Adduction of PRX3 by TS correlates with cytotoxicity.

(A) Human primary mesothelial, immortalized LP9 mesothelial, and HM and H2373 mesothelioma cell lines were incubated with 5 μM TS and lysates were collected at indicated time points over 24 hr. Formation of TS induced PRX3 dimers was visualized by reducing SDS-PAGE and immunoblotting with anti-PRX3 antibody. (B) Cell lines from (A) were incubated with indicated concentrations of TS (left) or GV (right) for 24 hr and total cell mass was determined by staining with crystal violet (Y axis values were normalized to untreated cells). The EC50 values for the indicated cell lines to TS or GV and the relative potency (REP) of TS and GV, as compared to primary mesothelial cells, are shown. (C) HM cells were treated with increasing concentrations of TS, GV, or TS + GV for 18 hr and extracts were resolved by reducing SDS-PAGE. PRX3 modification was assessed by immunoblotting as before. Note that GV accentuates modification of PRX3 by TS by blocking the activity of TRX2. (D) Extracts from cells treated as in panel C were resolved by non-reducing SDS-PAGE and PRX3 monomers (~23 kD) and disulfide-bonded dimers (~46 kD) were assessed by immunoblotting for PRX3. Note that GV markedly increased the level of disulfide-bonded PRX3 dimers, an indication of severe mitochondrial oxidative stress. (E) HMC2 and (F) HMC3 human primary mesothelial cell cultures were incubated with increasing doses of TS, GV or TS + GV for 18 hours, and assessed for PRX3 expression after reducing SDS-PAGE by immunoblotting. The formation of the modified species of PRX3 in response to TS was less evident in primary mesothelial cells.