11 research outputs found
Necrosis binding of Ac-Lys<sup>0</sup>(IRDye800CW)-Tyr<sup>3</sup>-octreotate: a consequence from cyanine-labeling of small molecules.
There is a growing body of nuclear contrast agents that are repurposed for fluorescence-guided surgery. New contrast agents are obtained by substituting the radioactive tag with, or adding a fluorescent cyanine to the molecular structure of antibodies or peptides. This enables intra-operative fluorescent detection of cancerous tissue, leading to more complete tumor resection. However, these fluorescent cyanines can have a remarkable influence on pharmacokinetics and tumor uptake, especially when labeled to smaller targeting vectors such as peptides. Here we demonstrate the effect of cyanine-mediated dead cell-binding of Ac-Lys <sup>0</sup> (IRDye800CW)-Tyr <sup>3</sup> -octreotate (800CW-TATE) and how this can be used as an advantage for fluorescence-guided surgery.
Binding of 800CW-TATE could be blocked with DOTA <sup>0</sup> -Tyr <sup>3</sup> -octreotate (DOTA-TATE) on cultured SSTR <sub>2</sub> -positive U2OS cells and was absent in SSTR <sub>2</sub> negative U2OS cells. However, strong binding was observed to dead cells, which could not be blocked with DOTA-TATE and was also present in dead SSTR <sub>2</sub> negative cells. No SSTR <sub>2</sub> -mediated binding was observed in frozen tumor sections, possibly due to disruption of the cells in the process of sectioning the tissue before exposure to the contrast agent. DOTA-TATE blocking resulted in an incomplete reduction of 61.5 ± 5.8% fluorescence uptake by NCI-H69-tumors in mice. Near-infrared imaging and dead cell staining on paraffin sections from resected tumors revealed that fluorescence uptake persisted in necrotic regions upon blocking with DOTA-TATE.
This study shows that labeling peptides with cyanines can result in dead cell binding. This does not hamper the ultimate purpose of fluorescence-guided surgery, as necrotic tissue appears in most solid tumors. Hence, the necrosis binding can increase the overall tumor uptake. Moreover, necrotic tissue should be removed as much as possible: it cannot be salvaged, causes inflammation, and is tumorigenic. However, when performing binding experiments to cells with disrupted membrane integrity, which is routinely done with nuclear probes, this dead cell-binding can resemble non-specific binding. This study will benefit the development of fluorescent contrast agents
Bisphosphonates induce apoptosis in human breast cancer cell lines
Breast cancer has a prodigious capacity to metastasize to bone. In women with advanced breast cancer and bone metastases, bisphosphonates reduce the incidence of hypercalcaemia and skeletal morbidity. Recent clinical findings suggest that some bisphosphonates reduce the tumour burden in bone with a consequent increase in survival, raising the possibility that bisphosphonates may have a direct effect on breast cancer cells. We have investigated the in vitro effects of bisphosphonates zoledronate, pamidronate, clodronate and EB 1053 on growth, viability and induction of apoptosis in three human breast cancer cell lines (MDA-MB-231, Hs 578T and MCF-7). Cell growth was monitored by crystal violet dye assay, and cell viability was quantitated by MTS dye reduction. Induction of apoptosis was determined by identification of morphological features of apoptosis using time-lapse videomicroscopy, identifying morphological changes in nucleis using Hoechst staining, quantitation of DNA fragmentation, level of expression of bcl-2 and bax proteins and identification of the proteolytic cleavage of Poly (ADP)-ribose polymerase (PARP). All four bisphosphonates significantly reduced cell viability in all three cell lines. Zoledronate was the most potent bisphosphonate with IC50values of 15, 20 and 3 ΌM respectively in MDA-MB-231, MCF-7 and Hs 578T cells. Corresponding values for pamidronate were 40, 35 and 25 ΌM, whereas clodronate and EB 1053 were more than two orders of magnitude less potent. An increase in the proportion of cells having morphological features characteristic of apoptosis, characteristic apoptotic changes in the nucleus, time-dependent increase in the percentage of fragmented chromosomal DNA, down-regulation in bcl-2 protein and proteolytic cleavage of PARP, all indicate that bisphosphonates have direct anti-tumour effects on human breast cancer cells. © 2000 Cancer Research Campaig
Inhibition of Gsk3 beta in cartilage induces steoarthritic features through activation of the canonical Wnt signaling pathway
Objective: In the past years, the canonical Wnt/\u3b2-catenin signaling pathway has emerged as a critical regulator of cartilage development and homeostasis. In this pathway, glycogen synthase kinase-3\u3b2 (GSK3\u3b2) down-regulates transduction of the canonical Wnt signal by promoting degradation of \u3b2-catenin. In this study we wanted to further investigate the role of Gsk3\u3b2 in cartilage maintenance. Design: Therefore, we have treated chondrocytes ex vivo and in vivo with GIN, a selective GSK3\u3b2 inhibitor. Results: In E17.5 fetal mouse metatarsals, GIN treatment resulted in loss of expression of cartilage markers and decreased chondrocyte proliferation from day 1 onward. Late (3. days) effects of GIN included cartilage matrix degradation and increased apoptosis. Prolonged (7. days) GIN treatment resulted in resorption of the metatarsal. These changes were confirmed by microarray analysis showing a decrease in expression of typical chondrocyte markers and induction of expression of proteinases involved in cartilage matrix degradation. An intra-articular injection of GIN in rat knee joints induced nuclear accumulation of \u3b2-catenin in chondrocytes 72. h later. Three intra-articular GIN injections with a 2. days interval were associated with surface fibrillation, a decrease in glycosaminoglycan expression and chondrocyte hypocellularity 6. weeks later. Conclusions: These results suggest that, by down-regulating \u3b2-catenin, Gsk3\u3b2 preserves the chondrocytic phenotype, and is involved in maintenance of the cartilage extracellular matrix. Short term \u3b2-catenin up-regulation in cartilage secondary to Gsk3\u3b2 inhibition may be sufficient to induce osteoarthritis-like features in vivo
Nitric oxide and bone
Nitric oxide (NO) is a free radical which has important effects on bone cell function. The endothelial isoform of nitric oxide synthase (eNOS) is widely expressed in bone on a constitutive basis, whereas inducible NOS is only expressed in response to inflammatory stimuli. It is currently unclear whether neuronal NOS is expressed by bone cells. Pro-inflammatory cytokines such as IL-1 and TNF cause activation of the iNOS pathway in bone cells and NO derived from this pathway potentiates cytokine and inflammation induced bone loss. These actions of NO are relevant to the pathogenesis of osteoporosis in inflammatory diseases such as rheumatoid arthritis, which are characterized by increased NO production and cytokine activation. Interferon gamma is a particularly potent stimulator of NO production when combined with other cytokines, causing very high concentrations of NO to be produced. These high levels of NO inhibit bone resorption and formation and may act to suppress bone turnover in severe inflammation. The eNOS isoform seems to play a key role in regulating osteoblast activity and bone formation since eNOS knockout mice have osteoporosis due to defective bone formation. Other studies have indicated that the NO derived from the eNOS pathway acts as a mediator of the effects of oestrogen in bone. eNOS also mediates the effects of mechanical loading on the skeleton where it acts along with prostaglandins, to promote bone formation and suppress bone resorption. Pharmacological NO donors have been shown to increase bone mass in experimental animals and preliminary evidence suggests that these agents may also influence bone turnover in man. These data indicate that the l-arginine/NO pathway represents a novel target for therapeutic intervention in the prevention and treatment of bone diseases