Nilotinib reduced the viability of human ovarian cancer cells via mitochondria-dependent apoptosis, independent of JNK activation

Highlights

• Nilotinib induced apoptosis in various human ovarian cancer cells.

• Disrupted mitochondrial membrane potentiation leads to apoptosis by nilotinib.

• Increased intracellular peroxide and JNK by nilotinib

• NAC and JNK inhibitors did not inhibit apoptosis induced by nilotinib.

• A more potent cytotoxicity on ovarian cancer than non-cancerous cells by nilotinib

Abstract

Nilotinib (AMN) induces apoptosis in various cancer cells; however the effect of AMN on human ovarian cancer cells is still unclear. A reduction in cell viability associated with the occurrence of apoptotic characteristics was observed in human SKOV-3 ovarian cancer cells under AMN but not sorafenib (SORA) or imatinib (STI) stimulation. Activation of apoptotic pathway including increased caspase (Casp)-3 and poly(ADP-ribose) polymerase 1 (PARP1) protein cleavage by AMN was detected with disrupted mitochondrial membrane potential (MMP) accompanied by decreased Bcl-2 protein and increased cytosolic cytochrome (Cyt) c/cleaved Casp-9 protein expressions was found, and AMN-induced cell death was inhibited by peptidyl Casp inhibitors, VAD, DEVD and LEHD. Increased phosphorylated c-Jun N-terminal kinase (JNK) protein expression was detected in AMN- but not SORA- or STI-treated SKOV-3 cells, and the JNK inhibitors, SP600125 and JNKI, showed slight but significant enhancement of AMN-induced cell death in SKOV-3 cells. The intracellular peroxide level was elevated by AMN and H₂O₂, and N-acetylcysteine (NAC) prevented H₂O₂- but not AMN-induced peroxide production and apoptosis in SKOV-3 cells. AMN induction of apoptosis with increased intracellular peroxide production and JNK protein phosphorylation was also identified in human A2780 ovarian cancer cells, cisplatin-resistant A2780CP cells, and clear ES-2 cells. The evidence supporting AMN effectively reducing the viability of human ovarian cancer cells via mitochondrion-dependent apoptosis is provided.

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.