Garlic Extract Participates in the Proliferation and Apoptosis of Nonsmall Cell Lung Cancer Cells Via Endoplasmic Reticulum Stress Pathway

Objective To investigate the effect of garlic extract (GE) on the proliferation and apoptosis of cell lines A549 and H1299 in lung cancer (LC). Methods A549 and H1299 cells with well-developed logarithmic growth were added with GE at a concentration of 0 μg/ml, 25 μg/ml, 50 μg/M, 75 μg/ml, and 100 μg/ml, respectively. The inhibition of A549 cell proliferation was detected using CCK-8 after cultured for 24 h, 48 h, and 72 h. The apoptosis of A549 cells was analyzed via flow cytometry (FCM) after 24 h of cultivation. In vitro migration of A549 and H1299 cells was determined by cell wound scratch assay after 0 h and 24 h culture. The caspase-3 and caspase-9 protein expression levels in A549 and H1299 cells were evaluated through western blot after 24 h of cultivation. Results Colony formation and EdU assays revealed that Z-ajoene could inhibit cell viability and cell proliferation in NSCLC cells. After 24 h culture, there was no significant difference in the proliferation rate of A549 and H1299 cells with different GE concentrations (P > 0.05). A remarkable proliferation rate difference emerged between A549 and H1299 cells with different GE concentrations after 48 and 72 hours of cultivation. The proliferation rate of A549 and H1299 cells in the experiment group was significantly lower than that in the control group. With an elevated level of GE concentration, the proliferation rate of A549 and H1299 cells decreased (P < 0.05) while the apoptotic rate increased continuously. Conclusion GE could exert toxic effects on A549 and H1299 cells, inhibit cell proliferation, promote apoptosis, and attenuate cell migration. Meanwhile, it might induce apoptosis of A549 and H1299 cells through the caspase signal pathway, which is positively correlated with the mass action concentration and is expected to be a new drug for LC treatment.


Introduction
Lung cancer remains the most common malignancy and the leading cause of cancer-related death worldwide over the past decades [1], and nonsmall cell lung cancer (NSCLC) accounts for over 80% of all lung cancer diagnoses [2,3]. Despite the advances in management and diagnostics for NSCLC, the overall survival (OS) of NSCLC patients has not been signifcantly improved, with the 5-year OS rate of less than 20% [4][5][6]. Drug resistances, undesirable side efects of chemotherapy, and high metastatic rates are signifcant obstacles to successful treatment of NSCLC [7] as well as insufcient eforts in disease prevention and early diagnosis and integrated therapies for early remission, though enormous, which does not support a reduced survival rate [8]. It is still imperative to develop more efective drugs and more sensitive diagnostics for timely prevention and intervention to improve NSCLC survival.
ER is the main site for protein synthesis and folding in cells, and is responsible for other basic cellular activities, including intracellular protein maturation and translocation [9]. ER stress (ERS) is initiated when ER homeostasis disturbance results in unfolded/misfolded protein accumulation in the ER lumen. In this process, ERS triggers a series of accommodative mechanisms, known as the unfolded protein response (UPR), including attenuation of translation, elevated expressions of ER chaperones and associated proteins, and degradation of unfolded/misfolded proteins by a quality-control system [10]. However, once the restoration of ER function fails, apoptotic signalings will be induced by ERS [11,12].

Ajoene Extract and Z-Ajoene from
Garlic. Garlic (2 kg) was bought in the Korean retail market, divided into three parts, put into three diferent plastic buckets, and soaked in diferent concentrations of alcohol solution to extract the corresponding ingredients. Next, after incubating at room temperature for 1 hour, it was extracted with ethyl acetate (at 60°C for 8 hours). Te ethyl acetate extract was evaporated in vacuo to make ajoene extract for in vitro experiments. Te observed content of Z-ajoene determined by highperformance liquid chromatography (HPLC) was 11.1% (w/w), which was subsequently isolated and purifed by repeated column chromatography. As described previously, z-ajoene extract with a purity greater than 98% was obtained [22].

Cell
Culture, Treatment, and Transfection. Human NSCLC A549 and H1299 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Te cells were cultured in RPMI-1640 medium supplemented with 10% FBS in a humidifed environment after resuscitation, and the medium was replaced with a fresh medium every other day. Te cells were divided into fve groups: control, Z-ajoene, Z-ajoene + DLG1-specifc shRNA (sh-DLG1), Z-ajoene + YAP-specifc shRNA (sh-YAP), and Z-ajoene + sh-DLG1 + sh-YAP. Tose in the four treatment groups were incubated with Z-ajoene for 72 h at 37°C. Tree treatment groups were transfected with sh-DLG1 and/or sh-YAP using Lipofectamine 3000 reagent (Life Technologies, Carlsbad, CA, USA) to knock down DLG1 and/or YAP. Te cells transfected with sh-RNA-NC were used as negative controls (GenePharma, Guangzhou, China). Te target sequences for sh-DLG1 and sh-YAP are 5′-AGC GAU GGU CCA UTC UUG CAA-3′ and 5′-GGG UCT UGC AUT UGC ACU ATT-3′, respectively.

Cell Counting Kit-8 Assay.
Cell viability of each group was detected using the CCK-8 assay (Beyotime, Beijing, China). Te cells at an appropriate concentration were inoculated in 96-well plates and treated accordingly. CCK-8 solution was added to each well, followed by two h of incubation in the dark. Te optical density was measured at 450 nm.

Colony Formation
Assay. Cells (1,000 cells/well) were seeded in a 6-well plate and incubated for two weeks. Clones were fxed with methanol, stained with 0.1% crystal violet for four h at room temperature, and counted under a light microscope (Olympus, Tokyo, Japan).

5-Ethynyl-2′-Deoxyuridine (EdU) Assay.
Cell proliferation was measured using an EdU assay. A total of 2 × 10 5 cells were transferred into 24-well plates and allowed to adhere overnight. After transfection, the cells were incubated with 100 μL EdU for two h, fxed with 4% paraformaldehyde for 30 min, and stained using a Cell-LightTM EdU Apollo ® 488 In Vitro Imaging kit (Guangzhou RiboBio Co., Ltd.) according to the manufacturer's instructions.
2.6. Cell Apoptosis Assay. Flow cytometry was conducted to detect cell apoptosis. Te cells (1 × 10 6 cells/well) were seeded in 6-well plates. Tey were harvested after 24 h of treatment and stained with an annexin-V fuorescein isothiocyanate (FITC) and propidium iodide (PI) apoptosis detection kit (Invitrogen, USA) to detect cell apoptosis using a FACScalibur fow cytometer (BD Biosciences, CA, USA).

Quantitative Real-Time PCR.
Total RNA from cell samples was extracted using the TRIpure isolation reagent (Invitrogen, USA), and reverse transcription was performed using a PrimeScript RT kit (TaKaRa, Japan). After the master mix was prepared, DLG1 and YAP mRNA expression levels were detected using a real-time PCR assay with SYBR green, normalized to internal control ACTB.

Statistical Analysis.
All experiments were performed independently, in triplicate. Quantitative variables were presented as the mean ± standard deviation (SD) from three independent experiments. As appropriate, the statistics were performed using the Student's t-test or one-way ANOVA in SPSS (version 17.0). A P value of <0.05 was considered statistically signifcant.  of Z-ajoene treatment using the CCK-8 assay. A signifcant dose-dependent (0-25 μM) decrease in cell viability was observed in cells treated with Z-ajoene (Figure 1(b)), together with dose-dependent (0-10 μM) inhibition of cell proliferation in colony formation and cell proliferation (Figures 1(c) and 1(d)).

Z-Ajoene Stimulates ERS in NSCLC Cells.
Since Z-ajoene inhibited cell proliferation in NSCLC cells, we assessed whether Z-ajoene was involved in the ER stress pathway in NSCLC cells, and protein expressions of the pathway coSeminars in Interventional Radiologyponents were determined. Te results showed pronouncedly increased expressions of binding protein (BiP) and protein kinase R-like endoplasmic reticulum kinase (PERK) after the Z-ajoene treatment (Figure 2(a)), as expected, which prompted us to detect the downstream UPR targets of PERK, activating transcription factor 4 (ATF4) and homologous protein (CHOP). ATF4 and CHOP proteins were signifcantly elevated with the Z-ajoene treatment compared to the control group. Z-ajoene was also very efective in inducing their expressions at the mRNA levels ( Figure 2(b)).

Z-Ajoene Induces NSCLC Cell Apoptosis.
Flow cytometry was performed to explore the efects of Z-ajoene on cell apoptosis in A549 and H1299 cells. Tumor cell apoptosis was markedly enhanced dose-dependently by the Z-ajoene treatment compared to that achieved in the control group (Figure 3(a)). Te Western blot of several essential apoptosis-associated proteins showed that expressions of pro-apoptotic Bax, cleaved caspase 3, and cleaved caspase 9 expressions signifcantly increased in cells treated with Zajoene with a low expression of the anti-apoptosis protein Bcl-2dose-dependently (Figure 3(b)).

Z-Ajoene-Induced NSCLC Cell Apoptosis Is Associated
with Enhanced ERS. Z-Ajoene has stimulated apoptosis and ERS in NSCLC cells; however, whether there was a potential relationship between them is worthy of further exploration. We added 4-phenylbutyric acid (4-PBA), an ERS inhibitor, to detect whether apoptosis could be rescued. Compared to the Z-ajoene group, a decrease in cell apoptosis was observed in both A549 and H1299 cells cotreated with Z-ajoene and 4-PBA (Figure 4(a)). Similarly, pro-apoptotic Bax, Cleaved caspase 3, and Cleaved caspase nine expressions markedly decreased, and anti-apoptoticBcl-2 was elevated in the Zajoene + 4-PBA group versus Z-ajoene-treated controls (Figure 4(b)).

Z-Ajoene-Induced NSCLC Cell Apoptosis Is Activated by
ERS Via DLG1/YAP Signaling Inhibition. Furthermore, we assessed whether Z-ajoene exerted its pro-apoptotic efect on NSCLC cells via the DLG1/YAP pathway, an essential mediator of ERS. Te Western blot assay showed that compared to the control group LATS2 and YAP protein expressions were signifcantly downregulated, and DLG1 levels upregulated dose-dependently in A549 and H1299 cells treated with Z-ajoene ( Figure 5(a)).
A549 and H1299 cells were transfected with sh-DLG1 and/or sh-DLG1 with pcDNA YAP before the Z-ajoene treatment, and DLG1 mRNA expression was sharply downregulated in cells transfected with sh-DLG1, and YAP remarkably upregulated in pcDNA YAP-transfected cells ( Figure 5(b)). Figure 5(c) shows that the cells treated with Zajoene alone showed the strongest apoptosis. A reduced apoptosis rate was detectable in sh-DLG1-transfected cells, whereas the opposite fnding was observed in sh-YAPtransfected cells. In cells co-transfected with sh-DLG1 and sh-YAP, the already inhibited apoptosis by sh-DLG1 was robustly reversed. We delineated that DLG1 knockdown remarkably suppressed Bip, PERK, ATF4, and CHOP expressions after the Z-ajoene treatment compared to negative controls, which were subsequently boosted after cotransfection with sh-YAP ( Figure 5(d)).

Discussion
NSCLC is the most prevalent lung cancer and the primary cause of cancer-associated mortality worldwide [23,24]. Some studies have proved the efciency of existing treatments for nonsmall cell lung cancer, but the survival rate of advanced nonsmall cell lung cancer has not improved, which remains virtually unchanged. Terefore, the identifcation of novel and efective agents for the management of advanced NSCLC is the priority. In the present study, we found that Zajoenedose-dependently inhibited cell viability or cytotoxicity and proliferation in NSCLC cells, showing an antiproliferative efect against NSCLC.
Te endoplasmic reticulum (ER) is an intricate organelle vital for cellular function and survival. When ER activity is hindered, the accumulation of unfolded proteins stimulates the transmembrane sensors to initiate the UPR, ultimately restoring ER homeostasis [25]. When the restoration of ER homeostasis or ERS attenuation fails, the UPR of ER activates signalings towards apoptosis. However, little research has been conducted on the specifc mechanism of ER homeostasis recovery and focusing on ERS-mediated apoptosis may create new treatment options for cancer patients [26]. For the involvement of ERS in Z-ajoene-mediated apoptosis, our results revealed strong ERS responses to Z-ajoene treatment in A549 and H1299 cells, together with increased expressions of the ERS pathway components, BiP, PERK, ATF4, and CHOP. Tis fnding demonstrates that the anticancer potential of Z-ajoene is triggered via ERS.
Apoptosis is strictly regulated by many proteins and pathways, of which the Bcl-2 family has a crucial role [27], including pro-apoptotic (Bax, Bim, Bcl-xs, Bak, Bid, Bad, and Bik) and anti-apoptotic (Bcl-2, Bcl-xl, Bcl-w, and A1) subfamilies [28]. Bax-type apoptotic proteins allow small molecules such as ions and cytochrome C to penetrate the mitochondrial membrane into the cytoplasm, leading to cell apoptosis. In our study, compared to untreated controls, Bax, caspase-3, and caspase-9 protein expressions were dosedependently upregulated and anti-apoptoticBcl-2 downregulated with the Z-ajoene treatment, which supports an apoptotic efect of Z-ajoene against NSCLC A549 and H1299 cells.
DLG1 is a crucial component gene of the cell polarity module SCRIB-LGL-DLG. A negative correlation of DLG1 expression with YAP activity, or the inhibitory of DLG1  Evidence-Based Complementary and Alternative Medicine 5 molecule on YAP activity, has been reported [29][30][31]. Te Hippo/YAP signaling pathway plays an important role in cell growth regulation and organ size control during biological growth and development [32]. In the Hippo signaling, the upstream activator of the core efector YAP, LATS1/2, is stimulated by MST1/2-induced phosphorylation to boost YAP phosphorylation. Phosphorylated YAP is maintained in the cytoplasm, ubiquitinated, and degraded to make the downstream pathway of YAP in a static state, thus inhibiting YAP-mediated gene transcription. YAP degradation can be inhibited by oxidative stress, ischemia or hypoxia, physical factors, G protein-coupled receptors, or other signalings to increase YAP activity, allowing activated YAP to bind to TAZ and enter the nucleus to initiate relevant  Evidence-Based Complementary and Alternative Medicine gene transcription [33]. Aberrant Hippo/Yap signaling may fuel cell proliferation and antagonize apoptosis for tumor occurrence and growth. In this study, Z-ajoene's regulation on YAP via DLG1 may be the mechanism responsible for its cytotoxic and apoptotic efects on NSCLC cells. Furthermore, our fnding of LATS2 and YAP downregulation and DLG1 upregulation in a dose-dependent manner after the Z-ajoene treatment in A549 and H1299 cells is consistent with YAP regulation by DLG1, which was verifed by YAP upregulation after DLG1 knockdown, together with a suppressed efect of Zajoene. Additional knockdown of the YAP gene reversed the efect of sh-DLG1 transfection into A549 and H1299 cells.
In summary, our study demonstrates that Z-ajoene induces ER stress through DLG1/YAP signal inhibition and promotes apoptosis in NSCLC cells. It is expected to ofer more options for developing novel natural monotherapy or combination regimens for NSCLC.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.  Evidence-Based Complementary and Alternative Medicine 9