Usenamine A triggers NLRP3/caspase-1/GSDMD-mediated pyroptosis in lung adenocarcinoma by targeting the DDX3X/SQSTM1 axis

Background: Usenamine A (C18H17NO6) is a newly developed, natural anticancer drug that reportedly exerts low toxicity. The therapeutic efficacy and underlying mechanisms of usenamine A in lung adenocarcinoma (LUAD) remain poorly understood. We aimed to explore the therapeutic effects and molecular mechanisms through which usenamine A inhibits LUAD tumorigenesis. Methods: We used LUAD cell lines H1299 and A549 in the present study. CCK-8 and colony formation assays were performed to analyze cell proliferation. Cell migration, invasion, and apoptosis were evaluated using wound-healing, transwell, and flow cytometric assays, respectively. Levels of reactive oxygen species were measured using a DCFH-DA probe. Inflammatory factors (lactate dehydrogenase, interleukin [IL]-1β, and IL-18) were detected using enzyme-linked immunosorbent assays. Western blotting was performed to determine the expression of NOD-like receptor pyrin 3 (NLRP3)/caspase-1/gasdermin D (GSDMD) pathway-related proteins. Pyroptosis was detected using transmission electron microscopy. The interaction and co-localization of DDX3X and sequestosome 1 (SQSTM1) were identified using co-immunoprecipitation and immunofluorescence assays, respectively. For in vivo assessment, we established a xenograft model to validate the usenamine A-mediated effects and mechanisms of action in LUAD. Results: Usenamine A inhibited the proliferation, migration, and invasion of LUAD cells. Furthermore, usenamine A induced NLRP3/caspase-1/GSDMD-mediated pyroptosis in LUAD cells. Usenamine A upregulated DDX3X expression to trigger pyroptosis. DDX3X interacted with SQSTM1, which is responsible for inducing pyroptosis. In vivo, usenamine A suppressed LUAD tumorigenesis by triggering NLRP3/caspase-1/GSDMD-mediated pyroptosis via the upregulation of the DDX3X/SQSTM1 axis. Conclusions: Usenamine A was found to induce NLRP3/caspase-1/GSDMD-mediated pyroptosis in LUAD by upregulating the DDX3X/SQSTM1 axis.


INTRODUCTION
Lung adenocarcinoma (LUAD) is the most common subtype of lung cancer, known for its high mortality rate [1,2].It is predicted that lung cancer will be responsible for 609,360 deaths in 2022 in the United States, equating to approximately 350 deaths daily [3].LUAD accounts for approximately 50% of all lung cancer cases worldwide, with a 5-year survival rate at only 22% [3,4].Currently, radiation and chemotherapy are first-line therapies for LUAD; however, these treatment strategies may cause severe adverse effects and afford a poor prognosis [5].Other therapeutic interventions, including lobectomy and segmentectomy, are mainly effective for early-stage LUAD [6].Therefore, it is crucial to explore effective adjuvant drugs and elucidate their mechanisms of action, contributing to developing novel therapeutic strategies for LUAD.
Pyroptosis is a form of proinflammatory programmed cell death that is increasingly recognized [7,8].Pyroptosis is mediated by caspase-gasdermin D (GSDMD) signaling pathways, which is activated by NOD-like receptor pyrin 3 (NLRP3) inflammasome [9].NLRP3/caspase-1/GSDMD-mediated pyroptosis is emerging as a promising therapeutic target in cancer treatment, thereby leading to the search for effective drugs and biomarkers for cancer treatment.A previous study revealed that circNEIL3 can regulate NLRP3/ caspase-1/GSDMD-mediated pyroptosis to influence radiotherapy targeting LUAD [8], demonstrating the pivotal role of pyroptosis in LUAD.Usenamine A (C18H17NO6), a compound derived from the Usnea plant in Yunnan (China), is a novel natural anticancer drug with low toxicity both in vitro and in vivo (Patent No.:201710388136.8).In vitro experiments have revealed that usenamine A can exert a notable inhibitory effect on lung, bladder, and liver cancers, and was found to be superior to cisplatin and paclitaxel.He et al. confirmed that usenamine A combined with scutellarin promotes apoptosis of human glioma cells and impairs glioma cell survival [10,11].However, the therapeutic effects of usenamine A on LUAD remain poorly explored.This study investigates whether usenamine A regulates NLRP3/caspase-1/GSDMDmediated pyroptosis in LUAD.DEAD-Box helicase 3 X-Linked (DDX3X), a stress granule protein, interacts with NLRP3 to influence the assembly of NLRP3 inflammasome [12].The initiation of pyroptosis via the NLRP3/caspase-1/GSDMD pathway critically depends on the involvement of DDX3X [12].Given its central role in inflammasomemediated cell death, and considering the frequent mutations of DDX3X found in various cancers, it presents as a potential target for anticancer therapies [13,14].Wu et al. showed that the loss of DDX3 can facilitate malignant tumor progression and indicates a poor prognosis of non-small cell lung cancer [15].In addition, DDX3X has been shown to promote sequestosome 1 (SQSTM1/p62) accumulation in pancreatic ductal adenocarcinoma [16], suggesting a regulatory relationship between DDX3X and SQSTM1.SQSTM1 is a critical autophagy receptor that regulates inflammatory response [17].Cao et al. showed that SQSTM1 accumulation could exacerbate NLRP3independent cell death via GSDMD-mediated pyroptosis [18].Moreover, elevated SQSTM1 expression was closely correlated with poor prognosis of LUAD [19].The specific mechanism of the DDX3X/SQSTM1 axis in NLRP3/caspase-1/GSDMD-mediated pyroptosis in LUAD remains largely unknown.
Herein, we hypothesized that usenamine A induces NLRP3/caspase-1/GSDMD-mediated pyroptosis in LUAD by targeting the DDX3X/SQSTM1 axis.Accordingly, we examined the function of usenamine A in LUAD considering the NLRP3/caspase-1/GSDMDmediated pyroptosis and determined the mechanism involved in the DDX3X/SQSTM1 axis.This exploration could pave the way for novel therapeutic strategies in the treatment of LUAD.
Reportedly, DDX3X induces epithelial-mesenchymal transition in pancreatic ductal adenocarcinoma by promoting SQSTM1 accumulation [16], indicating the interaction between DDX3X and SQSTM1.In the present study, Co-IP and immunofluorescence assays confirmed the interaction and co-localization of DDX3X and SQSTM1 in the cytoplasm.SQSTM1 is a ubiquitinbinding autophagy receptor that exhibits oncogenic activity in various cancers [16,36].Moreover, SQSTM1 accumulation can exacerbate NLRP3-and GSDMDmediated pyroptosis [18].Therefore, we speculate that usenamine A induces NLRP3/caspase-1/GSDMDmediated pyroptosis in LUAD by targeting the DDX3X/ SQSTM1 axis.Our results showed that DDX3X overexpression upregulated SQSTM1 expression and promoted NLRP3/caspase-1/GSDMD-mediated pyroptosis in LUAD cells.In addition, MCC950, an inhibitor of NLRP3 inflammasome, reversed the stimulatory effects of oe-DDX3X on SQSTM1 expression and pyroptosis.These findings confirm the essential positive role of DDX3X/SQSTM1 in the effects of usenamine A, triggering pyroptosis in LUAD.Finally, in vivo experiments validated that usenamine A impedes LUAD tumorigenesis and induces pyroptosis by upregulating the DDX3X/ SQSTM1 axis.
This study examines the anti-cancer effects of usenamine A in LUAD and identifies the underlying mechanism involving the activation of NLRP3/ caspase-1/GSDMD-mediated pyroptosis.However, there are some potential limitations and confounding variables.Firstly, further validation with additional LUAD cell lines and primary human tissue samples will strengthen the credibility of these findings.Second, the specificity of DDX3X-SQSTM1 binding needs to be explored to rule out any alternative contributors to pyroptosis regulation in LUAD cells.Lastly, the in vivo study was restricted to a single strain and dosing regimen, necessitating further investigations to account for inter-strain, gender-based, and dosage-specific differences.Despite these caveats, the findings provide encouraging evidence supporting the continued development of usenamine A as a treatment option for LUAD patients.

CONCLUSIONS
In summary, usenamine A suppresses LUAD progression by inducing NLRP3/caspase-1/GSDMDmediated pyroptosis.This process is achieved by upregulating the DDX3X/SQSTM1 axis.Our study revealed a promising auxiliary therapeutic drug for LUAD.Moreover, we elucidated the mechanism through which usenamine A induces pyroptosis in LUAD, specifically by targeting the DDX3X/SQSTM1 axis and activating the NLRP3/caspase-1/GSDMD pathway (Figure 9).Our study lays a strong foundation for developing novel therapeutic approaches for LUAD.We plan to expand upon our findings by conducting investigations on other LUAD cell lines and primary patient tissues, clarifying the mechanism regulating DDX3X/SQSTM1.

Cell culture and treatment
The normal human lung epithelial cell line (BEAS-2B), LUAD cell lines (H1299 and A549), and HEK-293T cells were obtained from the FuHeng Cell Center (Shanghai, China).All cells were maintained in Dulbecco's modified Eagle's medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, MA, USA) and 1% penicillin/streptomycin (Hyclone, MA, USA).All cells were cultured in a humidified atmosphere containing 5% CO2 at 37° C. All treatments were performed on cells at passage three to ensure uniformity.
Cell treatment 1: H1299 and A549 cells were treated with gradient concentrations of usenamine A (0, 1, 2, and 3 μg/mL) for 24 h.The concentrations of usenamine A used in our study were selected based on Patent No.: 201710388136.8and a previous study [37].Morphology of cells was observed using a microscope (Olympus, Japan).Usenamine A was provided by Xiaoqiong He, School of Public Health, Kunming Medical University.
The successful transfection of H1299 cells with sh-DDX3X, oe-DDX3X or NC was confirmed by RT-qPCR and Western blot analysis, ensuring the effectiveness of gene silencing or overexpression.

CCK-8 assay
The viability of H1299 and A549 cells was evaluated using the CCK-8 assay.Briefly, H1299 and A549 cells were seeded in 96-well plates (100 µL per well) at 37° C at an atmosphere of 5% CO2.After treatment at 24 and 48 h, CCK-8 solution (10 μL) was added into each well and incubated for 2 h.Cell viability was measured using a microplate reader (Wuxi Hiwell-Diatek, China) at 450 nm.

Colony formation assay
Cells were resuspended and then plated into a six-well plate (200 cells per well) to obtain a 14-day culture in a Heracell™ 150i CO2 incubator (Thermo Fisher Scientific, MA, USA).Next, cells were fixed in methanol (1 mL per well) for 15 min and then stained with crystal violet (Beyotime, China) for 20 min.Excess staining solution was washed with phosphate-buffered saline (PBS), and cells were captured by a digital camera (Olympus, Japan).

Wound-healing assay
Cell suspension was seeded into six-well plates (5 × 10 5 cells per well).At the second day, a pipette tip (10 μL) was applied to create equal-width linear scratches on the six-well plates.Cell fragments were washed with PBS, and cell images were captured at 0, 12, and 24 h under a DMI3000 B inverted fluorescence phase contrast microscope (Leica, Germany).Wound-healing rate (%) = average distance at (0 h-x h)/0 h × 100.

Transwell assay
At 48 h following treatment, cells were digested with trypsin and resuspended in serum-free medium to 1 × 10 5 /mL.For the migration assay, the upper chamber of the Transwell (24-well insert, 8 μm pores, Corning, NY, USA) was filled with 200 µL cell suspension, and the lower chamber with 10% FBS medium.Considering the invasion assay, the upper chamber was covered with Matrigel (BD Biosciences).After 48-h incubation, cells in the lower chamber were rinsed twice with PBS and then immobilized with methanol for 30 min.Next, cells were stained with crystal violet for 20 min and photographed using a fluorescence microscope (Olympus, Japan) in three random fields.

Flow cytometry
Apoptosis was examined using an Annexin V-FITC Apoptosis Detection Kit (Beyotime, China).Cells were digested with 0.25% trypsin and centrifuged for 5 min at 1,500 rpm to collect cells.After washing twice with PBS, 300 µL of binding buffer was used to resuspend cells.The cell suspension was incubated with annexin V-FITC (5 μL) for 15 min and with 10 μL propidium iodide for 10 min under dark conditions.Apoptotic cells were detected using a CytoFLEX S Flow Cytometer (Beckman Coulter, CA, USA) and analyzed using the CellQuest Pro software.

RT-qPCR
Total RNA was extracted from cells using TRIzol reagent (Invitrogen, CA, USA).Complementary DNA was obtained using the FastKing-RT SuperMix (Tiangen, China).RT-qPCR was carried out using SYBR Green PCR Master Mix (Lifeint, China) with the Mx3000P Multiplex Quantitative PCR (QPCR) System (Agilent Stratagene, CA, USA).The thermocycling conditions were 95° C for 3 min, followed by 40 cycles of 95° C for 12 s and 62° C for 40 s.The relative gene expression was obtained by the 2 -ΔΔCT method.Primer sequences used are presented in Table 1.

TEM
Cells were fixed with 2.5% glutaraldehyde, dehydrated with ethanol and acetone, embedded, and cut into slices.Next, sections were stained with 2% uranyl acetate and lead citrate.Finally, cells were observed under a Tecnai F30 transmission electron microscope (FEI, CA, USA) at a voltage of 80 kV.

Co-IP assay
To determine the interactions between DDX3X and SQSTM1, HEK-293T cells were lysed with IP buffer, and supernatant was obtained by centrifuging for 1 min at 1,000 ×g.The supernatant was incubated with beads carrying DDX3X and SQSTM1 antibodies for 12 h.Then, beads were washed with lysis buffer and mixed with sodium dodecyl sulfate (SDS) buffer for western blot analysis.

Tumor xenografts animal experiments
Animal study was approved by the Institutional Animal Care and Use Committee of Xiamen University (XMULAC20220034-9).Specific pathogen-free 6-8 weeks old BALB/c mice (n = 20) were obtained from GemPharmatech Co., Ltd.(Nanjing, China, animal license #: SCXK-2018-0027).All mice were acclimatized under a 12-h light/dark cycle at 22 ± 1° C.After one week, non-transfected or sh-NC/sh-DDX3Xtransfected H1299 cells (3 × 10 6 cells per mouse) were administered subcutaneously to mice.Tumor size was monitored every five days.Tumor volume measurements were determined using the formula V (mm 3 ) = (width) 2 × length/2.When the tumor volume reached 180-200 mm 3 , the mice were subcutaneously injected with 2 mg/kg usenamine A every three days.Control mice were injected with 0.9% normal saline.After four weeks or upon reaching humane endpoints, which were rigorously defined by clinical signs such as severe lethargy, inability to obtain food or water, significant weight loss, or tumor burden exceeding ethical limits, animals were euthanized using CO2 inhalation-a method aligned with the latest AVMA guidelines for humane euthanasia.The euthanasia process involved gradual filling of the chamber with CO2 to ensure minimal distress.Tumors were then collected for further analysis.

TUNEL staining
Apoptosis in tumor tissues was detected using the One-Step TUNEL Apoptosis Assay Kit (Beyotime, China).Briefly, tumor tissue sections were digested using proteinase K solution at 37° C for 30 min.After washing with PBS three times, samples were stained with TUNEL reagent and then counterstained with DAPI for 1 h at 37° C under dark conditions.TUNEL staining was captured using a fluorescence microscope (Olympus, Tokyo, Japan).

ROS detection
Cell suspensions and tumor tissue sections were incubated with DCFH-DA reagent (10 μM; Solarbio, AGING China) for 40 min and DAPI staining solution for 15 min at 37° C. Subsequently, the cells were photographed using a fluorescence microscope and analyzed using Image J software (National Institutes of Health, Bethesda, MD).

Enzyme-linked immunosorbent assay (ELISA)
Cells were plated in 96-well plates (8 × 10 3 cells per well) for 24 h.LDH release from cells was measured by the CyQUANT LDH Cytotoxicity Assay kit (Thermo Fisher Scientific, MA, USA).Levels of IL-1β and IL-18 in LUAD cells and serum were measured using IL-1β and IL-18 ELISA kits (Mlbio, China), respectively.

Western blot assay
Cells and tumor tissues were lysed by radioimmunoprecipitation assay (RIPA) buffer (Beyotime, China) with phenylmethanesulfonyl fluoride (PMSF) protease inhibitor (Beyotime) to extract total protein.Proteins (25 μg per well) were separated using 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Beyotime).Membranes were blocked with 5% skim milk for 1 h, and then incubated with primary antibodies overnight at 4° C.After washing with 1× TBST containing 0.1% Tween-20, membranes were incubated with a secondary antibody (1:2,000; ab6721, Abcam) in the dark for 1 h.Protein bands were visualized by the ECL chemiluminescence solution (APPLYGEN, China).The primary antibodies used for western blotting are presented in Supplementary Table 1.

Statistical analyses
Animal experiments were conducted with quintuplicate samples (n = 5), and cell-based assays were performed in triplicate (n = 3).Data values are expressed as mean ± standard deviation.Differences between pairs of groups were assessed with an unpaired two-tailed Student's ttest.For analyses involving more than two groups, we employed one-way ANOVA.Subsequent to ANOVA, we conducted post-hoc pairwise comparisons applying Tukey's Honest Significant Difference (HSD) test, which corrects for the multiple comparison problem.A P-value of < 0.05 was considered significant.
Usenamine A (C18H17NO6) is a novel natural anticancer drug with favorable antitumor effects and low toxicity in multiple cancers (Patent No.: 201710388136.8).The