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Silibinin modulates TNF-α and IFN-γ mediated signaling to regulate COX2 and iNOS expression in tumorigenic mouse lung
Alpna Tyagi1, Chapla Agarwal, Lori D Dwyer-Nield
1Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Denver, Aurora, Colorado 80045, USA.
Abstract:
Silibinin inhibits mouse lung tumorigenesis in part by targeting tumor microenvironment. Tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) can be pro- or anti-tumorigenic, but in lung cancer cell lines they induce pro-inflammatory enzymes cyclooxygenase 2 (COX2) and inducible nitric oxide synthase (iNOS). Accordingly, here we examined mechanism of silibinin action on TNF-α + IFN-γ (hereafter referred as cytokine mixture) elicited signaling in tumor-derived mouse lung epithelial LM2 cells. Both signal transducers and activators of the transcription (STAT)3 (tyr705 and ser727) and STAT1 (tyr701) were activated within 15 min of cytokine mixture exposure, while STAT1 (ser727) activated after 3 h. Cytokine mixture also activated Erk1/2 and caused an increase in both COX2 and iNOS levels. Pretreatment of cells with a MEK, NF-κB, and/or epidermal growth factor receptor (EGFR) inhibitor inhibited cytokine mixture-induced activation of Erk1/2, NF-κB, or EGFR, respectively, and strongly decreased phosphorylation of STAT3 and STAT1 and expression of COX2 and iNOS. Also, janus family kinases (JAK)1 and JAK2 inhibitors specifically decreased cytokine-induced iNOS expression, suggesting possible roles of JAK1, JAK2, Erk1/2, NF-κB, and EGFR in cytokine mixture-caused induction of COX2 and iNOS expression via STAT3/STAT1 activation in LM2 cells. Importantly, silibinin pretreatment inhibited cytokine mixture-induced phosphorylation of STAT3, STAT1, and Erk1/2, NF-κB-DNA binding, and expression of COX2, iNOS, matrix metalloproteinases (MMP)2, and MMP9, which was mediated through impairment of STAT3 and STAT1 nuclear localization. Silibinin also inhibited cytokine mixture-induced migration of LM2 cells. Together, we showed that STAT3 and STAT1 could be valuable chemopreventive and therapeutic targets within the lung tumor microenvironment in addition to being targets within tumor itself, and that silibinin inhibits their activation as a plausible mechanism of its efficacy against lung cancer.
Insights
Silibinin, a compound found in milk thistle, shows promise in lung cancer prevention and treatment. It works by inhibiting key signaling pathways like STAT3 and STAT1, which are crucial for tumor growth and spread.
Area of Science:
- Oncology
- Molecular Biology
- Pharmacology
Background:
- Tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) can promote lung cancer by inducing pro-inflammatory enzymes.
- Signal transducers and activators of transcription (STAT) proteins play critical roles in cellular signaling pathways relevant to cancer.
Purpose of the Study:
- To investigate the mechanism by which silibinin affects cytokine-induced signaling in lung cancer cells.
- To determine if STAT3 and STAT1 are viable therapeutic targets for lung cancer chemoprevention and treatment.
Main Methods:
- LM2 mouse lung epithelial cells were treated with a mixture of TNF-α and IFN-γ (cytokine mixture).
- The effects of silibinin, MEK, NF-κB, EGFR, and JAK inhibitors on STAT and downstream protein activation and gene expression were analyzed.
- Cell migration assays were performed to assess the impact of silibinin on cell motility.
Main Results:
- Cytokine mixture activated STAT3, STAT1, and Erk1/2, leading to increased expression of cyclooxygenase 2 (COX2) and inducible nitric oxide synthase (iNOS).
- Inhibitors of MEK, NF-κB, and EGFR modulated cytokine-induced signaling, highlighting the involvement of these pathways.
- Silibinin pretreatment inhibited cytokine-induced STAT3/STAT1 phosphorylation, NF-κB DNA binding, and expression of COX2, iNOS, MMP2, and MMP9, while also reducing cell migration.
Conclusions:
- STAT3 and STAT1 are key mediators of cytokine-induced pro-tumorigenic signaling in lung epithelial cells.
- Silibinin effectively inhibits STAT3 and STAT1 activation, nuclear localization, and downstream signaling, suggesting its potential as a chemopreventive and therapeutic agent for lung cancer.
- Targeting STAT3 and STAT1 within the lung tumor microenvironment offers a promising strategy for cancer therapy.
