The effects of quercetin on SW480 human colon carcinoma cells: a proteomic study

BACKGROUND: High fruit and vegetable intake is known to reduce the risk of colon cancer. To improve understanding of this phenomenon the action of different phytochemicals on colon cells has been examined. One such compound is quercetin that belongs to the group known as flavonoids. The purpose of this study was to determine the influence of quercetin on the proteome of the SW480 human colon adenocarcinoma cell line, specifically to identify proteins that could be the molecular targets of quercetin in its amelioration of the progression of colon cancer. To this end, two-dimensional gel electrophoresis and mass spectrometry were used to identify proteins that underwent a change in expression following treatment of the cells with 20 μM quercetin. This could elucidate how quercetin may reduce the progression of colon cancer. RESULTS: Quercetin treatment of the SW480 human colon cancer cells was found to result in the decreased expression of three proteins and the increased expression of one protein. The identified proteins with decreased expression were type II cytoskeletal 8 keratin and NADH dehydrogenase Fe-S protein 3. The other protein with decreased expression was not identified. The protein with increased expression belonged to the annexin family. CONCLUSION: Several proteins were determined to have altered expression following treatment with quercetin. Such changes in the levels of these particular proteins could underlie the chemo-protective action of quercetin towards colon cancer

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Determination of the dissociation constants of ropinirole and some impurities and their quantification using capillary zone electrophoresis

Ropinirole, 4-[2-(dipropylamino)ethyl]-1,3-dihydro-2H-indol-2-one, is a potent anti-Parkinson’s disease drug developed by SmithKline Beecham Pharmaceuticals. Capillary zone electrophoresis (CZE) was used for the determination of the dissociation constants of ropinirole and five structurally related impurities, potentially formed during its synthesis and for separation and quantification of these substances. The dissociation constants obtained from the CZE measurements were confirmed by UV spectrophotometry for some of the test compounds, obtaining a good agreement between the values. Careful optimization of the running buffer composition permitted base-line resolution of the six compounds in a borate buffer containing acetonitrile and magnesium sulfate (a 100 mM borate buffer containing 30 mM MgSO4 and 20 vol.% of acetonitrile). It was shown that CZE can determine the level of these impurities, down to a level of 0.05% of the main component within 15 min

Separation and quantification of ropinirole and some impurities using capillary liquid chromatography

Ropinirole, 4-[2-(dipropylamino)ethyl]-1,3-dihydro-2H-indol-2-one, is a potent anti-Parkinson’s disease drug developed by SmithKline Beecham Pharmaceuticals. Capillary liquid chromatography (CLC) was used for the separation and quantification of ropinirole and its five related impurities, potentially formed during its synthesis. A simultaneous optimization of three mobile phase parameters, i.e., pH, buffer concentration and acetonitrile content was performed employing an experimental design approach which proved a powerful tool in method development. The retention factors of the investigated substances in different mobile phases were determined. Baseline resolution of the six substances on a C18 reversed stationary phase was attained using a mobile phase with an optimized composition [acetonitrile–8.7 mM 2-(N-morpholino)ethanesulfonic acid adjusted to pH 6.0 (55:45, v/v)]. It was shown that CLC, operated in the isocratic mode under the mobile phase flow-rate of 4 µl/min, can determine the level of these impurities, down to a level of 0.06% of the main component within 25 min

An investigation concerning the stability of a sterically protected cyanopropyl modified silica substrate

The hydrolytic stability of a sterically protected cyanopropyl modified silica phase, used in reversed-phase mode, was studied. This Zorbax SB-CN stationary phase shows superior hydrolytic stability in comparison with conventional CN stationary phases against intensive flushing with aggressive eluents of high and low pH, plain and also with the addn. of an org. modifier. In addn. to chromatog. and elemental anal., solid-state NMR was also used to characterize the changes in the properties of these phases. Also, the effect of sample vol. and nature of the sample solvent on overloading of the SB-CN columns was studied. Vol. overloading was already obsd., when 10 mL of the sample solved in pure org. modifier was injected on the column. However, if the eluent was used as the sample solvent, no significant loss of column performance could be obsd., at least up to 50 mL of sample injection

An on-column amperometric detector for capillary liquid chomatography

An amperometric detector has been constructed for use with capillary LC columns. The micro glassy-carbon working electrode and the capillary column outlet form a micro wall-jet detector cell. This detector cell is immersed in a vessel containing an Ag/AgCl reference and a Pt counter electrode and has an effective volume between 1 and 0.1 nl. The operational parameters of the detector were determined using a chromatographic system with various fused-silica capillary columns (5 to 30 mum i.d.). The detector has been found to match the performance of 26 mum i.d. capillary columns in terms of sensitivity and variance. An example of application. the chromatographic determination of some phenols is presented