Thiazolide-induced apoptosis in colorectal cancer cells is mediated via the Jun kinase-Bim axis and reveals glutathione-S-transferase P1 as Achilles' heel

Glutathione-S-transferase of the Pi class (GSTP1) is frequently overexpressed in a variety of solid tumors and has been identified as a potential therapeutic target for cancer therapy. GSTP1 is a phase II detoxification enzyme and conjugates the tripeptide glutathione to endogenous metabolites and xenobiotics, thereby limiting the efficacy of antitumor chemotherapeutic treatments. In addition, GSTP1 regulates cellular stress responses and apoptosis by sequestering and inactivating c-Jun N-terminal kinase (JNK). Thiazolides are a novel class of antibiotics for the treatment of intestinal pathogens with no apparent side effects on the host cells and tissue. Here we show that thiazolides induce a GSTP1-dependent and glutathione-enhanced cell death in colorectal tumor cell lines. Downregulation of GSTP1 reduced the apoptotic activity of thiazolides, whereas overexpression enhanced it. Thiazolide treatment caused strong Jun kinase activation and Jun kinase-dependent apoptosis. As a critical downstream target of Jun kinase we identified the pro-apoptotic Bcl-2 homolog Bim. Thiazolides induced Bim expression and activation in a JNK-dependent manner. Downregulation of Bim in turn significantly blocked thiazolide-induced apoptosis. Whereas low concentrations of thiazolides failed to induce apoptosis directly, they potently sensitized colon cancer cells to TNF-related apoptosis-inducing ligand- and chemotherapeutic drug-induced cell death. Although GSTP1 overexpression generally limits chemotherapy and thus antitumor treatment, our study identifies GSTP1 as Achilles' heel and thiazolides as novel interesting apoptosis sensitizer for the treatment of colorectal tumors.

Genetic polymorphisms of manganese-superoxide dismutase and glutathione-S-transferase in chronic alcoholic pancreatitis

Chronic alcohol consumption is a major risk factor for the development of chronic pancreatitis. However, chronic pancreatitis occurs only in a minority of heavy drinkers. This variability may be due to yet unidentified genetic factors. Several enzymes involved in the degradation of reactive oxidants and xenobiotics, such as glutathione-S-transferase P1 (GSTP1) and manganese-superoxide dismutase (MnSOD) reveal functional polymorphisms that affect the antioxidative capacity and may therefore modulate the development of chronic pancreatitis and long-term complications like endocrine and exocrine pancreatic insufficiency. Two functional polymorphisms of the MnSOD and the GSTP1 gene were assessed by polymerase chain reaction and restriction fragment length polymorphism in 165 patients with chronic alcoholic pancreatitis, 140 alcoholics without evidence of pancreatic disease and 160 healthy control subjects. The distribution of GSTP1 and MnSOD genotypes were in Hardy-Weinberg equilibrium in the total cohort. Genotype and allele frequencies for both genes were not statistically different between the three groups. Although genotype MnSOD Ala/Val was seemingly associated with the presence of exocrine pancreatic insufficiency, this subgroup was too small and the association statistically underpowered. None of the tested genotypes affected the development of endocrine pancreatic insufficiency. Polymorphisms of MnSOD and GSTP1 are not associated with chronic alcoholic pancreatitis. The present data emphasize the need for stringently designed candidate gene association studies with well-characterized cases and controls and sufficient statistical power to exclude chance observations.

Tissue-dependent subcellular localization of Drosophila arginine methyl-transferase 4 (DART4), a coactivator whose overexpression affects neither viability nor differentiation

Drosophila arginine methyl-transferase 4 (DART4) belongs to the type I class of arginine methyltransferases. It catalyzes the methylation of arginine residues to monomethylarginines and asymmetrical dimethylarginines. The DART4 sequence is highly similar to mammalian PRMT4/CARM1, and DART4 substrate specificity has been conserved, too. Recently it was suggested that DART4/Carmer functions in ecdysone receptor mediated apoptosis of the polytene larval salivary glands and an apparent up-regulation of DART4/Carmer mRNA levels before tissue histolysis was reported. Here we show that in Drosophila larvae, DART4 is mainly expressed in the imaginal disks and in larval brains, and to a much lesser degree in the polytene larval tissue such as salivary glands. In glands, DART4 protein is present in the cytoplasm and the nucleus. The nuclear signal emanates from the extrachromosomal domain and gets progressively restricted to the region of the nuclear lamina upon pupariation. Surprisingly, DART4 levels do not increase in salivary glands during pupariation, and overexpression of DART4 does not cause precautious cell death in the glands. Furthermore, over- and misexpression of DART4 under the control of the alpha tubulin promoter do not lead to any major problem in the life of a fly. This suggests that DART4 activity is regulated at the posttranslational level and/or that it acts as a true cofactor in vivo. We present evidence that nuclear localization of DART4 may contribute to its function because DART4 accumulation changes from a distribution with a strong cytoplasmic component during the transcriptional quiescence of the young embryo to a predominantly nuclear one at the onset of zygotic transcription.

Thiazolides inhibit growth and induce glutathione-S-transferase Pi (GSTP1)-dependent cell death in human colon cancer cells

Thiazolides are a novel class of broad-spectrum anti-infective drugs with promising in vitro and in vivo activities against intracellular and extracellular protozoan parasites. The nitrothiazole-analogue nitazoxanide (NTZ; 2-acetolyloxy-N-(5-nitro 2-thiazolyl) benzamide) represents the thiazolide parent compound, and a number of bromo- and carboxy-derivatives with differing activities have been synthesized. Here we report that NTZ and the bromo-thiazolide RM4819, but not the carboxy-thiazolide RM4825, inhibited proliferation of the colon cancer cell line Caco2 and nontransformed human foreskin fibroblasts (HFF) at or below concentrations the compounds normally exhibit anti-parasitic activity. Thiazolides induced typical signs of apoptosis, such as nuclear condensation, DNA fragmentation and phosphatidylserine exposure. Interestingly, the apoptosis-inducing effect of thiazolides appeared to be cell cycle-dependent and induction of cell cycle arrest substantially inhibited the cell death-inducing activity of these compounds. Using affinity chromatography and mass spectrometry glutathione-S-transferase P1 (GSTP1) from the GST class Pi was identified as a major thiazolide-binding protein. GSTP1 expression was more than 10 times higher in the thiazolide-sensitive Caco2 cells than in the less sensitive HFF cells. The enzymatic activity of recombinant GSTP1 was strongly inhibited by thiazolides. Silencing of GSTP1 using siRNA rendered cells insensitive to RM4819, while overexpression of GSTP1 increased sensitivity to RM4819-induced cell death. Thiazolides may thus represent an interesting novel class of future cancer therapeutics.

Activity of the glycosylating enzyme, core 2 GlcNAc (beta1,6) transferase, is higher in polymorphonuclear leukocytes from diabetic patients compared with age-matched control subjects: relevance to capillary occlusion in diabetic retinopathy

The exact mechanism for capillary occlusion in diabetic retinopathy is still unclear, but increased leukocyte-endothelial cell adhesion has been implicated. We examined the possibility that posttranslational modification of surface O-glycans by increased activity of core 2 transferase (UDP-Glc:Galbeta1-3GalNAcalphaRbeta-N-acetylglucoaminyltr ansferase) is responsible for increased adhesion of leukocytes to vascular endothelium in diabetes. The mean activity of core 2 transferase in polymorphonuclear leukocytes isolated from type 1 and type 2 diabetic patients was higher compared with age-matched control subjects (1,638 +/- 91 [n = 42] vs. 249 +/- 35 pmol x h(-1) x mg(-1) protein [n = 24], P = 0.00013; 1,459 +/- 194 [n = 58] vs. 334 +/- 86 [n = 11], P = 0.01). As a group, diabetic patients with retinopathy had significantly higher mean activity of core 2 transferase compared with individuals with no retinopathy. There was a significant association between enzyme activity and severity of retinopathy in type 1 and type 2 diabetic patients. There was a strong correlation between activity of core 2 transferase and extent of leukocyte adhesion to cultured retinal capillary endothelial cells for diabetic patients but not for age-matched control subjects. Results from transfection experiments using human myelocytic cell line (U937) demonstrated a direct relationship between increased activity of core 2 transferase and increased binding to cultured endothelial cells. There was no relationship between activity of core 2 transferase and HbA(1c) (P = 0.8314), serum advanced glycation end product levels (P = 0.4159), age of the patient (P = 0.7896), and duration of diabetes (P = 0.3307). On the basis that branched O-glycans formed by the action of core 2 transferase participate in leukocyte adhesion, the present data suggest the involvement of this enzyme in increased leukocyte-endothelial cell adhesion and the pathogenesis of capillary occlusion in diabetic retinopathy.

Cholestasis in a patient with gallstones and a normal gamma-glutamyl transferase

Cholestasis with normal gamma glutamyl transferase characterizes functional deficiencies in the gene ABCB11, which encodes the bile salt export pump (BSEP), a liver-specific adenosine triphosphate (ATP)-binding cassette transporter. Here we report the case of a patient presenting with features of benign recurrent intrahepatic cholestasis associated with a heterozygous mutation in the ABCB11 gene. Immunohistochemistry showed a gradual decrease of BSEP from zone 1 to zone 3 of the liver lobule, suggesting that the mutation identified here may predispose patients to cholestasis through a delocalization process of BSEP at the lobular level. (HEPATOLOGY 2013;57:2539-2541).

Oxidized bases in the ribosome's peptidyl transferase center and their effects on translation

The ribosome is central to protein biosynthesis and the focus of extensive research. Recent biochemical and structural studies, especially detailed crystal structures and high resolution Cryo-EM in different functional states have broadened our understanding of the ribosome and its mode of action. However, the exact mechanism of peptide bond formation and how the ribosome catalyzes this reaction is not yet understood. Also, consequences of direct oxidative stress to the ribosome and its effects on translation have not been studied. So far, no conventional replacement or even removal of the peptidyl transferase center's bases has been able to affect in vitro translation. Significant contribution to the catalytic activity seems to stem from the ribose-phosphate backbone, specifically 2'OH of A2451. Using the technique of atomic mutagenesis, novel unnatural bases can be introduced to any desired position in the 23S rRNA, surpassing conventional mutagenesis and effectively enabling to alter single atoms in the ribosome. Reconstituting ribosomes in vitro using this approach, we replaced universally conserved PTC bases with synthetic counterparts carrying the most common oxidations 8-oxorA, 5-HOrU and 5-HOrC. To investigate the consequent effects on translation, the chemically engineered ribosomes were studied the in various functional assays. Incorporation of different oxidized bases into the 70S ribosome affected the ribosomes in different ways. Depending on the nucleobase modified, the reconstituted ribosomes exhibited radical deceleration of peptide bond formation, decrease of synthesis efficiency or even an increase of translation rate. These results may further our understanding of the residues involved in the peptide bond formation mechanism, as well as the disease-relevant effects of oxydative stress on the translation machinery.

Oxidized bases in the ribosome's peptidyl transferase center and their effects on translation

The ribosome is central to protein biosynthesis and the focus of extensive research. Recent biochemical and structural studies, especially detailed crystal structures and high resolution Cryo-EM in different functional states have broadened our understanding of the ribosome and its mode of action. However, the exact mechanism of peptide bond formation and how the ribosome catalyzes this reaction is not yet understood. Also, consequences of direct oxidative stress to the ribosome and its effects on translation have not been studied. So far, no conventional replacement or even removal of the peptidyl transferase center's bases has been able to affect in vitro translation. Significant contribution to the catalytic activity seems to stem from the ribose-phosphate backbone, specifically 2'OH of A2451. Using the technique of atomic mutagenesis, novel unnatural bases can be introduced to any desired position in the 23S rRNA, surpassing conventional mutagenesis and effectively enabling to alter single atoms in the ribosome. Reconstituting ribosomes in vitro using this approach, we replaced universally conserved PTC bases with synthetic counterparts carrying the most common oxidations 8-oxorA, 5-HOrU and 5-HOrC. To investigate the consequent effects on translation, the chemically engineered ribosomes were studied the in various functional assays. Incorporation of different oxidized bases into the 70S ribosome affected the ribosomes in different ways. Depending on the nucleobase modified, the reconstituted ribosomes exhibited radical deceleration of peptide bond formation, decrease of synthesis efficiency or even an increase of translation rate. These results may further our understanding of the residues involved in the peptide bond formation mechanism, as well as the disease-relevant effects of oxydative stress on the translation machinery.