Abstract
Background/Aim: Niclosamide is an antihe-minthic drug that has shown cytotoxic effects on non-small cell lung carcinoma (NSCLC) cells. However, the exact mechanisms underlying the anti-tumour activity of niclosamide in NSCLC cancer cells remains to be defined. The aim of this study was to evaluate the antitumor activity of niclosamide in human A549 and CL1-5 non-small cell lung cancer cells using in vitro and in vivo. Materials and Methods: We investigated the effects of niclosamide on cell viability, apoptosis, the mitochondrial membrane potential (MMP; Δϕm), and autophagy and apoptosis-related protein expression in human A549 and CL1-5 non-small cell lung cancer cells. Results: Niclosamide induced mainly caspase-independent apoptosis through apoptosis-inducible factor (AIF) translocation to the nucleus upon mitochondria damage. Moreover, niclosamide-induced autophagy may act as adaptive response against apoptosis. AMPK/AKT/mTOR pathway were involved in niclosamide-induced cell death and autophagy in response to ATP depletion. Furthermore, niclosamide efficiently suppressed tumor growth and induce autophagy in vivo. Conclusion: Niclosamide induced apoptosis by activating the intrinsic and caspase-independent pathway in human A549 and CL1-5 non-small cell lung cancer cells. Therefore, niclosamide is a potential candidate for anti-NSCLC therapy.
Lung cancer is a leading cause of death from cancer worldwide. Among lung cancers, non-small cell lung carcinomas (NSCLC) account for approximately 80 % of all lung cancer cases (1). Despite substantial improvements in overall survival through screening for early diagnosis and treatment of NSCLC, certain patients are still plagued by rapid recurrence and progression of metastases (2, 3). Thus, the search for novel, effective therapeutic approaches for lung cancer is urgently needed.
Niclosamide, whose systematic name is (5-choloro-N-2-chloro-4-nitrophenyl)-2-hydroxybenzamide, is an oral anthelminthic drug used for approximately 50 years for treating most tapeworm infection. It is also used as a molluscicide for water treatment in schitosomiasis control programs (4). The activity of niclosamide against these parasites is believed to be mediated by inhibition of mitochondrial oxidative phosphorylation and anaerobic ATP production (5). Recently, several reports have indicated that niclosamide is active against cancers without affecting normal cells. Therefore, a number of mechanisms have been proposed for its anticancer action (4). For example, Jin et al., have shown that niclosamide can inhibit the NF-kB pathway and increase ROS levels to induce caspase-dependent apoptosis in acute myelogenous leukemia (AML) (6). In addition, Ren et al., have reported that niclosamide potently inhibited the activation and transcriptional function of STAT3 and consequently induced G0/G1 phase arrest and apoptosis of several types of human cancer cells that exhibit higher levels of STAT3 constitutive activation (7). Moreover, Lu et al., have shown that niclosamide is a unique small molecule Wnt/b-catenin signaling inhibitor targeting the Wnt co-receptor LRP6 and triggering apoptosis in human prostate and breast cancer (8). These findings suggest potential clinical benefits of niclosamide and drive an increase in clinical trials for cancer.
Although a preliminary study has indicated cytotoxic effects of niclosamide on NSCLC cells, the exact mechanisms underlying the anti-tumour activity of niclosamide in NSCLC cancer cells remains to be defined. Therefore, in the present study, the anticancer effects of niclosamide were evaluated using two NSCLC cell lines, namely, A549 and CL1-5 human lung adenocarcinoma cells. In addition, the possible molecular mechanisms responsible for its anticancer activity were also investigated. We show here that niclosamide was a cytotoxic agent that induced autophagy in human NSCLC cells. Autophagy was followed by apoptotic cell death that was mitochondria-mediated caspase-dependent and independent.
Materials and Methods
Chemicals and reagents. Niclosamide, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2-(4-amidinophenyl)-1H - indole-6-carboxamidine (DAPI) and dosomorphin were purchased from Sigma-Aldrich (St. Louis, MO, USA). 3-Methyladenine, taxol and rapamycin were purchased from Tocris (Ellisville, MO, USA). All of these chemicals were dissolved in DMSO and stored in −20°C, except for 3-methyladenine, DAPI and MTT which were dissolved in water and stored at 4°C. Lyovec was purchased from InvivoGene (San Diego, CA, USA).
Antibodies. Anti-cleaved-caspase 3, anti-cleaved-caspase 9 and anti-cleaved PARP were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-LC-3, anti-Atg5 and were purchased from Novus Biological (Littleton, CO, USA). Anti-p-AMPKα, anti-p-ACC, anti-p-p70S6K and anti-β-actin were purchased from Santa Cruz (Santa Cruz, CA, USA). Goat anti-rabbit IgG and goat anti-mouse IgG were purchased from Jackson Laboratory (Bar Harbor, ME, USA).
Cell culture and transfection. The lung cancer cell line CL1-5 cells were culture in Dulbecco modified Eagle medium (DMEM) containing 10% fetal bovine serum [FBS; Gibco (Gland Island, NY, USA)] and 1% antibiotic antimycotic. A549 cells were cultured in Kaighn's medium (F12K) containing 10% fetal bovine serum and 1% antibiotic antimycotic.
Cell viability assay. CL1-5 and A549 cells were seeded in 24-well plates at a density of 3×104 cell/well, and incubated with various concentrations of niclosamide or its analogues for 24, 48 and 72 h. MTT was dissolved in PBS at the concentration of 5 mg/ml and 200 μl of 1 mg/ml MTT in DMEM was added to each well 4 h before the end of each incubation. The MTT solution was removed after 4 h. The adherent cells were lysed with 600ul DMSO and the optical density (OD) was obtained at 570 nm.
Analysis of apoptosis. Cells harvested by trypsinization were washed by ice-cold PBS. The cells were stained by Annexin V-FITC and propidium iodide (PI) at 37°C for 10-15 min in the dark. The FACSCalibur system (Becton Dickinson, Mountain View, CA, USA) was used to analyze early apoptotic cells, late apoptotic or necrotic cells.
Analysis of autophagy using GFP-LC3. One hundred μl Lyovec were mixed with 2 μg p-GFP-LC3 plasmid at room 37°C for 20 min to allow complex formation. The complex was added to cells for 24 h. Then, the transfected cells were treated with the indicated concentrations of niclosamide or 2 μg/ml rapamycin for another 12 and 24 h. Cells were washed with PBS twice, and then fixed with 4% paraformaldehyde for 30 min at room temperature. The nucleus was stained with DAPI. The LC-3II formation was analyzed using confocal microscopy (Leica, Wetzlar, Germany) at 63x magnification.
Western blot. Cells were lysed in 1×RIPA buffer with 10% proteasome inhibitor. All cell extracts were cleared at 2,700 × g in a microcentrifuge at 4°C for 20 min. Proteins were separated by 5, 10 or 15% SDS–PAGE and transferred onto PVDF membrane. The membrane was blocked in 5% non-fat dry milk in Tris-buffered saline with Tween 20 (TBST) buffer and then immunostaining was performed with anti-LC-3, anti-phospho–mTOR, anti-mTOR, anti–caspase3, anti–PARP, anti-Apoptosis-inducible factor (AIF), anti–Endonuclease G followed by HRP-conjugated anti-rabbit or anti-mouse IgG secondary antibodies. ECL western blotting reagent (GE Healthcare Life Sciences, Chalfont, UK) was used for protein detection.
Assessment of the mitochondrial membrane potential. Cell pellets were washed with PBS twice. Ten μg/ml 5,5’,6,6’-tetrachloro-1,10,3,3’-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Sigma-Aldrich, St. Louis, MO, USA) and serum free medium were mixed in the ratio of 1:500, and 1 ml of the mixture was added to each sample followed by incubation at 37°C for 15 min in the dark. Samples were washed with PBS once and analysed by FACSCalibur system.
Cytochrome C release. Cell pellets were washed by PBS twice. One hundred μl digitonin buffer was added on ice for 5 min and the samples were washed by 0.1 % BSA buffer and centrifuged at 270 x g in a microfuge at 4°C for 5 min. The pellets were re-suspended with 200 μl IC–fixation buffer and incubated at 37°C for 20 min in the dark and subsequently the supernatant was removed. One ml permeabilization buffer was added to each sample and centrifuged at 270 × g in a microfuge at 4°C for 5 min, then the supernatant was removed. Anti–cytochrome C–FITC and permeabilization buffer were mixed in the ratio of 1:50 and 50 μl of the mixture were added to each sample and stained on ice for 1 h in the dark. The stained samples were washed with permeabilization buffer and centrifuged at 270 × g in a microfuge at 4°C for 5 min. The sample pellets were resuspended in 1% paraformaldehyde. FACSCalibur system (Becton Dickinson) was used to analyze cytochrome C release.
Immunocytochemistry. Cells were growth on coverslips and treated with the indicated concentrations of niclosamide for 12 and 24 h. Cells were washed in PBS twice and fixed with 4% paraformaldehyde at 37°C for 30 min, then fixed cells were permeabilized in 0.5% triton X–100 for 10 min. Cells were incubated in 1% bovine serum albumin (BSA) for 1 h, and then incubated with primary antibody for AIF at 4°C for 18 h. Afterwards, cells were washed with PBS four times and incubated with secondary anti–rabbit–FITC antibodies diluted 1:200 in 1% bovine serum albumin (BSA) at 4°C for 18 h, then the nuclei were stained with 2 μg/ml DAPI at 37°C for 15 min. These samples were examined using Laser Scanning Confocal Microscope (Leica SP5).
Xenograft murine model of NSCLC. The animal-related procedures were approved by the Institutional Animal Care and Use Committee of National Chung Hsing University. The CL1–5 cells were mixed with Matrigel ((BD Biosciences, Franklin Lakes, NJ, USA) at a ratio of 1:1. The cells (1×107) were subcutaneously injected into the back of each nude mouse, and the tumors were allowed to grow for 8 days until they reached a size of 10 mm3. Then, the mice were randomized into two treatment groups (n=5 per group), namely the control group, and the niclosamide group (20 mg/kg of niclosamide). The mice were treated with the vehicle, 20 mg/kg of niclosamide through an intraperitoneal injection of 100 μl (total volume) over 27 days (days 7 to 27).
Statistical analysis. The data were analyzed with GraphPad Prism Version 6.0 (San Diego, CA, USA), and values of p<0.05 were considered statistically significant. For comparison between two groups, we used unpaired two-tailed t-test (Student's t-test). All values are expressed as the mean±SD.
Results
Niclosamide induces growth inhibition and delayed apoptotic responses in A549 and CL1-5 cells. To determine the effect of niclosamide on cell viability, A549 and CL1-5 cells exposed to various concentrations (0.6125-10 μM) of niclosamide for 24, 48, 72 h and their viability was analyzed using the MTT assay. As shown in Figure 1A, cells exhibited a lag period lasting over 24 h in their response to niclosamide, while a dose-dependent reduction of the viability of A549 and CL1-5 cells was observed at 48 and 72 h. After 48 and 72 h of treatment, the IC50 values were approximately 2.7 μM and 2.3 μM in A549; 2.2 μM and 1.7 μM in CL1-5 cells, respectively. At concentrations below 2.5 μM niclosamide showed minor toxicity for A549 and CL1-5 cells, while at concentrations over 2.5 μM significantly inhibited A549 and CL1-5 cell viability. Therefore, 2.5 μM niclosamide was used in the following experiments.
To further determine the mode of cell death induced by niclosamide in both cell lines, we analyzed the cell cycle distribution and apoptosis at 24, 48, 72 h after drug treatment using PI and annexin V staining, which stain phosphatidylserine (PS) residues and DNA, respectively. As shown in Figure 1B and C, annexin V positive and Sub G1, hallmark of apoptosis, were only observed after 48 h or 72 h of niclosamide (2.5 μM) treatment in both cell lines. Therefore, these observations suggest that the anti-proliferative effect of niclosamide in human NSCLC cells derives, at least in part, from its ability to induce apoptosis.
Niclosamide altered mitochondrial membrane potential (MMP) in A549 and CL1-5 cells. It is established that, at the early stage, apoptotic stimuli alter the mitochondrial membrane potential (MMP). To investigate whether niclosamide affected the mitochondrial membrane potential (Δψm), we stained treated cells with the fluorescent cationic dye JC-1. Loss of Δψm is an indicator of mitochondrial damage during apoptosis. After cells were treated with 2.5 μM niclosamide as described above, red fluorescence was detected in A549 and CL1-5 cells in a time-dependent manner, suggesting that niclosamide treatment led to a reduction in Δψm (Figure 2A). To confirm that mitochondria were involved in the mechanism of apoptosis, we also evaluated the cytochrome c release. Cytochrome c release from the mitochondria into the cytosol is also an important event in the mitochondrial apoptotic pathway (19). Flow cytometry analysis of cells treated with niclosamide showed significantly increased cytochrome c release (Figure 2B), indicating that mitochondrial dysfunction was involved in niclosamide-induced apoptosis.
Niclosamide-induced apoptosis involves the activation of caspases via the intrinsic mitochondrial pathway and caspase-independent apoptosis pathway via AIF translocation. Mitochondrial dysfunction results in the activation of caspase-9, which subsequently activates down-stream caspase-3 and the cleavage of PARP (9). To examine whether the above apoptotic proteins were involved in niclosamide-induced cell death, A549 and CL1-5 cells were exposed to niclosamide for 24, 48, 72 h and then lysed and analyzed by immunoblot analysis. We observed activation of caspase-9 and capsae-3, and cleavage of the caspase-3 substrate PARP after 48 h of niclosamide exposure in both A549 and CL1-5 cells (Figure 3A). It has been shown that mitochondria dysfunction not only induces caspase-dependent pathway but also activates caspase-independent pathway (10). AIF and endogenous G (endo G) are hallmarks of caspase-independent pathway which triggered apoptosis through translocating from mitochondria to nuclei (10, 11). We evaluated whether caspase-induced apoptosis was involved in niclosamide-induced cell death in A549 and CL1-5 cells by assessing the levels of the active form of AIF and endo G by western blotting. As shown in Figure 3B, niclosamide induced an increase in the amount active form of AIF in a dose and time dependent manner, whereas the expression of endo G was not significantly increased. AIF triggers apoptosis through chromatin condensation and DNA fragmentation by translocating from mitochondria to nuclei upon mitochondria damage. As shown in Figure 3C, by immunocytochemistry strong AIF labeling was observed around and within apoptotic nuclei upon niclosamide treatment. In the control group, AIF was only present in the peri-nuclear area.
Niclosamide induced autophagy in A549 and CL1-5 NSCLC cell lines. In view of the minimal level of apoptosis observed following 24 h of treatment with niclosamide, we examined whether autophagy was induced in A549 and CL1-5 cells with niclosamide treatment. We first examined levels of LC3-II and Atg5 (a selective target of autophagy) by western blotting. During autophagy, Atg5 is upregulated and LC3-I is converted to LC3-II for the initiation and formation of autophagosomes. As shown in Figure 4A, niclosamide induced a time dependent increase in the amount LC3-II and Atg5. This effect was already evident after 24 h of treatment, in contrast to the low levels of apoptosis at this time point. A recent study has reported that vincristine disruption of the microtube cytoskeleton may interfere with the fusion of autophagosomes with lysosomes (12). The expression of Atg5 and LC3-II correlates with the number of autophagosomes and both serve as good indicators of autophagosome formation. We therefore visualized autophagosome formation in A549 and CL1-5 cells using cells expressing the autophagosome-associated LC-3 protein fused to green fluorescent protein (GFP-LC3). Niclosamide induced a redistribution of GFP-LC3 form a diffuse to a vacuolar pattern when autophagosomes were formed at 24 h (Figure 4B).
Effects of niclosamide on the viability, apoptosis and cell cycle of A549 and CL1-5 cells. Cell viability of A) A549 and CL1-5 cells after treatment with various concentrations of niclosamide for 24-72 h. Cell viability was determined by the MTT assay. Data are presented as means±SD from triplicate samples for each treatment. B) Apoptosis of A549 and CL1-5 cells after niclosamide treatment for 24 and 48 h. Cell apoptosis was examined by flow cytometry analysis of Annexin V-FITC and PI double-stained cells. Data represent the average of the Annexin V positive apoptotic cells. C) cell-cycle distribution of A549 and CL1-5 cells examined after 24 and 48 h treatment with 2.5 μM niclosamide. The cells were stained with propidium iodide and flow cytometry analysis was used to assess cell cycle distribution.
Niclosamide induce AMPK activation through ATP depletion lead to inhibition of AKT/mTOR pathway. To further understand the molecular mechanisms of action of niclosamide-induced autophagy in A549 and CL1-5 cells, we examined the effect of niclosamide on autophagy suppressor mTOR and its upstream effectors PI3K/AKT. The PI3/K/AKT/mTOR signaling is one of the major intracellular signaling pathways activated in cancer cells, including lung cancer cells. This pathway plays a variety of physiological roles, including regulation of cell cycle and promotion of cell survival. Recent studies have indicated that inhibition of the PI3K/AKT/MTOR pathway is associated with triggering autophagy in cancer cells. As shown in Figure 5A, treatment of CL1-5 and A549 cells with niclosamide decreased the phosphorylation (at ser473) of AKT, mTOR (at ser 2448) and its substrate p70S6K (Thr389), while total AKT, mTOR and p70S6K levels were not affected by the treatment.
AMPK is a central regulator of metabolism, which restores energy balance during metabolic stress at physiological levels (13). Previous studies have reported that AMPK activation initiates the autophagy process through inhibiting mTOR phosphorylation which is associated with triggering caspase-independent apoptosis via up-regulating AIF activation (14). We further investigated whether niclosamide affects ATP levels and AMPK activation. As shown in Figure 5B, niclosamide treatment significantly reduced cellular ATP levels in A549 and CL1-5 cells. Moreover, AMPK phosphorylation was dramatically increased in A549 and CL1-5 cells treated with niclosamide (Figure 5C). Collectively, these findings suggest that niclosamide-induced autophagy in CL1-5 and A549 cells is mediated at least in part by the AMPK/mTOR/p70S6K signaling pathways via ATP depletion.
Inhibition of autophagy enhances niclosamide-induced apoptotic cell death. It has been reported that autophagy represents cell survival mechanism for cancer cells depending on the cancer type, size, and microenvironment, and inhibition of autophagy may trigger increased rates of apoptosis. By contrast, recent studies have also suggested that autophagy would lead to cellular self-degradation followed by apoptosis. As a consequence, both pro-survival and pro-apoptotic roles are reasonable (15, 16). However, the detail mechanisms of this dual role of autophagy remain largely unknown. We, therefore, explored the role niclosamide-induced autophagy in NSCLC cells. To examine whether the niclosamide-induced A549 and CL1-5 cell death could be attributed to autophagy, we blocked autophagy by 3-methyladenine (3-MA), a specific autophagy inhibitor targeting the autophagosome formation. As shown in Figure 6A, addition of 3-MA significantly increased the percentage of annexin V positive cells and Sub G1 proportion (Figure 6B) in A549 cell. In summary, these results suggested that autophagy may protect cells form niclosamide-induced caspase-dependent apoptotic cell death, and blocking autophagy by a pharmacologic inhibitor improves the cytotoxic effect of niclosamide in NSCLC cells through increased apoptosis.
Effect of niclosamide on mitochondrial membrane potential and cytochrome C release in A549 and CL1-5 cells. A) A549 and CL1-5 cells were treated with niclosamide followed by JC-1 staining and analyzed by flow cytometry. Red fluorescence represents cells with normal mitochondria membrane potential and green fluorescence represents those with depolarized mitochondrial membrane. B) Cytochrome c expression in cytosol was determined by anti-cytochrome c-FITC staining and flow cytometry analysis.
Effects of niclosamide on intrinsic mitochondria pathway and caspase-independent apoptosis pathway in A549 and CL1-5 cells. A) Western blot analysis of the expression of cleaved caspase-3, cleaved caspase-9, and cleaved PARP in A549 and CL1-5 cells treated with niclosamide for 24-72 h. B) Western blot analysis of the expression of AIF (active form) and endo G in A549 and CL1-5 cells treated with niclosamide for 24-72 h. C) A549 and CL1-5 cells were treated with 2.5 μM of niclosamide for 24 h. Immunocytochemistry was then performed using an anti-AIF antibody. The nuclei were stained with DAPI. Scale bar=10 μm.
Effects of niclosamide on autophagy in A549 and CL1-5 cells. A) Western blot analysis of the expression of LC3 and Atg5 in A549 and CL1-5 cells treated with niclosamide for 24-72 h. B) A549 and CL1-5 cells were transfected with GFP-LC-3 plasmid according to manufacturer's recommendations. Twenty-four h after transfection, cells were treated with 2.5 μM of niclosamide 24 h. Scale bar=10 μm.
Niclosamide inhibits CL1-5 xenograft growth and it is associated with increased autophagy and apoptosis in tumor tissue. The evident inhibitory effect of niclosamide on A549 and CL1-5 cell proliferation in vitro suggested that it may inhibit NSCLC tumor growth in vivo. To examine this hypothesis, the in vivo anti-tumor activity of niclosamide was evaluated in CL1-5 xenografts in nude mice. After i.p injection of 10 and 20 mg/kg niclosamide daily for 35 d, the tumor size was obviously decreased compared to the vehicle control group (p<0.05) (Figure 7A). Moreover, immunostaining of histological sections also showed that niclosamide-treated groups had significantly increased levels of LC-3-II and caspase-3 (Figure 7B). These in vivo data suggested that the niclosamide inhibits tumor growth and induces apoptosis and autophagy in human NSCSL.
Effects of niclosamide on the expression of AMPK/AKT/mTOR pathway and cellular ATP levels. A) Western blot analysis of the expression of phospho-AMPK, phospho-AKT, phospho-mTOR, AMPK, AKT and mTOR in A549 and CL1-5 cells treated with niclosamide for 24 h. B), C) The levels of ATP in A549 and CL1-5 cells treated with 2.5 μM niclosamide for 24 h. Data are presented as means±SD from triplicate samples for each treatment.
Discussion
Niclosamide is Food and Drug Administration (FDA) approved drug for the treatment of helminth infection. Accumulated evidence has demonstrated that niclosamide has anticancer activity in various cancer such as breast cancer, colon cancer and cancer stem cell through modulating multiple signaling pathway including NFκB, Wnt/β-catenne, Notch, Stat3 and mTOR (4, 5). In NSCLC, there have been several studies reporting that niclosamide is effective in promoting cell death via activating apoptosis and autophagy (17-19). However, the mechanism of its action is still elusive. In the present study, we have evaluated the anti-NSCLC effects of niclosamide in CL1-5 and A549 NSCLC lung cancer cells and demonstrated that niclosamide significantly inhibits cancer cell growth and proliferation in human lung cancer cells via inducing apoptosis and autophagy in vitro and in vivo.
Apoptosis plays a critical role in normal development and homeostasis of adult tissues (20). However, it has been shown that deregulation of apoptosis is involved in the pathogenesis of cancer (21). Thus, targeting apoptosis is one of the major anti-cancer strategies for drug development. Classical, caspase-dependent apoptosis is triggered by two major pathways including the extrinsic pathway and the intrinsic pathway, which is followed by activation of caspase-3 and cleavage of PARP (22-24). During the last decade, accumulating evidence has demonstrated that niclosamide induces apoptosis in a broad spectrum of cancer cell types (4, 5). For instance, niclosamide induces apoptosis through the mitochondrial intrinsic pathway in human thyroid cancer through disrupting mitochondrial membrane potential (25). Moreover, niclosamide has been shown to sensitize cervical cancer cells to paclitaxel through inducing oxidative stress via mTOR inhibition (26). However, our finding demonstrated that niclosamide slightly induces caspase-dependent apoptosis of CL1-5 and A549 through up-regulating cleaved caspase-3, caspase-9, and PARP. And the pan-caspase inhibitor failed to retard niclosamide induce cell death. These results suggested that there are other possible mechanisms involved in niclosamide induced cellular stress.
AIF and Endo G are caspase-independent death effectors that trigger chromatin condensation and DNA fragmentation to induce programmed cell death. It has been reported that intranuclear translocation of AIF and Endo G is involved in niclosamide-induced cell death (10, 11). For instance, growth inhibition of ovarian tumor-initiating cells by niclosamide has been associated with AIF activation (27). But whether this occurs in niclosamide-induced NSCLC cell death is unknown. In the present study, we demonstrated strong upregulation of AIF activated form but not Endo G in the niclosamide treated group compared with control group. Immunocytochemistry results demonstrated strong AIF labeling around and within apoptotic nuclei after niclosamide treatment, whereas AIF was only present in the peri-nuclear area but not within the nuclei in the control group. These results indicated translocation of AIF into the nuclei when CL1-5 and A549 were treated with niclosamide. Therefore, niclosamide induced-apoptosis is mediated by caspase-independent pathway through AIF translocation to the nucleus. To our knowledge, this is the first study demonstrating the role of AIF in niclosamide-induced cell death in NSCLC.
Recently, there have been many studies demonstrating that autophagy is a potential target of anti-NSCLC therapy (24, 28). For instance, inhibition of autophagy enhances the anti-tumor activity of afatinib in lung adenocarcinoma with activating EGFR mutation (29). Moreover, inhibition of autophagy promotes pemetrexed and simvastatin-induced apoptotic cell death in NSCLC cells (30). This evidence indicated that autophagy may function as a protective mechanism in NSCLC treated with anti-cancer drugs. Previous studies have illustrated that niclosamide induced autophagy in various cells. Balgi AD et al. have shown that niclosamide induced autophagy via inhibiting mTOR in breast cancer cells (31). In addition, Park SJ et al. have demonstrate that niclosamide induced apoptotic and autophagic cell death via mitochondria damage in HeLa cells (32). In the present study, our findings demonstrated that niclosamide significantly increased conversion of LC3-I to LC3-II, LC3-II puncta formation, protein expression of Beclin1 and reduced protein expression of p62 in both A549 and CL1-5 cell lines, which indicates that niclosamide acts as an autophagy inducer in NSCLC.
Moreover, we found that niclosamide promoted the early stage apoptosis under the condition of inhibiting niclosamide-induced autophagy via 3MA treatment. The cross-talk between apoptosis and autophagy is still complicated. Recently, Takano-Ohmuro et al. have demonstrated that fragmentation of mitochondria by autophagy is a process for embryonic cells to escape apoptosis via releasing cytochrome c from mitochondria (33).We suggest that niclosamide-induced autophagy is an adaptive response to maintain cellular energy homeostasis and escape apoptosis by engulfing mitochondria under the stress of niclosamide exposure in the early stage. Interestingly, niclosamide induced more apoptosis and significantly prevent mitochondria damage in p53-wild type A549 cell than p53-mutant CL1-5 cell under 3MA pretreatment condition. The data suggested that p53 may play a key role in modulating cellular response to niclosamide treatment under the condition of inhibiting autophagy. However, the mechanism has to be elucidated in the future.
AMPK is a highly conserved major modulator of metabolism, which restores energy balance during metabolic stress at the physiological levels (13). Previous studies have reported that AMPK activation not only contributed to initiation of autophagy process through inhibiting mTOR phosphorylation but also associated with triggering caspase-independent apoptosis via activating AIF (14). Moreover, there are reports demonstrating that targeting of the AMPK/mTOR is an attractive strategy for the development of therapeutic agents against NSCLC (34). For instance, Wu et al. have reported that activation of AMPK/mTOR signaling pathway contributed to resistance of lung adenocarcinoma to cisplatin treatment (35). D561-0775, an AMPK activator, has been shown to have a significant inhibitory effect on gefitinib-resistant NSCLC cell lines (36). Our findings demonstrated that niclosamide treatment significantly increased AMPK activation. We also found that niclosamide treatment inhibited AKT/mTOR pathway. In a previous study, Cui et al. have found that AMPK induced cell apoptosis by inhibiting the PI3K/AKT/mTOR pathway (37). Furthermore, AMPK activation appears to affect the nuclear translocation of AIF and subsequent neuronal death (14). Our results suggest that AMPK may act as a master regulator in niclosamide-induced apoptosis and autophagy.
Inhibition of autophagy by 3-MA promotes niclosamide-induced apoptotic cell death. A549 and CL1-5 cells were pretreated 1h with or without 3-MA and then treated with niclosamide (2.5 μM) for 24 h. A) Cell apoptosis was examined by flow cytometry analysis of Annexin V-FITC and PI double-staining. Data represent the average of the Annexin V positive apoptotic cells. B) Effects of niclosamide on cell cycle distribution of A549 and CL1-5 cells. The cells were stained with propidium iodide and flow cytometry analysis was used to assess cell cycle distribution.
Effect of niclosamide on CL1-5 tumor growth in nude mice. CL1-5 cells (1×107) were subcutaneously injected into mice, producing a visible tumor (approximately 10 mm3). Niclosamide (20 mg/kg) and vehicle control were administered intraperitoneally 2 times/week until day 27 (n=5). A) The graph shows the tumor volume changes with time. Quantified data are presented as mean±SD. B) Immunohistochemistry analysis of the expression of LC-3 and Atg5 in tumor tissues.
Activation of AMPK is related to decreased cellular ATP levels (38). Mitochondria not only play a critical role in controlling apoptosis pathway but also in ATP production through the TCA cycle. Previous studies have indicated that niclosamide has the ability to uncouple oxidative phosphorylation and subsequently to reduce ATP levels in human cells (32, 39). Our findings demonstrate that niclosamide significantly reduces cellular ATP levels in A549 and CL1-5 cell. Taken together, these results indicate that niclosamide-mediated cell death and autophagy is correlated to activation of AMPK via ATP reduction in NSCLC cell lines.
In conclusion, we demonstrated that niclosamide induces NSCLC cell death and autophagy in vitro and in vivo. Niclosamide-induced apoptosis is mediated by a caspase-independent pathway through AIF translocation to the nucleus. Moreover, niclosamide-induced autophagy may act as an adaptive response against apoptosis. The AMPK/AKT/mTOR pathway is involved in niclosamide-induced cell death under the condition of ATP depletion. Furthermore, niclosamide efficiently suppresses tumor growth and induces autophagy in vivo. In this study, we demonstrate that niclosamide is a potential candidate for anti-NSCLC therapy.
Acknowledgements
This study was supported by grants (107-CCH-HCR-027) from Changhua Christian Hospital, Taiwan.
Footnotes
↵* These Authors contributed equally to this work.
Authors' Contributions
W.-H.C., Y.-R.L. and C.-H.L conceived and designed the experiments. S.-H.L., Y.-H.C., C.-H.C. performed the experiments. P.-C.C. analyzed the data. Y.-R.L., and C.-H.L. wrote the manuscript. All Authors read and approved the final manuscript.
Conflicts of Interest
The Authors declare that they have no competing interests regarding this study.
- Received December 31, 2019.
- Revision received February 10, 2020.
- Accepted February 14, 2020.
- Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
