Fig. 6. GPx1 depletion suppresses the tumorigenic growth of MDA-MB-231 cells in vivo.

(a) The apoptosis rate was measured in siRNA-transfected HeLa cells. The transfected HeLa cells were unstimulated or stimulated with TNF-α (10 ng/ml) alone for 16 h. The data in the graph are the means ± SD of the percent of apoptotic cells (n = 3, ^*P < 0.0002). b The apoptosis rate was measured in a Dox-inducible MDA-MB-231 cell line harboring a small hairpin GPx1 (shGPx1) sequence. MDA-MB-231 stable cells were stimulated with and without TNF-α (10 ng/ml). The data in the graph are the mean ± SD of the percent of apoptotic cells (n = 3, ^*P < 0.001). A representative immunoblot shows the depletion of GPx1 upon treatment with doxycycline (Dox, 5 μg/ml) for 24 h. c, d Stable MDA-MB-231 cells were pretreated with Dox for 24 h and then subcutaneously injected into athymic nu/nu mice and grown for 40 days. The intravenous administration of recombinant mouse TNF-α (40 μg/kg) was started 7 days after cell injection (filled arrowhead) and continued twice per week for 30 days. The data in the graphs are the means ± SD of the tumor volume and weight (n = 8 mice per group, ^*P < 0.005, ^**P < 0.002 with repeated-measures ANOVA). A representative picture of tumor xenografts is shown (d). The systemic toxicity of TNF-α treatment was ruled out because no difference in the body weights of the mice in the two groups was observed for the duration of the experiments. e Caspase-3 activation was analyzed by immunoblotting the homogenates of tumor tissues obtained from two mice. GPx1 and IκB levels were immunoblotted as references. A representative immunoblot is shown. f Schematic model depicting the dual roles of GPx1 in ASK1 activation related to the TNF-α-induced apoptosis pathway. ASK1 is retained in the inactive form by interacting with the reduced form of thioredoxin 1 (Trx1_re). GPx1 maintains cellular TRAF2 proteins away from ASK1 through protein-protein interaction. Upon TNF-α stimulation, the blockade of NF-κB activation triggers mitochondria-derived H₂O₂ production, which can be adequately controlled by cytosolic GPx1. When GPx1 expression is depleted or inhibited, the cellular H₂O₂ concentration is increased to a level that can oxidize thioredoxin. Then, ASK1 is freed from oxidized Trx1 (Trx1_ox) and oligomerized upon association with the freed TRAF2. The resulting ASK1 activation induces the sustained activation of JNK (JNK^s), which is essential for apoptosis.