Influence of short-term glucocorticoid therapy on regulatory T cells in vivo.

BACKGROUND: Pre- and early clinical studies on patients with autoimmune diseases suggested that induction of regulatory T(T(reg)) cells may contribute to the immunosuppressive effects of glucocorticoids (GCs). OBJECTIVE: We readdressed the influence of GC therapy on T(reg) cells in immunocompetent human subjects and naïve mice. METHODS: Mice were treated with increasing doses of intravenous dexamethasone followed by oral taper, and T(reg) cells in spleen and blood were analyzed by FACS. Sixteen patients with sudden hearing loss but without an inflammatory disease received high-dose intravenous prednisolone followed by stepwise dose reduction to low oral prednisolone. Peripheral blood T(reg) cells were analyzed prior and after a 14 day GC therapy based on different markers. RESULTS: Repeated GC administration to mice for three days dose-dependently decreased the absolute numbers of T(reg) cells in blood (100 mg dexamethasone/kg body weight: 2.8±1.8×10(4) cells/ml vs. 33±11×10(4) in control mice) and spleen (dexamethasone: 2.8±1.9×10(5)/spleen vs. 95±22×10(5)/spleen in control mice), which slowly recovered after 14 days taper in spleen but not in blood. The relative frequency of FOXP3(+) T(reg) cells amongst the CD4(+) T cells also decreased in a dose dependent manner with the effect being more pronounced in blood than in spleen. The suppressive capacity of T(reg) cells was unaltered by GC treatment in vitro. In immunocompetent humans, GCs induced mild T cell lymphocytosis. However, it did not change the relative frequency of circulating T(reg) cells in a relevant manner, although there was some variation depending on the definition of the T(reg) cells (FOXP3(+): 4.0±1.5% vs 3.4±1.5%*; AITR(+): 0.6±0.4 vs 0.5±0.3%, CD127(low): 4.0±1.3 vs 5.0±3.0%* and CTLA4+: 13.8±11.5 vs 15.6±12.5%; * p<0.05). CONCLUSION: Short-term GC therapy does not induce the hitherto supposed increase in circulating T(reg) cell frequency, neither in immunocompetent humans nor in mice. Thus, it is questionable that the clinical efficacy of GCs is achieved by modulating T(reg) cell numbers.

Sunitinib Inhibits Cell Proliferation and Alters Steroidogenesis by Down-Regulation of HSD3B2 in Adrenocortical Carcinoma Cells.

The multi-tyrosine kinase inhibitor sunitinib is used in the treatment of several solid tumors. Animal experiments pointed to an adrenotoxic effect of sunitinib. Therefore, we evaluated the expression of key targets of sunitinib in human adrenocortical carcinoma (ACC) tumor samples and investigated its in vitro effects in ACC cell lines. We carried out immunohistochemistry for vascular endothelial growth factor (VEGF) and its receptor (VEGF-R2) in 157 ACC samples and nine normal adrenal glands. VEGF and VEGF-R2 protein were expressed in 72 and 99% of ACC samples, respectively. Using NCI-H295 and SW13 ACC cell lines, we investigated the effects of sunitinib on cell proliferation. Sunitinib reduced dose-dependently cell viability of both NCI-H295 and SW13 cells (SW13: 0.1 µM 96 ± 7%, 1 µM 90 ± 9%*, 5 µM 62 ± 6%*, controls 100 ± 9%; *p < 0.05). To determine sunitinib effects on steroidogenesis, we measured steroid hormones in cell culture supernatant by gas chromatography-mass spectrometry. We observed a pronounced decrease of cortisol secretion (1 µM 90.1 ± 1.5%*, 5 µM 57.2 ± 0.3%*, controls 100 ± 2.4%) and a concomitant increase in the DHEA/4-androstenedione and 17-hydroxypregnenolone/17-hydroxyprogesterone ratios, indicating specific inhibition of 3ß-hydroxysteroid dehydrogenase (HSD3B2). In yeast microsomes transformed with HSD3B2, no direct inhibition of HSD3B2 by sunitinib was detected. Sunitinib induced down-regulation of HSD3B2 mRNA and protein in ACC cell lines (mRNA: 1 µM 44 ± 16%*; 5 µM 22 ± 2%*; 10 µM 19 ± 4%*; protein: 1 µM 82 ± 8%; 5 µM 63 ± 8%*; 10 µM 55 ± 9%*). CYP11B1 was down-regulated at mRNA but not at protein level and CYP11A1 remained unchanged. In conclusion, target molecules of sunitinib are expressed in the vast majority of ACC samples. Sunitinib exhibits anti-proliferative effects in vitro, and appears to specifically block adrenal steroidogenesis by down-regulation of HSD3B2, rendering it a promising option for treatment of ACC.

High diagnostic and prognostic value of steroidogenic factor-1 expression in adrenal tumors.

CONTEXT: No immunohistochemical marker has been established to reliably differentiate adrenocortical tumors from other adrenal masses. A panel of markers like melan-A and inhibin-α is currently used for this purpose but suffers from limited diagnostic accuracy. We hypothesized that expression of steroidogenic factor-1 (SF-1), a transcription factor involved in adrenal development, is of value for the differential diagnosis of adrenal masses and predicts prognosis in adrenocortical carcinoma (ACC). PATIENTS AND METHODS: SF-1 protein expression was assessed by immunohistochemistry on tissue samples from 167 ACC, 52 adrenocortical adenomas (ACA), six normal adrenal glands, six normal ovaries and 73 neoplastic nonsteroidogenic tissues. In an independent cohort of 33 ACC and 58 ACA, SF-1 mRNA expression was analyzed. SF-1 expression was correlated with clinical outcome in patients with ACC. RESULTS: SF-1 protein staining was detectable in 158 of 161 (98%) evaluable ACC samples including 49 (30%) with strong SF-1 staining and in all normal and benign steroidogenic tissues. In addition, SF-1 mRNA expression was present in all 91 analyzed adrenocortical tumors. In contrast, SF-1 expression was absent in all nonsteroidogenic tumors. Strong SF-1 protein expression significantly correlated with poor clinical outcome: tumor stage-adjusted hazard ratio for death 2.46 [95% confidence interval (CI) = 1.30-4.64] and for recurrence 3.91 (95% CI = 1.71-8.94). Similar results were obtained in the independent cohort using RNA analysis [tumor stage-adjusted hazard ratio for death 4.69 (95% CI = 1.44-15.30)]. CONCLUSION: SF-1 is a highly valuable immunohistochemical marker to determine the adrenocortical origin of an adrenal mass with high sensitivity and specificity. In addition, SF-1 expression is of stage-independent prognostic value in patients with ACC.

Polymorphisms in the chemokine (C-X-C motif) ligand 10 are associated with invasive aspergillosis after allogeneic stem-cell transplantation and influence CXCL10 expression in monocyte-derived dendritic cells.

Patients after allogeneic stem-cell transplantation (alloSCT) have an increased risk for invasive aspergillosis (IA). Here, recipients of an allograft with IA (n=81) or without IA (n=58) were screened for 84 single nucleotide polymorphisms in 18 immune relevant genes. We found 3 markers in chemokine (C-X-C motif) ligand 10 (CXCL10, 4q21, 11,101 C>T, P=.007; 1642 C

Effects of HIF-1 inhibition by chetomin on hypoxia-related transcription and radiosensitivity in HT 1080 human fibrosarcoma cells.

BACKGROUND: Hypoxia-inducible factor-1 (HIF-1) overexpression has been linked to tumor progression and poor prognosis. We investigated whether targeting of HIF-1 using chetomin, a disrupter of the interaction of HIF-1 with the transcriptional coactivator p300, influences the radiosensitivity of hypoxic HT 1080 human fibrosarcoma cells. METHODS: Optimal dose of chetomin was determined by EGFP-HRE gene reporter assay in stably transfected HT 1080 cells. Cells were assayed for expression of the hypoxia-inducible genes carbonic anhydrase 9 (CA9) and vascular endothelial growth factor (VEGF) by RT-PCR and for clonogenic survival after irradiation with 2, 5 or 10 Gy, under normoxic or hypoxic (0.1% O2, 12 h) conditions in the presence or absence of chetomin (150 nM, 12 h, pre-treatment of 4 h). RESULTS: Chetomin treatment significantly reduced CA9 and VEGF mRNA expression in hypoxic cells to 44.4 +/- 7.2% and 39.6 +/- 16.0%, respectively, of untreated hypoxic controls. Chetomin clearly reduced the modified oxygen enhancement ratio (OER') compared to untreated cells, from 2.02 to 1.27, from 1.86 to 1.22 and from 1.49 to 1.06 at the 50%, 37% and 10% clonogenic survival levels, respectively. CONCLUSION: HIF-1 inhibition by chetomin effectively reduces hypoxia-dependent transcription and radiosensitizes hypoxic HT 1080 human fibrosarcoma cells in vitro.

Modulation of glucose metabolism inhibits hypoxic accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha).

BACKGROUND AND PURPOSE: The hypoxic accumulation of the transcription factor subunit hypoxia-inducible factor-1alpha (HIF-1alpha), a potential endogenous hypoxia marker and therapeutic target, has recently been shown to strongly depend on glucose availability. The aim of this study was to investigate the underlying mechanism of this effect. MATERIAL AND METHODS: HIF-1alpha protein levels were studied by Western blotting in HT 1080 human fibrosarcoma cells and in a hypoxia-responsive element green fluorescent protein (HRE-GFP) reporter assay in stably transfected HT 1080 cells treated with hypoxia (0.1% O(2), 12 h) and glycolysis inhibitors 2-deoxyglucose (2-DG) or iodoacetate (IAA). HIF-1alpha mRNA expression was quantified via real-time polymerase chain reaction (RT-PCR). RESULTS: Both inhibitors drastically reduced hypoxic HIF-1alpha accumulation (2-DG + hypoxia 2% mean HIF-1alpha protein level vs. 59% hypoxia alone; IAA + hypoxia 13% mean HIF-1alpha protein level vs. 96% hypoxia alone), an effect not rescued by the addition of pyruvate and confirmed in an HRE-GFP reporter assay in stably transfected HT 1080 cells. RT-PCR under identical conditions showed no effect of glycolysis inhibition on HIF-1alpha mRNA levels, suggesting a translational or posttranslational mechanism. CONCLUSION: The effect of glycolysis modulation on the HIF-1alpha levels in tumor cells may provide a novel approach to therapeutically target HIF-1alpha.