Cytotoxicity of naringenin induces Bax‐mediated mitochondrial apoptosis in human lung adenocarcinoma A549 cells

Abstract Naringenin (NGEN), a natural flavonoid has growth inhibition and apoptosis‐inducing activities in several cancer cells. However, the cytotoxicity mechanisms of NGEN in cell death of lung cancer cells have not been fully defined. In present study, treatment of human lung adenocarcinoma A549 cells with NGEN resulted in time‐ and dose‐dependent decreases in cell viability. Moreover, NGEN significantly induced apoptosis evidenced by morphological changes, DAPI staining, TUNEL assay and sub‐G1 population increase. In NGEN‐treated cells, intensely upregulated Bax and down‐regulated Bcl‐2 proteins were detected and the Bax protein associated with the mitochondrial membrane was analyzed by subcellular fractionation. Knockdown of the Bax expression by the shRNA method dramatically protected A549 cells against NGEN‐induced apoptosis. Treatment with the inhibitors of caspase‐3, ‐8, or ‐9 significantly reduced NGEN‐induced apoptotic deaths. Taken together, our results demonstrate that NGEN‐induced apoptosis may occur via a Bax‐activated mitochondrial pathway in lung adenocarcinoma A549 cells.


| INTRODUCTION
Naringenin (NGEN) is a flavanone, a type of flavonoid that is produced in citrus fruits in large quantities. NGEN has various beneficial activities including anti-diabetic, 1 antioxidant, 2,3 and anti-inflammatory, 4 and it has also provided neuroprotective activities in different experimental rodent models. 5,6 The in vitro, in vivo, and clinical trials reports have been nicely reviewed for NGEN therapeutic potential in medicine. 7 NGEN increased cell viability in isolated neurons that were obtained from the brains of Sprague-Dawley rats and decreased the rate of cell apoptosis. 8 These findings demonstrate that NGEN eliminates oxidative stress and recovers from mitochondrial abnormality through the Nrf2/ARE signaling pathway activation in neurons. 8 In contrast to the protection activities found in neuron, NGEN has also exhibited great chemopreventive potential against colon cancer in Wistar rats. 9 Administration of NGEN to rats with induced gastric carcinoma has markedly enhanced redox status and decreased the risk of cancer. 10 Therefore, in additional to the normal cell protection effect, Gwo-Tarng Sheu and Shur-Hueih Cherng contributed equally to this work.
NGEN also provides anti-proliferation and apoptosis-inducting activities for cancer cells. Moreover, NGEN induced apoptosis and enhanced reactive oxygen species (ROS) production in prostate cancer PC3 and LNCaP cells; in which, NGEN also induced mitochondrial membrane potential depletion, resulted in decreased Bcl-2 and increased Bax proteins in PC3 cells only, but similar results had not been found in LNCaP cells. 11 Mitochondria-mediated apoptosis is mediated by the Bcl-2 proteins family, which can strengthen (eg, Bax and Bid) or impede (eg, Bcl-2 and Bcl-xL) apoptosis. 12,13 Bcl-2 was originally found in a translocation chromosomal fragment in B-cell lymphoma and has been proven to be a proto-oncogene. 14,15 The members of the Bcl-2 family are separated into three major groups: the first is made of antiapoptotic proteins, including Bcl-2, Bcl-w, Bcl-xL, and Mcl-1. The second group has been classified as proapoptotic proteins, such as Bax, Bak and PUMA. The third group is named the apoptosis initiator proteins including Bad, Bid, Bim, PUMA, and NOXA and is made of BH3 domain-only. 16 In case of no apoptotic stress, Bcl-2 and Bcl-xL interact into heterodimers with Bax and Bak (proapoptotic) to preserve the integrity of the outer mitochondrial membrane and restraint mitochondrial apoptosis. However, when apoptotic stimulation occurs, it changes the ratios between antiapoptotic and proapoptotic groups, driving to the construction of Bax/Bak complexes followed by punching through the membrane of mitochondria. In previous research, it has been demonstrated that the cytochrome c was discharged from the mitochondria to the cytosol through the opened orifices and it formed an apoptosis multi-units with procaspase-9, apoptotic protease activation factor-1 (Apaf-1), and deoxyadenosine triphosphate (dATP) to activate caspase-9 and caspase-3 mediated apoptosis. 16,17 The cytotoxic effects of NGEN have been identified in different human cancer cell lines, including human lung cancer cells. Treatment of human lung cancer A549 cells with modified NGEN derivatives, including 7-O-benzyl NGEN (KUF-1) and KUF-7 induced significant apoptosis and intracellular ROS production. 18 In mice with pulmonary fibrosis, NGEN also significantly reduced lung metastases and increased their survival by repressing transforming growth factor-β1 and reducing regulatory T cells that might improve the immunosuppressive environment. Therefore, it has been suggested that NGEN could be used as a therapeutic agent to control lung cancer and lung fibrosis. 19 Treatment with a low dosage of NGEN combined with TRAIL induced apoptosis in TRAIL-resistant lung cancer cells by upregulating death receptor 5 also indicated NGEN might sensitizing extrinsic death pathway. 20 Furthermore, NGEN also inhibited the AKT signaling pathway and reduced MMP-2 and -9 activities to inhibit the migration of A549 cells. 21 Both anti-proliferation and apoptosis induction are significant functions of the response of cancers to chemotherapeutic agents. Therefore, it is crucially important to characterize the molecular mechanism of NGEN-induced effects. In present study, the human lung carcinoma cell line A549 was used to evaluate the molecular mechanism of NGEN-induced apoptosis. Our results may help to understand whether NGEN possesses therapeutic potential for lung cancer management. Reagents of caspase activity assay were purchased from R&D Systems (Minneapolis, MN). All other chemicals with analytical grade quality had been obtained through commercial sources.

| Cell culture and Trypan blue viability test
The human lung A549 cancer cell line that carries a wild-type p53 gene was bought from the American Type Culture Collection (Rockville, MD) and the cell line has been further authenticated by Mission Biotech (Taipei, Taiwan) with cell line STR locus DNA typing (Case number CID20150026) that matches all repeat numbers of the A549 profile (ATCC CCL-185). Cells were cultured in RPMI-1640 medium (Gibco-BRL, Gaithersburg, MD) added with 10% heat-inactivated fetal bovine serum (Hyclone, Logan, Utah), antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin), and 2 mM glutamine, at 37 C in a humidified incubator containing 95% air and 5% CO 2 . For growth inhibition assay, cells were cultivated into 12-well plates and incubated (24 hours), and then cells were treated with indicated concentrations of NGEN. The quantity of viable cells was counted by the Trypan blue dye exclusion method as previously described. 22 Briefly, cells were collected, and then incubation with 0.4% Trypan blue solution. The viable cells were then counted under a microscope using a hematocytometer. The viable cells (average percentage) were calculated from three independent experiments.

| Inhibition of Bax by VSV-G pseudo lentivirus-shRNA
Initially, lentivirus vectors at a multiplicity of infection of 2 were used to infect A549 cells followed by 24 hours incubation. To harvest stably infected cells, the cells were selected by puromycin (2 μg/mL, Sigma, P8833) with a fresh medium and incubated for 6 days continuously until all parental A549 cells died. The shLUC and shBax cells were used for further analysis. Lung cancer cells (8 × 10 4 /well) were seeded onto 24-well plates and incubated at 37 C for 24 hours, then cells were treated with NGEN (800 μM) for 24 hours. The number of viable cells was determined by the Trypan blue dye exclusion method. We purchased RNAi reagents from the National RNAi Core Facility (Institute of Molecular Biology/Genomic Research Center, Academia Sinica, Taiwan). The unique TRC number of each clone as following: shLuc TRCN0000072249 targeted to luciferase for vector control; clones of shBax (TRCN0000312625) targeted to Bax.

| Preparation of subcellular fractions
The procedures of cell fractionation have been described previously. 23 Concisely, cells were harvested at indicated time and followed by washing with PBS then stored at −80 C. The pellets were then thawed at 4 C and suspended in cytosol extraction buffer (20 mM HEPES, pH 7.5, 5 μg/mL leupeptin, 5 μg/mL aprotinin, 1 mM PMSF, 1 mM DTT 1 mM EDTA, 1 mM EGTA, 1.5 mM MgCl 2 , 10 mM KCl) at 4 C. Cell lysates was centrifuged at 100 000g (30 minutes) at 4 C to obtain the supernatant as the cytosolic fraction. The remained pellet was further suspended extensively in modified RIPA buffer at 4 C followed by centrifugation to collect the supernatant as the particulate fraction. The particulate fraction includes membrane-organelle associated proteins that represent mitochondria content. Protein concentrations were further determined by Bradford method and samples were applied to subsequent experiments.

| Statistical analysis
The calculated data from three separate experiments are indicated as mean ± SD Statistical differences were examined by the Student's t test. Multiple groups (viability test, caspase activity and inhibition) were compared using one-way ANOVA with Dunnett post-hoc test using SPSS version 12 (Chicago, IL) and considered significant at the *P < .05, **P < .01, or ***P < .001 level.

| NGEN reduces the viability of A549 cells in time and dose-dependent manner
To analyze the cytotoxicity of NGEN in A549 cells, cells were treated with various concentrations (100-800 μM) of NGEN for 24, 48, and 72 hours. When 100 and 200 μM of NGEN were used, the growth inhibition effect was observed with 24 hours exposure. When A549 cells were exposed with 400 and 800 μM of NGEN for 48 hours, viabilities of the cells were significantly reduced, indicating that cell death may be involved ( Figure 1A). The morphologies of A549 cells are compared in Figure 1B, which shows that more dense and round cells were observed in A549 cells treated with NGEN (800 μM) after 48 and 72 hours of exposure.

| NGEN induces DNA fragmentation and apoptosis of A549 cells
When cells suffering from apoptosis, they lose part of their DNA as a result of DNA fragmentation in later apoptosis. One of the assays to determine apoptosis by flow cytometry is the estimation of fractional DNA content to quantitate apoptosis. Using flow cytometry, part of propidium iodide (PI) stained cells stain less intensely and show a peak below the G1 peak, which is identified as the sub-G1 peak. 24 When A549 cells were treated with NGEN, the sub-G1 peak was increased from 3.66% to 11.89% after 48 and 72 hours of exposure, respectively ( Figure 2A). Furthermore, the margin of the nucleus is abnormal and the condensed chromosome is easily stained in the NGEN-treated apoptotic cells detected by DAPI staining. The DNA fragmentation of NGEN-treated cells was also observed by TUNEL assay (Figure 2B), showing that NGEN induces A549 cell death via an apoptosis associated death pathway.

| NGEN induces caspase-dependent apoptotic cell death
The mechanism of NGEN-induced apoptosis was analyzed by caspase activity assay to distinguish the association of unique caspases in NGEN-induced cell death. Significant caspase-3 activity was detected and caspase-9 was also activated in cells treated with 800 μM of NGEN ( Figure 3A). To assess the contribution of caspases in NGEN-induced cell death, the caspase-3 inhibitor, caspase-8 inhibitor, and caspase-9 inhibitor were applied individually in combination with NGEN, followed by counting of viable cells. The data showed caspase-3, caspase-9 and caspase-8 inhibitors mitigate NGEN-induced cell death with respective efficacy ( Figure 3B). According to these results, the intrinsic apoptosis through caspase-9 activated capspase-3 plays the major role in NGENinduced apoptotic death; however, the extrinsic apoptosis via caspase-8 activation is not affected as much as the intrinsic pathway.

| Mitochondrial translocation of Bax and cytochrome c release were induced by NGEN
The most of Bax is present in the cytosol, when apoptotic signaling is initiated; Bax undergoes a conformational rearrange and changes into a mitochondrial membrane associated protein. 28 To further verify that F I G U R E 2 NGEN induces DNA fragmentation as detected by flow cytometry and TUNEL assay. A, The cells were treated with 800 μM NGEN for 24, 48, and 72 hours, and were harvested for flow cytometry analysis of the sub-G1 population. Quantification of sub-G1 phase and cell population in each phase was determined using ModFITLT 2.0 software. B, A549 cells were treated without or with 800 μM of NGEN for 48 hours, and then TUNEL assay was performed, and nuclear DNA was stained using DAPI. The stained cells were examined by a fluorescence microscope with a magnification of ×200 (scale bar, 20 μm) [Color figure can be viewed at wileyonlinelibrary.com] the overexpression and relocation of Bax to mitochondria leads to mitochondrial depolarization and stimulation of caspases activities, we prepared subcellular fractions and determined the expression of Bad and cytochrome c in mitochondria and cytosol fractions by Western blot assay ( Figure 5). We used β-actin as control proteins for cytoplasmic fractions and cytochrome oxidase IV (Cyto ox IV) were used as F I G U R E 4 Characterization of NGEN regulated proteins associated with apoptosis. A, A549 cells were treated without or with NGEN (200 or 800 μM) for 24, 48, and 72 hours. After treatment, cells lyses were extracted, and the levels of Bcl-2 family proteins were analyzed by Western blot. B, A549-shLuc cells and A549-shBax cells were treated with 800 μM of NGEN for 24 hours. Cell viability was measured using direct cell counting by the Trypan blue dye exclusion method (upper panel). The expression of Bax was analyzed by Western blot (lower panel). Comparison was performed using Student's t test and considered significant at the *P < .05. Protein intensities were calculated using ImageJ (https://imagej. nih.gov/ij/) and the folds of change were as listed F I G U R E 3 NGEN activates caspases-mediated cell death. A, A549 cells were treated with or without NGEN (200 or 800 μM) for 24 hours. Extracts from untreated or NGEN-treated cells were assayed for caspase activities using fluorogenic peptide substrates. B, A549 cells were treated with NGEN (800 μM) after pretreatment of the caspase-3 (z-DEVD-fmk), caspase-8 (z-IETD-fmk), and caspase-9 (z-LEHD-fmk) inhibitors (100 μM) respectively for 1 hour. After 24 hours treatment, cell numbers were calculated using hemocytometers by the Trypan blue dye exclusion method. Comparisons were performed using one-way ANOVA with Dunnett post hoc test and considered significant at the *P < .05, **P < .01, or ***P < . and dATP). 16 PUMA is transactivated by p53 to transduce death signals primarily to the mitochondria and indirectly resists all recognized antiapoptotic Bcl-2 group members to induce mitochondrial abnormality and caspase activation. 25 Human lung cancer A549 cells contain a wildtype p53 that is able to induce PUMA activation upon NGEN treatment. The minimal activation of caspase-8 by a high concentration of NGEN was detected, along with inhibition of caspase-8 increased cell survival with NGEN treatment (Figure 3A,B). It seems that the receptormediated extrinsic apoptotic pathway could be involved in NGENinduced apoptosis. Indeed, a previous report has demonstrated that a low dose of NGEN (100 μM) enhances the expression of death receptor 5 and upregulates TRAIL-induced apoptosis in A549 cells. Importantly, treatment with low dosage of 100 μM NGEN and 100 ng/mL TRAIL for 12 hours, alone or in combination, did not affect expression levels of Bcl-2, Bcl-xL, and Bax proteins. 20 This observation is also supported by our data of Figure 4A. The levels of Bcl-2, Bcl-xL, and Bax proteins were not markedly altered when A549 cells were treated with 200 μM NGEN for 48 and 72 hours. Altogether, low concentrations of NGEN may induce death receptor 5 without mitochondria alteration; however, high concentrations of NGEN could trigger apoptosis through the intrinsic mitochondria-mediated caspases activation signaling pathway.

| CONCLUSION
In summary, the present study contributes original results to demonstrate the mechanism of NGEN-mediated apoptosis in human lung cancer cells and shows that cell death is due to mitochondrial membrane dysfunction, nuclear condensation and DNA fragmentation. We also demonstrated that cytotoxic NGEN induced the lung cancer cell cycle sub-G1 phase and caspase-3 activity. Our data rule out possible antioxidant activity of NGEN that may interfere with cancer management and further support the potential of NGEN can be a compound with chemotherapeutic and cytostatic activity in human lung cancer treatment. Therefore, NGEN may be useful for further practice in drug improvement.

ACKNOWLEDGMENT
This work was supported by grant from the Ministry of Science and Technology, Taiwan (MOST-104-2320-B-040-003).

CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.

AUTHOR CONTRIBUTIONS
Win-Long Lu analyzed the results and writing the manuscript. Chang-Tze Ricky Yu performed experimental assay and analyzed the results.
Hsiu-Man Lien analyzed the results and performed manuscript revision. Shur-Hueih Cherng and Gwo-Tarng Sheu designed and performed experimental assay and control data quality. All authors have read and confirmed the final manuscript.

DATA AVAILABILITY STATEMENT
The materials included in this manuscript, may be made freely available to any researchers who wish to use them for non-commercial purposes, while preserving any necessary confidentiality and anonymity.