Download In vitro and in vivo effect of SASP in pancreatic

2008 AACR meeting, San Diego, CA: Abstract No. 2293
The xc- cystine transporter as a target for sensitizing pancreatic cancer to
gemcitabine: Use of sulfasalazine
Maisie Lo, Victor Ling, Y.Z. Wang, Peter W. Gout
The amino acid cystine, or its reduced form, cysteine, is crucial for mammalian cell growth.
Cysteine, while required for protein synthesis, is particularly important as a rate-limiting
precursor in the biosynthesis of glutathione (L-γ-glutamyl-L-cysteinylglycine; GSH), a free
radical-scavenger essential for cellular protection from oxidative stress. Intracellular cysteine
deficiency readily leads to GSH depletion, with subsequent growth arrest and reduced drug
resistance. Some cancers cannot synthesize cysteine and are dependent on uptake of the amino
acid from their environment. The plasma membrane cystine transporter, xc-, plays a major role in
this process by (i) facilitating secretion of cysteine by somatic cells into the extracellular space
which can be readily taken up by cancer cells and (ii) mediating cystine uptake by xc--expressing
cancer cells. Pancreatic cancer is one of the most aggressive and therapy-resistant cancers
known. Although chemotherapy using gemcitabine (GEM) has increased patient survival, there
is no effective therapy for this disease. In this study we have examined whether inhibition of the
xc- transporter leading to GSH depletion can result in growth arrest and reduced GEM resistance
of human pancreatic cancer cells. To this end we used sulfasalazine (SASP), a drug first
identified by us as a potent xc- inhibitor, and Mia PaCa-2 and PANC-1 cell lines as pancreatic
cancer models, both in vitro (in MEM/10% FBS containing 0.1 mM cystine) and in vivo (as
subrenal capsule xenografts in Rag-2M mice). As shown previously, these cells express xc- (like
normal pancreatic tissue) and depend on extracellular cystine/cysteine. Cell viability was
determined by Neutral Red assay; GSH by colorimetric assay; TNFα-induced NFκB activity by
luciferase reporter assay. Incubation of the cell lines with 0.15 mM SASP (a patient-tolerated
plasma level) led to marked reduction both in total GSH levels (~70% at 24 h) and cell survival
(~85% at 72 h). Both reductions were prevented by 60 µM 2-mercaptoethanol (2-ME; allowing
cystine uptake via a route bypassing the xc- transporter), indicating they had resulted from
cysteine starvation. Combined use of SASP and GEM showed that pretreatment of cells in vitro
with 0.10 mM SASP greatly increased effectiveness of 10 nM GEM by >95% and ~50% for Mia
PaCa-2 and PANC-1 cells, respectively. In vivo, major sensitization was obtained for PANC-1
tumors in particular. SASP also inhibited NFκB activation. However, this property does not
appear to underlie the growth-arresting activity of SASP. Thus Mia PaCa-2 cultures containing
both SASP (0.2 mM) and 2-ME (66 µM) exhibited normal growth in spite of reduced NFκB
activity (39% at 24 h). This study shows that SASP, an FDA-approved drug with relatively low
toxicity, may be useful for combination chemotherapy of pancreatic cancer. Supported by CIHR,
BC Cancer Foundation (PWG/YZW) and Cdn Cancer Society - NCI-C (VL).