Vitamin K3 (Menadione)-Induced Oncosis Associated with Keratin 8 Phosphorylation and Histone H3 Arylation

Abstract

The vitamin K analog menadione (K3), capable of both redox cycling and arylating nucleophilic substrates by Michael addition, has been extensively studied as a model stress-inducing quinone in both cell culture and animal model systems. Exposure of keratin 8 (k-8) expressing human breast cancer cells (MCF7, T47D, SKBr3) to K3 (50 -100 M) induced rapid, sustained, and site-specific k-8 serine phosphorylation (pSer73) dependent on signaling by a single mitogen activated protein kinase (MAPK) pathway, MEK1/2. Normal nuclear morphology and k-8 immunofluorescence coupled with the lack of DNA laddering or other features of apoptosis indicated that K3-induced cytotoxicity, evident within 4 h of treatment and delayed but not prevented by MEK1/2 inhibition, was due to a form of stress-activated cell death known as oncosis. Independent of MAPK signaling was the progressive appearance of K3-induced cellular fluorescence, principally nuclear in origin and suggested by in vitro fluorimetry to have been caused by K3 thiol arylation. Imaging by UV transillumination of protein gels containing nuclear extracts from K3-treated cells revealed a prominent 17-kDa band shown to be histone H3 by immunoblotting and mass spectrometry (MS). K3 arylation of histones in vitro followed by electrospray ionization-tandem MS analyses identified the unique Cys110 residue within H3, exposed only in the open chromatin of transcriptionally active genes, as a K3 arylation target. These findings delineate new pathways associated with K3-induced stress and suggest a potentially novel role for H3 Cys110 as a nuclear stress sensor.

Apoptosis inducing factor (AIF) mediates lethal redox stress induced by menadione

“…In the past, studying an endothelial cell death model, Chang et al [29] suggested that the protective effect of GSH could be explained by its capacity to conjugate with menadione outside of the cell and reduce the cellular uptake of the unconjugated form of the quinone. By using GSH as a thiol donor in an in vitro acellular fluorometric assay [28], we confirmed that the formation of conjugates between menadione and GSH led to the appearance of a fluorescent arylation product (Figure 6). Thus, fluorescence spectra analysis revealed that the addition of menadione to the GSH solution sufficed to generate a fluorescence that was undetectable with menadione or GSH alone (Figure 6).…”

“…Both GSH depletion and the menadione-induced fluorescence are facilitated by the expression of AIF. This kind of menadione-induced fluorescence has been reported to result from the arylation of cellular proteins including nuclear histones [27, 28]. One argument in favor of this possibility is the observation that exogenous GSH, which is not cell membrane-permeable (contrary to its ethyl ester that enters cells), protected cells from the cytotoxic effect of menadione and abolished the fluorescent signal (Figure 5C and 5D).…”

Initial approach to the cellular irradiation injury of human pancreatic carcinoma cell line MIA PaCa-2 by high dose per fraction

“…Zheng et al [14] irradiated cylindromatous SACC-83 cells in vitro with different doses (2,5,10,15,20,25,30 Gy) of X-ray. They found cellular apoptosis at 2-15 Gy and obvious necrosis at more than 15 Gy.…”

“…Some studies [20][21][22][23] have shown that treatment through inducing oncotic necrosis of tumor cells can effectively control tumors, if apoptosis process has been inhibited. Oncosis is an important factor in the process of tumor occurrence and development.…”

Novel Pathways Associated with Quinone-Induced Stress in Breast Cancer Cells

“…Nonetheless, recent studies indicate that such non-arylating vitamin K quinones can potentially activate intracellular cell growth and survival signals by their γ-carboxyglutamic acid modification of receptor ligands and membrane lipoproteins (Saxena et al, 2001). We have been exploring the cellular and subcellular consequences of short-term (≤8 h) exposure to the redox-cycling and arylating K3 quinone on both hormone-independent (ER-negative SKBr3) and hormone-dependent (ER-positive MCF7 and T47D) cell line models of the human breast (Liang et al, 1998;Scott et al, 2005).…”