Silibinin modulates TNF-α and IFN-γ mediated signaling to regulate COX2 and iNOS expression in tumorigenic mouse lung epithelial LM2 cells
Lori D. Dwyer-Nield1,2,
Rana P. Singh1,3,
Alvin M. Malkinson1,2,
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.
NB Silibinin (INN), also known as silybin, is the major active constituent of silymarin, standardized extract of the milk thistle seeds (Silybum marianum)
Growth Inhibition and Regression of Lung Tumors by Silibinin: Modulation of Angiogenesis by Macrophage-Associated Cytokines and Nuclear Factor-κB and Signal Transducers and Activators of Transcription 3
Alpna Tyagi1,Rana P. Singh1,3,Kumaraguruparan Ramasamy1,Komal Raina1,Elizabeth F. Redente1,Lori D. Dwyer-Nield1,Richard A. Radcliffe1,
Alvin M. Malkinson1,2 and Rajesh Agarwal1,2
Abstract The latency period for lung tumor progression offers a window of opportunity for therapeutic intervention. Herein, we studied the effect of oral silibinin (742 mg/kg body weight, 5 d/wk for 10 weeks) on the growth and progression of established lung adenocarcinomas in A/J mice. Silibinin strongly decreased both tumor number and tumor size, an antitumor effect that correlates with reduced antiangiogenic activity. Silibinin reduced microvessel size (50%, P < 0.01) with no change in the number of tumor microvessels and reduced (by 30%, P < 0.05) the formation of nestin-positive microvessels in tumors. Analysis of several proteins involved in new blood vessel formation showed that silibinin decreased the tumor expression of interleukin-13 (47%) and tumor necrosis factor-α (47%), and increased tissue inhibitor of metalloproteinase-1 (2-fold) and tissue inhibitor of metalloproteinase-2 (7-fold) expression, without significant changes in vascular endothelial growth factor levels. Hypoxia- inducible factor-1α expression and nuclear localization were also decreased by silibinin treatment. Cytokines secreted by tumor cells and tumor-associated macrophages regulate angiogenesis by activating nuclear factor-κB (NF-κB) and signal transducers and activators of transcription (STAT). Silibinin decreased the phosphorylation of p65NF-κB (ser276, 38%; P < 0.01) and STAT-3 (ser727, 16%; P < 0.01) in tumor cells and decreased the lung macrophage population. Angiopoietin-2 (Ang-2) and Ang-receptor tyrosine kinase (Tie-2) expression were increased by silibinin. Therapeutic efficacy of silibinin in lung tumor growth inhibition and regression by antiangiogenic mechanisms seem to be mediated by decreased tumor-associated macrophages and cytokines, inhibition of hypoxia-inducible factor-1α, NF-κB, and STAT-3 activation, and up-regulation of the angiogenic inhibitors, Ang-2 and Tie-2.
MORE: Lung cancer is the leading cause of cancer death in both men and women in the United States, with an estimated 213,380 new lung cancer cases and 160,390 associated deaths in 2007 (1). The 5-year survival rate of 14% has shown little improvement over the last 30 years, even with the development of molecularly targeted therapies such as epidermal growth factor receptor inhibitors. Tobacco exposure has been implicated in 90% of lung carcinomas; compared with never smokers, smokers have a 20-fold greater risk of developing lung cancer (2). Because smoking is the major risk factor for developing lung cancer and most smokers have small pulmonary nodules, strategies for inducing nodule regression or preventing their further growth should decrease the number of patients diagnosed with advanced malignant disease.
Efforts are being made towards identifying dietary supplements to prevent and treat lung cancer. One such agent, silibinin, inhibits the growth of various cancer cell lines and primary tumors in several chemically induced rodent models, including mouse lung (3–7). Silibinin is a flavonolignan, a major component in the silymarin complex of flavonolignans and polyphenols present in milk thistle (Silybum marianum) seeds. Silymarin has been extensively used in patients with liver disease for decades (8). Silibinin has shown strong anticancer efficacy against SHP-77 and A549 lung cancer cells, in which it inhibits cell growth and induces cell cycle arrest (9). It also inhibits the invasion of lung cancer cells via down-regulating phosphoinositide 3-kinase-Akt and mitogen-activated protein kinase signaling pathways and decreased production of urokinase-plasminogen activator and matrix metalloproteinase-2 (10, 11). Silibinin inhibits the in vivo growth of A549 xenografts in nude mice, reduces systemic toxicity of doxorubicin, and reduces doxorubicin-induced chemoresistance by inhibiting nuclear factor-κB (NF-κB) signaling (12)...........
In summary, oral silibinin showed antitumor effects in urethane-induced and established lung adenocarcinomas, most likely by decreasing microvessel size and inhibiting newly formed microvessel growth in tumors. The decrease in TAM infiltration into lungs as well as the lower levels of angiogenic cytokines, and greater TIMP-1 and TIMP-2 concentrations, along with the inhibition of HIF-1α, NF-κB, and STAT3 activation, could account for the antiangiogenic effects of silibinin. Additionally, elevating levels of Ang-2 and Tie-2 without changing VEGF amounts could have led to microvessel regression in tumors by silibinin. Overall, our findings here, together with our earlier studies (7), suggest that silibinin is a promising agent for intervention in human lung cancer oncogenesis.
A randomized, controlled, double-blind, pilot study of milk thistle for the treatment of hepatotoxicity in childhood acute lymphoblastic leukemia (ALL)
Elena J. Ladas MS, RD1,
David J. Kroll PhD2,
Nicholas H. Oberlies PhD3,
Bin Cheng PhD4,
Deborah H. Ndao MPH1,
Susan R. Rheingold MD5,
Kara M. Kelly MD1,*,
Abstract BACKGROUND:Despite limited preclinical and clinical investigations, milk thistle (MT) is often used for the treatment of chemotherapy-associated hepatotoxicity. Limited treatment options exist for chemotherapy-related hepatoxicity. Given the wide use of MT, the authors investigated MT in both the laboratory and a clinical setting.
METHODS:In a double-blind study, children with acute lymphoblastic leukemia (ALL) and hepatic toxicity were randomized to MT or placebo orally for 28 days. Liver function tests were evaluated during the study period. To assess MT in vitro, the authors evaluated supratherapeutic concentrations in an ALL cell line.
RESULTS:Fifty children were enrolled. No significant differences in frequency of side effects, incidence and severity of toxicities, or infections were observed between groups. There were no significant changes in mean amino alanine transferase (ALT), aspartate amino transferase (AST), or total bilirubin (TB) at Day 28. At Day 56, the MT group had a significantly lower AST (P = .05) and a trend toward a significantly lower ALT (P = .07). Although not significantly different, chemotherapy doses were reduced in 61% of the MT group compared with 72% of the placebo group. In vitro experiments revealed no antagonistic interactions between MT and vincristine or L-asparaginase in CCRF-CEM cells. A modest synergistic effect with vincristine was observed.
CONCLUSIONS:In children with ALL and liver toxicity, MT was associated with a trend toward significant reductions in liver toxicity. MT did not antagonize the effects of chemotherapy agents used for the treatment of ALL. Future study is needed to determine the most effective dose and duration of MT and its effect on hepatotoxicity and leukemia-free survival.