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  • Autophagic flux blockage by accumulation of weakly basic tenovins leads to elimination of B-Raf mutant tumour cells that survive vemurafenib.

Autophagic flux blockage by accumulation of weakly basic tenovins leads to elimination of B-Raf mutant tumour cells that survive vemurafenib.

PloS one (2018-04-24)
Marcus J G W Ladds, Andrés Pastor-Fernández, Gergana Popova, Ingeborg M M van Leeuwen, Kai Er Eng, Catherine J Drummond, Lars Johansson, Richard Svensson, Nicholas J Westwood, Anna R McCarthy, Fredrik Tholander, Mihaela Popa, David P Lane, Emmet McCormack, Gerald M McInerney, Ravi Bhatia, Sonia Laín
摘要

Tenovin-6 is the most studied member of a family of small molecules with antitumour activity in vivo. Previously, it has been determined that part of the effects of tenovin-6 associate with its ability to inhibit SirT1 and activate p53. However, tenovin-6 has also been shown to modulate autophagic flux. Here we show that blockage of autophagic flux occurs in a variety of cell lines in response to certain tenovins, that autophagy blockage occurs regardless of the effect of tenovins on SirT1 or p53, and that this blockage is dependent on the aliphatic tertiary amine side chain of these molecules. Additionally, we evaluate the contribution of this tertiary amine to the elimination of proliferating melanoma cells in culture. We also demonstrate that the presence of the tertiary amine is sufficient to lead to death of tumour cells arrested in G1 phase following vemurafenib treatment. We conclude that blockage of autophagic flux by tenovins is necessary to eliminate melanoma cells that survive B-Raf inhibition and achieve total tumour cell kill and that autophagy blockage can be achieved at a lower concentration than by chloroquine. This observation is of great relevance as relapse and resistance are frequently observed in cancer patients treated with B-Raf inhibitors.

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TWEEN ® 20, for molecular biology, viscous liquid
Sigma-Aldrich
胰蛋白酶-EDTA 溶液, 10 ×, sterile-filtered, BioReagent, suitable for cell culture, 5.0 g porcine trypsin and 2 g EDTA • 4Na per liter of 0.9% sodium chloride
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McCoy 5A培养基, Modified, with sodium bicarbonate, without L-glutamine, liquid, sterile-filtered, suitable for cell culture
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DL-二硫苏糖醇, ≥99.0% (RT)
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核糖核酸酶A 来源于牛胰腺, (Solution of 50% glycerol, 10mM Tris-HCL pH 8.0)
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三氯乙酸, BioXtra, ≥99.0%
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低限量Eagle培养基, with Earle′s Balanced Salts, with non-essential amino acids, without L-glutamine, liquid, sterile-filtered, suitable for cell culture