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. 2008 Aug 5;99(3):464-72.
doi: 10.1038/sj.bjc.6604485. Epub 2008 Jul 22.

The xc- cystine/glutamate antiporter: a mediator of pancreatic cancer growth with a role in drug resistance

M Lo  1 , V Ling, Y Z Wang, P W Gout
Affiliations

The xc- cystine/glutamate antiporter: a mediator of pancreatic cancer growth with a role in drug resistance

M Lo et al. Br J Cancer. .

Abstract

The x(c)(-) cystine transporter enhances biosynthesis of glutathione, a tripeptide thiol important in drug resistance and cellular defense against oxidative stress, by enabling cellular uptake of cystine, a rate-limiting precursor. Because it is known to regulate glutathione levels and growth of various cancer cell types, and is expressed in the pancreas, we postulate that it is involved in growth and drug resistance of pancreatic cancer. To examine this, we characterised expression of the x(c)(-) transporter in pancreatic cancer cell lines, MIA PaCa-2, PANC-1 and BxPC-3, as subjected to cystine-depletion and oxidative stress. The results indicate that these cell lines depend on x(c)(-)-mediated cystine uptake for growth, as well as survival in oxidative stress conditions, and can modulate x(c)(-) expression to accommodate growth needs. Immunohistochemical analysis showed that the transporter was differentially expressed in normal pancreatic tissues and overexpressed in pancreatic cancer tissues from two patients. Furthermore, gemcitabine resistance of cells was associated with elevated x(c)(-) expression and specific x(c)(-) inhibition by monosodium glutamate led to growth arrest. The results suggest that the x(c)(-) transporter by enhancing glutathione biosynthesis plays a major role in pancreatic cancer growth, therapy resistance and represents a potential therapeutic target for the disease.

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Figures

Figure 1
Figure 1
Pancreatic cancer cells depend on extracellular cystine for growth. Neutral red uptake assay for cell proliferation in MIA PaCa-2, PANC-1, and BxPC-3 cells incubated in medium in the presence or absence of methionine (0.1 mM), cystine (0.1 mM), and/or cystathionine (0.15 mM), in the combinations indicated for 72 h. Data represent the mean±s.e.m. from three independent experiments. *P<0.01.
Figure 2
Figure 2
A negative correlation exists between extracellular cystine deprivation and expression of the xc− transporter. (A) Q-RT–PCR for expression of xCT or 4F2hc mRNA in MIA PaCa-2, PANC-1, and BxPC-3 cell lines incubated with various cystine concentrations (0.01, 0.1 or 1 mM) for 72 h. Data represent the mean±s.e.m. from three independent experiments. *P⩽0.05. (B) Western blot for expression of 4F2hc or tubulin in MIA PaCa-2, PANC-1, and BxPC-3 cell lines incubated with various cystine concentrations (0.01, 0.1 or 1 mM) for 72 h. Results are representative of two independent experiments.
Figure 3
Figure 3
Oxidative stress increases xc− transporter expression and GSH levels. (A) Intracellular GSH levels in MIA PaCa-2, PANC-1, and BxPC-3 cell lines either untreated or treated with 1 mM DEM for 24 h. Data represent the mean±s.e.m. from at least two independent experiments. *P<0.05 using the Wilcoxon rank-sum test. (B) Q-RT–PCR for xCT and 4F2hc mRNA expression in MIA PaCa-2, PANC-1, and BxPC-3 cell lines either untreated or treated with 1 mM DEM for 24 h. Data represent the mean±s.e.m. from at least two independent experiments. *P<0.05 (C) Western blot for expression of 4F2hc and tubulin in MIA PaCa-2, PANC-1, and BxPC-3 cell lines either untreated or treated with 1 mM DEM for 24 h. Results are representative of two independent experiments. (D) Immunofluorescence for xCT (red) and DAPI (blue) in MIA PaCa-2, PANC-1, and BxPC-3 cell lines either untreated or treated with 1 mM DEM for 24 h. Results are representative of two independent experiments.
Figure 4
Figure 4
Expression of the xc− transporter in primary human pancreatic cancer specimens. Immunofluorescence for xCT (red) and DAPI (blue) in normal ductal cells, normal acinar cells, and pancreatic ductal adenocarcinoma cells from two primary human pancreatic cancer patient specimens.
Figure 5
Figure 5
A positive correlation exists between the expression level of xCT and resistance towards GEM. (A) Q-RT–PCR for xCT expression in MIA PaCa-2, PANC-1, and BxPC-3 cell lines. Data represent the mean±s.e.m. from three independent experiments. *P<0.05. (B) Neutral red uptake assay for cell proliferation in Mia PaCa-2, PANC-1, and BxPC-3 cultures treated with increasing concentrations of GEM for 72 h. Data represent the mean±s.e.m. from three independent experiments. *P<0.01.
Figure 6
Figure 6
Overexpression of transfected xCT increases cystine uptake and increases GEM resistance. (A) RT–PCR for xCT and 4F2hc expression in MIA PaCa-2 cells transfected with pcDNA3.1 empty vector, pcDNA3.1-xCT plasmid, or pcDNA3.1-4F2hc plasmid. (B, C) Q-RT–PCR for xCT (B) or 4F2hc (C) expression in MIA PaCa-2 cells transfected with pcDNA3.1 empty vector, pcDNA3.1-xCT plasmid, or pcDNA3.1-4F2hc plasmid. *P<0.001. (D) [3H]-glutamate uptake assay in MIA PaCa-2 and PANC-1 cells transfected with pcDNA3.1 empty vector, pcDNA3.1-xCT plasmid, and/or pcDNA3.1-4F2hc plasmid. *P<0.01 compared with respective cell line pcDNA3.1 control. (E) Neutral red uptake assay for survival of untreated or GEM-treated MIA PaCa-2 cells transfected with pcDNA3.1 empty vector, pcDNA3.1-xCT plasmid, and/or pcDNA3.1-4F2hc plasmid. *P⩽0.05 compared with pcDNA3.1 control; #P⩽0.05 compared with pcDNA3.1 GEM treatment. All data represent the mean±s.e.m. from three independent experiments.
Figure 7
Figure 7
Targeting xc− transporter function with MSG. Neutral red uptake assay for cell proliferation in MIA PaCa-2, PANC-1, and BxPC-3 cultures treated with MSG (4.0 mM) or 2-ME (66 μM) in various combinations for 72 h. *P⩽0.05. All data represent the mean±s.e.m. from three independent experiments.
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