Sulfhydryl chemistry plays a vital role in normal biology and in defense of cells against oxidants, free radicals, and electrophiles. Modification of critical cysteine residues is an important mechanism of signal transduction, and perturbation of thiol-disulfide homeostasis is an important consequence of many diseases. A prevalent form of cysteine modification is reversible formation of protein mixed disulfides (protein-SSG) with glutathione (GSH). The abundance of GSH in cells and the ready conversion of sulfenic acids and S-nitroso derivatives to S-glutathione mixed disulfides suggests that reversible S-glutathionylation may be a common feature of redox signal transduction and regulation of the activities of redox sensitive thiol-proteins. The glutaredoxin enzyme has served as a focal point and important tool for evolution of this regulatory mechanism, because it is a specific and efficient catalyst of protein-SSG de-glutathionylation. However, mechanisms of control of intracellular GRx activity in response to various stimuli are not well understood, and delineation of specific mechanisms and enzyme(s) involved in formation of protein-SSG intermediates requires further attention. A large number of proteins have been identified as potentially regulated by reversible S-glutathionylation, but only a few studies have documented glutathionylation-dependent changes in activity of specific proteins in a physiological context. Oxidative stress is a hallmark of many diseases which may interrupt or divert normal redox signaling and perturb protein-thiol homeostasis. Examples involving changes in S-glutathionylation of specific proteins are discussed in the context of diabetes, cardiovascular and lung diseases, cancer, and neurodegenerative diseases.

In murine embryonic fibroblasts, N-acetyl-L-cysteine (NAC), a GSH generating agent, enhances hypoxic apoptosis by blocking the NFB survival pathway (Qanungo, S., Wang, M., and Nieminen, A. L. (2004) J. Biol. Chem. 279, 50455-50464). Here, we examined sulfhydryl modifications of the p65 subunit of NFB that are responsible for NFB inactivation. In MIA PaCa-2 pancreatic cancer cells, hypoxia increased p65-NFB DNA binding and NFB transactivation by 2.6-and 2.8-fold, respectively. NAC blocked these events without having an effect on p65-NFB protein levels and p65-NFB nuclear translocation during hypoxia. Pharmacological inhibition of the NFB pathway also induced hypoxic apoptosis, indicating that the NFB signaling pathway is a major protective mechanism against hypoxic apoptosis. In cell lysates after hypoxia and treatment with N-ethylmaleimide (thiol alkylating agent), dithiothreitol (disulfide reducing agent) was not able to increase binding of p65-NFB to DNA, suggesting that most sulfhydryls in p65-NFB protein were in reduced and activated forms after hypoxia, thereby being blocked by N-ethylmaleimide. In contrast, with hypoxic cells that were also treated with NAC, dithiothreitol increased p65-NFB DNA binding. Glutaredoxin (GRx), which specifically catalyzes reduction of protein-SSG mixed disulfides, reversed inhibition of p65-NFB DNA binding in extracts from cells treated with hypoxia plus NAC and restored NFB activity. This finding indicated that p65-NFB-SSG was formed in situ under hypoxia plus NAC conditions. In cells, knock-down of endogenous GRx1, which also promotes protein glutathionylation under hypoxic radical generating conditions, prevented NAC-induced NFB inactivation and hypoxic apoptosis. The results indicate that GRx-dependent S-glutathionylation of p65-NFB is most likely responsible for NAC-mediated NFB inactivation and enhanced hypoxic apoptosis.Tumor hypoxia is strongly associated with tumor propagation, malignant progression, and resistance to chemo-and radiation therapy (1). NFB 2 is a redox-regulated transcription factor that is activated during hypoxia (2, 3). NFB belongs to the Rel family, which includes five mammalian Rel/NFB proteins: RelA (p65), c-Rel, RelB, NFB1 (p50/p105), and NFB2 (p52/ p100) (4). The inactive form of NFB is localized in the cytoplasm as p65:p50 (the most abundant form) or p50:cRel heterodimers through interaction with IB repressor proteins (IB␣, IB␤, IB␥, and IB⑀) (5). Once activated, NFB translocates to the nucleus, where it binds to DNA and activates various target genes including Bcl-xL, Bcl-2, a hematopoieticspecific Bcl-2 homologue A1, caspase-8-FADD-like interleukin-1␤-converting enzyme inhibitory protein, tumor necrosis factor receptor-associated factors 1 and 2, cellular inhibitors of apoptosis, and X chromosome-linked inhibitor of apoptosis (XIAP/hILP) (6, 7).NFB family proteins have a conserved domain of ϳ300 amino acids in the amino-terminal region known as the Rel homology region. The Rel homology region consists of a DNA binding domain, a dimerization d...

Polyphenols such as epigallocatechin-3-gallate (EGCG) from green tea extract can exert a growth-suppressive effect on human pancreatic cancer cells in vitro. In pursuit of our investigations to dissect the molecular mechanism of EGCG action on pancreatic cancer, we observed that the antiproliferative action of EGCG on pancreatic carcinoma is mediated through programmed cell death or apoptosis as evident from nuclear condensation, caspase-3 activation and poly-ADP ribose polymerase (PARP) cleavage. EGCG-induced apoptosis of pancreatic cancer cells is accompanied by growth arrest at an earlier phase of the cell cycle. In addition, EGCG invokes Bax oligomerization and depolarization of mitochondrial membranes to facilitate cytochrome c release into cytosol. EGCG-induced downregulation of IAP family member X chromosome linked inhibitor of apoptosis protein (XIAP) might be helpful to facilitate cytochrome c mediated downstream caspase activation. On the other end, EGCG elicited the production of intracellular reactive oxygen species (ROS), as well as the c-Jun N-terminal kinase (JNK) activation in pancreatic carcinoma cells. Interestingly, inhibitor of JNK signaling pathway as well as antioxidant N-acetyl-L-cysteine (NAC) blocked EGCG-induced apoptosis. To summarize, our studies suggest that EGCG induces stress signals by damaging mitochondria and ROS-mediated JNK activation in MIA PaCa-2 pancreatic carcinoma cells.

Background Hypertension doubles coronary heart disease (CHD) risk. Treating hypertension only reduces CHD risk ~25%. Treating hypercholesterolemia in hypertensive patients reduces residual CHD risk >35%. Methods and Results To assess progress in concurrent hypertension and hypercholesterolemia control, National Health and Nutrition Examination Surveys 1988–1994, 1999–2004, and 2005–2010 were analyzed. Hypertension was defined by blood pressure (BP) ≥140/≥90 mmHg, current medication treatment, and twice-told hypertension status; BP <140/<90 defined control. Hypercholesterolemia was defined by ATP III criteria based on 10-yr CHD risk, low-density lipoprotein cholesterol (LDL-C) and non-high(H)DL-C; values below diagnostic thresholds defined control. Across surveys, 60.7%–64.3% of hypertensives were hypercholesterolemic. From 1988–1994 to 2005–2010, control of LDL-C rose (9.2% [6.6%–11.9%] to 45.4% [42.6%–48.3%]), concomitant hypertension and LDL-C (5.0% [3.3%–6.7%] to 30.7% [27.9%–33.4%]) and combined hypertension, LDL-C, and non-HDL-C (1.8% [0.4%–3.2%] to 26.9% [24.4%–29.5%]). By multivariable logistic regression, factors associated with concomitant hypertension, LDL-C and non-HDL-C control (odds ratio [95% CI]) were statin (10.7 [8.1–14.3]) and antihypertensive (3.32 [2.45–4.50]) medications, age (0.77 [0.69–0.88/10-yr increase), ≥2 healthcare visits/yr (1.96 [1.23–3.11]) black race (0.59 [0.44–0.80]), Hispanic ethnicity (0.62 [0.43–0.90]), cardiovascular disease ([CVD] 0.44 [0.34–0.56]), and diabetes mellitus (0.54 [0.42–0.70]). Conclusions Despite progress, opportunities for improving concomitant hypertension and hypercholesterolemia control persist. Prescribing antihypertensive and anti-hyperlipidemic medications to achieve treatment goals, especially for older, minority, diabetic and CVD patients, and accessing healthcare at least biannually could improve concurrent risk factor control and CHD prevention.