RETRACTED ARTICLE: Flavokawain C suppresses nephroblastoma growth by inducing autophagy-mediated downregulation of FABP4 via AMPK/mTOR pathway

Abstract

Nephroblastoma is the most common pediatric kidney cancer. Flavokawain C (FKC) is a naturally occurring chalcone which has been reported to inhibit the growth of several cancers. However, whether FKC has therapeutic potential for nephroblastoma remains unclear. This study aimed to investigate the effect of FKC on nephroblastoma growth and reveal its possible mechanism. The viability, clone formation and proliferation of G401 cells under FKC treatment were evaluated by CCK-8, clone formation and EdU assays. The migration and invasion of G401 cells under FKC treatment were evaluated by transwell and wound healing assays. The effects of FKC on epithelial‐mesenchymal transition (EMT) markers, autophagy-related proteins, FABP4 expression and AMPK/mTOR pathway were evaluated by western blot. The bioinformatic tools were used to evaluate the expression of in tumor tissues. Furthermore, the interaction between FKC and FABP4 in autophagy was analyzed by Co-IP assay. The in vivo assay was conducted to observe the effect of FKC on tumor growth. FKC was found to inhibit the viability, clone formation and proliferation of G401 cells. Then, FKC was found to exert an inhibitory effect on EMT, migration and invasion of G401 cells in vitro. Furthermore, FABP4 was overexpressed in nephroblastoma tissues, which was closely related to the prognosis of nephroblastoma patients. And FKC was found to suppress FABP4 expression by which affect the proliferation, migration and invasion of G401 cells. Moreover, AMPK/mTOR pathway was involved in the autophagy formation induced by FKC and mediated the degradation of FABP4. The in vivo study further confirmed that FKC inhibited the growth of nephroblastoma. This study demonstrates that FKC inhibits the proliferation, migration, and invasion of nephroblastoma cells by inducing AMPK pathway-mediated autophagy, leading to the degradation of FABP4.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-42414-1.

Introduction

Nephroblastoma or Wilms tumor, is the most common pediatric kidney cancer, accounting for 90% of renal tumors in children¹. The treatments for nephroblastoma include radical resection, radiotherapy, and chemotherapy, which have improved the 5-year survival rate of nephroblastoma to over 90%². Although the prognosis of most patients is well, there is still 10% of patients with nephroblastoma having poor survival due to cancer recurrence and metastasis³. Thus, there exists a pressing need for novel therapeutic interventions in the management of nephroblastoma.

Chalcones are precursors of flavonoids, which can be found in a variety of fruits, vegetables, and medicinal plants⁴. They have shown several important biological properties including anti-cancer activities against several cancer cell lines^4,5. Flavokawain C (FKC) is a naturally occurring chalcone that can be found in Kava root. Previously, FKC has been reported to inhibit the growth of live cancer, nasopharyngeal carcinoma and colon carcinoma via inducing cellular apoptosis, triggering intense DNA damage, inhibiting angiogenesis, and inducing cell cycle arrest^6–9. However, the potential of FKC as an effective drug for nephroblastoma treatment, along with its associated mechanisms, remains uncertain.

Previous studies have shown that FKC possessed the ability to regulate mammalian target of rapamycin (mTOR)⁹, an important protein involved in autophagy activation. Autophagy is an important homeostatic pathway that facilitates the degradation and recycling of cellular material¹⁰. Abnormalities of autophagy often lead to human diseases, such as neurodegeneration and cancer¹¹. In cancers, dysregulated autophagy has been reported to promote cancer progression^10,12. Targeting autophagy has been suggested as a promising approach for advanced cancers¹³. Multiple signal transduction pathways have been reported to be involved in the autophagy regulation in cancers¹⁴. Among them, several pathways converge at mTOR, including adenosine-monophosphate activated-protein kinase (AMPK)/mTOR signaling. Targeting AMPK/mTOR signaling is a promising therapeutic strategy for autophagy-related diseases¹⁵. Previously, chalcones, such as flavokawain B, have been demonstrated to participate in AMPK activation¹⁶. These studies suggested that FKC might exhibit its anti-tumor effects through autophagy activation.

In cancers, autophagy has been reported to fight cancer cells through degrading oncoproteins¹⁷. Fatty acid-binding protein 4 (FABP4) is highly expressed in adipocytes, endothelial cells and immune cells, which plays an important role in the pathogenesis of a series of metabolic pathologies¹⁸. Recent studies reported that FABP4 in tumor microenvironment was involved in the progression of different types of cancers^19–21. Thus, FABP4 is regarded as a new driver of primary tumors. Previously, the relationship between AMPK and FABP4 has been established^22,23. It has been reported that the activation of AMPK pathway negatively affected FABP4 protein secretion²². Due to the relationship among FABP4, autophagy activation and AMPK pathway, we hypothesized that FKC could inhibit the growth, migration, and invasion of nephroblastoma by regulating FABP4 and autophagy. To validate this hypothesis, we conducted this study, with the aim to investigate the effect of FKC on nephroblastoma growth and reveal its possible mechanism.

Methods

Cell culture

G401 cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, USA) containing DMEM, which were kept in a 37 °C incubator with 5% CO₂ and passaged every 3–4 days.

Treatment

FKC (Cat. HY-N2445, 99.79% purity) was purchased from MedChemExpress (MCE, New Jersey, USA), which was dissolved in dimethylsulfoxide (DMSO) at a concentration of 1 mM as the primary stock and further diluted in medium to different concentration levels (1, 5, 10, and 15 μM) for use. Chloroquine (CQ) is the inhibitor of autophagy. For the combined treatment of FKC and CQ (20 μM, Cat. HY-17589A, MCE), G401 cells were pre-treated with CQ for 6 h, followed by stimulation with FKC. BAY-3827 is a selective AMPK inhibitor²⁴. In pathway inhibition test, cells were pretreated with BAY-3827 (20 nM, Cat. HY-112083, MCE) for 1 h and then incubated with FKC.

Cell infection

For FABP4 overexpression and Atg5 knock-down, the FABP4 overexpressed (oe FABP4) and empty (oeNC) lentiviruses, and the specific Atg5 shRNA lentiviruses and scrambled control (sh-NC) were designed and synthesized by GenePharma Pharmaceutical Technology Co., Ltd (Shanghai, China). G401 cells infected with lentivirus at a multiplicity of infection (MOI) of 25 for 3 days were treated with different treatment for another 24 h.

Cell counting kit (CCK)-8 assay

The cell viability was detected by CCK-8 assay. G401 cells or infected cells were treated with different concentrations of FKC (0, 1, 5, 10, and 15 μM) and were added into 96-well plates (1000 per well). After incubation for 0, 24, 48 and 72 h, 10 μL of CCK-8 solution (Beyotime, Shanghai, China) was added into each well. The optical density (OD) value was evaluated at 450 nm.

Clone formation assay

G401 cells treated with different concentrations of FKC (0, 1, 5, 10 and 15 μM) were inoculated in 12-well plates for culture. When colonies were formed, the medium was removed and methanol fixative (Beyotime) was added into the well for fixation, followed by colony staining using crystal violet (Beyotime).

5‐Ethynyl‐20‐deoxyuridine (Edu) assay

G401 cells treated with or without 10 μM FKC were inoculated in 24-well plates for culture. When the cell density reached 60%-70%, the medium was removed and EdU (Beyotime) was added for 2 h. The cells were then stained and photographed under a microscope. The percentage of EdU-positive cells was calculated using the following formula: EdU-positive cell rate (%) = (Number of EdU-positive cells / Total number of DAPI-positive cells) × 100%.

At least three randomly selected fields per sample were analyzed for quantification.

Migration and invasion assays

Migration or invasion assays were performed using 24-well plates inserted by 8-μm pore size transwell filter insert (Corning, USA) with or without pre-coated diluted Matrigel (Becton Dickinson, USA). G401 cells or infected cells were placed into the upper chamber with FBS-free medium, and medium containing 10% FBS was added into the bottom chamber. After incubation with or without 10 μM FKC in 37 °C for 24 h (migration) or 48 h (invasion), cells on the membrane were immobilized and stained with crystal violet. The cells per microscopic field (× 400) were imaged and counted in 10 randomly chosen fields.

Wound healing assay

G401 cells or infected cells were cultured in 12‐well plates with or without 10 μM FKC. After 90% cell fusion, a linear wound was created using a 200 μL pipette tip. Then, the cells were incubated in 5% low‐serum medium, and the scratches were photographed under a microscope at 0 and 24 h. The migration rate (%) was calculated as follows: [(Area at 0 h – Area at 24 h) / Area at 0 h] × 100%. Data are presented as the mean ± standard deviation (SD) from at least three independent experiments.

mRFP-GFP-LC3 adenovirus double label assay

G401 cells were infected with mRFP‐GFP‐LC3 (Hanbio, Shanghai, China) and cultured for 24 h. Then, the cells were treated with 10 μM FKC or negative control for another 12 h. Subsequently, the cells were fixed with paraformaldehyde (PFA) and nuclei were stained with DAPI (Sigma). The images of cells were taken using under a confocal microscope (TCS SP5, Leica Microsystems, Wetzlar, Germany), and autophagic flux was quantified via GFP and mRFP puncta. The number of yellow spots representing autophagic bodies and red spots representing autophagic lysosomes were counted.

Western blot analysis

Protein samples from G401 cells, infected cells and tumor tissues were extracted using Minute Total Protein Extraction Kit (Invent Biotechnologies, USA). The content of each sample was determined by BCA protein assay kit (Beyotime). Subsequently, the proteins were separated and transferred to PVDF membranes (Millipore, USA). Following by blocking with 5% skimmed milk, the membranes were incubated with primary antibodies specific to FABP4 [1:1000, #50,699, Cell Signaling Technology (CST), Danvers, USA], E-cadherin (1:1000, #3195, CST), N-cadherin (1:1000, #13,116, CST), Vimentin (1:1000, #5741, CST), P62 (1:1000, #39,749, CST), microtubule-associated protein 1 light chain 3 (LC3, 1:1000, #12,741, CST), AMPK (1:1000, #2532, CST), phosphorylated (p)-AMPK (1:1000, #2535, CST), mTOR (1:1000, #2972, CST), p-mTOR (1:1000, #2971, CST), and β-tubulin (1:1000, #2146, CST). Then, the membranes were incubated with HRP-linked goat anti-rabbit IgG secondary antibodies (1:3000, # 7074, CST). ECL (Millipore) solution was used to observe the protein band. Relative protein expression was quantified by BioImaging Systems. All Western blot analyses were performed with three independent biological replicates. Quantitative data for protein expression were obtained by normalizing the band intensity of target proteins to that of β-tubulin, and the blots shown are representative.

Co-immunoprecipitation (Co-IP) assay

Processed G401 were collected and prepared according to the manufacturer’s instructions for the Co-IP kit (Thermo Scientiffc™ Pierce™, USA). Briefly, the washed Pierce Protein A/G magnetic beads were incubated with 100 µL of either anti-p62 antibody solution or negative control IgG solution and combined with the DSS crosslinker. After washed with elution buffer and the immunoprecipitation lysis/wash buffer, the beads were incubated overnight with the cell lysate at 4 °C, followed by a final wash in pure water. Subsequently, the bound antigen was eluted and analyzed following the previously described Western blotting method.

Patient samples and ethical statement

The human “normal” kidney tissue and “nephroblastoma” tissue samples used in this study were obtained from the General Hospital of Ningxia Medical University. All research procedures involving human participants (or their legal guardians) were conducted in strict accordance with the ethical standards of the Declaration of Helsinki and were approved by the Human Research Ethics Committee of Ningxia Medical University (Approval No.: KJ-LL-2022–100).Written informed consent was obtained from all participants or their legal guardians prior to tissue sample collection. For the nephroblastoma group, tumor tissue samples were collected from pediatric patients with a pathologically confirmed diagnosis of nephroblastoma, independently verified by two senior pathologists. The normal control tissues were obtained from the non-tumor parenchyma of patients who underwent nephrectomy for non-neoplastic reasons (such as traumatic injury or benign kidney disease), and were subsequently confirmed to be histologically normal by pathological examination.Following surgical resection, all samples were immediately snap-frozen in liquid nitrogen within 30 min to preserve RNA and protein integrity, and subsequently stored at -80 °C until further analysis.

Mouse xenograft model

Five-week-old BALB/c male nude mice were purchased from Charles River (Beijing, China). Mice were adapted to the breeding environment under standard laboratory conditions (ventilated room, 25 °C ± 1 °C, 60% ± 5% humidity, 12 h light/ dark cycle) for one week before the experiment. All animal experiments conducted for this study were approved by the Laboratory Animal Ethics and Welfare Committee of the Laboratory Animal Center of Ningxia Medical University. A total of 5 × 10⁶ G401 cells infected with oeFABP4 or oeNC were suspended in 200 μL PBS mixed with Matrigel and then subcutaneously injected into nude mice to establish tumor xenograft. When tumors were visible to the naked eye, tumor-bearing mice were randomly divided into three groups (WT, oeNC + FKC and oeFABP4 + FKC) of 5 mice each. For the treatment groups, the tumor-bearing mice were given intraperitoneal injection (i.p.) of 3 mg/kg of FKC thrice weekly⁷. The control group (WT group) was given i.p. of vehicle solution (0.9% saline containing 4% DMSO and 5% Tween 80). Tumor volume was measured twice per week using vernier calipers, and the tumor volume calculated according to the formula: Volume = (Length × (Width)2)/2. After 4 weeks, mice were sacrificed, and tumors in each group were excised, weighed and photographed. Protein samples from tumor tissues were extracted and examined by western blot and the other fixed in 4% paraformaldehyde for subsequent pathological examination. All methods were performed in accordance with the relevant guideline and regulations. The animal study is reported in accordance with ARRIVE guidelines. Euthanasia was conducted via cervical dislocation subsequent to deep isoflurane anesthesia, in strict compliance with the AVMA Guidelines for the Euthanasia of Animals. When performing cervical dislocation, the weight range of the animals used is from 19.0 g to 22.3 g.

Immunohistochemistry (IHC) analysis

IHC was used to detect the expression of FABP4, Vimentin and proliferation marker (Ki67). Briefly, tumor tissue samples were fixed in 10% formalin, embedded in paraffin, cut into sections, and stained with hematoxylin and eosin (H&E) solution. Then, the sections were incubated with primary antibodies of FABP4 (1:500, #ab219595, Abcam, San Francisco, CA, USA), Vimentin (1:200, #5741, CST) and Ki67 (1:400, #12,202, CST) overnight at 4 °C. The following day, the slides were incubated with biotinylated pan-specific universal secondary antibody (Vector laboratories) for 10 min. Signals were detected by adding 3,3′-diaminobenzidine (DAB) substrate hydrogen peroxide.

Statistical analysis

All data were analyzed by SPSS 22.0. The measurement data were described as mean ± Standard Deviation (SD). The differences among groups were evaluated by a Student’s t-test (for 2 groups) or ANOVA (for more than 2 groups) followed by a LSD post hoc test. p < 0.05 was considered statistically significant.

Results

FKC inhibited the proliferation of G401 cells

Previously, FKC has shown to inhibit the growth of various kinds of tumor cells^6–9. In this study, we evaluated the role of FKC on G401 cells. G401 cells were treated with different concentrations of FKC (0, 1, 5, 10, and 15 μM), and the cell viability was detected by CCK-8 assay. Our results indicated that FKC inhibited the viability of G401 cells in a dose and time-dependent manner (Fig. 1A–B). Then, the clone formation assay was conducted after G401 cells were treated with different concentrations of FKC (0, 1, 5, 10 and 15 μM). The results showed that the cloning formation rate of G401 cells was significantly reduced with the increase of FKC concentrations (Fig. 1C). In addition, the EdU assay further demonstrated that the proliferation of G401 cells was significantly attenuated after FKC treatment (Fig. 1D). Taken together, the above findings suggested that FKC treatment could inhibit the viability and proliferation of G401 cells.

Fig. 1.

FKC inhibited the growth of G401 cells. (A) The cell viability of G401 treated with different concentrations of FKC (0, 1, 5, 10, and 15 μM) and (B) Stimulated with 10 μM FKC at different time point was detected by CCK-8 assay. (C) The clone formation assay was conducted after G401 cells were treated with different dose of FKC (0, 1, 5, 10, and 15 μM). (D) The proliferation of G401 cells treated with or without FKC was examined by EdU assay. Data are presented as mean ± SD of three independent experiments; *P < 0.05, compared to the Control group. NS not significant.

FKC inhibited the migration and invasion of G401 cells

We next investigated whether FKC could affect the migration and invasion of G401 cells. G401 cells were treated with different concentrations of FKC (0, 1, 5 and 10 μM), and the expressions of epithelial-mesenchymal transition (EMT) markers was detected by western blot analysis. The results showed that FKC upregulated the expression of E-cadherin, and downregulated the expression of N-cadherin and Vimentin in a dose-dependent manner (Fig. 2A). Additionally, the capability of G401 cells on migration and invasion under FKC treatment were evaluated by wound healing and transwell assays. The wound healing assay revealed that the rate of wound closure decreased significantly when treated with FKC, comparing to the control (Fig. 2B). The transwell assays showed that FKC exerted an inhibitory effect on the migration and invasion of G401 cells, comparing to the control. (Fig. 2C-D). The above results indicated that FKC exerted an inhibitory effect on EMT, migration and invasion of G401 cells in vitro.

Fig. 2.

FKC restrained the migration and invasion of G401 cells. (A) G401 cells were treated with different concentrations of FKC (0, 1, 5, and 10 μM), and the expression of epithelial-mesenchymal transition (EMT) markers was detected by western blot analysis. (B) G401 cells were stimulated with 10 μM FKC, and wound closure was detected by wound healing assay. (C, D) The inhibitory effect of FKC on the migration and invasion of G401 cells was detected by transwell assay. The bar graphs show the relative protein expression levels normalized to β-tubulin. Data are presented as mean ± SD of three independent experiments; *P < 0.05 , compared to the Control group.

FKC inhibited the proliferation, migration and invasion of G401 cells by inhibiting FABP4 expression

Emerging studies have showed that upregulation of FABP4 is critical for tumor proliferation, metastasis and drug resistance, which is closely correlated with poor prognosis of cancer patients¹⁸. To determine whether FABP4 is upregulated in nephroblastoma, the expression of FABP4 in nephroblastoma tissues and normal tissues was detected by western blot. The results showed that FABP4 levels were much higher in nephroblastoma tissues than in normal samples (Fig. 3A). To validate whether FKC could regulate FABP4 expression, G401 cells were treated with different concentrations of FKC (0, 1, 5, and 10 μM), and the expressions of FABP4 were evaluated by western blot. Additively, G401 cells were infected with oeFABP4 and oeNC lentivirus and then treated with 10 μM FKC. The western blot showed that FKC dose‐dependently down-regulated the expression of FABP4, compared to the control. (Fig. 3B). Furthermore, we found that oeFABP4 showed an antagonistic action against FKC, increasing FABP4 expression in FKC-treated cells significantly (Fig. 3C). The CCK-8 assay showed that oeFABP4 abolished the inhibition of FKC treatment on cell proliferation (Fig. 3D). Moreover, in FKC-treated cells, oeFABP4 resulted in lower levels of E-cadherin and higher levels of N-cadherin and Vimentin comparing to oeNC (Fig. 3E). Transwell and wound healing assays showed that oeFABP4 enhanced the migration and invasion abilities of G401 cells under the treatment of FKC comparing to oeNC (Fig. 3F-H). Taken together, the above results suggested that FKC inhibited the proliferation, migration and invasion of G401 cells by regulating FABP4 expression.

Fig. 3.

FKC suppressed growth and metastasis of G401 cells by decreasing FABP4 expression. The expression of FABP4 in nephroblastoma tissues and normal tissues was analyzed by western blot (A). G401 cells were treated with different concentrations of FKC (0, 1, 5, and 10 μM), and the expression of FABP4 was examined by Western blotting (B). G401 cells infected with oe-NC and oe-FABP4 lentivirus, then stimulated with or without FKC (10 μM). The efficiency of overexpression lentivirus was identified by Western blotting (C). The cell viability was assessed by CCK8 (D). Expression of E-cadherin, N-cadherin and Vimentin were assessed by Western blotting (E). The migration and invasion of G401 cells was were evaluated by transwell (F, G) and wound healing assays (H). The bar graphs show the relative protein expression levels normalized to β-tubulin. Data are presented as mean ± SD of three independent experiments; *P < 0.05. NS not significant.

FKC induced autophagy in G401 cells

mTOR is a well-known initiation factor involved in regulating autophagy. FKC treatment has been shown to regulate mTOR phosphorylation⁹. Thus, we hypothesized that FKC might be involved in autophagy activation. G401 cells were treated with 10 μM FKC for different time (0, 4, 8, 12, 18 and 24 h), and the expression of LC3 was detected by western blot. We found that the level of LC3II showed a trend of increasing first and then decreasing with the increase of the FKC exposure time, while the time point inducing the highest level of LC3II was 8 h (Fig. 4A). Then, G401 cells were treated with FKC and/or CQ, and the expressions of LC3 and P62 were evaluated by western blot. CQ blocks the autophagic flux at late stage by inhibiting the fusion with lysosomes, thus determining the accumulation of autophagic machinery such as LC3II and P62 proteins in treated cells²⁵. Compared with FKC treatment group, western blot showed that the co-treatment of FKC and CQ further increased the ratio of LC3II, simultaneously increased protein expression of P62 in G401 cells (Fig. 4B), indicating impaired autophagic degradation due to lysosomal blockade. mRFP-GFP-LC3 assay revealed that FKC increased yellow puncta (autophagosomes) and red puncta (autolysosomes) (Fig. 4C). The increase in red puncta (autolysosomes) is a key indicator of successful autophagosome maturation and degradation. Collectively, these findings demonstrate that FKC triggers the initiation of autophagy and facilitates the complete autophagic flux, leading to an increase in both autophagosome formation and subsequent lysosomal degradation.

Fig. 4.

FKC induced autophagy in G401 cells. G401 cells were treated with 10 μM FKC for different time (0, 4, 8, 12, 18, and 24 h), and the expression of LC3 was detected by western blot (A). G401 cells were treated with 10 μM FKC and/or 5 µM CQ for 12 h, and the expressions of LC3 and P62 were quantified by western blot (B). The autophagy flux after different treatments was observed by mRFP-GFP-LC3 adenovirus double label assay (C). The bar graphs show the relative protein expression levels normalized to β-tubulin. Data are presented as mean ± SD of three independent experiments; *P < 0.05. NS not significant.

FKC induced autophagy mediated the degradation of FABP4

To establish the functional role of the interaction between FKC and FABP4 in the autophagic program of G401 cells, sh-Atg5 was used to inhibit the autophagy in G401 cells. Compared with sh-NC + FKC, Atg5 knockdown reduced LC3II expression in FKC-treated G401 cells (Fig. 5A). Then, the expressions of FABP4 after sh-Atg5 infection were evaluated by western blot analysis. The results showed that the decrease of FABP4 expression in G401 cells after FKC treatment was successfully abolished by Atg5 knockdown (Fig. 5B). Furthermore, Co-IP experiments were utilized to determine the role of autophagy mediated FABP4 degradation by analyzing the interaction between FABP4 and autophagic adaptor protein P62 in G401 stimulated with FKC. The results indicated that the obvious interaction between p62 and FABP4 in FKC -treated G401 (Fig. 5C). Collectively, these findings suggested that FABP4 protein degradation relies on autophagy induced by FKC in G401, and autophagy suppression could alleviate the effects of FKC on FABP4 expression.

Fig. 5.

Inhibition of autophagy attenuated the inhibitory effect of FKC on FABP4 expression. G401 cells transfected with sh-Atg5 or sh-NC were treated with FKC. The expression of autophagy-related proteins was detected by western blot (A). The expressions of FABP4 after sh-Atg5 transfection were evaluated by western blot analysis (B). The interaction between p62 and FABP4 in FKC -treated G401 was evaluated by Co-IP assay (C). The bar graphs show the relative protein expression levels normalized to β-tubulin. Data are presented as mean ± SD of three independent experiments; *P < 0.05.

FKC induced autophagy in G401 cells via the AMPK/mTOR signaling pathway

AMPK is the central pathway that regulates autophagy initiation and autophagosome formation²⁶. Thus, we focused on this pathway to clarify the mechanism of autophagy regulation by FKC in G401 cells. By western blot analysis, we observed that FKC upregulated p-AMPK/AMPK and subsequently downregulated p-mTOR/mTOR in G401 cells comparing to NC (Fig. 6A). BAY-3827 is a selective AMPK inhibitor. G401 cells were treated with FKC, FKC + BAY-3827 or negative control for 24 h, followed by western blot analysis to detect the expressions of autophagic markers and FABP4. We found that BAY-3827 showed an antagonistic action against FKC to increase the expressions of p-mTOR and FABP4, and decrease the expressions of p-AMPK and LC3II (Fig. 6B). Taken together, our findings demonstrated that FKC downregulated the expression of FABP4 in G401 cells by autophagy via AMPK/mTOR pathway.

Fig. 6.

FKC induced autophagy in G401 cells via the AMPK/mTOR signaling pathway. G401 cells were treated with or without FKC for 30 min, and the expressions of proteins in AMPK/mTOR pathway were detected by western blot (A). G401 cells were treated with FKC and/or BAY-3827 for 24 h, followed by western blot analysis to detect the expressions of autophagic markers and FABP4 (B). The bar graphs show the relative protein expression levels normalized to β-tubulin. Data are presented as mean ± SD of three independent experiments; *P < 0.05.

FKC inhibited the growth of nephroblastoma in vivo

Tumor xenografts were constructed to investigate the effect of FKC on nephroblastoma growth in vivo. We found that the tumor growth was significantly repressed in the oeNC and oeFABP4 combined with FKC administration groups comparing to the control (Fig. 7A-C). Furthermore, the tumor volume and tumor weight in oeFABP4 group were significantly larger than those in oeNC group under FKC treatment (p < 0.05, Fig. 7A-C), indicating that FABP4 could reverse effects of FKC on tumor growth. Hematoxylin–eosin staining revealed that tumors had a loose structure, with increased infammatory cell infltration in tumor tissues treated with FKC, while oeFABP4 reversed this phenomenon (Fig. 7D). The IHC analysis showed that the expression of Ki67, FABP4 and Vimentin in oeFABP4 group was higher than that in oeNC group (Fig. 7D), consistent with the tendency of tumor growth in vivo. The western blot analysis showed that oeFABP4 group expressed higher levels of FABP4 and Vimentin, while lower levels of E-cadherin compared to oeNC group (p < 0.05, Fig. 7E). Taken together, these results suggested that the level of FABP4 expression determined the function of FKC inhibited the growth of nephroblastoma in vivo.

Fig. 7.

FKC inhibited the growth and metastasis of nephroblastoma in vivo. Nude mice were subcutaneously injected with normal and oeNC or oeFABP4 infected G401 cells. One week after cell injection, mice in the treatment group were intraperitoneally administered 3 mg/kg FKC thrice a week for 4 weeks; control mice received the same volume of normal saline. Images of tumor masses from each group (A). The value of tumor volume and weight was quantified in each group (B–C). Tumor tissue sections stained with hematoxylin–eosin, the expression of Ki67, FABP4 and Vimentin by immunohistochemistry (D). Western blot was performed to detect the expression of FABP4, E-Cadherin and Vimentin in animal samples (E). The bar graphs show the relative protein expression levels normalized to β-tubulin. All data are presented as mean ± SD. *P < 0.05, NS not significant.

Discussion

Although the prognosis of most nephroblastoma patients is well, there is still 10% of patients having poor survival due to cancer recurrence and metastasis³. Finding a novel therapeutic intervention is necessary for nephroblastoma management. FKC is a naturally occurring chalcone, which has been reported to inhibit the growth of several cancers. In the present study, FKC was demonstrated to inhibit the proliferation, migration, and invasion of nephroblastoma cells. FABP4, a novel adipokine, was found to be regulated by FKC. For mechanisms, we found that FKC exerted its anti-tumor effects by autophagy-mediated degradation of FABP4 via the activation of AMPK/mTOR pathway.

Chalcone has been shown to exhibit anticancer activity by targeting a variety of molecular pathways, such as apoptosis, cell cycle, and NF-kappa B, with low toxicity to normal cells⁹. FKC is a naturally occurring chalcone found in Kava. Recently, FKC has shown to inhibit the growth of several kinds of cancer cells including human colon carcinoma cells⁵, liver cancer cells⁸, and nasopharyngeal carcinoma cells⁹. In the present study, we found that FKC could significantly inhibit the viability, clone formation and proliferation of G401 cells in vitro, exhibiting a similar inhibitory effect on G401 cells like other cancer cell lines^5,8,9.

EMT is a process in which epithelial cancer cells lose their typical epithelial characteristics and acquire mesenchymal features, which is thought to be closely related to the invasion and metastasis progression of tumors²⁷. This transition is characterized by the increase of mesenchymal markers such as N-cadherin and vimentin; and the downregulation of the epithelial markers such as E-cadherin. Previous studies have indicated that the inhibition of EMT could repress the progression of nephroblastoma^28,29. In the present study, we found that FKC could upregulate the expression of E-cadherin, and downregulate the expression of N-cadherin and Vimentin, indicating that FKC exerted an inhibitory effect on EMT. Furthermore, wound healing and transwell assays further demonstrated that FKC could inhibit the migration and invasion of G401 cells in vitro. The similar results were obtained from the in vivo study. Thus, our results indicated that FKC possessed the ability to inhibit the malignant progression of nephroblastoma.

FABP4 are members of the adipokine family of multifunctional proteins that are related to fatty acid metabolism. Emerging studies have shown that FABP4, which is critical for tumor proliferation, metastasis and drug resistance, is upregulated in several cancers including breast, prostate and ovarian cancers^19,30,31. The upregulation of FABP4 has been reported to be closely correlated with poor prognosis of cancer patients¹⁸. In this study, we found that FABP4 was also upregulated in nephroblastoma tissues. It has been reported that FABP4 is involved in the proliferation and metastasis of cancer cells^19,20. To validate whether FABP4 participated in the regulation role of FKC on nephroblastoma cells, we firstly investigated the effect of FKC on FABP4 expression. As expected, we found that FKC suppressed the expression of FABP4 at the protein level in a dose-dependent manner. Subsequently, we infected oeFABP4 in G401 cells to overexpress FABP4 and observed the effects of FABP4 on FKC-treated cells. Our results suggested that oeFABP4 abolished the inhibition of FKC treatment on cell proliferation, migration and invasion, indicating FABP4 participated in the regulation role of FKC on nephroblastoma cells. Moreover, our in vivo study confirmed that FABP4 could reverse effects of FKC on tumor growth and metastasis in tumor models, consistent with the in vitro findings.

Autophagy is an intracellular self-degradative mechanism that is critical for maintaining cellular homeostasis during stress conditions. It has been reported that autophagy regulates multiple processes involved in cancer development, including cell apoptosis, cell metastasis, cell proliferation and cell cycle³². Deregulation of autophagy has been found in various cancers including nephroblastoma³³. Targeting autophagy has been suggested as a promising approach to treat nephroblastoma^33,34. mTOR is a highly conserved kinase important for autophagy regulation, which plays a negative role in autophagy³⁵. In nasopharyngeal carcinoma, FKC has been reported to regulate mTOR phosphorylation⁹. Furthermore, previous studies have demonstrated that other chalcones, such as flavokawain B and xanthoangelol, repressed tumor growth through inducing autophagy^36–38. Thus, it is rational to hypothesize that FKC has the potential to modulate autophagy in cancer cells. As expected, our study successfully demonstrated that FKC had autophagy induction effect on G401 cells. Atg5 in an important autophagy-related protein, which is indispensable for autophagic vesicle formation³⁹. Knocking down or knocking out Atg5 can result in downregulation or total inhibition of autophagy³⁹. In the present study, sh-Atg5 was used to knockdown Atg5 and inhibit autophagy. We found that the decrease of FABP4 expression in G401 cells after FKC treatment was successfully abolished by Atg5 knockdown, providing direct genetic evidence that the degradation of FABP4 is dependent on a functional autophagic pathway.

To further elucidate the molecular mechanism by which FABP4 is targeted for degradation, we investigated the role of the selective autophagy receptor p62. Co-immunoprecipitation analysis revealed a direct interaction between FABP4 and p62 in FKC-treated cells. This interaction is critical, as p62 acts as a bridge that recognizes ubiquitinated cargo (such as FABP4) and delivers it to the growing autophagosome by binding to LC3. Our results suggest that FKC-induced autophagy does not occur through bulk degradation but rather through a p62-mediated selective autophagic process that specifically targets FABP4 for destruction.

AMPK/mTOR pathway is an important signaling pathway participating in autophagy. mTOR is a highly conserved kinase important for autophagy regulation⁴⁰. Previous studies demonstrated that mTOR suppressed the function of ATG1, an autophagy-initiating kinase, and negatively regulated autophagy⁴¹. AMPK is a key energy sensor and regulates cellular metabolism, which inhibits mTOR expression and its substrate p70S6K⁴². During energy depletion, AMPK is activated and mTOR is suppressed, which results in autophagy activation⁴³. AMPK and FABP4 are both involved in metabolic homeostasis. It has been reported that AMPK activation could down-regulate the expressions of adipogenic proteins including FABP4^22,23. In the present study, FKC was found to active AMPK/mTOR pathway in G401 cells in vitro. Using BAY-3827 to inhibit AMPK/mTOR pathway, the effects of FKC on FABP4 expression and autophagy were alleviated. Furthermore, we also found that FKC could active AMPK signaling pathways like other chalcones reported in previous studies¹⁶. Taken together, our findings demonstrate that FKC downregulates the expression of FABP4 in G401 cells through an AMPK/mTOR-autophagy-p62 axis: FKC activates AMPK and inhibits mTOR signaling, thereby inducing autophagy initiation; concurrently, this process facilitates the recognition of FABP4 by the autophagy receptor p62, leading to its selective degradation via autophagy.

This study has several limitations. First, we only used the G401 nephroblastoma cell line to evaluate the anti-tumor effects of FKC. Although G401 is a well-established model for pediatric nephroblastoma, the tumor is highly heterogeneous, and our results may not fully represent all subtypes. Future studies should validate these findings in other nephroblastoma cell lines, such as WiT49 and HFWT, to comprehensively assess the efficacy and underlying mechanisms of FKC. Second, we acknowledge the absence of a control group consisting of oeFABP4 animals without FKC treatment. This omission limits our ability to fully delineate the independent effect of FABP4 overexpression on tumor progression, separate from the therapeutic effect of FKC. Future studies should include this control group to provide a more comprehensive understanding of the specific role of FABP4.

In summary, this study is the first to demonstrate that FKC as a nature chalcone was involved in inhibiting the proliferation, migration and invasion of G401 cells by autophagy-mediated degradation of FABP4. Furthermore, AMPK/mTOR pathway contributed to the FKC induced autophagy and FABP4 expression. Thus, this study expands current understanding of FKC, providing a new potential treatment or adjuvant therapy for nephroblastoma.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

H.L. contributed the original draft preparation. Y.T. provides the conceptualization. G.L., Z.S. and S.Z. contributed to the interpretation of the data. W.F. reviewed and edited the article. Y.G. provided the methodology. Q.L. provided funding for the project. X.M. supervised work and wrote manuscripts. All the authors have contributed to the paper and have reviewed the manuscript and agree with its contents.

Funding

This work was supported by grants from the Ningxia Natural Science Foundation Project (2023AAC03727). Ningxia Natural Science Joint Fund (2022AAC03573).

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.