Silibinin inhibits cytokine-induced signaling cascades and down-regulates inducible nitric oxide synthase in human lung carcinoma A549 cells

Abstract

Recently, we reported that silibinin inhibits primary lung tumor growth and progression in mice and down-regulates inducible nitric oxide synthase (iNOS) expression in tumors; however, the mechanisms of silibinin action are largely not understood. Also, the activation of signaling pathways inducing various transcription factors are associated with lung carcinogenesis and their inhibition could be an effective strategy to prevent and/or treat lung cancer. Herein, we used human lung epithelial carcinoma A549 cells to explore the potential mechanisms and observed strong iNOS expression by cytokine mixture (containing 100 units/mL IFN-gamma + 0.5 ng/mL interleukin-1beta + 10 ng/mL tumor necrosis factor-alpha). We also examined the cytokine mixture-activated signaling cascades, which could potentially up-regulate iNOS expression, and then examined the effect of silibinin (50-200 mumol/L) on these signaling cascades. Silibinin treatment inhibited, albeit to different extent, the cytokine mixture-induced activation of signal transducer and activator of transcription 1 (Tyr(701)), signal transducer and activator of transcription 3 (Tyr(705)), activator protein-1 family of transcription factors, and nuclear factor-kappaB. The results for activator protein-1 were correlated with the decreased nuclear levels of phosphorylated c-Jun, c-Jun, JunB, JunD, phosphorylated c-Fos, and c-Fos. Further, silibinin also strongly decreased cytokine mixture-induced phosphorylation of extracellular signal-regulated kinase 1/2 but only marginally affected JNK1/2 phosphorylation. Silibinin treatment also decreased constitutive p38 phosphorylation in the presence or absence of cytokine mixture. Downstream of these pathways, silibinin strongly decreased cytokine mixture-induced expression of hypoxia-inducible factor-1alpha without any considerable effect on Akt activation. Cytokine mixture-induced iNOS expression was completely inhibited by silibinin. Overall, these results suggest that silibinin could target multiple cytokine-induced signaling pathways to down-regulate iNOS expression in lung cancer cells and that could contribute to its overall cancer preventive efficacy against lung tumorigenesis.

Figure 1

Time course effect of CM on the activation of STAT, AP-1 and NF-κB in human lung epithelial A549 cells. A, Time course (30 min to 24h) expression of pSTAT1 (Tyr-701), STAT1, pSTAT3 (Tyr-705), pSTAT3 (Ser-727) and STAT3 with CM treatment in total cell lysate as described in the ‘Materials and Methods’. First lane is control i.e. without CM. In all cases, membranes were stripped and reprobed with anti-β-actin antibody for protein loading correction. The densitometry data presented below the bands are ‘fold change’ as compared with control after normalization with respective loading control value. B, DNA binding of AP-1 at various time points with CM treatment as measured by EMSA. C, Gel-super shift assay was performed to examine the specific constituents of AP-1 after CM treatment for 24h. D, DNA binding of NF-κB at various time points with CM treatment as measured by EMSA. Gel super-shift assay was performed to examine the constituents of NF-κB after 30 min treatment with CM. The data shown are representative of at least two independent experiments.

Figure 2

Time course effect of CM on the activation of MAPKs and Akt, and expression of HIF-1α and iNOS in A549 cells. A-C, Time course (30 min to 24h) expression of pERK1/2, ERK1/2, pJNK1/2, JNK1/2, p-p38, p38, pAkt, Akt, HIF-1α and iNOS with CM treatment in total cell lysate as described in the ‘Materials and Methods’. In all cases, membranes were stripped and reprobed with anti-β-actin antibody for protein loading correction. The densitometry data presented below the bands are ‘fold change’ as compared with control after normalization with respective loading control value. The data shown are representative of at least two independent experiments. ND: Not detectable.

Figure 3

Effect of silibinin on CM-induced STAT activation in human lung epithelial A549 cells. A-C, The effect of silibinin pre-treatment for 2h on the expression of pSTAT 1 (Tyr-701), STAT1, pSTAT 3 (Tyr-705), pSTAT 3 (Tyr-727) and STAT3 in the presence (for 30 min) or absence of CM in total-, nuclear- and cytoplasmic lysates. In all cases, membranes were stripped and reprobed with anti-β-actin antibody for protein loading correction. The densitometry data presented below the bands are ‘fold change’ as compared with control after normalization with respective loading control value. The data shown are representative of at least two independent experiments. The line in the middle of the blots is to separate the effect of silibinin on constitutive (left) or CM modulated (right) expression of the molecules shown. ND: Not detectable.

Figure 4

Effect of silibinin on CM-induced AP-1 activation and expression of AP-1 constituents in human lung epithelial A549 cells. A, EMSA was performed in the nuclear extract to analyze the effect of silibinin pre-treatment for 2h on AP-1 activation in the presence (for 30 min) or absence of CM as described in the ‘Materials and Methods’. B, Nuclear extract was also analyzed for the expression of p-c-Jun, c-Jun, Jun B, Jun D, p-c-Fos and c-Fos. The densitometry data presented below the bands are ‘fold change’ as compared with control after normalization with respective loading control value. Blots shown are representative of at least two independent experiments. The line in the middle of the blots is to separate the effect of silibinin on constitutive (left) or CM modulated (right) expression of the molecules shown. ND: Not detectable.

Figure 5

Effect of silibinin on CM-induced NF-κB activation and expression of NF-κB constituents in human lung epithelial A549 cells. A, EMSA was performed in the nuclear extracts to analyze the effect of silibinin pre-treatment for 2h on NF-κB activation in the presence (for 30 min) or absence of CM as described in the ‘Materials and Methods’. B &C, Nuclear and cytoplasmic extracts were also analyzed for the expression of p50 and p65. The densitometry data presented below the bands are ‘fold change’ as compared with control after normalization with respective loading control value. Blots shown are representative of at least two independent experiments. The line in the middle of the blots is to separate the effect of silibinin on constitutive (left) or CM modulated (right) expression of the molecules shown.

Figure 6

Inhibitory effect of silibinin on CM-modulated ERK1/2, p38, HIF-1α and iNOS expression in A549 cells. A-C, Effect of silibinin pre-treatment for 2h on the expression of pERK1/2, ERK1/2, p-p38, p38, HIF-1α and iNOS in the presence (for 30 min) or absence of CM. The densitometry data presented below the bands are ‘fold change’ as compared with control after normalization with respective loading control value. The data shown are representative of at least two independent experiments. The line in the middle of the blots is to separate the effect of silibinin on constitutive (left) or CM modulated (right) expression of the molecules shown. D, The proposed mechanism for the inhibitory effects of silibinin on cytokine-induced signaling cascades regulating iNOS expression in A459 cells. Abbreviations: IFN-γ, interferon-γ; IL-1β, interleukin-1β; TNF-α, tumor necrosis factor-α; JAK, janus activated kinase; STAT, signal transducer and activator of transcription; MAPK, mitogen-activated protein kinase; IKK, inhibitor of kappa B kinase; IκB, inhibitor of kappa B; NF-κB, nuclear factor kappa B; HIF-1α, hypoxia inducing factor-1α; AP-1, activator protein-1; iNOS, inducible nitric oxide synthase. ND: Not detectable.