Sigma-2 ligands induce tumour cell death by multiple signalling pathways

BACKGROUND: The sigma-2 receptor has been identified as a biomarker of proliferating cells in solid tumours. In the present study, we studied the mechanisms of sigma-2 ligand-induced cell death in the mouse breast cancer cell line EMT-6 and the human melanoma cell line MDA-MB-435. METHODS: EMT-6 and MDA-MB-435 cells were treated with sigma-2 ligands. The modulation of multiple signaling pathways of cell death was evaluated. RESULTS: Three sigma-2 ligands (WC-26, SV119 and RHM-138) induced DNA fragmentation, caspase-3 activation and PARP-1 cleavage. The caspase inhibitor Z-VAD-FMK partially blocked DNA fragmentation and cytotoxicity caused by these compounds. These data suggest that sigma-2 ligand-induced apoptosis and caspase activation are partially responsible for the cell death. WC-26 and siramesine induced formation of vacuoles in the cells. WC-26, SV119, RHM-138 and siramesine increased the synthesis and processing of microtubule-associated protein light chain 3, an autophagosome marker, and decreased the expression levels of the downstream effectors of mammalian target of rapamycin (mTOR), p70S6K and 4EBP1, suggesting that sigma-2 ligands induce autophagy, probably by inhibition of the mTOR pathway. All four sigma-2 ligands decreased the expression of cyclin D1 in a time-dependent manner. In addition, WC-26 and SV119 mainly decreased cyclin B1, E2 and phosphorylation of retinoblastoma protein (pRb); RHM-138 mainly decreased cyclin E2; and 10 μ siramesine mainly decreased cyclin B1 and pRb. These data suggest that sigma-2 ligands also impair cell-cycle progression in multiple phases of the cell cycle. CONCLUSION: Sigma-2 ligands induce cell death by multiple signalling pathways

Identification of the PGRMC1 protein complex as the putative sigma-2 receptor binding site

The sigma-2 receptor, whose gene remains to be cloned, has been validated as a biomarker for tumor cell proliferation. Here we report the use of a novel photoaffinity probe, WC-21, to identify the sigma-2 receptor binding site. WC-21, a sigma-2 ligand containing both a photoactive moiety azide and a fluorescein isothiocyanate group, irreversibly labels sigma-2 receptors in rat liver; the membrane-bound protein was then identified as PGRMC1 (progesterone receptor membrane component-1). Immunocytochemistry reveals that both PGRMC1 and SW120, a fluorescent sigma-2 receptor ligand, colocalizes with molecular markers of the endoplasmic reticulum and mitochondria in HeLa cells. Overexpression and knockdown of the PGRMC1 protein results in an increase and a decrease in binding of a sigma-2 selective radioligand, respectively. Th

The Golgin GMAP210/TRIP11 Anchors IFT20 to the Golgi Complex

Eukaryotic cells often use proteins localized to the ciliary membrane to monitor the extracellular environment. The mechanism by which proteins are sorted, specifically to this subdomain of the plasma membrane, is almost completely unknown. Previously, we showed that the IFT20 subunit of the intraflagellar transport particle is localized to the Golgi complex, in addition to the cilium and centrosome, and hypothesized that the Golgi pool of IFT20 plays a role in sorting proteins to the ciliary membrane. Here, we show that IFT20 is anchored to the Golgi complex by the golgin protein GMAP210/Trip11. Mice lacking GMAP210 die at birth with a pleiotropic phenotype that includes growth restriction, ventricular septal defects of the heart, omphalocele, and lung hypoplasia. Cells lacking GMAP210 have normal Golgi structure, but IFT20 is no longer localized to this organelle. GMAP210 is not absolutely required for ciliary assembly, but cilia on GMAP210 mutant cells are shorter than normal and have reduced amounts of the membrane protein polycystin-2 localized to them. This work suggests that GMAP210 and IFT20 function together at the Golgi in the sorting or transport of proteins destined for the ciliary membrane

Chronological gene expression of parathyroid hormone-related protein (PTHrP) in the stellate reticulum of the rat: implications for tooth eruption

OBJECTIVE: Tooth eruption is a localized event that requires the expression of certain molecules at precise times to regulate bone resorption and bone formation. Parathyroid hormone-related protein (PTHrP) may be one of those molecules. Although PTHrP is produced in the stellate reticulum (SR) of the tooth and exerts its effect on the adjacent dental follicle, its expression pattern in the SR is unknown. Thus, it was the objectives of this study to determine the chronology of expression of PTHrP, and then to determine its effect on vascular endothelial growth factor (VEGF) expression for osteoclastogenesis and on bone morphogenetic protein-2 (BMP-2) for bone growth. DESIGN: Laser capture microdissection and RT-PCR were used to determine the chronological expression of PTHrP in vivo. In vitro, dental follicle cells were incubated with PTHrP and RT-PCR was conducted to determine its effect on VEGF and BMP-2 gene expression. RESULTS: PTHrP was maximally expressed at day 7 postnatally in the SR with the level of expression still high at day 9. In vitro, PTHrP upregulated VEGF120 and VEGF164 expression after 4h of incubation with a maximum effect at 6h. PTHrP upregulated BMP-2 gene expression with a maximal effect at 2h. CONCLUSIONS: Because the secondary burst of osteoclastogenesis needed for eruption occurs around day 10, it is possible that PTHrP is stimulating this osteoclastogenesis by upregulating VEGF. Concurrently, the upregulation of BMP-2 by PTHrP may stimulate bone growth at the base of the bony crypt to promote eruption

Effect of vascular endothelial growth factor on RANK gene expression in osteoclast precursors and on osteoclastogenesis

OBJECTIVE: The aim of this study was to determine if vascular endothelial growth factor (VEGF) can upregulate the gene expression of receptor activator of nuclear factor kappa B (RANK) in osteoclast precursors, as does CSF-1. A secondary aim was to determine if VEGF can promote osteoclastogenesis in vitro comparable to CSF-1. DESIGN: Osteoclast precursors (mononuclear cells) were incubated with different concentrations of VEGF, CSF-1, or a combination of the two, and the gene expression of RANK was determined by RT-PCR. A TRAP assay also was conducted to determine their effect on osteoclastogenesis. An Alamar blue assay was done to analyse the effect of the molecules on proliferation of the osteoclast precursors. RESULTS: VEGF upregulated RANK expression in osteoclast precursors as effectively as CSF-1. VEGF did not promote osteoclastogenesis, as did CSF-1. A combination of the two did. CSF-1 enhanced proliferation of the osteoclast precursors but VEGF did not. However, VEGF in combination with CSF-1 did increase proliferation. CONCLUSIONS: At the time of the secondary burst of osteoclastogenesis prior to tooth eruption, VEGF expression in the dental follicle is high but the expression of CSF-1 is low. This study demonstrates that VEGF can fully substitute for CSF-1 to upregulate the RANK expression in osteoclast precursors that is needed for osteoclastogenesis. However, VEGF alone neither can promote osteoclastogenesis nor stimulate proliferation of the osteoclast precursors in vitro. For proliferation and osteoclastogenesis, a low dose of CSF-1 in combination with VEGF is needed

TNF-alpha upregulates expression of BMP-2 and BMP-3 genes in the rat dental follicle--implications for tooth eruption

The dental follicle appears to regulate both the alveolar bone resorption and bone formation needed for tooth eruption. Tumor necrosis factor-alpha (TNF-alpha) gene expression is maximally upregulated at postnatal day 9 in the rat dental follicle of the first mandibular molar, a time that correlates with rapid bone growth at the base of the tooth crypt, as well as a minor burst of osteoclastogenesis. TNF-alpha expression is correlated with the expression of bone morphogenetic protein-2 (BMP-2), a molecule expressed in the dental follicle that can promote bone formation. Because BMP-2 signaling may be augmented by bone morphogenetic protein-3 (BMP-3), our objective in this study was to determine if the dental follicle expresses BMP-3 and if TNF-alpha stimulates the dental follicle cells to express BMP-2 and BMP-3. Dental follicles were collected from different postnatal ages of rat pups. Dental follicle cells were incubated with TNF-alpha to study its dosage and time-course effects on gene expression of BMP-2 and BMP-3, as determined by real-time RT-PCR. Next, immunostaining was conducted to confirm if the protein was synthesized and ELISA of the conditioned medium was conducted to determine if BMP-2 was secreted. We found that BMP-3 expression is correlated with the expression of TNF-alpha in the dental follicle and TNF-alpha significantly increased BMP-2 and BMP-3 expression in vitro. Immunostaining and ELISA showed that BMP-2 and BMP-3 were synthesized and secreted. This study suggests that TNF-alpha can upregulate the expression of bone formation genes that may be needed for tooth eruption

Non-alcoholic fatty liver disease is an influencing factor for the association of SHBG with metabolic syndrome in diabetes patients

Abstract Metabolic syndrome (MS) and non-alcoholic fatty liver disease (NAFLD) have been identified as risk factors affecting serum sex hormone binding globulin (SHBG) levels. We conducted this cross-sectional study to delineate whether MS or NAFLD has more impact on circulating SHBG levels in type 2 diabetes (T2D) patients. Anthropometric and biochemical parameters including serums SHBG, testosterone (TT), liver enzymes, lipids, insulin, C-peptide and plasma glucose were measured. Regardless of the MS status, SHBG level was significantly lower in NAFLD patients than in non-NAFLD patients (P < 0.001). In the multiple linear regression analysis, lower serum SHBG level was strongly correlated with a higher incidence of NAFLD, but not MS components. In logistic regression analyses, after adjusted for age, sex, duration of diabetes, smoking status, and alcohol use, the ORs and 95%CI for presence of MS was 2.26 (95%CI 1.91–2.68) and for presence of NAFLD was 6.36 (95%CI 4.87–8.31) with per one SD decrease in serum SHBG (both P < 0.001). In conclusion, lower serum SHBG is associated with a higher prevalence of NAFLD, compared with MS and other metabolic disorders, in T2D patients. NAFLD might be an important influencing factor for the association of circulating SHBG with MS in T2D patients