Ion-trap tandem mass spectrometric analysis of Amadori-glycated phosphatidylethanolamine in human plasma with or without diabetes

Peroxidized phospholipid-mediated cytotoxicity is involved in the pathophysiology of diseases [i.e., an abnormal increase of phosphatidylcholine hydroperoxide (PCOOH) in plasma of type 2 diabetic patients]. The PCOOH accumulation may relate to Amadori-glycated phosphatidylethanolamine (Amadori-PE; deoxy-D-fructosyl phosphatidylethanolamine), because Amadori-PE causes oxidative stress. However, the occurrence of lipid glycation products, including Amadori-PE, in vivo is still unclear. Consequently, we developed an analysis method of Amadori-PE using a quadrupole/linear ion-trap mass spectrometer, the Applied Biosystems QTRAP. In positive ion mode, collision-induced dissociation of Amadori-PE produced a well-characterized diglyceride ion ([M+H-303]+) permitting neutral loss scanning and multiple reaction monitoring (MRM). When lipid extract from diabetic plasma was infused directly into the QTRAP, Amadori-PE molecular species could be screened out by neutral loss scanning. Interfacing liquid chromatography with QTRAP mass spectrometry enabled the separation and determination of predominant plasma Amadori-PE species with sensitivity of approximately 0.1 pmol/injection in MRM. The plasma Amadori-PE level was 0.08 mol% of total PE in healthy subjects and 0.15-0.29 mol% in diabetic patients. Furthermore, plasma Amadori-PE levels were positively correlated with PCOOH (a maker for oxidative stress). These results show the involvement between lipid glycation and lipid peroxidation in diabetes pathogenesis.     

Synthetically prepared Aamadori-glycated phosphatidylethanolaminecan trigger lipid peroxidation via free radical reactions

This study for the first time confirmed the peroxidative role of the Amadori product derived from the glycation of phosphatidylethanolamine (PE), namely Amadori-PE. The product was synthesized from the reaction of dioleoyl PE with D-glucose, and then purified by a solid-phase extraction procedure, which was a key step in the next HPLC technique for the isolation of essentially pure Amadori-PE. When the synthetically prepared Amadori-PE was incubated with linoleic acid in the presence of Fe(3+) in micellar system, a remarkable formation of thiobarbituric acid reactive substances was observed together with increases in lipid hydroperoxides. In addition, the lipid peroxidation caused by Amadori-PE was effectively inhibited by superoxide dismutase, mannitol, catalase and metal chelator. These results indicated that Amadori-PE triggers oxidative modification of lipids via the generation of superoxide, and implied the involvement of 'lipid glycation' along with membrane lipid peroxidation in the pathogenesis of diabetes and aging.     

Amadori-glycated phosphatidylethanolamine induces angiogenic differentiations in cultured human umbilical vein endothelial cells

Glycation has been implicated in the endothelial dysfunction that contributes to both diabetes- and aging-associated vascular complications. The aim of the present study was to determine whether Amadori-glycated phosphatidylethanolamine (Amadori-PE), a lipid-linked glycation compound that is formed at an increased rate in hyperglycemic states, affected proliferation, migration and tube formation of cultured human umbilical vein endothelial cells (HUVEC). Amadori-PE at a low concentration of less than 5 microM significantly enhanced these three factors involved in angiogenesis. Furthermore, stimulation of HUVEC with Amadori-PE resulted in secretion of matrix metalloproteinase 2 (MMP-2), a pivotal enzyme in the initial step of angiogenesis. Our results demonstrated for the first time that Amadori-PE may be an important compound that promotes vascular disease as a result of its angiogenic activity on endothelial cells. We also demonstrated that MMP-2 is a primary mediator of Amadori-PE-driven angiogenesis.     

Targeting critical steps of cancer metastasis and recurrence using telomerase template antagonists

Metastasis, tumor relapse, and drug resistance remain major obstacles in the treatment of cancer. Therefore, more research on the mechanisms of these processes in disease is warranted for improved treatment options. Recent evidence suggests that the capability to sustain tumor growth and metastasis resides in a subpopulation of cells, termed cancer stem cells or tumor-initiating cells. Continuous proliferation and self-renewal are characteristics of stem/progenitor cells. Telomerase and the maintenance of telomeres are key players in the ability of stem and cancer cells to bypass senescence and be immortal. Therefore, telomerase inhibitors have the therapeutic potential for reducing tumor relapse by targeting cancer stem cells and other processes involved in metastasis. Herein we review the role of telomerase in the immortal phenotype of cancer and cancer stem cells, targeting telomerase in cancer, and discuss other opportunities for telomerase inhibitors to target critical steps in cancer metastasis and recurrence.     

端粒、端粒酶与肿瘤的相关研究进展

端粒是真核细胞染色体末端的DNA序列,在维持染色体的稳定中起着重要的作用。快速生长的细胞通过端粒酶来合成端 粒重复序列以弥补其损耗。在人类恶性肿瘤细胞中,85%以上能检测到端粒酶的活性,使其成为一个几乎普遍的癌标志物,而在大 多数正常体细胞中,端粒酶是阴性的。端粒酶与肿瘤之间的最新研究已经在肿瘤生物学领域开辟了新的途径,可能会彻底改变抗 癌疗法。在这篇文章中,我们将会总结端粒和端粒酶在癌细胞中的作用。随着科技的发展,端粒和端粒酶拥有巨大的潜力,必将能 够为肿瘤的治疗带来更多的方法。     

Aminophospholipid glycation and its inhibitor screening system: a new role of pyridoxal 5'-phosphate as the inhibitor

Peroxidized phospholipid-mediated cytotoxity is involved in the pathophysiology of a number of diseases [i.e., the abnormal increase of phosphatidylcholine hydroperoxide (PCOOH) found in the plasma of type 2 diabetic patients]. The PCOOH accumulation may relate to Amadori-glycated phosphatidylethanolamine (deoxy-D-fructosyl PE, or Amadori-PE), because Amadori-PE causes oxidative stress. However, lipid glycation inhibitor has not been discovered yet because of the lack of a lipid glycation model useful for inhibitor screening. We optimized and developed a lipid glycation model considering various reaction conditions (glucose concentration, temperature, buffer type, and pH) between PE and glucose. Using the developed model, various protein glycation inhibitors (aminoguanidine, pyridoxamine, and carnosine), antioxidants (ascorbic acid, alpha-tocopherol, quercetin, and rutin), and other food compounds (L-lysine, L-cysteine, pyridoxine, pyridoxal, and pyridoxal 5'-phosphate) were evaluated for their antiglycative properties. Pyridoxal 5'-phosphate and pyridoxal (vitamin B(6) derivatives) were the most effective antiglycative compounds. These pyridoxals could easily be condensed with PE before the glucose/PE reaction occurred. Because PE-pyridoxal 5'-phosphate adduct was detectable in human red blood cells and the increased plasma Amadori-PE concentration in streptozotocin-induced diabetic rats was decreased by dietary supplementation of pyridoxal 5'-phosphate, it is likely that pyridoxal 5'-phosphate acts as a lipid glycation inhibitor in vivo, which possibly contributes to diabetes prevention.     

An introduction to telomeres and telomerase

It is now more than a dozen years since the enzyme telomerase was discovered, and since that time, key studies have characterized the structural components of the enzyme and the associated telomeric proteins. Since the original discovery of telomerase, a clear association with cancer has been demonstrated. In normal somatic cells the telomeres at the ends of chromosomes shorten with every cell division, whereas in cancer cells telomere length is often maintained by reactivation of the enzyme telomerase. These discoveries have led to the proposal that telomerase expression can be used as a helpful marker for diagnostic and prognostic purposes in humans. Another area of research that has developed as a result of improving knowledge and understanding of the role of telomerase in malignancy is that of cancer therapeutics. This article is an introduction to the field of telomere and telomerase research, with an introduction to recent attempts to develop novel cancer treatments based on telomerase structure and function.     

Functional genetics and experimental models of human cancer

Abundant evidence supports the hypothesis that cancer arises from normal cells through the stepwise accumulation of genetic mutations. The study of cells obtained from patients with cancer has identified numerous molecules and pathways that fundamentally contribute to malignant transformation; however, cancer cell lines are often difficult to isolate or maintain, and the cell lines that are available for experimentation represent only a small subset of late-stage human cancers. Recent work has elucidated the role of telomerase in regulating human cell lifespan and has enabled the development of new experimental systems to study human cancer. This review highlights the recent progress in combining genetic methods and primary human cells to understand the role of specific genes and pathways in cancer pathogenesis.     

Targeting telomerase-expressing cancer cells

The role of telomeres and telomerase as a target for cancer therapeutics is an area of continuing interest. This review is intended to provide an update on the field, pointing to areas in which our knowledge remains deficient and exploring the details of the most promising areas being advanced into clinical trials. Topics that will be covered include the role of dysfunctional telomeres in cellular aging and how replicative senescence provides an initial barrier to the emergence of immortalized cells, a hallmark of cancer. As an important translational theme, this review will consider possibilities for selectively targeting telomeres and telomerase to enhance cancer therapy. The role of telomerase as an immunotherapy, as a gene therapy approach using telomerase promoter driven oncolytic viruses and as a small oligonucleotide targeted therapy (Imetelstat) will be discussed.     

Telomere and telomerase in oncology

Telomere and cell replicative senescenceTelomeres, which are located at the end of chro-mosome, are crucial to protect chromosome againstdegeneration, rearrangment and end to end fusion[1].Human telomeres are tandemly repeated units of thehexanucleotide TTAGGG. The estimated length oftelomeric DNA varies from 2 to 20 kilo base pairs,depending on factors such as tissue type and hu-man age. The buck of telomeric DNA is double-stranded, but the end of telomeric DNA consists of3' overhang of…     

Lipid glycation and protein glycation in diabetes and atherosclerosis

Recent instrumental analyses using a hybrid quadrupole/linear ion trap spectrometer in LC-MS/MS have demonstrated that the Maillard reaction progresses not only on proteins but also on amino residues of membrane lipids such as phosphatidylethanolamine (PE), thus forming Amadori-PE (deoxy-d-fructosyl PE) as the principal products. The plasma Amadori-PE level is 0.08 mol% of the total PE in healthy subjects and 0.15–0.29 mol% in diabetic patients. Pyridoxal 5′-phosphate and pyridoxal are the most effective lipid glycation inhibitors, and the PE-pyridoxal 5′-phosphate adduct is detectable in human red blood cells. These findings are beneficial for developing a potential clinical marker for glycemic control as well as potential compounds to prevent the pathogenesis of diabetic complications and atherosclerosis. Glucose and other aldehydes, such as glyoxal, methylglyoxal, and glycolaldehyde, react with the amino residues of proteins to form Amadori products and Heynes rearrangement products. Because several advanced glycation end-product (AGE) inhibitors such as pyridoxamine and benfotiamine inhibit the development of retinopathy and neuropathy in streptozotocin (STZ)-induced diabetic rats, AGEs may play a role in the development of diabetic complications. In the present review, we describe the recent progress and future applications of the Maillard reaction research regarding lipid and protein modifications in diabetes and atherosclerosis.     

Recombination in telomere-length maintenance

Although telomerase is the major mechanism for telomere elongation in most cells, telomerase-independent mechanisms of telomere maintenance can allow cell survival. Yeast cells that lack telomerase maintain telomere length through a form of recombination known as gene conversion. Understanding the role that telomeric recombination might play in mammalian cells has important implications for cancer therapeutics.     

Telomere dysfunction and telomerase activation in cancer--a pathological paradox?

Telomerase is expressed in more than 90% of human cancers. Telomere maintenance by this enzyme is believed to safeguard genomic integrity in neoplastic cells. Nevertheless, many telomerase-expressing tumours exhibit chromosomal instability triggered by short, dysfunctional telomeres, implying that active telomerase is not sufficient for preserving a functional telosomic nucleoprotein complex in cancer cells. We here examine three possible solutions to this ostensible paradox. First, prior to telomerase activation, telomere erosion may have evolved to a level where telomeric repeat sequences are too short to provide a functional substrate for telomerase enzyme activity. Second, mechanisms other than the continuous telomere erosion counteracted by telomerase may contribute to rapid shortening of telomere repeats. Third, dysfunction of telomere-regulating proteins may result in direct telomere uncapping. Moreover, telomerase may contribute to tumour development also through mechanisms unrelated to telomere length maintenance. Taken together, the available data on the role of telomerase in cancer strongly support that inhibition of this enzyme is a feasible strategy for cancer therapy.     

Knock-Down of the 37kDa/67kDa Laminin Receptor LRP/LR Impedes Telomerase Activity

Cancer has become a major problem worldwide due to its increasing incidence and mortality rates. Both the 37kDa/67kDa laminin receptor (LRP/LR) and telomerase are overexpressed in cancer cells. LRP/LR enhances the invasiveness of cancer cells thereby promoting metastasis, supporting angiogenesis and hampering apoptosis. An essential component of telomerase, hTERT is overexpressed in 85–90% of most cancers. hTERT expression and increased telomerase activity are associated with tumor progression. As LRP/LR and hTERT both play a role in cancer progression, we investigated a possible correlation between LRP/LR and telomerase. LRP/LR and hTERT co-localized in the perinuclear compartment of tumorigenic breast cancer (MDA_MB231) cells and non-tumorigenic human embryonic kidney (HEK293) cells. FLAG^® Co-immunoprecipitation assays confirmed an interaction between LRP/LR and hTERT. In addition, flow cytometry revealed that both cell lines displayed high cell surface and intracellular LRP/LR and hTERT levels. Knock-down of LRP/LR by RNAi technology significantly reduced telomerase activity. These results suggest for the first time a novel function of LRP/LR in contributing to telomerase activity. siRNAs targeting LRP/LR may act as a potential alternative therapeutic tool for cancer treatment by (i) blocking metastasis (ii) promoting angiogenesis (iii) inducing apoptosis and (iv) impeding telomerase activity.     

hTERT cDNA片段的克隆及其单克隆抗体与喉癌的发病机制

端粒酶的激活及其调控机制至今不仍不清楚。为研究喉癌发生中端粒酶表达规律及激活的可能机制,我们克隆了hTERT cDNA片段并制备了抗hTERT单克隆抗体。应用此抗体对喉癌组织进行了免疫组化检测,发现喉癌分化程度降低与癌组织中hTERT阳性细胞率增高有关;而c-Myc表达与hTERT表达呈明显正相关,提示c-Myc可能对喉癌发生过程中端粒酶的激活起着重要作用。这些研究表明,喉癌的发生可能是由于c-Myc的过度表达使端粒酶表达上调,从而使喉鳞状上皮细胞达到永生化,这一机制不仅存在于喉癌发生的早期,而且贯穿于喉癌的发展过程。     

The MAPK Pathway Signals Telomerase Modulation in Response to Isothiocyanate-Induced DNA Damage of Human Liver Cancer Cells

4-methylthiobutyl isothiocyanate (MTBITC), an aliphatic, sulphuric compound from Brassica vegetables, possesses in vitro and in vivo antitumor activity. Recently we demonstrated the potent growth inhibitory potential of the DNA damaging agent MTBITC in human liver cancer cells. Here we now show that MTBITC down regulates telomerase which sensitizes cells to apoptosis induction. This is mediated by MAPK activation but independent from production of reactive oxygen species (ROS). Within one hour, MTBITC induced DNA damage in cancer cells correlating to a transient increase in hTERT mRNA expression which then turned into telomerase suppression, evident at mRNA as well as enzyme activity level. To clarify the role of MAPK for telomerase regulation, liver cancer cells were pre-treated with MAPK-specific inhibitors prior to MTBITC exposure. This clearly showed that transient elevation of hTERT mRNA expression was predominantly mediated by the MAPK family member JNK. In contrast, activated ERK1/2 and P38, but not JNK, signalled to telomerase abrogation and consequent apoptosis induction. DNA damage by MTBITC was also strongly abolished by MAPK inhibition. Oxidative stress, as analysed by DCF fluorescence assay, electron spin resonance spectroscopy and formation of 4-hydroxynonenal was found as not relevant for this process. Furthermore, N-acetylcysteine pre-treatment did not impact MTBITC-induced telomerase suppression or depolarization of the mitochondrial membrane potential as marker for apoptosis. Our data therefore imply that upon DNA damage by MTBITC, MAPK are essential for telomerase regulation and consequent growth impairment in liver tumor cells and this detail probably plays an important role in understanding the potential chemotherapeutic efficacy of ITC.