Cysteine endoprotease activity of human ribosomal protein S4 is entirely due to the C-terminal domain, and is consistent with Michaelis-Menten mechanism.

BACKGROUND: It is known that tandem domains of enzymes can carry out catalysis independently or by collaboration. In the case of cysteine proteases, domain sequestration abolishes catalysis because the active site residues are distributed in both domains. The validity of this argument is tested here by using isolated human ribosomal protein S4, which has been recently identified as an unorthodox cysteine protease. METHODS: Cleavage of the peptide substrate Z-FR↓-AMC catalyzed by recombinant C-terminal domain of human S4 (CHS4) is studied by fluorescence-monitored steady-state and stopped-flow kinetic methods. Proteolysis and autoproteolysis were analyzed by electrophoresis. RESULTS: The CHS4 domain comprised of sequence residues 116-263 has been cloned and ovreexpressed in Escherichia coli. The purified domain is enzymatically active. Barring minor differences, steady-state kinetic parameters for catalysis by CHS4 are very similar to those for full-length human S4. Further, stopped-flow transient kinetics of pre-steady-state substrate binding shows that the catalytic mechanism for both full-length S4 and CHS4 obeys the Michaelis-Menten model adequately. Consideration of the evolutionary domain organization of the S4e family of ribosomal proteins indicates that the central domain (residues 94-170) within CHS4 is indispensable. CONCLUSION: The C-terminal domain can carry out catalysis independently and as efficiently as the full-length human S4 does. SIGNIFICANCE: Localization of the enzyme function in the C-terminal domain of human S4 provides the only example of a cysteine endoprotease where substrate-mediated intramolecular domain interaction is irrelevant for catalytic activity.

Arrested cell proliferation through cysteine protease activity of eukaryotic ribosomal protein S4.

S4 is an integral protein of the smaller subunit of cytosolic ribosome. In prokaryotes, it regulates the synthesis of ribosomal proteins by feedback inhibition of the α-operon gene expression, and it facilitates ribosomal RNA synthesis by direct binding to RNA polymerase. However, functional roles of S4 in eukaryotes are poorly understood, although its deficiency in humans is thought to produce Turner syndrome. We report here that wheat S4 is a cysteine protease capable of abrogating total protein synthesis in an actively translating cell-free system of rabbit reticulocytes. The translation-blocked medium, imaged by atomic force microscopy, scanning electron microscopy, and transmission electron microscopy, shows dispersed polysomes, and the disbanded polyribosome elements aggregate to form larger bodies. We also show that human embryonic kidney cells transfected with recombinant wheat S4 are unable to grow and proliferate. The mutant S4 protein, where the putative active site residue Cys 41 is replaced by a phenylalanine, can neither suppress protein synthesis nor arrest cell proliferation, suggesting that the observed phenomenon arises from the cysteine protease attribute of S4. The results also inspire many questions concerning in vivo significance of extraribosomal roles of eukaryotic S4 performed through its protease activity.

C-Phycocyanin inhibits MDR1 through reactive oxygen species and cyclooxygenase-2 mediated pathways in human hepatocellular carcinoma cell line.

The effects of C-Phycocyanin (C-PC), a biliprotein from Spirulina platensis on the regulation of multidrug resistance-1 (MDR1), a poly glycoprotein in human hepatocarcinoma cell line, HepG2 were reported. The results revealed that a significant down regulation of MDR1 expression in C-PC treated HepG2 cells was through reactive oxygen species and cyclooxygenase-2 (COX-2) mediated pathways. C-PC in a concentration dependent manner increased the accumulation of doxorubicin in HepG2 cells and enhanced sensitivity of the cells to doxorubicin by 5 folds. The induction of MDR1 expression by PGE2 and its down regulation by C-PC and DPI (Diphenylene iodonium, NADPH oxidase inhibitor) or by COX-2 knockdown suggest that the enhanced sensitivity of HepG2 cells to doxorubicin by C-PC is mediated by the down regulation of MDR1 expression. Further studies reveal the involvement of NF-κB and AP-1 in the C-PC induced down regulation of MDR1. Also the inactivation of the signal transduction pathways involving Akt, ERK, JNK and p38 by C-PC was observed. The present study thus demonstrates the efficacy of C-PC in overcoming the MDR1 mediated drug resistance in HepG2 cells by the down regulation of reactive oxygen species and COX-2 pathways via the involvement of NF-κB and AP-1.

Celecoxib inhibits MDR1 expression through COX-2-dependent mechanism in human hepatocellular carcinoma (HepG2) cell line.

The role of COX-2 in the regulation of the expression of MDR1, a P-glycoprotein involved in hepatocellular carcinoma cell line, HepG2, was studied in the present investigation. Celecoxib, a selective inhibitor of COX-2, at 25 microM concentration increased the accumulation of doxorubicin in HepG2 cells and enhanced the sensitivity of the cells to doxorubicin by tenfold. The induction of MDR1 expression by PGE2 and its downregulation by celecoxib or by COX-2 knockdown suggests that the enhanced sensitivity of HepG2 cells to doxorubicin by celecoxib is mediated by the downregulation of MDR1 expression, through COX-2-dependent mechanism. Further studies revealed the involvement of AP-1 in the celecoxib-induced downregulation of MDR1 expression. These experimental studies correlated well with in silico predictions and further suggested the inactivation of the signal transduction pathways involving ERK, JNK and p38. The present study thus demonstrates the usefulness of COX-2 intervention in overcoming the drug resistance in HepG2 cells.

Inhibition of 12-LOX and COX-2 reduces the proliferation of human epidermoid carcinoma cells (A431) by modulating the ERK and PI3K-Akt signalling pathways.

Eicosanoids, the oxygenated metabolites of arachidonic acid (AA), mediate a variety of human diseases, such as cancer, inflammation and arthritis. To evaluate the role of eicosanoids in epidermoid carcinoma, the expression of AA metabolizing enzymes, such as lipoxygenases (LOXs) and cyclooxygenases (COXs), was analysed in a human epidermoid carcinoma cell line (A431). These studies revealed overexpression of 12-R-LOX and COX-2 in A431 cells. Baicalein (a 12-LOX inhibitor) and celecoxib (a COX-2 inhibitor) significantly reduced thymidine incorporation, whereas 12-(R)-HETE and 12-(S)-HETE (12-LOX metabolites) and PGE(2) (COX-2 metabolite) significantly enhanced thymidine incorporation, suggesting a role for these enzymes in the regulation of A431 cell proliferation. Further studies on the mechanism of cell death by baicalein and celecoxib revealed that the induction of apoptosis in A431 cells was associated with reduction in the Bcl-2/Bax ratio, release of cytochrome c, activation of caspase-3 and PARP cleavage. The apoptosis induced by baicalein and celecoxib was mediated by down regulation of ERK and PI3K-Akt pathways. Further, 12-(R)-HETE, 12-(S)-HETE and PGE(2) upregulated the p-ERK and p-Akt levels, suggesting the involvement of ERK and Akt pathways in the 12-LOX- and COX-2-mediated regulation of growth in A431 cells. Our findings suggest that 12-R-LOX and COX-2 play a critical role in the regulation of growth in epidermoid carcinoma and that their inhibitors may be of potential therapeutic importance.

Na(+)-stimulated Na+/H+ exchange and an unfavorable Ca2+ homeostasis initiate the cycloxygenase-2 inhibitors-induced apoptotic signals in colonic epithelial cells during the early stage of colon carcinogenesis.

Evidence suggests that nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit cycloxygenase (COX) and production of the proinflammatory prostaglandin, PGE2, and thus prevent carcinogenesis in the colon. Indeed, one of the specific COX-2 inhibitors, celecoxib, had been accepted by the US FDA for the treatment of familial adenomatous polyposis. However, the molecular mechanism of such inhibition is not clear, although apoptosis appears to be the dominant antiproliferative end effect. The present study delineates the intracellular ionic milieu in the colonocytes that could generate strong apoptotic signals where DMH-induced carcinogenesis was studied in the initiation stage in rats and its regression with the COX inhibitors. While DMH treatment produced a significant elevation in the Na+/H+ exchanger activity and resultant proton efflux, this was reversed by the NSAIDs, particularly so with celecoxib and etoricoxib compared to aspirin. Similarly, the intracellular pH was changed, with more alkalosis noted in DMH, which was reversed by NSAIDs. Also, an intracellular Ca2+ build up was noted by Fura 2 AM, which was also supported by a reduced Ca2+ ATPase and an enhanced inward movement of Ca2+. Further, mitochondrial dysfunction-related cyt C release, increased DNA ladder formation, activation of caspase-3, and cleavage product of poly (ADP-ribose) polymerase (PARP) were not seen in DMH but well noted in NSAIDs. Our results indicate that NSAIDs can induce apoptosis through a change in the colonic Na+/H+ exchange, intracellular pH, and an unfavorable Ca2+ homeostasis.

Effects of (15S)-hydroperoxyeicosatetraenoic acid and (15S)-hydroxyeicosatetraenoic acid on the acute- lymphoblastic-leukaemia cell line Jurkat: activation of the Fas-mediated death pathway.

The antiproliferative effects of 15-LOX (15-lipoxygenase) metabolites of arachidonic acid {(15S)-HPETE [(15S)-hydroperoxyeicosatetraenoic acid] and (15S)-HETE [(15S)-hydroxyeicosatetraenoic acid]} and the mechanism(s) involved were studied in the human T-cell leukaemia cell line Jurkat. (15S)-HPETE, the hydroperoxy metabolite of 15-LOX, inhibited the growth of Jurkat cells 3 h after exposure and with an IC(50) value of 10 microM. The hydroxy metabolite of 15-LOX, (15S)-HETE, on the other hand, inhibited the growth of Jurkat cells after 6 h of exposure and with an IC(50) value of 40 microM. The cells exposed to 10 microM (15S)-HPETE for 3 h or to 40 microM (15S)-HETE for 6 h showed increased expression of Fas ligand and FADD (Fas-associated death domain), caspase 8 activation, Bid (BH3-interacting domain death agonist) cleavage, decrease in mitochondrial membrane potential, cytochrome c release, caspase 3 activation, PARP-1 [poly(ADP-ribose) polymerase-1] cleavage and DNA fragmentation, suggesting the involvement of both extrinsic and intrinsic death pathways. Further studies on ROS (reactive oxygen species) generation revealed the involvement of NADPH oxidase. In conclusion, the present study indicates that NADPH oxidase-induced ROS generation activates the Fas-mediated death pathway.

Selective inhibition of cyclooxygenase-2 (COX-2) by 1alpha,25-dihydroxy-16-ene-23-yne-vitamin D3, a less calcemic vitamin D analog.

Inducible cyclooxygenase-2 (COX-2) has been implicated to play a role in inflammation and carcinogenesis and selective COX-2 inhibitors have been considered as anti-inflammatory and cancer chemopreventive agents. 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3), the active hormonal form of vitamin D3 also has been considered to be a cancer chemopreventive agent in addition to its important role in maintaining calcium homeostasis. Based on these observations, we studied the direct effect of 1alpha,25(OH)2D3 and one of its less calcemic synthetic analogs, 1alpha,25(OH)2-16-ene-23-yne-D3 on the activity of both COX-1 and COX-2 in an in vitro enzyme assay. Preliminary data indicated that both 1alpha,25(OH)2D3 and 1alpha,25(OH)2-16-ene-23-yne-D3 inhibited selectively the activity of COX-2 with no effect on the activity of COX-1. Out of the two compounds, 1alpha,25(OH)2-16-ene-23-yne-D3 was found to be more effective with an IC50 of 5.8 nM. Therefore, the rest of the experiments were performed using 1alpha,25(OH)2-16-ene-23-yne-D3 only. 1alpha,25(OH)2-16-ene-23-yne-D3 inhibited the proliferation of lipopolysaccharide (LPS) stimulated mouse macrophage cells (RAW 264.7) with a reduction in the expression of COX-2 along with other inflammatory mediators like inducible nitric oxide synthase (iNOS) and interleukin-2 (IL-2). Furthermore, 1alpha,25(OH)2-16-ene-23-yne-D3 also inhibited carrageenan induced inflammation in an air pouch of a rat and effectively reduced the expression of COX-2, iNOS, and IL-2 in the tissues of the same air pouch. In both cases, 1alpha,25(OH)2-16-ene-23-yne-D3 did not show any effect on the expression of COX-1. In summary, our results indicate that 1alpha,25(OH)2-16-ene-23-yne-D3, a less calcemic vitamin D analog, exhibits potent anti-inflammatory effects and is a selective COX-2 inhibitor.

C-Phycocyanin ameliorates 2-acetylaminofluorene induced oxidative stress and MDR1 expression in the liver of albino mice.

AIM: To study the effect of C-Phycocyanin (C-PC), a biliprotein isolated from Spirulina platensis, on 2-acetylaminofluorene (2-AAF) induced oxidative stress and MDR1 expression in the liver of albino mice. METHODS: In the present study, albino mice aged 40-60 days were used. The mice were randomly assigned to four groups of six animals each. The first group was treated with the vehicle (absolute alcohol), the second group was treated with C-PC (50 mg/kg body weight), the third group was treated with 2-AAF (25 mg/kg body weight) and the fourth group was treated with C-PC (50 mg/kg body weight) and 2-AAF, daily for 3 days. The mice were sacrificed and the tissues were collected and stored for histology and biochemical studies. RESULTS: 2-AAF induced liver tissue damage in albino mice. 2-AAF treatment resulted in upregulation of MDR1 expression and enhanced the generation of reactive oxygen species (ROS). It also induced phosphorylation of Akt and nuclear translocation of NF-kappaB. Co-administration of C-PC and 2-AAF inhibited the expression of MDR1 by preventing ROS generation, Akt phosphorylation and NF-kappaB nuclear translocation. CONCLUSION: 2-AAF-induced oxidative stress is reduced by C-PC treatment. C-PC inhibited the 2-AAF induced expression of MDR1 by interfering at the level of ROS generation, Akt phosphorylation and NF-kappaB translocation. This study reveals the usefulness of C-PC in preventing oxidative stress and downregulation of MDR1 induced by xenobiotics like 2-AAF.

Imatinib-resistant K562 cells are more sensitive to celecoxib, a selective COX-2 inhibitor: role of COX-2 and MDR-1.

Selective inhibition of the BCR/ABL tyrosine kinase by imatinib (STI571, Glivec/Gleevec) is the therapeutic strategy in patients with chronic myelogenous leukemia (CML). Despite significant hematologic and cytogenetic responses with imatinib, mainly due to the mutations in the Abl kinase domain, resistance occurs in patients with advanced disease. In the present study on imatinib-resistant K562 cells (IR-K562), however, no such mutations in the Abl kinase domain were observed. Further studies revealed the over-expression of COX-2 and MDR-1 in IR-K562 cells suggesting the possible involvement of COX-2 in the development of resistance to imatinib. So, we sought to examine the effect of celecoxib, a selective COX-2 inhibitor, on IR-K562 cells. The results clearly indicate that celecoxib is more effective in IR-K562 cells with a lower IC50 value of 10 microM compared to an IC50 value of 40 microM in K562 cells. This increase in the sensitivity of IR-K562 cells towards celecoxib suggests that the development of resistance in IR-K562 cells is COX-2 dependent. Further studies revealed down-regulation of MDR-1 by celecoxib and a decline in p-Akt levels. Celecoxib-induced apoptosis of IR-K562 cells led to release of cytochrome c, PARP cleavage and decreased Bcl2/Bax ratio. Also, celecoxib at 1 microM concentration induced apoptosis in IR-K562 cells synergistically with imatinib by reducing the IC50 value of imatinib from 10 to 6 microM. In conclusion, the present study indicates over-expression of COX-2 and MDR-1 in IR-K562 cells and celecoxib, a COX-2 specific inhibitor, induces apoptosis by inhibiting COX-2 and down-regulating MDR-1 expression through Akt/p-Akt signaling pathway.