10-HDA Induces ROS-Mediated Apoptosis in A549 Human Lung Cancer Cells by Regulating the MAPK, STAT3, NF-κB, and TGF-β1 Signaling Pathways

10-Hydroxy-2-decenoic acid (10-HDA), also known as royal jelly acid, has a variety of physiological functions, and recent studies have shown that it also has anticancer effects. However, its anticancer mechanisms have not been clearly defined. In this study, we investigated the underlying mechanisms of 10-HDA in A549 human lung cancer cells. We used Cell Counting Kit-8 assay, scratch wound healing assay, flow cytometry, and western blot analysis to investigate its apoptotic effects and underlying mechanism. Our results showed that 10-HDA inhibited the proliferation of three types of human lung cancer cells and had no significant toxic effects on normal cells. Accompanying reactive oxygen species (ROS), 10-HDA induced A549 cell apoptosis by regulating mitochondrial-associated apoptosis, and caused cell cycle arrest at the G0/G1 phase in a time-dependent manner. Meanwhile, 10-HDA also regulated mitogen-activated protein kinase (MAPK), signal transducer and activator of transcription 3 (STAT3), and nuclear factor kappa B (NF-κB) signaling pathways by increasing the expression levels of phosphorylated c-Jun N-terminal kinase, p-p38, and I-κB, and additionally, by decreasing the expression levels of phosphorylated extracellular signal-regulated kinase, p-STAT3, and NF-κB. These effects were blocked by MAPK inhibitors and N-acetyl-L-cysteine. Furthermore, 10-HDA inhibited cell migration by regulating transforming growth factor beta 1 (TGF-β1), SNAI1, GSK-3β, E-cadherin, N-cadherin, and vimentin. Taken together, the results of this study showed that 10-HDA induced cell cycle arrest and apoptosis in A549 human lung cancer cells through ROS-mediated MAPK, STAT3, NF-κB, and TGF-β1 signaling pathways. Therefore, 10-HDA may be a potential therapy for human lung cancer.


Introduction
Lung cancer is a very serious illness, and its morbidity and mortality rank first among all cancer types [1]. In China, it ranks first in mortality, accounting for 24.41% of cancerrelated deaths, and its mortality rate has shown an increasing trend [2]. The clinical manifestations of lung cancer are com-plex; the presence, severity, and appearance of symptoms and signs depend on the location of the tumor, the type of pathology, the presence or absence of metastases and complications, and the patient's response and tolerance [3]. At present, the main treatment methods for lung cancer include surgery, chemotherapy, radiation therapy, and molecular targeted therapy [4]. However, the mortality rate remains high, and these therapies often have side effects [5]. Therefore, it is urgent to find a natural anticancer drug that is safe and has high efficiency and low toxicity.
At present, the primary way for various active ingredients to exert anticancer activity is by triggering the apoptotic pathway in cancer cells, which leads to cell death [6][7][8]. Apoptosis is a programmed cell death mechanism controlled by genes and proteins, usually manifested as nuclear condensation, wrinkling, membrane foaming, and DNA fragmentation [9]. It is not a passive process, but is an active process that involves the activation, expression, and regulation of a series of intracellular proteins and complex signaling pathways [10][11][12]. Apoptosis occurs in the human body all the time, and the ultimate executor of apoptosis is caspase, which transmits signals from the point of origin to the various pathways of the cell, so that the cell reaches a globally consistent state of apoptosis, and finally decomposes the cell into small fragments [13]. Many studies have shown that some signaling pathways help promote cancer cell apoptosis, including MAPK, STAT3, and NF-κB signaling pathways, and these pathways play key roles in cell apoptosis through the activation or inhibition of ROS [14,15].
Recently, natural products have attracted the attention of many researchers for their potential anticancer properties [16]. 10-HDA, also known as royal jelly acid, is an organic acid compound extracted from royal jelly and is one of the main active ingredients [17,18]. It has a variety of physiological functions such as antibacterial, anti-inflammatory, blood lipid lowering, immunity enhancing, and anticancer effects [19][20][21][22][23][24]. However, its pharmacodynamic effect and pharmacological mechanism of action in cancer remain unknown.
The main goal of this study was to reveal the target and pharmacological mechanism of 10-HDA in A549 lung cancer cells. Specifically, we measured its effects on cell viability, cell cycle, apoptosis, intracellular ROS production, inhibition of cell migration, potential molecular mechanisms, and related signaling pathways in lung cancer cells.

Cell Apoptosis
Analysis. A549 cells were seeded onto cell slides in 6-well plates (1 × 10 5 cells/well) and treated with 30 μM 10-HDA for different time periods (3, 6, 12, and 24 h). After washing twice with phosphate-buffered saline (PBS), cells were resuspended in 195 μL binding buffer, and dual staining was performed with 3 μL Hoechst 33342 and 2 μL propidium iodide (PI). The staining solution was evenly distributed by shaking and incubated at 37°C for 3-5 min. The change in fluorescence intensity was observed using the EVOS FL Auto Cell Imaging System (Thermo Fisher Scientific, Waltham, MA, USA) at a magnification of 400x. Meanwhile, the effects of 10-HDA on the apoptosis of A549 cells were quantified using the Annexin V-FITC Apoptosis Detection Kit. A549 cells were treated with 30 μM 10-HDA for different time periods (3, 6, 12, and 24 h). A549 cells were resuspended in 195 μL Annexin V-FITC binding solution followed by the addition of 3 μL Annexin V-FITC and 2 μL PI and gentle mixing. Then, cells were incubated at 4°C for 30 min, and the cell suspension was transferred to a flow cytometer (Beckman Coulter, Brea, CA, USA) for quantitative apoptosis detection.  2.8. Cell Migration Analysis. A549 cells were seeded onto cell slides in 6-well plates (1 × 10 5 cells/well). When the cells were completely fused, a wound was made by a 10 μL pipette tip and washed twice with 1 mL PBS to remove cellular debris. Culture medium and 10-HDA (30 μM) were added to continue the culture (3, 6, 12, and 24 h); then, the A549 cells were observed using the EVOS FL Auto Cell Imaging System at a magnification of 100x.

Results
3.1. 10-HDA Inhibits the Proliferation of Human Lung Cancer Cells. As shown in Table 1  3.2. 10-HDA Induces Apoptosis in A549 Human Lung Cancer Cells. As shown in Figure 2(a), the cells became rounded, and the dead cells floated to the surface of the medium in the 10-HDA treatment groups. Meanwhile, as shown in Figure 2(b), the flow cytometry results showed that with an increase in 10-HDA treatment time, the number of early and late apoptotic cells increased to varying degrees, from 6.17% to 54.79%. In addition, when cell apoptosis occurred, the mitochondrial membrane potential (MMP) disappeared, membrane permeability changed, and a series of changes occurred. As shown in Figure 2(c), with increased treatment time, the fluorescence intensity of the cells continuously increased, and the proportion of depolarized cells was increased from 13.84% to 38.52%. As shown in Figure 2(d), the results indicated that 10-HDA treatment led to the downregulation of Bcl-2 and upregulation of BAX, cyto-c, caspase-3, and PARP in A549 cells. These results suggested that 10-HDA inhibits the proliferation of A549 human lung cancer cells through mitochondrial-dependent apoptosis.
3.3. 10-HDA Induces Apoptosis by Regulating MAPK, STAT3, and NF-κB Signaling Pathways in A549 Human Lung Cancer Cells. As shown in Figure 3(a), the results indicated that 10-HDA treatment led to the upregulation of p-p38, p-JNK, and I-κB and the downregulation of p-ERK, p-STAT3, and NF-κB in A549 cells. As shown in Figure 3(b), when a JNK inhibitor (SP600125) and a p38 inhibitor (SB203580) were added, the inhibitory effect of 10-HDA was alleviated and p-STAT3 and NF-κB were upregulated. There was a limited activation effect of 10-HDA and a limited downregulation  3.4. 10-HDA Induces Apoptosis by Regulating Intracellular ROS Generation in A549 Human Lung Cancer Cells. As shown in Figure 4(a), with 10-HDA treatment, intracellular ROS levels in the human lung cancer cells were significantly increased from 40.94% to 70.16% in a time-dependent manner, and intracellular ROS levels in IMR90 human normal lung cancer cells were significantly decreased from 59.08% to 34.39% in a time-dependent manner. As shown in Figure 4(b), after incubation with 10-HDA+NAC, compared with 10-HDA treatment alone, the number of apoptotic cells was significantly reduced from 42.49% to 25.27%. Meanwhile, as shown in Figure 4(c), compared with the control group, 10-HDA significantly led to the upregulation of p-p38, p-JNK, I-κB, and caspase-3 in a time-dependent manner, and it also led to the downregulation of p-ERK, p-STAT3, and NF-κB. NAC treatment alone also showed no significant changes compared to the control group. However, after incubation with 10-HDA+NAC, compared with 10-HDA treatment alone, scavenging of ROS by NAC significantly blocked MAPK, STAT3, and NF-κB signaling pathways and decreased the caspase-3 levels. These results suggested that 10-HDA increased the levels of ROS in A549 cells, leading to apoptosis.
3.5. 10-HDA Triggers G0/G1 Phase Cell Cycle Arrest in A549 Human Lung Cancer Cells. As shown in Figure 5(a), with increased 10-HDA treatment time, the percentage of cells in the G0/G1 phase increased over time, from 62.97% to 80.54%. As shown in Figure 5(b), 10-HDA treatment led to the downregulation of AKT, CDK2/4/6, and cyclin D1/E, and it also led to the upregulation of p21 and p27 in A549 cells. As shown in Figure 5(c), upon treatment with 10-HDA alone, compared to incubation with 10-HDA+NAC, the percentage of cells in the G0/G1 phase decreased, from 80.01% to 70.57%. As shown in Figure 5(d), compared with the control group, 10-HDA significantly led to the downregulation of p-AKT, CDK2/4/6, and cyclin D1/E, and it also led to the upregulation of p21 and p27. NAC treatment alone also showed no significant changes compared to the control group. After incubation with 10-HDA+NAC, compared with 10-HDA treatment alone, 10-HDA+NAC induced increased expression of p-AKT, CDK2/4/6, and cyclin D1/E, and it also induced decreased expression of p21 and p27. These results demonstrated that 10-HDA induced cell cycle arrest by regulating cell cycle-associated protein expression in A549 cells.

10-HDA Inhibits Cell Migration by Regulating TGF-β1
Signaling Pathways in Human Lung Cancer A549 Cells. As shown in Figure 6(a), compared with the control group, the human lung cancer cell intracellular migration was inhibited obviously under 10-HDA treatment in a time-dependent manner. As shown in Figure 6(b), the expression levels of TGF-β1, SNAI1, GSK-3β, N-cadherin, and vimentin were decreased, and the expression level of E-cadherin was also increased. These results indicated that 10-HDA can inhibit the migration of human lung cancer cells.

Discussion
10-HDA has garnered wide attention in recent years because of its various biological and pharmacological activities. In   7 BioMed Research International this study, we investigated the effects of 10-HDA on inhibiting cell proliferation, cell cycle arrest, and the induction of apoptosis in lung cancer cells. We evaluated the cytotoxic effects of 10-HDA on human lung cancer A549, NCI-H460, and NCI-H23 cells and found that 10-HDA significantly inhibited the proliferation of A549, NCI-H460, and NCI-H23 cells and had little cytotoxicity in normal IMR90, L-02, and GES-1 cells.
Apoptosis is process of programmed cell death with spontaneous characteristics. There are two ways to activate apoptosis: through intrinsic and extrinsic pathways [25]. Millions of cells in the human body undergo programmed cell death every hour; at the same time, millions of new proliferating cells replace these apoptotic cells, allowing tissues and organs to maintain their physiological functions for a long time. During the apoptotic process, it is mediated by the antiapoptotic protein Bcl-2 and proapoptotic protein BAX, which increases membrane permeability. Cyto-c is released into the cytosol and subsequently participates in the process leading to caspase-9 and caspase-3 activation [26]. Our results showed that after 10-HDA treatment of A549 human lung cancer cells, the expression level of antiapoptotic protein Bcl-2 decreased, and the expression of proapoptotic proteins BAX, cyto-c, caspase-3, and PARP increased in a time-dependent manner. These results suggest that 10-HDA can regulate the expression of Bcl-2 and BAX, and induce caspase-3-dependent apoptosis via the mitochondrial pathway.
Studies have reported that there is a wide range of interaction mechanisms between the MAPK STAT3 and NF-κB   10 BioMed Research International signaling pathways [27,28]. The MAPK family involves three major subgroups including ERK1/2, JNK, and p38 kinase. ERK1/2 is activated primarily by mitogenic stimuli such as growth factors leading to cell growth and survival [29,30].
Here, we showed that 10-HDA increased the phosphorylation of p38 and JNK, and decreased the phosphorylation of ERK in A549 cells in a time-dependent manner. These results confirm that 10-HDA activates the p38 and JNK signaling pathways through protein phosphorylation, and inhibits the ERK signaling pathway, further inhibiting STAT3 and NF-κB activity, resulting in cell apoptosis. ROS, as a natural by-product of aerobic respiration, is closely related to cell apoptosis, cell cycle, signal transduction cascade, protein phosphorylation, and cytoskeleton formation [31,32]. Increased ROS stimulates the cancer-related signal transduction pathway and enhances the survival and proliferation of cancer cells [33]. ROS can also be used as a signaling molecule to transduce extracellular stimulus signals, directly inducing apoptosis or indirectly participating in intracellular signal transduction [34]. In this study, 10-HDA induced the generation of ROS in A549 cells and inhib-ited the generation of ROS in normal cells, both in a timedependent manner. However, when the cells were treated with NAC and 10-HDA, NAC had no effect on the control group, but had a strong inhibitory effect on the 10-HDA group. Thus, 10-HDA stimulated ROS after entering the cells. It mediated the pathways of MAPK, STAT3, and NF-κB to inhibit A549 cell proliferation and control the growth and metastasis of cells, thereby achieving anticancer effects.
The uncontrolled proliferation of cancer cells is closely related to the regulatory mechanism of cell cycle progression, and it is also a significant feature of accelerating tumor growth [35]. In the present study, we demonstrated that 10-HDA induced G0/G1 cell cycle arrest in A549 cells by flow cytometry, as well as the molecular mechanisms underlying the regulation of the cell cycle processes. The cyclin D1 gene, which is tightly correlated with cancerous cell proliferation, has been considered a marker molecule [36]. Similarly, cyclin E and CDK2/4/6 also play key roles in the G0/G1 transition in the cell cycle [37][38][39][40]. Furthermore, p21 and p27 act by binding CDK in the G1 phase of the cell cycle, leading to inhibition of the phosphorylation of other proteins such as   11 BioMed Research International retinoblastoma, which is necessary for cell cycle progression [41,42]. In our study, protein analysis showed the decreased expression of cyclin D1/E and CDK2/4/6, and increased expression of p21 and p27 proteins. However, 10-HDA cotreatment with NAC led to the decrease in Akt. These results suggest that ROS generation can inhibit the phosphorylation of Akt, thereby activating formation of the CDK2/4/6 and cyclin D1/E kinase complex [43]. These results indicated that 10-HDA triggered cell cycle arrest at the G0/G1 phase in A549 human lung cancer cells by downregulating the expression of Akt, CDK2/4/6, and cyclin D1/E and also by upregulating p21 and p27.
It is well known that inhibiting cancer cell migration has important significance for the treatment of cancer. The TGF-β1 signaling pathway has been confirmed to modulate numerous physiologic processes, including proliferation, migration, and invasion of tumors [44,45]. Activation of TGF-β1 signaling may affect the crucial role in cells through activating downstream factors (GSK-3β) [46,47]. Furthermore, TGF-β1 signaling was the most enriched pathway by ectopic expression of Akt and MAPK pathways, all of which were engaged in cell proliferation and migration, and some studies also found that the inhibition of cell migration induced by the TGF-β1 signaling pathway presumably attributed to the suppression of ROS-dependent mechanisms [48,49]. In our study, we found that the expression level of E-cadherin was upregulated, and those of TGF-β1, SANI 1, GSK-3β, N-cadherin, and vimentin were decreased to different extents. These results showed that 10-HDA regulated the signal transduction pathways in A549 cells, so that the cells could not proliferate normally and limiting their range of activities, thereby inhibiting the migration of lung cancer cells.
In the present study, we demonstrated that 10-HDA induces ROS-mediated apoptosis in A549 human lung cancer cells by regulating the MAPK, STAT3, NF-κB, and TGF-β1 signaling pathways. The effects of 10-HDA demonstrated in vivo should be evaluated in future studies.  Figure 6: Effects of cell migration after treatment of human lung cancer cells with 10-HDA. (a) A549 cells were treated 3, 6, 12, and 24 h with 10-HDA, using the EVOS FL Auto Cell Imaging System at a magnification of 100x to observe. (b) A549 cells were treated with 10-HDA for 3, 6, 12, and 24 h to detect the levels of apoptotic proteins (TGF-β1, SNAI1, GSK-3β, E-cadherin, N-cadherin, and vimentin) and were normalized to αtubulin. The expression levels of proteins were analyzed by ImageJ software. * p < 0:05, * * p < 0:01, and * * * p < 0:001 vs. the control group.

Conclusion
In conclusion, 10-HDA induced apoptosis and cell cycle arrest of A549 human lung cancer cells through ROSmediated modulation of the MAPK, STAT3, NF-κB, and TGF-β1 signaling pathways, and it also induced eventual mitochondrial-dependent apoptosis (Figure 7). These findings indicate that 10-HDA may be a potential therapy for human lung cancer.

Data Availability
The data used to support the findings of this study are available from the corresponding authors upon request.

Conflicts of Interest
The authors declare that there are no conflicts of interest.