Background
Materials and methods
Culture of B16-F10 melanoma cells
Animals
Transfection of red fluorescent protein
Experimental metastases
Isolation of B16-F10 melanoma cells from metastatic foci
Measurement of adrenocorticotropin hormone, corticosterone, and norepinephrine levels
CRH expression in the brain (in situ hybridization)
Measurement of IL-6 levels
DNA binding activity of NF-κB, CREB, AP-1, and NF-IL-6
Transfection of anti-IL-6 small interfering RNA
GSH determination
Cell death and cell cycle analysis
Compartmentation of B16-F10 cells
Cellular electroporation
RT-PCR and detection of mRNA
Expression of results and statistical analyses
Results
Stress hormones in metastatic tumor-bearing mice
Corticosterone and noradrenaline stimulate IL-6 expression and secretion in metastatic cells
Tumor cells | Additions | Expression (-fold change) | Secretion (pg/106cells x 24 h) |
---|---|---|---|
B16-F10 | None | 1.0 ± 0.1 | 833 ± 170 |
Corticosterone | 2.5 ± 0.4* | 1654 ± 266* | |
NORA | 1.7 ± 0.2* | 1257 ± 214* | |
Corticosterone+NORA | 2.4 ± 0.3* | 1712 ± 247* | |
B16-F10-RFP | None | 3.0 ± 0.3*+ | 1966 ± 331*+ |
Corticosterone | 3.3 ± 0.4*+ | 2184 ± 287*+ | |
NORA | 3.0 ± 0.3*+ | 1763 ± 294*+ | |
Corticosterone+NORA | 3.6 ± 0.5*+ | 2477 ± 315*+ |
Transcriptional regulation of the IL-6 gene by corticosterone and noradrenaline
Tumor-derived IL-6 facilitates activation of the pituitary-adrenal axis
Lung metastases | ||
---|---|---|
B16-F10 | B16-F10/IL-6-siRNA | |
Tumor IL-6 expression (-fold change) | 1.0 ± 0.2 | 0.15 ± 0.05* |
Serum IL-6 (pg/ml) | 347 ± 75 | 132 ± 34* |
Hypothalamic CRH expression (AU) | 40 ± 12 | 85 ± 16* |
Plasma ACTH (pg/ml) | 240 ± 50 | 255 ± 63 |
Treatment | ||||||||
---|---|---|---|---|---|---|---|---|
Physiological saline | RU-486 | Propranolol | IL-6-siRNA | |||||
Metastases | Lung | Liver | Lung | Liver | Lung | Liver | Lung | Liver |
Tumor IL-6 expression (-fold change) | 1.0 ± 0.1 | 1.0 ± 0.2 | 0.4 ± 0.1** | 0.3 ± 0.15** | 0.6 ± 0.2** | 0.7 ± 0.15* | 0.15 ± 0.05** | 0.2 ± 0.1** |
Serum IL-6 (pg/ml) | 355 ± 81 | 432 ± 69 | 123 ± 36** | 154 ± 49** | 206 ± 77* | 255 ± 60** | 107 ± 40** | 124 ± 33** |
Liver GSH (μmol/g of tissue) | 4.5 ± 05 | 3.8 ± 0.4 | 6.8 ± 0.6** | 5.9 ± 0.5** | 6.2 ± 0.5** | 5.5 ± 0.5** | 7.3 ± 0.6** | 7.1 ± 0.5** |
Plasma NORA (ng/ml) | 406 ± 65 | 447 ± 72 | 337 ± 59 | 412 ± 77 | 426 ± 68 | 394 ± 59 | 433 ± 67 | 360 ± 45* |
Plasma NORA (ng/ml) | 7.9 ± 1.5 | 8.7 ± 1.4 | 7.3 ± 1.2 | 8.1 ± 1.6 | 7.5 ± 1.1 | 8.5 ± 1.7 | 8.2 ± 1.3 | 9.2 ± 1.6 |
Metastases volume (%) | 6.4 ± 09 | 8.5 ± 1.2 | 4.0 ± 0.6** | 5.0 ± 0.7** | 4.9 ± 0.5** | 5.8 ± 0.9** | 3.0 ± 0.7** | 3.7 ± 0.8** |
Treatment with glucocorticoid receptor or β adrenoceptor blockers inhibits metastatic growth
Corticosterone induces cell death in metastatic cells with low GSH content
Culture time | |||||||
---|---|---|---|---|---|---|---|
12 h | 48 h | ||||||
B16-F10 cells | Additions | GSH (nmol/106cells) | Cell number (106cells) | GSH (nmol/106cells) | Cell number (106cells) | ||
Viable | Dead | Viable | Dead | ||||
High GSH content | |||||||
No addition | 40 ± 5 | 2.01 ± 0.26 | 0.02 ± 0.01 | 46± 7 | 5.24 ± 0.36* | 0.15 ± 0.04* | |
+ Corticosterone | 5.06 ± 0.17 | 0.18 ± 0.03 | |||||
+NORA | 5.57 ± 0.43 | 0.14 ± 0.03 | |||||
+Corticosterone+NORA | 4.82 ± 0.30 | 0.20 ± 0.05 | |||||
Low GSH content | |||||||
No addition | 14 ± 3+ | 1.93 ± 0.15 | 0.05 ± 0.01+ | 8 ± 2 | 2.24 ± 0.45+ | 0.15 ± 0.03* | |
+ Corticosterone | 0.86 ± 0.15+ | 0.76 ± 0.14+ | |||||
+NORA | 2.07 ± 0.26+ | 0.13 ± 0.05 | |||||
+Corticosterone+NORA | 0.75 ± 0.18+ | 0.80 ± 0.17+ |
Parameters | Controls | + Corticosterone | +Corticosterone |
---|---|---|---|
+GSH ester | |||
H2O2 (nmol/106 cells) | 0.45 ± 0.09 | 1.74 ± 0.35* | 0.73± 0.20*+ |
O2.- (ΔFL1) | 2.10 ± 0.39 | 4.33 ± 0.41* | 2.65 ± 0.27+ |
mtGSH (nmol/106 cells) | 3.2 ± 0.5 | 1.6 ± 0.4* | 6.4 ± 1.0*+ |
MMP (TPM accumulation ration ratio, %) | 95 ± 4 | 52 ± 9 | 90 ± 7+ |
mtATP (mM) | 1.07 ± 0.24 | 0.66 ± 0.23 | 0.95 ± 0.18 |
Cytosolic cytochrome C (% of control) | 100 ± 11 | 165 ± 27 | 112 ± 15+ |
Caspase 3 (pmol/106 cells x min) | 1.36 ± 0.21 | 3.15 ± 0.77 | 1.79 ± 0.26+ |