1  Introduction
PDAC is one of the most fatal human malignant cancers, because it is often diagnosed at middle or late stage. It is currently the fourth leading cause of cancer death worldwide with a less than 5% 5-year survival rate [1], [2]. Although some effective treatment measures are used, PDAC death rate is still on the rise. The development of chemoresistance is a major reason leading to chemotherapy failure in pancreatic cancer. Gemcitabine, a deoxycytidine analog that inhibits DNA replication and thereby arrests tumor growth, is widely used single-agent chemotherapy for pancreatic cancer, but high rate of chemoresistance reduce the effectiveness of its clinical treatment [3]. Therefore, it is necessary to find potential adjuvants to reverse the gemcitabine resistance in gemcitabine-resistant pancreatic cancer.
Nrf2, a basic leucine zipper transcription factor, participates in protecting cells from electrophilic or oxidative stresses through regulating cellular redox homeostasis [4], [5]. Nrf2 regulates the expression of its downstream genes such as glutamate cysteine ligase (GCL), NADP(H): quinone oxidoreductase (NQO), heme oxygenase-1 (HO-1) and several ATP-dependent drug efflux pumps through binding to antioxidant-response elements (AREs) [6], [7], [8], [9]. Kelch-like ECH-associated protein 1 (Keap1), a substrate adaptor protein, connects Nrf2 and Cul3-dependent E3 ubiquitin ligase to form complex, suppresses Nrf2 activity under basal condition [10]. When the intracellular stable environment is changed, electrophiles and oxidants inhibit the Keap1-mediated proteasomal degradation, causing the translocation of Nrf2 to the nucleus. Then Nrf2 binds to AREs and enhances transcription of its target genes.
Recently, some studies suggested that overactivation of Nrf2 signaling was one of the reasons for the drug resistance during chemotherapy [11], [12]. Frequent mutations of Keap1 in human cancers such as breast and lung cancer result in the upregulation of Nrf2 signaling [13]. We previously reported that Nrf2 and its downstream genes were highly expressed in MCF-7/DOX cells, and using Nrf2 siRNA to knockdown Nrf2 could reverse chemoresistance [14]. Similarly, tamoxifen and imatinib-resistant cancer cells also exhibited overactivation of Nrf2 signaling [15], [16]. Moreover, Hong et al. found that drug resistance was increased or decreased in pancreatic cancer cells with overexpression or knockdown of Nrf2, respectively [17]. Therefore, Nrf2 may be expected to become a pharmacological target to reverse chemoresistance in drug-resistant cancers with overactivation of Nrf2 signaling. It is necessary to find adjuvants that have inhibitory effect of Nrf2 activity and such adjuvants combined with chemotherapy drugs might be useful to reverse chemoresistance.
Cardiac glycosides, a class of glycosides with strong cardiac functions, are mainly used in the treatment of chronic cardiac insufficiency and heart failure through inhibiting plasma membrane Na+/K+-ATPase. Among them, digoxin is mainly used to treat heart failure [18], [19] and several studies have reported that digoxin exerted anti-tumor activities by inhibition of proliferation, induction of apoptosis, supporting its potential use for cancer therapy [20], [21]. Choi et al. found that digoxin was able to inhibit activity of the Nrf2-ARE luciferase reporter gene in A549-ARE cells [22], suggesting that digoxin may be a potent Nrf2 inhibitor.
Here, we demonstrated that digoxin, a potent Nrf2 inhibitor, reversed drug resistance of gemcitabine in SW1990/Gem and Panc-1/Gem cells. Mechanistically, digoxin inhibited the activity of Nrf2 through suppressing phosphatidylinositol-3 kinase (PI3K)/Akt signaling pathway. Thus, digoxin might be a promising agent to reverse gemcitabine resistance in gemcitabine-resistant pancreatic cancer cells via inhibiting Nrf2 signaling.