Binuclear palladacycles with ionisable and non-ionisable tethers as anticancer agents.

The search for novel anticancer agents to replace the current platinum-based treatments remains an ongoing process. Palladacycles have shown excellent promise as demonstrated by our previous work which yielded BTC2, a binuclear palladadycle with a non-ionisable polyethylene glycol (PEG) tether. Here, we explore the importance of the PEG-tether length on the anticancer activity of the binuclear palladacycles by comparing three analogous binuclear palladacycles, BTC2, BTC5 and BTC6, in the oestrogen receptor positive MCF7 and triple-negative MDA-MB-231 breast cancer cell lines. In addition, these are compared to another analogue with an ionisable morpholine tether, BTC7. Potent anticancer activity was revealed through cell viability studies (MTT assays) revealed that while BTC6 showed similar potent anticancer activity as BTC2, it was less toxic towards non-cancerous cell lines. Interestingly, BTC7 and BTCF were less potent than the PEGylated palladacycles but showed significantly improved selectivity towards the triple-negative breast cancer cells. Cell death analysis showed that BTC7 and BTCF significantly induced apoptosis in both the cancer cell lines while the PEGylated complexes induced both apoptosis and secondary necrosis. Furthermore, experimental and computational DNA binding studies indicated partial intercalation and groove binding as the modes of action for the PEGylated palladacycles. Similarly, experimental and computational BSA binding studies indicated and specific binding sites in BSA dependent on the nature of the tethers on the complexes.

Maillard reactions by alpha-oxoaldehydes: detection of glyoxal-modified proteins.

Proteins can be chemically modified by sugars by glycation, or the Maillard reaction. The Maillard reaction produces irreversible adducts on proteins that are collectively known as advanced glycation end products, or AGEs. Recent studies indicate that several alpha-dicarbonyl compounds, including glyoxal (GXL), are precursors of AGEs in vivo. We developed antibodies against a GXL-modified protein (GXL-AGE) and purified a mixture of GXL-AGE-specific antibodies by chromatography on GXL-modified bovine serum albumin (BSA-GXL) coupled to EAH-Sepharose. This preparation was then processed on a human serum albumin-carboxymethyllysine (HSA-CML)-NHS-Sepharose to remove CML-specific antibodies. We used the resulting purified antibody in a competitive ELISA to probe GXL-AGEs in vitro and in vivo. We found increasingly greater antibody binding with increasing concentrations of GXL-modified BSA, but the antibody failed to react with either free CML or protein-bound CML. Incubation experiments with BSA revealed that glyceraldehyde, ribose and threose could be precursors of GXL-AGEs as well. Experiments in which GXL was incubated with N-alpha-acetyl amino acids showed that the antibody reacts mostly with lysine modifications. The GXL-derived lysine-lysine crosslinking structure, GOLD was found to be one of the antigenic epitopes for the antibody. Analysis of human plasma proteins revealed significantly higher levels of GXL-AGE antigens in type II diabetic subjects compared with normal controls (P<0.0001). We also found GXL-AGEs in human lens proteins. Bovine aortic endothelial cells cultured for 7 days with 30 mM glucose did not accumulate intracellular GXL-AGEs. These studies underscore the importance of GXL for extracellular AGE formation (except in lens where it is likely to be formed intracellularly) and suggest that changes associated with age and diabetes might be prevented by alteration of GXL-AGE formation.

Early glycation products produce pentosidine cross-links on native proteins. novel mechanism of pentosidine formation and propagation of glycation.

Bovine lens alpha-crystallin was immobilized on EAH-Sepharose gel and glycated using d-ribose. Incubation with 500 and 100 mm d-ribose for 2 and 15 days produced short-term glycated (STGP gel) and long-term glycated proteins (LTGP gel). Both STGP and LTGP gels produced oxygen free radicals. Hydroxyl radical production was twice that in STGP gel compared with the LTGP gel. Incubation with the glycated gels produced pentosidine in a mixture of N-alpha-acetylarginine + N-alpha-acetyllysine, bovine lens proteins (BLP), and lysozyme; the amounts measured with STGP gel were higher than those with LTGP gel. Reactive oxygen species scavengers decreased the formation of pentosidine. Pentosidine was also formed in BLP when incubated with water-insoluble proteins extracted from aged or brunescent human lenses. Early glycated proteins from aged or diabetic lenses were bound to a boronate affinity column, the protein-containing gel was incubated with BLP, and pentosidine was measured in the incubation mixtures. With this method we found that diabetic lens proteins produced more pentosidine on BLP than did aged lens proteins. Further investigation indicates that two and three carbon carbohydrates possibly formed from oxidative cleavage of early glycation products are involved in pentosidine formation. Based on our findings, we propose a novel pathway for pentosidine formation on native proteins from glycated proteins.

Protein crosslinking by the Maillard reaction: dicarbonyl-derived imidazolium crosslinks in aging and diabetes.

alpha-Dicarbonyl compounds that arise from various metabolic pathways react with proteins to form a variety of adducts in a reaction known as the Maillard reaction. These adducts are collectively known as advanced glycation end products or AGEs. Methylglyoxal (MG) and glyoxal (GXL) are two such dicarbonyls. They react with proteins to produce lysine-lysine imidazolium crosslinking AGEs. The imidazolium crosslinks derived from MG (MOLD-methylglyoxal-lysine dimer) and GXL (GOLD-glyoxal-lysine dimer) are present in human tissue proteins. In this study, we report an HPLC method for the simultaneous quantification of GOLD and MOLD in biological specimens. The method consists of reverse-phase HPLC of acid-hydrolyzed proteins, collection of eluate-containing imidazoliums, phenylisothiocyanate derivatization, followed by a second reverse-phase HPLC. This method was linear for both the imidazolium compounds in the range of 0.5-300 pmol. The levels of GOLD and MOLD in aging lenses (20 to 80 years) were trace-8.4 pmol and 15-93 pmol per milligram of protein, respectively. Cataractous lenses showed significantly higher levels of both GOLD and MOLD (mean +/- SD, 14.5 +/- 1.8 and 141 +/- 18.4 pmol per milligram of protein, P < 0.05). Brunescent lenses had the highest levels of imidazolium crosslinks (GOLD, 18.36 +/- 2.5; and MOLD, 179. 2 +/- 32.3 pmol per milligram of protein, P < 0.05). The GOLD and MOLD levels were higher in diabetic plasma proteins when compared to that of normal (GOLD, 17.5 +/- 6.34 pmol per milligram of protein vs 43.5 +/- 15.96 pmol per milligram of protein; and MOLD, 172.5 +/- 32. 53 pmol per milligram of protein vs 273 +/- 62.67 pmol per milligram of protein, P < 0.05). GOLD and MOLD are significant in terms of tissue damage in aging and diabetes because they represent protein crosslinking by compounds that are major precursors of AGEs. Our method can be used for quantification of imidazolium crosslinks in tissue proteins to assess alpha-dicarbonyl-mediated protein damage in vivo.

Chromatographic quantification of argpyrimidine, a methylglyoxal-derived product in tissue proteins: comparison with pentosidine.

Methylglyoxal (MG), an alpha-dicarbonyl compound, can be produced in vivo by several metabolic pathways and the Maillard reaction. It reacts rapidly with proteins to form advanced glycation end products or AGEs. We previously isolated and characterized a blue fluorescent product of the reaction between MG and arginine, which we named argpyrimidine. We found that argpyrimidine was stable to acid hydrolysis, which allowed us to hydrolyze tissue proteins with 6 N HCl and quantify argpyrimidine by high-performance liquid chromatography. Here we report argpyrimidine concentrations in human lens and serum proteins as determined by HPLC. We have also measured pentosidine, a fluorescent AGE derived from pentose sugars, and compared the concentrations of pentosidine and argpyrimidine. We found two- to threefold higher argpyrimidine concentrations in diabetic serum proteins than in nondiabetic controls (9.3 +/- 6.7 vs 4.4 +/- 3.4 pmol/mg). We found a significant correlation (P = 0.0001) between serum protein argpyrimidine and glycosylated hemoglobin. Argpyrimidine concentrations were approximately seven times greater in brunescent cataractous lenses than in aged noncataractous lenses. Pentosidine concentrations in serum and lens proteins were much lower than argpyrimidine concentrations; in general, argpyrimidine levels were 10--25 times higher than pentosidine. Results from our study confirm that MG-mediated arginine modifications occur in vivo and provide a method for assessing protein-arginine modification by MG in aging and diabetes.