Nilotinib reduced the viability of human ovarian cancer cells via mitochondria-dependent apoptosis, independent of JNK activation
Introduction
Ovarian cancer is one of the most lethal gynecological cancers worldwide, and tumors of patients are often already malignant at the time they are diagnosed (Salzberg et al., 2005). Up to 70% of ovarian cancer patients have moderate to advanced disease, and 20%–40% of patients have stage III or IV cancer, which results in a low 5-year survival rate (Davis et al., 2014). The current treatment options for ovarian cancer mainly rely on surgery and chemotherapy, a common anticancer treatment that uses antiproliferative molecules to kill cancer cells. However, some chemotherapeutic agents affect healthy cells, which lead to severe side effects, and resistance of ovarian cancer cells to chemotherapeutic agents such as cisplatin has been found (Lin et al., 2012). Therefore, development of a new generation of chemotherapeutic drugs is an important issue in treating ovarian cancers.
Inducing apoptosis is a promising way to treat cancers, and at least two apoptotic pathways, including intrinsic and extrinsic cascades, in response to apoptotic stimuli are known (Chien et al., 2012, Shen et al., 2012). Previous studies showed that apoptosis induced by chemotherapeutic agents mostly occurs through activation of mitochondrial apoptotic pathways, and the release of mitochondrial cytochrome (Cyt) c, which initiates activation of caspase (Casp)-9 leading to activation of effector caspases such as Casp-3, and its substrate was identified to be poly(ADP ribose) polymerase (PARP) (Chappell et al., 2012, Ko et al., 2005, Ko et al., 2007). Additionally, both proapoptotic (e.g., Bax and Bak) and antiapoptotic (e.g., Bcl-2 and Bcl-xL) Bcl-2 family proteins may participate in regulating the mitochondrial membrane potential (MMP) in apoptosis, and decreases in antiapoptotic and increases in proapoptotic Bcl-2 family proteins contribute to apoptosis of cancer cells under chemical stimulation (Czabotar et al., 2014). Therefore, disruption of the MMP via altering the Bcl-2/Bax balance to activate Casp-9 and − 3 plays a critical role in apoptosis induced by chemotherapeutic agents. Reactive oxygen species (ROS), which are secondary mediators, participate in various physiological responses including proliferation, differentiation, and apoptosis (Morgan and Liu, 2010, Schippers et al., 2012). ROS-dependent and -independent apoptosis was reported in several different cells under various treatments through activation of mitochondrion-dependent pathway. Mitogen-activated protein kinase (MAPK) was implicated in regulating survival and cell death responses of tumor cells, and alternative MAPK activation in cancer deregulation was found. ROS were shown to activate MAPK by inducing its phosphorylation, and anticancer drugs, such as paclitaxel and nocodazol, induced apoptosis accompanied by induction of MAPK activation (Chen, 2012, Davies and Tournier, 2012). In contrast to apoptosis, activation of MAPK also contributes to the proliferation of cancer cells under growth factors, such as epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) stimulation and prevents apoptosis (Chen and Khalil, 2006, Chien et al., 2009). Differential roles of MAPK activation in apoptosis and proliferation of cancer cells were indicated.
BCR–ABL caused by the translocation of t(9, 22) with increased tyrosine kinase activity to activate multiple downstream signaling pathways leads to the proliferation of chronic myeloid leukemia cells (Yang and Fu, 2015). Nilotinib (AMN), a second-generation tyrosine kinase inhibitor (TKI), functions via ATP-competitive inhibition, and possesses an in vitro Bcr–Abl-binding potency higher than its predecessor, imatinib (STI) (Martinelli et al., 2007). AMN inhibits a broad spectrum of kinases, including KIT, MAPK, ZAK, and PDGF receptor (PDGFR) with less potency, which makes it further applicable for treating other types of cancers such as gastrointestinal stromal tumors, breast cancer, and melanomas (Manley et al., 2010). AMN exerted antiproliferative effects against the MCF-7 breast cancer cell line and metastatic melanoma cells (Weigel et al., 2012). AMN's reduction of viability was mediated by inducing apoptosis in several cancer cells including K562 leukemic cells, HaCaT keratinocytes, and Jurkat T cells. Yu et al. (2013) indicated that AMN induces autophagy, but not apoptosis, in hepatocellular carcinoma cells (Yu et al., 2013). Although AMN-reduced viability was reported in various cancer cells, the effect of AMN against the viability of ovarian cancer cells is still unclear. In this study, we explored whether AMN exerts antitumor activity against various human ovarian cancer cells including SKOV-3, ES-2, A2780, and cisplatin-resistant A2780CP cells. Our data showed that AMN-induced cell death in these ovarian cancer cells was mediated by the activation of apoptosis. The mechanisms, including disruption of the MMP and induction of c-Jun N-terminal kinase (JNK) protein phosphorylation and intracellular peroxide production, of AMN were observed in human ovarian cancer cells.
Section snippets
Cell culture
High-grade human epithelial ovarian cancer cell lines, SKOV3, ES-2, A2780 and its daughter line, A2780CP (which is resistant to cisplatin), murine macrophages RAW264.7 rat astrocyte CTX TNA2, and human colon epithelial cells FHC (fetal human colon) were obtained from the American Type Culture Collection (Rockville, MD, USA). Human keratinocyte HaCaT was kindly gifted by Prof. Wan-Louh Lee (Department of Dermatology, Taipei Medical University, Taipei, Taiwan; originated from CLS; Cell Lines
AMN reduces the viability of human SKOV-3 ovarian cancer cells, but not non-cancerous cells, by inducing apoptosis
In order to examine the effect of AMN on the viability of human SKOV-3 ovarian cancer cells, morphological observations, a flow cytometric analysis, and an MTT assay were applied in the present study. As shown in Fig. 1A, induction of apoptotic morphology in SKOV-3 cells by AMN, but not sorafenib (SORA) or STI, was observed microscopically via Giemsa staining. Data of the MTT assay showed that AMN concentration-dependently reduced the viability of SKOV-3 cells; however, no change in the
Discussion
To overcome the emergence of drug resistance by ovarian cancer cells, investigations have increasingly focused on new therapeutic strategies to reverse tumor resistance and increase the therapeutic effects of chemotherapy. AMN was developed for the selective inhibition of several tyrosine kinases in malignant cells; however, the effect of AMN on ovarian cancers is still unknown. This study showed that AMN induces cytotoxicity via apoptosis in human ovarian cancer cells including SKOV-3, A2780,
Conflict of interest
The authors who took part in this study declare that they have nothing to disclose regarding funding or conflicts of interest with respect to this manuscript.
Author contributions
Conceived and designed the experiments: YC Chen. Performed the experiments: TC Chen, MC Yu, and CC Chien. Analyzed the data: MS Wu, and YC Lee. Wrote the paper: TC Chen, MC Yu, and YC Chen.
Acknowledgments
This study was supported by the Ministry of Sciences and Technology of R.O.C. (MOST104-2320-B-038-003 and MOST101-2320-B-038-025-MY3), Taipei Medical University-Wan Fang Hospital (104TMU-WFH-02-2), and Taipei Medical University-Chi Mei Hospital (104CM-TMU-05).
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Dr. Chen and Dr. Yu equally contribute to the present work.