- Research Article
Ets-1 expression and gemcitabine chemoresistance in pancreatic cancer cells
Cellular & Molecular Biology Letters volume 16, pages 101–113 (2011)
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.
2′,2′-difluorodeoxycytidine 5′-triphosphate (gemcitabine triphosphate)
Dulbecco’s modified Eagle’s medium
avian erythroblastosis virus
E26 transformation specific sequence-1
fetal bovine serum
multiple drug resistance-1
matrix metalloproteinase-1 (collagenase-1)
matrix metallopeptidase-1 (stromelysin-1)
pancreatic ductal adenocarcinoma
reverse transcriptase real-time quantitative PCR
small interfering RNA
urokinase-type plasminogen activator
viral transforming gene of E26
Li, D., Xie, K., Wolff, R. and Abbruzzese, J.L. Pancreatic cancer. Lancet 363 (2004) 1049–1057.
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.
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.
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.
Galmarini, C.M., Mackey, J.R. and Dumontet, C. Nucleoside analogues: mechanisms of drug resistance and reversal strategies. Leukemia 15 (2001) 875–890.
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.
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.
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.
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.
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.
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.
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.
Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65 (1983) 55–63.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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
- MIA PaCa2