TCF4 silencing sensitizes the colon cancer cell line to oxaliplatin as a common chemotherapeutic drug Fatemeh Gheidari a,b , Behnaz Bakhshandeh a , Ladan Teimoori-Toolabi b , Amirhosein Mehrtash b , Mahdis Ghadir b and Sirous Zeinali b Colon cancer is among the most prevalent cancers worldwide. Although the main modality of treatment is surgery, resistance to chemoradiotherapy raises concerns. Hence, we aimed to determine the effect of RNA-mediated silencing of tcf4, the downstream effector of the wnt signaling pathway, on the response of the SW480 cell line to oxaliplatin, a common chemotherapeutic drug. For this, two different silencing sequences against TCF4 mRNA were selected and cloned into pSilencer neo2.1. The SW480 cell line was stably transfected with the silencing constructs (namely p1396, p1874, and p silencer containing a scrambled sequence) and labeled SW1396, SW1874, and SW-Sc, respectively. Subsequently, the effect of oxaliplatin (from 0 to 11.25 lmol/l) on these cells was studied using 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide proliferation assay. Suppression of tcf4 expression in stable transfected cells with p1396 and p1874 was confirmed by quantitative reverse transcription-PCR and western blot analysis. Although oxaliplatin was not toxic to SW480 and SW-sc in the range tested, in SW1396 and SW1874 cells, a toxic effect was evident at 3.75 and 4.375 lmol/l. Also, SW1396 and SW1874 cells appeared to have a round shape in comparison with SW480 and SW-Sc cells. Only for SW1396, the number of apoptotic cells was significantly different before and after the addition of oxaliplatin (LC50 of oxaliplatin). The proliferating cells in SW480, SW1874, and SW-Sc increased after treatment with oxaliplatin; however, this was not observed in SW1396. Although silencing the tcf4 gene would confer sensitivity to oxaliplatin in SW1874 and especially SW1396, in SW480 and SW-Sc, the lethal effect of oxaliplatin was compensated by its effect in increasing the proliferation of cells. This sensitization effect may be because of different mechanisms including TCF4 motifs in the ABCB1 promoter or defects in nucleotide excision repair or double-strand break repair systems after tcf4 silencing. Anti-Cancer Drugs 25:908–916  c 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. Anti-Cancer Drugs 2014, 25:908–916 Keywords: gene silencing, human, neoplasia, oxaliplatin, RNA, small interfering, TCF4 protein a Department of Biotechnology, College of Science, University of Tehran and b Molecular Medicine Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran Correspondence to Ladan Teimoori-Toolabi, MD, PhD, Molecular Medicine Department, Biotechnology Research Center, Pasteur Institute of Iran, 69th Pasteur Avenue, Kargar Street, Tehran 13169-43551, Iran Tel: + 98 216 695 3311-19 x2455; fax: + 98 216 648 0780; e-mail: lteimoori@pasteur.ac.ir Received 13 October 2013 Revised form accepted 13 March 2014 Introduction Colorectal cancer is the third most common cancer in men and the second most common cancer among women worldwide. About 60% of new cases of colorectal cancer (CRC) occur in the developed countries [1]. Five-year survival can be expected in more than 90% of patients if the disease is diagnosed in early stages. Unfortunately, only 39% of CRCs are detected in the early stages. With the most optimal medical management, the mean survival of patients with metastatic CRC (mCRC) is about 6 months [2]. Although different regimens of chemo/radiotherapy can be tested on mCRC patients to identify the most suitable ones, resistance to chemotherapeutic drugs raises serious concerns in the treatment of these patients. This resistance is mainly because of a phenomenon called multidrug resistance (MDR). MDR is mainly associated with overexpression of ABC transporters [3]. ABCB1 coding P-glycoprotein was the first cloned human ATP- binding cassette transporter or ABC transporter [4]. These transporters act by pumping drugs out of the cells and reducing their intracellular concentration [5]. Reports on the existence of T cell factor4 or TCF4-binding motifs in the ATP-binding cassette transporter sub-family B member 1 or the ABCB1 promoter suggest that the b-catenin/TCF4 complex, the operator of the Wnt pathway, may also exert an effect on MDR [6]. The Wnt signaling pathway is hyperactivated in about 90% of CRCs. This pathway plays roles in controlling stem cell proliferation, differentiation, renewal, and embryonic development, whereas in adults it is involved in intestinal homeostasis. Owing to the inappropriate activation of this pathway, indefinite proliferation and finally neoplasia may be observed in intestinal cells. The Wnt signaling pathway is also responsible for angio- genesis, tissue invasion, and metastasis [7]. Activated Wnt signaling pathway exerts its effects by the accumulation of b-catenin in the nucleus, leading to activation of TCF4 and transcription of a set of genes such as c-Myc (cellular avian myelocytomatosis virus) and cyclin D1. When the Wnt signaling pathway is repressed, b-catenin is 908 Preclinical report 0959-4973  c 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins DOI: 10.1097/CAD.0000000000000118 Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.