Employing Pancreatic Tumor γ‑Glutamyltransferase for Therapeutic Delivery

γ-Glutamyltransferase (γGT) is a cell surface enzyme that catalyzes hydrolysis of the bond linking the glutamate and cysteine residues of glutathione and glutathione-S-conjugates. We have observed that human pancreatic tumor cells and tumor-associated stellate cells express high levels of this enzyme when compared to normal pancreatic epithelial and stellate cells. Detection of the protein in tumor sections correlated with γGT activity on the surface of the cultured tumor and stellate cells. We tested whether the tumor γGT could be employed to deliver a therapeutic to the tumor endothelial cells. GSAO is a glutathione-S-conjugate of a trivalent arsenical that is activated to enter endothelial cells by γGT cleavage of the γ-glutamyl residue. The arsenical moiety triggers proliferation arrest and death of the endothelial cells by targeting the mitochondria. Human pancreatic tumor and stellate cell γGT activated GSAO in culture and γGT activity positively correlated with GSAO-mediated proliferation arrest and death of endothelial cells in Transwell and coculture systems. A soluble form of γGT is found in blood, and we measured the rate of activation of GSAO by this enzyme. We calculated that systemically administered GSAO would circulate through the pancreatic blood supply several times before appreciable activation by normal blood levels of γGT. In support of this finding, tumor γGT activity positively correlated with GSAO-mediated inhibition of pancreatic tumor angiogenesis and tumor growth in mice. Our findings indicate that pancreatic tumor γGT can be used to deliver a therapeutic to the tumor

The effect of long-acting β₂-agonists and fluticasone, alone or in combination, on repression of TNFα-induced IL-6 and IL-8 secretion.

<p>Growth-arrested ASM cells were pretreated for 1 h with vehicle, salmeterol (100 nM) or formoterol (10 nM), alone or in combination with fluticasone (1 nM), before stimulation for 24 h with TNFα (10 ng/ml). (A) IL-6 and (B) IL-8 secretion was measured by ELISA and results expressed as (A) % TNFα-induced IL-6 secretion or (B) % TNFα-induced IL-8 secretion (where TNFα-induced IL-6 and IL-8 protein secretion was 5153.9±618.9 and 5913.1±480.0 pg/ml, respectively (mean+SEM values from n = 6 primary ASM cell cultures)). Statistical analysis was performed using repeated measures ANOVA with Bonferroni’s multiple comparison test where * denotes a significant increase in IL-6 secretion and § denotes a significant repression of TNFα-induced cytokine secretion (<i>P</i><0.05).</p

Formoterol increases fluticasone-induced MKP-1 protein upregulation.

<p>(A) Growth-arrested ASM cells were treated with vehicle, formoterol (10 nM), fluticasone (1 nM), formoterol (10 nM)+fluticasone (1 nM), for 0, 0.5, 1, 2, 4, 8, and 24 h. MKP-1 protein (molecular weight:∼39 kDa) was quantified by Western blotting, using α-tubulin as the loading control (molecular weight:∼55 kDa), where (A) illustrates representative Western blots and (B) demonstrates densitometric analysis where results are expressed as fold increase over 0 h and results for fluticasone (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059635#pone-0059635-g001" target="_blank">Figure 1B</a>) shown graphically for comparative purposes (mean+SEM values from n = 3–8 primary ASM cell cultures). Statistical analysis was performed using two-way ANOVA then Bonferroni's post-test (where * indicates a significant effect of formoterol or fluticasone on MKP-1 protein upregulation, compared to vehicle-treated cells, and § indicates a significant effect of formoterol on fluticasone-induced MKP-1 (<i>P</i><0.05)).</p

Salmeterol increases fluticasone-induced MKP-1 protein upregulation.

<p>(A) Growth-arrested ASM cells were treated with vehicle, salmeterol (100 nM), fluticasone (1 nM), salmeterol (100 nM)+fluticasone (1 nM), for 0, 0.5, 1, 2, 4, 8, and 24 h. MKP-1 protein (molecular weight:∼39 kDa) was quantified by Western blotting, using α-tubulin as the loading control (molecular weight: 55∼kDa), where (A) illustrates representative Western blots and (B) demonstrates densitometric analysis where results are expressed as fold increase over 0 h (mean+SEM values from n = 6–8 primary ASM cell cultures). Statistical analysis was performed using two-way ANOVA then Bonferroni's post-test (where * indicates a significant effect of salmeterol or fluticasone on MKP-1 protein upregulation, compared to vehicle-treated cells, and § indicates a significant effect of salmeterol on fluticasone-induced MKP-1 (<i>P</i><0.05)).</p

Effect of MKP-1 siRNA.

<p>ASM cells were transiently transfected using nucleofection with scrambled control or MKP-1 siRNA, growth-arrested, then pretreated for 1 h with formoterol (10 nM)+fluticasone (1 nM) (along with relevant controls) before stimulation for 24 h with TNFα (10 ng/ml). Supernatants were then removed for IL-6 and IL-8 protein secretion by ELISA and lysates utilized for MKP-1 Western blotting (compared to α-tubulin as a loading control). (A) illustrates a representative Western blot, while (B, C) demonstrates results expressed as % TNFα-induced (B) IL-6 or (C) IL-8 secretion in cells transfected with scrambled control siRNA (mean+SEM values from n = 6 primary ASM cell cultures).</p

Effect of long-acting β₂-agonists on IL-6 and IL-8 mRNA expression and secretion.

<p>(A, B, C, D) Growth-arrested ASM cells were treated with vehicle, salmeterol (100 nM) or formoterol (10 nM). (A) IL-6 and (B) IL-8 mRNA expression was quantified by real-time RT-PCR at 1 h and results expressed as fold increase compared to vehicle-treated cells (mean+SEM values from n = 9 primary ASM cell cultures). (C) IL-6 and (D) IL-8 secretion was measured by ELISA at 24 h and results are expressed as fold increase compared to vehicle-treated cells (where IL-6 and IL-8 protein levels in cells treated with vehicle were 295.0±34.8 and 26.3±4.6 pg/ml, respectively (mean+SEM values from n = 6 primary ASM cell cultures)). Statistical analysis was performed using repeated measures ANOVA with Bonferroni’s multiple comparison test (where * denotes a significant increase in mRNA expression or secretion (<i>P</i><0.05)). (E, F) Growth-arrested ASM cells were treated with vehicle, dibutyryl cAMP (1 mM), forskolin (10 µM) and (E) IL-6 and (F) IL-8 mRNA expression was quantified by real-time RT-PCR at 1 h. Results are expressed as fold increase compared to vehicle-treated cells (mean+SEM values from n = 3 primary ASM cell cultures). Statistical analysis was performed using the Student's unpaired <i>t</i> test where * denotes a significant increase in mRNA expression or secretion (<i>P</i><0.05).</p

Long-acting β₂-agonists increase fluticasone-induced MKP-1 mRNA expression.

<p>Growth-arrested ASM cells were treated for 1 h with vehicle, fluticasone (1 nM), salmeterol (100 nM), formoterol (10 nM), salmeterol (100 nM)+fluticasone (1 nM), formoterol (10 nM)+fluticasone (1 nM). MKP-1 mRNA expression was quantified by real-time RT-PCR and results expressed as fold increase compared to vehicle-treated cells (mean+SEM values from n = 9 primary ASM cell cultures). Statistical analysis was performed using repeated measures ANOVA with Bonferroni’s multiple comparison test (where * denotes a significant increase in MKP-1 mRNA expression and § denotes a significant effect of the β₂-agonists on fluticasone-induced MKP-1 mRNA expression (<i>P</i><0.05)).</p