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New naphthalene derivatives induce human lung cancer A549 cell apoptosis via ROS-mediated MAPKs, Akt, and STAT3 signaling pathways
Chemico-Biological Interactions 304 (2019) 148–157
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New naphthalene derivatives induce human lung cancer A549 cell apoptosis via ROS-mediated MAPKs, Akt, and STAT3 signaling pathways
T
Wan-Ting Xua,1, Gui-Nan Shena,1, Ying-Hua Luob,1, Xian-Ji Piaoc, Jia-Ru Wanga, Hao Wanga, Yi Zhanga, Jin-Qian Lia, Yu-Chao Fengd, Yu Zhanga, Tong Zhanga, Shi-Nong Wanga, Chang-Yuan Wangd,**, Cheng-Hao Jina,d,* a
Department of Biochemistry and Molecular Biology, College of Life Science & Technology, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang, 163319, China College of Animal Science & Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang, 163319, China c Department of Gynaecology and Obstetrics, The Fifth Affiliated Hospital of Harbin Medical University, Daqing, Heilongjiang, 163316, China d College of Food Science, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang, 163319, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: 1,4-Naphthoquinone derivatives Lung cancer cells Apoptosis Reactive oxygen species MAPKs STAT3
1,4-Naphthoquinone compounds are a class of organic compounds derived from naphthalene. They exert a wide variety of biological effects, but when used as anticancer drugs, have varying levels of side effects. In the present study, in order to reduce toxicity and improve the antitumor activity, we synthesized two novel 1,4-naphthoquinone derivatives, 2-(butane-1-sulfinyl)-1,4-naphthoquinone (BSQ) and 2-(octane-1-sulfinyl)-1,4-naphthoquinone (OSQ). We investigated the antitumor effects of BSQ and OSQ in human lung cancer cells and the underlying molecular mechanisms of these effects, focusing on the relationship between these compounds and reactive oxygen species (ROS) production. MTT assay and trypan blue exclusion assay results showed that BSQ and OSQ had significant cytotoxic effects in human lung cancer cells. Flow cytometry results indicated that the number of apoptotic cells and the intracellular ROS levels significantly increased after treatment with BSQ and OSQ. However, cell apoptosis was inhibited by pretreatment with the ROS scavenger N-acetyl-L-cysteine (NAC). Western blotting results showed that BSQ and OSQ increased the expression levels of p-p38 kinase and p-c-Jun N-terminal kinase (p-JNK), and decreased the expression levels of p-extracellular signal-regulated kinase (pERK), p-protein kinase B (p-Akt), and p-signal transducer and activator of transcription-3 (p-STAT3). These phenomena were blocked by mitogen-activated protein kinase (MAPK) inhibitors, Akt inhibitors and NAC. In conclusion, BSQ and OSQ induce human lung cancer A549 cell apoptosis by ROS-mediated MAPKs, Akt, and STAT3 signaling pathways. Therefore, BSQ and OSQ may be therapeutic potential agents for the treatment of human lung cancer.
1. Introduction Lung cancer is one of the most prevalent cancers worldwide, with a 5-year overall survival rate less than 15% [1]. The current treatment methods for lung cancer include surgical resection, chemotherapy, and radiation. At present, various chemotherapeutic drugs, including 5fluorouracil (5-FU) and cisplatin, have been used for the treatment of lung cancer [2,3]; although these drugs have a certain effect on lung cancer, their toxic side effects on normal cells are even more pronounced. Therefore, it is necessary to develop less toxic, more efficient, and more inexpensive drugs for the treatment of lung cancer.
Apoptosis is the process of programmed cell death, which is an important active and orderly death process, and relates to the evolution and development of the organism. The process of apoptosis is synergistic, involving a variety of intracellular proteins and complex signaling pathways [4,5]. An increasing number of studies have shown that signal transducer and activator of transcription-3 (STAT3), protein kinase B (Akt), and mitogen-activated protein kinase (MAPK) signaling pathways play key roles in cell apoptosis through reactive oxygen species (ROS) activation or inhibition. 1,4-Naphthoquinone is an organic compound derived from naphthalene. Many 1,4-naphthoquinone derivatives are known to show
*
Corresponding author. College of Life Science and Technology, Heilongjiang Bayi Agricultural University, 5 Xinfa Street, Daqing, Heilongjiang, 163319, China. Corresponding author. E-mail addresses: [email protected] (C.-Y. Wang), [email protected] (C.-H. Jin). 1 Contributed equally to this study as first authors. **
https://doi.org/10.1016/j.cbi.2019.03.004 Received 27 January 2019; Received in revised form 28 February 2019; Accepted 7 March 2019 Available online 11 March 2019 0009-2797/ © 2019 Elsevier B.V. All rights reserved.
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analyzed by flow cytometry (Beckman Coulter, CA, USA) [15]. Detection of mitochondrial membrane potential (MMP). The MMP of human lung cancer A549 cells was measured by the JC-1 assay. A549 cells were plated in 6-well plates and treated with 5 μmol/l BSQ and OSQ for different time periods (3, 6, 12, and 24 h). Cells were washed one time with PBS, and then collected. The cells were incubated with JC-1 working solution (Solarbio, China) 20 min at 37 °C. Then discarded the supernatant, and the cells were washed with 1 × JC-1 staining buffer solution twice. The data were analyzed by flow cytometry [16]. Western blotting. Equal amounts of proteins (30 μg) were loaded per well on a SDS-10%–12% PAGE gel and transferred onto NC membranes (Millipore, Billerica, MA). The transferred NC membranes were subsequently blocked with 5% skim milk in Tris-Buffered Saline Tween-20 (TBST) at room temperature for 1 h. NC membranes were placed in solutions of specific primary antibodies and incubated overnight at 4 °C. All antibodies were obtained from Santa Cruz Biotechnology, Inc., Dallas, TX, USA. These included rabbit polyclonal antibodies against ERK2, p38α/β, Akt1/2/3, and p-Akt1/2/3, and mouse monoclonal antibodies against α-tubulin, Bax, Bcl-2, Poly (ADP-ribose) polymerase1 (PARP-1), cleaved-caspase-3, p-ERK, p-JNK, JNK, p-p38, p-STAT3, and STAT3. This step was followed by treatment with horseradish peroxidase-conjugated secondary antibodies, peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG and Goat Anti-Rabbit IgG. Chemiluminescence was detected using ECL kits (Bio-Rad, Hercules, CA). Band intensity was measured using software Image J 1.42q [17]. Detection of ROS. A549 cells were plated in 6-well plates at a density of 1 × 105 cells per well and treated with 5 μmol/l BSQ and OSQ for different times (3, 6, 12, and 24 h). Cells were centrifuged at 5000 rpm for 5 min and washed with PBS two times. 2′,7′-Dichlorofluorescein diacetate (DCFH-DA; MERCK, Shanghai, China) was used to treat cells for 30 min at 37 °C. Cells were then washed with PBS two times. The intracellular ROS levels in the cell samples were analyzed by flow cytometry [18]. Statistical analysis. All experiments were performed three times and data were represented as the means ± standard error. Continuous data were analyzed by one-way analysis of variance followed by Tukey's post hoc tests using SPSS version 18.0 statistical software. P < 0.05 was set as statistical significance and is shown in the figures as aP < 0.05, b P < 0.01, or cP < 0.001 [19].
obvious biological activities including anticancer, anti-inflammatory, and antibacterial activities [6,7]. Recently, increasing attention has been focused on the application of 1,4-naphthoquinone derivatives in cancer treatment. It has been reported that 1,4-naphthoquinone derivatives could induce cancer cell apoptosis in colon cancer cells, breast cancer cells, and melanoma cells [8–10]. However, these compounds have many side effects and are therefore not suitable for use as chemotherapeutic drugs [11]. In a previous study, we found that two 1,4naphthoquinone derivatives, 2-(butane-1-sulfinyl)-1,4-naphthoquinone (BSQ) and 2-(octane-1-sulfinyl)-1,4-naphthoquinone (OSQ), have a proliferation-inhibiting effect in human liver cancer Hep3B cells with lower toxic side effects than other compounds [12]. In the present study, we elucidated the antitumor effect of BSQ and OSQ in human lung cancer cells. We also examined the relationship between ROS, MAPKs, Akt, and the STAT3 signaling pathways in BSQand OSQ-induced apoptosis. 2. Materials and methods Chemical and reagents. All the solvents used in the study were of analytical grade. 5-FU was purchased from MedChem Express and stored at −20 °C. In the previous study, we synthesized BSQ and OSQ, and then dissolved at a concentration of 10 mM in DMSO as a stock solution and stored at −20 °C [12]. They were diluted freshly to the desired concentration with the culture medium before use. The final concentration of DMSO did not exceed 0.05% throughout the experiment. Cell line and cell culture. Human lung cancer A549, NCI-H23, and NCI-H460 cell lines were procured from American Type Culture Collection (Manassas, VA, USA) and were maintained in DMEM (Gibco, MA, USA). The human normal lung IMR-90, the normal liver L-02 and normal gastric GES-1 cell lines were obtained from Saiqi Biological Engineering Co., Ltd. (Shanghai, China). These cells were maintained in RPMI medium 1640 (Gibco). All cell lines were supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco). The cells were cultured in a 5% CO2humidified atmosphere at 37 °C until they reached 70%–80% confluence, and then were subcultured. MTT assay. Cells were plated in 96-well plates in 200 μl of medium per well at a density of 5000 cells per well and incubated for 24 h. The cells were treated with different concentrations of 5-FU, BSQ, and OSQ (1, 3, 10, 30, and 100 μmol/l) for 24 h. We added 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to every well of the cells (20 μl; 5 mg/ml), and incubated the samples 2 h in a 5% CO2-humidified atmosphere at 37 °C. The resulting formazan crystals in each well were dissolved with 100 μl of DMSO and incubated 15 min at 37 °C, followed by measuring the absorbance at 490 nm in a microplate reader. Cell viability was calculated using the following formula: (Atreatment/Acontrol) × 100 [13]. Trypan blue exclusion assay. The lung cancer A549, NCI-H23, NCIH460 cells, normal lung IMR-90, liver L-02 and stomach GES-1 cells were seeded in 12-well plates. According to the 50% inhibitory concentration (IC50) value of BSQ in lung cancer and normal cells, then cells were treated with 5-FU, BSQ and OSQ in different doses (2, 6, 3, 10, 10 and 10 μM) for 24 h. After cells were collected, then we added 10 μl trypan blue dye (Shanghai, China) for 3 min at room temperature and the dead cells were counted by the hemocytometer [14]. Cell apoptosis analysis. Cells were seeded in 6-well plates at a density of 1 × 105 cells per well and incubated for 24 h. Then they were treated with 5 μmol/l 5-FU, BSQ and OSQ for different time periods (3, 6, 12, and 24 h). After washing the cells with PBS once, we added 195 μl binding buffer, 2 μl PI, and 3 μl Annexin V-FITC to every tube in the dark for 15 min at room temperature. The fluorescence intensities of cells were observed using an EVOS FL Auto Cell Imaging System (Thermo Fisher Scientific Inc., Waltham, MA, USA) with 200 × magnification, and the percentages of early and late apoptotic cells were
3. Results Cytotoxic effects of BSQ and OSQ in human lung cancer cells. The chemical structure of BSQ and OSQ are shown in Fig. 1A. To investigate whether BSQ and OSQ have cytotoxic effects in human lung cancer cells, the cell viabilities of A549, NCI-H23, and NCI-H460 were measured by the MTT assay. Compared with the 5-FU group, BSQ and OSQ had significant, dose-dependent, cytotoxic effects in lung cancer cells (Fig. 1B). Meanwhile, after BSQ and OSQ treatment, the survival rate of human normal IMR-90, L-02, and GES-1 cells was significantly higher than in the 5-FU group (Fig. 1C). Among these, the A549 cells were more sensitive to BSQ and OSQ than were the NCI-H23 and NCIH460 cells, IC50 values of 40.01 for 5-FU, 2.08 for BSQ, and 4.69 for OSQ. In addition, As shown in Fig. 1D, BSQ and OSQ had significant cytotoxic effects in lung cancer cells, and lower toxicity in normal cells than 5-FU (Fig. 1E). These results suggested that BSQ and OSQ have significant cytotoxic effects in lung cancer cells. There were no obvious cytotoxic effects in the normal cells. Apoptosis induced by BSQ and OSQ in human lung cancer A549 cells. To evaluate whether BSQ and OSQ could induce human lung cancer cell apoptosis, the fluorescence intensity was measured by fluorescence microscopy. As shown in Fig. 2A, the fluorescence intensities of Annexin V-FITC and PI were markedly increased in a time-dependent manner. A549 cells showed the morphological features of early apoptosis, such as bright, nuclear condensation, and similar results were observed under fluorescence microscopy after 5-FU, BSQ, and OSQ 149
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Fig. 1. BSQ and OSQ had good anticancer effects in human lung cancer cells. (A) Chemical structure of BSQ and OSQ. (B) MTT assay was used to determine the effects of 5-FU, BSQ, and OSQ on A549, NCI-H23 and NCI-H460 cell viabilities. (C) MTT assay was used to determine the effects of 5-FU, BSQ, and OSQ on IMR-90, L02 and GES-1 cell viabilities. (D) Trypan blue exclusion assay was used to determine the effects of 5-FU, BSQ, and OSQ on A549, NCI-H23 and NCI-H460 cell viabilities. (E) Trypan blue exclusion assay was used to determine the effects of 5-FU, BSQ, and OSQ on IMR-90, L-02 and GES-1 cell viabilities. Data are expressed as means ± SD. aP < 0.05, bP < 0.01, cP < 0.001 vs. 5-FU.
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Fig. 2. BSQ and OSQ induced apoptosis in human lung cancer A549 cells. (A) The fluorescence intensities and morphology changes of cells were observed under a fluorescence microscope (original magnification, 200 × ). (B) Left panel: The percentages of apoptotic cells were detected by flow cytometry; Right panel: Quantification of apoptotic cells by flow cytometry. (C) Left panel: The mitochondrial membrane potential was detected by flow cytometry; Right panel: Quantification of mitochondrial membrane potential by flow cytometry. (D) The protein expression levels were measured by western blotting after treatment of A549 cells with BSQ and OSQ. The percentages of apoptotic cells represent mean ± SD; α-tubulin was used as the loading control. aP < 0.05, bP < 0.01, c P < 0.001 vs. 5-FU; aP < 0.05, bP < 0.01, cP < 0.001 vs. Control.
Apoptosis induced by BSQ and OSQ via MAPKs, Akt, and STAT3 signaling pathways in human lung cancer A549 cells. To further determine the molecular mechanism of BSQ- and OSQ-induced apoptosis in human lung cancer A549 cells, the apoptosis related protein expression levels were detected with western blotting. As shown in Fig. 3A–E, the phosphorylation levels of p38 and JNK were significantly increased. The phosphorylation levels of ERK, Akt, and STAT3 were significantly decreased in a time- and dose-dependent manner. Furthermore, to further investigate whether MAPKs and Akt induce apoptosis by regulating the STAT3 signaling pathway in human lung cancer cells, A549 cells were pretreated with SB203580 (pharmacological inhibitor of P38), SP600125 (pharmacological inhibitor of JNK), FR180204 (pharmacological inhibitor of ERK) and HY10249 (pharmacological inhibitor of Akt), and protein expression levels of the MAPKs, Akt and STAT3 were measured by western blotting. As shown in Fig. 4A, After incubation with BSQ + SB203580 and OSQ + SB203580, compared with BSQ or OSQ treatment alone, the expression levels of p38, cle-c-3,
treatment (Supplementary Figure 1). In addition, the apoptotic cell numbers were measured by flow cytometry. As shown in Fig. 2B, the percentages of early and late apoptotic cells were increased in a timedependent manner. These results indicated that BSQ and OSQ could induce lung cancer A549 cell apoptosis. Mitochondrial-dependent apoptosis induced by BSQ and OSQ in human lung cancer A549 cell. To investigate whether BSQ and OSQ induced apoptosis in human lung cancer A549 cells via the mitochondrial pathway, the MMP was detected with flow cytometry. As shown in Fig. 2C, BSQ and OSQ significantly reduced the MMP. Meanwhile, mitochondrial-dependent pathway related protein expression levels were measured by western blotting. BSQ and OSQ significantly increased the protein expression levels of Bax, cleaved-caspase-3 (cle-c-3) and cleaved-PARP (cle-PARP), decreasing the protein expression levels of Bcl-2 in a time-dependent manner (Fig. 2D). These results indicated that BSQ and OSQ induce apoptosis via the mitochondrial-dependent pathway in human lung cancer A549 cells.
Fig. 3. BSQ- and OSQ-induced A549 cell apoptosis depended on the MAPKs, Akt, and STAT3 signaling pathways. (A) Left panel: p38 protein expression levels were measured by western blotting after treatment of A549 cells with BSQ and OSQ; Right panel: Quantification of western blotting. (B) Left panel: JNK protein expression levels were measured by western blotting after treatment of A549 cells with BSQ and OSQ; Right panel: Quantification of western blotting. (C) Left panel: ERK protein expression levels were measured by western blotting after the treatment of A549 cells with BSQ and OSQ; Right panel: Quantification of western blotting. (D) Left panel: Akt protein expression levels were measured by western blotting after the treatment of A549 cells with BSQ and OSQ; Right panel: Quantification of western blotting. (E) Left panel: STAT3 protein expression levels were measured by western blotting after the treatment of A549 cells with BSQ and OSQ; Right panel: Quantification of western blotting. αtubulin was used as a loading control. aP < 0.05, b P < 0.01, cP < 0.001 vs. Control group.
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Fig. 4. Effects of BSQ and OSQ on the MAPKs, Akt and STAT3 signaling pathways in human lung cancer A549 cells. The expression levels of p-p38, p-JNK, p-ERK, pAkt, p-STAT3, Bcl-2, cle-c-3, and cle-PARP were detected by western blotting. (A) Left panel: Expression levels of p-p38, p-STAT3, Bcl-2, cle-c-3, and cle-PARP proteins in BSQ or OSQ-treated and p38 inhibitor SB203580-treated A549 cells; Right panel: Quantification of western blotting. (B) Left panel: Expression levels of pJNK, p-STAT3, Bcl-2, cle-c-3, and cle-PARP proteins in BSQ or OSQ-treated and JNK inhibitor SP600125-treated A549 cells; Right panel: Quantification of western blotting. (C) Left panel: Expression levels of p-ERK, p-STAT3, Bcl-2, cle-c-3, and cle-PARP proteins in BSQ or OSQ-treated and ERK inhibitor FR180204-treated A549 cells; Right panel: Quantification of western blotting. (D) Left panel: Expression levels of p-Akt, p-STAT3, Bcl-2, cle-c-3, and cle-PARP proteins in BSQ or OSQtreated and Akt inhibitor HY10249-treated A549 cells; Right panel: Quantification of western blotting. aP < 0.05, bP < 0.01, cP < 0.001 vs. inhibitor + BSQ or OSQ group.
Fig. 5. BSQ and OSQ induce apoptosis in human lung cancer A549 cells via intracellular ROS generation. (A) Left panel: The intracellular ROS levels were measured by flow cytometry; Right panel: Quantification of intracellular ROS levels by flow cytometry. (B) Left panel: A549 cells were treated with NAC, BSQ, and/or OSQ. Then the percentages of apoptotic cells were measured by flow cytometry; Right panel: Quantification of apoptotic cells by flow cytometry. (C) A549 cells were treated with BSQ or OSQ and NAC, after which the expression levels of MAPKs, Akt, STAT3, cle-c-3, and cle-PARP were detected by western blotting. aP < 0.05, b P < 0.01, cP < 0.001 vs. the NAC + BSQ or the OSQ group.
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our previous experiments, we found that the newly synthesized compounds had marked killing effects in human liver cancer Hep3B cells and gastric cancer AGS cells. In addition, they had no obvious side effects on normal cells [12,27]. In the present study, we found that BSQ and OSQ had significant, dose-dependent, cytotoxic effects on lung cancer cells, with no obvious cytotoxic effects in normal cells. Therefore, we concluded that BSQ and OSQ could induce apoptosis in human lung cancer A549 cells. Apoptosis is a cellular defense mechanism that eliminates cancer cells and plays an important role in the prevention of tumorigenesis. Recent studies have shown that many anticancer drugs work mainly by regulating apoptosis-related signals to induce apoptosis [28]. In this study, we investigated whether BSQ and OSQ induced apoptosis through a mitochondrial-dependent pathway in human lung cancer cells. Annexin V/FITC and PI were used to stain the cells to distinguish between viable, apoptotic, and necrotic cells [29]. From the experimental results, we saw that the morphological changes of cells over the BSQ and OSQ treatment time gradually increased, and then flow cytometry showed that the percentages of early and late apoptotic cells were increased. Further, JC-1 assay showed that the mitochondrial membrane potential was decreased. The process of apoptosis in cancer cells involves a large number of changes in the apoptotic proteins. The caspase family and the Bcl-2 family are important components of mitochondrial apoptotic pathways. Our results showed that BSQ and OSQ markedly induced apoptosis of A549 cells by upregulation of the expression levels of the apoptotic protein Bax and downregulation of the expression levels of the anti-apoptotic protein Bcl-2, as well as by activating both cle-c-3 and cle-PARP. These results suggested that BSQ and OSQ induce mitochondrial-dependent apoptosis. Intracellular ROS can mediate cell proliferation, differentiation, apoptosis, and other physiological and pathological reactions [30,31]. Furthermore, ROS may be carcinogenic to human beings and play an important role as regulators of tumor cell proliferation and survival. On the contrary, normal cells can avoid the accumulation of ROS and oxidative stress due to their complete antioxidant system, so that the normal metabolic activities of cells are less affected [32]. Some studies have indicated that chimaphilin, a 1,4-naphthoquinone derivative, can induce apoptosis in human breast cancer MCF-7 cells through an ROSmediated mitochondrial pathway. Shikonin induced apoptosis in human promyelocytic leukemia HL-60 cells through an ROS-mediated mitochondrial pathway [33–36]. Our results showed that BSQ and OSQ significantly increased the intracellular ROS levels. NAC has been recommended for the treatment of diseases caused by oxidative stress. Several studies have reported efficient antioxidant activity of NAC [37]. Our results showed that NAC could significantly inhibit BSQ- and OSQinduced apoptosis by upregulating the levels of ROS. These results further demonstrated that BSQ and OSQ induced apoptosis by increasing the generation of ROS in human lung cancer A549 cells. An increasing number of studies have identified the important role of MAPKs, Akt, and STAT3 signaling pathways in cell apoptosis. The MAPKs are serine–threonine protein kinases that are one of the most classic signaling pathways in the regulation of many cellular processes, including cell growth, proliferation, and apoptosis. The MAPKs consist of growth factor-regulated ERK, JNK, and p38 MAPKs [38,39]. Akt is an anti-apoptotic kinase that promotes cell survival and blocks apoptosis; in addition, the Akt pathway is an intracellular regulator which modulates multiple pathways including apoptosis, metabolism, and cell proliferation [40]. STAT3 plays an important role in cells, such as transferring extracellular signals into the nucleus and inducing target genes to express the biological stimulus. Because tumorigenesis is a multistage process, STAT3 is considered to be a key molecule in the complex environment of the organism [41,42]. Therefore, we have investigated the molecular mechanism of BSQ- and OSQ-induced apoptosis in lung cancer A549 cells. Our research showed that after BSQ and OSQ treatment, the JNK and p38 signaling pathways and the mitochondrial-dependent pathway were activated and the ERK, Akt, and
and cle-PARP were decreased, and the expression of STAT3 and Bcl-2 were increased. Next, After incubation with BSQ + SP600125 and OSQ + SP600125, compared with BSQ or OSQ treatment alone, the expression of JNK, cle-c-3, and cle-PARP were decreased, and the expression of STAT3 and Bcl-2 were increased (Fig. 4B). In addition, After incubation with BSQ + FR180204 and OSQ + FR180204, compared with BSQ or OSQ treatment alone, the expression of cle-c-3 and clePARP were increased, and the expression of ERK, STAT3, and Bcl-2 were decreased (Fig. 4C). After incubation with BSQ + HY10249 and OSQ + HY10249, compared with BSQ or OSQ treatment alone, the expression of cle-c-3 and cle-PARP were increased, and the expression of Akt, STAT3, and Bcl-2 were decreased (Fig. 4D). These results indicated that BSQ and OSQ induce apoptosis via MAPKs, Akt, and STAT3 signaling pathways. BSQ and OSQ induced ROS-mediated apoptosis in human lung cancer A549 cells. To investigate the underlying relationship between the two compounds and ROS generation, the levels of ROS was detected with flow cytometry. As shown in Fig. 5A, the intracellular ROS levels were significantly increased under BSQ and OSQ treatment in a time-dependent manner. After incubation with BSQ + NAC and OSQ + NAC, compared with BSQ or OSQ treatment alone, the number of apoptotic cells was reduced significantly (Fig. 5B). Furthermore, western blotting results showed that after incubation with BSQ + NAC and OSQ + NAC the expression levels of p-p38, p-JNK, cle-c-3, and cle-PARP were decreased, and the expression levels of p-ERK, p-Akt, and p-STAT3 were increased (Fig. 5C). These results suggested that BSQ and OSQ induce apoptosis in human lung cancer A549 cells through ROS-mediated MAPKs, Akt, and the STAT3 signaling pathway (Fig. 6). 4. Discussion 1,4-Naphthoquinone is an organic compound extracted from naphthalene, with naphthoquinone derivatives having several pharmacological activities [20–22]. Some 1,4-naphthoquinone derivatives, such as juglone and shikonin, inhibit cell viabilities and have significant cytotoxic effects on cervical ME-180 cells and HepG2 cells [23,24]. Although the 1,4-naphthoquinone derivatives have good anticancer effects, their side effects on normal cells have limited their clinical application [25]. Many studies have shown that the product obtained by oxidation of MCPBA is less side effects than the unoxidized 1,4naphthoquinone derivative [26]. Therefore, in order to reduce the toxicity of 1,4-naphthoquinones and increase their antitumor activity, we oxidized the sulfhydryl group to sulfoxide by MCPBA oxidation in the final step of the synthesis to reduce the toxicity of the compound. In
Fig. 6. The underlying mechanism of BSQ and OSQ anticancer activity in human lung cancer A549 cells. 155
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STAT3 signaling pathways were inhibited. These results showed that BSQ and OSQ induce apoptosis by activating the activity of the JNK and p38 signaling pathways and the mitochondria-dependent pathway, inhibiting the activity of ERK, Akt, and STAT3 signaling pathways in lung cancer A549 cells. In the present study, our results indicated that BSQ and OSQ inhibit the STAT3 signaling pathway by activating the MAPKs signaling pathway and inhibiting the Akt signaling pathway. The STAT3 signaling pathway was regulated by MAPKs and Akt in BSQ- and OSQ-induced apoptosis in human lung cancer A549 cells. Meanwhile, NAC also inhibited the activation of MAPKs, AKT, and STAT3 signaling pathways. Our results suggested that BSQ and OSQ could induce apoptosis via ROS-mediated MAPKs, Akt, and STAT3 signaling pathways in lung cancer A549 cells. Furthermore, although two compounds act on the same target, but the pro-apoptotic ability of BSQ is more effective than OSQ in A549 cells. We speculate that the reason for this result may be due to the fact that the substituent of BSQ is shorter than the substituent of OSQ. This possibility will be investigated in further studies. In conclusion, the present study demonstrated the molecular mechanisms underlying the antitumor effects of BSQ and OSQ in human lung cancer A549 cells. BSQ and OSQ induced apoptosis via ROSmediated MAPK, Akt, and STAT3 signaling pathways. BSQ and OSQ may, thus, be potential anticancer drugs for human lung cancer therapy.
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Conflicts of interest The authors declare they have no conflict of interest.
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Acknowledgments [14]
This work was funded by the Multigrain Production and Processing Characteristic Discipline Construction Project, the Postdoctoral Scientific Research Foundation of Heilongjiang Province of China (LBHQ13132) and National key research and development plan “Regulation and activity retention technology and application of coarse cereals active components in processing” (2017YFD0401203). In addition, we thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.
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Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.cbi.2019.03.004.
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Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cbi.2019.03.004.
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New naphthalene derivatives induce human lung cancer A549 cell apoptosis via ROS-mediated MAPKs, Akt, and STAT3 signaling pathways
T
Wan-Ting Xua,1, Gui-Nan Shena,1, Ying-Hua Luob,1, Xian-Ji Piaoc, Jia-Ru Wanga, Hao Wanga, Yi Zhanga, Jin-Qian Lia, Yu-Chao Fengd, Yu Zhanga, Tong Zhanga, Shi-Nong Wanga, Chang-Yuan Wangd,**, Cheng-Hao Jina,d,* a
Department of Biochemistry and Molecular Biology, College of Life Science & Technology, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang, 163319, China College of Animal Science & Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang, 163319, China c Department of Gynaecology and Obstetrics, The Fifth Affiliated Hospital of Harbin Medical University, Daqing, Heilongjiang, 163316, China d College of Food Science, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang, 163319, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: 1,4-Naphthoquinone derivatives Lung cancer cells Apoptosis Reactive oxygen species MAPKs STAT3
1,4-Naphthoquinone compounds are a class of organic compounds derived from naphthalene. They exert a wide variety of biological effects, but when used as anticancer drugs, have varying levels of side effects. In the present study, in order to reduce toxicity and improve the antitumor activity, we synthesized two novel 1,4-naphthoquinone derivatives, 2-(butane-1-sulfinyl)-1,4-naphthoquinone (BSQ) and 2-(octane-1-sulfinyl)-1,4-naphthoquinone (OSQ). We investigated the antitumor effects of BSQ and OSQ in human lung cancer cells and the underlying molecular mechanisms of these effects, focusing on the relationship between these compounds and reactive oxygen species (ROS) production. MTT assay and trypan blue exclusion assay results showed that BSQ and OSQ had significant cytotoxic effects in human lung cancer cells. Flow cytometry results indicated that the number of apoptotic cells and the intracellular ROS levels significantly increased after treatment with BSQ and OSQ. However, cell apoptosis was inhibited by pretreatment with the ROS scavenger N-acetyl-L-cysteine (NAC). Western blotting results showed that BSQ and OSQ increased the expression levels of p-p38 kinase and p-c-Jun N-terminal kinase (p-JNK), and decreased the expression levels of p-extracellular signal-regulated kinase (pERK), p-protein kinase B (p-Akt), and p-signal transducer and activator of transcription-3 (p-STAT3). These phenomena were blocked by mitogen-activated protein kinase (MAPK) inhibitors, Akt inhibitors and NAC. In conclusion, BSQ and OSQ induce human lung cancer A549 cell apoptosis by ROS-mediated MAPKs, Akt, and STAT3 signaling pathways. Therefore, BSQ and OSQ may be therapeutic potential agents for the treatment of human lung cancer.
1. Introduction Lung cancer is one of the most prevalent cancers worldwide, with a 5-year overall survival rate less than 15% [1]. The current treatment methods for lung cancer include surgical resection, chemotherapy, and radiation. At present, various chemotherapeutic drugs, including 5fluorouracil (5-FU) and cisplatin, have been used for the treatment of lung cancer [2,3]; although these drugs have a certain effect on lung cancer, their toxic side effects on normal cells are even more pronounced. Therefore, it is necessary to develop less toxic, more efficient, and more inexpensive drugs for the treatment of lung cancer.
Apoptosis is the process of programmed cell death, which is an important active and orderly death process, and relates to the evolution and development of the organism. The process of apoptosis is synergistic, involving a variety of intracellular proteins and complex signaling pathways [4,5]. An increasing number of studies have shown that signal transducer and activator of transcription-3 (STAT3), protein kinase B (Akt), and mitogen-activated protein kinase (MAPK) signaling pathways play key roles in cell apoptosis through reactive oxygen species (ROS) activation or inhibition. 1,4-Naphthoquinone is an organic compound derived from naphthalene. Many 1,4-naphthoquinone derivatives are known to show
*
Corresponding author. College of Life Science and Technology, Heilongjiang Bayi Agricultural University, 5 Xinfa Street, Daqing, Heilongjiang, 163319, China. Corresponding author. E-mail addresses: [email protected] (C.-Y. Wang), [email protected] (C.-H. Jin). 1 Contributed equally to this study as first authors. **
https://doi.org/10.1016/j.cbi.2019.03.004 Received 27 January 2019; Received in revised form 28 February 2019; Accepted 7 March 2019 Available online 11 March 2019 0009-2797/ © 2019 Elsevier B.V. All rights reserved.
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analyzed by flow cytometry (Beckman Coulter, CA, USA) [15]. Detection of mitochondrial membrane potential (MMP). The MMP of human lung cancer A549 cells was measured by the JC-1 assay. A549 cells were plated in 6-well plates and treated with 5 μmol/l BSQ and OSQ for different time periods (3, 6, 12, and 24 h). Cells were washed one time with PBS, and then collected. The cells were incubated with JC-1 working solution (Solarbio, China) 20 min at 37 °C. Then discarded the supernatant, and the cells were washed with 1 × JC-1 staining buffer solution twice. The data were analyzed by flow cytometry [16]. Western blotting. Equal amounts of proteins (30 μg) were loaded per well on a SDS-10%–12% PAGE gel and transferred onto NC membranes (Millipore, Billerica, MA). The transferred NC membranes were subsequently blocked with 5% skim milk in Tris-Buffered Saline Tween-20 (TBST) at room temperature for 1 h. NC membranes were placed in solutions of specific primary antibodies and incubated overnight at 4 °C. All antibodies were obtained from Santa Cruz Biotechnology, Inc., Dallas, TX, USA. These included rabbit polyclonal antibodies against ERK2, p38α/β, Akt1/2/3, and p-Akt1/2/3, and mouse monoclonal antibodies against α-tubulin, Bax, Bcl-2, Poly (ADP-ribose) polymerase1 (PARP-1), cleaved-caspase-3, p-ERK, p-JNK, JNK, p-p38, p-STAT3, and STAT3. This step was followed by treatment with horseradish peroxidase-conjugated secondary antibodies, peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG and Goat Anti-Rabbit IgG. Chemiluminescence was detected using ECL kits (Bio-Rad, Hercules, CA). Band intensity was measured using software Image J 1.42q [17]. Detection of ROS. A549 cells were plated in 6-well plates at a density of 1 × 105 cells per well and treated with 5 μmol/l BSQ and OSQ for different times (3, 6, 12, and 24 h). Cells were centrifuged at 5000 rpm for 5 min and washed with PBS two times. 2′,7′-Dichlorofluorescein diacetate (DCFH-DA; MERCK, Shanghai, China) was used to treat cells for 30 min at 37 °C. Cells were then washed with PBS two times. The intracellular ROS levels in the cell samples were analyzed by flow cytometry [18]. Statistical analysis. All experiments were performed three times and data were represented as the means ± standard error. Continuous data were analyzed by one-way analysis of variance followed by Tukey's post hoc tests using SPSS version 18.0 statistical software. P < 0.05 was set as statistical significance and is shown in the figures as aP < 0.05, b P < 0.01, or cP < 0.001 [19].
obvious biological activities including anticancer, anti-inflammatory, and antibacterial activities [6,7]. Recently, increasing attention has been focused on the application of 1,4-naphthoquinone derivatives in cancer treatment. It has been reported that 1,4-naphthoquinone derivatives could induce cancer cell apoptosis in colon cancer cells, breast cancer cells, and melanoma cells [8–10]. However, these compounds have many side effects and are therefore not suitable for use as chemotherapeutic drugs [11]. In a previous study, we found that two 1,4naphthoquinone derivatives, 2-(butane-1-sulfinyl)-1,4-naphthoquinone (BSQ) and 2-(octane-1-sulfinyl)-1,4-naphthoquinone (OSQ), have a proliferation-inhibiting effect in human liver cancer Hep3B cells with lower toxic side effects than other compounds [12]. In the present study, we elucidated the antitumor effect of BSQ and OSQ in human lung cancer cells. We also examined the relationship between ROS, MAPKs, Akt, and the STAT3 signaling pathways in BSQand OSQ-induced apoptosis. 2. Materials and methods Chemical and reagents. All the solvents used in the study were of analytical grade. 5-FU was purchased from MedChem Express and stored at −20 °C. In the previous study, we synthesized BSQ and OSQ, and then dissolved at a concentration of 10 mM in DMSO as a stock solution and stored at −20 °C [12]. They were diluted freshly to the desired concentration with the culture medium before use. The final concentration of DMSO did not exceed 0.05% throughout the experiment. Cell line and cell culture. Human lung cancer A549, NCI-H23, and NCI-H460 cell lines were procured from American Type Culture Collection (Manassas, VA, USA) and were maintained in DMEM (Gibco, MA, USA). The human normal lung IMR-90, the normal liver L-02 and normal gastric GES-1 cell lines were obtained from Saiqi Biological Engineering Co., Ltd. (Shanghai, China). These cells were maintained in RPMI medium 1640 (Gibco). All cell lines were supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco). The cells were cultured in a 5% CO2humidified atmosphere at 37 °C until they reached 70%–80% confluence, and then were subcultured. MTT assay. Cells were plated in 96-well plates in 200 μl of medium per well at a density of 5000 cells per well and incubated for 24 h. The cells were treated with different concentrations of 5-FU, BSQ, and OSQ (1, 3, 10, 30, and 100 μmol/l) for 24 h. We added 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to every well of the cells (20 μl; 5 mg/ml), and incubated the samples 2 h in a 5% CO2-humidified atmosphere at 37 °C. The resulting formazan crystals in each well were dissolved with 100 μl of DMSO and incubated 15 min at 37 °C, followed by measuring the absorbance at 490 nm in a microplate reader. Cell viability was calculated using the following formula: (Atreatment/Acontrol) × 100 [13]. Trypan blue exclusion assay. The lung cancer A549, NCI-H23, NCIH460 cells, normal lung IMR-90, liver L-02 and stomach GES-1 cells were seeded in 12-well plates. According to the 50% inhibitory concentration (IC50) value of BSQ in lung cancer and normal cells, then cells were treated with 5-FU, BSQ and OSQ in different doses (2, 6, 3, 10, 10 and 10 μM) for 24 h. After cells were collected, then we added 10 μl trypan blue dye (Shanghai, China) for 3 min at room temperature and the dead cells were counted by the hemocytometer [14]. Cell apoptosis analysis. Cells were seeded in 6-well plates at a density of 1 × 105 cells per well and incubated for 24 h. Then they were treated with 5 μmol/l 5-FU, BSQ and OSQ for different time periods (3, 6, 12, and 24 h). After washing the cells with PBS once, we added 195 μl binding buffer, 2 μl PI, and 3 μl Annexin V-FITC to every tube in the dark for 15 min at room temperature. The fluorescence intensities of cells were observed using an EVOS FL Auto Cell Imaging System (Thermo Fisher Scientific Inc., Waltham, MA, USA) with 200 × magnification, and the percentages of early and late apoptotic cells were
3. Results Cytotoxic effects of BSQ and OSQ in human lung cancer cells. The chemical structure of BSQ and OSQ are shown in Fig. 1A. To investigate whether BSQ and OSQ have cytotoxic effects in human lung cancer cells, the cell viabilities of A549, NCI-H23, and NCI-H460 were measured by the MTT assay. Compared with the 5-FU group, BSQ and OSQ had significant, dose-dependent, cytotoxic effects in lung cancer cells (Fig. 1B). Meanwhile, after BSQ and OSQ treatment, the survival rate of human normal IMR-90, L-02, and GES-1 cells was significantly higher than in the 5-FU group (Fig. 1C). Among these, the A549 cells were more sensitive to BSQ and OSQ than were the NCI-H23 and NCIH460 cells, IC50 values of 40.01 for 5-FU, 2.08 for BSQ, and 4.69 for OSQ. In addition, As shown in Fig. 1D, BSQ and OSQ had significant cytotoxic effects in lung cancer cells, and lower toxicity in normal cells than 5-FU (Fig. 1E). These results suggested that BSQ and OSQ have significant cytotoxic effects in lung cancer cells. There were no obvious cytotoxic effects in the normal cells. Apoptosis induced by BSQ and OSQ in human lung cancer A549 cells. To evaluate whether BSQ and OSQ could induce human lung cancer cell apoptosis, the fluorescence intensity was measured by fluorescence microscopy. As shown in Fig. 2A, the fluorescence intensities of Annexin V-FITC and PI were markedly increased in a time-dependent manner. A549 cells showed the morphological features of early apoptosis, such as bright, nuclear condensation, and similar results were observed under fluorescence microscopy after 5-FU, BSQ, and OSQ 149
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Fig. 1. BSQ and OSQ had good anticancer effects in human lung cancer cells. (A) Chemical structure of BSQ and OSQ. (B) MTT assay was used to determine the effects of 5-FU, BSQ, and OSQ on A549, NCI-H23 and NCI-H460 cell viabilities. (C) MTT assay was used to determine the effects of 5-FU, BSQ, and OSQ on IMR-90, L02 and GES-1 cell viabilities. (D) Trypan blue exclusion assay was used to determine the effects of 5-FU, BSQ, and OSQ on A549, NCI-H23 and NCI-H460 cell viabilities. (E) Trypan blue exclusion assay was used to determine the effects of 5-FU, BSQ, and OSQ on IMR-90, L-02 and GES-1 cell viabilities. Data are expressed as means ± SD. aP < 0.05, bP < 0.01, cP < 0.001 vs. 5-FU.
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Fig. 2. BSQ and OSQ induced apoptosis in human lung cancer A549 cells. (A) The fluorescence intensities and morphology changes of cells were observed under a fluorescence microscope (original magnification, 200 × ). (B) Left panel: The percentages of apoptotic cells were detected by flow cytometry; Right panel: Quantification of apoptotic cells by flow cytometry. (C) Left panel: The mitochondrial membrane potential was detected by flow cytometry; Right panel: Quantification of mitochondrial membrane potential by flow cytometry. (D) The protein expression levels were measured by western blotting after treatment of A549 cells with BSQ and OSQ. The percentages of apoptotic cells represent mean ± SD; α-tubulin was used as the loading control. aP < 0.05, bP < 0.01, c P < 0.001 vs. 5-FU; aP < 0.05, bP < 0.01, cP < 0.001 vs. Control.
Apoptosis induced by BSQ and OSQ via MAPKs, Akt, and STAT3 signaling pathways in human lung cancer A549 cells. To further determine the molecular mechanism of BSQ- and OSQ-induced apoptosis in human lung cancer A549 cells, the apoptosis related protein expression levels were detected with western blotting. As shown in Fig. 3A–E, the phosphorylation levels of p38 and JNK were significantly increased. The phosphorylation levels of ERK, Akt, and STAT3 were significantly decreased in a time- and dose-dependent manner. Furthermore, to further investigate whether MAPKs and Akt induce apoptosis by regulating the STAT3 signaling pathway in human lung cancer cells, A549 cells were pretreated with SB203580 (pharmacological inhibitor of P38), SP600125 (pharmacological inhibitor of JNK), FR180204 (pharmacological inhibitor of ERK) and HY10249 (pharmacological inhibitor of Akt), and protein expression levels of the MAPKs, Akt and STAT3 were measured by western blotting. As shown in Fig. 4A, After incubation with BSQ + SB203580 and OSQ + SB203580, compared with BSQ or OSQ treatment alone, the expression levels of p38, cle-c-3,
treatment (Supplementary Figure 1). In addition, the apoptotic cell numbers were measured by flow cytometry. As shown in Fig. 2B, the percentages of early and late apoptotic cells were increased in a timedependent manner. These results indicated that BSQ and OSQ could induce lung cancer A549 cell apoptosis. Mitochondrial-dependent apoptosis induced by BSQ and OSQ in human lung cancer A549 cell. To investigate whether BSQ and OSQ induced apoptosis in human lung cancer A549 cells via the mitochondrial pathway, the MMP was detected with flow cytometry. As shown in Fig. 2C, BSQ and OSQ significantly reduced the MMP. Meanwhile, mitochondrial-dependent pathway related protein expression levels were measured by western blotting. BSQ and OSQ significantly increased the protein expression levels of Bax, cleaved-caspase-3 (cle-c-3) and cleaved-PARP (cle-PARP), decreasing the protein expression levels of Bcl-2 in a time-dependent manner (Fig. 2D). These results indicated that BSQ and OSQ induce apoptosis via the mitochondrial-dependent pathway in human lung cancer A549 cells.
Fig. 3. BSQ- and OSQ-induced A549 cell apoptosis depended on the MAPKs, Akt, and STAT3 signaling pathways. (A) Left panel: p38 protein expression levels were measured by western blotting after treatment of A549 cells with BSQ and OSQ; Right panel: Quantification of western blotting. (B) Left panel: JNK protein expression levels were measured by western blotting after treatment of A549 cells with BSQ and OSQ; Right panel: Quantification of western blotting. (C) Left panel: ERK protein expression levels were measured by western blotting after the treatment of A549 cells with BSQ and OSQ; Right panel: Quantification of western blotting. (D) Left panel: Akt protein expression levels were measured by western blotting after the treatment of A549 cells with BSQ and OSQ; Right panel: Quantification of western blotting. (E) Left panel: STAT3 protein expression levels were measured by western blotting after the treatment of A549 cells with BSQ and OSQ; Right panel: Quantification of western blotting. αtubulin was used as a loading control. aP < 0.05, b P < 0.01, cP < 0.001 vs. Control group.
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Fig. 4. Effects of BSQ and OSQ on the MAPKs, Akt and STAT3 signaling pathways in human lung cancer A549 cells. The expression levels of p-p38, p-JNK, p-ERK, pAkt, p-STAT3, Bcl-2, cle-c-3, and cle-PARP were detected by western blotting. (A) Left panel: Expression levels of p-p38, p-STAT3, Bcl-2, cle-c-3, and cle-PARP proteins in BSQ or OSQ-treated and p38 inhibitor SB203580-treated A549 cells; Right panel: Quantification of western blotting. (B) Left panel: Expression levels of pJNK, p-STAT3, Bcl-2, cle-c-3, and cle-PARP proteins in BSQ or OSQ-treated and JNK inhibitor SP600125-treated A549 cells; Right panel: Quantification of western blotting. (C) Left panel: Expression levels of p-ERK, p-STAT3, Bcl-2, cle-c-3, and cle-PARP proteins in BSQ or OSQ-treated and ERK inhibitor FR180204-treated A549 cells; Right panel: Quantification of western blotting. (D) Left panel: Expression levels of p-Akt, p-STAT3, Bcl-2, cle-c-3, and cle-PARP proteins in BSQ or OSQtreated and Akt inhibitor HY10249-treated A549 cells; Right panel: Quantification of western blotting. aP < 0.05, bP < 0.01, cP < 0.001 vs. inhibitor + BSQ or OSQ group.
Fig. 5. BSQ and OSQ induce apoptosis in human lung cancer A549 cells via intracellular ROS generation. (A) Left panel: The intracellular ROS levels were measured by flow cytometry; Right panel: Quantification of intracellular ROS levels by flow cytometry. (B) Left panel: A549 cells were treated with NAC, BSQ, and/or OSQ. Then the percentages of apoptotic cells were measured by flow cytometry; Right panel: Quantification of apoptotic cells by flow cytometry. (C) A549 cells were treated with BSQ or OSQ and NAC, after which the expression levels of MAPKs, Akt, STAT3, cle-c-3, and cle-PARP were detected by western blotting. aP < 0.05, b P < 0.01, cP < 0.001 vs. the NAC + BSQ or the OSQ group.
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our previous experiments, we found that the newly synthesized compounds had marked killing effects in human liver cancer Hep3B cells and gastric cancer AGS cells. In addition, they had no obvious side effects on normal cells [12,27]. In the present study, we found that BSQ and OSQ had significant, dose-dependent, cytotoxic effects on lung cancer cells, with no obvious cytotoxic effects in normal cells. Therefore, we concluded that BSQ and OSQ could induce apoptosis in human lung cancer A549 cells. Apoptosis is a cellular defense mechanism that eliminates cancer cells and plays an important role in the prevention of tumorigenesis. Recent studies have shown that many anticancer drugs work mainly by regulating apoptosis-related signals to induce apoptosis [28]. In this study, we investigated whether BSQ and OSQ induced apoptosis through a mitochondrial-dependent pathway in human lung cancer cells. Annexin V/FITC and PI were used to stain the cells to distinguish between viable, apoptotic, and necrotic cells [29]. From the experimental results, we saw that the morphological changes of cells over the BSQ and OSQ treatment time gradually increased, and then flow cytometry showed that the percentages of early and late apoptotic cells were increased. Further, JC-1 assay showed that the mitochondrial membrane potential was decreased. The process of apoptosis in cancer cells involves a large number of changes in the apoptotic proteins. The caspase family and the Bcl-2 family are important components of mitochondrial apoptotic pathways. Our results showed that BSQ and OSQ markedly induced apoptosis of A549 cells by upregulation of the expression levels of the apoptotic protein Bax and downregulation of the expression levels of the anti-apoptotic protein Bcl-2, as well as by activating both cle-c-3 and cle-PARP. These results suggested that BSQ and OSQ induce mitochondrial-dependent apoptosis. Intracellular ROS can mediate cell proliferation, differentiation, apoptosis, and other physiological and pathological reactions [30,31]. Furthermore, ROS may be carcinogenic to human beings and play an important role as regulators of tumor cell proliferation and survival. On the contrary, normal cells can avoid the accumulation of ROS and oxidative stress due to their complete antioxidant system, so that the normal metabolic activities of cells are less affected [32]. Some studies have indicated that chimaphilin, a 1,4-naphthoquinone derivative, can induce apoptosis in human breast cancer MCF-7 cells through an ROSmediated mitochondrial pathway. Shikonin induced apoptosis in human promyelocytic leukemia HL-60 cells through an ROS-mediated mitochondrial pathway [33–36]. Our results showed that BSQ and OSQ significantly increased the intracellular ROS levels. NAC has been recommended for the treatment of diseases caused by oxidative stress. Several studies have reported efficient antioxidant activity of NAC [37]. Our results showed that NAC could significantly inhibit BSQ- and OSQinduced apoptosis by upregulating the levels of ROS. These results further demonstrated that BSQ and OSQ induced apoptosis by increasing the generation of ROS in human lung cancer A549 cells. An increasing number of studies have identified the important role of MAPKs, Akt, and STAT3 signaling pathways in cell apoptosis. The MAPKs are serine–threonine protein kinases that are one of the most classic signaling pathways in the regulation of many cellular processes, including cell growth, proliferation, and apoptosis. The MAPKs consist of growth factor-regulated ERK, JNK, and p38 MAPKs [38,39]. Akt is an anti-apoptotic kinase that promotes cell survival and blocks apoptosis; in addition, the Akt pathway is an intracellular regulator which modulates multiple pathways including apoptosis, metabolism, and cell proliferation [40]. STAT3 plays an important role in cells, such as transferring extracellular signals into the nucleus and inducing target genes to express the biological stimulus. Because tumorigenesis is a multistage process, STAT3 is considered to be a key molecule in the complex environment of the organism [41,42]. Therefore, we have investigated the molecular mechanism of BSQ- and OSQ-induced apoptosis in lung cancer A549 cells. Our research showed that after BSQ and OSQ treatment, the JNK and p38 signaling pathways and the mitochondrial-dependent pathway were activated and the ERK, Akt, and
and cle-PARP were decreased, and the expression of STAT3 and Bcl-2 were increased. Next, After incubation with BSQ + SP600125 and OSQ + SP600125, compared with BSQ or OSQ treatment alone, the expression of JNK, cle-c-3, and cle-PARP were decreased, and the expression of STAT3 and Bcl-2 were increased (Fig. 4B). In addition, After incubation with BSQ + FR180204 and OSQ + FR180204, compared with BSQ or OSQ treatment alone, the expression of cle-c-3 and clePARP were increased, and the expression of ERK, STAT3, and Bcl-2 were decreased (Fig. 4C). After incubation with BSQ + HY10249 and OSQ + HY10249, compared with BSQ or OSQ treatment alone, the expression of cle-c-3 and cle-PARP were increased, and the expression of Akt, STAT3, and Bcl-2 were decreased (Fig. 4D). These results indicated that BSQ and OSQ induce apoptosis via MAPKs, Akt, and STAT3 signaling pathways. BSQ and OSQ induced ROS-mediated apoptosis in human lung cancer A549 cells. To investigate the underlying relationship between the two compounds and ROS generation, the levels of ROS was detected with flow cytometry. As shown in Fig. 5A, the intracellular ROS levels were significantly increased under BSQ and OSQ treatment in a time-dependent manner. After incubation with BSQ + NAC and OSQ + NAC, compared with BSQ or OSQ treatment alone, the number of apoptotic cells was reduced significantly (Fig. 5B). Furthermore, western blotting results showed that after incubation with BSQ + NAC and OSQ + NAC the expression levels of p-p38, p-JNK, cle-c-3, and cle-PARP were decreased, and the expression levels of p-ERK, p-Akt, and p-STAT3 were increased (Fig. 5C). These results suggested that BSQ and OSQ induce apoptosis in human lung cancer A549 cells through ROS-mediated MAPKs, Akt, and the STAT3 signaling pathway (Fig. 6). 4. Discussion 1,4-Naphthoquinone is an organic compound extracted from naphthalene, with naphthoquinone derivatives having several pharmacological activities [20–22]. Some 1,4-naphthoquinone derivatives, such as juglone and shikonin, inhibit cell viabilities and have significant cytotoxic effects on cervical ME-180 cells and HepG2 cells [23,24]. Although the 1,4-naphthoquinone derivatives have good anticancer effects, their side effects on normal cells have limited their clinical application [25]. Many studies have shown that the product obtained by oxidation of MCPBA is less side effects than the unoxidized 1,4naphthoquinone derivative [26]. Therefore, in order to reduce the toxicity of 1,4-naphthoquinones and increase their antitumor activity, we oxidized the sulfhydryl group to sulfoxide by MCPBA oxidation in the final step of the synthesis to reduce the toxicity of the compound. In
Fig. 6. The underlying mechanism of BSQ and OSQ anticancer activity in human lung cancer A549 cells. 155
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STAT3 signaling pathways were inhibited. These results showed that BSQ and OSQ induce apoptosis by activating the activity of the JNK and p38 signaling pathways and the mitochondria-dependent pathway, inhibiting the activity of ERK, Akt, and STAT3 signaling pathways in lung cancer A549 cells. In the present study, our results indicated that BSQ and OSQ inhibit the STAT3 signaling pathway by activating the MAPKs signaling pathway and inhibiting the Akt signaling pathway. The STAT3 signaling pathway was regulated by MAPKs and Akt in BSQ- and OSQ-induced apoptosis in human lung cancer A549 cells. Meanwhile, NAC also inhibited the activation of MAPKs, AKT, and STAT3 signaling pathways. Our results suggested that BSQ and OSQ could induce apoptosis via ROS-mediated MAPKs, Akt, and STAT3 signaling pathways in lung cancer A549 cells. Furthermore, although two compounds act on the same target, but the pro-apoptotic ability of BSQ is more effective than OSQ in A549 cells. We speculate that the reason for this result may be due to the fact that the substituent of BSQ is shorter than the substituent of OSQ. This possibility will be investigated in further studies. In conclusion, the present study demonstrated the molecular mechanisms underlying the antitumor effects of BSQ and OSQ in human lung cancer A549 cells. BSQ and OSQ induced apoptosis via ROSmediated MAPK, Akt, and STAT3 signaling pathways. BSQ and OSQ may, thus, be potential anticancer drugs for human lung cancer therapy.
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Conflicts of interest The authors declare they have no conflict of interest.
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Acknowledgments [14]
This work was funded by the Multigrain Production and Processing Characteristic Discipline Construction Project, the Postdoctoral Scientific Research Foundation of Heilongjiang Province of China (LBHQ13132) and National key research and development plan “Regulation and activity retention technology and application of coarse cereals active components in processing” (2017YFD0401203). In addition, we thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.
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Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.cbi.2019.03.004.
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Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cbi.2019.03.004.
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