Gurmit Singh

Increased expression of mitochondrial glycerophosphate dehydrogenase and antioxidant enzymes in prostate cancer cell lines/cancer

The involvement of mitochondrial glycerophosphate dehydrogenase (mGPDH) has previously been established in the production of ROS in prostate cancer cell lines (LNCaP, DU145, PC3 and CL1). The current study demonstrates that the mRNA level of mGPDH in prostate cancer cells is 3.3–8.9-fold higher compared to the normal prostate epithelial cell line, PNT1A. This is consistent with the enzymatic activity and protein level of mGPDH. However, cytochrome c oxidase (COX) activity is 2.9–3.2-fold down-regulated in androgen-independent prostate cancer cell lines. The level of antioxidant enzymes, catalase, MnSOD and CuZnSOD are up-regulated in prostate cancer cell lines. Furthermore, it was observed that the activity of mGPDH is significantly higher in liver tissues from all mice with cancer compared to liver tissues from control mice. These data suggest that the up-regulation of mGPDH, due to a highly glycolytic environment, contributes to the overall increase in ROS generation and may result in the progression of the cancer.

Collagenase expression and activity in the stromal cells from giant cell tumour of bone

The characteristic bone destruction in giant cell tumour of bone (GCT) is largely attributed to the osteoclast-like giant cells. However, experimental analyses of bone resorption by cells from GCT often fail to exclude the neoplastic spindle-like stromal cells, and several studies have demonstrated that bone resorption by GCT cells is increased in the presence of stromal cells. The spindle-like stromal cells from GCT may therefore actively contribute to the bone resorption observed in the tumour. Type I collagen, a major organic constituent of bone, is effectively degraded by three matrix metalloproteinases (MMPs) known as the collagenases: MMP-1, MMP-8 and MMP-13. We established primary cell cultures from nine patients with GCT and the stromal cell populations were isolated in culture. The production of collagenases by primary cultures of GCT stromal cells was determined through real-time PCR, western blot analysis and a multiplex assay system. Results show that the cells produce MMP-1 and MMP-13 but not MMP-8. Immunohistochemistry confirmed the presence of MMP-1 and MMP-13 in paraffin-embedded GCT tissue samples. Medium conditioned by the stromal cell cultures was capable of proteolytic activity as determined by MMP-1 and MMP-13-specific standardized enzyme activity assays. The spindle-like stromal cells from GCT may therefore actively participate in the bone destruction that is characteristic of the tumour.

Glutamate signaling in healthy and diseased bone

Bone relies on multiple extracellular signaling systems to maintain homeostasis of its normal structure and functions. The amino acid glutamate is a fundamental extracellular messenger molecule in many tissues, and is used in bone for both neural and non-neural signaling. This review focuses on the non-neural interactions, and examines the evolutionarily ancient glutamate signaling system in the context of its application to normal bone functioning and discusses recent findings on the role of glutamate signaling as they pertain to maintaining healthy bone structure. The underlying mechanisms of glutamate signaling and the many roles glutamate plays in modulating bone physiology are featured, including those involved in osteoclast and osteoblast differentiation and mature cell functions. Moreover, the relevance of glutamate signaling systems in diseases that affect bone, such as cancer and rheumatoid arthritis, is discussed, and will highlight how the glutamate system may be exploited as a viable therapeutic target. We will identify novel areas of research where knowledge of glutamate communication mechanisms may aid in our understanding of the complex nature of bone homeostasis. By uncovering the contributions of glutamate in maintaining healthy bone, the reader will discover how this complex molecular signaling system may advance our capacity to treat bone pathologies.

Evaluation of single amino acid chelate derivatives and regioselective radiolabelling of a cyclic peptide for the urokinase plasminogen activator receptor

INTRODUCTIONnThe aim of this work was to investigate the relative radiolabelling kinetics and affinity of a series of ligands for the [(99m)Tc(CO)(3)](+) core, both in the absence and in the presence of competing donors. This information was used to select a suitable ligand for radiolabelling complex peptide-based targeting vectors in high yield under mild conditions.nnnMETHODSnA series of alpha-N-Fmoc-protected lysine derivatives bearing two heterocyclic donor groups at the epsilon-amine (1a, 2-pyridyl; 1b, quinolyl; 1c, 6-methoxy-2-pyridyl; 1d, 2-thiazolyl; 1e, N-methylimidazolyl; 1f, 3-pyridyl) were synthesized and labelled with (99m)Tc. A resin-capture purification strategy for the separation of residual ligand from the radiolabelled product was also developed. The binding affinities of targeted peptides 4, 5a and 5b for uPAR were determined using flow cytometry.nnnRESULTSnVariable temperature radiolabelling reactions using 1a-1f and [(99m)Tc(CO)(3)](+) revealed optimal kinetics and good selectivity for compounds 1a and 1d; in the case of 1a, 1d, and 1e, the labelling can be conducted at ambient temperature. The utility of this class of ligands was further demonstrated by the radiolabelling of a cyclic peptide that is known to target the serine protease receptor uPAR; essentially quantitative incorporation of (99m)Tc occurred exclusively at the SAAC site, despite the presence of a His residue, and without disruption of the disulfide bond.nnnCONCLUSIONnA series of single amino acid chelate (SAAC) ligands have been evaluated for their ability to incorporate (99m)Tc into peptides. The lead agent to emerge from this work is the thiazole SAAC derivative 1d which has demonstrated the ability to regioselectively label the widest range of peptides.

A by-product of glutathione production in cancer cells may cause disruption in bone metabolic processes

Bone is a frequent site for metastasis of breast and prostate cancers, often resulting in pathologic changes in bone metabolism and severe pain. The mechanisms involved are not well understood, but tumour cells may release factors that interfere with bone homeostasis. Several observations have led us to hypothesize that the functional disruptions in bone metastasis are the result of a biological process common to many cell types. The high metabolic activity characteristic of cancer cells often upregulates oxidative stress protection mechanisms such as the antioxidant molecule glutathione. In maintaining redox balance, this normal metabolic response may result in unintended pathologic effects in certain sensitive organ sites. Malignant glioma cells kill surrounding neurons in the brain specifically by secreting the amino acid glutamate, an obligatory waste product of glutathione synthesis. We suggest that glutamate release is a plausible mechanism that may account for the pathologic changes in bone metastasis, since bone, like brain, is also highly sensitive to glutamatergic disruption. This report reviews the available evidence to draw a mechanistic connection between tumour cell oxidative stress and the pathology seen in patients with bone metastasis.

Abstract 3694: The mitochondrial FAD-linked glycerol-3-phosphate dehydrogenase as an inhibitory target for cancer therapeutics

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DCnnThe hypoxic and glycolytic environment of malignant cells facilitates high levels of reactive oxygen species (ROS) production. Breast, ovarian and prostate cancer etiology is associated with the ROS production, which are thought to play an important role in regulating the cancers cell growth, survival and metastasis. A potent and major cellular, however functional limited characterized source of ROS, is the mitochondrial FAD-linked glycerol-3-phosphate dehydrogenase (GPD2). GPD2 is an essential component of the glycerol-3-phosphate shuttle and necessarily expressed in all tissues. In several cancer cell lines, GPD2 is highly expressed and we estimated it accounts for up to 70% of the total ROS production within the prostate cancer cell line PC3. In this study we have utilized this knowledge, to complete a cell-based high throughput-screening (HTS) assay, aimed to identify small molecule inhibitors of the GPD2 dependent ROS formation in PC3 cell lines.nnWe employed the amplex red hydrogen peroxidase detection assay and adapted it to the HTS format. 133 inhibitory compounds from the Canadian Compound Collection were identified to reduce the ROS production activity within the PC3 cells. In order to validate these hits and to determine their mode of action, we PCR amplified GPD2 form PC3-cell cDNA and purified recombinant human GPD2 from a bacterial overproducing strain to homogeneity by employing Strep-tactin affinity-chromatography. The heterologously expressed enzyme, devoid of its mitochondrial target sequence, displayed glycerol-3-phosphate dependent resazurin, dichlorophenolindophenol, as well as tetrazole/phenazine methosulfate oxidoreductase activity. Follow up enzymatic assays of the primary HTS hits with the artificial electron acceptors identified several GPD2 activity specific inhibitors. Subsequent concentration response studies of the HTS derived inhibitors elucidated their in vitro half maximal inhibitory concentration at the range of 10 μM. In order to map GPD2s ROS production site and determine the inhibitor binding sites within the enzyme, we bioinformatically derived the proteins tertiary structure.nnConsidering that GPD2 dependent ROS formation may drive the prostate cancer proliferation, we aim by exploiting the results of this work to generate novel lead molecules as promising potential anti-proliferating cancer drug candidates.nn(This work was supported by a grant from the Canadian Institutes of Health Research to Gurmit Singh)nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3694.

Cell Adhesion Molecules in Tumor Metastasis

When tumors progress to greater malignancy, cells within the tumor develop an increasing ability to detach from neighboring cells and invade through surrounding tissues and tissue boundaries to form new growths (metastasis) at sites distinct from that of the primary tumor. The molecular mechanisms involved in the metastatic process are diverse and not completely understood; however, the processes of cell-cell and cell-extracellular matrix (ECM) adhesion and degradation of the extracellular matrix are accepted as critical. In this chapter, we will focus on the current knowledge of the roles of cell-cell and cell-ECM adhesions in the initiation and maintenance of metastasis at new sites.

Role of TGF-β and IGF in Tumor Progression and Bone Metastases

Metastatic disease contributes to a large proportion of cancer-related deaths, and bone is among the most common sites for metastases for tumors originating in the breast and prostate. The propensity for these cancers to form bone metastases is not completely understood; however, it undoubtedly involves a number of unique characteristics of both the tumor cells and the bone microenvironment. Such an explanation was proposed more than a decade ago with Paget’s “seed and soil” hypothesis, which suggested that meta-static cells are dispersed throughout the body, yet they will only survive and grow upon reaching tissues that are optimal for their growth (reviewed in ref. 1).

COMPARATIVE MORPHOMETRIC STUDY ON BONE REMODELING IN HUMAN SPECIMENS AND IN EXPERIMENTAL MODELS OF METASTATIC BONE DISEASE

Metastatic cancer is the most common malignant tumor affecting bone and is accompanied by significant clinical morbidity, including pain, osteolysis, pathological fracture, spinal compression syndromes and hypercalcemia. Bone destruction is a marked clinical feature and the major source of morbidity associated with bone metastases. Most of these clinical features can be related to structural changes in bone that are caused by effects of the tumor on normal processes of bone remodeling.