Doxorubicin-Mediated Bone Loss in Breast Cancer Bone Metastases Is Driven by an Interplay between Oxidative Stress and Induction of TGFβ
Figure 5
Doxorubicin mediates bone loss by elevating oxidative stress.
(A) C400 oxidation show increase in reactive oxygen species (ROS) upon in vitro doxorubicin (0.01 ug/ml, 20 hours) treatment increases oxidative stress in the mouse bone marrow stromal cells, which was decreased by concomitant treatment with 1D11(25 µg/ml). Data represents average percentage of C400 positive cells from triplicate samples. (B) RT-PCR showing a decrease in SOD1 (copper zinc superoxide dismutase 1) and GPx expression was noted in MC3T3 mouse osteoblast cells upon treatment with doxorubicin (0.01 µg/ml, 20 hours), which was returned to normal level by co-treatment with anti-TGFβ antibody 1D11(25 ug/ml). (C) SOD1 expression normalized against GAPDH expression, quantified by Image J. (D) GPx expression normalized against GAPDH expression, quantified by Image J. (E) SOD1 activity was performed using MC3T3 cells as described in Materials and Methods section. The inhibition of SOD1 activity was measured by formation of NBT-diformazan from NBT following 20 hours treatment in either serum free alpha-MEM media alone, or supplemented with 0.01 ug/ml doxorubicin, 25 µg/ml anti-TGFβ antibody and a combination of both doxorubicin and anti-TGFβ antibody. A drastic inhibition of SOD1 activity was noted following doxorubicin treatment which was restored by anti-TGFβ antibody. (F) Calvarial osteoblasts from wild type mice (3–4 days old pups) were cultured until confluent and treated with osteoblast differentiation media supplemented with doxorubicin (0.01 µg/ml), N-acetyl cysteine (NAC, 20 mM) treatment, or a combination of both, or media alone until mineralized matrix was formed. Quantification of Von Kossa staining images from at least 3 different fields were done using Metamorph software.