after cisplatin treatment.Sánchez-Pérez et al. reported that cisplatin induced transient activation of the JNK pathway within a few hours via the regulation of its upstream-kinase MEKK1 [16,17].Therefore, there is a possibility that the JNK pathway may also be involved in the development of cisplatininduced renal failure at an early stage.
Several studies have shown that ROS leads to increases in the phosphorylation of ERK and p38 MAPK [26][27][28].Cisplatin generates hydrogen peroxide (H 2 O 2 ), a relatively long-lived species of ROS, in a variety of cells including macrophages [29,30].From these findings, we hypothesized that cisplatin-induced TNF-α production in murine macrophages may be due to an increase in ERK Copyright © 2013 SciRes.PP and p38 MAPK phosphorylation by the H 2 O 2 generated by cisplatin.We demonstrated that DMTU inhibited the expression of TNF-α mRNA and also decreased cisplatin-induced phosphorylation of ERK.Since DMTU, an agent with a long half-life and the ability to scavenge H 2 O 2 and hydroxyl radicals selectively, can be highly and rapidly permeable to cell membranes, it has the ability to inhibit the inflammatory response caused by ROS generated within cells [31].Taken together, ERK activation, induced by H 2 O 2 and hydroxyl radicals and generated by cisplatin, is essential for the expression of the TNF-α mRNA and production of the TNF-α protein.
We found that although DMTU slightly inhibited the phosphorylation of p38 MAPK, a significant inhibitory effect was not observed (Figure 4).The link between the TNF receptor (TNFR) and p38 MAPK activation has been previously reported [32,33].Ligations of the TNFR phosphorylate MKK3/MKK6, upstream of p38 MAPK, have also been shown [34,35].Therefore, we speculate that autocrine activation by TNF-α caused the phosphorylation of p38 MAPK.
In conclusion, our study showed that macrophages require the ERK pathway for cisplatin-induced TNF-α mRNA expression and that this pathway is phosphorylated by cisplatin-induced ROS.

Figure 2 .
Figure 2. The effects of cisplatin on MAPK pathway activation.ERK, p38 MAPK, and JNK phosphorylation in cells treated with 1 μM cisplatin or vehicle for 24 h were analyzed by an ELISA assay kit.Phosphorylation in RAW264 cells treated with a respective inhibitor alone was defined as the value of control levels.Data are shown as the mean ± S.E.M. (n = 3 -4).*** p < 0.001 significantly different from the control group.

Figure 3 .
Figure 3.The effects of MAPK inhibitors on cisplatin-induced TNF-α production.RAW264 cells were treated with 1 μM PD98059 and 1 μM SB203580 between 0.5 h before and 72 h after 1 μM cisplatin stimulation.TNF-α levels in the supernatant were determined using an ELISA assay kit.Data are shown as the mean ± S.E.M. (n = 3).** p < 0.01 significantly different from the control group.# p < 0.05 and ## p < 0.01 significantly different from the cisplatin group.

Figure 4 .
Figure 4.The effects of a radical scavenger on cisplatin-induced TNF-α mRNA expression and MAPK pathway activation.RAW264 cells were treated with 100 μM DMTU or vehicle between 0.5 h before and 24 h after cisplatin (1 μM) or vehicle stimulation.TNF-α mRNA expression was then analyzed by RT-PCR (A) and the phosphorylation of ERK and p38 MAPK were analyzed by an ELISA assay kit (B).The mean value of the vehicle control was set to 1.0 (A) and the phosphorylation value in the cisplatin treatment group was set to 1.0 (B).Data are shown as the mean ± S.E.M. (n = 3 -4).** p < 0.01 significantly different from the vehicle control group.# p < 0.05 significantly different from the cisplatin group (A).* p < 0.05 significantly different from the control group (B).