Nuclear factor (erythroid-derived 2)-like 2 regulates drug resistance in pancreatic cancer cells

Abstract

Objective: To investigate the molecular basis of drug resistance in pancreatic cancer.

Methods: The expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) levels in pancreatic cancer tissues and cell lines was analyzed. Clinical relevance between Nrf2 activation and drug resistance was demonstrated by measuring cell viability after Nrf2 and adenosine 5'-triphosphate-binding cassette, subfamily G member 2 (ABCG2) regulation by overexpression or knock-down of these genes. Activity of ABCG2 was measured by Hoechst 33342 staining.

Results: Abnormally elevated Nrf2 protein levels were observed in pancreatic cancer tissues and cell lines relative to normal pancreatic tissues. Increasing Nrf2 protein levels either by overexpression of exogenous Nrf2 or by activating endogenous Nrf2 resulted in increased drug resistance. Conversely, a reduction in endogenous Nrf2 protein levels or inactivation of endogenous Nrf2 resulted in decreased drug resistance. These changes in drug resistance or sensitivity were also positively correlated to the expression levels of Nrf2 downstream genes. Similarly, the expression of ABCG2 was correlated with drug resistance.

Conclusions: Because the intrinsic drug resistance of pancreatic cancers is, in part, due to abnormally elevated Nrf2 protein levels, further research on regulating Nrf2 activity may result in the development of novel pancreatic cancer therapies.

Figure 1. Differential expression of Nrf2 in human pancreatic tissues and cells

(A) Expression of Nrf2 in normal pancreatic tissue, which is restricted to the cytoplasm of acini and ductal epithelium. The cytoplasmic expression of acini shows mild to moderate intensity (arrow). The cytoplasm of ductal cells reveals focal and faint expression (arrowhead). (B–E) Nuclear immunoreactivity of Nrf2 in pancreatic carcinoma. The nuclear scoring is (B), no expression; (C), less than 5% of tumor cells; (D), 5–50% of tumor cells; and (E), more than 50% of tumor cells. (F–I) Cytoplasmic immunoreactivity of Nrf2 in pancreatic carcinoma. The cytoplasmic staining of tumor cells is divided into four categories by degree of intensity; (F) no expression, (G) mild or faint, (H) moderate, and (I) strong and diffuse intensity.

Figure 2

Human pancreatic cancer cell lines accumulate Nrf2. (A) Western blot (WB) analysis of total lysates reveal that pancreatic tumor cell lines (AsPC-1, Colo-357 and PANC-1) have elevated level of Nrf2 and its target proteins when compared with normal pancreatic cell lines (HPDE6-C7 and HPDE6-C11). (B) WB analysis of nuclear and cytosolic fractions for Nrf2 protein levels show that three pancreatic cancer cell lines accumulate activated Nrf2 in the nuclei. (C) t-BHQ treatments (100 uM for 8 hrs) increased the relative amounts of GCLC and the slower migrating Nrf2 in the HPDE6-C11 and AsPC-1 cell lines. (D) Lambda phosphatase (λ-PPase) treatments (30 min) of whole cell lysates of t-BHQ treated (100uM for 16 hr) cells selectively reduced amount of the slower migrating Nrf2 species. (E) TransAM Nrf2 activity assay shows that DNA binding activities of nuclear Nrf2 are also elevated in pancreatic cancer cell lines.

Figure 3

Nrf2 over-expression increased drug resistance in pancreatic cancer cells. (A–D) AsPC-1 and Colo-357 transiently transfected with an Nrf2 expression vector (Flag-Nrf2) for 24 hrs showed increased resistance to Cisplatin (A and C) and Camptothecin (B and D) as compared with transfected with control vector. (E–H) Preincubation with 100 µM of t-BHQ for 24 hrs resulted in drug resistance of AsPC-1 and Colo-357 to Cisplatin (E and G) and Camptothecin (F and H). Indicated concentrations of Cisplatin or Camptothecin were treated for 24 hrs. Cell survival relative to untreated cells (set at 100%) was measured by a standard MTT assay. *, p < 0.05; **, p < 0.01

Figure 4

Inhibition of Nrf2 activity with dominant negative Nrf2 (DN-Nrf2) or knock-down with Nrf2 siRNA decreased drug resistance. (A–D) Transfection with DN-Nrf2 mutants increased sensitivity of AsPC-1 (A and B) and Colo-357 (C and D) to Cisplatin (A and C) and Camptothecin (B and D). (E-H) Nrf2 knock-down with Nrf2-specific siRNA increased sensitivity of AsPC-1 (E and F) and Colo-357 (G and H) to various concentrations of both Cisplatin (E and G) and Camptothecin (F and H). Indicated concentrations of Cisplatin or Camptothecin were incubated for 24 hrs. Cell survival relative to untreated cells (set at 100%) was measured by a standard MTT assay as described in Material and Methods. *, p < 0.05; **, p < 0.01.

Figure 5

Nrf2 knock-down reduces expression of several multidrug resistance genes. (A) Y-axis shows the percentage of mRNA expression levels of the Nrf2 and its target genes in the AsPC-1, Colo-357 and PANC-1 cells treated with Nrf2-siRNA, compared with those in the cells treated with control-siRNA. The mRNA was quantified by real-time PCR described in Material and Methods. (B) Western blotting shows that Nrf2 knock-down reduces Nrf2 and its target gene product GCLC protein levels.

Figure 6

Nrf2 Knock-down affects Hoechst 33342 efflux. (A) The cell numbers of Hoechst 33342 positive decreased after over-expression of Nrf2 or ABCG2. (B) The cell numbers of Hoechst 33342 positive increased after knock-down of Nrf2 or ABCG2. (C) Restoring ABCG2 decreased the numbers of Hoechst 33342 positive cells in Nrf2 knockdown cells. (D) Reduced cell viability on Camptothecin by knock-down of Nrf2 is recovered by over-expression of ABCG2. (E) Western blot shows changes of Nrf2 and ABCG2 protein levels after knock-down of Nrf2 and transient over-expression of ABCG2. **, p < 0.01; ***, p < 0.001.