Application of Mass Spectrometry Profiling to Establish Brusatol as an Inhibitor of Global Protein Synthesis

Abstract

The KEAP1/Nrf2 pathway senses and responds to changes in intracellular oxidative stress. Mutations that result in constitutive activation of Nrf2 are present in several human tumors, especially non-small cell lung cancer. Therefore, compounds that inhibit Nrf2 activity might be beneficial in treating patients whose tumors show activation of this pathway. Recent reports suggest that the natural product brusatol can potently and selectively inhibit Nrf2 activity, resulting in cell cytotoxicity, and can be effectively combined with chemotherapeutic agents. Here, we analyzed the effects of brusatol on the cellular proteome in the KEAP1 mutant non-small cell lung cancer cell line A549. Brusatol was found to rapidly and potently decrease the expression of the majority of detected proteins, including Nrf2. The most dramatically decreased proteins are those that display a short half-life, like Nrf2. This effect was confirmed by restricting the analysis to newly synthesized proteins using a labeled methionine analogue. Moreover, brusatol increased the expression of multiple components of the ribosome, suggesting that it regulates the function of this macromolecular complex. Finally, we show that brusatol induces its potent cellular cytotoxicity effects on multiple cancer cell lines in a manner independent of KEAP1/Nrf2 activity and with a profile similar to the protein translation inhibitor silvestrol. In conclusion, our data show that the activity of brusatol is not restricted to Nrf2 but,

Brusatol inhibits HIF-1 signaling pathway and suppresses glucose uptake under hypoxic conditions in HCT116 cells

“…Also, inhibition of overall protein synthesis was observed in some cell lines derived from leukemia, lung adenocarcinoma, Ehrlich carcinoma, and hepatoma when the concentration of BRU is higher than 500 nM2122. Recently, BRU has been identified as an unique inhibitor of the Nrf2 pathway at nanomolar concentrations without affecting protein synthesis1423, enhancing the anti-tumor efficacy of cisplatin1424.…”

Nrf2 in cancers: A double‐edged sword

“…Brusatol, a component of Brucea javanica seeds, strongly reduces the protein level of Nrf2 in A549 cells, sensitizing them to cisplatin and other chemotherapeutic drugs . Nevertheless, Brusatol was proved lately to be a nonspecific inhibitor as it rapidly and significantly reduces the level of a large amount of proteins, demonstrating its potential role as a protein translation machinery inhibitor . Ochratoxin A (OTA), produced by Aspergillus and Penicillium species, is both a nephrotoxin and a renal carcinogen.…”

The protein synthesis inhibitor brusatol normalizes high‐fat diet‐induced glucose intolerance in male C57BL/6 mice: role of translation factor eIF5A hypusination

“…In our previous in vitro study, brusatol protected against cytokine-induced b-cell dysfunction and death (15). Brusatol was therefore provided to C57BL/6J mice in their drinking water at 1 mg/kg per day to determine whether HFD-induced glucose intolerance was affected.…”

“…In addition, brusatol and other quassinoids inhibited total protein synthesis at micromolar concentrations (13,14), as well as at nanomolar concentrations, as more recently observed (15). It was therefore proposed that brusatol is primarily an inhibitor of protein synthesis, and that its ability to block different de novo responses, for example the induction of the Nrf-2 response, occurs via this mechanism (15).…”