HSF1-dependent upregulation of Hsp70 by sulfhydryl-reactive inducers of the KEAP1/NRF2/ARE pathway.

The KEAP1/NRF2/ARE pathway and the heat shock response are inducible cytoprotective systems regulated by transcription factors NRF2 and HSF1, respectively. We report that structurally distinct small molecule NRF2 activators, all of which react with sulfhydryl groups but differ in potency by 15,000-fold, upregulate Hsp70, a prototypic HSF1-dependent gene. Hsp70 upregulation requires HSF1 but is NRF2 independent. We further demonstrate that a sulfoxythiocarbamate inducer conjugates to the negative regulator of HSF1, Hsp90. The differential concentration dependence of the two responses suggests that activation of NRF2 precedes that of HSF1: the KEAP1/NRF2/ARE pathway is at the forefront of cellular defense, protecting against instant danger; the heat shock response closely follows to resolve subsequent potentially devastating damage, saving the proteome. This uncovered duality undoubtedly contributes to the cytoprotective effects of such molecules in models of carcinogenesis, cardiovascular disease, and neurodegeneration.

Bis(2-hydroxybenzylidene)acetone, a potent inducer of the phase 2 response, causes apoptosis in mouse leukemia cells through a p53-independent, caspase-mediated pathway.

Bis(2-hydroxybenzylidene)acetone is a potent inducer of the phase 2 response through the Keap1-Nrf2-ARE pathway. This double Michael reaction acceptor reacts directly with Keap1, the sensor protein for inducers, leading to enhanced transcription of phase 2 genes and protection against oxidant and electrophile toxicities. In our efforts to identify potent chemoprotective agents, we found that in rapidly growing murine leukemia cells (L1210) low concentrations (in the submicromolar range) of bis(2-hydroxybenzylidene)acetone markedly increased the activities of NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1) and glutathione reductase, and the levels of total glutathione, three markers of the phase 2 response. In contrast, at high concentrations (in the micromolar range) the same compound caused G2/M cell cycle arrest and apoptosis. Importantly, a mutant L1210 cell line (Y8), selected for resistance to deoxyadenosine and lacking expression of p53 protein, was considerably more sensitive to the apoptotic effects of bis(2-hydroxybenzylidene)acetone. When caspase activities were evaluated in cell-free extracts prepared from treated wild type or mutant L1210 cells, the activities of caspase-3, the terminal caspase in the cascade leading to apoptosis, and caspase-10 were found to be markedly elevated. The activities of other caspases measured, caspase-1, -6 and -8, were not appreciably affected. Thus, both induction of the phase 2 response and p53-independent, caspase-3-mediated apoptosis could act cooperatively in chemoprotection. The concentration-dependent differential effects on these two pathways should be carefully considered in mechanistic explanations and strategic designs.

Effect of the chalcone analog E,E-bis(2-hydroxybenzylidene) acetone on the 7,12-dimethylbenz[a]anthracene-induced Ha-ras gene activity in vivo.

The chalcone analog E,E-bis(2-hydroxybenzylidene)acetone (HBA) was found to display strong NAD(P)H:quinone reductase (NQO1) inducer potency in Hepa 1c1c7 cells. In order to determine whether this promising chemopreventive activity would extend to anticarcinogenic properties, the effect of HBA on the 7,12-dimethylbenz[a]anthracene (DMBA)-induced expression of the Ha-ras gene in isolated RNA from liver, lung, kidney, spleen, thymus, lymph nodes and bone marrow of CBA/Ca inbred mice was investigated. DMBA is a well-known chemical carcinogen, which can act as initiator by causing point mutations in certain oncogenes and tumor suppressor genes. According to the previous results, elevated Ha-ras expression has been noted even 24 h after DMBA treatment. Administration of HBA simultaneously with DMBA resulted in a decrease of the DMBA-induced Ha-ras gene expression in all the investigated tissues. This observation suggests metabolic interaction of HBA and DMBA. Administration of HBA 24 h prior to the DMBA treatment reduced the Ha-ras gene expression in all the tissues but the liver, where a slight elevation could be detected. This latter effect could be the result of a possible CYPIA inducer and pro-oxidant effects of HBA. The pro-oxidant effect of HBA can be taken into consideration based on its previously demonstrated GSH-reactivity and the present results obtained by investigation of the time-course of Fenton reaction-initiated degradation of 2-deoxyribose in the presence of HBA.