S-glutathionylated serine proteinase inhibitors as plasma biomarkers in assessing response to redox-modulating drugs

Abstract

Many cancer drugs impact cancer cell redox regulatory mechanisms and disrupt redox homeostasis. Pharmacodynamic biomarkers that measure therapeutic efficacy or toxicity could improve patient management. Using immunoblot analyses and mass spectrometry, we identified that serpins A1 and A3 were S-glutathionylated in a dose- and time-dependent manner following treatment of mice with drugs that alter reactive oxygen or nitrogen species. Tandem mass spectrometry analyses identified Cys(256) of serpin A1 and Cys(263) of serpin A3 as the S-glutathionylated residues. In human plasma from cancer patients, there were higher levels of unmodified serpin A1 and A3, but following treatments with redox active drugs, relative S-glutathionylation of these serpins was higher in plasma from normal individuals. There is potential for S-glutathionylated serpins A1 and A3 to act as pharmacodynamic biomarkers for evaluation of patient response to drugs that target redox pathways.

Figure 1

NOV-002 treatment induces serpin A1 and A3 S-glutathionylation in vivo in mouse plasma and ex vivo in human plasma. Immunoblot analyses of S-glutathionylated proteins (PSSG) from wild-type mice treated in vivo with an intravenous bolus of 25 mg/kg NOV-002 at time points following 1 hour (A) and wild-type mouse plasma treated ex vivo with various concentrations of NOV-002 (B). MALDI-TOF mass spectrometry identified S-glutathionylated mouse plasma proteins (C). Human plasma was treated ex vivo with 40 µmol/L NOV-002 for 0 to 240 minutes and evaluated by immunoblot for S-glutathionylated proteins (D). Albumin immunoblotting determined equal protein loading. For immunoprecipitation (IP), human plasma was treated with 40 µmol/L NOV-002 for 1 hour and biotinylated serpin A1 and A3 antibodies were used to pull-down total unmodified and modified serpins. S-glutathionylated proteins were evaluated by immunoblot (E). Blots were stripped and reprobed with anti-serpin A1 or A3 as a loading control. Due to high homology, there is significant cross reactivity of anti-A1 and A3. IB, immunoblot.

Figure 2

S-glutathionylation of serpins A1 and A3 is time and dose dependent and impacts protein structure. Recombinant serpin A1 and A3 were treated with 1 mmol/L GSH and 40 µmol/L of PABA/NO for 0 to 30 minutes (A) or 0 to 100 µmol/L of PABA/NO for 30 minutes (B). Immunoblot analyses were used to detect S-glutathionylation (PSSG) and total serpin. Spectroscopic analyses of secondary and tertiary (quaternary) structure of native (control; solid curves) and PABA/ NO + GSH–treated (Glut; dashed curves) A1 (top curves, dark gray) and A3 (bottom curves, light gray) protein were done in vitro using tryptophanyl fluorescence (C) and circular dichroism (D). IB, mmunoblot.

Figure 3

Serpin A1 is S-glutathionylated at Cys²⁵⁶ and serpin A3 is S-glutathionylated at Cys²⁶³. Recombinant serpin A1 and A3 proteins were treated with 1 mmol/LGSH and 100 µmol/L PABA/NO for 30 minutes. To identify specific cysteine residues susceptible to S-glutathionylation, control unmodified serpin A1 (A) PABA/NO-treated serpin A1 (B); control unmodified serpin A3 (C) and PABA/NO-treated serpin A3 (D) were digested under nonreducing conditions and analyzed via LC–ESI–MS/MS to identify modification GSH addition. A single S-glutathionylated modification [+305.6] was detected at of Cys²⁵⁶ of serpin A1 (B) and Cys²⁶³ of treated serpin A3 (D). Arrows indicate modified peaks. The 2 arrows in D represent both forward and reverse analyses.

Figure 4

Unmodified serpin is elevated in certain cancers, whereas the ratio of S-glutathionylated to unmodified serpin is decreased. Human plasma samples from cancer-free patients (n = 8; represented in lanes 1,3,5 of A) and cancer patients undergoing chemotherapeutic treatment (n = 47; represented in lanes 2,4,6 of A) were analyzed by immunoblotting for unmodified serpin A1 and A3 proteinlevels(B)as well as relative levels of S-glutathionylated serpin compared with total unmodified serpin (C). Relative quantities of unmodified serpins and S-glutathionylated serpin A1 (PSSGa and PSSGc), and serpin A3 (PSSGb) were determined by densitometry measurements normalized to both albumin loading controls and an internal standard incorporated on all gels. Data are ± SEM. IB, immunoblot.

Figure 5

Ex vivo treatment with NOV-002 induces serpin A1 and A3 S-glutathionylation and results in greater relative increases in serpin A1 glutathionylation in cancer-free human plasma. Cancer (samples 2–8) and cancer-free (sample 1) human plasma samples were treated with 100 µmol/L NOV-002 for 60 minutes (A). S-glutathionylation (PSSG), serpin A1, serpin A3, and albumin levels (loading control) were evaluated by immunoblot. Levels of S-glutathionylated serpin A1 (PSSGa and PSSGc) and serpin A3 (PSSGb) after NOV-002 treatment (both cancer and cancer free) were determined by densitometry normalized to both albumin loading controls and an internal standard on all gels (B). Relative S-glutathionylation levels in reference to total unmodified serpin levels induced in response to NOV-002 treatment in cancer patient plasma as compared with those induced in cancer-free plasma were analyzed using 2-way ANOVAs (C). To confirm that S-glutathionylation of serpins does not alter serpin A1 and A3 protein stability, plasma was treated with 40 µmol/L NOV-002 for 0 to 240 minutes (D). The relative quantity of serpins A1 and A3 in treated and untreated samples (both cancer and cancer free) after 100 µmol/L NOV-002 treatment for 1 hour was normalized to both albumin loading controls and an internal standard on all gels(E). Data are mean for 8 cancer-free and 47 cancer samples ± SEM. IB, immunoblot.

Figure 6

Cancer patient plasma samples have decreased levels of GSTP protein and elevated levels of Grx1. Human plasma samples from cancer-free patients (represented in lanes 1 and 3) and cancer patients undergoing chemotherapeutic treatment (represented in lanes 2 and 4) were evaluated for GSTP and Grx1 using immunoblots. Relative quantities of GSTP and Grx1 were normalized to both albumin loading controls and an internal standard on all gels. Data are mean for 8 cancer-free and 47 cancer samples ± SEM. IB, immunoblot.