Celecoxib, a selective inhibitor of the enzyme cyclooxygenase-2 (COX-2), has been shown to be a promising chemoprevention agent. The chemopreventive efficacy of celecoxib is believed to be a consequence of its COX-2-dependent and COX-2-independent effects on a variety of cellular processes including proliferation, apoptosis, angiogenesis, and immunosurveillance. In an attempt to identify proteomic markers modulated by celecoxib that are independent of its inhibitory effect on COX-2, the colorectal cancer cell line HCT-116, a nonexpresser of COX-2, was treated with celecoxib. We used the powerful, state-of-the-art two-dimensional difference gel electrophoresis technology coupled with mass spectrometric sequencing to compare global proteomic profiles of HCT-116 cells before and after treatment with celecoxib. Among the differentially expressed proteins identified following celecoxib treatment were proteins involved in diverse cellular functions including glycolysis, protein biosynthesis, DNA synthesis, mRNA processing, protein folding, phosphorylation, redox regulation, and molecular chaperon activities. Our study presents a comprehensive analysis of large-scale celecoxib-modulated proteomic alterations, at least some of which may be mechanistically related to the COX-2-independent chemopreventive effect of celecoxib. (Cancer Epidemiol Biomarkers Prev 2006;15(9):1598 -606)

Ruminococcus albus is an important fibrolytic bacterium in the rumen. Cellobiose is metabolized by this organism via hydrolytic and well as phosphorylytic enzymes, but the relative contributions of each pathway were not clear. The cellobiose consumption rate by exponentially growing cells was less than that of crude extracts (75 versus 243 nmol/min/mg protein). Cellobiose phosphorolytic cleavage was much greater than hydrolytic activity (179 versus 19 nmol/min/mg protein) indicating that phosphorylases were key enzymes in the initial metabolism of the soluble products of cellulose degradation. Cellodextrin phosphorylase appeared to be active against substrates as large as cellohexaose. Phosphorylase activities were cytoplasmic, but hydrolytic activities were associated with both the membrane and cytoplasmic fractions. Free glucose was phosphorylated with a GTP-dependent glucokinase, and this enzyme showed 20-fold higher activity with GTP or ITP (>324 nmol/min/mg protein) than with ATP, UTP, CTP, GDP, or PEP. The activity was decreased at least 57% when mannose, 2-deoxyglucose, or fructose was used as substrate compared with glucose. The Kms for glucose and GTP were 321 and 247 microM, respectively. Since phosphorolytic cleavage conserves more metabolic energy than simple hydrolysis, it is likely that such pathways provide for more efficient growth of R. albus in substrate-limiting conditions like those found in the rumen.

In bacteria, cellobiose and cellodextrins are usually degraded by either hydrolytic or phosphorolytic cleavage. Prevotella ruminicola B 1 4 is a noncellulolytic ruminal bacterium which has the ability to utilize the products of cellulose degradation. In this organism, cellobiose hydrolytic cleavage activity was threefold greater than phosphorolytic cleavage activity (113 versus 34 nmol/min/mg of protein), as measured by an enzymatic assay. Cellobiose phosphorylase activity (measured as the release of P i) was found in cellobiose-, mannose-, xylose-, lactose-, and cellodextrin-grown cells (>92 nmol of P i /min/mg of protein), but the activity was reduced by more than 74% for cells grown on fructose, L-arabinose, sucrose, maltose, or glucose. A small amount of cellodextrin phosphorylase activity (19 nmol/min/mg of protein) was also detected, and both phosphorylase activities were located in the cytoplasm. Degradation involving phosphorolytic cleavage conserves more metabolic energy than simple hydrolysis, and such degradation is consistent with substrate-limiting conditions such as those often found in the rumen. MATERIALS AND METHODS Organisms and cell growth. P. ruminicola B 1 4 was obtained from J. B. Russell, Cornell University, Ithaca, N.Y. The organism was cultured in an anaerobic medium containing the following (per liter): 292 mg of K 2 HPO 4 , 240 mg of KH 2 PO 4 , 480 mg of Na 2 SO 4 , 480 mg of NaCl, 100 mg of MgSO 4 ⅐ 7H 2 O, 64 mg of CaCl 2 ⅐ H 2 O, 600 mg of cysteine, 4 g of Na 2 CO 3 , 1 g of Trypticase (Becton Dickinson Microbiological Systems, Cockeysville, Md.), 500 mg of yeast extract, 100 mg of resazurin, 22.1 mM acetate, 6.0 mM propionate, 2.4 mM butyrate, 0.68 mM valerate, 0.68 mM isovalerate, 0.81 mM isobutyrate, and 0.66 mM 2-methylbutyrate. The medium was adjusted to pH 6.7 with NaOH and prepared and maintained under a carbon dioxide atmosphere. Carbohydrates were added as separate solutions, and cultures were grown at 39ЊC. Cell fractionation. Cells were harvested during logarithmic growth by centrifugation (15,000 ϫ g for 10 min at 4ЊC) and washed twice with either 50 mM PIPES [piperazine-N,NЈ-bis(2-ethanesulfonic acid) (pH 6.8)] for cellobiose phosphorylase and ␤-glucosidase assays or 50 mM Tris-maleate-NaOH (pH 6.8) for cellodextrin phosphorylase assays. Washed cells were passed through a French pressure cell (1,120 kg/cm 2) two times, and debris and whole cells were removed by centrifugation (15,000 ϫ g for 10 min at 4ЊC). Portions of the cell extracts were kept at 4ЊC for assay the same day. The remaining extract was centrifuged (140,000 ϫ g for 30 min at 4ЊC), yielding cytosol (supernatant) and membrane (pellet) fractions. The membrane pellets were washed with the appropriate buffer. Phosphoglucomutase activity was used as a cytosoplasmic marker and assayed as previously described (12). ATPase activity served as a membrane marker and was assayed as described by Kinoshita et al. (11). Cellobiose degradation by crude extracts. The total cellobiose cleavage activity was determined in an...

Prevotella ruminicola is an important ruminal bacteria. In maltose-grown cells, nearly 60% of cell dry weight consisted of high-molecular-weight (>2 ؋ 10 6 ) glycogen. The ratio of glycogen to protein (grams per gram) was relatively low (1.3) during exponential growth, but when cell growth slowed during the transition to the stationary phase, the ratio increased to 1.8. As much as 40% of the maltose was converted to glycogen during cell growth. Glycogen accumulation in glucose-grown cells was threefold lower than that in maltose-grown cells. In continuous cultures provided with maltose, much less glycogen was synthesized at high (>0.2 per h) than at low dilution rates, where maltose was limiting (28 versus 60% of dry weight, respectively). These results indicated that glycogen synthesis was stimulated at low growth rates and was also influenced by the growth substrate. In permeabilized cells, glycogen was synthesized from [ 14 C]glucose-1-phosphate but not radiolabelled glucose, indicating that glucose-1-phosphate is the initial precursor of glycogen formation. Glycogen accumulation may provide a survival mechanism for P. ruminicola during periods of carbon starvation and may have a role in controlling starch fermentation in the rumen.

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