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. 2025 Apr 2;14(2):tfaf047. doi: 10.1093/toxres/tfaf047

Astragaloside IV inhibits nasopharyngeal carcinoma progression by suppressing the SATB2/Wnt signaling axis

Yinping Zeng 1, Tingting Duan 2, Jiajun Huang 3, Xiaofeng Wang 4,✉
PMCID: PMC11964083  PMID: 40177383

Abstract

 

Astragaloside IV (AS-IV), a major bioactive component of Astragalus membranaceus, exhibits anti-cancer and anti-inflammatory properties. However, its precise role in nasopharyngeal carcinoma (NPC) remains unclear. This study investigated the effects of AS-IV on NPC progression and its relationship with Special AT-rich binding protein-2 (SATB2), a diagnostic marker for NPC. AS-IV treatment reduced NPC cell viability in a dose-dependent manner, as assessed by CCK-8 assays. Functional experiments, including transwell, immunofluorescence, and flow cytometry assays, demonstrated that AS-IV inhibited cell migration, invasion, and autophagy while promoting apoptosis. Western blot analysis showed that SATB2 expression was significantly elevated in NPC cells, particularly in C666–1 and HK-1 cells. Overexpression of SATB2 partially reversed AS-IV’s inhibitory effects on NPC progression. Further analysis revealed that AS-IV suppressed the Wnt signaling pathway by downregulating SATB2 expression, while SATB2 overexpression restored Wnt pathway activation. This effect was reversed upon treatment with the Wnt pathway inhibitor DKK-1. In vivo, AS-IV administration inhibited tumor growth in a nude mouse subcutaneous xenograft model, reduced Ki-67 positivity, and lowered LC3B expression, indicating decreased proliferation and autophagy. However, these effects were diminished upon SATB2 overexpression. These findings suggest that AS-IV exerts anti-tumor effects in NPC by downregulating SATB2 and suppressing Wnt pathway activation, highlighting its potential as a therapeutic agent for NPC.

Highlights

  1. Astragaloside IV (AS-IV) reduces nasopharyngeal carcinoma (NPC) cell vitality, suppresses cell migration, invasion and autophagy, and fosters apoptosis.

  2. SATB2 exhibits notably high levels in NPC cells.

  3. Overexpression of SATB2 counteracts the inhibition of NPC malignant progression by AS-IV.

  4. AS-IV impedes NPC progression by decreasing SATB2 and thereby hindering the Wnt pathway.

  5. AS-IV deters NPC tumor growth in nude mice.

Keywords: NPC, AS-IV, SATB2, Wnt pathway, autophagy

Graphical Abstract

Graphical Abstract.

Graphical Abstract

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Introduction

Arising from nasopharyngeal epithelium, nasopharyngeal carcinoma (NPC) exhibits distinct geographic distribution characteristics. For instance, this condition is often identified in Southeast Asia and East Asia, especially in southern China.1,2 Differentiated poorly, NPC has the top metastatic rate among head–neck tumors.3,4 Although mere radical chemotherapy can cure around 90% of early-stage NPC patients, over 70% of patients have entered the middle or late stage or have exhibited distant metastasis at their first diagnosis. The reason is that this condition onsets insidiously and is much more aggressive.5,6 Additionally, the molecular mechanisms driving the occurrence, progression, and metastasis of NPC remain elusive, hence the demand for developing new and efficient drugs to curb the malignant progression of NPC.

Astragalus membranaceus is a traditional Chinese medicine enriched with such active ingredients as terpenoids, flavonoids, and polysaccharides.7 The extracts from Huang Qi hold numerous pharmacological activities, e.g. antioxidative, anti-inflammatory, anticancer, and immunomodulatory effects.8,9 Among the primary bioactive substances in Astragalus membranaceus, Astragaloside IV (AS-IV) is a terpenoid, possessing multiple pharmacological effects.10,11 AS-IV can enhance breast cancer’s chemosensitivity to paclitaxel by targeting caveolin-1, as reported by Zheng et al..12 Moreover, AS-IV can impair the aggravation of many cancers, like cervical, prostate, and non-small-cell lung cancer.13–15 Nevertheless, the impact of AS-IV treatment on NPC and the detailed mechanism remain unclear.

Special AT-rich binding protein-2 (SATB2) functions as a transcription regulatory factor.16 Numerous studies have demonstrated a remarkable upregulation of SATB2 in many cancers, enhancing the aggravation of such carcinomas as renal cell carcinoma, oral squamous cell carcinoma, and melanoma.17–19 Furthermore, SATB2 is heightened greatly in NPC and can serve as a diagnostic marker for this disease.20 Wnt pathway is a highly conserved cellular signaling system that is crucial in embryonic development, maintenance of organ and tissue homeostasis, and has been associated with the pathogenesis of many human diseases.21 Currently, numerous studies have demonstrated the involvement of the Wnt pathway in regulating the resistance to chemotherapy, proliferation and migration of NPC cells.22–24 Notably, SATB2 can regulate the Wnt signaling pathway.25,26

This research sought to investigate the regulatory role of AS-IV on SATB2 expression and its effect on the malignant progression of NPC, with a view to offer a new reference for NPC treatment.

Methodology & Materials

Cultivating and transfecting cells

Human nasopharyngeal epithelial cells (NP-69, SNL-565) and NPC cells C666–1 (SNL-516), HK-1 (SNL-563) were supplied from Sunncell Biotechnology Co., Ltd (Wuhan, Hubei, China). NPC cell line HNE-3 (Cs-010-006177) was purchased from Tongwei Biotechnology Co.,Ltd (Beijing, China), and NPC43 (CVCL_UH64) were obtained from BioVector Science Lab,Inc. (Beijing, China). The above cells were grown in DMEM medium (11,965,092, Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (F0193, Sigma-Aldrich, St. Louis, MO, USA) and 1% penicillin/streptomycin mixture (15,140,122, Gibco). The fluid change interval was 3 days, the passaging ratio was 1:3. The temperature was set to 37 °C and placed in an environment with saturated humidity and 5% volume CO2.

SATB2 overexpressing vector (OE-SATB2) and its control (OE-NC) were produced by Sangon Biotech (Shanghai, China). Referring to the instructions of Lipofectamine 3,000 (L3000001, Invitrogen, Austin, TX, USA), control and experimental vectors were transfected into NPC cells. After 48 h of transfection, the transfection efficiency was reflected by detecting the expression level of SATB2 in cells.

In the AS-IV cytotoxicity assay, the concentration of AS-IV (HY-N0431, MedChemExpress, Monmouth Junction, NJ, USA) was 50, 100, 200, 400, or 800 μM, and the treatment time was 24 h. Based on the findings of the toxicity assay, 100, 200 and 400 μM of AS-IV were subsequently selected to treat NPC cells. In the signaling axis probe experiment, the Control+OE-NC group was transfected with OE-NC in NPC cells. The AS-IV + OE-NC group was exposed to AS-IV (400 μM) for 24 h after transfection of OE-NC in NPC cells. The AS-IV + OE-SATB2 group was exposed to AS-IV (400 μM) in NPC cells for 24 h after transfection of OE-SATB2 in the NPC cells. For the AS-IV + OE-SATB2 + DKK1 group, after transfection of OE-SATB2 in NPC cells, the NPC cells were exposed to AS-IV (400 μM) for 4 h, and then exposed to Dickkopf-related protein 1 (Dkk-1, 100 ng/mL, HY-P72968, MedChemExpress), a Wnt pathway inhibitor, treatment for 24 h.

Cell counting Kit-8 (CCK-8) assay

NPC cells were inoculated in 96-well cell culture plates (1.5 × 104 cells/well). After different treatments, each well was filled with 10% CCK-8 reagent (96,992, Sigma-Aldrich). The cells were maintained for 2 h at 37 °C, and then the OD450 values of the cells in each group were determined by using an enzyme marker (1,410,101, Thermo Fisher Scientific, Waltham, MA, USA).

Transwell assays

Matrigel (HY-K6002, MedChemExpress) was melted overnight at 4 °C in a refrigerator protected from light and subsequently diluted with serum-free DMEM medium. Transwell chambers (Corning, Tewksbury, MA, USA) were placed into 24-well plates and 100 μL of the above dilution gel was aspirated and spread on the bottom of each chamber and placed in the incubator overnight. The following day, the remaining liquid in the chambers was aspirated. Cell suspension (200 μL, 1.5 × 105 cells/mL) was seed into the upper section, followed by the addition of the complete medium in the lower section. Following a 48- incubation, the cell suspension in the chambers was discarded, and then exposed to 4% paraformaldehyde (HY-Y0333, MedChemExpress) for 30 min. 0.1% Crystal violet (32,675, Sigma-Aldrich) staining was performed for 10 min. Followed by washing twice in PBS, gently scraping the inner wall cells with a cotton swab, and drying naturally in a ventilated area for 2 h. The field of view was randomly selected and captured using microscope (XK-DZ004, SINICO Optical Instrument Co., LTD, Shenzhen, China) to count the amount of invasive cells.

The Transwell migration test does not require Matrigel matrix gel, and the rest of the manipulation and analytical steps are consistent with the invasion test.

Autophagic flux measurement

To assess the impact of AS-IV on autophagic flux in NPC cells, this study used microtubule-associated protein light chain 3 (LC3) adenovirus (Ad-mRFP-GFP-LC3, Hanbio Biotechnology Co., Shanghai, China) co-labeled with cherry red fluorescent protein (mRFP) and green fluorescent protein (GFP) to transfect the cells.27,28 Cells were seed in 6-well plates (6 × 104/well) and cultured for 24 h. When the cell confluence reached more than 70%, added Ad-mRFP-GFP-LC3 into each well, and after transfection for 4 h, added fresh medium, and left to incubate for 24 h. Viral transfection was observed using a fluorescence microscope (DM IL LED, Leica, Heidelberg, Germany), and the number of autophagosomes and fusion of autophagosomes with lysosomes in the cells was counted by Image J software (version 1.54 h, Wavne Resband, National Institute of Mental Health, USA).

Flow cytometry

After various treatments, NPC cells were gathered, rinsed twice with PBS and gently mixed by adding 500 μL Binding Buffer. After that, Annexin-V-APC (5 μL, AP107, MULTI SCIENCE, Hangzhou, Zhejiang, China) and propidium iodide (5 μL, HY-D0815, MedChemExpress) were introduced and left to incubate for 15 min away from light. Transferred to a flow cytometer (BD FACSCaliburTM, BD biosciences, San Jose, CA, USA) for detection and analysed for cell apoptosis by FlowJo software (v10.8, BD biosciences).

Nude mice subcutaneous tumor model

Balb/c nude mice were procured from Vitalriver (Beijing, China), housed at a constant temperature of 22 °C, 55% to 60% humidity, with an age range of 6 ~ 8 wk. The facilities were disinfected regularly, and feeding and drinking were performed autonomously. After 1 wk of acclimatisation, mice were divided into five groups in a random manner (n = 3). The OE-NC group, Control+OE-NC group and AS-IV + OE-NC group of mice received subcutaneous inoculation with 0.2 mL of C666–1 cell suspension transfected with OE-NC (1 × 107 cells/mL); the OE-SATB2 group and AS-IV + OE-SATB2 group of mice were subcutaneously inoculated with 0.2 mL of C666–1 cell suspension transfected with OE-SATB2 (1 × 107 cells/mL). The AS-IV + OE-NC and AS-IV + OE-SATB2 group of mice were intraperitoneally injected with AS-IV (0.3 mL, 40 mg/kg) daily; the Control+OE-NC group of mice were given the same quantity of saline solution. The said dosing was carried out once a day, for a total of 15 days. Since the commencement of the first dosing, the subcutaneous tumors were measured with a caliper every 3 days. After 15 days of dosing, mice were executed through cervical dislocation, and the tumors were completely stripped with tissue shears, weighed and photographed to record the tumour mass.

Immunohistochemistry

Nude mice tumor tissues were exposed to 4% paraformaldehyde, routinely dehydrated, sectioned after paraffin embedding (thickness of 4 ~ 5 μm), deparaffinized with xylene (247,642, Sigma-Aldrich), and repaired antigen. The sections were incubated in 3% H2O2 solution for 25 min, rinsed twice with PBS, and evenly covered with drops of bovine serum albumin (BSA, 5%, V900933, Sigma-Aldrich) for 30 min. Ki67 primary antibody (ab15580, 1:100, Abcam Inc., Cambridge, UK) was added dropwise and incubated at 37 °C for 90 min. The tissues were covered with HRP-labeled goat anti-rabbit IgG (31,460, 1:10000, Invitrogen) for 20 min. DAB (DA1010, Solarbio, Beijing, China) developed the color and tap water terminated the color development. Mayer Hematoxylin (MHS16, Sigma-Aldrich) was re-stained, neutral gum (C0173, Beyotime, Shanghai, China) was used to seal, observed under an inverted fluorescence microscope.

Immunofluorescence

NPC cells after different treatments were inoculated in 12-well plates. When the density reached 50%–60%, the cells were fixed with 4% paraformaldehyde for 15 min. Paraffin sections of tumor tissues from nude mice were taken, deparaffinized with xylene and then subjected to antigenic repair. The surface of cells and tumor tissue sections were covered with 0.3% Triton X-100 solution (X100, Sigma-Aldrich) for 10 min for permeabilization and then blocked using a solution containing 5% BSA for 30 min. The cells and tumor tissue sections were placed at 4 °C for an overnight incubation with primary antibody anti-LC3B (ab48394, 1:1000, Abcam). On the following day, the cells were incubated at 37 °C in darkness for 1 hour in a solution of goat anti-rabbit IgG (F-2765, 1:100, Invitrogen). Finally, DAPI staining solution (C0060, Solarbio) was added to stain the cells in the dark for 10 min, observed by fluorescence microscopy, and the fluorescence intensity was analyzed by Image J software.

TUNEL staining

NPC cells after different treatments were exposed to 4% paraformaldehyde for 30 min. Added 0.3% Triton X-100 and incubated for 5 min. Paraffin sections of tumor tissues from nude mice were taken, deparaffinized with xylene and hydrated with gradient ethanol. The surface of tumor tissue sections were covered with a solution containing DNase-free proteinase K (20 μg/mL, ST532, Beyotime) for 30 min. TUNEL assay solution (C1086, Beyotime) was added dropwise to evenly cover the cells and tissue sections and incubated in darkness for 90 min. Subsequently, treated with DAPI staining solution and incubated in the dark for 10 min. After sealing with AntiFade mounting medium (HY-K1042, MedChemExpress), the cells were observed by microscope and photographed.

Western blot

Each group of cells or tissues received the treatment of RIPA lysis buffer (P0013B, Beyotime) to obtain total protein. The protein content was determined using the BCA assay kit (P0012, Beyotime). Next, sample proteins underwent electrophoresis on SDS-PAGE gels (12%, Invitrogen), then shifted to a PVDF membranes (Invitrogen), blocked with BSA for 3 h. After rinsing the membranes, placed at 4 °C for an overnight incubation with LC3 primary antibody (ab51520, 1:3000, Abcam), Beclin1 primary antibody (PA1–16857, 1:2000, Invitrogen), caspase-3 primary antibody (700,182, 1:1500, Invitrogen), Cleaved caspase-3 primary antibody (ab32042, 1:1000, Abcam), Bcl-2 primary antibody (ab59348, 1:500, Abcam), SATB2 primary antibody (ab34735, 1:100, Abcam), cytochrome C (cytc) primary antibody (PA5–87380, 1:1000, Invitrogen), Bax primary antibody (PA5–11378, 1:2000, Invitrogen), glycogen synthase kinase 3 β (GSK3β) primary antibody (MA5–15109, 1: 1000, Invitrogen), p-GSK3β primary antibody (44-604G, 1:1000, Invitrogen) and β-catenin primary antibody (71–2,700, 1:1000, Invitrogen). After three repetitions of membrane washing the next day, the membranes were cultured with secondary antibody goat anti-rabbit IgG (31,460, 1:10000, Invitrogen) for 2 h. The chemiluminescent agent ECL (HY-K1005, MedChemExpress) was evenly dripped onto the membrane and scanned with a gel imaging system (iBright CL1500, Invitrogen). Bands were analyzed in gray value using Image J software, and the relative expression was expressed as its ratio to GAPDH (PA1–987, 1:1000, Invitrogen).

Statistical processing

For each assay, a minimum of three repetitions were performed, with the data being displayed as mean ± standard deviation. For statistical analysis of data, we employed SPSS 26.0 software (IBM SPSS Statistics 26). The normality of the data distribution was checked by a post hoc test (Tukey’s test). If the normality test indicated that the data were normally distributed, a one-way ANOVA was used; if not, a nonparametric ANOVA was utilized. P < 0.05 representing a significant difference. Graphpad Prism 9.0 software was utilized for drawing graphs.

Results

AS-IV treatment inhibits NPC cell migration, invasion and autophagy

Through CCK-8 assays, the actions of AS-IV on cellular viability were investigated. AS-IV (50, 100, 200 and 400 μM) treatment did not significantly affect NP-69 cell viability, but when the concentration increased to 800 μM, NP-69 cell viability was significantly reduced, which indicates that high concentrations of AS-IV have certain cytotoxicity (Fig. 1A). AS-IV at 50 μM exerted no considerable influence on the viability of C666–1 and HK-1 cells, whereas AS-IV (100, 200 and 400 μM) markedly reduced the viability of NPC cells (Figs. 1B and C). In subsequent assays, 100, 200, and 400 μM of AS-IV were adopted. Transwell assays revealed that AS-IV alleviated the transference and penetration of C666–1 and HK-1 cells markedly, showing dose-dependent effects (Figs. 1D and E). LC3 is a hallmark of intracellular autophagosome activation during.29,30 Ad-mRFP-GFP-LC3 transfection results demonstrated that the amount of autophagosomes in C666–1 and HK-1 cells was markedly declined after AS-IV treatment (Figs. 1F–H). By immunofluorescence, a notable decrease in the fluorescence intensity of LC3 in NPC cells after AS-IV treatment could be observed, indicating that AS-IV inhibited autophagy (Fig. 1I). Western blot assay results similarly indicated that autophagy-related proteins LC3II/I and Beclin 1 levels were markedly diminished after AS-IV treatment (Figs. 1J–L). The results above indicated that AS-IV treatment inhibited the migration, invasion and autophagy of NPC cells.

Figure 1.

Figure 1

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AS-IV suppresses NPC cell proliferation, migration, invasion, and autophagy. (A-C) CCK-8 assay examined the impacts of various levels of AS-IV on the viability of different cells, and confirmed that AS-IV (100, 200 and 400 μM) was able to reduce the cell viability of C666–1 and HK-1 cells, but did not affect NP-69 cells. (D-E) Transwell assay confirmed that AS-IV reduced the migration and invasion cell numbers (20 ×, bar = 100 μm). (F-H) The impact of AS-IV on autophagy in NPC cells was assessed by transfection of ad-mRFP-GFP-LC3 (40 ×, bar = 50 μm). (I) Immunofluorescence measured that AS-IV reduced LC3B expression and inhibited cellular autophagy (40 ×, bar = 50 μm). (J-L) examining LC3II/I and Beclin 1 protein levels through western blot after AS-IV treatment. n = 3. (*P < 0.05, **P < 0.01, ***P < 0.001).

AS-IV treatment promotes NPC cell apoptosis

By TUNEL staining, we observed a notable rise in the number of TUNEL positivity cells after AS-IV treatment, suggesting that AS-IV promotes apoptosis in NPC cells (Fig. 2A). The interference of AS-IV with cell apoptosis was analyzed using flow cytometry, revealing a considerable elevation in the apoptosis rates of NPC cells post-treatment with AS-IV (Fig. 2B). In addition, apoptosis-associated proteins cytc, Bax and Cleaved-caspase 3/caspase 3 levels were markedly elevated, and apoptosis inhibition-associated protein Bcl-2 was declined by AS-IV treatment (Figs. 2C–G), which further indicated that AS-IV promoted apoptosis in NPC cells.

Figure 2.

Figure 2

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AS-IV treatment promotes NPC cell apoptosis. (A) TUNEL staining confirmed that AS-IV increased the number of TUNEL-positive cells (40 ×, bar = 50 μm). (B) AS-IV treatment increased apoptosis rates as assessed by flow cytometry. (C-G) western blot and quantitative analysis confirmed that AS-IV increased cytc, cleaved-caspase 3/caspase 3 and Bax levels, and declined Bcl-2 level. n = 3. (*P < 0.05, **P < 0.01, ***P < 0.001).

AS-IV down-regulates SATB2 expression to inhibit malignant progression of NPC cells

Next, we assessed the expression of SATB2 in NPC cell lines (C666–1, HK-1, HNE-3, and NPC43) and normal nasopharyngeal epithelial cells (NP-69). As visualized in western blots, NPC cells held notably higher SATB2 levels than NP-69 cells did; SATB2 levels were found to be highest in HK-1 cells, with C666–1 cells ranking second in expression (Fig. 3A). Consequently, HK-1 and C666–1 cells were adopted to do further assays. The trend in SATB2 gene expression post-treatment with AS-IV (400 μM) was ascertained by transfecting OE-SATB2 into HK-1 and C666–1 cells. Followed by, the SATB2 overexpression efficiency was tested through western blot assays. As an outcome, NPC cells exhibited greatly heightened SATB2 after OE-SATB2 transfection (Fig. 3B). According to CCK-8 assay findings, overexpressing SATB2 partially relieved the AS-IV-induced viability suppression in NPC cells, improved cell viability (Fig. 3C). Transwell assay findings indicated that SATB2 overexpression moderately alleviated the AS-IV-induced prevention of HK-1 and C666–1 cell transference and penetration (Figs. 3D and E). In addition, overexpression SATB2 partially attenuated the suppressive role of AS-IV on autophagy in NPC cells, increased autophagic flux, and elevated the fluorescence intensity of LC3B, LC3II/I and Beclin 1 levels (Figs. 3F–L). These findings demonstrated that AS-IV may inhibit NPC cell migration, invasion, and autophagy by decreasing SATB2 level.

Figure 3.

Figure 3

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AS-IV reduces SATB2 expression to inhibit NPC progression. (A) Western blot measured that SATB2 protein was elevated in NPC cell lines, with HK-1 cells showing the highest expression, followed by C666–1. (B) OE-NC and OE-SATB2 were transfected in NPC cells, and SATB2 level was elevated after transfection with OE-SATB2. (C) CCK-8 assay measured that transfection of OE-SATB2 declined the inhibitory impact of AS-IV treatment on cell viability. (D-E) Transwell measured that transfection of OE-SATB2 increased the migration and invasion cell numbers (20 ×, bar = 100 μm). (F-H) ad-mRFP-GFP-LC3 transfection results confirmed that overexpression SATB2 reduced the suppressive impact of AS-IV treatment on cellular autophagy (40 ×, bar = 50 μm). (I) Overexpression SATB2 reduced the impact of AS-IV and increased LC3B expression as measured by immunofluorescence (40 ×, bar = 50 μm). (J–L) examining LC3II/I and Beclin 1 protein levels through western blot after overexpression SATB2. n = 3. (***P < 0.001vs control+OE-NC; ##P < 0.01, ###P < 0.001vs AS-IV + OE-NC).

AS-IV reduces SATB2 gene expression to promote apoptosis in NPC cells

The application of AS-IV was found to enhance apoptosis of NPC cells and increased TUNEL positive numbers, as determined by flow cytometry and TUNEL staining, though this effect was attenuated significantly following OE-SATB2 transfection (Figs. 4A and B). Furthermore, AS-IV caused a notable rise in cytc, Bax and Cleaved-caspase 3/caspase 3 levels and a notable decline in Bcl-2. In contrast, overexpression of SATB2 attenuated this phenomenon by decreasing cytc, Bax and Cleaved-caspase 3/caspase 3 levels and increasing Bcl-2 expression (Figs. 4C-G), suggesting that AS-IV may promote NPC apoptosis by decreasing SATB2 expression.

Figure 4.

Figure 4

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AS-IV reduces SATB2 expression to promote apoptosis in NPC cells. (A) TUNEL staining confirmed that overexpression SATB2 reduced TUNEL-positive cell numbers and inhibited apoptosis (40 × , bar = 50 μm). (B) Overexpression SATB2 attenuated the impact of AS-IV treatment and declined the apoptosis rates as assessed by flow cytometry. (C-G) western blot and quantitative analysis confirmed that overexpression SATB2 reduced the impacts of AS-IV, downregulated cytc, cleaved-caspase 3/caspase 3 and Bax, and increased Bcl-2 level. n = 3. (***P < 0.001vs control+OE-NC; #P < 0.05, ##P < 0.01, ###P < 0.001vs AS-IV + OE-NC).

AS-IV blocks the Wnt pathway by down-regulating SATB2

Further, the Wnt pathway inhibitor DKK-1 was administered to intervene in NPC cells with SATB2 overexpressed post-treatment with AS-IV to ascertain if AS-IV’s targeted action on SATB2 and further blocking of NPC worsening is mediated by impairing the Wnt pathway. Subsequently, the levels of Wnt pathway proteins p-GSK3β/GSK3β and β-catenin were tested. In consequence, the western blots revealed that after AS-IV treatment, C666–1 cells witnessed considerable attenuation in p-GSK3β/GSK3β and β-catenin production; the production of these two proteins was boosted somewhat by SATB2 overexpression, which trend however was overturned partially with the addition of DKK-1 (Figs. 5A–C). The said variation trends were also detected in HK-1 cells (Figs. 5D–F). This suggested that AS-IV could block the Wnt pathway, whereas overexpression SATB2 activated the Wnt pathway, and AS-IV may inactivate the Wnt pathway by down-regulating SATB2.

Figure 5.

Figure 5

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AS-IV blocks the Wnt pathway by down-regulating SATB2. (A) Protein bands related to the Wnt pathway detected in C666–1 cells. (B-C) quantitative analysis confirmed that AS-IV treatment reduced p-GSK3β/GSK3β and β-catenin levels in C666–1 cells, and these proteins was elevated after transfection with OE-SATB2, whereas the effect of overexpression SATB2 was attenuated by the Wnt pathway inhibitor, DKK1. (D) Protein bands related to the Wnt pathway detected in HK-1 cells. (E-F) Ecamining p-GSK3β/GSK3β and β-catenin levels in HK-1 cells through western blot. n = 3. (***P < 0.001vs control+OE-NC; ###P < 0.001vs AS-IV + OE-NC; &&P < 0.01, &&&P < 0.001vs AS-IV + OE-SATB2).

AS-IV inhibits the Wnt pathway by decreasing SATB2 level to suppress NPC progression

Per CCK-8 assay outcomes, AS-IV treatment substantially diminished the viability of NPC cells; overexpression SATB2 heightened this cell viability, which trend was reversed partially after injecting DKK-1 (Fig. 6A). AS-IV treatment deterred the transference and penetration abilities of NPC cells; following SATB2 overexpression, the said abilities were enhanced, which trend however was countered with the DKK-1 administration (Figs. 6B and C). Besides, overexpression SATB2 attenuated the effect of AS-IV, leading to a marked increase in autophagic flux in NPC cells (Figs. 6D–F), LC3B fluorescence intensity and LC3II/I and Beclin 1 protein levels were elevated, which was reversed by adding DKK-1 (Figs. 6G–J). The mentioned findings demonstrated that AS-IV hindered the Wnt pathway and further suppresses the migration, invasion, and autophagy of NPC cells by downregulating SATB2.

Figure 6.

Figure 6

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AS-IV hinders the Wnt pathway by decreasing SATB2 level to suppress NPC progression. (A) CCK-8 assay indicated that DKK1 reduced the effect of transfection with OE-SATB2 on cell viability. (B and C) Transwell measured that DKK1 attenuated the effect of overexpression of SATB2 and reduced the number of migrating and invading cells (20 ×, bar = 100 μm). (D–F) ad-mRFP-GFP-LC3 transfection results confirmed that DKK1 reduced the promoting effect of overexpressed SATB2 on cellular autophagy (40 ×, bar = 50 μm). (G) Immunofluorescence measured that DKK1 reduced the effect of overexpression SATB2 and could decline LC3B expression (40 ×, bar = 50 μm). (H-J) examining LC3II/I and Beclin 1 protein levels through western blot after DKK1 treatment. n = 3. (***P < 0.001vs control+OE-NC; ##P < 0.01, ###P < 0.001vs AS-IV + OE-NC; &P < 0.05, &&P < 0.01, &&&P < 0.001vs AS-IV + OE-SATB2).

AS-IV promotes apoptosis in NPC cells by hindering the Wnt pathway through decreasing SATB2

Post-administration of AS-IV, there was a notable increase in TUNEL positive numbers and apoptosis rates of NPC cells, overexpression SATB2 weakened the treatment effect of AS-IV, which was reversed by adding DKK-1, which increased TUNEL positive numbers and apoptosis rates (Figs. 7A and B). Not only that, overexpression SATB2 caused a marked decrline in cytc, Cleaved-caspase 3/caspase 3 and Bax levels, and an up-regulation of Bcl-2, whereas DKK-1 attenuated the effect of overexpression SATB2 (Figs. 7C–G). The above outcomes further emphasized that AS-IV inactivates the Wnt pathway and further accelerates apoptosis of NPC cells by dampening the SATB2 production.

Figure 7.

Figure 7

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AS-IV promotes apoptosis in NPC cells via hindering the Wnt pathway through decreasing SATB2. (A) TUNEL staining confirmed that DKK1 reduced the effect of overexpression SATB2 and elevated TUNEL-positive cell numbers (40 ×, bar = 50 μm). (B) DKK1 increased apoptosis rates as assessed by flow cytometry. (C-G) western blot and quantitative analysis confirmed that DKK1 increased cytc, cleaved-caspase 3/caspase 3 and Bax levels, while declined Bcl-2. n = 3. (***P < 0.001 vs control+OE-NC; ###P < 0.001 vs AS-IV + OE-NC; &&P < 0.01, &&&P < 0.001 vs AS-IV + OE-SATB2).

AS-IV inhibits Wnt pathway activation by down-regulating SATB2, thereby inhibits NPC tumor growth

Injection of C666–1 cells transfected with OE-SATB2 caused a notable increase in SATB2 level in tumors tissues, which indicated that injection of C666–1 cells transfected with OE-SATB2 effectively regulated SATB2 level in nude mice in vivo and allowed for subsequent experiments (Fig. 8A). The injection of AS-IV caused a notable decline in SATB2, p-GSK3β/GSK3β and β-catenin levels in the tumor tissues, suggesting that AS-IV can down-regulate SATB2 expression and block the Wnt pathway. In contrast, SATB2, p-GSK3β/GSK3β and β-catenin levels were markedly increased in AS-IV + OE-SATB2 group, suggesting that overexpression SATB2 activated the Wnt pathway, further confirming that AS-IV blocked the Wnt pathway by down-regulating SATB2 (Figs. 8B-E). Mice that received AS-IV exhibited a considerable reduction in tumor volume and mass, while overexpression SATB2 partially reversed the tumor-suppressing effects of AS-IV (Figs. 8F–H). Not only that, injection of AS-IV declined Ki-67 expression and increased the amount of TUNEL-positive cells, whereas overexpression SATB2 weakened the impact of AS-IV (Figs. 8I and J). In addition, LC3B fluorescence intensity and LC3II/I and Beclin 1 levels were notaby reduced in tumor tissues after injection of AS-IV, suggesting that AS-IV could effectively inhibit autophagy, and overexpression SATB2 partially reversed the suppressive impact of AS-IV on autophagy (Figs. 8K–N). The above results confirmed that AS-IV down-regulated SATB2 in tumor tissues and hindered the Wnt pathway, which in turn effectively inhibited the growth of NPC tumors in nude mice.

Figure 8.

Figure 8

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AS-IV inhibits Wnt pathway activation by down-regulating SATB2, thereby inhibits NPC tumor growth. (A) C666–1 cells transfected with OE-NC or OE-SATB2 were introduced into nude mice through subcutaneous injection, and SATB2 protein was up-regulated in tumor tissues after transfection with OE-SATB2. (B–E) western blot measured that injection of AS-IV decreased SATB2 and Wnt pathway-related protein levels in tumor tissues, while transfection of OE-SATB2 increased these proteins. (F) The size of the subcutaneous tumors was gauged using a Vernier caliper on days 3, 6, 9, 12, and 15. On d 15, mice were anesthetized and executed, followed by the removal and imaging of tumors (G), and tumor weights were recorded (H). (I) Immunohistochemistry measured a decline in Ki-67 levels after AS-IV injection, and Ki-67 was upregulated after transfection with OE-SATB2 (20 ×, bar = 100 μm). (J) TUNEL staining confirmed an increase in TUNEL-positive cell numbers after injection of AS-IV and a decrease after transfection with OE-SATB2 (40 ×, bar = 50 μm). (K) Immunofluorescence measured a decline in LC3B fluorescence level in tumor tissues after injection of AS-IV and an rise in LC3B fluorescence intensity after transfection with OE-SATB2 (40 ×, bar = 50 μm). (L-N) examining LC3II/I and Beclin 1 levels in tumor tissues after injection of AS-IV and transfection of OE-SATB2 through western blot. n = 3. (***P < 0.001 vs control+OE-NC; ###P < 0.001 vs AS-IV + OE-NC).

Discussion

AS-IV presents as a major active ingredient in Huang Qi. As demonstrated in the literature,31 AS-IV is able to curtail the expansion and metastasis of gastric cancer cells and hinder tumor growth in mice. Research by Cheng et al. unraveled that AS-IV prevents lung cancer cells from expanding, transferring, penetrating, and epithelial-mesenchymal transition through the PKC-α-ERK1/2-NF-κB pathway.32 In our study, AS-IV held up the viability of NPC cells but did not mediate the viability of normal nasopharyngeal epithelial cells. Cleaved-caspase 3/caspase 3, cytc and Bax are hallmark proteins of apoptosis, whereas Bcl-2 is an apoptosis inhibitor protein.33 AS-IV treatment declined the migration and invasive ability of NPC cells, increased Cleaved-caspase 3/caspase 3, cytc, and Bax levels, while down-regulated Bcl-2, and these effects depended on the dosages of AS-IV. Moreover, the tumor transplantation and formation experiment in nude mice also validated that the expansion of NPC cells can be impeded notably by the administration of AS-IV, suggesting that AS-IV could serve as a promising medication for NPC treatment, warranting additional research in the future.

Autophagy is a self-digestion process, encapsulating improperly folded or accumulated proteins, damaged or aged organelles like mitochondria and the endoplasmic reticulum, as well as pathogens within cells, into autophagosomes, followed by merging with lysosomes, resulting in the breakdown of the encapsulated materials.34,35 Accumulating evidence confirms that autophagy performs dual roles in cancer: one is to impede tumor expansion at the early stage of cancer; the other one is to speed up tumor aggression and metastasis by supplying energy for angiogenesis.36,37 LC3A, LC3B, and LC3C are three variants of a major autophagy-related gene LC3 in mammals.38 LC3B within autophagosomes is degraded as the encapsulated materials are broken down once the autophagosomes merge with lysosomes. For this reason, LC3B is regarded as a marker for autophagy.39,40 In this study, AS-IV treatment declined LC3B expression of in NPC cells and tissues and down-regulated autophagy-related proteins LC3II/I, Beclin 1, confirming that AS-IV can inhibit autophagy. Similarly, Liu et al. indicated that AS-IV reduced LC3 and Beclin 1 levels and inhibited cellular autophagy in non-small cell lung cancer cells.41

The production of SATB2 is augmented in NPC, making SATB2 a likely marker for identifying NPC, as evidenced in the literature.20 From the western blots of SATB2 in this survey, we discovered that SATB2 is generated much higher in NPC cells than in normal cells, which aligns with the previous research findings of others. Post-transfection of OE-SATB2 into NPC cells, we delved into the intervention of overexpressed SATB2 in such NPC aggravation as refrained by AS-IV. Consequently, SATB2, when generated over much, somewhat attenuated AS-IV’s impeding action on the transference, penetration, and autophagy of NPC cells and partially alleviated the NPC cell apoptosis caused by AS-IV treatment. This trend evidenced that AS-IV is likely to prevent NPC from aggravating by dampening the SATB2 production.

The emergence and growth of tumors involve complex interactions within multiple signaling pathways.42 Within the Wnt signaling pathway, there are proteins vital for both embryonic development and adult tissue equilibrium. Many diseases including cancers are often caused by detuning of the Wnt signal.21,43 Transcription factor β-catenin is a key part of the Wnt pathway. The phosphorylation of β-catenin is mediated by GSK3β and casein kinase 1α to promote the β-catenin’s ubiquitination and subsequent degradation via proteasomes.44 Research by Jiang et al. uncovered that AS-IV prevents hepatocellular carcinoma cells from expanding, transferring, and penetrating and remarkably dampened the Wnt and β-catenin expressions both in laboratory settings and live models. This demonstrated that AS-IV can mediate the Wnt/β-catenin pathway to block the aggravation of liver cancer.45 Furthermore, He et al. surveyed and revealed that silencing miR-25 can deactivate the Wnt/β-catenin pathway, further hold up NPC cell expansion, and accelerate NPC cell apoptosis.46 Through the present work, it was unraveled as follows: following AS-IV treatment, there was a marked fall in the contents of p-GSK3β/GSK3β and β-catenin proteins in NPC cells; after SATB2 overexpression, these two proteins were expressed to higher levels; however, this heightening in these protein levels was countered post-treatment with a Wnt/β-catenin pathway inhibitor. These variations proved that AS-IV can disable the Wnt pathway by mediating SATB2 directly, which further aggravates the NPC.

Conclusion

To conclude, AS-IV can disable the Wnt pathway by dampening SATB2 production, which in turn diminishes NPC cell vitality, curtails cell transference, penetration, autophagy, and fosters apoptosis. What’s more, AS-IV has shown efficacy in curbing NPC growth in mice, suggesting its therapeutic potential for this tumor. This work only uncovered initial insights into the anti-cancer mechanisms of AS-IV. Further investigation should be made to unravel whether AS-IV is toxic to the heart, liver, and kidney and whether it is likely to pose multidrug resistance. Overall, AS-IV is a highly promising anti-cancer agent, deserving of further research in the future.

Consent to publish

The manuscript has neither been previously published nor is under consideration by any other journal. The authors have all approved the content of the paper.

Acknowledgments

None.

Contributor Information

Yinping Zeng, Department of Otorhinolaryngology Head and Neck Surgery, The First Affiliated Hospital of Hainan Medical University, 31 Longhua Road, Longhua District, Haikou 570102, Hainan Province, China.

Tingting Duan, Department of Otorhinolaryngology Head and Neck Surgery, The First Affiliated Hospital of Hainan Medical University, 31 Longhua Road, Longhua District, Haikou 570102, Hainan Province, China.

Jiajun Huang, Department of Otorhinolaryngology Head and Neck Surgery, The First Affiliated Hospital of Hainan Medical University, 31 Longhua Road, Longhua District, Haikou 570102, Hainan Province, China.

Xiaofeng Wang, Department of Otorhinolaryngology Head and Neck Surgery, The First Affiliated Hospital of Hainan Medical University, 31 Longhua Road, Longhua District, Haikou 570102, Hainan Province, China.

Author contribution

[Yinping Zeng]: Developed and planned the study, performed experiments, and interpreted results. Edited and refined the manuscript with a focus on critical intellectual contributions.

[Tingting Duan, Jiajun Huang]: Participated in collecting, assessing, and interpreting the date. Made significant contributions to date interpretation and manuscript preparation.

[Xiaofeng Wang]: Provided substantial intellectual input during the drafting and revision of the manuscript.

Funding

  1. Effect of SMG-1 on Radiosensitivity of Head and Neck Squamous Cell Carcinoma and its Signaling Pathway and Regulation of Immune Microenvironment of Head (No. 820MS141)

  2. Neck Squamous Cell Carcinoma by SERS Probes Via RIG-NFkB Axis Reprogramming TAM. (No. 824QN381)

Conflicts of interest

The authors declare that they have no financial conflicts of interest.

Ethic approval

This experiment was approved by The First Affiliated Hospital of Hainan Medical University Ethics Committee.

References

  • 1. Chen  YP  et al.  Nasopharyngeal carcinoma. Lancet. 2019:394:64–80. 10.1016/s0140-6736(19)30956-0 [DOI] [PubMed] [Google Scholar]
  • 2. Tang  LL  et al.  The Chinese Society of Clinical Oncology (CSCO) clinical guidelines for the diagnosis and treatment of nasopharyngeal carcinoma. Cancer commun (London, England). 2021:41:1195–1227. 10.1002/cac2.12218 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3. Juarez-Vignon Whaley  JJ  et al.  Recurrent/metastatic nasopharyngeal carcinoma treatment from present to future: where are we and where are we heading?  Curr Treat Options in Oncol. 2023:24:1138–1166. 10.1007/s11864-023-01101-3 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. Toumi  N, Ennouri  S, Charfeddine  I, Daoud  J, Khanfir  A. Prognostic factors in metastatic nasopharyngeal carcinoma. Braz J Otorhinol. 2022:88:212–219. 10.1016/j.bjorl.2020.05.022 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5. Huang  H  et al.  Immunotherapy for nasopharyngeal carcinoma: current status and prospects (review). Int J Oncol. 2023:63:97. 10.3892/ijo.2023.5545 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6. Guan  S, Wei  J, Huang  L, Wu  L. Chemotherapy and chemo-resistance in nasopharyngeal carcinoma. Eur J Med Chem. 2020:207:112758. 10.1016/j.ejmech.2020.112758 [DOI] [PubMed] [Google Scholar]
  • 7. Abd Elrahim Abd Elkader  HT, Essawy  AE, Al-Shami  AS. Astragalus species: Phytochemistry, biological actions and molecular mechanisms underlying their potential neuroprotective effects on neurological diseases. Phytochemistry. 2022:202:113293. 10.1016/j.phytochem.2022.113293 [DOI] [PubMed] [Google Scholar]
  • 8. Li  W  et al.  Characterization and anti-tumor bioactivity of astragalus polysaccharides by immunomodulation. Int J Biol Macromol. 2020:145:985–997. 10.1016/j.ijbiomac.2019.09.189 [DOI] [PubMed] [Google Scholar]
  • 9. Dong  M, Li  J, Yang  D, Li  M, Wei  J. Biosynthesis and pharmacological activities of flavonoids, triterpene Saponins and polysaccharides derived from Astragalus membranaceus. Molecules. 2023:28:5018. 10.3390/molecules28135018 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10. Zhang  J, Wu  C, Gao  L, Du  G, Qin  X. Astragaloside IV derived from Astragalus membranaceus: a research review on the pharmacological effects. Adv Pharmacol. 2020:87:89–112. 10.1016/bs.apha.2019.08.002 [DOI] [PubMed] [Google Scholar]
  • 11. Gong  F, Qu  R, Li  Y, Lv  Y, Dai  J. Astragalus Mongholicus: a review of its anti-fibrosis properties. Front Pharmacol. 2022:13:976561. 10.3389/fphar.2022.976561 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12. Zheng  Y  et al.  Astragaloside IV enhances taxol chemosensitivity of breast cancer via caveolin-1-targeting oxidant damage. J Cell Physiol. 2019:234:4277–4290. 10.1002/jcp.27196 [DOI] [PubMed] [Google Scholar]
  • 13. Zhang  L, Zhou  J, Qin  X, Huang  H, Nie  C. Astragaloside IV inhibits the invasion and metastasis of SiHa cervical cancer cells via the TGF-β1-mediated PI3K and MAPK pathways. Oncol Rep. 2019:41:2975–2986. 10.3892/or.2019.7062 [DOI] [PubMed] [Google Scholar]
  • 14. Jia  L, Lv  D, Zhang  S, Wang  Z, Zhou  B. Astragaloside IV inhibits the progression of non-small cell lung cancer through the Akt/GSK-3β/β-catenin pathway. Oncol Res. 2019:27:503–508. 10.3727/096504018x15344989701565 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15. You  X  et al.  Astragalus-scorpion drug pair inhibits the development of prostate cancer by regulating GDPD4-2/PI3K/AKT/mTOR pathway and autophagy. Front Pharmacol. 2022:13:895696. 10.3389/fphar.2022.895696 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16. Huang  X  et al.  SATB2: a versatile transcriptional regulator of craniofacial and skeleton development, neurogenesis and tumorigenesis, and its applications in regenerative medicine. Genes & diseases. 2022:9:95–107. 10.1016/j.gendis.2020.10.003 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. Gao  J  et al.  SATB2 overexpression promotes oral squamous cell carcinoma progression by up-regulating NOX4. Cell Signal. 2021:82:109968. 10.1016/j.cellsig.2021.109968 [DOI] [PubMed] [Google Scholar]
  • 18. Jin  J  et al.  YAP-activated SATB2 is a coactivator of NRF2 that amplifies Antioxidative capacity and promotes tumor progression in renal cell carcinoma. Cancer Res. 2023:83:786–803. 10.1158/0008-5472.Can-22-1693 [DOI] [PubMed] [Google Scholar]
  • 19. Fazio  M  et al.  SATB2 induction of a neural crest mesenchyme-like program drives melanoma invasion and drug resistance. elife. 2021:10:10. 10.7554/eLife.64370 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20. Uccella  S  et al.  Transcription factor expression in Sinonasal neuroendocrine neoplasms and olfactory Neuroblastoma (ONB): Hyams' grades 1-3 ONBs expand the Spectrum of SATB2 and GATA3-positive neoplasms. Endocr Pathol. 2022:33:264–273. 10.1007/s12022-022-09715-3 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21. Liu  J  et al.  Wnt/β-catenin signalling: function, biological mechanisms, and therapeutic opportunities. Signal Transduction Targeted Ther. 2022:7:3. 10.1038/s41392-021-00762-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22. Zhang  J  et al.  Melatonin reverses nasopharyngeal carcinoma cisplatin chemoresistance by inhibiting the Wnt/β-catenin signaling pathway. Aging (Albany N Y). 2020:12:5423–5438. 10.18632/aging.102968 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23. Chen  B  et al.  N(7)-methylguanosine tRNA modification promotes tumorigenesis and chemoresistance through WNT/β-catenin pathway in nasopharyngeal carcinoma. Oncogene. 2022:41:2239–2253. 10.1038/s41388-022-02250-9 [DOI] [PubMed] [Google Scholar]
  • 24. Li  Z, Cai  X. Baicalein targets STMN1 to inhibit the progression of nasopharyngeal carcinoma via regulating the Wnt/β-catenin pathway. Environ Toxicol. 2024:39:3003–3013. 10.1002/tox.24173 [DOI] [PubMed] [Google Scholar]
  • 25. Xin  T  et al.  A novel mutation of SATB2 inhibits odontogenesis of human dental pulp stem cells through Wnt/β-catenin signaling pathway. Stem Cell Res Ther. 2021:12:595. 10.1186/s13287-021-02660-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26. Ouyang  X, Li  S, Ding  Y, Xin  F, Liu  M. Mechanism of miRNA-31 regulating Wnt/β-catenin Signaling pathway by targeting Satb2 in the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells. J Musculoskelet Neuronal Interact. 2023:23:346–354. [PMC free article] [PubMed] [Google Scholar]
  • 27. Wen  J  et al.  LTF induces Radioresistance by promoting autophagy and forms an AMPK/SP2/NEAT1/miR-214-5p feedback loop in lung squamous cell carcinoma. Int J Biol Sci. 2023:19:1509–1527. 10.7150/ijbs.78669 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28. Li  K  et al.  Targeting STK26 and ATG4B: miR-22-3p as a modulator of autophagy and tumor progression in HCC. Transl Oncol. 2025:51:102214. 10.1016/j.tranon.2024.102214 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29. Runwal  G  et al.  LC3-positive structures are prominent in autophagy-deficient cells. Sci Rep. 2019:9:10147. 10.1038/s41598-019-46657-z [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30. Zhou  B  et al.  Ferroptosis is a type of autophagy-dependent cell death. Semin Cancer Biol. 2020:66:89–100. 10.1016/j.semcancer.2019.03.002 [DOI] [PubMed] [Google Scholar]
  • 31. Li  F, Cao  K, Wang  M, Liu  Y, Zhang  Y. Astragaloside IV exhibits anti-tumor function in gastric cancer via targeting circRNA dihydrolipoamide S-succinyltransferase (circDLST)/miR-489-3p/ eukaryotic translation initiation factor 4A1(EIF4A1) pathway. Bioengineered. 2022:13:10112–10123. 10.1080/21655979.2022.2063664 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32. Cheng  X  et al.  Astragaloside IV inhibits migration and invasion in human lung cancer A549 cells via regulating PKC-α-ERK1/2-NF-κB pathway. Int Immunopharmacol. 2014:23:304–313. 10.1016/j.intimp.2014.08.027 [DOI] [PubMed] [Google Scholar]
  • 33. Ashrafizadeh  M. Cell death mechanisms in human cancers: molecular pathways, therapy resistance and therapeutic perspective. J Cancer Biomol Ther. 2024:1:17–40. 10.62382/jcbt.v1i1.13 [DOI] [Google Scholar]
  • 34. Glick  D, Barth  S, Macleod  KF. Autophagy: cellular and molecular mechanisms. J Pathol. 2010:221:3–12. 10.1002/path.2697 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35. Mizushima  N, Komatsu  M. Autophagy: renovation of cells and tissues. Cell. 2011:147:728–741. 10.1016/j.cell.2011.10.026 [DOI] [PubMed] [Google Scholar]
  • 36. Onorati  AV, Dyczynski  M, Ojha  R, Amaravadi  RK. Targeting autophagy in cancer. Cancer. 2018:124:3307–3318. 10.1002/cncr.31335 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37. Li  YJ  et al.  Autophagy and multidrug resistance in cancer. Chin J Cancer. 2017:36:52. 10.1186/s40880-017-0219-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38. Schaaf  MB, Keulers  TG, Vooijs  MA, Rouschop  KM. LC3/GABARAP family proteins: autophagy-(un)related functions. FASEB J. 2016:30:3961–3978. 10.1096/fj.201600698R [DOI] [PubMed] [Google Scholar]
  • 39. Hwang  HJ, Kim  YK. The role of LC3B in autophagy as an RNA-binding protein. Autophagy. 2023:19:1028–1030. 10.1080/15548627.2022.2111083 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40. Hwang  HJ  et al.  LC3B is an RNA-binding protein to trigger rapid mRNA degradation during autophagy. Nat Commun. 2022:13:1436. 10.1038/s41467-022-29139-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41. Liu  J  et al.  AS-IV enhances the antitumor effects of propofol in NSCLC cells by inhibiting autophagy. Open med (Warsaw, Poland). 2023:18:20230799. 10.1515/med-2023-0799 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42. Park  JH, Pyun  WY, Park  HW. Cancer metabolism: phenotype, Signaling and therapeutic targets. Cells. 2020:9:e12480. 10.3390/cells9102308 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43. Yu  F  et al.  Wnt/β-catenin signaling in cancers and targeted therapies. Signal Transduction Targeted Ther. 2021:6:307. 10.1038/s41392-021-00701-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44. Zhang  Y, Wang  X. Targeting the Wnt/β-catenin signaling pathway in cancer. J Hematol Oncol. 2020:13:165. 10.1186/s13045-020-00990-3 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45. Jiang  Z, Mao  Z. Astragaloside IV (AS-IV) alleviates the malignant biological behavior of hepatocellular carcinoma via Wnt/β-catenin signaling pathway. RSC Adv. 2019:9:35473–35482. 10.1039/c9ra05933d [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  • 46. He  H, Yuan  K, Chen  W. Effect of miR-25 on proliferation of nasopharyngeal carcinoma cells through Wnt/β-catenin Signaling pathway. Biomed Res Int. 2021:2021:9957161. 10.1155/2021/9957161 [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]

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