Background
Lung cancer is the leading cause of cancer-associated deaths worldwide [
1]. Non–small cell lung cancer (NSCLC) represents 80–85% of all lung cancers and is further subtyped based on defined genetic abnormalities. These genetic aberrations have also enabled the development of targeted therapeutic approaches. In particular, therapies targeting tumors carrying mutations in epidermal growth factor receptor (EGFR) or a fusion of echinoderm microtubule-associated protein-like 4 (EML4) and anaplastic lymphoma kinase (ALK) genes have been clinically successful as first-line therapies [
2‐
5].
Crizotinib is a small-molecule inhibitor of ALK [
6], c-Met [
7], ROS1 [
8], and is approved by US Food and Drug Administration for the treatment of advanced NSCLC with ALK rearrangements. Crizotinib has shown promise in targeting NSCLC and has clearly improved the prognosis and quality of life for patients [
9]. Recent studies have highlighted the importance of crizotinib enantiomers in inhibiting different targets. ALK, c-Met, and ROS1 inhibition is attributed to (R)-crizotinib, whereas inhibition of these targets by the (S)-enantiomer of crizotinib is negligible. Interestingly, (S)-crizotinib inhibits human mutT homologue (MTH1) at nanomolar doses which is approximately 20 times more potent than the (R)-enantiomer. MTH1 is a member of the nudix phosphohydrolase superfamily of enzymes. MTH1 hydrolyzes oxidized nucleotides and prevents their incorporation into replicating DNA [
10]. In colon cancer cells, (S)-crizotinib was shown to induce DNA single-strand breaks and activate DNA repair mechanisms through MTH1 inhibition. The result was suppressed tumor growth in mice, implicating MTH1 as the target of (S)-crizotinib. However, recent studies have questioned the specificity of MTH1 inhibitors including (S)-crizotinib [
11‐
13]. What these developments have exposed is that there may be multiple mechanisms by which (S)-crizotinib mediates anti-tumor activities.
Expression of MTH1 is thought to protect cancer cells from the cytotoxic effect of high levels of reactive oxygen specifies (ROS) [
14]. Although cancer cells exhibit intrinsically high levels of ROS compared to their normal counterparts, MTH1 may sanitize oxidized dNTPs by converting 8-oxo-dGTP and 2-OH-dATP into monophosphates and therefore, prevent incorporation of oxidized nucleotides into DNA [
15]. Therefore, this suggests that (S)-crizotinib may allow ROS-mediated cell proliferation and survival [
16,
17] while preventing the adverse effects of ROS such as promotion of cell death [
18]. Whether these mechanisms are involved in NSCLC in not known. In this study, we have tested the hypothesis that (S)-crizotinib inhibits NSCLC growth by a MTH1-independent mechanism. In culture studies and in tumor xenografts produced in mice, we found that (S)-crizotinib induces apoptosis in NSCLC cells through the elevation of ROS and subsequent activation of the ER stress pathway. We also show that these activities are independent of MTH1.
Methods
Reagents
(S)-crizotinib (Selleck Chemical, Shanghai, China) was reconstituted in dimethyl sulfoxide (DMSO) and stored at −20 °C. The antioxidants N-acetyl-L-cysteine (NAC), glutathione (GSH), and superoxide dismutase (SOD) were purchased from Beyotime Biotech (Nantong, China). Antibodies against B cell lymphoma 2 (Bcl-2), Bcl-2 associated protein x (Bax), Ki67, GAPDH, and horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against activating transcription factor 4 (ATF4), phosphorylated and total eukaryotic initiation factor 2α (eIF2α), and CCAAT/−enhancer-binding protein homologous protein (CHOP) were purchased from Cell Signaling Technology (Danvers, MA). FITC Annexin V apoptosis Detection Kit I and Propidium Iodide (PI) were purchased from BD Pharmingen (Franklin Lakes, NJ).
Cells and cell culture
Human NSCLC cell lines (NCI-H460, H1975, and A549) were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. NCI-H460 and H1975 cells were cultured in RPMI-1640 medium (Gibco, Eggenstein, Germany), whereas A549 were grown in F12 K medium (Gibco, Eggenstein, Germany). All media formulations were supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Eggenstein, Germany).
Cell viability assay
To measure viability, cells were first seeded at a density of 8 × 103 per well in 96-well plates and cultured for 24 h in normal growth media to allow attachment. Cells were then treated with (S)-crizotinib. Initial studies were performed with 24 h culture of cells with final concentrations of (S)-crizotinib at 0.625, 1.25, 2.5, 5, 10, 20,40,60,and 80 μM. All other studies were performed with concentrations and time of exposure as indicated. Following treatments, media was replaced with fresh media containing 0.5 mg/mL MTT reagent. Cells were cultured for an additional 4 h. DMSO (100 μL) was added to dissolve the formazan product and absorbance was measured at 490 nm using RT 6000 microplate reader. The percent inhibition rate was calculated as [1 − (treated/control)] × 100. The IC50 values were obtained using the Logit method.
Measurement of reactive oxygen species generation in cells
Cellular ROS generation was measured by flow cytometry. Briefly, 5 × 105 cells were plated in 6-well culture dishes and allowed to attach overnight. Cells were then treated with (S)-crizotinib at concentrations and times indicated. Cells were stained with 10 μM DCFH-DA or 5 μM DAF-FM-DA (Beyotime Biotech, Nantong, China) at 37 °C for 30 min. Cells were harvested and then washed three times with ice-cold PBS and fluorescence was measured by flow cytometry (FACSCalibur, BD Biosciences, CA). In some experiments, cells were pretreated with 5 mM NAC, 5 mM GSH, or 300 U/mL SOD for 1 h. In all experiments, 8000 viable cells were analyzed.
Cell apoptosis analysis
NCI-H460, H1975 and A549 cells were plated at 2.5 × 105 cells/well in 6-well culture dishes for 24 h and treated with (S)-crizotinib (10, 20 or 30 μM) for 24 h. Cells were then collected, washed with ice-cold PBS, and evaluated for apoptosis by double staining with FITC conjugated Annexin V and Propidium Iodide (PI). Data were collected and analyzed using FACSCalibur flow cytometer.
Western blot analysis
Cells or tumor tissues were homogenized in protein lysis buffer, and debris was removed by centrifugation for 10 min at 4 °C. Protein concentrations in all samples were determined by using Bradford protein assay (Bio-Rad, Hercules, CA). Protein samples were separated using 6–12% sodium dodecyl sulfate-polyacrylamide gels and transferred to PVDF membranes. Five percent nonfat milk was used to block nonspecific binding for 1 h at room temperature. Blots were then probed with specific primary antibodies. Horseradish peroxidase-conjugated secondary antibodies and ECL kit (Bio-Rad, Hercules, CA) were used for detection.
siRNA knockdown experiments
The siRNA duplexes used in this study were purchased from Invitrogen (Carlsbad, CA, USA). CHOP (NM_004083, sense 5′-GAGCUCUGAUUGACCGAAUGGUGAA-3′), and MTH1 (NCBI accession: sense 5′-CGACGACAGCUACUGGUUUTT-3′) siRNA were custom-designed. Negative Universal Control (Invitrogen) was used as the control. NCI-H460 cells were seeded at 3 × 105/well into 6-well plates and cultured for 24 h. Cells were then transfected with siRNA (100 nM final concentration) or control siRNA by lipofectamine 2000 (Invitrogen). Forty-eight hours after transfection, levels of CHOP and MTH1 proteins were detected by Western blot.
ER electron microscopy
NCI-H460 cells were exposed to vehicle control (DMSO) or (S)-crizotinib (30 μM), with or without 5 mM NAC pretreatment for 1 h. Cells were then fixed with 2.5% glutaraldehyde overnight at 4 °C, post-fixed in 1% OsO4 at room temperature for 60 min, stained with 1% uranylacetate, and embedded in epon. Areas containing cells were block-mounted and cut into 70 nm sections and examined with an electron microscope (H-7500, Hitachi, Ibaraki, Japan).
In vivo xenograft model
Animal studies were reported in compliance with the ARRIVE guidelines [
19,
20]. All animal experiments complied with the Wenzhou Medical University’s Policy on the Care and Use of Laboratory Animals. Protocols for animal studies were approved by the Wenzhou Medical College Animal Policy and Welfare Committee (Approved documents: 2016/APWC/0046) and followed the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). Five-week-old, athymic BALB/c nu/nu female mice (17-19 g, totally
n = 15) were purchased from Vital River Laboratories (Beijing, China). Mice were housed at a constant room temperature with a 12 h:12 h light/dark cycle and fed a standard rodent diet and given water ad lib. The mice were divided into three experimental groups on randomization and blinding. NCI-H460 cells were injected subcutaneously into the right flank of mice at 1 × 10
7 cells in 150 μL of PBS. When tumors reached a volume of 50–100 mm
3 (approximately 6 d), mice were treated by intraperitoneal injections of (S)-crizotinib once daily (7.5, or 15 mg/kg) for 10 days. The tumor volumes were determined by measuring length (l) and width (w) and calculating volume (V = 0.5 × l × w
2) at the indicated time points. At the end of treatment, the animals were sacrificed after being anesthetized by i.p. injection with pentobarbital sodium (50 mg·kg
−1) and the tumors were isolated by surgery in a room separated from the other mice, and then the tumors were removed and weighed for use in histology and proteins expression studies.
ROS determination in tumor tissues
In order to analyze ROS levels in tumor tissue, we stained tissue sections with 10 μM dihydroethdium (DHE) and 50 μM DCFH-DA for 30 min. Images were captured under a fluorescence microscope (Nikon, Japan).
Malondialdehyde (MDA) assay
Tumors samples from mice were homogenized and sonicated. Tissue lysates were then centrifuged at 12,000 xg for 10 min at 4 °C to collect the supernatant. Total protein content was determined by the Bradford assay. Malondialdehyde (MDA) levels were measured by using Lipid Peroxidation MDA assay kit (Beyotime Institute of Biotechnology).
Immunohistochemistry
Tumor tissues were fixed in 10% formalin at room temperature, processed and embedded in paraffin. Parraffin-embedded tissues were sectioned at 5 μm thickness. Tissue sections were rehydrated and incubated with primarily antibodies at 4 °C overnight. Conjugated secondary antibodies and diaminobenzidine (DAB) were used for detection. Images were captured using Image-Pro Plus 6.0 (Media Cybernetics, Inc., Bethesda, MD). Liver, kidney, and heart tissues from mice were subjected to routine hematoxylin and eosin (H&E) staining.
Statistical analysis
All experiments are randomized and blinded. The data and statistical analysis in this study complies with the recommendations on experimental design and analysis in pharmacology [
21]. All data are shown as mean ± SEM. Statistical analysis was performed with GraphPad Prism 5.0 software (San Diego, CA, USA). In accordance with Journal policy, statistical analysis was performed only when a minimum of
n = 5 independent samples was acquired. One-way ANOVA followed by Dunnett’s post hoc test was used for comparing more than two groups of data and one-way ANOVA, non-parametric Kruskal–Wallis test, followed by Dunn’s post hoc test was used when comparing multiple independent groups. All other results were analysed using unpaired t-test for comparison between two groups. Values of at least
P < 0.05 were considered statistically significant. Post-tests were run only if F achieved
P < 0.05 and there was no significant variance in homogeneity.
Discussion
Crizotinib is probably one of the most successful anticancer drugs of the past decade. Crizotinib shows high tumor response rate and prolonged progression-free survival compared to traditional chemotherapy [
3,
29‐
31]. Crizotinib mediates these beneficial activities through targeting c-Met [
7], CD74-ROS1 [
8], and EML4-ALK [
6]. In addition, recent reports indicate that crizotinib targets the nudix phosphohydrolase superfamily member, MTH1 [
10]. In the latter case, (S)-crizotinib is found to be more potent than (R)-crizotinib [
32]. Importantly, inhibiting MTH1 function by RNAi-mediated knockdown [
10] or by (S)-crizotinib treatment [
33] led to selective cell death in cancer cells but not normal counterparts. A number of MTH1 inhibitors have been reported to exert anti-proliferation effects, including (S)-crizotinb [
33]. Interestingly, a recent study aimed at identifying potent MTH1 inhibitors showed that two compounds, NPD7155 and NPD9948, which inhibited MTH1 failed to show appreciable toxicity in cervical cancer HeLa cells [
11]. Similarly, a series of MTH1 inhibitors were developed and tested on various cells alongside (S)-crizotinib [
12]. Results of this study also showed limited effect of (S)-crizotinib on viability of a panel of cells. The investigators concluded that the reported effects of (S)-crizotinib may be ‘off-target’ effects, or in other words, independent of MTH1 inhibition. Therefore, the underlying molecular mechanism which contributes to (S)-crizotinib-induced death of human NSCLC cells remains enigmatic.
We set out to identify the mechanism of (S)-crizotinib-induced cell death in NSCLC. We hypothesized that (S)-crizotinib may exhibit antiproliferative activity in NSCLC cells independent of MTH1. We also postulated that the mechanism may involve generation of ROS, based on a few observations. First, crizotinib has been shown to increase oxidative stress in ovarian cancer cells [
24]. Second, it is well established that cancer cells have higher basal levels of endogenous ROS compared to normal cell counterparts. ROS in cancer cells promotes cell proliferation and survival and accelerates mutations and genomic instability [
16,
17]. The adverse effects of ROS such as promotion of cell death [
18] is kept in check in cancer cells through high levels of antioxidant activities [
34]. It is, therefore, possible that (S)-crizotinib increases ROS and unmasks the adverse, anti-proliferative effects of ROS in NSCLC cells. Indeed, our results show that (S)-crizotinib induces ROS generation in NSCLC cells within 3 h of exposure. This induction is mediated by mechanisms which are independent of MTH1 alteration or inhibition as knockdown of MTH1 with or without the (S)-crizotinib had no alter of ROS levels. Overproduction of ROS may lead to the induction of apoptosis and cell senescence through DNA damage [
35,
36]. Incorporation of oxidized nucleotides into genomic DNA plays a crucial role in ROS-induced apoptosis [
37]. Therefore, inhibition of MTH1 by (S)-crizotinib may amplify ROS toxicity by rendering cells unable to clear and sanitize oxidized nucleotides. We know that ROS is the central player as allowing cells to brace for the oxidative injury through pretreatment with NAC (as well as GSH) prevents (S)-crizotinib induced apoptosis in NSCLC cells.
In response to oxidative stress generated through elevation of ROS, (S)-crizotinib treatment activated the ER stress response [
38]. ER stress manifests as increased phosphorylation of eIF2α which results in global repression of protein translation. One exception is ATF4 which is required for CHOP induction [
39]. CHOP is considered as a marker of ‘commitment’ in the ER stress-induced apoptotic pathway [
40,
41]. We have shown that (S)-crizotinib increases ROS levels and activates the ER stress pathway in NSCLC cells. However, when we knocked down the expression of CHOP, we noted significant but not complete normalization of cell death. However, NAC was able to completely attenuate (S)-crizotinib-induced cell death. These results suggest that ER stress pathway activation is a part of the mechanism but not the only path in cell death observed. The central player remains ROS but the mechanisms downstream may be multifactorial. It is possible that part of the mechanism may involve mitochondrial dysfunction. In a number of human cancers, we have shown that induction of ROS mediates apoptosis partly through producing mitochondrial deficits [
42‐
45]. Whether (S)-crizotinib also causes mitochondrial alterations in NSCLC cells remains to be determined.
Conclusions
In this study, we here investigated the potential usefulness and novel molecular mechanism of (S)-crizotinib in the treatment of NSCLCs. In our in vitro and in vivo data, we show that (S)-crizotinib treatment resulted in significant increases in ROS levels, and accumulation of ROS caused the activation of the ER stress apoptotic pathway. Moreover, changes in cell survival and ROS production triggered by (S)-crizotinib were carried out independently of its inhibitory effect on MTH1. We demonstrated that (S)-crizotinib remains a promising candidate for the treatment of NSCLCs and acts as a ROS regulator. Furthermore, ROS-mediated ER stress apoptotic pathway may be an unrecognized mechanism underlying the biological activity of (S)-crizotinib. Despite the advancement made in this study, the direct molecular target of (S)-crizotinib and mechanisms inducing the levels of ROS remain unclear. Further studies are necessary to establish this novel concept.