Notch3 induces epithelial–mesenchymal transition and attenuates carboplatin-induced apoptosis in ovarian cancer cells Nidhi Gupta a, b , Zhihua Xu a, b , Ahmed El-Sehemy b , Helen Steed a, b , YangXin Fu a, b, ⁎ a Department of Obstetrics and Gynecology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada b Department of Oncology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada HIGHLIGHTS • Notch3 induces EMT and confers resistance to carboplatin in ovarian cancer OVCA429 cells. • Blocking ERK phosphorylation attenuates carboplatin-induced apoptosis in OVCA429 cells. • Notch3 activation attenuates carboplatin-induced ERK phosphorylation and apoptosis in OVCA429 cells. abstract article info Article history: Received 9 December 2012 Accepted 21 March 2013 Available online 29 March 2013 Keywords: Ovarian cancer Notch3 Carboplatin ERK Objective. Notch3 is implicated in chemoresistance of ovarian cancer, yet the molecular mechanism un- derlying Notch3-mediated drug resistance remains to be elucidated. Here, we investigated the role of Notch3 in carboplatin-induced apoptosis in ovarian cancer cells. Methods. Ovarian cancer cell line OVCA429 cells were stably transduced with an empty vector or a retro- viral vector expressing the Notch3 intracellular domain (NICD3, the constitutively active form of Notch3) to generate OVCA429/vector and OVCA429/NICD3 cells. Epithelial–mesenchymal transition (EMT) was deter- mined by morphological change and expression of the EMT markers. Carboplatin-induced cytotoxicity was determined by the neutral red uptake assay. Apoptosis was determined by Annexin V staining and Western blotting. Carboplatin-induced phosphorylation of extracellular signal-regulated kinase (ERK) was identified by a phospho-kinase array and confirmed by Western blotting. Results. Activation of Notch3 in OVCA429 cells causes a spindle and fibroblast-like morphology, induces the expression of smooth muscle α-actin, Slug and Snail, but decreases the expression of E-cadherin, indicating that Notch3 activation induces EMT in OVCA429 cells. Furthermore, Notch3 activation renders OVCA429 cells more resistant to carboplatin-induced cytotoxicity and attenuates carboplatin-induced apoptosis in these cells. Our re- sults indicate that phosphorylation of ERK is a positive regulator of carboplatin-induced apoptosis in OVCA429 cells. Interestingly, carboplatin-induced ERK phosphorylation is inhibited by Notch3 activation. Conclusions. Notch3 activation induces EMT and attenuates carboplatin-induced apoptosis in OVCA429 cells. ERK phosphorylation plays a pro-apoptotic role in carboplatin-induced apoptosis in OVCA429 cells. Interestingly, Notch3 activation attenuates carboplatin-induced ERK phosphorylation in these cells. © 2013 Elsevier Inc. All rights reserved. Introduction Ovarian cancer is the fifth leading cause of cancer deaths in women and the leading cause of death related to gynecologic cancers [1]. Although an early diagnosis can result in a cure in 90% of all cases, approximately 75% of ovarian cancer patients are diagnosed at ad- vanced stages when tumors have spread beyond the ovaries and metastasized to other parts of the peritoneal cavity [2]. Advanced ovarian cancer patients are generally treated by a debulking surgery and a platinum–paclitaxel combination regimen as the first-line treatment [3]. Despite an initial positive response, relapse occurs in most ovarian cancer patients and recurrent diseases are resistant to current chemotherapy regimens. Thus, the 5-year survival rate for advanced stage III/IV ovarian cancer patients is only 15 to 25% [2].A better understanding of the molecular mechanisms underlying the chemoresistance of ovarian cancer will help us develop novel thera- peutic strategies and improve the management of ovarian cancer. The Notch signaling pathway determines cell fate by regulating multiple cellular processes [4,5]. Notch signaling is activated through Gynecologic Oncology 130 (2013) 200–206 ⁎ Corresponding author at: Department of Oncology, University of Alberta, 5142M, Katz Building, 114th St & 87th Ave., Edmonton, AB, T6G 2E1, Canada. Fax: +1 780 492 8160. E-mail address: yangxin@ualberta.ca (Y. Fu). 0090-8258/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ygyno.2013.03.019 Contents lists available at SciVerse ScienceDirect Gynecologic Oncology journal homepage: www.elsevier.com/locate/ygyno