Abstract
B16 melanoma F10 (B16-F10) cells with high glutathione (GSH) content show high metastatic activity in vivo. An intertissue flow of GSH, where the liver is the main reservoir, can increase GSH content in metastatic cells and promote their growth. We have studied here possible tumor-derived molecular signals that could activate GSH release from hepatocytes. GSH efflux increases in hepatocytes isolated from mice bearing liver or lung metastases, thus suggesting a systemic mechanism. Fractionation of serum-free conditioned medium from cultured B16-F10 cells and monoclonal antibody-induced neutralization techniques facilitated identification of interleukin (IL)-6 as a tumor-derived molecule promoting GSH efflux in hepatocytes. IL-6 activates GSH release through a methionine-sensitive/organic anion transporter polypeptide 1- and multidrug resistance protein 1-independent channel located on the sinusoidal site of hepatocytes. Specific siRNAs were used to knock down key factors in the main signaling pathways activated by IL-6, which revealed a STAT3-dependent mechanism. Our results show that IL-6 (mainly of tumor origin in B16-F10-bearing mice) may facilitate GSH release from hepatocytes and its interorgan transport to metastatic growing foci.
Highlights
Studies on the organ distribution of metastatic cells showed that less than 0.1% of circulating cells survive to cause secondary metastatic growth [5]
By comparing B16 cells cultured to low versus high density, which have different GSH contents and different metastatic activities, we found that NO was tumoricidal in the presence of H2O2 [10]
We found that GGT overexpression in B16-F10 cells, by degrading extracellular GSH, facilitates their metastatic growth [15]
Summary
Culture of B16-F10 Melanoma Cells—Murine B16-F10 melanoma cells (from the ATCC, Manassas, VA) were cultured in serum-free Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen), pH 7.4, supplemented with 10 mM HEPES, 40 mM NaHCO3, 100 units/ml penicillin, and 100 g/ml streptomycin [15]. Cells were harvested by incubation for 5 min with 0.05% (w/v) trypsin (Sigma) in PBS (10 mM sodium phosphate, 4 mM KCl, 137 mM NaCl), pH 7.4, containing 0.3 mM EDTA, followed by the addition of 10% calf serum to inactivate the trypsin. Cells were incubated for 4 h with the polyethyleneimine-DNA complex in 5% of their initial culture medium (DMEM containing 10% fetal calf serum) volume. Cells were harvested (as above) 4 days after transfection and subcultured into selective medium that contained 200 g/ml Geneticin (Invitrogen). Serial dilutions of mAbs in PBS, pH 7.4, were added to melanoma cell cultures, and standard protocols from eBioscience (San Diego, CA) for in vitro cytokine neutralization were followed. The significance test refers, for both groups, to the comparison of rates in the absence or in the presence of amino acids (**, p Ͻ 0.01), and to the difference between results for the B16-F10 group and the non-tumor-bearing group (†, p Ͻ 0.05; ††, p Ͻ 0.01)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
