Skip to main content

Ets-1 expression and gemcitabine chemoresistance in pancreatic cancer cells

Abstract

Gemcitabine, a novel pyrimidine nucleoside analog, has become the standard chemotherapeutic agent for pancreatic cancer patients. The clinical impact of gemcitabine remains modest owing to the high degree of inherent and acquired resistance. There are various lines of evidence that confirm the role of Ets-1, a proto-oncoprotein, in tumor invasion, progression, and chemoresistance. This study examines a hypothesis that implicates Ets-1 in the development of gemcitabine-resistance in pancreatic cancer cells. Ets-1 protein expression was assessed in parental pancreatic cancer cells and their gemcitabine-resistant clones. Western blot analysis revealed elevated levels of Ets-1 protein expression in gemcitabine-resistant PANC1GemRes (4.8-fold increase; P < 0.05), MIA PaCa2GemRes (3.2-fold increase; P < 0.05), and Capan2GemRes (2.1-fold increase; P < 0.05) cells as compared to their parental counterparts. A time course analysis was conducted to determine the change in Ets-1 expression in the parental cells after incubation with gemcitabine. Reverse transcriptase quantitative real-time PCR (RT-qPCR) and Western blot analysis revealed a significant increase in Ets-1 expression. All the three parental cells incubated with gemcitabine showed elevated Ets-1 protein expression at 6 h. By 24 h, the expression level had decreased. Using small interfering RNA (siRNA) against Ets-1 in gemcitabine-resistant cells, we demonstrated a reversal in gemcitabine chemosensitivity and also detected a marked reduction in the expression of the Ets-1 target genes MMP1 and uPA. Our novel finding demonstrates the significance of Ets-1 in the development of gemcitabine chemoresistance in pancreatic cancer cells. Based on these results, a new siRNA-based therapeutic strategy targeting the Ets-1 genes can be designed to overcome chemoresistance.

Abbreviations

cDNA:

complementary DNA

Ct:

cycle threshold

dFdCTP:

2′,2′-difluorodeoxycytidine 5′-triphosphate (gemcitabine triphosphate)

DMEM:

Dulbecco’s modified Eagle’s medium

DMSO:

dimethyl sulfoxide

E26:

avian erythroblastosis virus

Ets-1:

E26 transformation specific sequence-1

FBS:

fetal bovine serum

GAPDH:

glyceraldehydes-3-phosphate dehydrogenase

MDR1:

multiple drug resistance-1

MMP1:

matrix metalloproteinase-1 (collagenase-1)

MMP3:

matrix metallopeptidase-1 (stromelysin-1)

MTT:

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

PDAC:

pancreatic ductal adenocarcinoma

RT-qPCR:

reverse transcriptase real-time quantitative PCR

siRNA:

small interfering RNA

uPA:

urokinase-type plasminogen activator

v-ets:

viral transforming gene of E26

References

  1. Li, D., Xie, K., Wolff, R. and Abbruzzese, J.L. Pancreatic cancer. Lancet 363 (2004) 1049–1057.

    CAS  Article  PubMed  Google Scholar 

  2. Klein, B., Sadikov, E., Mishaeli, M., Levin, I. and Figer, A. Comparison of 5-FU and leucovorin to gemcitabine in the treatment of pancreatic cancer. Oncol. Rep. 7 (2000) 875–877.

    CAS  PubMed  Google Scholar 

  3. Burris, H.A., Moore, M.J., Andersen. J., Green, M.R., Rothenberg, M.L., Modiano, M.R., Cripps, M.C., Portenoy, R.K., Storniolo, A.M., Tarassoff, P., Nelson, R., Dorr, F.A., Stephens, C.D. and Von Hoff, D.D. Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: a randomized trial. J. Clin. Oncol. 15 (1997) 2403–2413.

    CAS  PubMed  Google Scholar 

  4. Ueno, H., Kiyosawa, K. and Kaniwa, N. Pharmacogenomics of gemcitabine: can genetic studies lead to tailor-made therapy. Br. J. Cancer 97 (2007) 145–151.

    CAS  Article  PubMed  Google Scholar 

  5. Galmarini, C.M., Mackey, J.R. and Dumontet, C. Nucleoside analogues: mechanisms of drug resistance and reversal strategies. Leukemia 15 (2001) 875–890.

    CAS  Article  PubMed  Google Scholar 

  6. Leprince, D., Gegonne, A., Coll, J., De Taisne, C., Schneeberger, A., Lagrou, C. and Stehel, D. A putative second cell-derived oncogene of the avian leukaemia retrovirus E26. Nature 306 (1983) 395–397.

    CAS  Article  PubMed  Google Scholar 

  7. Nunn, M.F. and Hunte, T. The Ets sequence is required for induction of erythroblastosis in chickens by avian retrovirus E26. J. Virol. 63 (1989) 398–402.

    CAS  PubMed  Google Scholar 

  8. Koizumi S; Fisher R.J., Fujiwara S., Jorcyk C., Bhat N.K., Seth A. Isoforms of the human ets-1 protein: generation by alternative splicing and differential phosphorylation. Oncogene 81(1990) 675–681.

    Google Scholar 

  9. Oda, N., Abe, M. and Sato, Y. Ets-1 converts endothelial cells to the angiogenic phenotype by inducing the expression of matrix metalloproteinases and integrin beta3. J. Cell Physiol. 178 (1999) 121–132.

    CAS  Article  PubMed  Google Scholar 

  10. Nakada, M., Yamashita, J., Okada, Y. and Sato, H. Ets-1 positively regulates expression of urokinase-type plasminogen activator (uPA) and invasiveness of astrocytic tumors. J. Neuropathol. Exp. Neurol. 58 (1999) 329–334.

    CAS  Article  PubMed  Google Scholar 

  11. Sampath, J., Sun, D., Kidd, V.J., Grenet, J., Gandhi, A., Shapiro, L.H., Wang, Q., Zambetti, G.P. and Schuetz, J.D. Mutant p53 cooperates with Ets and selectively up-regulates human MDR1 not MRP1. J. Biol. Chem. 276 (2001) 39359–39367.

    CAS  Article  PubMed  Google Scholar 

  12. Kars, M.D., Işeri, O.D., Gunduz, U., Ural, A.U., Arpaci, F. and Molnar, J. Development of rational in vitro models for drug resistance in breast cancer and modulation of MDR by selected compounds. Anti Can. Res. 26 (2006) 4559–4568.

    CAS  Google Scholar 

  13. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65 (1983) 55–63.

    CAS  Article  PubMed  Google Scholar 

  14. Liu, X., Zhou, B., Xue, L., Yen, F., Chu, P., Un, F. and Yen, Y. Ribonucleotide reductase subunits M2 and p53R2 are potential biomarkers for metastasis of colon cancer. Clin. Colorectal Canc. 6 (2007) 374–381.

    CAS  Article  Google Scholar 

  15. Stephanie, G., Melanie, M., Jerome, P., Olivier, V.W., Catherine, B. and Laurent, G. Normalization of qRT-PCR data: the necessity of adopting a systematic, experimental conditions-specific, validation of references J. Exp. Bot. 60 (2009) 487–493.

    Article  Google Scholar 

  16. Lefter, P., Dima, S., Sunamura, M., Furukawa, T., Sato, Y., Abe, M., Chivu, M., Popescu, I. and Horii, A. Transcriptional silencing of Ets-1 efficiently suppresses angiogenesis of pancreatic cancer. Cancer Gene Ther. 16 (2009) 137–148.

    CAS  Article  PubMed  Google Scholar 

  17. Maroni, P., Bendinelli, P., Matteucci, E. and Desiderio, M.A. HGF induces CXCR4 and CXCL12-mediated tumor invasion through Ets-1 and NF-B. Carcinogenesis 28 (2007) 267–279.

    CAS  Article  PubMed  Google Scholar 

  18. Fujimoto, J., Aoki, I., Toyoki, H., Khatun, S., Sato, E., Sakaguchi, H., and Tamaya, T. Clinical implications of expression of Ets-1 related to angiogenesis in metastatic lesions of ovarian cancers. Oncology 66 (2004) 420–428.

    CAS  Article  PubMed  Google Scholar 

  19. Wernert, N., Gilles, F., Fafeur, V. Bouali, F., Raes, M.-B., Pyke, C., Dupressoir, T., Seitz, G., Vandenbunder, B. and Stéhelin, D. Stromal expression of c-Ets-1 transcription factor correlates with tumor invasion. Cancer Res. 54 (1994) 5683–5688.

    CAS  PubMed  Google Scholar 

  20. Wilson, L.A., Yamamoto, H. and Singh, G. Role of the transcription factor Ets-1 in cisplatin resistance. Mol. Cancer Ther. 3 (2004) 823–830.

    CAS  PubMed  Google Scholar 

  21. Kitange, G., Shibata, S., Tokunaga, Y., Yagi, N., Yasunaga, A., Kishikawa, M. and Naito, S. Ets-1 transcription factor mediated urokinase-type plasminogen activator expression and invasion in glioma cells stimulated by serum and basic fibroblast growth factors. Lab. Invest. 79 (1999) 407–516.

    CAS  PubMed  Google Scholar 

  22. Naotaka, H., Nobuo, J., Motokuni, A., Kunio, M., Toshikazu, N., Yasufumi, S., Nahoko, O., Toshio, O., Yasufumi, K. and Ryuichi M. In vivo evidence of angiogenesis induced by transcription factor Ets-1: Ets-1 is located upstream of angiogenesis cascade. Circulation 109 (2004) 3035–3041.

    Article  Google Scholar 

  23. Hiromichi, I., Mark, D., Eric, B., Thomas, E. C., Michael, J. Z., Stanley, W. A. and Edward, E. W. Prostaglandin E2 Enhances Pancreatic Cancer Invasiveness through an Ets-1-Dependent Induction of Matrix Metalloproteinase-2. Cancer Res. 64 (2004) 7439–7446.

    Article  Google Scholar 

Download references

Author information

Affiliations

Authors

Corresponding author

Correspondence to Giridharan Periyasamy.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Khanna, A., Mahalingam, K., Chakrabarti, D. et al. Ets-1 expression and gemcitabine chemoresistance in pancreatic cancer cells. Cell Mol Biol Lett 16, 101–113 (2011). https://doi.org/10.2478/s11658-010-0043-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.2478/s11658-010-0043-z

Key words

  • Gemcitabine
  • Ets-1
  • MMP1
  • uPA
  • PANC1
  • MIA PaCa2
  • Capan2