Silibinin is a suppressor of the metastasis-promoting transcription factor ID3.

BACKGROUND: ID3 (inhibitor of DNA binding/differentiation-3) is a transcription factor that enables metastasis by promoting stem cell-like properties in endothelial and tumor cells. The milk thistle flavonolignan silibinin is a phytochemical with anti-metastatic potential through largely unknown mechanisms. HYPOTHESIS/PURPOSE: We have mechanistically investigated the ability of silibinin to inhibit the aberrant activation of ID3 in brain endothelium and non-small cell lung cancer (NSCLC) models. METHODS: Bioinformatic analyses were performed to investigate the co-expression correlation between ID3 and bone morphogenic protein (BMP) ligands/BMP receptors (BMPRs) genes in NSCLC patient datasets. ID3 expression was assessed by immunoblotting and qRT-PCR. Luciferase reporter assays were used to evaluate the gene sequences targeted by silibinin to regulate ID3 transcription. In silico computational modeling and LanthaScreen TR-FRET kinase assays were used to characterize and validate the BMPR inhibitory activity of silibinin. Tumor tissues from NSCLC xenograft models treated with oral silibinin were used to evaluate the in vivo anti-ID3 effects of silibinin. RESULTS: Analysis of lung cancer patient datasets revealed a top-ranked positive association of ID3 with the BMP9 endothelial receptor ACVRL1/ALK1 and the BMP ligand BMP6. Silibinin treatment blocked the BMP9-induced activation of the ALK1-phospho-SMAD1/5-ID3 axis in brain endothelial cells. Constitutive, acquired, and adaptive expression of ID3 in NSCLC cells were all significantly downregulated in response to silibinin. Silibinin blocked ID3 transcription via BMP-responsive elements in ID3 gene enhancers. Silibinin inhibited the kinase activities of BMPRs in the micromolar range, with the lower IC50 values occurring against ACVRL1/ALK1 and BMPR2. In an in vivo NSCLC xenograft model, tumoral overexpression of ID3 was completely suppressed by systematically achievable oral doses of silibinin. CONCLUSIONS: ID3 is a largely undruggable metastasis-promoting transcription factor. Silibinin is a novel suppressor of ID3 that may be explored as a novel therapeutic approach to interfere with the metastatic dissemination capacity of NSCLC.

Estimating the capability of microalgae to physiological acclimatization and genetic adaptation to petroleum and diesel oil contamination.

There is increasing scientific interest in how phytoplankton reacts to petroleum contamination, since crude oil and its derivatives are generating extensive contamination of aquatic environments. However, toxic effects of short-term petroleum exposure are more widely known than the adaptation of phytoplankton to long-term petroleum exposure. An analysis of short-term and long-term effects of petroleum exposure was done using experimental populations of freshwater (Scenedesmus intermedius and Microcystis aeruginosa) and marine (Dunaliella tertiolecta) microalgae isolated from pristine sites without crude oil product contamination. These strains were exposed to increased levels of petroleum and diesel oil. Short-term exposure to petroleum or diesel oil revealed a rapid inhibition of photosynthetic performance and cell proliferation in freshwater and marine phytoplankton species. A broad degree of inter-specific variation in lethal contamination level was observed. When different strains were exposed to petroleum or diesel oil over the long-term, the cultures showed massive destruction of the sensitive cells. Nonetheless, after further incubation, some cultures were able to grow again due to cells that were resistant to the toxins. By means of a fluctuation analysis, discrimination between cells that had become resistant due to physiological acclimatization and resistant cells arising from rare spontaneous mutations was accomplished. In addition, an analysis was done as to the maximum capacity of adaptation to a gradual contamination process. An experimental ratchet protocol was used, which maintains a strong selection pressure in a temporal scale up to several months over very large experimental populations of microalgae. Microalgae are able to survive to petroleum contamination as a result of physiological acclimatization without genetic changes. However, when petroleum concentration exceeds the physiological limits, survival depends exclusively on the occurrence on mutations that confer resistance and subsequent selection of these mutants. Finally, it is certain that further mutations and selection will ultimately determine adaptation of microalgae to the environmental forcing.