Ilya Pinchuk

Celecoxib and Curcumin Synergistically Inhibit the Growth of Colorectal Cancer Cells

Purpose: Multiple studies have indicated that cyclooxygenase-2 (COX-2) inhibitors may prevent colon cancer, which is one of the leading causes of cancer death in the western world. Recent studies, however, showed that their long-term use may be limited due to cardiovascular toxicity. This study aims to investigate whether curcumin potentiates the growth inhibitory effect of celecoxib, a specific COX-2 inhibitor, in human colon cancer cells. Experimental Design: HT-29 and IEC-18-K-ras (expressing high levels of COX-2), Caco-2 (expressing low level of COX-2), and SW-480 (no expression of COX-2) cell lines were exposed to different concentrations of celecoxib (0-50 μmol/L), curcumin (0-20 μmol/L), and their combination. COX-2 activity was assessed by measuring prostaglandin E2 production by enzyme-linked immunoassay. COX-2 mRNA levels were assessed by reverse transcription-PCR. Results: Exposure to curcumin (10-15 μmol/L) and physiologic doses of celecoxib (5 μmol/L) resulted in a synergistic inhibitory effect on cell growth. Growth inhibition was associated with inhibition of proliferation and induction of apoptosis. Curcumin augmented celecoxib inhibition of prostaglandin E2 synthesis. The drugs synergistically down-regulated COX-2 mRNA expression. Western blot analysis showed that the level of COX-1 was not altered by treatment with celecoxib, curcumin, or their combination. Conclusions: Curcumin potentiates the growth inhibitory effect of celecoxib by shifting the dose-response curve to the left. The synergistic growth inhibitory effect was mediated through a mechanism that probably involves inhibition of the COX-2 pathway and may involve other non–COX-2 pathways. This synergistic effect is clinically important because it can be achieved in the serum of patients receiving standard anti-inflammatory or antineoplastic dosages of celecoxib.

The mechanism of action of antioxidants against lipoprotein peroxidation, evaluation based on kinetic experiments

Peroxidation of blood lipoproteins is regarded as a key event in the development of atherosclerosis. Hence, attenuation of the oxidative modification of lipoproteins by natural and synthetic antioxidants in vivo is considered a possible way of prevention of cardiovascular disorders. The assessment of the susceptibility of lipoproteins to oxidation is commonly based on in vitro oxidation experiments. Monitoring of oxidation provides the kinetic profile characteristic for the given lipoprotein preparation. The kinetic profile of peroxidation is characterized by three major parameters: the lag preceding rapid oxidation, the maximal rate of oxidation (V(max)) and the maximal accumulation of oxidation products (OD(max)). Addition of antioxidants alters this pattern, affecting the kinetic parameters of oxidation. In particular, antioxidants may prolong the lag and/or decrease the V(max) and/or decrease the OD(max). Such specific variation of the set of kinetic parameters may provide important information on the mechanism of the inhibitory action of a given antioxidant (scavenging free radicals, metal-binding or other mechanisms). Numerous natural and synthetic compounds were reported to inhibit oxidation of lipoproteins. Based on the analysis of reported effects and theoretical considerations, we propose a simple protocol that relates the kinetic effects of a given antioxidant to the mechanism of its action.

Peroxidation of liposomal lipids

Free radicals, formed via different mechanisms, induce peroxidation of membrane lipids. This process is of great importance because it modifies the physical properties of the membranes, including its permeability to different solutes and the packing of lipids and proteins in the membranes, which in turn, influences the membranes’ function. Accordingly, much research effort has been devoted to the understanding of the factors that govern peroxidation, including the composition and properties of the membranes and the inducer of peroxidation. In view of the complexity of biological membranes, much work was devoted to the latter issues in simplified model systems, mostly lipid vesicles (liposomes). Although peroxidation in model membranes may be very different from peroxidation in biological membranes, the results obtained in model membranes may be used to advance our understanding of issues that cannot be studied in biological membranes. Nonetheless, in spite of the relative simplicity of peroxidation of liposomal lipids, these reactions are still quite complex because they depend in a complex fashion on both the inducer of peroxidation and the composition and physical properties of the liposomes. This complexity is the most likely cause of the apparent contradictions of literature results. The main conclusion of this review is that most, if not all, of the published results (sometimes apparently contradictory) on the peroxidation of liposomal lipids can be understood on the basis of the physico-chemical properties of the liposomes. Specifically: (1) The kinetics of peroxidation induced by an “external” generator of free radicals (e.g. AAPH) is governed by the balance between the effects of membrane properties on the rate constants of propagation (kp) and termination (kt) of the free radical peroxidation in the relevant membrane domains, i.e. in those domains in which the oxidizable lipids reside. Both these rate constants depend similarly on the packing of lipids in the bilayer, but influence the overall rate in opposite directions. (2) Peroxidation induced by transition metal ions depends on additional factors, including the binding of metal ions to the lipid–water interface and the formation of a metal ions-hydroperoxide complex at the surface. (3) Reducing agents, commonly regarded as “antioxidants”, may either promote or inhibit peroxidation, depending on the membrane composition, the inducer of oxidation and the membrane/water partitioning. All the published data can be explained in terms of these (quite complex) generalizations. More detailed analysis requires additional experimental investigations.

Kinetic analysis of copper-induced peroxidation of LDL

We have employed our recently developed spectroscopic method of continuous monitoring of lipid oxidation to study the formation and decomposition of hydroperoxides in the time course of LDL oxidation. The results show satisfactory agreement with simulated time courses based on the following assumptions: (a) Both the rates of formation and decomposition of hydroperoxides depend on the ratio of bound copper to LDL as computed under the assumption that each LDL particle has 17 equivalent copper binding sites characterized by a dissociation constant K = 1 microM. (b) Peroxidation is initiated by copper-catalyzed decomposition of hydroperoxides (LOOH) into peroxy radicals (LOO.) and other products, including dienals. Under these assumptions, the rate of accumulation of LOOH can be computed from the equation (equation in text). The agreement between the simulated and experimentally-observed kinetics supports the assumptions used for simulations. The close agreement between the values of lipid oxidizability (kp/square root 2kt) obtained for LDL (0.035 (Ms)[-1/2]) and previously published data on the oxidizability of linoleates (0.02-0.11 (Ms)[-1/2]) lends further support for these assumptions.

Decision analysis supports the paradigm that indiscriminate supplementation of vitamin E does more harm than good.

Objectives—For many years, the prevailing concept was that LDL oxidation plays a central role in atherogenesis. As a consequence, supplementation of antioxidants, particularly vitamin E, became very popular. Unfortunately, however, the major randomized clinical trials have yielded disappointing results on the effects of vitamin E on both mortality and morbidity. Moreover, recent meta-analyses have concluded that vitamin E supplementation increases mortality. This conclusion has raised much criticism, most of it relating to three issues: (1) the choice of clinical trials to be included in the meta-analyses; (2) the end point of these meta-analyses (only mortality); and (3) the heterogeneity of the analyzed clinical trials with respect to both population and treatment. Our goal was to bring this controversy to an end by using a Markov-model approach, which is free of most of the limitations involved in using meta-analyses. Methods and Results—We used a Markov model to compare the vitamin E supplemented virtual cohorts with nonsupplemented cohorts derived from published randomized clinical trials that were included in at least one of the major meta-analyses. The difference between the virtual supplemented and nonsupplemented cohorts is given in terms of a composite end point denoted quality-adjusted life year (QALY). The vitamin E supplemented virtual cohort had 0.30 QALY (95%CI 0.21 to 0.39) less than the nontreated virtual cohort. Conclusions—Our study demonstrates that in terms of QALY, indiscriminate supplementation of high doses of vitamin E is not beneficial in preventing CVD. Selective supplementation of vitamin E to individuals under oxidative stress requires further investigation.

The effect of albumin on copper-induced LDL oxidation

In an attempt to gain deeper understanding of the mechanism or mechanisms responsible for the protective effect of serum albumin against Cu(2+)-induced peroxidation of low density lipoprotein (LDL), we have examined the influence of the concentrations of bovine serum albumin (BSA), Cu2+ and LDL on the kinetics of peroxidation. Since the common method of monitoring the oxidation by continuous recording of the absorbance of conjugated dienes at 234 nm cannot be used at high BSA-concentrations because of the intensive absorption of BSA, we have monitored the time-dependent increase of absorbance at 245 nm. At this wavelength, conjugated dienes absorb intensely, whereas the background absorbance of BSA is low. Using this method, as well as the TBARS assay for determination of malondialdehyde, over a large range of BSA concentrations, we show that in many cases the influence of BSA on the kinetics of oxidation can be compensated for by increasing the concentration of copper. This reconciles the apparent contradiction between previously published data. Detailed studies of the kinetic profiles obtained under different conditions indicate that binding of Cu2+ to albumin plays the major role in its protective effect while other mechanisms contribute much less than copper binding. This conclusion is consistent with the less pronounced effect of BSA on the oxidation induced by the free radical generator AAPH. It is also shown that the copper-albumin complex is capable of inducing LDL oxidation, although the kinetics of the latter process is very different from that of copper-induced oxidation. Nevertheless, when compared to copper induced oxidation at similar concentration of the oxidation-promotor, the kinetics of oxidation induced by copper-albumin complex is very different and is consistent with a tocopherol mediated peroxidation, characteristic under low radical flux. Similar kinetics was observed for copper-induced oxidation only at much lower copper concentrations.

Polyphenol-induced dissociation of various amyloid fibrils results in a methionine-independent formation of ROS☆

Fibrillization of amyloid polypeptides is accompanied by formation of reactive oxygen species (ROS), which, in turn, is assumed to further promote amyloid-related pathologies. Different polyphenols, all of which are established antioxidants, cause dissociation of amyloid fibrils. This study addresses the latter, poorly understood process. Specifically, we have investigated the dissociation of Abeta(42) fibrils by six different polyphenols, using electron microscopy and spectrofluorometric analysis. Simultanously, we have monitored the production of ROS using electron spin resonance (ESR) and the commercially available peroxide assay kit. Using the same methods we found that curcumin, one of the most potent destabilizing agents of Abeta(42), induced dissociation of fibrils of other amyloid polypeptides [Abeta(40), Abeta(42)Nle35, islet amyloid polypeptide and a fragment of alpha-synuclein]. When the solution contained traces of transition metal, all the dissociation reactions were accompanied by ROS formation, independent of the presence of a methionine residue. Kinetic studies show that the formation of ROS lags behind dissociation, indicating that if casual relationship exists between these two processes, then ROS formation may be considered a consequence and not a cause of dissociation. These findings open new avenues in amyloid research that will be required to gain further understanding of our results and of their implications.

Peroxidation of liposomal palmitoyllinoleoylphosphatidylcholine (PLPC), effects of surface charge on the oxidizability and on the potency of antioxidants

Peroxidation of membrane phospholipids is an important determinant of membrane function. Previously we studied the kinetics of peroxidation of the polyunsaturated fatty acid (PUFA) residues in model membranes (liposomes) made by sonication of palmitoyllinoleoylphosphatidylcholine (PLPC). Since most biomembranes are negatively-charged, we have now studied the effect of negative surface charge on the kinetics of peroxidation of liposomes made of PLPC and 9% of one of the negatively-charged phospholipids phosphatidylserine (PS) or phosphatidic acid (PA). Peroxidation was initiated by either CuCl2 or AAPH and continuously monitored spectrophotometrically. The following results were obtained: (i) The negative charge had only a slight effect on AAPH-induced peroxidation, but accelerated markedly copper-induced peroxidation of the liposomes, probably by increasing the binding of copper to the membrane surface. (ii) Ascorbic acid (AA) inhibited AAPH-induced but promoted copper-induced peroxidation in all the studied liposomes, probably by enhancing the production of free radicals upon reduction of Cu(II) to Cu(I). (iii) alpha-tocopherol (Toc) inhibited AAPH-induced peroxidation in all the studied liposomes, whereas the effect of tocopherol on copper-induced peroxidation varied from being pro-oxidative in PA-containing liposomes, to being extremely anti-oxidative in PS-containing liposomes, even at very low tocopherol concentrations. The significance of the latter unusual protective effect, which we attribute to recycling of tocopherol by a PS-Cu complex, requires further investigation.

No evidence supports vitamin E indiscriminate supplementation

For many years, the prevailing concept was that LDL oxidation plays the central role in atherogenesis. As a consequence, supplementation of antioxidants, particularly vitamin E, became very popular. Unfortunately, major randomized clinical trials yielded disappointing results and recent meta‐analyses concluded that indiscriminate, high dose vitamin E supplementation results in increased mortality. This conclusion raised (quite reasonable) criticism, much of which referred to the characteristics of meta‐analysis. In our recent study, we used a Markov‐model approach, which is free of most of the limitations of meta‐analyses. Our major finding was that the average quality‐adjusted life years (QALY) of vitamin E‐ supplemented individuals was 0.30 QALY (95%CI 0.21 to 0.39) less than that of untreated people. In our view, this supports the view that indiscriminate supplementation of high dose vitamin E can not be recommended to the general public.

Copper-induced LDL peroxidation: interrelated dependencies of the kinetics on the concentrations of copper, hydroperoxides and tocopherol.

Excessive uptake of oxidized low density lipoprotein plays a role in the onset of atherosclerosis. Lipid‐associated antioxidants, the most abundant of which is tocopherol (vitamin E), are therefore believed to have anti‐atherogenic properties. By contrast, hydroperoxides enhance the peroxidation of low density lipoprotein. We demonstrate that none of these compounds markedly affect the maximal rate of oxidation of low density lipoprotein, whereas the lag preceding rapid oxidation is prolonged by tocopherol but shortened by hydroperoxides. The corresponding ‘prolongation’ and ‘shortening’ can be compensated by each other in low density lipoprotein preparations enriched with both these compounds. The dependence of the balance between the effects of tocopherol and hydroperoxides on the copper concentration indicates that the antioxidative effect of vitamin E increases with the oxidative stress.