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Arsenic trioxide (ATO) is one of the most potent drugs in cancer chemotherapy, and is highly effective in treating both newly diagnosed and relapse patients with acute promyelocytic leukemia (APL). Despite a number of reports regarding the molecular mechanisms by which ATO promotes anti-tumor or pro-apoptotic activity in hematological and other solid malignancies, the effects of ATO on immune responses remain poorly understood. To further understand and clarify the effects of ATO on immune responses, we sought to examine whether ATO affects the production of nitric oxide (NO) in a lipopolysaccharide (LPS)-stimulated mouse macrophage cell line, RAW 264.7. Arsenic trioxide was found to prevent NO production in a dose-dependent manner. Arsenic trioxide significantly inhibited the increase in inducible nitric oxide synthase (iNOS) at both the mRNA and protein levels. Furthermore, our analyses revealed that the inhibitory effect of ATO on iNOS expression was ascribed to the prevention of IRF3 phosphorylation, interferon (IFN)-β expression, and STAT1 phosphorylation, but not the prevention of the MyD88-dependent pathway. Taken together, our results indicate that ATO prevents NO production by inhibiting the TIR-domain-containing adaptor protein inducing IFN-β (TRIF)-dependent pathway, thus highlighting an anti-inflammatory property of ATO in innate immunity.
Arsenic, an ancient drug used in traditional Chinese medicine, has attracted worldwide interest because it shows substantial anticancer activity in patients with acute promyelocytic leukemia (APL).[1, 2] Arsenic trioxide (ATO) has been shown to exert anti-cancer activity against APL by directly binding to promyelocytic leukemia retinoic acid receptor α (PML-RARα), an onco-protein regarded to be crucial for the pathogenesis of APL, which leads to subsequent degradation of PML-RARα protein.[3-5] Of note, a number of studies have demonstrated the pro-apoptotic activity of ATO. Indeed, ATO induced apoptosis by increasing CASPASE-10 expression, which is also associated with histone H3 phosphoacetylation. Second, ATO suppressed the expression of anti-apoptotic genes such as hTERT, MYC and C17, which are associated with Sp1 oxidation via production of reaction oxygen species. Finally, ATO induced apoptosis of ATRA-treated NB4 cells associated with the inhibition of nuclear factor (NF)-κB activation and the enhancement of JNK activation.
Bobé P et al. reported the therapeutic effects of ATO on the severe autoimmune disorders manifested in MRL/lpr mice, in which ATO significantly reduced lymphoproliferation, suppressed skin lesions, and reduced the serum concentration of immunoglobulins, Th1 cytokines, and NO. Thus, it is likely that ATO may play an important role in the regulation of inflammatory responses. However, the cellular and molecular mechanisms by which ATO regulates immune response remains poorly understood. Nitric oxide, a product of macrophages activated by cytokines, microbial compounds or both, is derived from the amino acid l-arginine by the enzymatic activity of inducible nitric oxide synthase (iNOS). It functions as a tumoricidal and anti-microbial molecule both in vitro and in vivo.[10-15] Nitric oxide also plays a diverse role in the pathogenesis of osteoclastogenesis,[16, 17] pleurisy, and asthma,[19, 20] indicating that NO is one of the most versatile molecules in the immune system. Sodium arsenite (SA), which forms a trivalent molecule (As3+), was reported to inhibit LPS-induced NO production in mouse macrophages. Therefore, it is of particular interest to examine whether ATO affects NO production.
In this study, we show the inhibitory effect of ATO on LPS-induced NO production in the mouse macrophage cell line RAW 264.7. We also discuss the molecular mechanisms underlying the inhibitory effect of ATO on iNOS gene expression.
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In the present study, we demonstrate that ATO inhibits iNOS expression, leading to the reduction of NO production in LPS-stimulated RAW mouse macrophage cells by inhibiting the TRIF-dependent pathway. It is unlikely that the inhibition of iNOS expression by ATO is due to its cytotoxicity or its alteration of TLR4 or CD14 expression levels. Rather, this inhibition is due to the impairment of the LPS-inducible intracellular signal transduction pathway. After LPS recognition, TLR4 initiates distinct signaling pathways depending on the adapters MyD88 and TRIF, leading to the activation of the transcription factors NF-κB, AP-1, ATF2, and IRF3 (Fig. 4). Activation of these transcription factors consequentially induces the production of pro-inflammatory cytokines such as type I interferons. It is well known that iNOS expression is induced by the activation of the TLR4 signaling pathway.[25, 27] It is also known that NF-κB acts as a transcriptional factor for iNOS gene expression through the MyD88-dependent pathway in LPS-stimulated mouse macrophages.[25, 28] Chakravortty et al. previously reported that sodium arsenite inhibits iNOS expression via prevention of NF-κB activity. These studies prompted us to examine whether ATO affects the activation of NF-κB. However, in this study, ATO did not exhibit any inhibitory effects on the transcriptional activity of NF-κB. The transcriptional factors AP-1 and ATF2, which are activated through MyD88 in the TLR4 signaling pathway, are known to play an important role in the transcriptional regulation of the iNOS gene. Therefore, we examined whether ATO affects the activation of AP-1 and ATF-2, and the activation of MAP kinases after LPS stimulation. Again, ATO did not show any inhibitory effects on the phosphorylation levels of AP-1 and ATF-2 or the phosphorylation levels of MAP kinases, including ERK1/2, p38, and JNK/SAPK. Thus, our results indicate that the inhibitory effect of ATO on iNOS expression is not mediated by the prevention of the activation of the MyD88-dependent pathway.
Figure 4. A possible mechanism by which arsenic trioxide (ATO) inhibits nitric oxide (NO) production in the lipopolysaccharide (LPS)/TLR4 signaling pathway. The induction of the iNOS mRNA after LPS stimulation is mediated by the MyD88-dependent and/or TRIF-dependent pathway. Nuclear factor (NF)-κB is independently activated and acts as the transcriptional factor in inducing pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, through the MyD88-dependent pathway. In the TRIF-dependent pathway, IRF3 is phosphorylated, forms homodimers, and acts as a transcriptional activator of interferon (IFN)-β expression. Subsequently, IFN-β induces the phosphorylation of STAT1α/β, which acts as a transcriptional activator of the iNOS gene expression. In this study, ATO was found to inhibit NO production in LPS-stimulated RAW cells through the inhibition of the TRIF-dependent pathway.
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Apart from the MyD88-dependent pathway, TLR4 triggers the TRIF-dependent signaling pathway to induce IFN-β (Fig. 4). In the TRIF-dependent pathway, IRF3 is phosphorylated, forms homodimers, and acts as a transcriptional activator of IFN-β expression. Finally, IFN-β induces the phosphorylation of STAT1α/β, which acts as a crucial transcriptional factor of the iNOS gene in LPS-stimulated mouse macrophages.[26, 30] Meraz MA et al. and Ohmori Y et al. [31, 32] have independently reported that STAT1 knockout mice failed to produce NO from macrophages after LPS/IFN stimulation. Collectively, these reports prompted us to examine the involvement of the TRIF-dependent pathway in the inhibitory effect of ATO on iNOS expression. Our results demonstrated that ATO treatment decreases both IFN-β mRNA and protein expression, and decreases the phosphorylation levels of IRF3. Finally, knockdown of STAT1 resulted in the reduction of iNOS expression, strongly indicating that STAT1 can act as one of the key transcriptional activators of iNOS gene expression. Arsenic trioxide was found to decrease STAT1α/β phosphorylation. This decrease in STAT1 phosphorylation can be partly explained by the prevention of IRF3 activation by ATO. Since IRF3 activation is known to occur through its phosphorylation by IκB kinase ε, it is possible that ATO may prevent the activation of IRF3 by inhibiting this upstream kinase.
Arsenic trioxide has been shown to have pro-apoptotic activity by affecting a number of signal transduction pathways in APL cells.[3-8, 33, 34] However, the relationship between NO production and apoptosis in APL cells remains poorly understood. In this study, we found that ATO prevents NO production in LPS-stimulated RAW cells. Therefore, it will be interesting to examine whether ATO exerts pro-apoptotic activity in APL cells by preventing NO production.
In conclusion, we demonstrate that ATO prevents NO production, at least in part, through inhibition of the TRIF-dependent pathway. This finding raises the possibility that ATO can regulate TLR4-inducible pro-inflammatory gene expression and modulate the TLR4-mediated innate immune systems. In this study, we have done the experiments by using only one cell line, RAW 264.7. Thus, it will be interesting to examine whether ATO prevents NO production in either mouse or human naive macrophages. Further studies are certainly warranted to contribute to the understanding of the molecular action of ATO in immune defense systems.