The anticancer properties of cruciferous vegetables are well known and attributed to an abundance of isothiocyanates (ITCs) such as benzyl ITC (BITC) and phenethyl ITC (PEITC). While many potential targets of ITCs have been proposed, a full understanding of the mechanisms underlying their anticancer activity has remained elusive. Here we report that BITC and PEITC effectively inhibit deubiquitinating enzymes (DUBs), including the enzymes USP9x and UCH37, which are associated with tumorigenesis, at physiologically relevant concentrations and time scales. USP9x protects the anti-apoptotic protein Mcl-1 from degradation, and cells dependent on Mcl-1 were especially sensitive to BITC and PEITC. These ITCs increased Mcl-1 ubiquitination and either ITC treatment or RNAi-mediated silencing of USP9x decreased Mcl-1 levels, consistent with the notion that USP9x is a primary target of ITC activity. These ITCs also increased ubiquitination of the oncogenic fusion protein Bcr-Abl, resulting in degradation under low ITC concentrations and aggregation under high ITC concentrations. USP9x inhibition paralleled the decrease in Bcr-Abl levels induced by ITC treatment, and USP9x silencing was sufficient to decrease Bcr-Abl levels, further suggesting that Bcr-Abl is a USP9x substrate. Overall, our findings suggest that USP9x targeting is critical to the mechanism underpinning the well established anticancer activity of ITC. We propose that the ITC-induced inhibition of DUB may also explain how ITCs affect inflammatory and DNA repair processes, thus offering a unifying theme in understanding the function and useful application of ITCs to treat cancer as well as a variety of other pathological conditions.

Putting a number on it: Cleavable linkers are widely utilized in proteomics applications. In particular, the azobenzene-based linker cleaves under mild conditions that are mass-spectrometry-compatible. Here, we adapt this linker for quantitative proteomic applications by incorporating an isotopic label. These light- and heavy-tagged linkers enable the identification and quantitation of labeled peptides from multiple proteomes.

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Excessive inflammation after traumatic brain injury (TBI) is a major cause of secondary TBI. Though several inflammatory biomarkers have been postulated as the risk factors of TBI, there has not been any comprehensive description of them. Fingolimod, a new kind of immunomodulatory agent which can diminish various kinds of inflammatory responses, has shown additional therapeutic effects in the treatment of intracranial cerebral hematoma (ICH), ischemia, spinal cord injury (SCI), and many other CNS disorders. However, its therapeutic application has not been confirmed in TBI. Thus, we hypothesized that a 3-day consecutive fingolimod administration could broadly modulate the inflammatory reactions and improve the outcomes of TBI. The TBI models of C57/BL6 mice were established with the controlled cortical impact injury (CCI) system. A 3-day consecutive fingolimod therapy (given at 1, 24, and 48 h post injury) was performed at a dose of 1 mg/kg. The flow cytometry, immunoflourence, cytokine array, and ELISA were all applied to evaluate the immune cells and inflammatory markers in the injured brains. Immunohistochemical staining with anti-APP antibody was performed to assess the axonal damage. The neurological functions of these TBI models were assessed by mNSS/Rota-rod and Morris water maze (MWM). The brain water content and integrity of the blood-brain barrier (BBB) were also observed. On the 3rd day after TBI, the accumulation of inflammatory cytokines and chemokines reached the peak and administration of fingolimod reduced as many as 20 kinds of cytokines and chemokines. Fingolimod decreased infiltrated T lymphocytes and NK cells but increased the percentage of regulatory T (Treg) cells, and the concentration of IL-10 on the 3rd day after TBI. Fingolimod also notably attenuated the general activated microglia but augmented the M2/M1 ratio accompanied by decreased axonal damage. The neurological functions were improved after the fingolimod treatment accompanied with alleviation of the brain edema and BBB damage. This study suggests that the 3-day consecutive fingolimod administration extensively modulates multiple immuno-inflammatory responses and improves the neurological deficits after TBI, and therefore, it may be a new approach to the treatment of secondary TBI.